Method for capturing and purification of biologics

Abstract

To provide methods for the continuous production, capture, and purification of biologics such as recombinant proteins.SOLUTION: A method for manufacturing biologics is provided. The method comprises: culturing host cells that secrete biologics in a bioreactor; removing media and / or cell suspension from the bioreactor; processing the media and / or cell suspension to separate a filtrate containing the biologics; loading the filtrate onto at least two capture columns; eluting a first biologic product from the capture columns; loading the first biologic product onto one or more purification columns; and eluting a second biologic product from the one or more purification columns; wherein the bioreactor has a bioreactor volume, the at least two capture columns have a total capture column volume, and a ratio of the bioreactor volume to the total capture column volume is in the range of about 500:1 to about 10:1.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] Principles and embodiments of the present invention relate generally to the production of biologics, particularly lysosomal enzymes, with a high content of mannose-6-phosphate. [Background technology]

[0002] Lysosomal storage disorders are a group of autosomal recessive disorders characterized by the accumulation of molecular substrates, such as glycosphingolipids, glycogen, or mucopolysaccharides, in intracellular compartments called lysosomes. Individuals with these disorders possess mutated genes encoding enzymes defective in catalyzing the hydrolysis of one or more of these substrates, resulting in their accumulation in lysosomes. For example, Pompe disease, also known as acid maltase deficiency or type II glycogen storage disease, is one of several lysosomal storage disorders. Other examples of lysosomal disorders include Gaucher disease, GM1-gangliosidosis, fucosidosis, mucopolysaccharidoses, Hurler syndrome, Niemann-Pick A and B disease, and Fabry disease. Pompe disease is also classified as a neuromuscular or metabolic myopathy.

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

[0004] Recent treatment options for lysosomal storage diseases include enzyme replacement therapy (ERT) using recombinant enzymes. For example, one treatment option for Pompe disease is ERT using recombinant human acid α-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 replacement enzymes via intravenous infusion. The replacement enzyme is then transported through the circulation and enters intracellular lysosomes, where it acts to break down accumulated substrates (e.g., glycogen), compensating for the deficient activity of the endogenous defective mutant enzyme and thereby alleviating the symptoms of the disease. In patients with infantile-onset Pompe disease, treatment with alglucosidase alfa has been shown to significantly improve survival compared with historical controls, and in late-onset Pompe disease, alglucosidase alfa has been shown to have modest, but statistically significant, effects on 6-minute walk test (6MWT) and forced vital capacity (FVC) outcomes compared with placebo.

[0005] However, most subjects' condition remains stable or continues to worsen while undergoing treatment with alglucosidase alfa. The reasons for the apparently suboptimal effects of alglucosidase alfa-based ERT are unclear but may be due in part to the progressive nature of the underlying muscle lesion or the inadequate tissue targeting of current ERTs. For example, the infused enzyme is not stable at neutral pH, including that of plasma (approximately pH 7.4), and may be irreversibly inactivated in the circulation. Furthermore, infused alglucosidase alfa exhibits poor uptake in critical disease-related muscle, likely due to insufficient glycosylation with mannose-6-phosphate (M6P) residues. Such residues bind to the cation-independent mannose-6-phosphate receptor (CIMPR) on the cell surface, allowing the enzyme to enter cells and lysosomes. Therefore, high doses of enzyme may be required for effective treatment to ensure sufficient amounts of active enzyme reach the lysosomes, making treatment costly and time-consuming.

[0006] rhGAA has seven potential N-linked glycosylation sites. Because of the heterogeneity of N-linked oligosaccharide (N-glycan) types present at each glycosylation site, rhGAA consists of a complex mixture of proteins and N-glycans with varying binding affinities for the M6P receptor and other carbohydrate receptors. High-mannose-containing rhGAA with N-glycans bearing one M6P group (mono-M6P) binds to CIMPR with low affinity (approximately 7,000 nM), whereas 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 bisM6P, 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 the mannose receptor, resulting in non-productive clearance of ERT (Figure 2B).

[0007] Other types of N-glycans, glycoconjugates containing galactose and sialic acid, are also present in rhGAA. Because complex N-glycans are not phosphorylated, they have no affinity for CIMPR. However, complex N-glycans with exposed galactose residues have moderate to high affinity for the asialoglycoprotein receptor on hepatocytes, leading to rapid, nonproductive clearance of rhGAA (Figure 2B). Summary of the Invention [Problem to be solved by the invention]

[0008] Current manufacturing methods used to make conventional rhGAA, such as Myozyme®, Lumizyme®, or alglucosidase alfa, have not significantly increased the content of M6P or bis-M6P because the processing of cellular carbohydrates is inherently complex and extremely difficult to engineer. Therefore, further improvements in enzyme replacement therapy for the treatment of Pompe disease, including new methods for producing, capturing, and purifying rhGAA, are needed.

[0009] Similarly, other recombinant proteins targeted to lysosomes, such as other lysosomal enzymes, also bind to CIMPR. However, current production methods used to generate other conventional recombinant proteins targeted to lysosomes do not provide recombinant proteins with high M6P or bis-M6P content. Therefore, further improvements are needed in the production, capture, and purification methods of these other recombinant proteins. [Means for solving the problem]

[0010] One aspect of the present invention relates to a method for producing a biologic. In various embodiments of this aspect, the method includes culturing host cells in a bioreactor and loading a biologic-containing fluid (e.g., filtrate) onto at least two capture columns, the at least two capture columns having a total capture column volume, wherein the ratio of bioreactor volume to total capture column volume is in the range of about 500:1 to about 10:1, e.g., about 100:1 to about 20:1. In various embodiments of this aspect, the total capture column residence time (i.e., the quotient of the total capture column volume and the volumetric flow rate loading the capture columns) is in the range of about 0.5 minutes to 200 minutes, e.g., about 10 to 70 minutes.

[0011] In one or more embodiments, the method includes culturing host cells in a bioreactor that produce and optionally secrete a biologic; removing the culture medium and / or cell suspension from the bioreactor; processing the culture medium and / or cell suspension to isolate a filtrate containing the biologic; loading the filtrate onto at least two capture columns to capture the biologic; eluting a first biological product from the at least two capture columns; loading the first biological product onto one or more purification columns; and eluting a second biological product from the one or more purification columns, wherein the bioreactor has a bioreactor volume and the at least two capture columns have a total capture column volume, and the ratio of the bioreactor volume to the total capture column volume is in the range of about 500:1 to about 10:1, e.g., about 100:1 to about 20:1. Alternatively, in one or more embodiments, the biologic is not secreted but is removed after lysing the cells.

[0012] In one or more embodiments, the biologic comprises one or more of a recombinant protein, a viral particle, or an antibody.

[0013] In one or more embodiments, the recombinant protein is a secreted protein, a membrane protein, or an intracellular protein produced by a host cell. In one or more embodiments, the recombinant protein is separated from the cells and / or organelles into a filtrate. In one or more embodiments, the filtrate is separated by filtration or centrifugation.

[0014] In one or more embodiments, at least two trap columns are loaded in series to provide for sequential loading of the filtrate onto the at least two trap columns.

[0015] In one or more embodiments, the filtrate is loaded onto at least two trapping columns at a filtrate loading rate ranging from about 0.5 to about 100 column volumes (CV) / hr, such as from about 1 to about 40 CV / hr.

[0016] In one or more embodiments, the filtrate is loaded onto at least two trap columns to provide a trap column loading time of less than 48 hours, for example less than 24 hours, for each trap column.

[0017] In one or more embodiments, the biologic comprises a recombinant human lysosomal protein.

[0018] In one or more embodiments, the at least two capture columns comprise at least two anion exchange chromatography (AEX) columns. In one or more embodiments, the at least two capture columns comprise at least two affinity chromatography columns. The affinity chromatography columns can be one or more of a Protein A column and a Protein Z column. In one or more embodiments, the at least two capture columns comprise at least two cation exchange chromatography (CEX) columns. In one or more embodiments, the at least two capture columns comprise at least two immobilized metal affinity chromatography (IMAC) columns. In one or more embodiments, the at least two capture columns comprise at least two size exclusion chromatography columns. In one or more embodiments, the at least two capture columns comprise at least two hydrophobic interaction chromatography (HIC) columns.

[0019] In one or more embodiments, the one or more purification columns comprise one or more anion exchange chromatography (AEX) columns. In one or more embodiments, the one or more purification columns comprise one or more affinity chromatography columns. The affinity chromatography column can be one or more of a Protein A column and a Protein Z column. In one or more embodiments, the one or more purification columns comprise one or more cation exchange chromatography (CEX) columns. In one or more embodiments, the one or more purification columns comprise one or more immobilized metal affinity chromatography (IMAC) columns. In one or more embodiments, the one or more purification columns comprise one or more size exclusion chromatography columns. In one or more embodiments, the one or more purification columns comprise one or more hydrophobic interaction chromatography (HIC) columns. In one or more embodiments, the one or more purification columns comprise one or more immobilized metal affinity chromatography (IMAC) columns.

[0020] In one or more embodiments, the second biological product is eluted from the one or more purification columns within 48 hours of removing the medium and / or cell suspension from the bioreactor.

[0021] In one or more embodiments, the one or more purification columns have a total purification column volume, and the ratio of bioreactor volume to total purification column volume ranges from about 5,000:1 to about 50:1.

[0022] In one or more embodiments, the ratio of total capture column volume to total purification column volume ranges from about 20:1 to about 1:1.

[0023] Another aspect of the present disclosure describes a method for producing a recombinant human lysosomal protein. In some embodiments, the method includes culturing host cells that produce the recombinant human lysosomal protein in a bioreactor, removing culture medium and / or a cell suspension from the bioreactor, processing the culture medium and / or cell suspension to separate a filtrate containing the lysosomal protein, loading the filtrate onto at least two anion exchange chromatography (AEX) columns to capture the lysosomal protein, eluting a first biological product from the at least two AEX columns, loading the first biological product onto one or more immobilized metal affinity chromatography (IMAC) columns, and eluting a second biological product from the one or more IMAC columns, wherein the bioreactor has a bioreactor volume, the at least two AEX columns have a total AEX column volume, and the ratio of the bioreactor volume to the total AEX column volume is in the range of about 500:1 to about 10:1, for example, a ratio of about 100:1 to about 20:1.

[0024] In one or more embodiments, the lysosomal protein is a secreted protein, a membrane protein, or an intracellular protein produced by a host cell. In some embodiments, the intracellular protein is separated into a filtrate by lysing the cells to prepare a cell lysate. In some embodiments, the cell lysate is separated from the filtrate by filtration or centrifugation.

[0025] In some embodiments, at least two AEX columns are loaded in series to provide for sequential loading of the filtrate onto the at least two AEX columns for producing recombinant human lysosomal proteins.

[0026] In one or more embodiments, the filtrate is loaded onto at least two AEX columns at a filtrate loading rate ranging from about 0.5 to about 100 column volumes (CV) / hr, such as from about 1 to about 40 CV / hr.

[0027] In some embodiments, the filtrate is loaded onto at least two AEX columns to provide an AEX loading time of less than 48 hours, for example less than 24 hours, for each AEX column.

[0028] In some embodiments, each AEX column has a column volume of 50 L or less.

[0029] In one or more embodiments, the second biological product is eluted from the one or more IMAC columns within 48 hours of removing the medium and / or cell suspension from the bioreactor.

[0030] In one or more embodiments, the one or more IMAC columns have a total IMAC column volume, and the ratio of bioreactor volume to total IMAC column volume ranges from about 5,000:1 to about 50:1.

[0031] In some embodiments, the ratio of total AEX column volume to total IMAC column volume ranges from about 20:1 to about 1:1.

[0032] In some embodiments, each IMAC column has a column volume of 20 L or less.

[0033] In one or more embodiments, the method further comprises storing the second biological. In one or more embodiments, the second biological is stored at a temperature between 0°C and 10°C for a period of 24 hours to 105 days. In one or more embodiments, the second biological is stored for up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 days. In one or more embodiments, the second biological is stored at a temperature between 15°C and 30°C for a period of 1 hour to 3 days.

[0034] In one or more embodiments, the method further includes loading the second biological product onto a third chromatography column; and eluting the third biological product from the third chromatography column. In one or more embodiments, the third chromatography column is selected from an anion exchange chromatography (AEX) column, an affinity chromatography column, a cation exchange chromatography (CEX) column, an immobilized metal affinity chromatography (IMAC) column, a size exclusion chromatography (SEC) column, and a hydrophobic interaction chromatography (HIC) column.

[0035] In one or more embodiments, the filtrate is separated by filtering the medium and / or cell suspension through one or more of alternating tangential flow filtration (ATF) and tangential flow filtration (TFF).

[0036] In one or more embodiments, the method further comprises inactivating viruses in one or more of the first biological product, the second biological product, and the third biological product.

[0037] In one or more embodiments, the method further comprises filtering the second biological product or the third biological product to provide a filtered product and filling a vial with the filtered product.

[0038] In one or more embodiments, the method further comprises lyophilizing the filtered product.

[0039] In one or more embodiments, the biologic comprises rhGAA. In one or more embodiments, the rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

[0040] In one or more embodiments, the host cells comprise Chinese hamster ovary (CHO) cells. In one or more embodiments, the host cells comprise CHO cell lines GA-ATB-200 or ATB-200-001-X5-14, or subcultures thereof.

[0041] In one or more embodiments, (i) at least 90% of the first biological, or the second biological, or the third biological binds to CIMPR, and / or (ii) at least 90% of the first biological, or the second biological, or the third biological contain N-glycans bearing mono-M6P or bis-M6P.

[0042] In one or more embodiments, rhGAA contains seven potential N-glycosylation sites, at least 50% of the rhGAA molecules contain N-glycan units having two mannose-6-phosphate residues at the first site, at least 30% of the rhGAA molecules contain N-glycan units having one mannose-6-phosphate residue at the second site, at least 30% of the rhGAA molecules contain N-glycan units having two mannose-6-phosphate residues at the fourth site, and at least 20% of the rhGAA molecules contain N-glycan units having one mannose-6-phosphate residue at the fourth site.

[0043] In one or more embodiments, 40% to 60% of the N-glycans in rhGAA are complex-type N-glycans; and the rhGAA contains 3.0 to 5.0 moles of M6P residues per mole of rhGAA.

[0044] Another aspect of the invention relates to a method for producing a biologic. In various embodiments of this aspect, the method includes culturing cells in a bioreactor and loading at least two AEX columns with a biologic-containing fluid, the at least two AEX columns having a total AEX column volume, wherein the ratio of bioreactor volume to total AEX column volume is in the range of about 500:1 to about 10:1, e.g., about 100:1 to about 20:1. In various embodiments of this aspect, the total AEX column residence time (i.e., the quotient of the total AEX column volume and the volumetric flow rate loading the AEX columns) is in the range of about 0.5 minutes to 200 minutes, e.g., about 10 to 70 minutes.

[0045] In one or more embodiments, the method includes culturing host cells that secrete a recombinant human lysosomal protein in a bioreactor; removing culture medium from the bioreactor; filtering the culture medium to provide a filtrate; loading the filtrate onto at least two AEX columns to capture the lysosomal protein; eluting a first protein product from the at least two AEX columns; loading the first protein product onto one or more IMAC columns; and eluting a second protein product from the one or more IMAC columns, wherein the bioreactor has a bioreactor volume and the at least two AEX columns have a total AEX column volume, and the ratio of the bioreactor volume to the total AEX column volume is in the range of about 500:1 to about 10:1, e.g., a ratio of about 100:1 to about 20:1.

[0046] In one or more embodiments, at least two AEX columns are packed in series to provide for sequential loading of the filtrate onto the at least two AEX columns.

[0047] In one or more embodiments, the filtrate is loaded onto at least two AEX columns at a filtrate loading rate ranging from about 0.5 to about 100 CV / hr, such as from about 1 to about 40 CV / hr.

[0048] In one or more embodiments, the filtrate is loaded onto at least two AEX columns to provide an AEX loading time of less than 48 hours, for example less than 24 hours, for each AEX column.

[0049] In one or more embodiments, each AEX column has a column volume of 50 L or less.

[0050] In one or more embodiments, the second protein product is eluted from the one or more IMAC columns within 48 hours of removing the medium from the bioreactor.

[0051] In one or more embodiments, the one or more IMAC columns have a total IMAC column volume, and the ratio of bioreactor volume to total IMAC column volume ranges from about 5,000:1 to about 50:1.

[0052] In one or more embodiments, the ratio of total AEX column volume to total IMAC column volume ranges from about 20:1 to about 1:1.

[0053] In one or more embodiments, each IMAC column has a column volume of 20 L or less.

[0054] In one or more embodiments, the method further comprises storing the second protein product. In one or more embodiments, the second protein product is stored at a temperature between 0°C and 10°C for a period of 24 hours to 105 days. In one or more embodiments, the second protein product is stored for up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 days. In one or more embodiments, the second protein product is stored at a temperature between 15°C and 30°C for a period of 1 hour to 3 days.

[0055] In one or more embodiments, the method further comprises loading the second protein product onto a third chromatography column; and eluting the third protein product from the third chromatography column. In one or more embodiments, the third chromatography column is selected from a CEX column and a SEC column.

[0056] In one or more embodiments, the filtration of the medium is selected from ATF and TFF.

[0057] In one or more embodiments, the method further comprises inactivating virus in one or more of the first protein product, the second protein product, and the third protein product.

[0058] In one or more embodiments, the method further comprises filtering the second protein product or the third protein product to provide a filtered product, and filling the filtered product into vials.

[0059] In one or more embodiments, the method further comprises lyophilizing the filtered product.

[0060] In one or more embodiments, the recombinant human lysosomal protein is rhGAA. In one or more embodiments, the rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2.

[0061] In one or more embodiments, the host cells comprise CHO cells. In one or more embodiments, the host cells comprise CHO cell lines GA-ATB-200 or ATB-200-001-X5-14, or subcultures thereof.

[0062] In one or more embodiments, (i) at least 90% of the first protein product, or the second protein product, or the third protein product binds to CIMPR, and / or (ii) at least 90% of the first protein product, or the second protein product, or the third protein product contain N-glycans bearing mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P).

[0063] In one or more embodiments, rhGAA contains seven potential N-glycosylation sites, at least 50% of the rhGAA molecules contain N-glycan units having two mannose-6-phosphate residues at the first site, at least 30% of the rhGAA molecules contain N-glycan units having one mannose-6-phosphate residue at the second site, at least 30% of the rhGAA molecules contain N-glycan units having two mannose-6-phosphate residues at the fourth site, and at least 20% of the rhGAA molecules contain N-glycan units having one mannose-6-phosphate residue at the fourth site.

[0064] In one or more embodiments, 40% to 60% of the N-glycans in rhGAA are complex-type N-glycans; and the rhGAA contains 3.0 to 5.0 moles of M6P residues per mole of rhGAA.

[0065] Another aspect of the invention pertains to a biologic produced by any of the methods described herein.

[0066] Another aspect of the invention relates to a pharmaceutical composition comprising a biological agent and a pharmaceutically acceptable carrier.

[0067] Yet another aspect of the present invention relates to a method for treating a lysosomal storage disease, comprising administering the pharmaceutical composition to a patient in need thereof.

[0068] In one or more embodiments, the lysosomal storage disease is Pompe disease and the biologic is rhGAA. In one or more embodiments, the patient is co-administered with a pharmacological chaperone for α-glucosidase within 4 hours of administering the pharmaceutical composition containing the rhGAA product. 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.

[0069] Various embodiments are listed below, and it will be understood that the embodiments listed below may be combined in other suitable combinations in accordance with the scope of the present invention, not just those listed below.

[0070] Further features of the present invention will become apparent from the following specification and accompanying drawings. [Brief explanation of the drawings]

[0071] [Figure 1A] Non-phosphorylated high mannose glycans, mono-M6P glycans and bis-M6P glycans are shown. [Figure 1B] The chemical structure of the M6P group is shown. [Figure 2A] Productive targeting of rhGAA via M6P-bearing glycans to target tissue (e.g., muscle tissue of subjects with Pompe disease) is described. [Figure 2B] This could be explained by non-productive drug clearance to non-target tissues (e.g., liver and spleen) or binding of non-M6P glycans to non-target tissues. [Figure 3A] 1 is a graphical representation of the CIMPR receptor (also known as the IGF2 receptor) and the domains of this receptor. [Figure 3B] 1 shows the binding affinity (nanomolar) of glycans bearing bis- and mono-M6P to CIMPR, the binding affinity of high-mannose glycans to the mannose receptor, and the binding affinity of desialylated complex glycans to the asialoglycoprotein receptor. RhGAA bearing glycans bearing M6P and bis-M6P can productively bind to CIMPR on muscle. [Figure 4] 1 shows a DNA construct for transforming CHO cells with DNA encoding rhGAA. CHO cells were transformed with a DNA construct encoding rhGAA. [Figure 5] FIG. 1 is a schematic diagram of an exemplary prior art process for recombinant protein production, capture, and purification. [Figure 6] FIG. 1 is a schematic diagram of an exemplary process for the production, capture, and purification of a biologic according to one or more embodiments of the present invention. [Figure 7] An exemplary first sequence of events in the production, capture and purification of a biologic is described, where filtrate containing the biologic is loaded onto capture column 1. [Figure 8] Described below is a second exemplary sequence of events in the production, capture, and purification of a biologic, in which a captured biologic in capture column 1 is eluted and loaded onto a purification column. During this process, the filtrate containing the biologic is loaded onto capture column 2. [Figure 9]An exemplary third sequence of events in the production, capture, and purification of a biologic is described, in which the captured biologic in capture column 2 is eluted and loaded onto a purification column. During this process, the filtrate containing the biologic is loaded onto capture column 1. [Figure 10] An exemplary fourth sequence of events is described in the production, capture, and purification of a biologic, where the biologic is eluted from a purification column. [Figure 11] An exemplary sequence of events in the production, capture and purification of rhGAA is described, where an AEX column is used as the capture column and an IMAC column is used as the purification column. [Figure 12] FIG. 1 is a schematic diagram of another exemplary process for the production, capture, and purification of a biologic according to one or more embodiments of the present invention. [Figure 13] 1 shows the elution profiles from two AEX trapping columns in a batch purification process. [Figure 14] 1 shows the elution profiles from two AEX capture columns in a sequential purification process. [Figure 15] 1 shows a comparison of the elution profiles from an IMAC purification column between batch and continuous purification processes. [Figure 16] 16 shows a magnified image of the elution profile from the IMAC purification column between the batch and continuous purification processes from FIG. 15. DETAILED DESCRIPTION OF THE INVENTION

[0072] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or carried out in various ways.

[0073] The present disclosure describes methods for producing, capturing, and purifying biologics. In one or more embodiments, the biologic comprises one or more of a recombinant protein, a viral particle, or an antibody. In one or more embodiments, the recombinant protein is targeted to the lysosome. In one or more embodiments, the recombinant protein is recombinant human α-galactosidase A (rhGAA).

[0074] In one or more embodiments, the recombinant protein undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine may form pyroglutamic acid. As another example, asparagine residues may undergo deamidation 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.

[0075] Although specific reference is made to rhGAA, those skilled in the art will appreciate that the methods and systems described herein can be used to produce, capture, and purify other recombinant proteins. In various embodiments, other recombinant proteins also target the lysosome, including, but not limited to, the lysosomal enzyme α-galactosidase A. The methods and systems described herein can also be used to produce, capture, and purify other biologics, such as antibodies and viral particles (e.g., for gene therapy).

[0076] Some current methods for the production of rhGAA use large AEX columns, such as AEX columns with dimensions of 1 meter diameter x 30 cm bed height (i.e., each AEX column volume = 236 L). These large AEX columns have long packing times (e.g., 96 hours), and due to the low stability of rhGAA under AEX conditions, AEX is performed in a low-temperature room with a controlled temperature of 2 °C to 8 °C. The eluate from the large AEX column is then manually packed into a large IMAC column, such as an IMAC column with dimensions of 60 cm diameter x 20 cm bed height (i.e., each IMAC column volume = 56.5 L). These manufacturing systems, including large AEX and IMAC columns, also have several drawbacks: large equipment footprint; low productivity (enzyme produced per liter of resin per hour); high operator involvement (increasing the risk of human error); and high product loss / rejection rates if there is any malfunction in the AEX cycle or the upstream processing in IMAC is delayed.

[0077] However, it has been surprisingly discovered that a relatively compact manufacturing system can provide one or more of the following advantages: reduced capture column (e.g., AEX) packing time; elimination of cold room processing; reduced equipment footprint; increased productivity; reduced operator involvement through direct processing from the capture column (e.g., AEX) to the purification column (e.g., IMAC); and minimized product loss / rejection rates in the event of any malfunction in the biologic capture (e.g., AEX) cycle. Additionally, the system utilizes smaller column sizes, which facilitate achieving better separation of the final product from other proteins.

[0078] Accordingly, various aspects of the present invention relate to novel methods for the production, capture, and purification of biologics (e.g., recombinant proteins, including recombinant human lysosomal proteins, such as rhGAA). Other aspects of the present invention relate to biologics (e.g., recombinant proteins) produced by the methods described herein, as well as pharmaceutical compositions, methods of treatment, and uses of such biologics (e.g., recombinant proteins).

[0079] definition The terms used herein generally have their ordinary meaning in the art, with respect to the present invention and in the specific context in which each term is used. Certain terms are explained below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the present invention and how to make and use them.

[0080] In this specification, for literal explanation or necessary implication, unless the context otherwise requires, the word "comprises" or variations such as "comprises" or "comprising" are used in the inclusive sense, i.e., to specify the presence of stated features, and do not exclude the presence or addition of further features in various embodiments of the invention.

[0081] As used herein, the term "bioreactor volume" refers to the working volume (i.e., liquid volume) within a bioreactor. The working volume within a bioreactor can be less than 10,000 liters, less than 5,000 liters, less than 4,000 liters, less than 3,500 liters, less than 3,000 liters, less than 2,500 liters, less than 2,000 liters, less than 1,500 liters, less than 1,000 liters, less than 500 liters, or less than 250 liters.

[0082] As used herein, the term "capture column" refers to a chromatography column that captures a desired biological product produced from a bioreactor. The present disclosure describes methods that use at least two capture columns. In various embodiments, the at least two capture columns are packed in series to provide for the continuous loading of filtrate onto the at least two capture columns.

[0083] As used herein, the term "purification column" refers to a chromatography column used to further purify a desired biological product after it has been captured on a capture column.

[0084] As used herein, the term "column volume" refers to the packed bed volume of a chromatography column.

[0085] As used herein, the terms "total... column volume," such as "total biologic capture column volume," total AEX column volume, etc., refer to the combined column volume of all columns of a particular type.

[0086] As used herein, the term "total column residence time" refers to the quotient of the combined column volume of all columns of a particular type and the volumetric flow rate used to pack the columns.

[0087] As used herein, the term "recombinant DNA" refers to DNA that is artificially created by combining genetic material from multiple sources (e.g., different organisms).

[0088] As used herein, the terms "biological product" or "biological product" refer to a complex molecule or mixture of molecules produced in a biological system. Biologics are often produced in cell-based systems using recombinant DNA technology. Examples of biologics include, but are not limited to, recombinant proteins, viral particles, and antibodies.

[0089] As used herein, the term "recombinant protein" refers to a protein encoded by a gene in recombinant DNA cloned into a system that supports expression of the gene. In one or more embodiments, the recombinant protein is a secreted or intracellular protein produced in a host cell. The host cell can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells and eukaryotic cells, including insect cells, yeast cells, and mammalian cells. Particularly desirable host cells are selected from any mammalian species, including, but not limited to, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells (expressing a functional adenovirus E1), Saos, C2C12, L cells, HT1080, HepG2, and primary fibroblasts, hepatocytes, and myoblasts, derived from mammals, including humans, monkeys, mice, rats, rabbits, and hamsters. The choice of mammalian species from which the cells are provided is not a limitation of the present invention; neither is the type of mammalian cell, ie, fibroblasts, hepatocytes, tumor cells, etc.

[0090] In some embodiments, the recombinant protein may be a secreted protein, a membrane protein, or an intracellular protein. Secreted proteins may be separated into a filtrate by either filtration or centrifugation. Intracellular proteins may be separated into a filtrate by first lysing the cells and then either filtration or centrifugation. Membrane proteins may be separated by lysing the cells, separating the recombinant-containing membranes by ultracentrifugation, solubilizing the membrane proteins with a suitable detergent, and preparing a filtrate by ultracentrifugation to separate the insoluble membrane proteins. The detergent may be anionic, cationic, or amphoteric in nature.

[0091] As used herein, the term "lysosomal protein" refers to any protein targeted to the lysosome, such as a lysosomal enzyme. Examples of lysosomal enzymes and associated diseases include, but are not limited to, those shown in Table 1 below.

[0092] Table 1

[0093] In one or more embodiments, the lysosomal protein is selected from the group consisting of α-galactosidase (A or B), β-galactosidase, β-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B), β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., α-L), acetyl-CoA:α-glucosaminide N-acetyltransferase, glycosaminoglycan α-L-iduronohydrolase, heparan N-sulfatase, N-acetyl-α-D-glucosaminidase, iduronate-2-sulfatase, and the like. In some embodiments, the therapeutic protein is selected from the group consisting of: α-galactosamine-6-sulfatase, galactosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase, α-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, α-glucosidase, α-D-mannosidase, β-D-mannosidase, aspartylglucosaminidase, α-L-fucosidase, battenin, palmitoyl protein thioesterase, and other Batten disease-related proteins (e.g., neuronal ceroid lipofuscinosis protein 6). In some embodiments, the therapeutic protein is α-galactosidase. In some embodiments, the enzyme is a palmitoyl protein thioesterase (PPT), including palmitoyl protein thioesterases 1 and 2 (PPT1 and PPT2, respectively). In some embodiments, the enzyme is palmitoyl protein thioesterase 1.In some embodiments, the therapeutic protein is associated with a genetic disorder selected from the group consisting of CDKL5 deficiency, cystic fibrosis, alpha- and beta-thalassemia, sickle cell anemia, Marfan syndrome, fragile X syndrome, Huntington's disease, hemochromatosis, congenital deafness (non-syndromic), Tay-Sachs disease, familial hypercholesterolemia, Duchenne muscular dystrophy, Stargardt disease, Usher syndrome, congenital choroideremia, color blindness, X-linked retinoschisis, hemophilia, Wiskott-Aldrich syndrome, X-linked chronic granulomatous disease, aromatic L-amino acid decarboxylase deficiency, recessive dystrophic epidermolysis bullosa, alpha-1 antitrypsin deficiency, Hutchinson-Gilford progeria syndrome (HGPS), Noonan syndrome, and X-linked severe combined immunodeficiency (X-SCID). In some embodiments, the therapeutic protein is selected from the group consisting of CDKL5, connexin 26, hexosaminidase A, LDL receptor, dystrophin, CFTR, beta-globin, HFE, huntingtin, ABCA4, myosin VIIA (MYO7A), Rab escort protein-1 (REP1), cyclic nucleotide-gated channel beta 3 (CNGB3), retinoschisin 1 (RS1), hemoglobin subunit beta (HBB), factor IX, WAS, cytochrome B-245 beta chain, dopa decarboxylase (DDC), type VII collagen alpha 1 chain (COL7A1), serpin family A member 1 (SERPINA1), LMNA, PTPN11, SOS1, RAF1, KRAS, and IL2 receptor gamma genes.

[0094] In one or more embodiments, the genetic disease (e.g., lysosomal storage disease) is selected from the group consisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, and Schindler disease type II. In some embodiments, the lysosomal storage disease is activator deficiency, GM2-gangliosidosis; GM2-gangliosidosis, AB variant; α-mannosidosis (type 2, moderate; type 3, neonatal, severe); β-mannosidosis; lysosomal acid lipase deficiency; cystinosis (late-onset juvenile or adolescent nephropathic; childhood nephropathic); Shanarin-Dorfman syndrome; neutral lipid storage disease with myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease type II, late-onset; Farber disease; Farber lipogranulomatosis; fucosidosis; galactosialidosis (neuraminidase and β-galactosidase deficiency). Combined cerebroside sulfatase deficiency; Gaucher disease; Type II Gaucher disease; Type III Gaucher disease; Type IIIC Gaucher disease; Atypical Gaucher disease, due to saposin C deficiency; GM1-gangliosidosis (late-onset infantile / juvenile GM1-gangliosidosis; adult / chronic GM1-gangliosidosis); Globoid cell leukodystrophy, Krabbe disease (late-onset infantile; juvenile; adult); Krabbe disease, atypical, due to saposin A deficiency; Metachromatic leukodystrophy (juvenile; adult); Partial cerebroside sulfatase deficiency; Pseudoarylsulfatase A deficiency; Metachromatic leukodystrophy due to saposin B deficiency; Mucopolysaccharidosis disorders: MPS I, Hurler syndrome; MPS I, Hurler-Shea syndrome; MPS I, Shea syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome;Sanfilippo syndrome type A / MPS IIIA; Sanfilippo syndrome type B / MPS IIIB; Sanfilippo syndrome type C / MPS IIIC; Sanfilippo syndrome type D / MPS IIID; Morquio syndrome type A / MPS IVA; Morquio syndrome type B / MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamy syndrome; MPS VII Sly syndrome; Mucolipidosis I, Sialidosis II; I-cell disease, Leroy disease, Mucolipidosis II; Pseudo-Hurler polydystrophy / Mucolipidosis III; Mucolipidosis IIIC / ML III GAMMA; mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pick disease (type B; type C1 / chronic neuronopathic; type C2; ​​type D / Nova Scotia); neuronal ceroid lipofuscinosis: CLN6 disease - atypical late-onset infantile, late-onset variant, early-juvenile; Batten-Spielmeyer-Voigt / juvenile NCL / CLN3 disease; Finnish variant late-onset infantile CLN5; Jansky-Bielschowski disease / late-onset infantile CLN2 / TPP1 disease; Kuhus disease / adult NCL / CLN4 disease (type B); Northern epilepsy / variant late-onset infantile CLN8; Santavoli-Hartier disease / infantile CLN1 / PPT disease; Pompe disease (glycogenosis type II); late-onset Pompe disease; pyknodysostosis; Sandhoff disease / GM2 gangliosidosis; Sandhoff disease / GM The present invention is selected from the group consisting of: 2 gangliosidosis; Sandhoff disease / GM2 gangliosidosis; Schindler disease (type III / intermediate, variable); Kanzaki disease; Salla disease; infantile free sialic acid storage disease (ISSD); spinal muscular atrophy with progressive myoclonic epilepsy (SMAPME); Tay-Sachs disease / GM2 gangliosidosis; juvenile Tay-Sachs disease; late-onset Tay-Sachs disease; Christianson syndrome; Rowe oculocerebrorenal syndrome; Charcot-Marie-Tooth disease type 4J, CMT4J; Eunice-Baron syndrome; bilateral temporo-occipital polymicrogyria (BTOP); X-linked hypercalciuric nephrolithiasis, Dent disease type 1; and Dent disease type 2, adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis.

[0095] As used herein, the term "Pompe disease," also known as acid maltase deficiency, glycogenic storage disease type II (GSDII), and glycogenic disease type II, is intended to refer to a genetic lysosomal storage disorder characterized by mutations in the GAA gene, which encodes the human acid α-glucosidase enzyme. The term includes, but is not limited to, early- and late-onset forms of Pompe disease, including, but not limited to, infantile, juvenile, and adult-onset forms.

[0096] As used herein, the term "acid α-glucosidase" is intended to refer to a lysosomal enzyme that hydrolyzes the α-1,4 bond between D-glucose units of glycogen, maltose, and isomaltose. Alternative names include, but are not limited to, lysosomal α-glucosidase (EC:3.2.1.20); glucoamylase; 1,4-α-D-glucan glucohydrolase; amyloglucosidase; gamma-amylase; and exo-1,4-α-glucosidase. Human acid α-glucosidase is encoded by the GAA gene (National Centre for Biotechnology Information (NCBI) Gene ID 2548), which is mapped to the long arm of chromosome 17 (location 17q25.2-q25.3). More than 500 mutations have now been identified in the human GAA gene, many of which are associated with Pompe disease. Mutations that result in misfolding or misprocessing of the acid α-glucosidase enzyme include T1064C (Leu355Pro) and C2104T (Arg702Cys). Additionally, GAA mutations that affect enzyme maturation and processing include Leu405Pro and Met519Thr. The conserved hexapeptide WIDMNE at amino acid residues 516-521 is required for activity of the acid α-glucosidase protein. As used herein, the abbreviation "GAA" is intended to refer to the acid α-glucosidase enzyme, and the italicized abbreviation "GAA" is intended to refer to a 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, a rat or mouse gene, and the abbreviation "Gaa" is intended to refer to a non-human acid α-glucosidase enzyme. Thus, the abbreviation "rhGAA" is intended to refer to recombinant human acid alpha-glucosidase enzyme.

[0097] As used herein, the term "alglucosidase alfa" is intended to refer to recombinant human acid α-glucosidase identified as [199-arginine, 223-histidine] prepro-α-glucosidase (human); Chemical Abstracts Registry Number 420794-05-0. Alglucosidase alfa has been approved for commercial sale in the United States by Genzyme as of January 2016 under the trademarks Lumizyme® and Myozyme®.

[0098] As used herein, the term "ATB200" is intended to refer to the recombinant human acid α-glucosidase described in PCT patent application PCT / US2015 / 053252 (now issued as U.S. Pat. No. 10,208,299), the disclosure of which is incorporated herein by reference in its entirety. Methods for producing recombinant lysosomal proteins (including rhGAA, such as ATB200) are described in U.S. Pat. No. 10,227,577, which is also incorporated herein by reference in its entirety. Formulations and methods using rhGAA are described in co-pending application publication numbers U.S. 2017 / 0333534 and U.S. 2018 / 0228877, which are also incorporated herein by reference in their entireties.

[0099] As used herein, the term "glycan" is intended to refer to a polysaccharide chain covalently bound 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 bound to an amino acid residue on a protein or polypeptide via a nitrogen atom of the amino acid residue. For example, an N-glycan may be covalently bound to the side chain nitrogen atom of an asparagine residue. A glycan may contain one or several monosaccharide units, and the monosaccharide units may be covalently linked to form a linear or branched chain. In at least one embodiment, the N-glycan unit bound to ATB200 may contain one or more monosaccharide units independently selected from N-acetylglucosamine, mannose, galactose, or sialic acid. The N-glycan units on a protein may be determined by any suitable analytical technique, such as mass spectrometry. In some embodiments, N-glycan units can be determined by liquid chromatography-tandem mass spectrometry (LC-MS / MS) using instruments such as a Thermo Scientific Orbitrap Velos Pro™ mass spectrometer, a Thermo Scientific Orbitrap Fusion Lumos Tribid™ mass spectrometer, or a Waters Xevo® G2-XS QTof mass spectrometer.

[0100] As used herein, the term "high mannose N-glycan" is intended to refer to an N-glycan having one to six or more mannose units. In at least one embodiment, a high mannose N-glycan unit contains a bis(N-acetylglucosamine) chain attached to an asparagine residue, which may be further attached to a branched polymannose chain. As used interchangeably herein, the term "M6P" or "mannose-6-phosphate" is intended to refer to a mannose unit that is phosphorylated at the 6-position, i.e., has a phosphate group attached to the hydroxyl group at the 6-position. In at least one embodiment, one or more mannose units of one or more N-glycan units are phosphorylated at the 6-position to form a mannose-6-phosphate unit. In at least one embodiment, the term "M6P" or "mannose-6-phosphate" refers to both a mannose phosphodiester having an N-acetylglucosamine (GlcNAc) as a "cap" on the phosphate group and a mannose unit with an exposed phosphate group that lacks a GlcNAc cap. In at least one embodiment, the N-glycans of the protein can have multiple M6P groups, with at least one M6P group having a GlcNAc cap and at least one other M6P group lacking a GlcNAc cap.

[0101] As used herein, the term "complex N-glycan" is intended to refer to 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 mannose units are further attached to one or more monosaccharide units independently selected from N-acetylglucosamine, galactose, and sialic acid.

[0102] As 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: [ka]

[0103] One formulation of miglustat is marketed under the trade name Zavesca® as a monotherapy for type 1 Gaucher disease.

[0104] As described below, pharmaceutically acceptable salts of miglustat may also be used in the present invention. When a salt of miglustat is used, the dose of the salt is adjusted so that the dose of miglustat received by the patient is equivalent to the dose they would receive if the free base of miglustat were used.

[0105] As used herein, the compound duvoglustat, also known as 1-deoxynojirimycin or DNJ or (2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol, has the following chemical formula: [ka]

[0106] When a salt of duvoglustat is used, the dose of the salt is adjusted so that the dose of duvoglustat received by a patient is equivalent to the dose they would receive if the free base of duvoglustat were used.

[0107] As used herein, the term "pharmacological chaperone," or sometimes simply the term "chaperone," is intended to refer to a molecule that specifically binds to a protein (e.g., a natural or recombinant protein) and has one or more of the following effects: Promotes the formation of stable molecular conformations of proteins; · Facilitating proper transport of proteins from the endoplasmic reticulum to another cellular location, preferably their native cellular location, in order to prevent endoplasmic reticulum-associated degradation of the protein; · Prevents aggregation of structurally unstable or misfolded proteins; Restoring and / or improving the wild-type function, stability and / or activity of at least a portion of a protein; and / or Improving the phenotype or function of cells that contain the protein.

[0108] Thus, pharmacological chaperones include lysosomal proteins. For example, chaperones for acid α-glucosidase are molecules that bind to acid α-glucosidase and result in its proper folding, transport, non-aggregation, and activity. As used herein, the term includes, but is not limited to, active site-specific chaperones (ASSCs), inhibitors or antagonists, and agonists that bind to the active site of the enzyme. In at least one embodiment, a pharmacological chaperone can 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 blocks, inhibits, reduces, or neutralizes the activity of acid α-glucosidase. In at least one embodiment, a pharmacological chaperone is miglustat. Another non-limiting pharmacological chaperone for acid α-glucosidase is duvoglustat.

[0109] As used herein, the term "active site" is intended to refer to a region of a protein that is associated with and necessary for a specific biological activity of the protein. In at least one embodiment, the active site may be the site that binds a substrate or other binding partner and contributes amino acid residues that are directly involved in forming and breaking chemical bonds.

[0110] As used herein, "therapeutically effective dose" and "effective amount" are intended to refer to the amount of recombinant protein (e.g., rhGAA) and / or chaperone and / or combination thereof that results in a therapeutic response in a subject. A therapeutic response can be any response that a user (e.g., a clinician) recognizes as an effective response to treatment, including any surrogate clinical marker or symptom described herein and known in the art. Thus, in at least one embodiment, a therapeutic response can be an improvement or suppression of one or more symptoms or markers of Pompe disease, such as those known in the art. Symptoms or markers of Pompe disease include, but are not limited to, cardiomyopathy, cardiac hypertrophy, progressive muscle weakness, especially in the trunk or lower limbs, severe hypotension, macroglossia (and possibly tongue protrusion), difficulty swallowing, sucking, and / or feeding, respiratory failure, hepatomegaly (moderate), facial muscle relaxation, areflexia, exercise intolerance, exertional dyspnea, orthopnea, sleep apnea, morning headache, somnolence, lordosis and / or scoliosis, decreased deep tendon reflexes, lower back pain, and failure to achieve developmental motor milestones. 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 purposes of the present invention due to dilution (and consequent altered binding due to shifts in equilibrium), bioavailability, and metabolism of the chaperone upon in vivo administration.

[0111] As used herein, the term "enzyme replacement therapy" or "ERT" is 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 can be obtained from a natural source or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual who otherwise requires or would benefit from the administration of the purified enzyme. In at least one embodiment, such an individual suffers from an enzyme deficiency. The introduced enzyme can be a purified recombinant enzyme produced in vitro or a protein purified from an isolated tissue or fluid, such as the placenta or milk of an animal, or from a plant.

[0112] As used herein, the term "combination therapy" is intended to refer to any therapy in which two or more individual therapies are administered simultaneously or sequentially. In at least one embodiment, the results of the combination therapy are enhanced compared to the effects of each therapy when administered individually. Enhancement may include any improvement in the effects of the various therapies that may result in advantageous results compared to the results achieved by the therapies when administered alone. Enhanced effects or results may include synergistic enhancement, where the enhanced effect is greater than the additive effect of each therapy when administered alone; additive enhancement, where the enhanced effect is substantially equal to the additive effect of each therapy when administered alone; or subsynergistic enhancement, where the enhanced effect is less than the additive effect of each therapy when administered alone, but is superior to the effect of each therapy when administered alone. An enhanced effect may be measured by any means known in the art that can measure treatment effect or outcome.

[0113] As used herein, the term "viral particle" is intended to include genetic material (e.g., DNA or RNA) surrounded by a protein coat known as a capsid. Examples of viral particles include, but are not limited to, adeno-associated viruses (AAV), retroviruses, lentiviruses, herpes simplex viruses, and adenoviruses. In one or more embodiments, the viral particle comprises recombinant DNA encoding a recombinant protein. In one or more embodiments, the viral particle comprises additional elements to increase expression and / or stabilize the vector, such as a promoter (e.g., a hybrid CBA promoter (CBh) and a human synapsin 1 promoter (hSyn1)), a polyadenylation signal (e.g., a bovine growth hormone polyadenylation signal (bGHpolyA)), a stabilizing element (e.g., a woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE)), and / or an SV40 intron. In one or more embodiments, the vector may contain a polynucleotide sequence flanked by regions that promote homologous recombination at a desired site in the genome, thereby providing expression of a desired protein (see Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA, 86:8932-8935; Zijlstra et al., 1989, Nature 342:435-438; U.S. Pat. No. 6,244,113 to Zarling et al.; and U.S. Pat. No. 6,200,812 to Pati et al.).

[0114] As used herein, the term "antibody" refers to an immunoglobulin, which includes natural and / or recombinant immunoglobulins. The source of natural immunoglobulins can be mammals, including humans, domestic and farm animals and laboratory animals, zoo animals, sport animals, or pets, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. The source can be naturally or artificially exposed to a specific antigen to induce an immunogenic response, resulting in antibody production. Alternatively, recombinant immunoglobulins can be produced in suitable host cells.

[0115] As used herein, the term "pharmaceutically acceptable" is intended to refer to molecular entities and compositions that are physiologically tolerable and typically do not produce adverse reactions when administered to humans. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0116] As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, at least in one embodiment, are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0117] As used herein, the term "subject" or "patient" is intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0118] As used herein, the term "anti-drug antibodies" is intended to refer to antibodies that specifically bind to a drug administered to a subject and are 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 can result in immune responses ranging from mild to severe, including, but not limited to, life-threatening immune responses, including, but not limited to, anaphylaxis, cytokine release syndrome, and cross-reactive neutralization of endogenous proteins that mediate critical functions. Additionally or alternatively, the presence of anti-drug antibodies in a subject can reduce the effectiveness of the drug.

[0119] As used herein, the term "neutralizing antibody" is intended to refer to an anti-drug antibody that acts to neutralize the function of the drug. In at least one embodiment, a therapeutic protein drug product is a counterpart of an endogenous protein whose expression is reduced or absent in a subject. In at least one embodiment, a neutralizing antibody can act to neutralize the function of the endogenous protein.

[0120] As used herein, the terms "about" and "approximately" are intended to refer to an acceptable degree of error for the measured quantity, given the nature or precision of the measurement. For example, the degree of error can be indicated by the number of significant figures provided for a measurement, as understood in the art, and includes, but is not limited to, a variation of ±1 of the most precise significant figure reported for a measurement. Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical values ​​set forth herein are approximate unless otherwise specified, and the terms "about" or "approximately" mean that they can be inferred when not explicitly stated.

[0121] The term "concurrently," as used herein, is intended to mean at the same time or within a reasonably short time before or after, as understood by those skilled in the art. For example, if two treatments are administered simultaneously with each other, one treatment may be administered before or after the other to allow time needed to prepare for the later of the two treatments. Thus, "concurrent administration" of two treatments includes, but is not limited to, one treatment following the other by 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.

[0122] The term "pharmaceutically acceptable salt," as used herein, is intended to mean a salt that, within the scope of sound medical judgment, is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., and is generally water- or oil-soluble or dispersible, and effective for its intended use, in accordance with a reasonable benefit / risk ratio. This term includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts. Lists of suitable salts can be found, for example, in S. M. Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19, which is incorporated herein by reference.

[0123] The term "pharmaceutically acceptable acid addition salts," as used herein, refers to salts of inorganic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, which retain the biological effectiveness and properties of the free base and which are not biologically or otherwise undesirable, as well as acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphate, hemisulfonic acid, hexane ... It is intended to mean salts formed with organic acids, including, but not limited to, 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, undecanoic acid, and the like.

[0124] The term "pharmaceutically acceptable acid addition salts," as used herein, refers to salts of inorganic acids, including, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, phosphoric acid, and the like, which retain the biological effectiveness and properties of the free base and which are not biologically or otherwise undesirable, as well as acetic acid, trifluoroacetic acid, adipic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, butyric acid, camphorsulfonic acid, cinnamic acid, citric acid, digluconic acid, ethanesulfonic acid, glutamic acid, glycolic acid, glycerophosphate, hemisulfonic acid, hexane ... It is intended to mean salts formed with organic acids, including, but not limited to, 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, undecanoic acid, and the like.

[0125] ATB200 rhGAA In at least one embodiment, the recombinant protein (e.g., a recombinant protein such as rhGAA) is expressed in Chinese hamster ovary (CHO) cells and comprises an increased content of N-glycan units having one or more mannose-6-phosphate residues compared to the content of N-glycan units having one or more mannose-6-phosphate residues in conventional recombinant proteins, such as alglucosidase alfa. In at least one embodiment, the acid α-glucosidase is a recombinant human acid α-glucosidase, referred to herein as ATB200, as described in U.S. Pat. No. 10,208,299. ATB200 has a high affinity (K D It binds to the cation-independent mannose-6-phosphate receptor (CIMPR) at ~2-4 nM and is expressed in Pompe fibroblasts and skeletal myoblasts (K 取り込みATB200 has been characterized in vivo and has been shown to be efficiently internalized by alglucosidase alfa (t 1 / 2 Its apparent plasma half-life (t ) is shorter than 60 min. 1 / 2 It was shown that the solubility of the solubility is approximately 45 minutes.

[0126] In at least one embodiment, the recombinant human acid α-glucosidase is an enzyme having the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2. [ka] [ka] [ka] [ka]

[0127] In at least one embodiment, the recombinant human acid α-glucosidase has the wild-type GAA amino acid sequence set forth in SEQ ID NO:1, as described in U.S. Patent No. 8,592,362, and has GenBank accession number AHE24104.1 (GI:568760974). In at least one embodiment, the recombinant human acid α-glucosidase is glucosidase alpha, the human acid α-glucosidase enzyme encoded by the most prevalent of the nine observed haplotypes of the GAA gene.

[0128] In at least one embodiment, the recombinant human acid α-glucosidase is initially expressed as having the full-length 952 amino acid sequence of wild-type GAA set forth in SEQ ID NO: 1, and the recombinant human acid α-glucosidase undergoes intracellular processing to remove some of the amino acids, e.g., the first 56 amino acids. Thus, the recombinant human acid α-glucosidase secreted by the host cell can have a shorter amino acid sequence than the recombinant human acid α-glucosidase initially expressed within the cell. In at least one embodiment, the shorter protein can have the amino acid sequence set forth in SEQ ID NO: 2, which differs from SEQ ID NO: 1 only in that the first 56 amino acids, including the signal peptide and precursor peptide, have been removed, thus resulting in a protein having 896 amino acids. Other variations in the number of amino acids are also possible, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions relative to the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the rhGAA product comprises a mixture of recombinant human acid α-glucosidase molecules having different amino acid lengths.

[0129] In at least one embodiment, the recombinant human acid α-glucosidase undergoes post-translational and / or chemical modifications at one or more amino acid residues in the protein. For example, methionine and tryptophan residues may undergo oxidation. As another example, N-terminal glutamine may form pyroglutamic acid. As 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 as having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2, and the enzyme undergoes one or more of these post-translational and / or chemical modifications. Such modified forms are also within the scope of the present disclosure.

[0130] Polynucleotide sequences encoding GAA and such mutant human GAA are also contemplated and may be used to recombinantly express rhGAA according to the present invention.

[0131] Preferably, no more than 70, 65, 60, 55, 45, 40, 35, 30, 25, 20, 15, 10, or 5% of all recombinant protein (e.g., rhGAA) molecules lack N-glycan units with 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% of the recombinant protein (e.g., rhGAA) molecules contain at least one N-glycan unit with one or more mannose-6-phosphate residues or are capable of binding to CIMPR.

[0132] A recombinant protein (e.g., rhGAA) molecule can have one, two, three, or four mannose-6-phosphate (M6P) groups on its glycans. For example, only one N-glycan in a recombinant protein molecule can have M6P (mono-phosphorylated), a single N-glycan can have two M6P groups (bis-phosphorylated), or two different N-glycans in the same recombinant protein molecule can each have a single M6P group. A recombinant protein molecule can also have N-glycans that do not have M6P groups. In another embodiment, the N-glycans contain, on average, more than 3 moles / mole of M6P and more than 4 moles / mole of sialic acid, such that the protein contains, on average, at least 3 moles of mannose-6-phosphate residues per mole of recombinant protein and at least 4 moles of sialic acid per mole of recombinant protein. On average, at least about 3, 4, 5, 6, 7, 8, 9, or 10% of the total glycans in the recombinant protein may be in the form of mono-M6P glycans, e.g., about 6.25% of the total glycans may have a single M6P group, on average, at least about 0.5, 1, 1.5, 2.0, 2.5, 3.0% of the total glycans in the recombinant protein are in the form of bis-M6P glycans, and on average, less than 25% of the total recombinant protein does not contain phosphorylated glycans that are bound to CIMPR.

[0133] The recombinant protein (e.g., rhGAA) can have an average content of M6P-bearing N-glycans in the range of 0.5 to 7.0 moles / mole recombinant lysosomal protein, or any intermediate value in a subrange including 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, or 7.0 moles / mole recombinant lysosomal protein. Recombinant lysosomal protein can be fractionated to provide recombinant lysosomal protein preparations with different average numbers of M6P-containing or bis-M6P-containing glycans, allowing for further customization of recombinant lysosomal protein targeting to lysosomes in target tissues by selecting specific fractions or selectively combining different fractions.

[0134] In some embodiments, the recombinant protein (e.g., rhGAA) has an average of 2.0 to 8.0 moles of M6P per mole of recombinant protein (e.g., rhGAA), including all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles M6P / mole recombinant protein (e.g., rhGAA).

[0135] Up to 60% of the N-glycans on a recombinant protein (e.g., rhGAA) can be fully sialylated, e.g., up to 10%, 20%, 30%, 40%, 50%, or 60% of the N-glycans can be fully sialylated. In some embodiments, 4-20% of the total N-glycans are fully sialylated. In other embodiments, no more than 5%, 10%, 20%, or 30% of the N-glycans on a recombinant protein (e.g., rhGAA) have sialic acid and a terminal galactose residue (Gal). This range includes all intermediate values ​​and subranges, e.g., 7-30% of the total N-glycans on a recombinant protein can carry sialic acid and terminal galactose. In still other embodiments, no more than 5, 10, 15, 16, 17, 18, 19, or 20% of the N-glycans on a recombinant protein have only terminal galactose and no sialic acid. This range includes all intermediate values ​​and subranges, for example, 8-19% of all N-glycans on the recombinant protein in the composition may have only terminal galactose and no sialic acid.

[0136] In other embodiments of the present invention, 40, 45, 50, 55-60% of the total N-glycans in the recombinant protein (e.g., rhGAA) are complex N-glycans; or 1, 2, 3, 4, 5, 6, 7% or less of the total N-glycans in the recombinant protein (e.g., rhGAA) are hybrid N-glycans; 5, 10, or 15% or less of the high-mannose N-glycans in the recombinant protein (e.g., rhGAA) are non-phosphorylated; at least 5% or 10% of the high-mannose N-glycans in the recombinant protein (e.g., rhGAA) are phosphorylated mono-M6P; and / or at least 1 or 2% of the high-mannose N-glycans in the recombinant protein (e.g., rhGAA) are phosphorylated bis-M6P. These values ​​include all intermediate values ​​and subranges. A recombinant protein (e.g., rhGAA) may satisfy one or more of the above content ranges.

[0137] In some embodiments, the recombinant protein (e.g., rhGAA) has an average of 2.0 to 8.0 moles of sialic acid residues per mole of recombinant protein (e.g., rhGAA). This range includes all intermediate values ​​and subranges, including 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0 moles of residues per mole of recombinant protein (e.g., rhGAA). Without being bound by theory, it is believed that the presence of N-glycan units with sialic acid residues may prevent non-productive clearance of the recombinant protein (e.g., rhGAA) by the asialoglycoprotein receptor.

[0138] In one or more embodiments, the recombinant protein (e.g., rhGAA) has M6P and / or sialic acid units at certain N-glycosylation sites of the recombinant 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 sequence ASN-X-SER or ASN-X-THR in the protein amino acid sequence indicates a potential glycosylation site, with the exception that X cannot be HIS or PRO.

[0139] In various embodiments, the rhGAA has a particular N-glycosylation profile. In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the first N-glycosylation site (e.g., N84 of SEQ ID NO: 2 and N140 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the first N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a mono-M6P unit at the first N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a bis-M6P unit at the first N-glycosylation site.

[0140] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the second N-glycosylation site (e.g., N177 of SEQ ID NO: 2 and N223 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the second N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA 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 the rhGAA is phosphorylated at the third N-glycosylation site (e.g., N334 of SEQ ID NO:2 and N390 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the third N-glycosylation site. For example, the third N-glycosylation site may have a mixture of non-phosphorylated high-mannose glycans, di-, tri-, and tetra-antennary complex glycans, and hybrid glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the rhGAA is sialylated at the third N-glycosylation site.

[0141] In one or more embodiments, at least 20% of the rhGAA is phosphorylated at the fourth N-glycosylation site (e.g., N414 of SEQ ID NO: 2 and N470 of SEQ ID NO: 1). For example, at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA can be phosphorylated at the fourth N-glycosylation site. This phosphorylation can be the result of mono-M6P and / or bis-M6P units. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a mono-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA have a bis-M6P unit at the fourth N-glycosylation site. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, or 25% of the rhGAA is sialylated at the fourth N-glycosylation site.

[0142] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the fifth N-glycosylation site (e.g., N596 of SEQ ID NO:2 and N692 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the fifth N-glycosylation site. For example, the fifth N-glycosylation site may have a fucosylated di-antennary complex glycan as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the fifth N-glycosylation site.

[0143] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the sixth N-glycosylation site (e.g., N826 of SEQ ID NO:2 and N882 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the sixth N-glycosylation site. For example, the sixth N-glycosylation site may have a mixture of di-, tri-, and tetra-antennary complex glycans as the predominant species. In some embodiments, at least 3%, 5%, 8%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the rhGAA is sialylated at the sixth N-glycosylation site.

[0144] In one or more embodiments, at least 5% of the rhGAA is phosphorylated at the seventh N-glycosylation site (e.g., N869 of SEQ ID NO:2 and N925 of SEQ ID NO:1). In other embodiments, less than 5%, 10%, 15%, 20%, or 25% of the rhGAA is phosphorylated at the seventh N-glycosylation site. In some embodiments, less than 40%, 45%, 50%, 55%, 60%, or 65% of the rhGAA has any glycan at the seventh N-glycosylation site. In some embodiments, at least 30%, 35%, or 40% of the rhGAA has a glycan at the seventh N-glycosylation site.

[0145] In one or more embodiments, 40% to 60% of the N-glycans in rhGAA are complex-type N-glycans; and the rhGAA contains 3.0 to 5.0 moles of M6P residues per mole of rhGAA.

[0146] In various embodiments, the rhGAA has an average fucose content of 0-5 moles per mole rhGAA, a GlcNAc content of 10-30 moles per mole rhGAA, a galactose content of 5-20 moles per mole rhGAA, a mannose content of 10-40 moles per mole rhGAA, an M6P content of 2-8 moles per mole rhGAA, and a sialic acid content of 2-8 moles per mole rhGAA. In various embodiments, the rhGAA has an average fucose content of 2-3 moles per mole rhGAA, a GlcNAc content of 20-25 moles per mole rhGAA, a galactose content of 8-12 moles per mole rhGAA, a mannose content of 22-27 moles per mole rhGAA, an M6P content of 3-5 moles per mole rhGAA, and a sialic acid content of 4-7 moles per mole rhGAA.

[0147] Recombinant proteins (e.g., rhGAA) are preferably produced in Chinese hamster ovary (CHO) cells, such as the CHO cell lines GA-ATB-200 or ATB-200-001-X5-14, or subcultures or derivatives of such CHO cell cultures. DNA constructs expressing allelic variants of acid α-glucosidase or other variant acid α-glucosidase amino acid sequences, such as those 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 variant acid α-glucosidase amino acid sequences can contain deletions, substitutions, and / or insertions relative to SEQ ID NO: 1, e.g., 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 set forth in SEQ ID NO: 1 or SEQ ID NO: 2. Those skilled in the art will be able to select alternative vectors suitable for transformation of CHO cells for the production of such DNA constructs.

[0148] Various alignment algorithms and / or programs can be used to calculate identity between two sequences, including FASTA or BLAST, 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 and polynucleotides encoding such polypeptides with at least 90%, 95%, 98%, or 99% identity to a particular polypeptide described herein, preferably exhibiting substantially the same function, are contemplated. Unless otherwise indicated, similarity scores are based on the use of BLOSUM62. When BLASTP is used, the percentage similarity is based on the BLASTP positive score, and the percentage sequence identity is based on the BLASTP identity score. BLASTP "identity" indicates the number and percentage of total residues in a high-scoring sequence pair that are identical; and BLASTP "positive" indicates the number and percentage of residues that have a positive alignment score and are similar to each other. Amino acid sequences with these degrees of identity or similarity, or any intermediate degree of similarity to the amino acid sequences disclosed herein, are contemplated and encompassed by this disclosure. The polynucleotide sequence of a similar polypeptide can be deduced using the genetic code and obtained by conventional means, in particular by back-translating the amino acid sequence using the genetic code.

[0149] As described in U.S. Patent No. 10,208,299, recombinant human acid α-glucosidase with superior targeting ability to the cation-independent mannose-6-phosphate receptor (CIMPR) and cellular lysosomes, as well as a glycosylation pattern that reduces non-productive clearance in vivo, can be produced using Chinese hamster ovary (CHO) cells. These cells can be induced to express recombinant human acid α-glucosidase with significantly higher levels of N-glycan units with one or more mannose-6-phosphate residues than conventional recombinant human acid α-glucosidase products, such as alglucosidase alfa. The recombinant human acid α-glucosidase produced by these cells, as exemplified by ATB200, has significantly more muscle cell-targeting mannose-6-phosphate (M6P) and bis-mannose-6-phosphate N-glycan residues than conventional acid α-glucosidases, such as Lumizyme®. Without being bound by theory, it is believed that this extensive glycosylation allows the ATB200 enzyme to be more effectively taken up by target cells and therefore more efficiently removed from the circulation than other recombinant human acid α-glucosidases, such as alglucosidase alfa, which has a much lower M6P and bis-M6P content. ATB200 has been shown to bind efficiently to CIMPR, be efficiently taken up by skeletal and cardiac muscle, and have a glycosylation pattern that provides a favorable pharmacokinetic profile and reduces non-productive clearance in vivo.

[0150] It is also contemplated that the extensive glycosylation of ATB200 may contribute to the reduced immunogenicity of ATB200 compared to, for example, alglucosidase alfa. 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 shielding protein immunogenic epitopes from the immune system (Guidance for Industry-Immunogenicity Assessment for Therapeutic Protein Products, US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research, August 2014). Thus, in at least one embodiment, administration of recombinant human acid α-glucosidase does not induce anti-drug antibodies. In at least one embodiment, administration of recombinant human acid alpha-glucosidase reduces the incidence of anti-drug antibodies in a subject below the level of anti-drug antibodies induced by administration of alglucosidase alfa.

[0151] As described in U.S. Patent No. 10,208,299, cells such as CHO cells can be used to produce the rhGAA described therein, which can be used in the present invention. Examples of such CHO cell lines are GA-ATB-200 or ATB-200-001-X5-14, or subcultures thereof that produce the rhGAA compositions described therein. Such CHO cell lines can contain multiple copies of the gene, such as 5, 10, 15, 20, or more copies per polynucleotide encoding GAA.

[0152] High M6P and bis-M6P rhGAA, such as ATB200 rhGAA, can be produced by transforming CHO cells with a DNA construct encoding GAA. Although CHO cells have previously been used to make rhGAA, it was not found that transformed CHO cells could be cultured and selected to produce rhGAA with a high content of M6P and bis-M6P glycans that target CIMPR.

[0153] Surprisingly, it has been found that it is possible to transform a CHO cell line, select transformants that produce rhGAA containing a high content of glycans with M6P or bis-M6P targeting CIMPR, and stably express this high-M6P rhGAA. Accordingly, a method for producing these CHO cell lines is also described in U.S. Patent No. 10,208,299. This method includes transforming CHO cells with DNA encoding GAA or a GAA variant, stably integrating the DNA encoding GAA into their chromosomes, selecting CHO cells that stably express GAA, selecting CHO cells that express GAA with a high content of glycans with M6P or bis-M6P, and optionally selecting CHO cells with N-glycans that have a high sialic acid content and / or a low non-phosphorylated high mannose content.

[0154] These CHO cell lines can be used to produce rhGAA and rhGAA compositions by culturing the CHO cell lines and recovering the compositions from the culture of the CHO cells.

[0155] Biologics Production, Capture, and Purification Various embodiments of the present invention relate to methods for the production and / or capture and / or purification of biologics (e.g., recombinant proteins, including recombinant human lysosomal proteins such as rhGAA, antibodies, and viral particles). An exemplary prior art process 600 for producing, capturing, and purifying a biologic is shown in Figure 5. Exemplary processes for producing, capturing, and purifying a biologic according to one or more embodiments of the present invention are shown in Figures 6 and 7. Figure 6 shows a configuration with two capture columns (e.g., AEX columns) and one purification column (e.g., IMAC column), while Figure 7 shows a configuration with two capture columns (e.g., AEX columns) and two purification columns (e.g., IMAC columns).

[0156] In Figures 5-13, arrows indicate the direction of movement of various liquid phases containing a biologic (e.g., a recombinant human lysosomal protein such as rhGAA). Bioreactor 601 contains a culture of cells, such as CHO cells, that produce the biologic (e.g., rhGAA). Biologics include recombinant proteins, antibodies, and viral particles. The recombinant protein can be a secreted protein, a membrane protein, or an intracellular protein. Bioreactor 601 can be any suitable bioreactor for culturing cells, such as a perfusion, batch, or fed-batch bioreactor. In various embodiments, the bioreactor has a volume of about 1 L to about 20,000 L. Exemplary bioreactor volumes include 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, Including about 900 L, about 1,000 L, about 1,500 L, about 2,000 L, about 2,500 L, about 3,000 L, about 3,500 L, about 4,000 L, about 5,000 L, about 6,000 L, about 7,000 L, about 8,000 L, about 9,000 L, about 10,000 L, about 15,000 L and about 20,000 L.

[0157] As shown in Figures 5-13, the medium and / or cell suspension may be removed from the bioreactor. Such removal may be continuous, as in a perfusion bioreactor, or batchwise, as in a batch or fed-batch reactor. The medium and / or cell suspension is processed by a cell suspension processing system 603 to separate a filtrate containing the biologic. In one or more embodiments, the cell suspension processing system includes one or more steps of cell lysis, filtration, centrifugation, and membrane solubilization. In some embodiments, the biologic is a secreted recombinant protein. In some embodiments, the cells removed from the medium are reintroduced into the bioreactor, and the medium containing the secreted recombinant protein may be further processed. In certain embodiments, the cell suspension processing system 603 includes a filtration system. The filtration system may be any suitable filtration system, including an alternating tangential flow filtration (ATF) system, a tangential flow filtration (TFF) system, a centrifugal filtration system, etc. In various embodiments, the filtration system utilizes filters having a pore size of about 10 nanometers to about 2 micrometers. Typical filter pore sizes include about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm, about 400 nm, about 500 nm, about 600 nm, about 700 nm, about 800 nm, about 900 nm, about 1 μm, about 1.5 μm, and about 2 μm.

[0158] In various embodiments, the medium and / or cell suspension removal rate is from about 1 L / day to about 20,000 L / day. Exemplary medium and / or cell suspension removal rates are about 1 L / day, about 10 L / day, about 20 L / day, about 30 L / day, about 40 L / day, about 50 L / day, about 60 L / day, about 70 L / day, about 80 L / day, about 90 L / day, about 100 L / day, about 150 L / day, about 200 L / day, about 250 L / day, about 300 L / day, about 350 L / day, about 400 L / day, about 500 L / day, about 600 L / day, about 700 L / day, about Examples of suitable rates of removal include about 800 L / day, about 900 L / day, about 1,000 L / day, about 1,500 L / day, about 2,000 L / day, about 2,500 L / day, about 3,000 L / day, about 3,500 L / day, about 4,000 L / day, about 5,000 L / day, about 6,000 L / day, about 7,000 L / day, about 8,000 L / day, about 9,000 L / day, about 10,000 L / day, about 15,000 L / day, and about 20,000 L / day. Alternatively, the medium removal and / or cell suspension removal rates can be expressed as a function of bioreactor volume, such as from about 0.1 to about 3 reactor volumes / day. Exemplary media and / or cell suspension removal rates include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5, and about 3 reactor volumes.

[0159] For continuous or fed-batch processing, the rate at which fresh medium is fed into the bioreactor can be from about 1 L / day to about 20,000 L / day. Exemplary medium introduction rates are about 1 L / day, about 10 L / day, about 20 L / day, about 30 L / day, about 40 L / day, about 50 L / day, about 60 L / day, about 70 L / day, about 80 L / day, about 90 L / day, about 100 L / day, about 150 L / day, about 200 L / day, about 250 L / day, about 300 L / day, about 350 L / day, about 400 L / day, about 500 L / day, about 600 L / day, about 700 L / day, and about 800 L / day. The medium introduction rate may be about 900 L / day, about 1,000 L / day, about 1,500 L / day, about 2,000 L / day, about 2,500 L / day, about 3,000 L / day, about 3,500 L / day, about 4,000 L / day, about 5,000 L / day, about 6,000 L / day, about 7,000 L / day, about 8,000 L / day, about 9,000 L / day, about 10,000 L / day, about 15,000 L / day, and about 20,000 L / day. Alternatively, the medium introduction rate can be expressed as a function of bioreactor volume, such as from about 0.1 to about 3 reactor volumes / day. Exemplary medium introduction rates include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 2, about 2.5 and about 3 reactor volumes.

[0160] After being processed by the cell suspension processing system, the collected filtrate is loaded into capture system 605. Capture system 605 may include one or more chromatography columns.

[0161] In FIG. 6, capture system 605 includes two capture columns 605a and 605b. FIG. 13 shows two chromatography systems, each including a single capture column; i.e., capture column 605a is part of one chromatography system and capture column 605b is part of a separate but identical chromatography system. In both FIGS. 6 and 12, the capture columns are parallel so that the effluent from capture column 605a does not flow to capture column 605b. Rather, once capture column 605a is packed, valve 604 redirects the filtrate flow to the second capture column 605b instead of capture column 605a. In one or more embodiments, the packing of capture columns 605a and 605b cycles back and forth between the columns to provide continuous loading of media from the bioreactor to the capture columns. When more than two capture columns are used, the columns can be packed sequentially or in different orders.

[0162] Figures 7-10 depict two capture columns, capture column 1 and capture column 2, and a purification column operating in series for the continuous purification of a biologic. In Figure 7, capture column 1 is loaded with filtrate containing the biologic. In Figure 8, the captured biologic is eluted from capture column 1 and loaded onto a purification column while the filtrate is loaded onto capture column 2. In Figure 9, the captured biologic is eluted from capture column 2 and loaded onto a purification column while the filtrate is loaded onto capture column 1. In Figure 10, the purified biologic is eluted from the purification column.

[0163] In various embodiments, the capture system 605 includes one or more capture columns (e.g., AEX) for direct product capture of biologics. In some embodiments, the capture column is an AEX column, and the biologic is a secreted recombinant protein, particularly a lysosomal protein with a high M6P content. While not wishing to be bound by any particular theory, it is believed that using AEX chromatography to capture recombinant proteins from filtered media ensures that the captured recombinant protein product has a higher M6P content due to the more negative charge of recombinant proteins bearing one or more M6P groups. As a result, unphosphorylated recombinant protein and host cell impurities do not bind to the column resin and the highly phosphorylated recombinant protein, while the unphosphorylated recombinant protein 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 for proteins with more M6P) due to the high affinity of M6P-containing proteins for the AEX resin.

[0164] Without wishing to be bound by any particular theory, it is believed that direct product capture of recombinant proteins using AEX chromatography ensures that recombinant proteins with high M6P content are removed from medium containing proteases and other enzymes that can degrade and / or dephosphorylate proteins, thereby preserving high quality product.

[0165] Suitable AEX chromatography columns have chemical functional groups that bind to negatively charged molecules, such as negatively charged proteins. Exemplary functional groups include, but are not limited to, primary, secondary, tertiary, and quaternary ammonium or amine groups. These functional groups can bind to membranes (e.g., cellulose membranes) or conventional chromatography resins. Exemplary column media include SP, CM, Q, and DEAE Sepharose® Fast Flow media from GE Healthcare Lifesciences.

[0166] Other capture columns may be used depending on the biologic (e.g., recombinant protein) of interest. For example, CEX, hydrophobic interaction chromatography (HIC), and / or IMAC columns may also be used as capture columns. Other capture columns include those containing antibodies specific to the biologic. In some embodiments, affinity chromatography columns may be used to capture antibodies. In some embodiments, affinity chromatography columns may be used to capture viral particles. In some embodiments, affinity chromatography columns include Protein A and Protein Z columns. In some embodiments, size exclusion chromatography columns may be used as capture columns.

[0167] The volume of the trapping column (eg, AEX column) can be any suitable volume, such as 0.1 L to 1,000 L. Exemplary column volumes include about 0.1L, about 0.2L, about 0.3L, about 0.4L, about 0.5L, about 0.6L, about 0.7L, about 0.8L, about 0.9L, about 1L, about 2L, about 3L, about 4L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 20L, about 30L. , about 40L, about 50L, about 60L, about 70L, about 80L, about 90L, about 100L, about 150L, ​​about 200L, about 250L, about 300L, about 350L, about 400L, about 500L, about 600L, about 700L, about 800L, about 900L and about 1,000L.

[0168] In one or more embodiments, the capture column (e.g., an AEX column) is relatively small compared to the bioreactor size and / or the flow rate of the filtrate loaded onto the capture column. In one or more embodiments, the ratio of bioreactor volume to total capture column volume ranges from about 500:1 to about 10:1. Exemplary ratios include about 500:1, about 450:1, about 400:1, about 350:1, about 300:1, about 250:1, about 200:1, about 150:1, about 100:1, about 90:1, about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1, about 20:1, and about 10:1.

[0169] In one or more embodiments, the total trapping column residence time (e.g., total AEX column residence time) ranges from 0.5 minutes to 200 minutes. Exemplary total trapping column residence times include 0.5 minutes, 1 minute, 1.5 minutes, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 75 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes, and 200 minutes.

[0170] In one or more embodiments, the filtrate is loaded onto the at least two trap columns at a filtrate load rate ranging from about 0.5 to about 100 CV / hr. Exemplary filtrate load rates include about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, and about 100 CV / hr.

[0171] In one or more embodiments, the filtrate is loaded onto the at least two trap columns at a filtrate load rate ranging from about 10 to about 10,000 mL / min. Exemplary filtrate load rates include about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1500, about 2000, about 2500, about 3000, about 3500, about 4000, about 4500, about 5000, about 5500, about 6000, about 6500, about 7000, about 75 ... 00, about 750, about 800, about 850, about 900, about 950, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000 and about 10,000 mL / min.

[0172] Exemplary conditions for the AEX column are shown in Table 2 below.

[0173] [Table 2]

[0174] After the biologic containing filtrate is loaded into capture system 605, the biologic is eluted from the column by changing the pH and / or salt content in the column.

[0175] The eluted biologic may be subjected to further purification and / or quality assurance steps. For example, as shown in FIG. 5, the eluted biologic may be subjected to a virus kill step 607. Such virus kill 607 may include one or more of low pH kill, detergent kill, or other techniques known in the art. In one or more embodiments, the biologic is a virus particle (e.g., AAV) that is more robust than other undesired viruses. In such embodiments, a virus kill step may be further performed to selectively kill undesired viruses.

[0176] Any of the steps shown in Figure 5 may also be applied to the systems shown in Figures 6 and 7, including, but not limited to, virus kill, additional chromatography systems, additional filtration, final product adjustments, etc.

[0177] The biologic from virus killing step 607 can be introduced into a second chromatography system 609 to further purify the biologic. Alternatively, the biologic eluted from capture system 605 can be fed directly to second chromatography system 609. In various embodiments, second chromatography system 609 comprises one or more purification columns. In some embodiments, the purification column comprises an IMAC column for further removal of impurities. Exemplary metal ions include cobalt, nickel, copper, iron, zinc, or gallium. In some embodiments, the purification column comprises an AEX column for further removal of impurities. In some embodiments, the purification column comprises a CEX column for further removal of impurities. In some embodiments, the purification column comprises an affinity column (e.g., a Protein A column or a Protein Z column) for further removal of impurities. In some embodiments, the purification column comprises a size exclusion column for further removal of impurities. In some embodiments, the purification column comprises a hydrophobic interaction chromatography (HIC) column for further removal of impurities.

[0178] The volume of the second chromatography column (e.g., an IMAC column) can be any suitable volume, such as from 0.1 L to 100 L. Exemplary column volumes are about 0.01 L, about 0.02 L, about 0.03 L, about 0.04 L, about 0.05 L, about 0.06 L, about 0.07 L, about 0.08 L, about 0.09 L, about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, about 1L, about 1.5L, about 2L, about 2.5L, about 3L, about 3.5L, about 4L, about 4.5L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about Including 15L, about 20L, about 25L, about 30L, about 35L, about 40L and about 50L, about 60L, about 70L, about 80L, about 90L and about 100L.

[0179] In one or more embodiments, the eluate from the capture column is loaded onto one or more purification columns at a loading rate ranging from about 10 to about 30,000 mL / min. Exemplary purification column loading rates include about 10, about 11, about 12, about 13, about 14, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about about 850, about 900, about 950, about 1,000, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, about 4,000, about 4,500, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, about 20,000, about 25,000 and about 30,000 mL / min.

[0180] In one or more embodiments, the ratio of bioreactor volume to total purification column volume ranges from about 5,000: 1 to about 50: 1. Exemplary ratios include about 5,000: 1, about 4,500: 1, about 4,000: 1, about 3,500: 1, about 3,000: 1, about 2,500: 1, about 2,000: 1, about 1,500: 1, about 1,000: 1, about 900: 1, about 800: 1, about 700: 1, about 600: 1, about 500: 1, about 400: 1, about 300: 1, about 200: 1, about 150: 1, about 100: 1, about 90: 1, about 80: 1, about 70: 1, about 60: 1, and about 50: 1.

[0181] In one or more embodiments, the ratio of total capture column volume to total purification column volume ranges from about 20: 1 to about 1: 1. Exemplary ratios include about 20: 1, about 15: 1, about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4.5: 1, about 4: 1, about 3.5: 1, about 3: 1, about 2.5: 1, about 2: 1, about 1.9: 1, about 1.8: 1, about 1.7: 1, about 1.6: 1, about 1.5: 1, about 1.4: 1, about 1.3: 1, about 1.2: 1, about 1.1: 1, and about 1: 1.

[0182] Exemplary conditions for the IMAC column are shown in Table 3 below.

[0183] [Table 3]

[0184] After the filtrate containing biologic is loaded into a second chromatography system 609, the biologic is eluted from the column. As shown in Figure 5, the eluted biologic may be subjected to a virus kill step 611. As with virus kill 607, virus kill 611 may include one or more of low pH kill, detergent kill, or other techniques known in the art. In some embodiments, only one of virus kill 607 or 611 is used, or multiple virus kills are performed at the same stage in the purification method.

[0185] In one or more embodiments, the eluate from the second chromatography system 609 may be stored. For example, rhGAA, such as ATB200, may be particularly stable in IMAC eluate. In one or more embodiments, the eluate from the second chromatography system (e.g., IMAC eluate) is stored at a temperature between 0°C and 10°C for a period of 24 hours to 105 days. In one or more embodiments, the eluate from the second chromatography system is stored for up to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or 105 days. In one or more embodiments, the eluate from the second chromatography system is stored at a temperature between 15°C and 30°C for a period of 1 hour to 3 days.

[0186] 5, the biologic from virus killing step 611 can be introduced into a third chromatography system 613 to further purify the biologic. Alternatively, the eluted biologic from second chromatography system 609 can be fed directly to third chromatography system 613. In various embodiments, third chromatography system 613 includes one or more AEX columns, CEX columns, size exclusion columns, affinity columns, hydrophobic interaction chromatography columns, and / or SEC columns for further removal of impurities. The biologic is then eluted from third chromatography system 613.

[0187] The volume of the third chromatography column (e.g., a CEX or SEC column) can be any suitable volume, such as from 0.1 L to 200 L. Exemplary column volumes are about 0.01 L, about 0.02 L, about 0.03 L, about 0.04 L, about 0.05 L, about 0.06 L, about 0.07 L, about 0.08 L, about 0.09 L, about 0.1 L, about 0.2 L, about 0.3 L, about 0.4 L, about 0.5 L, about 0.6 L, about 0.7 L, about 0.8 L, about 0.9 L, about 1 L, and about 1. 5L, about 2L, about 2.5L, about 3L, about 3.5L, about 4L, about 4.5L, about 5L, about 6L, about 7L, about 8L, about 9L, about 10L, about 15L, about 20L, Including about 25L, about 30L, about 35L, about 40L and about 50L, about 60L, about 70L, about 80L, about 90L, about 100L, about 150L and about 200L.

[0188] Exemplary conditions for the CEX column are shown in Table 4 below.

[0189] [Table 4]

[0190] The biological 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 filters with pore sizes between 5 nm and 50 μm. Other product processing may include a product conditioning step 617, in which the biological product is sterilized, filtered, concentrated, stored, and / or may have additional components added to the final product formulation. For example, the biological product may be concentrated by a factor of 2-10. This final product may be used to fill vials or may be lyophilized for future use.

[0191] Administration of biologic agents The biologic or its pharmaceutically acceptable salt can be formulated according to conventional procedures as a pharmaceutical composition adapted for administration to humans. For example, in a preferred embodiment, the composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the injection site. Generally, the ingredients are supplied separately or mixed together in a unit dosage form, or as a lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet indicating the quantity of active ingredient. When the composition is administered by infusion, it can 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 for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0192] In some embodiments, the biologic (e.g., a recombinant protein (such as rhGAA) (or a composition or medicament containing the biologic) is administered by an appropriate route. In one embodiment, the biologic is administered intravenously. In other embodiments, the biologic (e.g., rhGAA) is administered by direct administration to a target tissue, such as the heart or skeletal muscle (e.g., intramuscularly) or the nervous system (e.g., direct injection into the brain; intracerebroventricularly; intrathecally). Multiple routes can be used simultaneously, if desired.

[0193] The biologic (e.g., recombinant protein (such as rhGAA) (or composition or medicament containing the biologic) is administered in a therapeutically effective amount (e.g., a dosage sufficient, when administered regularly, to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or reducing the severity or frequency of symptoms of the disease). The therapeutically effective amount for treating a disease will depend on the nature and extent of the effect of the disease, and can be determined by standard clinical techniques. Additionally, in vitro or in vivo assays can be optionally used to assess the efficacy and safety of the biologic. and can be used to facilitate identification of optimal dosage ranges. The precise dose employed will also depend on the route of administration and the severity of the disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may 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 a dose of about 1 mg / kg to about 100 mg / kg, for example, about 5 mg / kg to about 30 mg / kg, typically about 5 mg / kg to about 20 mg / kg. In one embodiment, the biologic is a recombinant protein. In some embodiments, the recombinant human acid alpha-glucosidase is administered by intravenous infusion at a dose of about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 50 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, or about 100 mg / kg. In at least one embodiment, the recombinant human acid alpha- The glucosidase is administered by intravenous infusion at a dose of about 20 mg / kg. In at least one embodiment, the recombinant human acid α-glucosidase is administered by intravenous infusion at a dose of about 20 mg / kg. The effective dose for a particular individual can be varied (e.g., increased or decreased) over time, depending on the individual's needs. For example, the amount can be increased during physical illness or stress, or if anti-acid α-glucosidase antibodies become present or increase, or if disease symptoms worsen.

[0194] A therapeutically effective amount of recombinant human acid α-glucosidase (or a composition or medicament containing recombinant human acid α-glucosidase) is administered periodically and continuously, depending on the nature and severity of the disease. As used herein, "periodic" administration refers to a therapeutically effective amount being administered periodically (as distinguished from a single administration). 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 need not be fixed and can vary over time depending on the individual's needs. For example, the administration interval can be reduced during physical illness or stress, if anti-recombinant human acid α-glucosidase antibodies are present or increase, or if disease symptoms worsen. In some embodiments, a therapeutically effective amount of 5, 10, 20, 50, 100, or 200 mg enzyme / kg body weight is administered twice weekly, weekly, or every other week, with or without a chaperone.

[0195] The biologic (e.g., recombinant protein, such as rhGAA) can be prepared for later use, such as in a unit-dose vial or syringe or in a bottle or bag for intravenous administration. Kits containing the biologic (e.g., recombinant protein, such as rhGAA) and optional excipients or other active ingredients, such as chaperones or other drugs, can be enclosed in packaging material and accompanied by instructions for reconstitution, dilution, or administration to treat a subject in need of treatment, such as a patient suffering from Pompe disease.

[0196] Combination treatment of rhGAA and pharmacological chaperones In various embodiments, rhGAA (e.g., ATB200) produced by the methods described herein can be used in combination therapy with a pharmacological chaperone, such as miglustat or duvoglustat.

[0197] In at least one embodiment, the pharmacological chaperone (e.g., miglustat) is administered orally. In at least one embodiment, miglustat is administered at an oral dose of about 200 to about 400 mg, or at an oral dose of about 200 mg, about 250 mg, about 300 mg, about 350 mg, or about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 233 mg to about 400 mg. In at least one embodiment, miglustat is administered at an oral dose of about 250 to about 270 mg, or at an oral dose of about 250 mg, about 255 mg, about 260 mg, about 265 mg, or about 270 mg. In at least one embodiment, miglustat is administered as an oral dose of about 260 mg.

[0198] Those skilled in the art will appreciate that for an adult patient weighing an average of about 70 kg, an oral dose of miglustat in the range of about 200 mg to 400 mg, or any smaller range, may be appropriate. For patients weighing significantly less than about 70 kg, including, but not limited to, infants, children, or underweight adults, a lower dose may be deemed appropriate by a physician. Thus, in at least one embodiment, miglustat is administered as an oral dose of about 50 mg to about 200 mg, or at oral doses of about 50 mg, about 75 mg, about 100 mg, 125 mg, about 150 mg, about 175 mg, or about 200 mg. In at least one embodiment, miglustat is administered as an oral dose of about 65 mg to about 195 mg, or at oral doses of about 65 mg, about 130 mg, or about 195 mg.

[0199] In at least one embodiment, miglustat is administered in a pharmaceutically acceptable dosage form suitable for oral administration, including, but not limited to, tablets, capsules, ovules, elixirs, solutions or suspensions, gels, syrups, mouthwashes, or dry powders for reconstitution with water or other suitable solvents prior to use, optionally with flavoring and coloring, for immediate-release, delayed-release, modified-release, sustained-release, pulsed-release, or controlled-release applications. Solid compositions such as tablets, capsules, lozenges, troches, pills, boluses, powders, pastes, granules, bullets, dragees, or premixed preparations can 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 contemplated that miglustat may be administered as four dosage forms or a total dose of 260 mg of miglustat. However, for patients with a body weight significantly lower than the average adult weight of 70 kg, including, but not limited to, infants, children, or underweight adults, miglustat may be administered as one dosage form (total dose of 65 mg miglustat), two dosage forms (total dose of 130 mg miglustat), or three dosage forms (total dose of 195 mg miglustat).

[0200] Solid and liquid compositions for oral use can be prepared according to methods well known in the art. Such compositions can also contain one or more pharmaceutically acceptable carriers and excipients, which can be in solid or liquid form. Tablets or capsules can 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 known in the art and include, but are not limited to, pregelatinized starch, polyvinylpyrrolidone, povidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), sucrose, gelatin, gum arabic, lactose, microcrystalline 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, dibasic calcium phosphate, glycine croscarmellose sodium, and silicate complexes. Tablets may be coated by methods well known in the art. In at least one embodiment, miglustat is administered in the formulation marketed as Zavesca® (Actelion Pharmaceuticals).

[0201] 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 administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 3 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 2 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 2 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1.5 hours before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 1 hour before administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 50 to about 70 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 55 to about 65 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 30 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 25 to about 35 minutes prior to administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered about 27 to about 33 minutes prior to administration of recombinant human acid α-glucosidase.

[0202] 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 within 20 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 15 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 10 minutes before or after the administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered within 5 minutes before or after the administration of recombinant human acid α-glucosidase.

[0203] In at least one embodiment, miglustat is administered after administration of recombinant human acid α-glucosidase. In at least one embodiment, miglustat is administered up to 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.

[0204] Another aspect of the present invention provides a kit for combination treatment of Pompe disease in a patient in need thereof. The kit includes a pharmaceutically acceptable dosage form comprising miglustat, a pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase as defined herein, and instructions for administering the pharmaceutically acceptable dosage form comprising miglustat and the pharmaceutically acceptable dosage form comprising recombinant acid α-glucosidase to a patient in need thereof. In at least one embodiment, the pharmaceutically acceptable dosage form comprising miglustat is an oral dosage form described herein, including, but not limited to, a tablet or capsule. In at least one embodiment, the pharmaceutically acceptable dosage form comprising 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 comprising miglustat before administering the pharmaceutically acceptable dosage form comprising recombinant human acid α-glucosidase by intravenous infusion, as described herein.

[0205] Without being bound by theory, miglustat is believed to act as a pharmacological chaperone of recombinant human acid α-glucosidase ATB200, binding to its active site. For example, it has been found that miglustat reduces the proportion of unfolded ATB200 protein, stabilizes the active conformation of ATB200, prevents denaturation and irreversible inactivation at the neutral pH of plasma, and allows it to survive in the circulation long enough to reach and be taken up by tissues. However, the binding of miglustat to the active site of ATB200 may also result in inhibition of the enzymatic activity of ATB200 by preventing the 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 the plasma and tissues are stabilized until ATB200 is taken up into the tissue and targets the lysosomes. However, because the clearance of miglustat is rapid, hydrolysis of glycogen by ATB200 in the lysosomes is not excessively inhibited by the presence of miglustat, and the enzyme is thought to retain sufficient activity to be therapeutically useful.

[0206] All of the above embodiments may be combined, including specific embodiments relating to: The properties of pharmacological chaperones, e.g., miglustat; and the active site for which they are specific; · type of pharmaceutical composition, including dosage, route of administration of pharmacological chaperone (e.g., miglustat) and nature of carrier and use of commercially available compositions; a therapeutic protein drug product of medicinal nature, for example, which may be a counterpart of an endogenous protein whose expression is reduced or absent in a subject, preferably a recombinant protein (e.g., rhGAA), such as recombinant human acid alpha-glucosidase expressed in Chinese hamster ovary (CHO) cells and comprising an increased content of N-glycan units with one or more alpha mannose-6-phosphate residues when compared to the content of N-glycan units with one or more alpha mannose-6-phosphate residues of alglucosidase; and preferably having the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; · The number and type of N-glycan units on the recombinant protein (e.g., rhGAA), such as complex N-glycans formed from N-acetylglucosamine, galactose, sialic acid, or combinations thereof, attached to the recombinant protein; · the degree of phosphorylation of mannose units on the recombinant protein (e.g., rhGAA) to form mannose-6-phosphate and / or bis-mannose-6-phosphate; · The dosage and route of administration (e.g., intravenous administration, particularly intravenous infusion or direct administration to the target tissue) of the replacement enzyme (e.g., recombinant human acid α-glucosidase) and the type of formulation, including carrier and therapeutically effective amount; · Administration interval of pharmacological chaperone (miglustat) and recombinant human acid α-glucosidase; · the nature of the treatment response and the outcome of the combined treatment (e.g., enhanced outcome compared with the effect of each treatment administered individually); Timing of administration of combination treatments, e.g., simultaneous or sequential administration of miglustat and recombinant human acid alpha-glucosidase, e.g., when miglustat is administered before recombinant human acid alpha-glucosidase, after recombinant human acid alpha-glucosidase, or within a certain time period before or after administration of recombinant human acid alpha-glucosidase; and The nature of the patient to be treated (e.g., a mammal such as a human) and the medical condition from which the individual is suffering (e.g., an enzyme deficiency).

[0207] Any embodiment in the above list can be combined with one or more of the other embodiments in the list. [Example]

[0208] Further features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.

[0209] Example 1: Preparation of CHO cells producing ATB200 rhGAA with a high content of mono- or bis-M6P-containing N-glycans CHO cells were transfected with DNA that expresses rhGAA, followed by selection of transformants that produced rhGAA. The DNA construct for transforming CHO cells with DNA encoding rhGAA is shown in Figure 4. CHO cells were transfected with DNA that expresses rhGAA, followed by selection of transformants that produced rhGAA.

[0210] After transfection, DG44 CHO (DHFR-) cells containing a stably integrated GAA gene were selected in hypoxanthine / thymidine-deficient (-HT) medium.

[0211] Amplification of GAA expression in these cells was induced by methotrexate treatment (MTX, 500 nM). Cell pools expressing large amounts of GAA were identified by GAA enzyme activity assay and used to establish individual clones producing rhGAA. Individual clones were generated on semi-solid medium plates, picked 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. Conditioned medium for determining GAA activity was prepared using 4-MU-α-glucosidase substrate. Clones producing higher levels of GAA, as measured by the GAA enzyme assay, were further evaluated for viability, growth potential, GAA productivity, N-glycan structure, and stable protein expression. CHO cell lines, including the CHO cell line GA-ATB-200, expressing rhGAA with enhanced mono-M6P or bis-M6P N-glycans were isolated using this procedure.

[0212] Example 2: Proof-of-concept design for capture and purification of ATB200 rhGAA Cells expressing ATB200 rhGAA are cultured in a bioreactor. The cell culture medium is removed, filtered, and frozen for later use. The bulk container containing the thawed harvest is then used in batch mode to pack two AEX columns, each with a column volume of 15.7 mL (1 cm diameter x 20 cm bed height). The AEX columns are packed at a flow rate of 1.57 mL / min. The total AEX column residence time (i.e., the quotient of the total AEX column volume and the volumetric flow rate at which the AEX columns are packed) is 20 minutes.

[0213] The continuous process was carried out according to the following protocol. Run two sequences (equal to the same number of trials as the control condition) Each AEX eluate is processed sequentially on an IMAC column. i. Sequence 1 1. Fill AEX1 → Send AEX1 eluate to IMAC via STP → Collect IMAC eluate (IMACa) 2. Fill AEX2 → Send AEX2 eluate to IMAC via STP → Collect IMAC eluate (IMACb) ii. Sequence 2 1. Fill AEX1 → Send AEX1 eluate to IMAC via STP → Collect IMAC eluate (IMACa) 2. Fill AEX2 → Send AEX2 eluate to IMAC via STP → Collect IMAC eluate (IMACb)

[0214] A batch process was also run as a control according to the following protocol. Run 4 tests (2 tests for each AEX column: AEX1, AEX2) Each AEX eluate is collected and processed on an IMAC column. i. Control test 1 = Load AEX1 → Collect AEX1 eluate → Load AEX1 eluate into IMAC → Collect IMAC eluate ii. Control test 2 = Load AEX2 → Collect AEX2 eluate → Load AEX2 eluate into IMAC → Collect IMAC eluate iii. Control test 3 = Load AEX1 → Collect AEX1 eluate → Load AEX1 eluate into IMAC → Collect IMAC eluate iv. Control Test 4 = Load AEX2 → Collect AEX2 eluate → Load AEX2 eluate into IMAC → Collect IMAC eluate

[0215] The process conditions for each AEX column are shown in Table 5 below.

[0216] [Table 5]

[0217] The dissolution profiles for the control and continuous processes are shown in Figures 13 and 14, respectively.

[0218] The AEX eluate is then loaded onto a single IMAC column with a column volume of 3.9 mL (1 cm diameter x 5 cm bed height). The IMAC column is loaded at a flow rate of 0.65 mL / min. The ratio of total AEX column volume to IMAC column volume is approximately 8:1. Elution profiles for both the batch and continuous purification processes were recorded and are shown in Figures 15 and 16. The process conditions for the IMAC column are shown in Table 6 below.

[0219] [Table 6]

[0220] The rhGAA produced according to the continuous process of this example is comparable to the rhGAA produced according to the control (previous batch mode) process, thus indicating no reduction in product quality using the continuous process in this Example 2. However, the proposed equipment footprint to implement the continuous process of this Example 2 is estimated to result in a 4-5 fold reduction in equipment footprint.

[0221] The final products from batch and continuous purification were analyzed and the data are listed in Tables 7-9.

[0222] [Table 7]

[0223] [Table 8]

[0224] [Table 9]

[0225] Example 3: Commercial-scale plant for capture and purification of ATB200 rhGAA Cells expressing ATB200 rhGAA are cultured in a large bioreactor (e.g., 500-2,000 L). The cell culture medium is continuously removed, filtered, and loaded onto two AEX columns, each with a column volume of 5-25 L. The AEX columns are configured as capture columns, as shown in Figure 6. The ratio of bioreactor volume to total AEX column volume ranges from about 100:1 to about 20:1. The AEX eluate is loaded directly onto an IMAC column. The IMAC column volume ranges from 1-10 L, and the ratio of total AEX column volume to IMAC column volume ranges from 2:1 to 10:1. Figure 11 shows an exemplary operating sequence for the AEX and IMAC columns during rhGAA purification.

[0226] 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 present invention. It is intended to include various modifications and variations that are consistent with the present specification as a whole and are readily apparent to those skilled in the art. The scope of the appended claims should not be limited by the specific embodiments described in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

[0227] Patents, patent applications, publications, product descriptions, GenBank accession numbers, and protocols are cited throughout this application, the disclosures of which are incorporated herein by reference in their entireties for all purposes.

Claims

1. 1. A method of producing a biologic, comprising: Cultivating host cells in a bioreactor that produce and optionally secrete a biologic; removing the medium and / or cell suspension from the bioreactor; processing the medium and / or cell suspension to isolate a filtrate containing the biologic; loading the filtrate onto at least two capture columns to capture the biologic; eluting the first biological agent from the at least two capture columns; loading the first biological product onto one or more purification columns; eluting the second biological product from the one or more purification columns; wherein the bioreactor has a bioreactor volume, the at least two trapping columns have a total trapping column volume, and the ratio of the bioreactor volume to the total trapping column volume is in the range of about 500:1 to about 10:

1.

2. 10. The method of claim 1, wherein the biologic comprises one or more of a recombinant protein, a viral particle, or an antibody.

3. 3. The method of claim 2, wherein the recombinant protein is a secreted protein, a membrane protein, or an intracellular protein produced by the host cell.

4. 4. The method of claim 3, wherein the recombinant protein is separated from cells and organelles in the filtrate.

5. The method according to any one of claims 1 to 4, wherein the filtrate is separated by filtration or centrifugation.

6. 6. The method of any one of claims 1 to 5, wherein the at least two trap columns are loaded in series to provide for sequential loading of the filtrate onto the at least two trap columns.

7. 7. The method of any one of claims 1 to 6, wherein the filtrate is loaded onto the at least two trap columns at a filtrate loading rate ranging from about 0.5 to about 100 column volumes (CV) per hour.

8. 8. The method of any one of claims 1 to 7, wherein the filtrate is loaded onto the at least two trap columns to provide a trap column loading time of less than 48 hours for each trap column.

9. The method of any one of claims 1 to 8, wherein the biologic comprises a recombinant human lysosomal protein.

10. The method of any one of claims 1 to 9, wherein the at least two trapping columns comprise at least two anion exchange chromatography (AEX) columns.

11. The method of any one of claims 1 to 9, wherein the at least two capture columns comprise at least two affinity chromatography columns.

12. 12. The method of claim 11, wherein the affinity chromatography column comprises one or more of a Protein A column and a Protein Z column.

13. The method of any one of claims 1 to 9, wherein the at least two trapping columns comprise at least two cation exchange chromatography (CEX) columns.

14. The method of any one of claims 1 to 9, wherein the at least two capture columns comprise at least two immobilized metal affinity chromatography (IMAC) columns.

15. The method of any one of claims 1 to 9, wherein the at least two trapping columns comprise at least two size exclusion chromatography columns.

16. The method of any one of claims 1 to 9, wherein the at least two capture columns comprise at least two hydrophobic interaction chromatography (HIC) columns.

17. 17. The method of any one of claims 1 to 9 or 11 to 16, wherein the one or more purification columns comprise one or more anion exchange chromatography (AEX) columns.

18. 17. The method of any one of claims 1 to 10 or 13 to 16, wherein the one or more purification columns comprise one or more affinity chromatography columns.

19. 20. The method of claim 18, wherein the affinity chromatography column comprises one or more of a Protein A column and a Protein Z column.

20. 17. The method of any one of claims 1 to 12 or 14 to 16, wherein the one or more purification columns comprise one or more cation exchange chromatography (CEX) columns.

21. 17. The method of any one of claims 1 to 13, or 15 or 16, wherein the one or more purification columns comprise one or more immobilized metal affinity chromatography (IMAC) columns.

22. 17. The method of any one of claims 1 to 14 or 16, wherein the one or more purification columns comprise one or more size exclusion chromatography columns.

23. The method of any one of claims 1 to 15, wherein the one or more purification columns comprise one or more hydrophobic interaction chromatography (HIC) columns.

24. 24. The method of any one of claims 1-23, wherein the second biological product is eluted from the one or more purification columns within 48 hours of removing the medium and / or cell suspension from the bioreactor.

25. 25. The method of any one of claims 1 to 24, wherein the one or more purification columns have a total purification column volume, and the ratio of the bioreactor volume to the total purification column volume ranges from about 5,000:1 to about 50:

1.

26. 26. The method of any one of claims 1 to 25, wherein the ratio of the total capture column volume to the total purification column volume ranges from about 20:1 to about 1:

1.

27. 1. A method for producing a recombinant human lysosomal protein, comprising: Culturing host cells in a bioreactor that produce and optionally secrete a recombinant human lysosomal protein; removing the medium and / or cell suspension from the bioreactor; treating the medium and / or cell suspension to isolate a filtrate containing the lysosomal protein; loading the filtrate onto at least two anion exchange chromatography (AEX) columns to capture the lysosomal proteins; eluting the first biological agent from the at least two AEX columns; loading the first biological product onto one or more immobilized metal affinity chromatography (IMAC) columns; eluting the second biological agent from the one or more IMAC columns; and wherein the bioreactor has a bioreactor volume, and the at least two AEX columns have a total AEX column volume, and wherein the ratio of the bioreactor volume to the total AEX column volume ranges from about 500:1 to about 10:

1.

28. 28. The method of claim 27, wherein the lysosomal protein is a secreted protein, a membrane protein, or an intracellular protein produced by the host cell.

29. 29. The method of claim 28, wherein the intracellular proteins are isolated in the filtrate by lysing the cells to prepare a cell lysate.

30. 30. The method of claim 28 or 29, wherein the cell lysate is separated from the filtrate by filtration or centrifugation.

31. 31. The method of any one of claims 27 to 30, wherein the at least two AEX columns are packed in series to provide for sequential loading of the filtrate onto the at least two AEX columns.

32. 32. The method of any one of claims 27 to 31, wherein the filtrate is loaded onto the at least two AEX columns at a filtrate loading rate ranging from about 0.5 to about 100 column volumes (CV) per hour.

33. 33. The method of any one of claims 27 to 32, wherein the filtrate is loaded onto the at least two AEX columns to provide an AEX loading time of less than 48 hours for each AEX column.

34. 34. The method of any one of claims 27 to 33, wherein each AEX column has a column volume of 50 L or less.

35. 35. The method of any one of claims 27-34, wherein the second biological product is eluted from the one or more IMAC columns within 48 hours of removing the medium and / or cell suspension from the bioreactor.

36. 36. The method of any one of claims 27-35, wherein the one or more IMAC columns have a total IMAC column volume, and the ratio of the bioreactor volume to the total IMAC column volume ranges from about 5,000:1 to about 50:

1.

37. 37. The method of any one of claims 27 to 36, wherein the ratio of the total AEX column volume to the total IMAC column volume ranges from about 20:1 to about 1:

1.

38. 38. The method of any one of claims 27 to 37, wherein each IMAC column has a column volume of 20 L or less.

39. 39. The method of any one of claims 1 to 38, further comprising storing the second biological product.

40. 40. The method of claim 39, wherein the second biological product is stored at a temperature between 0°C and 10°C for a period of between 24 hours and 105 days.

41. 41. The method of claim 40, wherein the second biological product is stored at a temperature of 15°C to 30°C for a period of 1 hour to 3 days.

42. loading the second biological product onto a third chromatography column; eluting the third biological product from the third chromatography column; and 42. The method of any one of claims 1 to 41, further comprising:

43. 43. The method of claim 42, wherein the third chromatography column is selected from an anion exchange chromatography (AEX) column, an affinity chromatography column, a cation exchange chromatography (CEX) column, an immobilized metal affinity chromatography (IMAC) column, a size exclusion chromatography (SEC) column, and a hydrophobic interaction chromatography (HIC) column.

44. 44. The method of any one of claims 1 to 43, wherein the filtrate is separated by filtering the culture medium and / or cell suspension through one or more of alternating tangential flow filtration (ATF) and tangential flow filtration (TFF).

45. 45. The method of any one of claims 1-44, further comprising inactivating viruses in one or more of the first biological product, the second biological product, and the third biological product.

46. 46. ​​The method of any one of claims 1-45, further comprising filtering the second biological product or the third biological product to provide a filtered product and filling a vial with the filtered product.

47. 47. The method of any one of claims 1 to 46, further comprising lyophilizing the filtered product.

48. 48. The method of any one of claims 1 to 47, wherein the biologic comprises recombinant human alpha-glucosidase (rhGAA).

49. 49. The method of claim 48, wherein the rhGAA comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

50. 50. The method of any one of claims 1 to 49, wherein the host cell comprises a Chinese hamster ovary (CHO) cell.

51. 51. The method of claim 50, wherein the host cell comprises CHO cell line GA-ATB-200 or ATB-200-001-X5-14, or a subculture thereof.

52. (i) at least 90% of the first biological agent, or the second biological agent, or the third biological agent binds to the cation-independent mannose-6-phosphate receptor (CIMPR); or (ii) the method of any one of claims 1-51, wherein at least 90% of the first biological product, or the second biological product, or the third biological product, contains N-glycans bearing mono-mannose-6-phosphate (mono-M6P) or bis-mannose-6-phosphate (bis-M6P).

53. 53. The method of any one of claims 48-52, wherein the rhGAA comprises seven potential N-glycosylation sites, at least 50% of the rhGAA molecules comprise N-glycan units having two mannose-6-phosphate residues at a first site, at least 30% of the rhGAA molecules comprise N-glycan units having one mannose-6-phosphate residue at a second site, at least 30% of the rhGAA molecules comprise N-glycan units having two mannose-6-phosphate residues at a fourth site, and at least 20% of the rhGAA molecules comprise N-glycan units having one mannose-6-phosphate residue at a fourth site.

54. A biological product produced by the method of any one of claims 1 to 53.

55. 55. A pharmaceutical composition comprising the biological preparation of claim 54 and a pharmaceutically acceptable carrier.

56. 56. A method for treating a lysosomal storage disease, comprising administering the pharmaceutical composition of claim 55 to a patient in need thereof.

57. 57. The method of claim 56, wherein the lysosomal storage disease is Pompe disease and the biologic is rhGAA.

58. 58. The method of claim 57, wherein the patient is co-administered with a pharmacological chaperone for α-glucosidase within 4 hours of administering the pharmaceutical composition comprising the rhGAA product.

59. 59. The method of claim 58, wherein the pharmacological chaperone is selected from 1-deoxynojirimycin and N-butyl-deoxynojirimycin.

60. 60. The method of claim 59, wherein the pharmacological chaperone is co-formulated with the rhGAA product.

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