Methods of increasing sialic acid levels in recombinant glycosylated proteins

Abstract

Provided herein are methods of increasing sialic acid levels in recombinant glycosylated proteins, recombinant glycosylated proteins produced by these methods, and methods of treating subjects in need thereof using these recombinant glycosylated proteins.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 461,211, filed April 21, 2023; the entirety of which is incorporated herein by reference.

[0002] SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format in which the file name is 47364-0050WO1_ST26_SL. The XML file was created on April 12, 2024 and is 1,210,951 bytes in size. The contents of the XML file are hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0003] The present disclosure relates to cell culture methods and biofabrication of recombinant glycosylated proteins. BACKGROUND

[0004] Lysosomal storage disorders (LSDs) are relatively rare, inherited metabolic diseases that arise from defects in lysosomal function. LSDs are typically caused by a deficiency in a single enzyme involved in the breakdown of metabolic products in the lysosome. Accumulation of products due to insufficient enzyme activity affects various organ systems and can lead to severe symptoms and early death. Most LSDs also have a significant neurological component, ranging from progressive neurodegeneration and severe cognitive impairment to seizures, behavioral, and psychiatric disorders. Recombinant versions of the deficient enzymes of LSDs can be used to treat the disorder.

[0005] LSD enzymes belong to a group of glycosylated proteins that can exhibit heterogeneity when produced in cell culture, in part due to the glycosylation pattern of the protein. The glycosylation pattern can be strongly influenced by cell culture conditions. In turn, the glycosylation (including galactosylation) profile of a particular protein can impact its biological activity due to variable effects on folding, stability, efficacy, and half-life. Various factors in cell culture are interconnected, so that one factor (e.g., addition of galactose to the culture medium) can positively influence the glycosylation pattern of a recombinant protein on one hand, but can negatively impact other properties of the protein, such as charge distribution, proportion of protein fragments, proportion of aggregates, and protein titer, on the other hand. In addition, cell viability can be impacted. Thus, there is a need to develop methods to improve the glycosylation pattern, such as increasing the sialic acid content in recombinantly produced glycosylated proteins. SUMMARY

[0006] Provided herein are methods of increasing sialic acid levels in a recombinant glycosylated protein comprising: culturing a mammalian host cell capable of expressing the recombinant glycosylated protein in a liquid culture medium, the liquid culture medium comprising one or more culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine, wherein the mammalian host cell has been previously transformed with one or more vectors encoding one or more sialyltransferases.

[0007] In some embodiments of any of the methods described herein, the method further comprises transforming a mammalian cell capable of expressing the recombinant glycosylated protein with one or more vectors encoding the one or more sialyltransferases prior to the culturing step to produce the mammalian host cell. In some embodiments, the mammalian host cell is a rodent cell. In some embodiments, the rodent cell is a CHO cell. In some embodiments of any of the methods described herein, the mammalian host cell is a human cell. In some embodiments of any of the methods described herein, the one or more sialyltransferases are selected from the group consisting of: ST6GAL1 and ST3GAL4 In some embodiments, one of the one or more vectors encodes ST6GAL1 .

[0008] In some embodiments of any of the methods described herein, the liquid culture medium comprises one of: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, or uridine. In some embodiments of any of the methods described herein, the liquid culture medium comprises two of: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine. In some embodiments of any of the methods described herein, the liquid culture medium comprises three of: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine. In some embodiments of any of the methods described herein, the liquid culture medium comprises hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine. In some embodiments of any of the methods described herein, the ManNAc is present in the liquid culture medium at a concentration of 10 mM to about 60 mM. In some embodiments of any of the methods described herein, the uridine is present in the liquid culture medium at a concentration of 2.5 mM to about 10 mM. In some embodiments of any of the methods described herein, the hydrocortisone is present in the liquid culture medium at a concentration of 5 μΜ to about 50 μΜ. In some embodiments of any of the methods described herein, the manganese is present in the liquid culture medium at a concentration of 2 μΜ to about 15 μΜ.

[0009] In some embodiments of any of the methods described herein, the recombinant glycosylated protein is a recombinant glycosylated enzyme. In some embodiments, the recombinant glycosylated enzyme comprises an enzyme replacement therapy (ERT) enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment. In some embodiments, the enzyme replacement therapy (ERT) enzyme comprises a lysosomal storage disease (LSD) enzyme. In some embodiments, the LSD enzyme is a-L-iduronidase (IDUA) or N-sulfoglucoamine sulfatohydrolase (SGSH). In some embodiments, the recombinant glycosylated enzyme is a fusion protein. In some embodiments, the fusion protein comprises (i) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment; and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide that is capable of specific binding to a transferrin receptor (TfR). In some embodiments, the fusion protein comprises (i) an enzyme replacement therapy (ERT) enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment; and (ii) a modified Fc dimer that is capable of specific binding to a transferrin receptor (TfR). In some embodiments, the fusion protein comprises an enzyme replacement therapy (ERT) enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment linked to an Fc polypeptide. In some embodiments, the Fc polypeptide is a modified Fc polypeptide. In some embodiments, the Fc polypeptide is capable of specific binding to a transferrin receptor (TfR).

[0010] In some embodiments of any of the methods described herein, the culture is a fed-batch culture. In some embodiments of any of the methods described herein, the culture is a batch culture. In some embodiments of any of the methods described herein, the culture is a perfusion culture.

[0011] In some embodiments of any of the methods described herein, the sialic acid level in the recombinant glycosylated protein is increased by at least 50% compared to the case of a recombinant glycosylated protein produced by a method that does not comprise one or both of (i) and (ii): (i) use of a mammalian host cell that is capable of expressing the recombinant glycosylated protein and comprises one or more vectors encoding the one or more sialyltransferases, and (ii) culturing the mammalian host cell that is capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0012] In some embodiments of any of the methods described herein, the sialic acid level in the recombinant glycosylated protein is increased at least 100% compared to the case of a recombinant glycosylated protein produced by a method that does not include one or both of (i) and (ii): (i) use of a mammalian host cell capable of expressing the recombinant glycosylated protein and comprising one or more vectors encoding the one or more sialyltransferases, and (ii) culturing the mammalian host cell capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0013] In some embodiments of any of the methods described herein, the sialic acid level in the recombinant glycosylated protein is increased at least 150% compared to the case of a recombinant glycosylated protein produced by a method that does not include one or both of (i) and (ii): (i) use of a mammalian host cell capable of expressing the recombinant glycosylated protein and comprising one or more vectors encoding the one or more sialyltransferases, and (ii) culturing the mammalian host cell capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0014] In some embodiments of any of the methods described herein, the sialic acid level in the recombinant glycosylated protein is increased at least 250% compared to the case of a recombinant glycosylated protein produced by a method that does not include one or both of (i) and (ii): (i) use of a mammalian host cell capable of expressing the recombinant glycosylated protein and comprising one or more vectors encoding the one or more sialyltransferases, and (ii) culturing the mammalian host cell capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0015] In some embodiments of any of the methods described herein, the method further comprises harvesting the recombinant glycosylated protein from the liquid culture medium and / or the mammalian host cell.

[0016] In some embodiments of any of the methods described herein, the method further comprises isolating the harvested recombinant glycosylated protein.

[0017] In some embodiments of any of the methods described herein, the method further comprises formulating the isolated recombinant glycosylated protein.

[0018] Also provided herein are recombinant glycosylated proteins produced by any of the methods described herein.

[0019] Also provided herein are methods of treating a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any of the recombinant glycosylated proteins described herein.

[0020] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs. Methods and materials are described herein for use in the present application; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0021] Other features and advantages of the present application will be apparent from the following detailed description, and from the drawings and claims. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a schematic showing the key pathways affecting sialylation and the evaluation areas tested for recombinant fusion protein production.

[0023] Figure 2 is a bar graph showing the percent sialic acid increase of SGSH-Fc fusion proteins produced in the presence of different media additives: 20 mM hydrocortisone, 40 mM ManNAc, 10 mM Mn, or 5 mM uridine.

[0024] Figure 3 is a bar graph including a graphical representation of the percent sialic acid increase of a pool expressing IDUA-Fc fusion protein (Pool-B) and its clones (Clone B-l and Clone B-2).

[0025] Figure 4 is a bar graph including a graphical representation of the percent sialic acid increase of a pool expressing IDUA-Fc protein under different culture conditions and in the presence or absence of sialyltransferase gene overexpression.

[0026] Figure 5 is a bar graph including a graphical representation of the percent sialic acid increase of a pool expressing IDUA-Fc protein under different culture conditions and in the presence or absence of galactosyltransferase and / or sialyltransferase gene overexpression. DETAILED DESCRIPTION

[0027] Provided herein are methods of increasing sialic acid levels in a recombinant glycosylated protein comprising: culturing a mammalian host cell capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more (e.g., 1, 2, 3, or 4) culture medium additives selected from the group consisting of hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine, wherein the mammalian host cell has been previously transformed with one or more vectors encoding one or more sialyltransferases. In some embodiments, the mammalian host cell has been previously transformed with one or more vectors encoding galactosyltransferases as well. Figure 1 Metabolic steps affecting sialylation and evaluation areas tested for recombinant fusion protein production are shown.

[0028] In some embodiments, the recombinant glycosylated protein is an ERT enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment. In some embodiments, the recombinant glycosylated protein is a fusion protein (e.g., any of the exemplary fusion proteins described herein, e.g., a fusion protein comprising an ERT enzyme). Various embodiments thereof are disclosed herein.

[0029] Definitions As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a recombinant glycosylated protein” can include two or more such recombinant glycosylated proteins, and the like.

[0030] As used herein, the terms “about” and “approximately” when used to modify a value or range of values specified in a quantity means that the value as well as a reasonable deviation from the value known to one of skill in the art, e.g., ±20%, ±10%, or ±5% are within the intended meaning of the recited value.

[0031] “Enzyme replacement therapy enzyme” or “ERT enzyme” refers to an enzyme that is deficient in a disorder (e.g., a lysosomal storage disorder). Examples of ERT enzymes include “lysosomal storage disorder (LSD) enzymes,” which refer to a class of enzymes that are deficient in lysosomal storage disorders. “Catalytically active ERT enzyme variant” refers to a functional variant of a wild-type ERT enzyme or catalytically active ERT enzyme fragment, including allelic variants and splice variants, wherein the ERT enzyme variant has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type ERT enzyme or catalytically active ERT enzyme fragment, e.g., when assayed under the same conditions. “Catalytically active ERT enzyme fragment” of an ERT enzyme refers to a portion of a full-length ERT enzyme or catalytically active ERT enzyme variant, wherein the catalytically active fragment has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length ERT enzyme or catalytically active ERT enzyme variant, e.g., when assayed under the same conditions.

[0032] “N-sulfogluconamidase sulfhydrylase” or “SGSH” as used herein refers to N-sulfogluconamidase sulfhydrylase (EC 3.10.1.1), which is an enzyme involved in lysosomal degradation of heparan sulfate. Mutations in this gene are associated with Sanfilippo syndrome A, a type of lysosomal storage disease mucopolysaccharidosis III, caused by impaired degradation of heparan sulfate. In some embodiments, SGSH can be a component of a recombinant glycosylated protein further comprising an Fc polypeptide (e.g., any of the exemplary Fc polypeptides or regions described herein). As used herein, SGSH encompasses catalytically active fragments of SGSH as well as functional variants of wild-type SGSH or fragments thereof, including allelic variants and splice variants. The sequence of human SGSH can be obtained at UniProt entry P51688 and is encoded by the human SGSH gene located at 17q25.3. The full-length sequence is provided at SEQ ID NO: 1. “Mature” SGSH sequence as used herein refers to a form of the polypeptide chain that lacks the signal sequence of the naturally occurring full-length polypeptide chain. The amino acid sequence of the mature human SGSH polypeptide is provided at SEQ ID NO: 2, which corresponds to amino acids 21-502 of the full-length human sequence. The structure of human SGSH has been well characterized. An exemplary structure can be obtained at PDB accession code 4MHX. Non-human primate SGSH sequences have also been described, including chimpanzee (UniProt entry K7C218). Mouse SGSH sequences can be obtained at UniProt entry Q9EQ08. A SGSH variant has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type SGSH or fragment thereof, e.g., when assayed under the same conditions. A catalytically active SGSH fragment has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length SGSH or variant thereof, e.g., when assayed under the same conditions.

[0033] “alpha-L-iduronase,” “iduronase alpha-L,” “L-iduronase,” “iduronase,” or “IDUA” as used herein refers to alpha-L-iduronase (EC 3.2.1.76), which is an enzyme involved in lysosomal degradation of glycosaminoglycans such as dermatan sulfate and heparan sulfate. IDUAMutations in the gene are associated with MPS I, which is caused by impaired degradation of heparan sulfate and dermatan sulfate. The term "IDUA" or "IDUA enzyme" as used herein (optionally as a component of a protein comprising an Fc polypeptide) encompasses catalytically active fragments of IDUA as well as functional variants, including allelic variants and splice variants, or fragments thereof. The sequence of human IDUA can be obtained at UniProt entry P35475, and is encoded by the human gene located at 4pl6.3. The full-length sequence is provided as SEQ ID NO: 3, which can have a H or Q at position 33, and / or an A or T at position 622, with the positions according to EU numbering. A "mature" IDUA sequence as used herein refers to a form of the polypeptide chain that lacks the signal sequence of the naturally occurring full-length polypeptide chain. One embodiment of the amino acid sequence of a mature human IDUA polypeptide is provided as SEQ ID NO: 8, which corresponds to amino acids 27-653 of the full-length human sequence. A "truncated" IDUA sequence as used herein refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. SEQ ID NO: 9 is one exemplary truncated IDUA sequence. The structure of human IDUA has been well characterized. Non-human primate IDUA sequences have also been described, including the chimpanzee (e.g., UniProt entry A0A2R9ALZ1 for Pan troglodytes / Pygmy chimpanzee / Bonobo). The mouse IDUA sequence can be obtained at UniProt entry P48441. An IDUA variant has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type IDUA or fragment thereof, e.g., when assayed under the same conditions. A catalytically active IDUA fragment has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length IDUA or variant thereof, e.g., when assayed under the same conditions. IDUA The gene encodes. The full-length sequence is provided as SEQ ID NO: 3, which can have a H or Q at position 33, and / or an A or T at position 622, with the positions according to EU numbering. A "mature" IDUA sequence as used herein refers to a form of the polypeptide chain that lacks the signal sequence of the naturally occurring full-length polypeptide chain. One embodiment of the amino acid sequence of a mature human IDUA polypeptide is provided as SEQ ID NO: 8, which corresponds to amino acids 27-653 of the full-length human sequence. A "truncated" IDUA sequence as used herein refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. SEQ ID NO: 9 is one exemplary truncated IDUA sequence. The structure of human IDUA has been well characterized. Non-human primate IDUA sequences have also been described, including the chimpanzee (e.g., UniProt entry A0A2R9ALZ1 for Pan troglodytes / Pygmy chimpanzee / Bonobo( Pan paniscus / Pygmy chimpanzee / Bonobo ) of the corresponding wild-type IDUA or fragment thereof, e.g., when assayed under the same conditions. A catalytically active IDUA fragment has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length IDUA or variant thereof, e.g., when assayed under the same conditions.

[0034] As used herein, “iduronate sulfatase,” “iduronate-2-sulfatase,” or “IDS” refers to iduronate-2-sulfatase (EC 3.1.6.13), an enzyme involved in the lysosomal degradation of the glycosaminoglycans heparan sulfate and dermatan sulfate. IDS deficiency is associated with mucopolysaccharidosis II (also known as Hunter syndrome). In some embodiments, IDS may be a component of a recombinant glycosylated protein that further comprises an Fc polypeptide (e.g., any exemplary Fc polypeptide or region described herein). As used herein, IDS encompasses the catalytically active fragment of IDS as well as functional variants of wild-type IDS or fragments thereof, including allelic variants and splice variants. The sequence of human IDS isotype I is a human sequence designated as a canonical sequence, which is available at UniProt entry P22304 and is encoded by the human IDS gene located at Xq28. The full-length sequence is provided as SEQ ID NO:5. As used herein, a “mature” IDS sequence refers to a polypeptide chain lacking the signal and propeptide sequences of the naturally occurring full-length polypeptide chain. The amino acid sequence of a mature human IDS polypeptide is provided as SEQ ID NO: 6, corresponding to amino acids 34-550 of the full-length human sequence. As used herein, a “truncated” IDS sequence refers to a catalytically active fragment of the naturally occurring full-length polypeptide chain. An exemplary truncated human IDS polypeptide amino acid sequence is provided as SEQ ID NO: 7, corresponding to amino acids 26-550 of the full-length human sequence. The structure of human IDS has been well characterized. Exemplary structures are available by PDB accession code 5FQL. Nat. Comm. The structure is also described in 8:15786 doi: 10.1038 / ncomms15786, 2017. Non-human primate IDS sequences have also been described, including those for chimpanzees (UniProt entry K7BKV4) and rhesus monkeys (UniProt entry H9FTX2). Mouse IDS sequences are available according to UniProt entry Q08890. IDS variants possess activity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding wild-type IDS or its fragments, for example, when measured under the same conditions. Catalytically active IDS fragments possess activity of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the activity of the corresponding full-length IDS or its variants, for example, when measured under the same conditions.

[0035] “Transferrin receptor” or “TfR” as used herein refers to transferrin receptor protein 1. The human transferrin receptor 1 polypeptide sequence is set forth in SEQ ID NO: 10. Transferrin receptor protein 1 sequences from other species are also known (e.g., chimpanzee, Accession No. XP_003310238.1; rhesus, NP_001244232.1; dog, NP_001003111.1; bovine, NP_001193506.1; mouse, NP_035768.1; rat, NP_073203.1; and chicken, NP_990587.1). The term “transferrin receptor” also encompasses allelic variants of exemplary reference sequences (e.g., the human sequence) encoded by genes located at the transferrin receptor protein 1 chromosomal locus. Full-length transferrin receptor proteins include a short N-terminal intracellular region, a transmembrane region, and a large extracellular domain. The extracellular domain is characterized by three domains: a protease-like domain, a helical domain, and an apical domain. The apical domain sequence of human transferrin receptor 1 is set forth in SEQ ID NO: 11.

[0036] “[ERT enzyme] fusion polypeptide” refers to a polypeptide linked (e.g., fused) to an ERT enzyme, catalytically active ERT enzyme variant, or catalytically active fragment thereof. The polypeptide can be linked to the ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment by a peptide bond or by a polypeptide linker. The fusion polypeptide can be a [ERT enzyme]-Fc fusion polypeptide, a [ERT enzyme]-antigen binding domain fusion polypeptide, a [ERT enzyme]-antibody heavy chain fusion polypeptide, a [ERT enzyme]-antibody heavy chain variable region fusion polypeptide, a [ERT enzyme]-antibody light chain fusion polypeptide, a [ERT enzyme]-antibody light chain variable region fusion polypeptide, a [ERT enzyme]-antibody fragment fusion polypeptide, and the like.

[0037] “[ERT enzyme]-Fc fusion polypeptide” as used herein refers to an Fc polypeptide linked (e.g., fused) to an ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment. The Fc polypeptide can be linked to the ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment by a peptide bond or by a polypeptide linker. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with another Fc polypeptide. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that confer the ability to bind to a transferrin receptor. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that reduce effector function. The Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that extend serum half-life. Exemplary modified Fc polypeptides are described, e.g., in WO 2019 / 070577.

[0038] In some embodiments, an [ERT enzyme]-Fc fusion protein can be a dimeric protein comprising a first Fc polypeptide linked (e.g., fused) to an ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment (i.e., a “[ERT]-Fc fusion polypeptide”); and a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide. The second Fc polypeptide can also be linked (e.g., fused) to an ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment. The first Fc polypeptide and / or the second Fc polypeptide can be linked to an ERT enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment by a peptide bond or by a polypeptide linker. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that promote its heterodimerization with another Fc polypeptide. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that confer the ability to bind to a transferrin receptor. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that reduce effector function. The first Fc polypeptide and / or the second Fc polypeptide can be a modified Fc polypeptide containing one or more modifications that extend serum half-life. Exemplary modified Fc polypeptides are described, e.g., in WO 2019 / 070577.

[0039] As used herein, the term “Fc polypeptide” refers to the C-terminal region of a naturally occurring immunoglobulin heavy chain polypeptide, characterized by an Ig fold into domains. An Fc polypeptide contains constant region sequences including at least a CH2 domain and / or a CH3 domain, and can contain at least part of a hinge region. In general, an Fc polypeptide does not contain a variable region.

[0040] A “modified Fc polypeptide” refers to an Fc polypeptide that has at least one mutation (e.g., substitution, deletion, or insertion) compared to a wild-type immunoglobulin heavy chain Fc polypeptide sequence, but retains the overall Ig fold or structure of the native Fc polypeptide.

[0041] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers. An amino acid polymer can comprise all L-amino acids, all D-amino acids, or a mixture of L and D amino acids.

[0042] The term “protein” as used herein refers to a dimer (i.e., two) or multimer (i.e., three or more) of a polypeptide or single chain polypeptide. The single chain polypeptides of a protein can be linked by covalent bonds (e.g., disulfide bonds) or non-covalent interactions.

[0043] The terms "conservative substitution," "conservative mutation," or "conservatively modified variants" refer to alterations that result in the substitution of an amino acid with another amino acid that is classified as having similar properties. Examples of categories of conservative amino acid substitutions that can be defined in this manner include: "charged / polar groups," including Glu (glutamic acid or E), Asp (aspartic acid or D), Asn (asparagine or N), Gin (glutamine or Q), Lys (lysine or K), Arg (arginine or R), and His (histidine or H); "aromatic groups," including Phe (phenylalanine or F), Tyr (tyrosine or Y), Trp (tryptophan or W), and (histidine or H); and "aliphatic groups," including Gly (glycine or G), Ala (alanine or A), Val (valine or V), Leu (leucine or L), He (isoleucine or I), Met (methionine or M), Ser (serine or S), Thr (threonine or T), and Cys (cysteine or C). Subgroups can also be identified within each group. For example, the group of charged or polar amino acids can be subdivided into subgroups including: a "positively charged subgroup" comprising Lys, Arg, and His; a "negatively charged subgroup" comprising Glu and Asp; and a "polar subgroup" comprising Asn and Gin. In another example, aromatic or cyclic groups can be subdivided into subgroups including: a "nitrogen ring subgroup" comprising Pro, His, and Trp; and a "phenyl subgroup" comprising Phe and Tyr. In a further example, aliphatic groups can be subdivided into subgroups, such as a "aliphatic nonpolar subgroup" comprising Val, Leu, Gly, and Ala; and a "aliphatic weakly polar subgroup" comprising Met, Ser, Thr, and Cys. Examples of categories of conservative mutations include amino acid substitutions of amino acids within the above-described subgroups, such as, but not limited to: Lys for Arg or vice versa, such that a positive charge can be maintained; Glu for Asp or vice versa, such that a negative charge can be maintained; Ser for Thr or vice versa, such that a free -OH can be maintained; and Gin for Asn or vice versa, such that a free -NH2 can be maintained. In some embodiments, a naturally occurring hydrophobic amino acid is substituted with a hydrophobic amino acid (e.g., at an active site), to maintain hydrophobicity.

[0044] The terms "identical" or percent "identity," in the context of two or more polypeptide sequences, refer to residues that are the same when aligned for maximum correspondence over a comparison window or designated comparison region, as measured using a sequence comparison algorithm or by manual alignment and visual inspection, for example at least 60% identity, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more.

[0045] For peptide sequence alignment, an amino acid sequence is typically used as a reference sequence against which candidate sequences are compared. Various methods available to those skilled in the art can be employed for alignment, such as visual alignment or using publicly available software employing known algorithms to achieve maximum alignment. Such programs include BLAST, ALIGN, ALIGN-2 (Genentech, South San Francisco, Calif.), or Megalign (DNASTAR). The parameters used for alignment to achieve maximum alignment can be determined by those skilled in the art. For the purposes of this application, for peptide sequence alignment, the BLASTP algorithm is used to align two protein sequences using the standard protein BLAST algorithm with default parameters.

[0046] When used to confirm a given amino acid residue in a polypeptide sequence, the terms "corresponds to," "reference...determined," or "reference...number" refer to the position of a residue in a specified reference sequence when the given amino acid sequence is best aligned with a reference sequence. Thus, for example, when best aligned with SEQ ID NO:1, an amino acid residue in the modified Fc polypeptide "corresponds to" an amino acid in SEQ ID NO:1, where that residue is aligned with that amino acid in SEQ ID NO:1. The polypeptide aligned with the reference sequence does not need to be the same length as the reference sequence.

[0047] As used herein, “binding affinity” refers to the strength of a non-covalent interaction between two molecules, such as the interaction between a single binding site on a polypeptide and its bound target (e.g., transferrin receptor). Therefore, for example, unless otherwise stated or obvious from the context, the term can refer to a 1:1 interaction between a polypeptide and its target. This can be measured by measuring the equilibrium dissociation constant (K0). D To quantify binding affinity, the equilibrium dissociation constant refers to the dissociation rate constant (k). d ,time -1 Divide by the binding rate constant (k) a ,time -1 M -1 K can be determined by measuring the kinetics of complex formation and dissociation. D For example, surface plasmon resonance (SPR) methods, such as the Biacore™ system; and kinetic exclusion methods, such as KinExA. ® ; and biological layer interferometry (e.g., using ForteBio) ® Octet ®platform). As used herein, "binding affinity" includes not only formal binding affinity, such as that reflecting a 1 : 1 interaction between a polypeptide and its target, but also apparent affinity, K D Can reflect avidity binding.

[0048] As used herein, when referring to an engineered TfR binding polypeptide, TfR binding peptide, or TfR binding antibody as described herein, the term "specifically binds" or "selectively binds" to a target (e.g., TfR) refers to a binding reaction of the engineered TfR binding polypeptide, TfR binding peptide, or TfR binding antibody to the target with greater affinity, avidity, and / or longer duration than to a structurally distinct target. In typical embodiments, the engineered TfR binding polypeptide, TfR binding peptide, or TfR binding antibody has at least 5-fold, 10-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold or greater affinity for a particular target (e.g., TfR) than for an unrelated target when assayed under identical affinity assay conditions. The term "specifically binds" a particular target (e.g., TfR), "binds specifically" to a particular target (e.g., TfR), or "has specificity for" a particular target (e.g., TfR) as used herein can manifest as, for example, an equilibrium dissociation constant K D is, for example, 10 -4 M or less, for example 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or 10 - 12 M. In some embodiments, the engineered TfR binding polypeptide, TfR binding peptide, or TfR binding antibody specifically binds to an epitope on TfR that is conserved across species (e.g., is structurally conserved across species, for example, is conserved across non-human primate and human species, for example, is structurally conserved across non-human primate and human species). In some embodiments, the engineered TfR binding polypeptide, TfR binding peptide, or TfR binding antibody can bind only to human TfR.

[0049] The terms "treatment" and "treating," as used herein, generally mean obtaining a desired pharmacologic and / or physiologic effect. "Treatment" can refer to successful treatment or amelioration of any indicia of a lysosomal storage disorder (e.g., mucopolysaccharidosis type I), Hunter syndrome (also known as "mucopolysaccharidosis type II" or "MPS II"), Sanfilippo syndrome A (also known as "mucopolysaccharidosis type IIIA," "MPS IIIA," or "Sanfilippo syndrome type A"), Niemann-Pick disease, Pompe disease, Gaucher disease, or Parkinson's disease, including any objective or subjective parameters, such as alleviation, remission, improvement in patient survival, increased survival time or survival rate, reduction in symptoms or making the disorder more tolerable to the patient, slowing in the rate of worsening, or improvement in the patient's physical or mental well-being. Treatment or amelioration can be based on objective or subjective parameters. The effects of the treatment can be compared to that of an individual or group not receiving the treatment, or to the same patient at a different time, such as prior to or during treatment.

[0050] The terms "subject," "individual," and "patient," as used interchangeably herein, refer to a mammal, including, but not limited to, a human, a non-human primate, a rodent (e.g., a rat, a mouse, and a guinea pig), a rabbit, a cow, a pig, a horse, and other mammalian species. In one embodiment, the patient is a human.

[0051] The term "pharmaceutically acceptable excipient" refers to a non-active pharmaceutical ingredient that is biologically or pharmacologically suitable for use in humans or animals, such as, but not limited to, a buffer, a carrier, or a preservative.

[0052] As used herein, a "therapeutic amount," "therapeutically effective amount," or "therapeutically effective concentration" of an agent is the amount or concentration of the agent that treats a sign or symptom of a disease (e.g., a LSD) in a subject (e.g., a mammal).

[0053] The term "administering" refers to methods of delivering an agent, compound, or composition to a desired site of biological action. These methods include, but are not limited to, topical delivery, parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, intrathecal delivery, colonic delivery, rectal delivery, or intraperitoneal delivery. In one embodiment, the polypeptides described herein are administered intravenously.

[0054] The term "expression" is used herein to refer to transcription and translation that occurs within a host cell. Protein encoded by a product gene can be quantified by assaying for the biological activity of the protein or by using assays that do not depend on such activity, such as Western blots or radioimmunoassays using antibodies that react with the protein. Sambrook et al., Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0055] The term "cell culture medium" as used herein refers to any cell culture medium used to culture cells without alteration by supplementation or by selective removal of certain components.

[0056] Nucleic acids, vectors, and host cells The methods described herein use mammalian host cells to produce recombinant glycosylated proteins. Accordingly, the present disclosure provides isolated nucleic acids comprising nucleic acid sequences encoding any of the recombinant glycosylated proteins described herein, and vectors comprising such nucleic acids, and mammalian host cells into which the nucleic acid(s) / vector(s) have been introduced, which host cells are used to replicate the nucleic acids encoding the recombinant glycosylated proteins and / or to express the recombinant glycosylated proteins.

[0057] Also provided herein are isolated nucleic acids comprising nucleic acid sequences encoding one or more sialyltransferases (e.g., any of the sialyltransferases described herein), and vectors comprising such nucleic acids, and mammalian host cells into which the nucleic acid(s) / vector(s) have been introduced, which host cells are used to replicate the nucleic acids encoding the sialyltransferases and / or to express the one or more sialyltransferases.

[0058] Also provided herein are mammalian host cells comprising (1) a nucleic acid encoding the recombinant glycosylated protein (e.g., any of the recombinant glycosylated proteins described herein) or a vector comprising such nucleic acid, and (2) one or more nucleic acids encoding one or more sialyltransferases (e.g., any of the exemplary sialyltransferases described herein or known in the art) or one or more vectors comprising such nucleic acid(s). In some embodiments, the mammalian host cell further comprises (3) a nucleic acid encoding a galactosyltransferase (e.g., a galactosyltransferase as described herein or known in the art).

[0059] In some embodiments, the one or more nucleic acids encoding the one or more sialyltransferases are selected from: ST6GAL1 and ST3GAL4 In some embodiments, the ST6GAL1 is humanST6GAL1 (e.g., NCBI Accession No. NP_001340845.1). In some embodiments, the ST3GAL4 is a human ST3GAL4 (e.g., NCBI Accession No. XP_047283377.1) or CHO ST3GAL4 (e.g., NCBI Accession No. NP_001233628.1).

[0060] In some embodiments, the galactosyltransferase is B4GALT1 In some embodiments, the B4GALT1 is a human B4GALT1 (e.g., NCBI Accession No. NP_001488.2) or CHO B4GALT1 (e.g., NCBI Accession No. NP_001233620.1).

[0061] In some embodiments, the mammalian host cell is a rodent cell (e.g., Chinese hamster ovary cell) or a human cell.

[0062] In some embodiments, the nucleic acid provided herein can be single-stranded or double-stranded. In some embodiments, the nucleic acid is DNA. In particular embodiments, the nucleic acid is cDNA. In some embodiments, the nucleic acid is RNA.

[0063] In some embodiments, the vector or vectors can be selected from the group consisting of a plasmid, a viral vector, a phagemid, a yeast chromosomal vector, and a non- episomal mammalian vector. In some embodiments, the vector or vectors can be a transposon.

[0064] In some embodiments, the nucleic acid is operably linked to one or more regulatory nucleotide sequences in an expression construct.

[0065] Exemplary vectors include plasmids of the following types: pBR322-derived plasmids for expression in prokaryotic cells (e.g., E. coli), pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids. pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg derived vectors. Alternatively, derivatives of viruses such as bovine papilloma virus (BPV-1) or Epstein-Barr virus (pHEBo, pREP derived and p205) can be used to transiently express proteins in eukaryotic cells. In some embodiments, it can be desirable to express a recombinant protein (e.g., a recombinant glycosylated protein) by using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (e.g., pVL 1392, pVL 1393, and pVL 941), pAcUW-derived vectors (e.g., pAcUWl), and pBlueBac-derived vectors. Additional expression systems include adenovirus, adeno-associated virus, and other viral vectors.

[0066] The vectors can be transformed into any suitable mammalian host cell. In some embodiments, the step of transforming the mammalian cell with the one or more vectors encoding the one or more sialyltransferases to produce the mammalian host cell comprises performing hypertransfection. Hypertransfection involves performing a first transfection of a mammalian cell with a first means for integrating a first gene into the genome of the mammalian cell (e.g., a first integrase, such as a first integrase, transposon, recombinase, etc.), and then performing a second transfection of the mammalian cell with a second means for integrating (e.g., the same as or different from the first means for integrating) a further copy of the first gene or a different gene without disrupting the DNA integrated in the first transfection.

[0067] In some cells, the vectors are expressed in mammalian host cells to express relatively large amounts of the recombinant glycosylated protein. Such host cells include mammalian cells, yeast cells, insect cells, and prokaryotic cells. In some embodiments, the mammalian host cell can be a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, an NSO cell, a YO cell, a HEK 293 cell, a COS cell, a Vero cell, or a HeLa cell. In some embodiments, the mammalian host cell is a rodent cell.

[0068] The recombinant glycosylated protein can be secreted and isolated from a mixture of mammalian host cells and liquid media containing the recombinant glycosylated protein. Alternatively, the recombinant glycosylated protein can remain in the cytoplasm or in a membrane fraction, which is then harvested, lysed, and the recombinant glycosylated protein isolated using a desired method. Exemplary embodiments of recombinant glycosylated proteins further comprising an Fc polypeptide are described in WO 2019 / 070577, incorporated by reference in its entirety.

[0069] Enzyme replacement therapy (ERT) enzymes Enzyme replacement therapy (ERT) enzymes are a class of enzymes that are deficient in some subjects. Lysosomal storage disorder (LSD) enzymes are a subclass of ERT enzymes that are deficient in subjects with LSDs. LSDs are inherited metabolic diseases characterized by the accumulation of undigested or partially digested macromolecules that ultimately lead to cellular dysfunction and clinical abnormalities. Traditionally, LSDs have been defined as lysosomal function defects, typically categorized according to the accumulated substrate, and include sphingolipidoses, oligosaccharidoses, mucolipidoses, mucopolysaccharidoses, lipoprotein storage disorders, neuronal ceroid lipofuscinoses, and others. The classification of these disorders has recently been expanded to include other protein deficiencies or defects that lead to the accumulation of macromolecules such as proteins necessary for normal post-translational modification of lysosomal enzymes or proteins important for correct lysosomal trafficking.

[0070] In some embodiments, the recombinant glycosylated protein can be an LSD enzyme or a catalytically active variant or catalytically active fragment thereof.

[0071] In some embodiments, the recombinant glycosylated protein can be an IDUA protein comprising the amino acid sequence of SEQ ID NO: 3, 4, 8, or 9. In some embodiments, the recombinant glycosylated protein can comprise a catalytically active variant or fragment of an IDUA enzyme having an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type SGSH enzyme.

[0072] In some embodiments, the recombinant glycosylated protein can be an SGSH protein comprising the amino acid sequence of SEQ ID NO: 1 or 2. In some embodiments, the recombinant glycosylated protein can comprise a catalytically active variant or fragment of an SGSH enzyme having an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type SGSH enzyme.

[0073] In some embodiments, the recombinant glycosylated protein can be an IDS protein comprising the amino acid sequence of any one of SEQ ID NOs: 5, 6, 7, 12, and 13. In some embodiments, the recombinant glycosylated protein can comprise a catalytically active variant or fragment of an IDS enzyme having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type IDS enzyme.

[0074] In some embodiments, the recombinant glycosylated protein is a fusion protein (e.g., any of the exemplary fusion proteins described herein or known in the art). In some embodiments, the fusion protein is an antigen-binding fusion protein comprising an antigen-binding protein and an ERT enzyme (e.g., LSD enzyme) linked thereto. The antigen-binding protein can be, for example, an antibody or antigen-binding fragment thereof, and encompasses Fab fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, dAb fragments, scFv fragments, chimeric antibodies, monoclonal antibodies, VNAR domains, and VHH domains.

[0075] In some embodiments, the fusion protein comprises: (i) an antibody heavy chain or fragment thereof, and (ii) an antibody light chain or fragment thereof, and an ERT enzyme (e.g., LSD enzyme) linked to (i) and / or (ii). In some embodiments, the ERT enzyme is linked to (i). In some embodiments, the ERT is linked to (ii). In some embodiments, the fusion protein contains at least two ERT enzyme units, wherein a first ERT enzyme unit is linked to (i) and a second ERT enzyme unit is linked to (ii). In some embodiments, the antigen-binding fusion protein is capable of specific binding to a blood-brain barrier (BBB) receptor (e.g., transferrin receptor (TfR)).

[0076] In some embodiments, the fusion protein is an ERT enzyme-Fc fusion protein comprising: (i) an Fc polypeptide, which can contain modifications (e.g., one or more modifications that promote heterodimerization) or can be a wild-type Fc polypeptide; and an ERT enzyme (e.g., a LSD enzyme); and (ii) an Fc polypeptide, which can contain modifications (e.g., one or more modifications that promote heterodimerization) or can be a wild-type Fc polypeptide; and an ERT enzyme (e.g., a LSD enzyme). In some embodiments, the fusion protein comprises (i) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment; and (ii) a second Fc polypeptide that forms an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide is a modified Fc polypeptide that is capable of specific binding to a blood-brain barrier (BBB) receptor (e.g., transferrin receptor (TfR)). In other embodiments, the fusion protein comprises (i) an enzyme replacement therapy (ERT) enzyme, catalytically active ERT enzyme variant, or catalytically active ERT enzyme fragment thereof; and (ii) a modified Fc dimer that is capable of specific binding to a transferrin receptor (TfR). In some embodiments, one or both Fc polypeptides can contain modifications that result in binding to a blood-brain barrier (BBB) receptor (e.g., transferrin receptor (TfR)).

[0077] In some embodiments, the ERT enzyme can be a LSD enzyme. The LSD enzyme incorporated into the fusion protein is catalytically active, i.e., it retains the enzyme activity that is deficient in the LSD. In some embodiments, the LSD enzyme is alpha-L-iduronidase (IDUA) that is deficient in Sanfilippo syndrome. In some embodiments, the LSD enzyme is alpha-L-iduronidase (IDUA) that is deficient in mucopolysaccharidosis type I. In some embodiments, the LSD enzyme is iduronate-2-sulfatase (IDS) that is deficient in Hunter syndrome. In some embodiments, the LSD enzyme is acid sphingomyelinase (ASM) that is deficient in Niemann-Pick disease. In some embodiments, the LSD enzyme is beta-glucocerebrosidase (GBA) that is deficient in Gaucher disease and Parkinson disease.

[0078] In some embodiments, the fusion protein comprising a LSD enzyme and a modified Fc polypeptide that optionally binds to a BBB receptor (e.g., a TfR-binding Fc polypeptide) comprises a catalytically active fragment or variant of SGSH or wild-type SGSH. In some embodiments, the SGSH is a catalytically active variant or catalytically active fragment of a SGSH protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 and 2. In some embodiments, the catalytically active variant or fragment of a SGSH protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type SGSH protein. In some embodiments, the fusion protein comprising a SGSH and a modified Fc polypeptide can comprise a first polypeptide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 14, 15, 16, 17, or 18 and a second polypeptide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 18, 19, 20, or 21.

[0079] In some embodiments, the fusion protein comprising a LSD enzyme and a modified Fc polypeptide that optionally binds to a BBB receptor (e.g., a TfR-binding Fc polypeptide) comprises a catalytically active fragment or variant of IDUA or wild-type IDUA. In some embodiments, the IDUA is a catalytically active variant or catalytically active fragment of an IDUA protein comprising the amino acid sequence of any one of SEQ ID NOs: 3, 4, 8, and 9. In some embodiments, the catalytically active variant or fragment of an IDUA protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type IDUA protein.

[0080] In some embodiments, the fusion protein comprising a IDUA and a modified Fc polypeptide can comprise a first polypeptide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 22 or 23 and a second polypeptide comprising a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to SEQ ID NO: 24 or 25.

[0081] In some embodiments, the fusion protein comprising an ERT enzyme and a modified Fc polypeptide that optionally binds to a BBB receptor (e.g., a TfR-binding Fc polypeptide) comprises a catalytically active fragment or variant of IDS or wild-type IDS. In some embodiments, the IDS is a catalytically active variant or catalytically active fragment of an IDS protein comprising the amino acid sequence of any one of SEQ ID NOs: 5, 6, 7, 12, and 13. In some embodiments, the catalytically active variant or fragment of an IDS protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type IDS protein.

[0082] In some embodiments, an ERT enzyme (e.g., IDUA, SGSH, or IDS) or catalytically active variant or fragment thereof present in a fusion protein described herein retains at least 25% of its activity as compared to its activity when it is not linked to an Fc polypeptide or TfR-binding Fc polypeptide. In some embodiments, an ERT enzyme or catalytically active variant or catalytically active fragment thereof retains at least 10% or at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of its activity as compared to its activity when it is not linked to an Fc polypeptide or TfR-binding Fc polypeptide. In some embodiments, an ERT enzyme or catalytically active ERT enzyme variant or catalytically active ERT enzyme fragment retains at least 80%, 85%, 90%, or 95% of its activity as compared to its activity when it is not linked to an Fc polypeptide or TfR-binding Fc polypeptide. In some embodiments, fusion to an Fc polypeptide does not decrease the activity of the ERT enzyme (e.g., IDUA, SGSH, or IDS) or catalytically active variant or fragment thereof. In some embodiments, fusion to a TfR-binding Fc polypeptide does not decrease the activity of the ERT enzyme.

[0083] Transferrin receptor-binding Fc polypeptides This section describes the generation of modified Fc polypeptides described herein that bind to the transferrin receptor (TfR) and are capable of being transported across the blood-brain barrier (BBB).

[0084] TfR binding Fc polypeptides comprising mutations in the CH3 domain In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises substitutions in the CH3 domain. In some embodiments, the modified Fc polypeptide comprises a human Ig CH3 domain, such as an IgG CH3 domain, modified to have TfR binding activity. The CH3 domain can be of any IgG subtype, i.e., from IgG1, IgG2, IgG3, or IgG4. Reference to a CH3 domain of an IgG antibody refers to the segment of amino acids from about position 341 to about position 447 according to the EU numbering scheme.

[0085] In some embodiments, the modified Fc polypeptide that specifically binds to TfR binds to the apical domain of TfR and can bind to TfR without blocking or otherwise inhibiting the binding of transferrin to TfR. In some embodiments, the binding of transferrin to TfR is not substantially inhibited. In some embodiments, the binding of transferrin to TfR is inhibited by less than about 50% (e.g., less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%). In some embodiments, the binding of transferrin to TfR is inhibited by less than about 20% (e.g., less than about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%).

[0086] In some embodiments, the modified Fc polypeptide that specifically binds to TfR comprises at least two, three, four, five, six, seven, eight, or nine substitutions at positions 384, 386, 387, 388, 389, 390, 413, 416, and 421 according to the EU numbering scheme. Exemplary substitutions that can be introduced at these positions are shown in Tables 4 and 5. In some embodiments, the amino acid at position 388 and / or 421 is an aromatic amino acid, e.g., Trp, Phe, or Tyr. In some embodiments, the amino acid at position 388 is Trp. In some embodiments, the aromatic amino acid at position 421 is Trp or Phe.

[0087] In some embodiments, at least one of the following positions is substituted: Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an acidic amino acid at position 387; an aromatic amino acid, such as Trp, at position 388; Val, Ser, or Ala at position 389; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Thr or an acidic amino acid at position 416; or Trp, Tyr, His, or Phe at position 421. In some embodiments, the modified Fc polypeptide can comprise conservative substitutions, such as at one or more positions in the set, the specified amino acid is substituted with an amino acid in the same charge grouping, hydrophobicity grouping, side chain ring structure grouping (e.g., aromatic amino acid), or size and / or polarity or nonpolarity grouping. Thus, for example, Ile can be present at position 384, 386, and / or position 413. In some embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Glu. In other embodiments, the acidic amino acid at one, two, or each of positions 387, 413, and 416 is Asp. In some embodiments, two, three, four, five, six, seven, or all eight of positions 384, 386, 387, 388, 389, 413, 416, and 421 have the amino acid substitutions specified in this paragraph.

[0088] In some embodiments, the Fc polypeptide modified as described in the previous two paragraphs comprises a native Asn at position 390. In some embodiments, the modified Fc polypeptide comprises Gly, His, Gin, Leu, Lys, Val, Phe, Ser, Ala, or Asp at position 390. In some embodiments, the modified Fc polypeptide further comprises one, two, three, or four substitutions at positions comprising 380, 391, 392, and 415 according to the EU numbering scheme. In some embodiments, Trp, Tyr, Leu, or Gin can be present at position 380. In some embodiments, Ser, Thr, Gin, or Phe can be present at position 391. In some embodiments, Gin, Phe, or His can be present at position 392. In some embodiments, Glu can be present at position 415.

[0089] In certain embodiments, the modified Fc polypeptide comprises two, three, four, five, six, seven, eight, nine, ten, or eleven positions selected from: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421. In some embodiments, the modified Fc polypeptide comprises all eleven of: Trp, Leu, or Glu at position 380; Tyr or Phe at position 384; Thr at position 386; Glu at position 387; Trp at position 388; Ser, Ala, Val, or Asn at position 389; Ser or Asn at position 390; Thr or Ser at position 413; Glu or Ser at position 415; Glu at position 416; and / or Phe at position 421.

[0090] In certain embodiments, the modified Fc polypeptide comprises Leu or Met at position 384; Leu, His, or Pro at position 386; Val at position 387; Trp at position 388; Val or Ala at position 389; Pro at position 413; Thr at position 416; and / or Trp at position 421. In some embodiments, the modified Fc polypeptide further comprises Ser, Thr, Gin, or Phe at position 391. In some embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu, or Gin at position 380 and / or Gin, Phe, or His at position 392. In some embodiments, Trp is present at position 380 and / or Gin is present at position 392. In some embodiments, the modified Fc polypeptide lacks Trp at position 380.

[0091] In other embodiments, the modified Fc polypeptide comprises Tyr at position 384; Thr at position 386; Glu or Val at position 387; Trp at position 388; Ser at position 389; Ser or Thr at position 413; Glu at position 416; and / or Phe at position 421. In some embodiments, the modified Fc polypeptide comprises the native Asn at position 390. In certain embodiments, the modified Fc polypeptide further comprises Trp, Tyr, Leu, or Gin at position 380; and / or Glu at position 415. In some embodiments, the modified Fc polypeptide further comprises Trp at position 380 and / or Glu at position 415.

[0092] In further embodiments, the modified Fc polypeptide further comprises one, two, or three substitutions at positions 414, 424, and 426, according to the EU numbering scheme. In some embodiments, position 414 is Lys, Arg, Gly, or Pro; position 424 is Ser, Thr, Glu, or Lys; and / or position 426 is Ser, Trp, or Gly.

[0093] In some embodiments, the modified Fc polypeptide comprises one or more of the following substitutions according to the EU numbering scheme: Trp at position 380; Thr at position 386; Trp at position 388; Val at position 389; Thr or Ser at position 413; Glu at position 415; and / or Phe at position 421.

[0094] In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to amino acids 111-217 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide has at least 70% identity, at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 384-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-390 and / or 413-421 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises amino acids at EU index positions 380-392 and / or 413-426 of any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) as described in WO 2019 / 070577, which is incorporated by reference in its entirety.

[0095] In some embodiments, the modified Fc polypeptide has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, or at least 95% identity to any one of SEQ ID NOs: 4-90, 97-100, and 105-108 (e.g., SEQ ID NOs: 34-38, 58, and 60-90) described in WO 2019 / 070577, which is incorporated by reference in its entirety, and further comprises at least five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or sixteen of the following positions numbered according to the EU Index: Trp, Tyr, Leu, Gin, or Glu at position 380; Leu, Tyr, Met, or Val at position 384; Leu, Thr, His, or Pro at position 386; Val, Pro, or an acidic amino acid at position 387; an aromatic amino acid, e.g., Trp, at position 388; Val, Ser, or Ala at position 389; Ser or Asn at position 390; Ser, Thr, Gin, or Phe at position 391; Gin, Phe, or His at position 392; an acidic amino acid, Ala, Ser, Leu, Thr, or Pro at position 413; Lys, Arg, Gly, or Pro at position 414; Glu or Ser at position 415; Thr or an acidic amino acid at position 416; Trp, Tyr, His, or Phe at position 421; Ser, Thr, Glu, or Lys at position 424; and Ser, Trp, or Gly at position 426.

[0096] In some embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 34-38, 58, and 60-90 as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In other embodiments, the modified Fc polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 34-38, 58, and 60-90 as described in WO 2019 / 070577, which is incorporated by reference in its entirety, but wherein one, two, or three amino acids are substituted.

[0097] In some embodiments, the modified Fc polypeptide comprises additional mutations, such as those described in Section VI below, including but not limited to a knob mutation (e.g., T366W, numbered with reference to the EU numbering scheme), a cavity mutation (e.g., T366S, L368A, and Y407V, numbered with reference to the EU numbering scheme), a mutation that modulates effector function (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S, numbered with reference to the EU numbering scheme), and / or a mutation that increases serum stability or serum half-life (e.g., (i) M252Y, S254T, and T256E, numbered with reference to the EU numbering scheme, or (ii) N434S, with or without M428L, numbered according to the EU numbering scheme). By way of illustration, SEQ ID NOs: 156-229 provide non-limiting examples of modified Fc polypeptides having mutations in the CH3 domain (e.g., clones CH3C.35.20.1, CH3C.35.23.2, CH3C.35.23.3, CH3C.35.23.4, CH3C.35.21.17.2, and CH3C.35.23), which comprise one or more of these additional mutations.

[0098] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, numbered with reference to the EU numbering scheme) and is at least 85% identical, at least 90% identical, or at least 95% identical to the sequence of any of SEQ ID NOs: 156, 168, 180, 192, 204, and 216, as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises the sequence of any of SEQ ID NOs: 156, 168, 180, 192, 204, and 216, as described in WO 2019 / 070577.

[0099] In some embodiments, the modified Fc polypeptide comprises a knob mutation (e.g., T366W, numbered with reference to the EU numbering scheme) and a mutation that modulates effector function (e.g., L234A and L235A; L234A, L235A, and P329G; or L234A, L235A, and P329S, numbered with reference to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, 218, 228, and 229 as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 157, 158, 169, 170, 181, 182, 193, 194, 205, 206, 217, and 218 as described in WO 2019 / 070577, which is incorporated by reference in its entirety.

[0100] In some embodiments, the modified Fc polypeptide comprises a cavity mutation (e.g., T366S, L368A, and Y407V, numbered with reference to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 162, 174, 186, 198, 210, and 222 as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 162, 174, 186, 198, 210, and 222 as described in WO 2019 / 070577, which is incorporated by reference in its entirety.

[0101] In some embodiments, the modified Fc polypeptide comprises cavity mutations (e.g., T366S, L368A, and Y407V, numbered with reference to the EU numbering scheme) and mutations that modulate effector function (e.g., L234A and L235A; L234A, L235A, and P329G; or L234S, L235A, and P329S, numbered with reference to the EU numbering scheme), and has at least 85% identity, at least 90% identity, or at least 95% identity to the sequence of any one of SEQ ID NOs: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224 as described in WO 2019 / 070577, which is incorporated by reference in its entirety. In some embodiments, the modified Fc polypeptide comprises the sequence of any one of SEQ ID NOs: 163, 164, 175, 176, 187, 188, 199, 200, 211, 212, 223, and 224 as described in WO 2019 / 070577, which is incorporated by reference in its entirety.

[0102] Sialyltransferase Sialyltransferases are enzymes that transfer sialic acid to oligosaccharides. Each sialyltransferase has specificity for a particular substrate. Sialyltransferases add sialic acid to the terminal portion of sialoglycolipids or to the N- or O-linked sugar chains of glycoproteins. Sialyltransferases belong to the glycosyltransferase family 29. There are twenty different sialyltransferases.

[0103] In some embodiments, the mammalian host cell has been previously transformed with a vector encoding one or more sialyltransferases (e.g., ST6GAL1 and / or ST3GAL4 ).

[0104] “ST6GAL1” as used herein refers to a beta-galactoside alpha-2,6-sialyltransferase 1 that transfers sialic acid from CMP-sialic acid to a galactose-containing acceptor substrate. The ST6GAL1 protein is normally found within the Golgi apparatus, but can be proteolytically processed into a soluble form. The sequence of human ST6GAL11 is the human sequence designated as canonical sequence, which can be obtained at UniProt entry P15907, and is encoded by the human ST6GAL1 gene located at 3q27.3. The full-length sequence is provided at SEQ ID NO: 18. There are 13 potential isoforms of ST6GAL1. Exemplary isoforms of ST6GAL1 can be obtained at UniProt entries C9J6X5, C9K0R8, C9JH16, and H7C472. Exemplary isoforms of ST6GAL1 are provided at SEQ ID NOs: 19-23. ST6GAL1 The gene can be obtained at NBCI accession number NP_001340845.1.

[0105] In some embodiments, the mammalian host cell has been previously transformed with a vector encoding ST6GAL1. In some embodiments, ST6GAL1 may have the amino acid sequence of any one of SEQ ID NOs: 26, 27, 28, 29, and 30, or a catalytically active variant or fragment thereof. In some embodiments, a catalytically active variant or fragment of a ST6GAL1 protein has an activity that is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type ST6GAL1 protein. In some embodiments, ST6GAL1 may comprise a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 26, 27, 28, 29, and 30.

[0106] “ST3GAL4” as used herein refers to CMP-N-acetylneuraminic acid-beta-galactosamide-alpha-2,3-sialyltransferase 4, which transfers sialic acid from CMP-Neu5Ac to acceptor Galbeta-(1->3)-GalNAc- and Galbeta-(1->4)-GlcNAc-terminated glycoconjugates via an alpha 2-3 linkage. ST3GAL4 protein is normally found within the Golgi apparatus, but can be proteolytically processed and secreted. The sequence of human ST3GAL4 is the human sequence designated as canonical sequence, which is available as UniProt entry Q11206, and is encoded by the human ST3GAL4 gene located at 11q24.2. The full-length sequence is provided as SEQ ID NO: 23. Exemplary ST3GAL4 The gene can be obtained under NBCI accession number XP_047283377.1 or NCBI accession number NP_001233628.1.

[0107] In some embodiments, the mammalian host cell has been previously transformed with a vector encoding ST3GAL4. In some embodiments, ST3GAL4 can have the amino acid sequence of any one of SEQ ID NOs: 30, 31, 32, 33, and 34, or a catalytically active variant or fragment thereof. In some embodiments, a catalytically active variant or fragment of a ST3GAL4 protein has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more of the activity of a wild-type ST3GAL4 protein. In some embodiments, ST3GAL4 can comprise a sequence that is at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, at least 99% identical, or 100% identical to any one of SEQ ID NOs: 30, 31, 32, 33, and 34.

[0108] Culture medium additives Liquid culture media (media) are known in the art. A liquid culture medium can comprise one or more culture medium additives. In some embodiments, a liquid culture medium comprises one or more (e.g., 1, 2, 3, or 4) culture medium additives selected from hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine. In some embodiments, a liquid culture medium comprises at least one (e.g., at least two, at least three) culture medium additives selected from hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine. In some embodiments, a liquid culture medium comprises hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0109] In some embodiments of any of the methods described herein, ManNAc is present in the liquid culture medium at a concentration of about 10 mM to about 60 mM (e.g., about 10 mM to about 50 mM, about 10 mM to about 40 mM, or about 10 mM to about 30 mM).

[0110] In some embodiments of any of the methods described herein, uridine is present in the liquid culture medium at a concentration of about 2.5 mM to about 10 mM (e.g., about 2.5 mM to about 8 mM, about 2.5 mM to about 6 mM, or about 2.5 mM to about 5 mM).

[0111] In some embodiments of any of the methods described herein, hydrocortisone is present in the liquid culture medium at a concentration of about 5 mM to about 50 mM (e.g., about 5 mM to about 40 mM, 5 mM to about 30 mM, or 5 mM to about 20 mM).

[0112] In some embodiments of any of the methods described herein, manganese is present in the liquid medium at a concentration of about 2 mM to about 15 mM (e.g., about 2 mM to about 10 mM or about 2 mM to about 5 mM).

[0113] In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM to about 60 mM (or any of the exemplary subranges described herein) of ManNAc and about 2.5 mM to about 10 mM (or any of the exemplary subranges described herein) of uridine. In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM of ManNAc and about 2.5 mM of uridine. In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM of ManNAc and about 5 mM of uridine.

[0114] In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM to about 60 mM (or any of the exemplary subranges described herein) of ManNAc and about 2 mM to about 15 mM (or any of the exemplary subranges described herein) of manganese. In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM of ManNAc and about 10 mM of manganese.

[0115] In some embodiments of any of the methods described herein, the liquid medium comprises about 2.5 mM to about 10 mM (or any of the exemplary subranges described herein) of uridine and about 2 mM to about 15 mM (or any of the exemplary subranges described herein) of manganese. In some embodiments of any of the methods described herein, the liquid medium comprises about 2.5 mM of uridine and about 10 mM of manganese. In some embodiments of any of the methods described herein, the liquid medium comprises about 5 mM of uridine and about 10 mM of manganese.

[0116] In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM to about 60 mM (or any of the exemplary subranges described herein) of ManNAc, about 2.5 mM to about 10 mM (or any of the exemplary subranges described herein) of uridine, and about 2 mM to about 15 mM (or any of the exemplary subranges described herein) of manganese. In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM of ManNAc, about 2.5 mM of uridine, and about 10 mM of manganese. In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM of ManNAc, about 20 mM of hydrocortisone, about 2.5 mM of uridine, and about 10 mM of manganese.

[0117] In some embodiments of any of the methods described herein, the liquid medium comprises about 10 mM to about 60 mM (or any of the exemplary subranges described herein) of ManNAc, about 2.5 mM to about 10 mM (or any of the exemplary subranges described herein) of uridine, about 5 μΜ to about 50 μΜ (or any of the exemplary subranges described herein) of hydrocortisone, and about 2 μΜ to about 15 μΜ (or any of the exemplary subranges described herein) of manganese.

[0118] The liquid medium used in any of the steps of any of the methods described herein can be any type of liquid medium known in the art.

[0119] Type of cultivation The term "batch culture" is a term of art meaning cultivating a cell culture in a vessel (e.g., a bioreactor) wherein cultivating the cell culture in the vessel does not include adding and / or removing a substantial amount of the liquid medium present in the vessel during the cell culture period. Batch culturing can be performed using temperatures, C02gas exposure, and / or agitation rates known to those of skill in the art. Those of skill in the art will appreciate that the length of time for batch culturing a cell culture will depend on the growth rate of the host cell, the recombinant glycosylated protein, and the starting cell culture density.

[0120] The term "fed-batch culture" is a term of art meaning cultivating a cell culture in a vessel (e.g., a bioreactor) wherein cultivating the cell culture in the vessel includes periodically or continuously adding fresh liquid medium and / or growth feed (e.g., glucose) to the vessel without removing a substantial amount of the liquid medium present in the vessel. The fresh liquid medium can be the same liquid medium as the liquid medium present in the vessel at the beginning of the cultivation period. In some examples of fed-batch culture, the fresh liquid medium is a different liquid medium, and / or includes one or more different medium additives (e.g., any of the medium additives described herein). Fed-batch culturing can be performed using temperatures, C02gas exposure, and / or agitation rates known to those of skill in the art. Those of skill in the art will appreciate that the length of time for fed-batch culturing a cell culture will depend on the growth rate of the host cell, the recombinant glycosylated protein, and the starting cell culture density.

[0121] The term "perfusion culture" is a term of art meaning culturing a cell culture in a vessel (e.g., a bioreactor) wherein culturing the cell culture in the vessel includes periodically or continuously removing liquid culture medium present in the vessel (e.g., at least 90% of the liquid culture medium free of host cells) during a cell culture period and simultaneously or shortly thereafter adding to the vessel a substantially identical volume of replacement liquid culture medium or fresh liquid culture medium. The replacement liquid culture medium or fresh liquid culture medium can be the same liquid culture medium as the liquid culture medium present in the vessel at the beginning of the culture period. In some examples of perfusion culture, the replacement liquid culture medium or fresh liquid culture medium is a different liquid culture medium and / or includes one or more different culture medium additives (e.g., any of the culture medium additives described herein). Perfusion culture can be performed using temperatures, C02gas exposure, and / or agitation rates known to those of skill in the art. Those of skill in the art will appreciate that the length of time for which a cell culture is perfusion cultured will depend on the growth rate of the host cells, the recombinant glycosylated protein, and the starting cell culture density.

[0122] Methods of determining the level of N-acetylneuraminic acid (NeuAc or sialic acid) in a recombinant glycosylated protein Methods of determining the level of sialic acid in a recombinant glycosylated protein are known in the art. In some embodiments, the methods provided herein result in at least a 50% (e.g., at least 75%, at least 95%, at least 100%, at least 150%, at least 200, at least 250%, 50%, 75%, 95%, 100%, 150%, 200%, or 250%) increase in the level of sialic acid in a recombinant glycosylated protein compared to a recombinant glycosylated protein produced by a method that does not include one or both of: (i) use of a mammalian host cell (e.g., any of the mammalian cells described herein) capable of expressing the recombinant glycosylated protein that comprises one or more vectors encoding one or more (e.g., 1, 2, 3, or 4) sialyltransferases (e.g., any of the sialyltransferases described herein), and (ii) culturing the mammalian host cell (e.g., any of the mammalian cells described herein) capable of expressing the recombinant glycosylated protein in a liquid culture medium comprising one or more (e.g., 1, 2, 3, or 4) culture medium additives selected from the group consisting of: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

[0123] In exemplary embodiments, the NeuAc (sialic acid) content in a recombinant glycosylated protein (e.g., produced using any of the methods described herein) can be measured by liquid chromatography-mass spectrometry analysis or by reverse phase liquid chromatography analysis. Preparation embodiments for samples used to measure NeuAc content are described in the examples disclosed herein.

[0124] Methods of harvesting and isolating recombinant glycosylated proteins In some embodiments of any of the methods described herein, the method further comprises harvesting the recombinant glycosylated protein from the liquid culture medium and / or the mammalian host cell (e.g., any of the mammalian host cells described herein). In some embodiments of any of the methods described herein, the method further comprises isolating the harvested recombinant glycosylated protein.

[0125] Recombinant glycosylated proteins can be recovered from the liquid culture medium by removing or otherwise physically separating the liquid culture medium from the mammalian host cell (e.g., any of the mammalian host cells described herein). A variety of different methods of removing liquid culture medium from mammalian host cells are known in the art, including, for example, centrifugation, filtration, pipetting, and / or aspiration. A variety of biochemical techniques can then be employed to recover and isolate the recombinant glycosylated protein from the liquid culture medium, including affinity chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography.

[0126] Methods of formulating isolated recombinant glycosylated proteins In some embodiments of any of the methods described herein, the method further comprises formulating the isolated recombinant glycosylated protein. Any isolated recombinant glycosylated protein can be formulated for parenteral (e.g., intravenous, intramuscular, intradermal, subcutaneous, intra-arterial, or intraperitoneal) administration in dosage unit form, i.e., physically discrete units with a predetermined mass of active recombinant glycosylated protein, suitable for administration and uniformity of dosage. Data obtained from cell culture assays and animal studies can be used in formulating appropriate dosages of any given isolated recombinant glycosylated protein for subjects (e.g., humans).

[0127] Methods of treatment Provided herein are recombinant glycosylated proteins (e.g., isolated recombinant glycosylated proteins) produced by any of the methods described herein. Also provided herein are methods of treating a subject in need thereof comprising administering to the subject a therapeutically effective amount of any recombinant glycosylated protein produced using the methods described herein. For example, recombinant glycosylated proteins comprising LSDase or a variant or catalytically active fragment thereof can be used to treat LSD. In some embodiments, patients having Hunter Syndrome are treated with recombinant glycosylated proteins comprising IDS or a variant or catalytically active fragment thereof. In some embodiments, patients having Mucopolysaccharidosis Type I are treated with recombinant glycosylated proteins comprising IDUA or a variant or catalytically active fragment thereof. In some embodiments, patients having Sanfilippo Syndrome A are treated with recombinant glycosylated proteins comprising SGSH or a variant or catalytically active fragment thereof.

[0128] Any recombinant glycosylated protein produced using any of the methods described herein can be administered to a subject in need thereof in a therapeutically effective amount or dose. The dose, however, can be varied depending upon several factors such as the route of administration selected, the formulation of the composition, the patient's response, the severity of the condition, the patient's body weight, and the judgment of the prescribing physician. The dose can be increased or decreased over time, depending on the needs of the individual patient. In some embodiments, a patient is initially given a low dose, which is then increased to an effective dose that the patient can tolerate.

[0129] In various embodiments, the recombinant glycosylated protein produced by the methods described herein is administered parenterally. In some embodiments, the recombinant glycosylated protein is administered intravenously.

[0130] Examples The application is further described in the following examples, which do not limit the scope of the application described in the claims.

[0131] Example 1. Effect of culture medium additives on expression of enzyme fusion proteins An exemplary enzyme fusion protein containing the SGSH enzyme ("SGSH-Fc fusion protein") was used to test the effect of different culture medium additives on sialic acid levels produced by clonal cells expressing the fusion protein.

[0132] Improved sialic acid levels by addition of media supplements Different concentrations of different culture medium additives were tested on clones expressing the SGSH-Fc fusion protein to determine whether sialic acid levels and other product quality attributes could be improved relative to production without the culture medium additives. As shown in Figure 2 As shown in Table 1, each of hydrocortisone, N-acetylmannosamine (ManNAc), manganese (Mn), and uridine resulted in increased sialic acid levels in the fusion protein relative to production without additives in the medium. For example, each of the following additives resulted in at least a 70% increase in sialic acid levels in the fusion protein: (1) hydrocortisone, 20 μΜ; (2) ManNAc, 40 μΜ; (3) Mn, 10 μΜ; and (4) uridine, 5 mM. The presence of uridine also contributed to an increase in protein titer (about a 42% increase).

[0133] Table 1. Effect of culture medium additives on sialic acid level content and titer in SGSH-Fc fusion protein

[0134] Recombinant protein expression and purification Stable CHO pools were generated by co-transfecting transposons encoding SGSH-Fc fusion protein and hSUMF1 (NCBI Accession No. NP_877437.2) at a mass ratio of 4:1 and transposase mRNA into GS-KO CHO-K1 cells (Horizon) by electroporation and subsequent selection in commercially available chemically defined, animal component free CHO growth medium without glutamine. Subsequently, stable pools were subjected to single cell cloning followed by clone screening to identify clones with desired titer and product quality attributes. The impact of media additives was evaluated during a 14-day fed-batch production process with the identified clones. Cells were seeded at a density of about 0.5 x 10 6 cells / mL in production medium at day 0. Cultures were maintained at 37°C, 5% CO2 and 85% relative humidity. When cultures reached a density of about 10-15 x 10 6 cells / mL, they were switched to 32°C for the remainder of the experiment. Commercial feed was added at days 3, 5, 7, 10 and 12. Media additives were added in 2-3 aliquots distributed over the course of the experiment. Cultures were harvested by centrifugation (5000 x g, 15-20 min) at day 14, followed by sterile filtration through a 0.22 pm filter and storage at 4°C.

[0135] The SGSH-Fc fusion protein was purified from cell culture supernatant using protein A affinity chromatography. Supernatant was loaded onto equilibrated (Tris acetate, NaCl pH 7.4) protein A affinity columns, which were then washed with high conductivity Tris acetate, NaCl pH 7.4 buffer. After re-equilibration of the column, bound protein was eluted using low pH sodium acetate buffer. Eluted protein was then neutralized to a final pH of 5.0 using Tris pH 8 buffer. The homogeneity of the SGSH-Fc fusion in the eluted fractions was assessed by multiple techniques including reducing and non-reducing SDS-PAGE and HPLC-SEC.

[0136] The expressed and purified SGSH-Fc fusion proteins comprise a first SGSH-Fc fusion polypeptide having the sequence of any one of SEQ ID NOs: 14 and 16 and a second SGSH-Fc fusion polypeptide that binds TfR having the sequence of any one of SEQ ID NOs: 19 and 20. During cell culture production, the SGSH-Fc fusion proteins can also be further processed such that the first SGSH-Fc fusion polypeptide has the sequence of SEQ ID NO: 15 or 17 and / or the second SGSH-Fc fusion polypeptide that binds TfR has the sequence of SEQ ID NO: 19 or 21. Thus, the fusion proteins can contain unprocessed sequences (i.e., SEQ ID NOs: 14, 16, 18, and 20); one or more processed sequences (i.e., selected from SEQ ID NOs: 15, 17, 19, and 21); or a mixture containing both processed and unprocessed protein molecules.

[0137] Measurement of N-acetylneuraminic acid (NeuAc or sialic acid) content The NeuAc (sialic acid) content in the SGSH-Fc fusion proteins was measured by one of two methods.

[0138] In the first method, the NeuAc (sialic acid) content was measured by liquid chromatography-mass spectrometry analysis. Five (5) pg of protein in PBS (lx, pH 7.4) was transferred to each well of a 96-well microplate. Two hundred (200) ng of internal standard (1,2,3- 13 C NeuAc, Omicronbio Inc, Catalog No. N-Acetyl-D-[1,2,3- 13C3] neuraminic acid (NEU-004)) was spiked into each well. The final volume of the sample was brought to 30 μΐ^by the addition of 1 M Tris HC1 buffer (final concentration 0.1 M) and ultrapure water. Two (2) μΐ^of SialEXO (10 units / μΐ^) (Genovis Inc. PN: G1-SM1-020) was added to each sample. The plate was then sealed and incubated at 37 °C with shaking for 2 hours. After the incubation period, the plate was centrifuged and 2 μΐ^of sample was directly injected onto the LC column. NeuAc analysis was performed on a UHPLC Vanquish (Thermo Scientific, CA, USA) connected to a UV / Vis and Q Exactive Orbitrap Electrospray Ionization Mass Spectrometer (Thermo Scientific, CA, USA) as described above for M6P analysis. Data were collected in negative ion mode using parallel reaction monitoring (PRM) including NeuAc and NeuAc internal standard (IS) with a time 1.0 to 1.8 minutes, precursors 308.0992 (NeuAc) and 311.1175 (NeuAc-IS). The number of molecules of NeuAc released from the protein was calculated using AUC ratio NeuAc / NeuAc-IS and the number of moles of NeuAc per mole of protein was obtained.

[0139] In a second method, the sialic acid content was determined using a reverse phase liquid chromatography method that measures the amount of N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). The SGSH-Fc test sample was desalted and sialic acids were released by acid hydrolysis using acetic acid. The released sialic acids were labeled with 4,5-dimethoxy-l,2-phenylenediamine dihydrochloride (DMB). Standard solutions of Neu5Ac and Neu5Gc were also labeled with DMB and used to generate a calibration curve. The DMB-labeled Neu5Ac and Neu5Gc were separated on a reverse phase C-18 HPLC column using a gradient elution and monitored using a fluorescence detector (FLD). The sialic acid content of the test sample was quantified by interpolation of the calibration curve and reported as a molar ratio (moles of sialic acid per mole of protein).

[0140] Example 2. Effect of combinations of media additives on enzyme fusion protein production An exemplary enzyme fusion protein containing the IDUA enzyme ("IDUA-Fc fusion protein") was used to test the effect of combinations of media additives on sialic acid levels produced by clonal cells expressing the fusion protein.

[0141] Improved sialic acid levels by combination of media supplements A combination of media additives (10 mM ManNAc, 10 μΜ Mn, and 2.5 mM uridine) was tested on clones expressing the IDUA-Fc fusion protein to determine if sialic acid levels and other product quality attributes could be improved relative to what was produced without the combination. As shown in Table 2, the combination of media additives resulted in at least an 80% improvement in sialic acid levels in the fusion protein relative to what was produced without the combination. Figure 3 As shown in Table 2, the combination of media additives resulted in at least an 80% improvement in sialic acid levels in the fusion protein relative to what was produced without the combination.

[0142] Table 2. Effect of media additive combination on sialic acid level content and product quality in IDUA-Fc fusion protein

[0143] Control = no additives Combination = 10 mM ManNAc, 10 μΜ Mn, and 2.5 mM uridine Recombinant protein expression and purification A stable CHO pool was generated by transfecting transposon and transposase mRNA encoding the IDUA-Fc fusion protein into GS-KO CHO-K1 cells (Horizon) by electroporation and subsequent selection in commercially available chemically defined, animal component free CHO growth media without glutamine. Subsequently, the stable pool was subjected to single cell cloning followed by clone screening to identify clones with desirable titer and product quality attributes (e.g., clones B-1, B-2). The effect of media additives was evaluated during a 14-day fed-batch production process with either the pool or clones. Cells were seeded at a density of 0.5 x 10 6 cells / mL in production media on day 0. Cultures were maintained at 37°C, 5% CO2, and 85% relative humidity. When cultures reached a density of 10-15 x 10 6 cells / mL, they were switched to 32°C for the remainder of the experiment. Commercial feed was added on days 3, 5, 7, 10, and 12. Media additives were added in 2-3 aliquots distributed throughout the experiment. Cultures were harvested by centrifugation (5000 x g, 15-20 minutes) on day 14, followed by sterile filtration through a 0.22 μιη filter, and stored at 4°C.

[0144] The IDUA-Fc fusion protein was purified from cell culture supernatant using protein A affinity chromatography. Supernatant was loaded onto equilibrated (PBS) protein A affinity columns, the columns were then re-equilibrated before bound protein was eluted using low pH sodium acetate buffer. Eluted protein was then neutralized to a final pH of 5.5 using Tris pH 8 buffer.

[0145] The expressed and purified IDUA-Fc fusion protein comprises a TfR binding modified Fc polypeptide having the sequence of SEQ ID NO: 22 and an IDUA-Fc fusion polypeptide having the sequence of SEQ ID NO: 24. The IDUA-Fc fusion protein can also be further processed during cell culture production such that the TfR binding modified Fc polypeptide has the sequence of SEQ ID NO: 23 and / or the IDUA-Fc fusion polypeptide has the sequence of SEQ ID NO: 25. Thus, the IDUA-Fc fusion protein can have unprocessed sequences (i.e., SEQ ID NOs: 22 and 24); one or more processed sequences (i.e., selected from SEQ ID NOs: 23 and 25); or a mixture comprising both processed and unprocessed protein molecules.

[0146] Measurement of N-acetylneuraminic acid (NeuAc or sialic acid) content The NeuAc (sialic acid) content in the IDUA-Fc fusion protein was measured by liquid chromatography-mass spectrometry analysis as described in Example 1.

[0147] Example 3. Effect of sialyltransferase gene overexpression and media additives on production of enzyme fusion protein Clonal cells expressing an enzyme fusion protein containing the IDUA enzyme ("IDUA-Fc fusion protein") were used to test the effect of (1) combinations of media additives and (2) sialyltransferase gene overexpression in the clonal cells on sialic acid levels in the fusion protein.

[0148] Improved sialic acid levels by combination of media supplements and ST6GAL1 gene overexpression Media additives were tested on the clonal cells expressing the IDUA-Fc fusion protein alone or in combination with ST6GAL1 or ST3GAL4 The combination of media additives (10 mM ManNAc, 10 μΜ Mn, and 5 mM uridine) was tested on the overexpression combination pool to determine if sialic acid levels and other product quality attributes were affected. As Figure 4 and shown in Table 3, ST6GAL1 Overexpression enhanced the effect of the media additives on sialic acid levels in the expressed fusion protein. In the presence of the media additives, ST6GAL1 The combination of overexpression increased sialic acid levels in the fusion protein by at least 290%. In contrast, the media additives alone increased sialic acid levels by about 188% over baseline. All sialic acid level comparisons were made relative to sialic acid levels in the fusion protein expressed in cells without sialyltransferase gene overexpression and without media additives. Slightly lower titers were observed in the clones expressing the sialyltransferase gene (titers decreased by about 12-24%).

[0149] Table 3. Sialic acid level content in IDUA-Fc fusion proteins and product quality

[0150] Recombinant protein expression and purification A stable CHO pool was generated by transfecting a transposon construct encoding an IDUA-Fc fusion protein and transposase mRNA into GS-KO CHO-K1 cells (Horizon) by electroporation and subsequent selection in commercially available chemically defined animal component free CHO growth medium without glutamine. This pool was then retransfected with a transposon construct encoding ST3GAL4 (NCBI Accession No. XP_047283377.1) or ST6GAL1 (NCBI Accession No. NP_001340845.1) and transposase mRNA and selected in medium containing different concentrations of puromycin to control the stringency of selection (“supertransfected” cells).

[0151] The impact of medium additives was evaluated in a 14-day fed-batch production process. On day 0, cells were seeded at a density of about 0.5 x 10 6 cells / mL in commercially available chemically defined animal component free CHO growth medium without glutamine. Cultures were maintained at 37°C, 5% CO2 and 85% relative humidity. When cultures reached a density of about 10-15 x 10 6 cells / mL, they were switched to 32°C for the remaining duration of the experiment. Commercial feed was added on days 3, 5, 7, 10 and 12. Medium additives were added in 2-3 aliquots distributed over the entire duration of the experiment. Cultures were harvested on day 14 by centrifugation (5000 x g, 15-20 min), followed by sterile filtration through a 0.22 pm filter and storage at 4°C.

[0152] IDUA-Fc fusion proteins were purified from cell culture supernatant using protein A affinity chromatography. Supernatant was loaded onto equilibrated (PBS) protein A affinity columns, which were then re-equilibrated before eluting bound proteins using low pH sodium acetate buffer. Eluted proteins were then neutralized to a final pH of 5.5 using Tris pH 8 buffer.

[0153] The expressed and purified IDUA-Fc fusion proteins are described in Example 2.

[0154] Measurement of N-acetylneuraminic acid (NeuAc or sialic acid) content NeuAc (sialic acid) content in IDUA-Fc fusion proteins was measured by liquid chromatography-mass spectrometry analysis as described in Example 1.

[0155] Example 4. Effect of galactosyltransferase and sialyltransferase gene overexpression on production of enzyme fusion proteins Clonal cells expressing enzyme fusion proteins containing IDUA enzyme (“IDUA-Fc fusion proteins”) were used to test the effect of the combination of sialyltransferase and galactosyltransferase gene overexpression on sialic acid levels in the fusion proteins. Experiments were performed in the presence and absence of media supplements (10 mM ManNAc, 10 mM Mn, and 2.5 mM uridine).

[0156] Improved sialic acid levels mainly by ST6GAL1 gene overexpression In clones expressing IDUA-Fc fusion proteins transformed to express sialyltransferase ST6GAL1 and / or ST3GAL4 galactosyltransferase B4GALT1 gene overexpression was tested to determine if sialic acid levels of the fusion proteins and other product quality attributes were affected. Experiments were performed in the presence and absence of a combination of media supplements (10 mM ManNAc, 10 mM Mn, and 2.5 mM uridine). As Figure 5 and shown in Table 4, B4GALT1 gene overexpression did not appear to affect sialic acid levels of the fusion proteins in clones that also overexpress ST6GAL1 and / or ST3GAL4 Galactosyltransferase ST6GAL1 gene overexpression and the presence of media supplements. All sialic acid level comparisons were made relative to sialic acid levels in the fusion proteins expressed in cells without galactosyltransferase and sialyltransferase gene overexpression and without media supplements.

[0157] Table 4. Sialic acid level content in IDUA-Fc fusion proteins and product quality

[0158] Recombinant protein expression and purification A stable CHO pool was generated by transfecting a transposon construct encoding an IDUA-Fc fusion protein and transposase mRNA into GS-KO CHO-K1 cells (Horizon) by electroporation and subsequent selection in commercially available chemically defined, animal component-free CHO growth medium without glutamine. This pool was then transduced with a lentivirus encoding B4GALT1 (NCBI Accession No. NP_001488.2), ST3GAL4 (NCBI Accession No. XP_047283377.1), or ST6GAL1The transposon construct and transposase mRNA of (NCBI accession number NP_001340845.1) were retransfected and selected in medium containing 8 μg / mL puromycin (“supertransfected” cells).

[0159] The effect of culture medium additives was evaluated during a 14-day fed-batch production process. On day 0, cells were fed at approximately 0.5 x 10⁻⁶ cells / day. 6 Inoculate at a density of 10⁻¹⁵ cells / mL in commercially available, chemically defined, animal-free CHO growth medium that is glutamine-free. Maintain the culture at approximately 37°C, 5% CO₂, and 85% relative humidity. When the culture reaches a density of approximately 10⁻¹⁵ x 10⁻¹⁰ cells / mL... 6 When the culture reaches 100 cells / mL, transfer them to approximately 32°C for the remaining duration of the experiment. Add commercially available feedstock on days 3, 5, 7, 10, and 12. Add culture supplement in 2–3 aliquots distributed throughout the experimental time. Harvest the culture on day 14 by centrifugation (5000 xg, 15–20 min), followed by aseptic filtration through a 0.22 µm filter and storage at 4°C.

[0160] The IDUA-Fc fusion protein was purified from cell culture supernatant using protein A affinity chromatography. The supernatant was loaded onto a pre-equilibrated (PBS) protein A affinity column, which was then reequilibrated. The bound protein was eluted with low-pH sodium acetate buffer. The eluted protein was then neutralized to a final pH of 5.5 using Tris pH 8 buffer.

[0161] The expressed and purified IDUA-Fc fusion protein is as described in Example 2.

[0162] Measurement of N-acetylneuraminic acid (NeuAc or sialic acid) content The NeuAc (sialic acid) content in the IDUA-Fc fusion protein was measured by liquid chromatography-mass spectrometry analysis as described in Example 1.

[0163] Other implementation plans It should be understood that although the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0164] sequence list SEQ ID NO: 1 – Human SGSH SEQ ID NO: 2 – Mature SGSH SEQ ID NO: 3 - (full-length human alpha-L-iduronidase polypeptide sequence, wherein X1 is H or Q; and X2 is A or T) SEQ ID NO: 4 - mature human IDUA SEQ ID NO: 5 - human IDS SEQ ID NO: 6 - mature human IDS SEQ ID NO: 7 - truncated human IDS SEQ ID NO: 8 - (mature human alpha-L-iduronidase polypeptide, wherein X1 is H or Q; and X2 is A or T) SEQ ID NO: 9 - (embodiment of truncated human alpha-L-iduronidase polypeptide sequence, wherein X1 is H or Q; X2 is A or T; and X3 is E or absent) SEQ ID NO: 10 - human transferrin receptor 1 SEQ ID NO: 11 - apical domain of human transferrin receptor 1 SEQ ID NO: 12 - human IDS variant SEQ ID NO: 13 - human IDS variant SEQ ID NO: 14 - SGSH-Fc fusion polypeptide SEQ ID NO: 15 - SGSH-Fc fusion polypeptide SEQ ID NO: 16 - SGSH-Fc fusion polypeptide SEQ ID NO: 17 - SGSH-Fc fusion polypeptide SEQ ID NO: 18 - SGSH-Fc fusion polypeptide with transferrin receptor binding site SEQ ID NO: 19 - SGSH-Fc fusion polypeptide with transferrin receptor binding site SEQ ID NO: 20 - SGSH-Fc fusion polypeptide with transferrin receptor binding site SEQ ID NO: 21 - SGSH-Fc fusion polypeptide with transferrin receptor binding site SEQ ID NO: 22 - Transferrin receptor binding Fc polypeptide SEQ ID NO: 23 - Transferrin receptor binding Fc polypeptide SEQ ID NO: 24 - IDUA-Fc fusion polypeptide SEQ ID NO: 25 - IDUA-Fc fusion polypeptide SEQ ID NO: 26 - Canonical human ST6GAL1 SEQ ID NO: 27 - Human ST6GAL1 isoform SEQ ID NO: 28 - Human ST6GAL1 isoform SEQ ID NO: 29 - Human ST6GAL1 isoform SEQ ID NO: 30 - Human ST6GAL1 isoform SEQ ID NO: 31 - Canonical human ST3GAL4 SEQ ID NO: 32 - human ST3GAL4 isoform SEQ ID NO: 33 - human ST3GAL4 isoform SEQ ID NO: 34 - human ST3GAL4 isoform SEQ ID NO: 35 - human ST3GAL4 isoform SEQ ID NO: 36 - human ST3GAL4 isoform SEQ ID NO: 37 - human ST3GAL4 isoform

Claims

1. A method for increasing sialic acid levels in recombinant glycosylated proteins, comprising: Mammalian host cells capable of expressing the recombinant glycosylated protein are cultured in a liquid culture medium containing one or more culture medium additives selected from the following: hydrocortisone, N-acetylmnosamine (ManNAc), manganese, and uridine. The mammalian host cells mentioned therein have been previously transformed with one or more vectors encoding one or more sialyl transferases.

2. The method of claim 1, wherein the method further comprises, prior to the culture step, transforming the mammalian cells capable of expressing the recombinant glycosylated protein with one or more vectors encoding the one or more sialic acid transferases to generate the mammalian host cells.

3. The method of claim 1 or 2, wherein the mammalian host cell is a rodent cell.

4. The method of claim 3, wherein the rodent cell is a CHO cell.

5. The method of claim 1 or 2, wherein the mammalian host cell is a human cell.

6. The method of claim 1 or 2, wherein the one or more sialyl transferases are selected from: ST6GAL1 and ST3GAL4 .

7. The method of claim 6, wherein the one or more carrier encodings ST6GAL1 .

8. The method of any one of claims 1-7, wherein the liquid culture medium comprises one of the following: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, or uridine.

9. The method of any one of claims 1-7, wherein the liquid culture medium comprises two of the following: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

10. The method of any one of claims 1-7, wherein the liquid culture medium comprises three of the following: hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

11. The method of any one of claims 1-7, wherein the liquid culture medium comprises hydrocortisone, N-acetylmannosamine (ManNAc), manganese, and uridine.

12. The method of any one of claims 1-11, wherein the ManNAc is present in the liquid culture medium at a concentration of 10 mM to about 60 mM.

13. The method of any one of claims 1-12, wherein the uridine is present in the liquid culture medium at a concentration of 2.5 mM to about 10 mM.

14. The method of any one of claims 1-13, wherein the hydrocortisone is present in the liquid culture medium at a concentration of 5 μM to about 50 μM.

15. The method of any one of claims 1-14, wherein the manganese is present in the liquid culture medium at a concentration of 2 μM to about 15 μM.

16. The method of any one of claims 1-15, wherein the recombinant glycosylated protein is a recombinant glycosylation enzyme.

17. The method of claim 16, wherein the recombinant glycosylation enzyme comprises an enzyme replacement therapy (ERT) enzyme, a catalytically active ERT enzyme variant, or a catalytically active ERT enzyme fragment.

18. The method of claim 17, wherein the enzyme replacement therapy (ERT) enzyme comprises lysosomal storage disease (LSD) enzyme.

19. The method of claim 18, wherein the LSD enzyme is α-L-iduronase (IDUA) or N-sulfoglucosamine sulfonylhydrolase (SGSH).

20. The method of claim 16, wherein the recombinant glycosylation enzyme is a fusion protein.

21. The method of claim 20, wherein the fusion protein comprises (i) a first Fc polypeptide linked to an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and (ii) a second Fc polypeptide forming an Fc dimer with the first Fc polypeptide, wherein the first Fc polypeptide and / or the second Fc polypeptide are modified Fc polypeptides capable of specifically binding to the transferrin receptor (TfR).

22. The method of claim 20, wherein the fusion protein comprises (i) an enzyme replacement therapy (ERT) enzyme, an ERT enzyme variant, or a catalytically active fragment thereof; and (ii) a modified Fc dimer capable of specifically binding to the transferrin receptor (TfR).

23. The method of claim 20, wherein the fusion protein comprises an enzyme replacement therapy (ERT) enzyme, an ERT variant, or a catalytically active fragment thereof linked to an Fc polypeptide.

24. The method of claim 23, wherein the Fc polypeptide is a modified Fc polypeptide.

25. The method of claim 24, wherein the Fc polypeptide is capable of specifically binding to the transferrin receptor (TfR).

26. The method of any one of claims 1-25, wherein the culture is a fed-batch culture.

27. The method of any one of claims 1-25, wherein the culture is a batch culture.

28. The method of any one of claims 1-25, wherein the culture is a perfusion culture.

29. The method of any one of claims 1-28, wherein the sialic acid level in the recombinant glycosylated protein is increased by at least 50% compared to the case of recombinant glycosylated proteins produced by methods excluding one or both of the following: (i) using mammalian host cells capable of expressing the recombinant glycosylated protein and containing a vector encoding one or more of the sialyl transferases, and (ii) culturing mammalian host cells capable of expressing the recombinant glycosylated protein in a liquid culture medium containing one or more culture medium additives selected from: hydrocortisone, N-acetylmnosamine (ManNAc), manganese, and uridine.

30. The method of any one of claims 1-28, wherein the sialic acid level in the recombinant glycosylated protein is increased by at least 100% compared to the case of recombinant glycosylated proteins produced by methods excluding one or both of the following: (i) using mammalian host cells capable of expressing the recombinant glycosylated protein and containing a vector encoding one or more sialyl transferases, and (ii) culturing mammalian host cells capable of expressing the recombinant glycosylated protein in a liquid culture medium containing one or more culture medium additives selected from: hydrocortisone, N-acetylmnosamine (ManNAc), manganese, and uridine.

31. The method of any one of claims 1-28, wherein the sialic acid level in the recombinant glycosylated protein is increased by at least 150% compared to the case of recombinant glycosylated proteins produced by methods excluding one or both of the following: (i) using mammalian host cells capable of expressing the recombinant glycosylated protein and containing a vector encoding one or more of the sialyl transferases, and (ii) culturing mammalian host cells capable of expressing the recombinant glycosylated protein in a liquid culture medium containing one or more culture medium additives selected from: hydrocortisone, N-acetylmnosamine (ManNAc), manganese, and uridine.

32. The method of any one of claims 1-28, wherein the sialic acid level in the recombinant glycosylated protein is increased by at least 250% compared to the case of recombinant glycosylated proteins produced by methods excluding one or both of the following: (i) using mammalian host cells capable of expressing the recombinant glycosylated protein and containing a vector encoding one or more sialyl transferases, and (ii) culturing mammalian host cells capable of expressing the recombinant glycosylated protein in a liquid culture medium containing one or more culture medium additives selected from: hydrocortisone, N-acetylmnosamine (ManNAc), manganese, and uridine.

33. The method of any one of claims 1-32, wherein the method further comprises harvesting the recombinant glycosylated protein from the liquid culture medium and / or the mammalian host cell.

34. The method of claim 33, wherein the method further comprises separating the harvested recombinant glycosylated protein.

35. The method of claim 34, wherein the method further comprises formulating the isolated recombinant glycosylated protein.

36. A recombinant glycosylated protein, produced by the method according to any one of claims 1-35.

37. A method of treating a subject in need, comprising administering to the subject a therapeutically effective amount of the recombinant glycosylated protein as claimed in claim 36.

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