Methods of manufacturing recombinant alkaline phosphatases

Abstract

Featured are methods of manufacturing recombinant alkaline phosphatases that provide more precise quality control over glycosylation patterns such as sialic acid, man nose, and fucose in the final product.

Description

[0001] PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0002] METHODS OF MANUFACTURING RECOMBINANT ALKALINE PHOSPHATASES

[0003] SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on March 8, 2024, is named 0667_US_P1_SL.xml and is 5,372 bytes in size.

[0005] BACKGROUND

[0006] Hypophosphatasia (HPP) is a life-threatening, genetic, metabolic disorder that results in a failure to produce functional tissue nonspecific alkaline phosphatase (TNSALP). It leads to the accumulation of unmineralized bone matrix (e.g., rickets and osteomalacia) and is characterized by hypo-mineralization of bones and teeth. When growing bone does not mineralize properly, impairment of growth disfigures joints and bones. This result in turn impacts motor performance, respiratory function, and potentially death. HPP includes perinatal, infantile, juvenile (or childhood), and adult HPP. Historically, six clinical forms were defined, most based in part upon age at symptom onset, including perinatal, benign prenatal, infantile, juvenile, adult, and odonto-HPP.

[0007] Recombinant alkaline phosphatases, such as asfotase alfa, have been used to treat HPP.

[0008] Recombinant alkaline phosphatases and engineered variants contain post-translation modifications, such as glycosylation (e.g., sialyation, mannosylation, and fucosylation). Production of commercial scale quantities of therapeutically effective alkaline phosphatases, such as asfotase alfa and other therapeutically active variants, involves a multi-step manufacturing process, the conditions of which significantly affect the final product. During one or more steps of the manufacturing process, post-translationally modified products can be exposed to glycosidases or other hydrolytic conditions that negatively impact the post-translational modifications, and can lead to loss of moieties (such as loss of mannose and fucose moieties form added sialic acid). These changes can reduce the half-life and enzymatic activity of large quantities of the batch product or negatively affect the batch-to-batch consistency.

[0009] While effective manufacturing processes have been developed for asfotase alfa, the same manufacturing processes are not suitable for other, specific variants. Improved methods of manufacturing alkaline phosphatases are needed to improve quality control of the final protein product and its glycosylation characteristics.

[0010] SUMMARY OF THE DISCLOSURE

[0011] Disclosed herein are manufacturing processes that can be used to improve quality control of glycosylation in the production of alkaline phosphatases. The methods can also be used for maintaining, preserving, modulating, and / or improving the enzymatic activity, and particularly maintaining, controlling, and / or improving half-life, of a recombinant protein, such as an alkaline phosphatase produced by cultured mammalian cells, particularly by cultured Chinese Hamster Ovary (CHO) cells. Such alkaline phosphatases are suitable for use in therapy. For example, alkaline phosphatases can be used for treatment of conditions PATENT ALEXION REF.: ALXN-0667-PCT01-NP associated with decreased alkaline phosphatase protein levels (e.g., HPP) and / or function (e.g., insufficient cleavage of inorganic pyrophosphate (PPi), etc.) in a subject, for example, a human subject in need thereof.

[0012] A first aspect of the disclosure features a method of producing a recombinant alkaline phosphatase (e.g., an alkaline phosphatase as defined herein, e.g., one having the amino acid sequence of SEQ ID NO: 2 or 3 and variants thereof). The method includes the step of (a) inoculating a bioreactor containing a culture medium with cells expressing a recombinant alkaline phosphatase. The pH of the culture medium is about 6.7 to about 7.1 (e.g., about pH 6.7, 6.8, 6.9, 7.0, or 7.1 , e.g., about pH 6.9). The method further includes the steps of (b) supplementing the culture medium with galactose, N-acetyl mannosamine, and fucose; (c) culturing the cells to express the recombinant alkaline phosphatase; and (d) harvesting the recombinant alkaline phosphatase from the culture medium.

[0013] In specific embodiments, step (b) includes adding of each of galactose, N-acetyl mannosamine, and fucose at a concentration of about 0.5 to about 5 g / L (e.g., about 0.5 g / L, about 1 .0 g / L, about 1 .5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, or about 5.0 g / L). For example, in certain embodiments step (b) includes adding about 4 g / L galactose, about 3 g / L N-acetyl mannosamine, and about 1 g / L fucose.

[0014] In some embodiments, the supplementing of step (b) is performed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after the inoculating step (a).

[0015] In some embodiments, the galactose, N-acetyl mannosamine, and / or fucose are added as a bolus.

[0016] In some embodiments, the method further includes adding glucose to the culture medium. For example, in specific embodiments glucose is added at a concentration of about 1 g / L to about 5 g / L (e.g., about 1 .0 g / L, about 1 .5 g / L, about 2.0 g / L, about 2.5 g / L, about 3.0 g / L, about 3.5 g / L, about 4.0 g / L, about 4.5 g / L, or about 5.0 g / L, e.g., about 2 g / L or about 4 g / L). In specific embodiments the glucose is added to the culture medium on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after the inoculating step (a).

[0017] In some embodiments, the method includes determining that a glucose concentration in the culture medium is less than or equal to 2 g / L in the bioreactor, and adding glucose to the culture medium at a concentration of about 2 g / L at about day 4, day 6, day 8, and / or day 10 after inoculation, and at a concentration of about 4 g / L at about day 1 , day 2, day 3, day 5, day 7, day 9, day 11 , day 12, and / or day 13 after inoculation.

[0018] In specific embodiments the culture medium includes or is composed of a suitable basal medium, such as a chemically defined Chinese hamster ovary advanced granulation technology (CD CHO AGT™) medium. In specific embodiments the culture medium includes CD CHO AGT™ medium at a concentration of 10 g / L to 100 g / L (e.g., about 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, or 100 g / L). In some embodiments, the culture medium includes CD CHO AGT™ medium at a concentration of about 24.1 g / L.

[0019] In specific embodiments the culture medium further includes supplemental metals or nonmetals, such as one or more of copper, zinc, magnesium, and chloride. For example, the culture medium in specific PATENT ALEXION REF.: ALXN-0667-PCT01-NP embodiments includes from 5 pM to 50 pM copper (e.g., about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM, e.g., about 20 pM copper).

[0020] In specific embodiments, the culture medium includes 5 mg / L to 100 mg / L zinc sulfate heptahydrate (e.g., 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L zinc sulfate hepta hydrate), 100 mg / L to 200 mg / L magnesium chloride hexahydrate (e.g., 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, 150 mg / L, 155 mg / L, 160 mg / L, 165 mg / L, 170 mg / L, 175 mg / L, 180 mg / L, 185 mg / L, 190 mg / L, 195 mg / L, or 200 mg / L magnesium chloride hexahydrate), and / or 5 mg / L to 100 mg / L calcium chloride dihydrate (e.g., 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L calcium chloride dihydrate). In some embodiments, the culture medium includes about 43 mg / L zinc sulfate heptahydrate, about 148 mg / L magnesium chloride hexahydrate, and about 55 mg / L calcium chloride dihydrate.

[0021] In specific embodiments the culture medium further includes or is supplemented with cysteine and / or tyrosine. In specific embodiments, basal medium is supplemented with 0.1 g / L to 0.5 g / L L-cysteine hydrochloride monohydrate (e.g., 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L L-cysteine hydrochloride monohydrate) and / or 0.1 g / L to 0.5 g / L L-tyrosine (e.g., 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L L-tyrosine). In some embodiments, the basal medium is supplemented with 0.342 g / L L-cysteine hydrochloride monohydrate and 0.269 g / L L-tyrosine.

[0022] In specific embodiments the culture medium includes or is supplemented with GLYCAN TUNE™ C+ (GTC+) feed. For example, the method in specific embodiments includes adding GTC+ feed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after the inoculating step (a). In specific embodiments the GTC+ feed is added at a concentration of 100 g / L to 200 g / L (e.g., 100 g / L, 125 g / L, 150 g / L, 175 g / L, or 200 g / L). In some embodiments, the GTC+ feed is added at a concentration of 175.4 g / L. In specific embodiments the GTC+ feed is added at a concentration of 1 % (v / v) to 10% (v / v) (e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v / v)) of the initial culture volume. In certain embodiments, the GTC+ feed is added at a concentration of 5% (v / v) of the initial culture volume. In certain embodiments, the GTC+ feed is added 3 or 4 equal boluses at a concentration of 5% (v / v) of the initial culture volume.

[0023] In some embodiments, step (c) includes culturing the cells at a dissolved oxygen (DO) concentration from 10% to 60%.

[0024] In some embodiments, step (a) includes inoculating the bioreactor with a cell density of about 0.3 x 106viable cells / mL to about 0.7 x 106viable cells / mL (e.g., about 0.3 x 106viable cells / mL, 0.4 x 106viable cells / mL, 0.5 x 106viable cells / mL, 0.6 x 106viable cells / mL, or 0.7 x 106viable cells / mL).

[0025] In some embodiments, the method includes culturing the cells at a first temperature until reaching a cell density of at least about 5 x 106viable cells / mL and shifting to a second temperature which is lower than the first temperature to express the recombinant alkaline phosphatase. In specific embodiments the first temperature is about 35 °C to about 38 °C (e.g., 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, or 38 °C, e.g., about 36.5 °C). In specific embodiments the second temperature is about 29 °C to about 36 °C (e.g., 29 °C, PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0026] 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, or 36 °C, e.g., about 34 °C to about 36 °C, e.g., about 35 °C). In some embodiments, the cell density is about 5 x 106viable cells / mL to about 25 x 106viable cells / mL (e.g., about 5 x 106viable cells / mL, 6 x 106viable cells / mL, 7 x 106viable cells / mL, 8 x 106viable cells / mL, 9 x 106viable cells / mL, 10 x 106viable cells / mL, 11 x 106viable cells / mL, 12 x 106viable cells / mL, 13 x 106viable cells / mL, 14 x 106viable cells / mL, 15 x 106viable cells / mL, 16 x 106viable cells / mL, 17 x 106viable cells / mL, 18 x 106viable cells / mL, 19 x 106viable cells / mL, 20 x 106viable cells / mL, 21 x 106viable cells / mL, 22 x 106viable cells / mL, 23 x 106viable cells / mL, 24 x 106viable cells / mL, 25 x 10sviable cells / mL, 26 x 106viable cells / mL, 27 x 106viable cells / mL, 28 x 106viable cells / mL, 29 x 106viable cells / mL, or 30 x 106viable cells / mL, e.g., about 6 x 106viable cells / mL to about 10 x 106viable cells / mL or about 15 x 106viable cells / mL to about 20 x 106viable cells / mL). In specific embodiments the temperature shift is performed on or about day 7 after the inoculating step.

[0027] In certain embodiments, step (d) includes harvesting the cells at a density of about 10 x 106viable cells / mL to about 30 x 106viable cells / mL (e.g., 10 x 106viable cells / mL, 11 x 106viable cells / mL, 12 x 106viable cells / mL, 13 x 106viable cells / mL, 14 x 106viable cells / mL, 15 x 106viable cells / mL, 16 x 106viable cells / mL, 17 x 106viable cells / mL, 18 x 106viable cells / mL, 19 x 106viable cells / mL, 20 x 106viable cells / mL, 21 x 106viable cells / mL, 22 x 10® viable cells / mL, 23 x 10® viable cells / mL, 24 x 106viable cells / mL, 25 x 10® viable cells / mL, 26 x 106viable cells / mL, 27 x 106viable cells / mL, 28 x 10® viable cells / mL, 29 x 106viable cells / mL, or 30 x 106viable cells / mL). In specific embodiments the harvesting is performed on about day 14 after the inoculating step (a). In certain embodiments the harvesting is performed within 24 hours of the cell viability being less than or equal to 80 % of the total cells.

[0028] In specific embodiments, the harvesting is performed by filtration (e.g., depth filtration) or centrifugation.

[0029] Any suitable cell can be used in the methods described herein. In specific embodiments as described herein, the cells are mammalian cells, such as Chinese Hamster Ovary (CHO) cells.

[0030] In specific embodiments the method includes using a bioreactor, e.g., having a volume of at least 2 L. In specific embodiments the bioreactor has a volume of at least 10 L, at least 100 L, at least 1 ,000 L, at least 10,000 L, at least 15,000 L, at least 20,000 L, or more (or any volume between 10 L and 20,000 L, or greater).

[0031] The methods described herein are useful for producing a recombinant alkaline phosphatase. In some embodiments, the recombinant alkaline phosphatase includes the structure of W-sALP-X-Fc-Y-Dn-Z, in which:

[0032] W is absent or is an amino acid sequence of at least one amino acid;

[0033] X is absent or is an amino acid sequence of at least one amino acid;

[0034] Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; Fc is a fragment crystallizable region;

[0035] Dn is a poly-aspartate, poly-glutamate, or combination thereof, in which n = 5 to 16 (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, such as n = 7 or n = 10); and PATENT ALEXION REF.: ALXN-0667-PCT01-NP sALP is a soluble alkaline phosphatase.

[0036] In some embodiments, the recombinant alkaline phosphatase includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 2 or 3. In some embodiments, the recombinant alkaline phosphatase comprises or consists of the amino acid sequence set forth in SEQ ID NO: 2 or 3.

[0037] In specific embodiments, the recombinant alkaline phosphatase comprises the amino acid sequence set forth in SEQ ID NO: 2 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 2). In specific embodiments the recombinant alkaline phosphatase is glycosylated (e.g., with one or more of sialic acid, fucose, and mannose) at one or more of N123, N254, N413, and N564. In specific embodiments the recombinant alkaline phosphatase comprises a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer (e.g., at least 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). In certain embodiments, the recombinant alkaline phosphatase comprises a total fucose content of at least 2.0 mol fucose / mol protein monomer. In certain embodiments, the recombinant alkaline phosphatase includes a total mannose content of at least 6.0 mol mannose / mol protein monomer. In specific embodiments the recombinant alkaline phosphatase comprises a dimer that comprises a first polypeptide and a second polypeptide, in which the first polypeptide and the second polypeptide have the amino acid sequence of SEQ ID NO: 2 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 2). In specific embodiments the dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide. In some embodiments, the first polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. In some embodiments, C102 of the first polypeptide and the second polypeptide is a free cysteine. In specific embodiments each of the first polypeptide and the second polypeptide coordinates two zinc ions, one magnesium ion, and one calcium ion.

[0038] In some embodiments, the recombinant alkaline phosphatase includes the amino acid sequence set forth in SEQ ID NO: 3 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 3). In specific embodiments the recombinant alkaline phosphatase is glycosylated (e.g., with one or more of sialic acid, fucose, and mannose) at one or more of N123, N213, N254, N286, N413, and N564. In specific embodiments the recombinant alkaline phosphatase includes a total sialic acid content (TSAC) of at least 1 .2 mol sialic acid / mol protein monomer (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). In specific embodiments the recombinant alkaline phosphatase comprises a total fucose content of at least 0.6 mol fucose / mol protein monomer. In specific embodiments the recombinant alkaline phosphatase comprises a total mannose content of at least 1 .8 mol mannose / mol protein monomer. In PATENT ALEXION REF.: ALXN-0667-PCT01-NP specific embodiments the recombinant alkaline phosphatase is a dimer that includes a first polypeptide and a second polypeptide, in which the first polypeptide and the second polypeptide have the amino acid sequence of SEQ ID NO: 3 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 3). The dimer may be linked by a first disulfide bond between 0494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and 0497 of the second polypeptide. In some embodiments, the first polypeptide includes disulfide bonds between C122 and C184, C472 and C480, 0528 and C588, and 0634 and C692, and the second polypeptide includes disulfide bonds between C122 and 0184, 0472 and C480, C528 and 0588, and C634 and C692. In some embodiments, C102 of the first polypeptide and the second polypeptide is a free cysteine. Each of the first polypeptide and the second polypeptide may coordinate two zinc ions, one magnesium ion, and one calcium ion.

[0039] Another aspect of the disclosure features a dimer of a recombinant alkaline phosphatase that includes a first polypeptide and a second polypeptide, in which the first polypeptide and the second polypeptide have the amino acid sequence of SEQ ID NO: 2 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 2). The dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between 0497 of the first polypeptide and C497 of the second polypeptide. The dimer is glycosylated at one or more of N123, N254, N413, and N564. The glycosylation includes one or more of sialic acid, fucose, and mannose. The recombinant alkaline phosphatase includes: (a) a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer (e.g., at least 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer); (b) a total fucose content of at least 2.0 mol fucose / mol protein monomer; and (c) a total mannose content of at least 6.0 mol mannose / mol protein monomer. In some embodiments, the first polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. In some embodiments, C102 of the first polypeptide and the second polypeptide is a free cysteine. Each of the first polypeptide and the second polypeptide may coordinate two zinc ions, one magnesium ion, and one calcium ion.

[0040] Another aspect of the disclosure features a dimer of a recombinant alkaline phosphatase including a first polypeptide and a second polypeptide, in which the first polypeptide and the second polypeptide have the amino acid sequence of SEQ ID NO: 3 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence set forth in SEQ ID NO: 3). The dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide. The dimer is glycosylated at one or more of N123, N213, N254, N286, N413, and N564. The glycosylation includes one or more of sialic acid, fucose, and mannose. The recombinant alkaline phosphatase includes: (a) a total sialic acid content (TSAC) of at least 1 .2 mol sialic acid / mol protein monomer (e.g., at least 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11 .5, 12.0, PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0041] 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer); (b) a total fucose content of at least 0.6 mol fucose / mol protein monomer; and (c) a total mannose content of at least 1.8 mol mannose / mol protein monomer. In some embodiments, the first polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. In some embodiments, C102 of the first polypeptide and the second polypeptide is a free cysteine. Each of the first polypeptide and the second polypeptide may coordinate two zinc ions, one magnesium ion, and one calcium ion.

[0042] Definitions

[0043] As used herein, the terms “about” and “approximately”, as applied to one or more particular cell culture conditions or numerical values, refer to a range of values that are + / - 10% of a subject value.

[0044] The term “amino acid,” as used herein, refers to any of the twenty naturally occurring amino acids that are normally used in the formation of polypeptides, or analogs or derivatives of those amino acids. Amino acids of the present disclosure can be provided in medium to cell cultures. The amino acids provided in the medium may be provided as salts or in hydrate form.

[0045] The term “bioreactor” as used herein refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). The bioreactor can be of any size so long as it is useful for the culturing of cells. Typically, the bioreactor will be at least 1 liter and may be 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,0000, 20,000, 22,000, 25,000, 30,000 liters or more, or any volume in between. The bioreactor may be, for example, 100 liters to 30,000 liters, 500 liters to 22,000 liters, 1 ,000 liters to 22,000 liters, 2,000 liters to 22,000 liters, 5,000 liters to 22,000 liters, or 10,000 liters to 22,000 liters. The maximum working volume of the bioreactor may vary by about 1% to 5%, e.g., may go up to about 22,250 liters or 33,000 liters. The internal conditions of the bioreactor, including, but not limited to pH and temperature, are typically controlled during the culturing period. The bioreactor can be composed of any material that is suitable for holding mammalian or other cell cultures suspended in media under the culture conditions of the present disclosure, including glass, plastic or metal. The term “production bioreactor” as used herein refers to the final bioreactor used in the production of the polypeptide or protein of interest. The volume of the large-scale cell culture production bioreactor is typically at least 500 liters and may be 1000, 2500, 5000, 8000, 10,000, 12,0000, 20,000 liters or more, or any volume in between.

[0046] The term “cell density,” as used herein, refers to the number of cells present in a given volume of medium.

[0047] The term “cell viability,” as used herein, refers to the ability of cells in culture to survive under a given set of culture conditions or experimental variations. The term as used herein also refers to that portion of cells which are alive at a particular time in relation to the total number of cells, living and dead, in the culture at that time.

[0048] The terms “culture” and “cell culture,” as used herein, refer to a cell population that is suspended in a medium (see definition of “medium” below) under conditions suitable for survival and / or growth of the cell PATENT ALEXION REF.: ALXN-0667-PCT01-NP population. As will be clear to those of ordinary skill in the art, these terms as used herein may refer to the combination including the cell population and the medium in which the population is suspended.

[0049] The term “fed-batch culture,” as used herein, refers to a method of culturing cells in which additional components are provided to the culture at some time subsequent to the beginning of the culture process. The provided components typically include nutritional supplements for the cells, which have been depleted during the culturing process. A fed-batch culture is typically stopped at some point and the cells and / or components in the medium are harvested and optionally purified. Fed-batch culture may be performed in the corresponding fed-batch bioreactor. In some embodiments, the method includes a fed-batch culture.

[0050] The term “fragment,” as used herein, refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or characteristic of that polypeptide. The term as used herein also refers to any discrete portion of a given polypeptide that retains at least a fraction of the activity of the full-length polypeptide. In some embodiments, the fraction of activity retained is at least 10% of the activity of the full- length polypeptide. In various embodiments, the fraction of activity retained is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the full-length polypeptide. In other embodiments, the fraction of activity retained is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide. In one embodiment, the fraction of activity retained is 100% of the activity of the full-length polypeptide. The term as used herein also refers to any portion of a given polypeptide that includes at least an established sequence element found in the full-length polypeptide. In some embodiments, the sequence element spans at least 4-5 amino acids of the full-length polypeptide. In some embodiments, the sequence element spans at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide.

[0051] The terms “glycoprotein,” as used herein, refers to a protein or polypeptide with carbohydrate groups (such as sialic acid) attached to the polypeptide chain.

[0052] The terms “medium”, “media”, “cell culture medium”, and “culture medium,” as used herein, refer to a solution containing nutrients which nourish growing mammalian cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and / or survival. The solution may also contain components that enhance growth and / or survival above the minimal rate, including hormones and growth factors. The solution may be, e.g., formulated to a pH and salt concentration optimal for cell survival and proliferation. In some embodiments, a culture medium may be a “defined media,” e.g., a serum-free media that contains no proteins, hydrolysates or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. In some embodiments, the culture medium is a basal medium, e.g., an undefined medium containing a carbon source, water, salts, a source of amino acids and nitrogen (e.g., animal, e.g., beef, or yeast extracts). Various mediums are commercially available and are known to those in the art. In some embodiments, the culture medium is selected from EX-CELL® 302 Serum- Free Medium (Sigma Aldrich, St. Louis, MO), CD DG44 Medium (ThermoFisher Scientific, Waltham, MA), BD Select Medium (BD Biosciences, San Jose, CA), Chinese hamster ovary advanced granulation technology (CD CHO AGT™) medium or a mixture thereof, or a mixture of BD Select Medium with SFM4CHO Medium (HYCLONE™, Logan UT). In some embodiments, the culture medium includes a combination of SFM4CHO PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0053] Medium and BD SELECT™ Medium. In some embodiments, the culture medium includes a combination of SFM4CHO Medium and BD SELECT™ Medium at a ratio selected from 90 / 10, 80 / 20, 75 / 25, 70 / 30, 60 / 40, or 50 / 50, which includes any intermediate ratio therebetween. In some embodiments, the culture medium includes a combination of SFM4CHO Medium and BD SELECT™ Medium at a ratio of 70 / 30 to 90 / 10. In some embodiments, the culture medium includes a combination of SFM4CHO Medium and BD SELECT™ Medium at a ratio 75 / 25. EX-CELL® 302 Serum-Free Medium contains 0.1 % PLURONIC® F68, 3.42 g / L glucose, 7.5 mM HEPES, and 1 .6 g / L sodium bicarbonate. BD SELECT™ Medium contains human recombinant insulin, hypoxanthine, thymidine, and low endotoxin (<5.0 EU / mL), at pH 7.1 + / - 0.2. CD DG44 Medium is a chemically defined, protein-free, hydrolysate-free medium that contains hypoxanthine and thymidine and L-glutamine without PLURONIC® F-68. In some embodiments, an alkaline phosphatase is produced by a process in which extra boluses of culture medium are added to the production bioreactor. For example, one, two, three, four, five, six, or more boluses of culture medium may be added. In one particular embodiment, three boluses of culture medium are added. In various embodiments, such extra boluses of culture medium may be added in various amounts. For example, such boluses of culture medium may be added in an amount of about 20%, 25%, 30%, 33%, 40%, 45%, 50%, 60%, 67%, 70%, 75%, 80%, 90%, 100%, 110%, 120%, 125%, 130%, 133%, 140%, 150%, 160%, 167%, 170%, 175%, 180%, 190%, 200%, or more, of the original volume of culture medium in the production bioreactor. In one particular embodiment, such boluses of culture medium may be added in an amount of about 33%, 67%, 100%, or 133% of the original volume. In various embodiments, such addition of extra boluses may occur at various times during the cell growth or protein production period. For example, boluses may be added at day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 1 1 , day 12, or later in the process. In one particular embodiment, such boluses of culture medium may be added in every other day (e g., at (1) day 3, day 5, and day 7; (2) day 4, day 6, and day 8; or (3) day 5, day 7, and day 9). In practice, the frequency, amount, time point, and other parameters of bolus supplements of culture medium may be combined freely according to the above limitation and determined by experimental practice.

[0054] The term “seeding,” as used herein, refers to the process of providing a cell culture to a bioreactor or another vessel. The cells may have been propagated previously in another bioreactor or vessel. Alternatively, the cells may have been frozen and thawed immediately prior to providing them to the bioreactor or vessel. The term refers to any number of cells, including a single cell. In various embodiments, alkaline phosphatase (e.g., a polypeptide of SEQ ID NO: 2 or 3) is produced by a process in which cells are seeded in a density of about 1 .0 x 105cells / mL, 1 .5 * 105cells / mL, 2.0 x 1 o5cells / mL, 2.5 x 1 o5cells / mL, 3.0 x 105cells / mL, 3.5 x 105cells / mL, 4.0 x 105cells / mL, 4.5 x 1 O5cells / mL, 5.0 x 105cells / mL, 5.5 x 105cells / mL, 6.0 x 1 o5cells / mL, 6.5 x 1 o5cells / mL, 7.0 x 1 o5cells / mL, 7.5 x 1 o5cells / mL, 8.0 x 1 o5cells / mL, 8.5 x 105cells / mL, 9.0 x 1 o5cells / mL, 9.5 x 1 o5cells / mL, 1 .0 x 1 o6cells / mL, 1 .5 x 1 o6cells / mL, 2.0 x 1 o5cells / mL, or a higher density. In one particular embodiment, in such process, cells are seeded in a density of about 4.0 x 105cells / mL, 5.5 x 105cells / mL or 8.0 * 105cells / mL.

[0055] The terms “total Sialic Acid Content” or “TSAC,” as used herein, refer to the amount of sialic acid (a carbohydrate) on a particular protein molecule. It is expressed as moles TSAC per mole of protein or PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0056] “mol / mol.” TSAC concentration is measured during the purification process. For example, one method of TSAC quantitation involves releasing TSAC from the alkaline phosphatase using acid hydrolysis and detecting the released TSAC via electrochemical detection using high-performance anion-exchange chromatography with pulsed amperometric detection technique (“HPAE-PAD”).

[0057] The term “sialic acid” refers generally to N- or O-substituted derivatives of neuraminic acid, a monosaccharide with a nine-carbon backbone. Sialic acid may also refer specifically to the compound N- acetylneuraminic acid and is sometimes abbreviated as Neu5Ac or NANA. The presence of sialic acid may affect absorption, serum half-life, and clearance of glycoproteins from the serum, as well as physical, chemical, and immunogenic properties of the glycoprotein (e.g., a recombinant alkaline phosphatase).

[0058] The term “sialy lation” refers to a specific type of glycosylation, e.g., the addition of one or more sialic acid molecules to biomolecules, particularly, the addition of one or more sialic acid molecules to proteins. In some embodiments of the present disclosure, sialylation is performed by a sialyltransferase enzyme. In some embodiments, sialyltransferases add sialic acid to nascent oligosaccharides and / or to N- or O-linked sugar chains of glycoproteins. In some embodiments, sialyltransferases are present natively in the cells producing recombinant alkaline phosphatase. In some embodiments, sialyltransferases are present in the cell culture medium and / or nutrient supplement used in culturing the cells producing recombinant alkaline phosphatase. In some embodiments, sialyltransferases are produced recombinantly, using recombinant protein expression methods known in the art. In some embodiments, recombinant sialyltransferases produced separately from the recombinant alkaline phosphatases are added exogenously to the cell culture.

[0059] The term “filtration” refers to a pressure driven process that uses membranes to separate components in a liquid solution or suspension based on their size and charge differences.

[0060] BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 is an illustration depicting asfotase alfa (SEQ ID NO: 1).

[0062] FIG. 2 is an illustration depicting the chemical structure of asfotase alfa variant A (SEQ ID NO: 2).

[0063] FIG. 3 is an illustration depicting the chemical structure of asfotase alfa variant B (SEQ ID NO: 3).

[0064] FIG. 4 is an illustration depicting two graphs outlining the pharmacokinetics (PK) of asfotase alfa

[0065] (SEQ ID NO: 1) and asfotase alfa variant A (SEQ ID NO: 2) when injected either intravenously (left panel) or subcutaneously (right panel) in cynomolgus monkeys.

[0066] FIG. 5 is an illustration depicting two graphs outlining the pharmacokinetics (PK) of asfotase alfa (SEQ ID NO: 1) and asfotase alfa variant B (SEQ ID NO: 3) when injected either intravenously (left panel) or subcutaneously (right panel) in monkeys.

[0067] DETAILED DESCRIPTION

[0068] The present disclosure provides improved methods of manufacturing recombinant glycoproteins, such as alkaline phosphatases, which contain post translational modifications (e.g., sialic acid, mannose, and fucose). The methods provide improved quality control over total sialic acid content (TSAC), as well as the PATENT ALEXION REF.: ALXN-0667-PCT01-NP mannose and fucose concentrations in the final product, by supplementing the culture media with galactose, mannose, and fucose. The methods allow modulation of TSAC, mannose, and fucose concentrations of the final product by using improved culture conditions and culture supplements. Ultimately, this method provides uniform properties of the recombinant alkaline phosphatase for commercial production. The disclosure also provides improved compositions containing the recombinant alkaline phosphatase with desired glycosylation patterns. These glycosylation patterns and the increased presence of sialic acid moieties results in improved pharmacokinetic (PK) properties and increased exposure upon administration to a subject.

[0069] Methods of Manufacture

[0070] The methods described herein include the steps of: inoculating a bioreactor containing a culture medium with cells (e.g., a mammalian cell, e.g., Chinese Hamster Ovary (CHO) cell) expressing a recombinant alkaline phosphatase. The pH of the culture medium may be, e.g., from about 6.7 to about 7.1 (e.g., about pH 6.7, 6.8, 6.9, 7.0, or 7.1 , e.g., about pH 6.9). The method further includes the steps of supplementing the culture medium with galactose, N-acetyl mannosamine, and fucose and culturing the cells to express the recombinant alkaline phosphatase. The method also includes harvesting the recombinant alkaline phosphatase from the culture medium.

[0071] The bioreactor may be inoculated with any suitable concentration of viable cells (e.g., CHO cells) that express an alkaline phosphatase (e.g., an alkaline phosphatase with the amino acid sequence of SEQ ID NO: 2 or 3 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 2 or 3). For example, the method may include inoculating the bioreactor with a cell density of about 0.3 x 106viable cells / mL to about 0.7 x 10® viable cells / mL (e.g., about 0.3 x 10® viable cells / mL, 0.4 x 10® viable cells / mL, 0.5 x 10® viable cells / mL, 0.6 x 10® viable cells / mL, or 0.7 x 106viable cells / mL).

[0072] The methods described herein include supplementing the culture medium with galactose, N-acetyl mannosamine, and fucose. The method may include adding each of galactose, N-acetyl mannosamine, and fucose to the culture at a concentration of about 0.5 to 5 g / L (e.g., about 0.5 g / L, 1 .0 g / L, 1 .5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, or 5.0 g / L. For example, the method may include adding about 4 g / L galactose, about 3 g / L N-acetyl mannosamine, and about 1 g / L fucose to the culture. The galactose, N- acetyl mannosamine, and fucose may be added as a bolus.

[0073] The supplementing step may occur on any day during the cell culture growth. Cell culture growth length is determined following inoculation of the bioreactor. The culturing may persist, e.g., for about 1 day to about 28 days (e.g., 1 day to 21 days, e.g., 1 day to 14 days, e.g., 5 days to 14 days, e.g., 7 days to 14 days, e.g., 10 days to 14 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days . For example, the supplementing step may be performed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after inoculation. PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0074] The methods described herein may further include adding glucose to the culture medium. For example, glucose may be added at a concentration of about 1 g / L to about 5 g / L (e.g., about 1 .0 g / L, 1 .5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, 4.5 g / L, or 5.0 g / L, e.g., about 2 g / L or about 4 g / L). Glucose may be added to the culture medium on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after inoculation.

[0075] The amount of glucose that is added to the culture medium may be determined by measuring a glucose concentration in the culture medium and supplementing the culture medium with glucose as needed. For example, if the glucose concentration is below a desired threshold (e.g., 2 g / L), then a higher concentration of glucose can be added, or more frequent administration (e.g., every other day to daily administration) of glucose can be performed. Alternatively, if the glucose concentration is above a desired threshold (e.g., 2 g / L), a lower concentration of glucose can be added, or less frequent administration (e.g., daily to every other day) of glucose can be performed. In other embodiments, the glucose concentration may be within a desired range, and the amount or frequency of glucose addition may remain the same. If the glucose concentration is less than or equal to 2 g / L in the bioreactor, glucose may be added daily. If the glucose concentration is more than 2 g / L, glucose may be added every other day. In one example, the glucose concentration in the culture medium is less than or equal to 2 g / L, and glucose is added to the culture medium at a concentration of about 2 g / L at about day 4, day 6, day 8, and / or day 10 after inoculation, and at a concentration of about 4 g / L at about day 1 , day 2, day 3, day 5, day 7, day 9, day 11 , day 12, and / or day 13 after inoculation.

[0076] The alkaline phosphatase protein described herein (e.g., a polypeptide of SEQ ID NO: 2 or 3) may be produced by mammalian or other cells, particularly CHO cells, using methods known in the art and as modified by the methods described herein. Such cells may be grown in culture dishes, flask glasses, or bioreactors. Specific processes for cell culture and producing recombinant proteins are known in the art, such as described in Nelson and Geyer, 1991 Bioprocess Technol. 13:112-143 and Rea et al., Supplement to BioPharm International March 2008, 20-25. Exemplary bioreactors include batch, fed-batch, and continuous reactors. In some embodiments, the alkaline phosphatase protein is produced in a fed-batch bioreactor.

[0077] Cell culture processes have variability caused by, for example, changes in the physicochemical environment, including but not limited to, changes in pH, temperature (e.g., the timing of temperature changes), cell culture media composition (e.g., changes in cell culture nutrient supplements), raw material lotto-lot variation, medium filtration material, bioreactor scale difference, gassing strategy (air, oxygen, and carbon dioxide), and the like. The cells may be cultured at a dissolved oxygen (DO) concentration of 10% to 60%. As disclosed herein, the yield, relative activity profile, and glycosylation profile of manufactured alkaline phosphatase protein may be affected and may be controlled within particular values by alterations in one or more of these parameters.

[0078] For recombinant protein production in cell culture, the recombinant gene with the necessary transcriptional regulatory elements is first transferred to a host cell by methods known in the biotechnological arts. Optionally, a second gene is transferred that confers to recipient cells a selective advantage. In the presence of the selection agent, which may be applied a few days after gene transfer, only those cells that PATENT ALEXION REF.: ALXN-0667-PCT01-NP express the selector gene survive. Two exemplary genes for such selection are dihydrofolate reductase (DHFR), an enzyme involved in nucleotide metabolism, and glutamine synthetase (GS). In both cases, selection occurs in the absence of the appropriate metabolite (hypoxanthine and thymidine, in the case of DHFR, and glutamine in the case of GS), preventing growth of any non-transformed cells. In general, for efficient expression of the recombinant protein, it is not important whether the biopharmaceutical-encoding gene and selector genes are on the same plasmid or not.

[0079] Following selection, surviving cells may be transferred as single cells to a second cultivation vessel, and the cultures are expanded to produce clonal populations. Eventually, individual clones are evaluated for recombinant protein expression, with the highest producers being retained for further cultivation and analysis. From these candidates, one cell line with the appropriate growth and productivity characteristics is chosen for production of the recombinant protein. A cultivation process is then developed that is determined by the production needs and the requirements of the final product.

[0080] Cells

[0081] Any mammalian cell or non-mammalian cell type, which can be cultured to produce a polypeptide, may be utilized in accordance with the present disclosure. Non-limiting examples of mammalian cells that may be used include, e.g., Chinese hamster ovary cells + / -DHFR (CHO, Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA, 77:4216); BALB / c mouse myeloma line (NSO / 1 , ECACC Accession No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., 1977 J. Gen Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-I 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., 1982, Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In a particular embodiment, culturing and expression of polypeptides and proteins occurs from a Chinese Hamster Ovary (CHO) cell line.

[0082] Additionally, any number of commercially and non-commercially available hybridoma cell lines that express polypeptides or proteins may be utilized in accordance with the present disclosure. One skilled in the art will appreciate that hybridoma cell lines might have different nutrition requirements and / or might require different culture conditions for optimal growth and polypeptide or protein expression and will be able to modify conditions as needed.

[0083] Seeding

[0084] In the present disclosure, the cells, e.g., Chinese Hamster Ovary (CHO) cells, are inoculated, e.g., seeded, into the culture medium of a bioreactor. Various seeding densities can be used. In some embodiments, a seeding density of about 1 x 105viable cells / mL to about 1 x 107viable cells / mL, e.g., 0.3 x PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0085] 106viable cells / mL to about 1 x 107viable cells / mL, e.g., 0.3 x 106viable cells / mL to about 0.7 x 106viable cells / mL (e.g., about 0.3 x 106viable cells / mL, 0.4 x 106viable cells / mL, 0.5 x 106viable cells / mL, 0.6 x 106viable cells / mL, or 0.7 x 106viable cells / mL).

[0086] Increased seeding density can impact fragmentation of alkaline phosphatase quality, e.g., as measured by SEC. The seeding density may be controlled when inoculating in order to reduce the risk of fragment generation. The seeding process may employ a seed train (e.g., a seed culture) to produce a suitable number of cells before inoculating the bioreactor.

[0087] The culture medium may be the same as the culture medium in the bioreactor during production of the seed culture. Alternatively, the culture medium may be different from the medium in the bioreactor or contain a different supplement. In one embodiment, the culture medium for producing the seed may include methionine sulfoximine (MSX). The seed culture medium may include, for example, from 5 pM to 50 pM MSX (e.g., about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM MSX, e.g., about 25 pM MSX).

[0088] Temperature

[0089] Temperature may have an impact on several parameters including growth rate, aggregation, fragmentation, and TSAC. The temperature may remain constant when culturing the cells, e.g., CHO cells, in the culture medium. The temperature may be from about 30 °C to about 40 °C, about 35 °C to about 40 °C, or about 37 °C to about 39 °C when culturing the cells, e.g., CHO cells, in the culture medium. The temperature may be about 30 °C, about 30.5 °C, about 31 °C, about 31 .5 °C, about 32 °C, about 32.5 °C, about 33 °C, about 33.5 °C, about 34 °C, about 34.5 °C, about 35 °C, about 35.5 °C, about 36 °C, about 36.5 °C, about 37 °C, about 37.5 °C, about 38 °C, about 38.5 °C, about 39 °C, about 39.5 °C, or about 40 °C when culturing the cells, e.g., CHO cells, in the culture medium. The temperature may be constant for 1 day to 10 days after inoculation. The temperature may be constant for 1 day to 9 days, 2 days to 8 days, or 3 days to 7 days after inoculation. The temperature may be constant for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after inoculation.

[0090] Temperature shifting

[0091] Run times of cell culture processes, especially non-continuous processes (e.g., fed-batch processes in bioreactors), may be limited by the remaining viability of the cells, which typically decline over the course of the run. Therefore, extending the length of time for cell viability is desired for improving recombinant protein production. Product quality concerns also offer a motivation for minimizing decreases in viable cell density and maintaining high cell viability, as cell death can release sialidases into the culture supernatant, which may reduce the sialic acid content of the protein expressed (e.g., by cleaving sialic acid moieties off the recombinantly produced protein in the culture medium). Protein purification concerns offer yet another motivation for minimizing decreases in viable cell density and maintaining high cell viability. Cell debris and the contents of dead cells in the culture can negatively impact the ability to isolate and / or purify the protein product at the end of the culturing run. Thus, by keeping cells viable for a longer period of time in culture, PATENT ALEXION REF.: ALXN-0667-PCT01-NP there is a reduction in the contamination of the culture medium by cellular proteins and enzymes (e.g., cellular proteases and sialidases) that may cause degradation and which could ultimately reduce the quality of the desired glycoprotein produced by the cells.

[0092] Temperature shifting may employ culturing the bioreactor at a first temperature until a desired cell density is reached and shifting to a second temperature, e.g., that is lower than the first temperature, e.g., to promote protein production. In one particular embodiment, the temperature for producing the recombinant alkaline phosphatase is first set at about 35 °C to about 38 °C (e.g., 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C,

[0093] 37.5 °C, or 38 °C, e.g., about 36.5 °C). When a reasonably high cell density is reached, the culturing temperature for the whole cell culture can then be shifted (e.g., decreased) to promote protein production. In most cases lowering temperature shifts the cells towards the non-growth G1 portion of the cell cycle, which may increase cell density and viability, as compared to the previous higher-temperature environment. In addition, a lower temperature may also promote recombinant protein production by increasing the cellular protein production rate, facilitating protein post-translational modification (e.g., glycosylation), decreasing fragmentation or aggregation of newly produced proteins, facilitating protein folding and formation of three- dimensional structure (thus maintaining activity), and / or decreasing degradation of newly produced proteins. The temperature may be decreased by about 0.5 °C, 1 °C, 1.5 °C, 2 °C, 2. 5 °C, 3 °C, 3.5 °C, 4 °C, 4.5 °C,

[0094] 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, or 10 °C during the temperature shift. For example, the temperature may be decreased to about 29 °C to about 36 °C (e.g., 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C, 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, or 36 °C e.g., about 34 °C to about 36 °C, e.g., about 35 °C) during the temperature shift.

[0095] The methods described herein may include culturing the cells at a first temperature until reaching a desired cell density and shifting to a second temperature that is lower than the first temperature to express the recombinant alkaline phosphatase. The first temperature may be about 35 °C to about 38 °C (e.g., 35 °C,

[0096] 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, or 38 °C, e.g., about 36.5 °C). The second temperature may be about 29 °C to about 36 °C (e.g., 29 °C, 29.5 °C, 30 °C, 30.5 °C, 31 °C, 31 .5 °C, 32 °C, 32.5 °C, 33 °C,

[0097] 33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, or 36 °C, e.g., about 34 °C to about 36 °C, e.g., about 35 °C).

[0098] The methods described herein may include culturing the cells at a first temperature until reaching a cell density of at least about 5 x 106viable cells / mL and shifting to a second temperature which is lower than the first temperature to express the recombinant alkaline phosphatase. The cell density reached before the temperature shift may be about 5 x 106viable cells / mL to about 25 x 106viable cells / mL (e.g., about 5 x 106viable cells / mL, about 6 x 106viable cells / mL, about 7 x 106viable cells / mL, about 8 x 106viable cells / mL, about 9 x 106viable cells / mL, about 10 x 106viable cells / mL, about 11 x 105viable cells / mL, about 12 x 106viable cells / mL, about 13 x 106viable cells / mL, about 14 x 106viable cells / mL, about 15 x 106viable cells / mL, about 16 x 106viable cells / mL, about 17 x 106viable cells / mL, about 18 x 106viable cells / mL, about 19 x 106viable cells / mL, about 20 x 106viable cells / mL, about 21 x 106viable cells / mL, about 22 x 106viable cells / mL, about 23 x 106viable cells / mL, about 24 x 106viable cells / mL, about 25 x 106viable cells / mL, about 26 x 106viable cells / mL, about 27 x 106viable cells / mL, about 28 x 106viable cells / mL, about 29 x 106viable cells / mL, PATENT ALEXION REF.: ALXN-0667-PCT01-NP or about 30 x 106viable cells / mL, e.g., about 6 x 106viable cells / mL to about 10 x 106viable cells / mL or about 15 x 106viable cells / mL to about 20 x 106viable cells / mL).

[0099] The temperature shift may be determined based on the cell density observed at inoculation. For example, the temperature shift may be performed from 1 day to 14 days after inoculation. The temperature shift may be performed from 2 days to 14 days, 2 days to 13 days, 2 days to 12 days, 3 days to 10 days, 3 days to 8 days after inoculation. The temperature shift may be performed about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after inoculation. The temperature shift may be performed on or about day 7 after the inoculating step. In other embodiments, multiple (e.g., more than one) steps of temperature shifting may be applied. The temperature after the temperature shift may be maintained until the cells, e.g., CHO cells, are harvested.

[0100] Culture Medium

[0101] Any suitable commercially available culture medium may be used in conjunction with the methods described herein. In some embodiments, fed batch is used, wherein one or more boluses of culture medium are added after inoculation. For example, two, three, four, five or six boluses of culture medium may be added after inoculation.

[0102] In various embodiments, alkaline phosphatase (e.g., a polypeptide of SEQ ID NO: 2 or 3) is produced by a process in which one or more boluses of culture medium are added to the production bioreactor. For example, one, two, three, four, five, six, seven, eight, nine, ten, or more boluses of culture medium may be added. In various embodiments, such extra boluses of culture medium may be added in various amounts. For example, such boluses of culture medium may be added in an amount of about 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 110%, 120%, 125%, 130%, 133%, 140%, 150%, 160%, 167%, 170%, 175%, 180%, 190%, 200%, or more, of the original volume of culture medium in the production bioreactor. Such addition of extra boluses may occur at various times during the cell growth or protein production period. For example, boluses may be added at day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, day 13, day 14, or later in the process. In one particular embodiment, such boluses of culture medium may be added in every other day (e.g., at day 2, day 4, day 6, day 8, day 10, day 12, and / or day 14; or at day 1 , day 3, day 5, day 7, day 9, day 11 , and / or day 13). In practice, the frequency, amount, time point, and other parameters of bolus supplements of culture medium may be combined freely according to the above limitation and determined by experimental practice.

[0103] Various culture mediums are available commercially. The culture medium may be selected from the group consisting of EX-CELL® 302 Serum-Free Medium; CD DG44 Medium; BD SELECT™ Medium; SFM4CHO Medium, Chinese hamster ovary advanced granulation technology (CD CHO AGT™) medium or a combination thereof. The culture medium may include a combination of commercially available mediums, e.g., SFM4CHO Medium and BD SELECT™ Medium. The culture medium may include a combination of commercially available mediums, e.g., SFM4CHO Medium and BD SELECT™ Medium, at a ratio selected from 90 / 10, 80 / 20, 75 / 25, 70 / 30, 60 / 40, or 50 / 50. PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0104] The culture medium may include or be composed of a suitable basal medium, such as a chemically defined Chinese hamster ovary advanced granulation technology (CD CHO AGT™) medium. The culture medium may include CD CHO AGT™ medium at a concentration of 10 g / L to 100 g / L (e.g., about 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, or 100 g / L). In some embodiments, the culture medium includes CD CHO AGT™ medium at a concentration of about 24.1 g / L. The culture medium may further include from 5 pM to 50 pM MSX (e.g., about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM MSX, e.g., about 25 pM MSX).

[0105] The culture medium may further include supplemental metals or nonmetals, such as one or more of copper, zinc, magnesium, and chloride. For example, the culture medium may include from 5 pM to 50 pM copper (e.g., about 5 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, or 50 pM copper, e.g., about 20 pM copper).

[0106] The culture medium may include 5 mg / L to 100 mg / L zinc sulfate heptahydrate (e.g., 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L zinc sulfate heptahydrate), 100 mg / L to 200 mg / L magnesium chloride hexahydrate (e.g., 100 mg / L, 105 mg / L, 110 mg / L, 115 mg / L, 120 mg / L, 125 mg / L, 130 mg / L, 135 mg / L, 140 mg / L, 145 mg / L, 150 mg / L, 155 mg / L, 160 mg / L, 165 mg / L, 170 mg / L, 175 mg / L, 180 mg / L, 185 mg / L, 190 mg / L, 195 mg / L, or 200 mg / L magnesium chloride hexahydrate), and / or 5 mg / L to 100 mg / L calcium chloride dihydrate (e.g., 5 mg / L, 10 mg / L, 15 mg / L, 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L, 45 mg / L, 50 mg / L, 55 mg / L, 60 mg / L, 65 mg / L, 70 mg / L, 75 mg / L, 80 mg / L, 85 mg / L, 90 mg / L, 95 mg / L, or 100 mg / L calcium chloride dihydrate). The culture medium may include about 43 mg / L zinc sulfate heptahydrate, about 148 mg / L magnesium chloride hexahydrate, and about 55 mg / L calcium chloride di hydrate.

[0107] The culture medium may further include or be supplemented with cysteine and / or tyrosine. For example, in specific embodiments, the basal medium is supplemented with 0.1 g / L to 0.5 g / L L-cysteine hydrochloride monohydrate (e.g., 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L L-cysteine hydrochloride monohydrate) and / or 0.1 g / L to 0.5 g / L L-tyrosine (e.g., 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.4 g / L, or 0.5 g / L L-tyrosine). The basal medium in specific embodiments is supplemented with about 0.342 g / L L-cysteine hydrochloride monohydrate and about 0.269 g / L L-tyrosine.

[0108] Nutrient Supplements

[0109] Various nutrient supplements are commercially available and are known to those of skill in the art. Nutrient supplements include a media (distinct from the culture media) added to a cell culture after inoculation has occurred. In some instances, the nutrient supplement can be used to replace nutrients consumed by the growing cells in the culture. The nutrient supplement may be added to optimize production of a desired protein, or to optimize activity of a desired protein. Numerous nutrient supplements have been developed and are available commercially. While the expressed purpose of the nutrient supplements is to increase an aspect of process development, no universal nutrient supplement exists that works for all cells and / or all proteins produced. The selection of a scalable and appropriate cell culture nutrient supplement that can work PATENT ALEXION REF.: ALXN-0667-PCT01-NP in combination with the desired cell line, protein produced, and a given base medium to achieve the desired titer and growth characteristics is not routine. The typical approach of screening multiple commercially available nutrient supplements and identifying the most appropriate supplement with a specific cell line, specific protein produced, and base medium combination may not be successful due to the myriad of variables present in the cell culture process. The nutrient supplement may be selected from the group consisting of Efficient Feed C+ AGT™ Supplement (Thermo Fisher Scientific, Waltham, MA), a combination of CELL BOOST™ 2 + CELL BOOST™ 4 (GE Healthcare, Sweden), a combination of CELL BOOST™ 2 + CELL BOOST™ 5 (GE Healthcare, Sweden), CELL BOOST™ 6 (GE Healthcare, Sweden), and CELL BOOST™ 7a + CELL BOOST™ 7b (GE Healthcare, Sweden), CHO feed bioreactor supplement (Sigma- Aldrich; e.g., product catalog no. C1615), GLYCAN TUNE™ C+ (GTC+) feed, or combinations thereof.

[0110] CELL BOOST™ 7a can be described as a first animal-derived component-free (ADCF) nutrient supplement containing one or more amino acids, vitamins, salts, trace elements, poloxamer and glucose, wherein the first ADCF nutrient supplement does not include hypoxanthine, thymidine, insulin, L-glutamine, growth factors, peptides, proteins, hydrolysates, phenol red and 2-mercaptoethanol. CELL BOOST™ 7a is a chemically defined supplement. The phrase “animal-derived component-free” or “ADCF” refers to a supplement in which no ingredients are derived directly from an animal source, e.g., are not derived from a bovine source. The nutrient supplement may be CELL BOOST™ 7a.

[0111] CELL BOOST™ 7b can be described as a second ADCF nutrient supplement containing one or more amino acids, wherein the second ADCF nutrient supplement lacks hypoxanthine, thymidine, insulin, L- glutamine, growth factors, peptides, proteins, hydrolysates, phenol red, 2-mercaptoethanol and poloxamer. CELL BOOST™ 7b is a chemically defined supplement. The nutrient supplement is CELL BOOST™ 7b.

[0112] The methods described herein may include using combinations of commercially available nutrient supplements. The term “nutrient supplement” refers to both a single nutrient supplement, as well as combinations of nutrient supplements. For example, a combination of nutrient supplements may include a combination of CELL BOOST™ 7a and CELL BOOST™ 7b.

[0113] In various embodiments, an alkaline phosphatase (e.g., an alkaline phosphatase having the sequence of SEQ ID NO: 2 or 3, or a variant thereof, as described herein) is produced by a process in which extra additions of nutrient supplement are added to the production bioreactor. The nutrient supplement may be added over a period of time, e.g., over a period of time ranging from, e.g., 1 minute to 2 hours. In some embodiments, the nutrient supplement is added in a bolus. For example, one, two, three, four, five, six, or more boluses of nutrient supplement may be added. The nutrient supplement may be added at more than 2 different times, e.g., 2 to 6 different times. In various embodiments, such extra boluses of nutrient supplement may be added in various amounts. For example, such boluses of nutrient supplement may be added in an amount of about 1% to 20%, 1% to 10% or 1 % to 5% (w / v) of the original volume of culture medium in the production bioreactor. In one particular embodiment, such boluses of nutrient supplement may be added in an amount of 1 % to 20%, 1% to 10% or 1 % to 5% (w / v) of the original volume.

[0114] A combination of nutrient supplements may be used, and the first nutrient supplement, e.g., CELL BOOST™ 7a, is added at a concentration of 0.5% to 4% (w / v) of the culture medium. In some embodiments, PATENT ALEXION REF.: ALXN-0667-PCT01-NP a combination of nutrient supplements is used, and the second nutrient supplement, e.g., CELL BOOST™ 7b, is added at a concentration of 0.05% to 0.8% (w / v) of the culture medium. In specific embodiments, such as the use of a combination of nutrient supplements that include CELL BOOST™ 7a and CELL BOOST™ 7b, boluses of nutrient supplement may be added in an amount of 1 % to 20%, 1% to 10%, or 1 % to 5% (w / v) of the original volume.

[0115] In various embodiments, such addition of extra boluses may occur at various times after inoculation. For example, boluses may be added at day 1 , day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11 , day 12, or later after inoculation. In practice, the frequency, amount, time point, and other parameters of bolus supplements of nutrient supplement may be combined freely according to the above limitation and determined by experimental practice.

[0116] The methods described herein may further include adding zinc into said culture medium during production of the recombinant polypeptide. Zinc may be added to provide a zinc concentration of from about 1 to about 300 pM in said culture medium. In one embodiment, zinc may be added to provide a zinc concentration of from about 10 to about 200 pM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 pM zinc) in the culture medium. For example, zinc may be added to provide a zinc concentration in the culture medium of from about 25 pM to about 150 pM or from about 60 pM to about 150 pM. In one embodiment, zinc is added to provide a zinc concentration in the culture medium of from about 30, 60, or 90 pM. The zinc may be added into the culture medium in a bolus, continuously, semi-continuously, or any combination thereof. The zinc may be added one day, two days, three days, four days, five days, six days, seven days, eight days, nine days, ten days, eleven days, twelve days, and / or thirteen days after inoculation.

[0117] The culture medium may include or be supplemented with GLYCAN TUNE™ C+ (GTC+) feed. For example, the methods described herein may include adding GTC+ feed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after inoculation. The GTC+ feed may be added at a concentration of 100 g / L to 200 g / L (e.g., 100 g / L, 125 g / L, 150 g / L, 175 g / L, or 200 g / L). The GTC+ feed may be added at a concentration of 175.4 g / L. The GTC+ feed may be added at a concentration of 1 % (v / v) to 10% (v / v) (e.g., 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (v / v)) of the initial culture volume. The GTC+ feed may be added at a concentration of 5% (v / v) of the initial culture volume. In some embodiments, the GTC+ feed is added 3 or 4 equal boluses at a concentration of 5% (v / v) of the initial culture volume.

[0118] Harvest

[0119] The methods described herein may include harvesting the cells or the proteins produced by the cells. Harvesting the cells can be performed by any suitable method known in the art. The harvesting may be performed by filtration (e.g., depth filtration). The harvesting may be performed by centrifugation.

[0120] The point at which the cells are harvested may depend on the number of viable cells in the bioreactor to prevent deterioration of the cells in the culture. Harvesting the cells may occur at a density of about 10 x 106viable cells / mL to about 30 x 106viable cells / mL (e.g., 10 x 106viable cells / mL, 11 x 106viable cells / mL, 12 x 106viable cells / mL, 13 x 106viable cells / mL, 14 x 106viable cells / mL, 15 x 106viable cells / mL, 16 x 106viable cells / mL, 17 x 106viable cells / mL, 18 x 106viable cells / mL, 19 x 106viable cells / mL, 20 x 106viable PATENT ALEXION REF.: ALXN-0667-PCT01-NP cells / mL, 21 x 106viable cells / mL, 22 x 106viable cells / mL, 23 x 106viable cells / mL, 24 x 106viable cells / mL, 25 x 106viable cells / mL, 26 x 106viable cells / mL, 27 x 106viable cells / mL, 28 x 106viable cells / mL, 29 x 106viable cells / mL, or 30 x 106viable cells / mL). The harvesting may be performed on or about day 10, 11 , 12, 13, 14, or later (e.g., day 14) after inoculation. The harvesting may be performed within 24 hours of the cell viability being less than or equal to 75% of the total cells, or of the cell viability being less than or equal to 80% of the total cells, or of the cell viability being between about 75% to about 80% of the total cells.

[0121] Determination of Glycosylation Content

[0122] The methods described herein may further include measuring the total sialic acid, mannose, and fucose content of the recombinant alkaline phosphatase produced by the methods herein. The measurement may be obtained from an aliquot that is removed from the culture medium. The aliquot may be obtained aseptically from the bioreactor in order to prevent contamination, e.g., to monitor glycosylation during culturing. Alternatively, or in addition, the aliquot may be obtained following culturing, e.g., during or immediately prior to, or following, the harvesting step. The aliquot may be from about 1 ml_ to about 1000 mL (e.g., from about 25 mL to about 500 ml_, e.g., from about 50 mL to about 300 ml_, e.g., about 100 ml_ or about 200 mL). Obtaining the aliquot may further include centrifuging the aliquot and / or removing the supernatant from the aliquot. This step may also include purifying the alkaline phosphatase from the supernatant using a chromatography column (e.g., a Protein A column, 1 cm Protein A column, e.g., a 1 mL HiTrap Protein A column or 600 pL Protein A Robocolumn). The alkaline phosphatase may be subject to a buffer exchange. The alkaline phosphatase may also be concentrated, e.g., before determining the total sialic acid, mannose, and / or fucose content.

[0123] Commercial methods of carbohydrate quantification are available, e.g., from ThermoFisher. Generally, sialic acid, mannose, and fucose are released from a glycoprotein, e.g., a polypeptide of SEQ ID NO: 2 or 3, using acid hydrolysis, and released sugars (e.g., sialic acid, mannose, and fucose) may be detected via electrochemical detection using column chromatography such as High-Performance Anion- Exchange Chromatography with Pulsed Amperometric Detection technique (HPAE-PAD). The resulting levels can be quantified per mole against an internal standard and expressed as a function of the total mol protein.

[0124] As described herein, the total sialic acid, mannose, and / or fucose content may impact the half-life or quality control of the recombinant alkaline phosphatase in physiological conditions, and thus serves as a critical quality attribute for recombinantly-produced alkaline phosphatases such as, e.g., a polypeptide of SEQ ID NO: 2 or 3. Tight control of the glycosylation content range is important for reproducibility and current good manufacturing practice (cGMP).

[0125] A recombinant alkaline phosphatase with the amino acid sequence of SEQ ID NO: 2 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 2) may include a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer (e.g., at least 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). The recombinant alkaline PATENT ALEXION REF.: ALXN-0667-PCT01-NP phosphatase may include a total fucose content of at least 2.0 mol fucose / mol protein monomer. The recombinant alkaline phosphatase may include a total mannose content of at least 6.0 mol mannose / mol protein monomer.

[0126] A recombinant alkaline phosphatase with the amino acid sequence of SEQ ID NO: 3 (or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 3) may include a total sialic acid content (TSAC) of at least 1 .2 mol sialic acid / mol protein monomer (e.g., at least 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). The recombinant alkaline phosphatase may include a total fucose content of at least 0.6 mol fucose / mol protein monomer. The recombinant alkaline phosphatase may include a total mannose content of at least 1.8 mol mannose / mol protein monomer.

[0127] In some embodiments, the TSAC is about 0.8 mol / mol to about 4.0 mol / mol recombinant alkaline phosphatase. For example, the TSAC may be about 0.9 mol / mol to about 3.0 mol / mol recombinant alkaline phosphatase. The TSAC may be about 1 .0 mol / mol to about 2.8 mol / mol recombinant alkaline phosphatase. The TSAC may be about 1.2 mol / mol to about 3.0 mol / mol recombinant alkaline phosphatase. The TSAC may be about 1 .2 mol / mol to about 2.4 mol / mol recombinant alkaline phosphatase. The TSAC may be about 0.9 mol / mol, about 1 .0 mol / mol, about 1 .1 mol / mol, about 1 .2 mol / mol, about 1 .3 mol / mol, about 1 .4 mol / mol, about 1 .5 mol / mol, about 1 .6 mol / mol, about 1 .7 mol / mol, about 1 .8 mol / mol, about 1 .9 mol / mol, about 2.0 mol / mol, about 2.1 mol / mol, about 2.2 mol / mol, about 2.3 mol / mol, about 2.4 mol / mol, about 2.5 mol / mol, about 2.6 mol / mol, about 2.7 mol / mol, about 2.8 mol / mol, about 2.9 mol / mol, or about 3.0 mol / mol recombinant alkaline phosphatase.

[0128] Recombinant Alkaline Phosphatases

[0129] The present disclosure relates to the manufacturing of an alkaline phosphatase protein (e.g., an alkaline phosphatase with the amino acid sequence of SEQ ID NO: 2 or 3, or a variant thereof with at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 2 or 3) in a recombinant cell culture. The alkaline phosphatase can be any polypeptide with alkaline phosphatase activity. For example, the alkaline phosphatase may have enzymatic activity toward natural alkaline phosphatase substrates, such as phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP).

[0130] In certain embodiments, such alkaline phosphatase protein, after being produced and then purified by the methods disclosed herein, can be used to treat or prevent alkaline phosphatase-related diseases or disorders. For example, such an alkaline phosphatase protein may be administered to a subject with a decreased level of endogenous alkaline phosphatase and / or with a dysfunctional endogenous alkaline phosphatase, or with an overabundance of (e.g., above normal levels) alkaline phosphatase substrates. The alkaline phosphatase may be a fusion protein. The alkaline phosphatase may specifically target a cell type, tissue (e.g., connective, muscle, nervous, or epithelial tissues), or organ (e.g., liver, heart, kidney, muscles, bones, cartilage, ligaments, tendons, etc.). For example, such an alkaline phosphatase may include a full- PATENT ALEXION REF.: ALXN-0667-PCT01-NP length alkaline phosphatase (ALP) or an enzymatically-active fragment of at least one alkaline phosphatase (ALP). The alkaline phosphatase may be a soluble ALP (sALP) linked to a bone-targeting moiety (e.g., a negatively-charged peptide, such as a polyaspartate or polyglutamate). The alkaline phosphatase may include a soluble ALP (sALP) linked to an immunoglobulin moiety (full-length or a fragment thereof). For example, such an immunoglobulin moiety may include a fragment crystallizable region (Fc). In some embodiments, the alkaline phosphatase includes a soluble ALP (sALP) linked to both a bone-targeting moiety (e.g., a negatively-charged peptide, such as a polyaspartate or polyglutamate) and an immunoglobulin moiety (full-length or fragment, such as a fragment crystallizable region (Fc)). For a more detailed description of the exemplary alkaline phosphatases, see e.g., PCT Publication Nos. WO 2005 / 103263, WO 2008 / 138131 , and WO 2021 / 119218, the teachings of which are incorporated by reference herein in their entirety.

[0131] The recombinant alkaline phosphatase proteins described herein may include the structure of W- sALP-X-Fc-Y-Dn-Z, in which W is absent or is an amino acid sequence of at least one amino acid; X is absent or is an amino acid sequence of at least one amino acid; Y is absent or is an amino acid sequence of at least one amino acid; Z is absent or is an amino acid sequence of at least one amino acid; Fc is a fragment crystallizable region; Dn is a poly-aspartate, poly-glutamate, or combination thereof, in which n = 5 to 16 (e.g., 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, or 16, such as n = 7 or n = 10); and sALP is a soluble alkaline phosphatase.

[0132] The fragment crystallizable (Fc) may be any Fc of an immunoglobulin molecule known in the art. The Fc may include a CH2 domain, a CH3 domain, and a hinge region. The Fc may be a constant domain of an immunoglobulin selected from the group consisting of lgG-1 , lgG-2, lgG-3, lgG-3 and lgG-4 or a chimera thereof (e.g., lgG-2 / 4 chimera). In one embodiment, the Fc is a constant domain of an immunoglobulin IgG- 1 . In one example, the Fc includes the sequence as set forth in D488-K714 of SEQ ID NO: 1 . In another embodiment, the Fc is an lgG-2 / 4 chimera. For example, the Fc domain may include the sequence as set forth in V492-K714 of SEQ ID NO: 2 or 3.

[0133] In one embodiment, the alkaline phosphatase includes the structure of TNALP-Fc-D7.

[0134] In another embodiment, the alkaline phosphatase includes the structure of TNALP-Fc-D10. Asparagine (N) residues (e.g., N123, N213, N254, N286, N413, and N564) correspond to potential glycosylation sites (relative to the amino acid sequences of one or more of SEQ ID NOs: 1-3). Amino acid residues (L486-K487 & D715-I716) correspond to linkers between sALP and Fc, and Fc and D10 domains, respectively.

[0135] The alkaline phosphatase polypeptide may be composed of three domains, optionally connected via a linker between the first and second domains and / or the second and third domains, respectively. In asfotase alfa, the polypeptide of SEQ ID NO: 1 , the first domain, the sALP, corresponds to amino acids L1-S485, which is the soluble part of the human tissue non-specific alkaline phosphatase enzyme; this domain contains the catalytic function. The polypeptide further contains a linker corresponding to amino acids L486-K487. The second domain, the Fc region, corresponds to amino acids D488-K714, which is the Fc of a human immunoglobulin gamma 1 (IgG 1 ) containing the hinge, CH2, and CH3 domains. The polypeptide further contains a linker corresponding to D715-I716. The fourth domain, which corresponds to amino acids D717- PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0136] D726 (D10), is a bone targeting moiety that allows the alkaline phosphatase to bind to the mineral phase of bone. In addition, each polypeptide chain contains six potential glycosylation sites and eleven cysteine (C) residues. C102 exists as free cysteine. Each polypeptide chain contains four intra-chain disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. The two polypeptide chains are connected by two inter-chain disulfide bonds between C493 on both chains and between C496 on both chains. In addition to these covalent structural features, mammalian alkaline phosphatases are thought to have four metal-binding sites on each polypeptide chain, including two sites for zinc, one site for magnesium and one site for calcium.

[0137] Asfotase alfa (SEQ ID NO: 1) can also be characterized as follows. From the N-terminus to the C terminus, asfotase alfa includes: (1) the soluble catalytic domain of human tissue non-specific alkaline phosphatase (TNSALP) (UniProtKB / Swiss-Prot Accession No. P05186), (2) the human immunoglobulin G1 Fc domain (UniProtKB / Swiss-Prot Accession No. P01857) and (3) a deca-aspartate peptide (D10) used as a bone-targeting domain (Nishioka et al. 2006 Mol Genet Metab 88:244-255). The protein associates into a homo-dimer from two primary protein sequences. This fusion protein contains 6 confirmed complex N- glycosylation sites. Five of these N-glycosylation sites are located on the sALP domain and one on the Fc domain. Another important post-translational modification present on asfotase alfa is the presence of disulfide bridges stabilizing the enzyme and the Fc-domain structure. A total of 4 intra-molecular disulfide bridges are present per monomer and 2 inter-molecular disulfide bridges are present in the dimer. One cysteine of the alkaline phosphatase domain is free.

[0138] Asfotase alfa has been used as an enzyme-replacement therapy for the treatment of hypophosphatasia (HPP). In patients with HPP, loss-of-function mutation(s) in the gene encoding TNSALP causes a deficiency in TNSALP enzymatic activity, which leads to elevated circulating levels of substrates, such as inorganic pyrophosphate (PPi) and pyridoxal-5’-phosphate (PLP). Administration of asfotase alfa to patients with HPP cleaves PPi, releasing inorganic phosphate for combination with calcium, thereby promoting hydroxyapatite crystal formation and bone mineralization, and restoring a normal skeletal phenotype. For more details on asfotase alfa and its uses in treatment, see PCT Publication Nos. WO 2005 / 103263 and WO 2008 / 138131

[0139] The methods described herein also provide an alkaline phosphatase (a polypeptide of SEQ ID NO: 2 or 3) having improved enzymatic activity of the produced alkaline phosphatase (e.g., a polypeptide of SEQ ID NO: 2 or 3) relative to an alkaline phosphatase produced by conventional methods, by minimizing the concentration of metal ions having potential negative impact on activity or increasing the concentration of metal ions having potential positive impact on activity or both as described herein. Activity may be measured by any known method. Such methods include, e.g., those in vitro and in vivo assays measuring the enzymatic activity of the produced alkaline phosphatase (e.g., a polypeptide of SEQ ID NO: 2 or 3) to substrates of an alkaline phosphatase, such as phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP).

[0140] The alkaline phosphatase disclosed herein may be encoded by a first polynucleotide which hybridizes under high stringency conditions to a second polynucleotide comparing the sequence completely PATENT ALEXION REF.: ALXN-0667-PCT01-NP complementary to a third polynucleotide encoding a polypeptide having the sequence as set forth in SEQ ID NO: 1 . Such high stringency conditions may include: pre-hybridization and hybridization in 6 x SSC, 5 x Denhardt's reagent, 0.5% SDS and 100 mg / ml of denatured fragmented salmon sperm DNA at 68°C; and washes in 2 x SSC and 0.5% SDS at room temperature for 10 minutes; in 2 x SSC and 0.1 % SDS at room temperature for 10 minutes; and in 0.1 x SSC and 0.5% SDS at 65°C three times for 5 minutes.

[0141] Variants of asfotase alfa (SEQ ID NO: 1) have been developed to optimize the structure, function, and therapeutic activity of the polypeptide. These variants include, for example, the polypeptides of SEQ ID NOs: 2 and 3. The alkaline phosphatase polypeptide (e.g., of SEQ ID NO: 2; variant A) may be composed of three domains, optionally connected via a linker between the first and second domains and / or the second and third domains, respectively. The first domain, the sALP, corresponds to amino acids L1-S485, which is the soluble part of the human tissue non-specific alkaline phosphatase enzyme and containing E108M, N213Q, and N286Q mutation; this domain contains the catalytic function. The polypeptide may include a linker containing amino acids A486-G487. The second domain, the Fc region, corresponds to amino acids S488- K714, which is a chimera Fc of a human immunoglobulin gamma 2 (lgG2) and gamma 4 (lgG4), containing the hinge, CH2, and CH3 domains. The third domain, corresponds to amino acids D715-D724 (D10), is a bone targeting moiety that allows the alkaline phosphatase of, e.g., SEQ ID NO: 2, to bind to the mineral phase of bone. In addition, each polypeptide chain contains six potential glycosylation sites and eleven cysteine (C) residues. C102 exists as free cysteine. Each polypeptide chain contains four intra-chain disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. The two polypeptide chains are connected by two inter-chain disulfide bonds between C494 on both chains and between C494 on both chains. In addition to these covalent structural features, mammalian alkaline phosphatases are thought to have four metal-binding sites on each polypeptide chain, including two sites for zinc, one site for magnesium and one site for calcium.

[0142] The alkaline phosphatase disclosed herein may be encoded by a first polynucleotide which hybridizes under high stringency conditions to a second polynucleotide comparing the sequence completely complementary to a third polynucleotide encoding a polypeptide having the sequence as set forth in SEQ ID NO: 2. Such high stringency conditions may include: pre-hybridization and hybridization in 6 x SSC, 5 x Denhardt's reagent, 0.5% SDS and 100 mg / ml of denatured fragmented salmon sperm DNA at 68°C; and washes in 2 x SSC and 0.5% SDS at room temperature for 10 minutes; in 2 x SSC and 0.1 % SDS at room temperature for 10 minutes; and in 0.1 x SSC and 0.5% SDS at 65°C three times for 5 minutes.

[0143] The alkaline phosphatase polypeptide (e.g., of SEQ ID NO: 3; variant B) may be composed of three domains, optionally connected via a linker between the first and second domains and / or the second and third domains, respectively. The first domain, the sALP, corresponds to amino acids L1-S485, which is the soluble part of the human tissue non-specific alkaline phosphatase enzyme and containing an E108M mutation; this domain contains the catalytic function. The polypeptide may include a linker containing amino acids A486- G487. The second domain, the Fc region, corresponds to amino acids S488-K714, which is a chimera Fc of a human immunoglobulin gamma 2 (lgG2) and gamma 4 (lgG4), containing the hinge, CH2, and CH3 domains. The third domain, corresponds to amino acids D715-D721 (D7), is a bone targeting moiety that PATENT ALEXION REF.: ALXN-0667-PCT01-NP allows the alkaline phosphatase of, e.g., SEQ ID NO: 3, to bind to the mineral phase of bone. In addition, each polypeptide chain contains four potential glycosylation sites and eleven cysteine (C) residues. C102 exists as free cysteine. Each polypeptide chain contains four intra-chain disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. The two polypeptide chains are connected by two inter-chain disulfide bonds between C494 on both chains and between C494 on both chains. In addition to these covalent structural features, mammalian alkaline phosphatases are thought to have four metalbinding sites on each polypeptide chain, including two sites for zinc, one site for magnesium and one site for calcium.

[0144] The alkaline phosphatase disclosed herein may be encoded by a first polynucleotide which hybridizes under high stringency conditions to a second polynucleotide comparing the sequence completely complementary to a third polynucleotide encoding a polypeptide having the sequence as set forth in SEQ ID NO: 3. Such high stringency conditions may include: pre-hybridization and hybridization in 6 x SSC, 5 x Denhardt's reagent, 0.5% SDS and 100 mg / ml of denatured fragmented salmon sperm DNA at 68°C; and washes in 2 x SSC and 0.5% SDS at room temperature for 10 minutes; in 2 x SSC and 0.1 % SDS at room temperature for 10 minutes; and in 0.1 x SSC and 0.5% SDS at 65°C three times for 5 minutes.

[0145] There are four known isozymes of ALP, namely tissue non-specific alkaline phosphatase (TNALP) further described below, placental alkaline phosphatase (PALP) (as described e.g., in GenBank Accession Nos. NP_112603 and NP_001623), germ cell alkaline phosphatase (GCALP) (as described, e.g., in GenBank Accession No. P10696) and intestinal alkaline phosphatase (IAP) (as described, e.g., in GenBank Accession No. NP_001622). These enzymes possess very similar three-dimensional structures. Each of their catalytic sites contains four metal-binding domains, for metal ions that are necessary for enzymatic activity, including two Zn and one Mg. These enzymes catalyze the hydrolysis of monoesters of phosphoric acid and also catalyze a transphosphorylation reaction in the presence of high concentrations of phosphate acceptors. Three known natural substrates for ALP (e.g., TNALP) include phosphoethanolamine (PEA), inorganic pyrophosphate (PPi) and pyridoxal 5'-phosphate (PLP) (Whyte et al., J Clin Invest 95: 1440-1445, 1995). An alignment between these isozymes is shown in Figure 30 of PCT Publication No. WO 2008 / 138131 , the teachings of which are incorporated by reference herein in their entirety.

[0146] An alkaline phosphatase protein of this disclosure may be in the form of a dimer or a multimer of any ALP protein, alone or in combination. Chimeric ALP proteins or fusion proteins may also be produced, such as the chimeric ALP protein that is described in Kiffer-Moreira et al. PLoS One 9:e89374, 2014, the entire disclosure of which is incorporated by reference herein in its entirety.

[0147] In one particular embodiment, an alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide with the sequence as set forth in SEQ ID NO: 2 or 3 (or a variant thereof, as described herein). In some embodiments, the alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide having at least 80%, 85%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 2 or 3. In some embodiments, the alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide having 95% or 99% identity to SEQ ID NO: 2 or 3. PATENT

[0148] ALEXION REF.: ALXN-0667-PCT01-NP

[0149] Table 1 below illustrates the differences between the polypeptides of SEQ ID NOs: 1-3, corresponding to asfotase alfa, variant A, and variant B, respectively.

[0150] Table 1. Alkaline phosphatase constructs

[0151] Asfotase alfa includes the sequence set forth in SEQ ID NO: 1 :

[0152] LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEM

[0153] DKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKS

[0154] VGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKT

[0155] DVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNN

[0156] VTDPSLSEMVWAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVT

[0157] ADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAV

[0158] PLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSLKDKTHTCPPCPAPEL

[0159] LGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[0160] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV

[0161] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKDID

[0162] DDDDDDDDD (SEQ ID NO: 1)

[0163] The alkaline phosphatase, e.g., produced by a method as described herein, may include the sequence set forth in SEQ ID NO: 2:

[0164] LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEM

[0165] DKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKS

[0166] VGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKQKT

[0167] DVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNQ

[0168] VTDPSLSEMVWAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTWT

[0169] ADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKWGGERENVSMVDYAHNNYQAQSAV

[0170] PLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAVECPPCPAPP

[0171] VAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSV

[0172] LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

[0173] VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKD DDDDDDDDD (SEQ ID NO: 2).

[0174] The alkaline phosphatase, e.g., produced by a method as described herein, may include the sequence set forth in SEQ ID NO: 3: PATENT ALEXION REF.: ALXN-0667-PCT01-NP

[0175] LVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEM DKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANMGTVGVSAATERSRCNTTQGNEVTSILRWAKDAGKS VGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKT DVEYESDEKARGTRLDGLDLVDTWKSFKPRYKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNN VTDPSLSEMVWAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVT ADHSHVFTFGGYTPRGNSIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAV PLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAVECPPCPAPP VAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKD DDDDDD (SEQ ID NO: 3).

[0176] The recombinant alkaline phosphatase may include the amino acid sequence set forth in SEQ ID NO: 2. The recombinant alkaline phosphatase may be glycosylated (e.g., with one or more of sialic acid, fucose, and mannose) at one or more of N123, N254, N413, and N564. The recombinant alkaline phosphatase may include a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer (e.g., at least 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). The recombinant alkaline phosphatase may include a total fucose content of at least 2.0 mol fucose / mol protein monomer. The recombinant alkaline phosphatase may include a total mannose content of at least 6.0 mol mannose / mol protein monomer. The recombinant alkaline phosphatase may be a dimer that includes a first polypeptide and a second polypeptide, each polypeptide including the amino acid sequence of SEQ ID NO: 2. The dimer may be linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide. The first polypeptide may include disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. C102 of the first polypeptide and the second polypeptide may be a free cysteine. Each of the first polypeptide and the second polypeptide may coordinate two zinc ions, one magnesium ion, and one calcium ion.

[0177] The recombinant alkaline phosphatase includes the amino acid sequence set forth in SEQ ID NO: 3. The recombinant alkaline phosphatase may be glycosylated (e.g., with one or more of sialic acid, fucose, and mannose) at one or more of N123, N213, N254, N286, N413, and N564. The recombinant alkaline phosphatase may include a total sialic acid content (TSAC) of at least 1 .2 mol sialic acid / mol protein monomer (e.g., at least 1 .3, 1 .4, 1 .5, 1 .6, 1 .7, 1 .8, 1 .9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, or more mol sialic acid / mol protein monomer). The recombinant alkaline phosphatase may include a total fucose content of at least 0.6 mol fucose / mol protein monomer. The recombinant alkaline phosphatase may include a total mannose content of at least 1 .8 mol mannose / mol protein monomer. The recombinant alkaline phosphatase may be a dimer that includes a first polypeptide and a second polypeptide, each polypeptide including the amino acid PATENT ALEXION REF.: ALXN-0667-PCT01-NP sequence of SEQ ID NO: 2. The dimer may be linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide. The first polypeptide may include disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide includes disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692. C102 of the first polypeptide and the second polypeptide may be a free cysteine. Each of the first polypeptide and the second polypeptide may coordinate two zinc ions, one magnesium ion, and one calcium ion.

[0178] EXAMPLES

[0179] Example 1 : Alkaline phosphatase manufacturing process

[0180] Asfotase alfa (SEQ ID NO: 1), variant A (SEQ ID NO: 2), and variant B (SEQ ID NO: 3) were manufactured using methods described herein. The dimeric molecules have precisely defined chemical structures with disulfide bonds connecting the two proteins (see, e.g., FIGS. 1-3). The manufacturing process parameters were optimized for growth and production temperatures, media, metal supplementation, sugar supplementation, pH, and other process variables. The optimized methods yielded a high sialylation process that was developed to enhance the protein expression and product quality attributes of Chinese hamster ovary (CHO) cell produced recombinant alkaline phosphatases. The high sialylation upstream process uses specialty media, feed, and sugar supplementation for optimal cell culture performance and product quality.

[0181] Metal supplementation of the production media with zinc, magnesium, and calcium promotes proper folding of the protein, and zinc additionally improves the enzymatic activity.

[0182] The use of the specialty feed, Glycan Tune C+, increased the total sialic acid content on the molecule. The timing and amount of feed were optimized to enhance protein expression.

[0183] Sugar supplementation with fucose, galactose, and N-acetyl-D-mannosamine improved the process robustness for the fucosylation, galactosylation, and sialylation of the alkaline phosphatase. The high sialylation upstream production process employed a temperature shift to sustain cell viability and extend the production phase while improving product quality attributes. The temperature shift and production temperatures were shown to impact glycosylation, enzymatic activity, aggregates, fragments, and charge variants. Specifically, an optimized temperature shift was implemented to control aggregate, fragment, and charge species, as well as enhancing sialylation and enzymatic activity.

[0184] In addition to production temperature, pH was identified as having a significant impact on product quality. An increased percentage of fragments and aggregates was observed at a higher pH. The culture pH was controlled by sparging CO2 gas and by the addition of sodium carbonate solution. Therefore, the pH of the culture was maintained at a level of 7.1 or lower to help reduce the development of fragments and aggregates.

[0185] A number of manufacturing protocol changes were made starting from the protocol used to produce asfotase alfa (SEQ ID NO: 1) in order to arrive at a suitable protocol used to produce variant A (SEQ ID NO: PATENT

[0186] ALEXION REF.: ALXN-0667-PCT01-NP

[0187] 2), and variant B (SEQ ID NO: 3). These changes are summarized in Table 2. Specific upstream production process details for each construct are shown in Tables 3-5.

[0188] Table 2. Upstream process comparison

[0189] Table 3. Variant A upstream production process PATENT

[0190] ALEXION REF.: ALXN-0667-PCT01-NP

[0191] Table 4. Variant B upstream production process PATENT

[0192] ALEXION REF.: ALXN-0667-PCT01-NP

[0193] Table 5. Asfotase alfa upstream production process PATENT

[0194] ALEXION REF.: ALXN-0667-PCT01-NP

[0195] Following production of each construct, product quality was assessed from two representative lots in each process (see Table 6). Similar purity was observed as determined by SEC-HPLC, CE-SDS (reduced and non-reduced). Similar quantity of charge variants as measured by anion exchange, and specific activities as measured by activity on pNPP hydrolysis, were observed among the lots produced by the three processes.

[0196] Significantly higher sialy lation as measured by total sialic acid content was observed in high sialy lation processes (variant A and variant B) relative to the total sialic acid content observed in the asfotase alfa manufacturing process (12.8-15.8 mol sialic acid per mol protein monomer as compared to 2.2 mol sialic acid per mol protein monomer for asfotase alfa, indicating the manufacturing methods impart substantially higher sialylation under the newly designed process.

[0197] Table 6. Purity, specific activity, and TSAC content of variant A, variant B, and asfotase alfa PATENT

[0198] ALEXION REF.: ALXN-0667-PCT01-NP PATENT ALEXION REF.: 0667 US P1

[0199] Example 2. Subcutaneous and intravenous dosing in cynomolgus monkeys

[0200] Additional in vivo experiments were performed on cynomolgus monkeys. Monkeys were intravenously or subcutaneously dosed with 3 mg / kg asfotase alfa (SEQ ID NO: 1), or 2 mg / kg, 6 mg / kg, or 20 mg / kg variant A (SEQ ID NO: 2), or 2 mg / kg, 20 mg / kg, or 50 mg / kg variant B (SEQ ID NO: 3). As shown in FIG. 4, all three dosages of variant A showed similar decline rates of drug concentration post administration, which were all significantly slower than asfotase alfa. Enhanced sialylation of variant A slowed drug clearance and extended the duration of plasma drug levels, contributing to the superior pharmacokinetics profile with significantly improved exposure and / or half-life of the product as compared to asfotase alfa. Similar results were observed with all three dosages of variant B as shown in FIG. 5.

[0201] OTHER EMBODIMENTS

[0202] The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The disclosure is not limited to the exact details shown and described, for variations apparent to one skilled in the art will be included within the disclosure defined by the claims.

[0203] Unless otherwise indicated, all numbers expressing quantities of components and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the disclosure. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0204] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0205] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0206] The complete disclosures of all patents, patent applications including provisional patent applications, publications including patent publications and non-patent publications, and electronically available material (including, for example, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference.

Claims

PATENTALEXION REF.: 0667 US P1CLAIMS1 . A method of producing a recombinant alkaline phosphatase comprising:(a) inoculating a bioreactor comprising a culture medium with cells expressing a recombinant alkaline phosphatase, wherein the pH of the culture medium prior to the inoculating is about 6.7 to about 7.1 ;(b) supplementing the inoculated culture medium with galactose, N-acetyl mannosamine, and fucose;(c) culturing the cells to express the recombinant alkaline phosphatase; and(d) harvesting the recombinant alkaline phosphatase from the inoculated, supplemented culture medium.

2. The method of claim 1 , wherein after step (b), the galactose, the N-acetyl mannosamine, and the mannosamine are each at a concentration from about 0.5 to 5 g / L in the inoculated culture medium.

3. The method of claim 2, wherein after step (b), the concentration in the inoculated culture medium of galactose is about 4 g / L, the concentration of N-acetyl mannosamine is about 3 g / L, and the concentration of fucose is about 1 g / L.

4. The method of any one of claims 1-3, wherein the supplementing of step (b) is performed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after the inoculating of step (a).

5. The method of any one of claims 1-4, wherein the supplementing of step (b) comprises adding the galactose, N-acetyl mannosamine, and fucose to the inoculated culture medium as a bolus.

6. The method of any one of claims 1-5, wherein the culture medium to which the cells are added in step (a) comprises a basal medium comprising chemically defined CHO advanced granulation technology (CD CHO AGT™) medium.

7. The method of any one of claims 1-6, wherein the culture medium to which the cells are added in step (a) further comprises one or more of zinc, magnesium, and chloride.

8. The method of claim 7, wherein copper is added to the inoculated culture medium as a bolus.

9. The method of claim 7 or 8, wherein the culture medium to which the cells are added in step (a) comprises 5 mg / L to 100 mg / L zinc sulfate heptahydrate, 100 mg / L to 200 mg / L magnesium chloride hexahydrate, and / or 5 mg / L to 100 mg / L calcium chloride dihydrate.PATENTALEXION REF.: 0667 US P110. The method of claim 7, wherein the culture medium to which the cells are added in step (a) comprises about 43 mg / L zinc sulfate heptahydrate, about 148 mg / L magnesium chloride hexahydrate, and about 55 mg / L calcium chloride dihydrate.

11. The method of any one of claims 1-10, wherein the culture medium to which the cells are added in step (a) comprises cysteine and / or tyrosine, or cysteine and / or tyrosine are added to the culture medium as a further supplementation of the supplementing step (b)12. The method of claim 11 , wherein the further supplementation of step (b) comprises adding the cysteine as L-cysteine.

13. The method of claim 11 , wherein the further supplementation of step (b) comprises adding the tyrosine as L-tyrosine.

14. The method of claim 1 , wherein the method further comprises adding the GTC+ feed on or about day 4, on or about day 6, on or about day 8, and / or on or about day 10 after the inoculating step (a).

15. The method of any one of claims 1-14, wherein step (c) comprises culturing the cells at a dissolved oxygen (DO) concentration of about 10% to about 60%.

16. The method of any one of claims 1-15, wherein step (a) comprises inoculating the bioreactor with a cell density of about 0.3 x 106viable cells / mL to about 0.7 x 106viable cells / mL17. The method of any one of claims 1 -17, wherein the method comprises culturing the cells at a first temperature until reaching a cell density of at least about 5 x 106viable cells / mL and shifting to a second temperature which is lower than the first temperature to express the recombinant alkaline phosphatase.

18. The method of claim 17, wherein the first temperature is about 35 °C to about 38 °C.

19. The method of claim 18, wherein the first temperature is about 36.5 °C.

20. The method of any one of claims 17-19, wherein the second temperature is about 29 °C to about 35 °C.21 . The method of claim 20, wherein the second temperature is about 34 °C to about 36 °C.

22. The method of claim 21 , wherein the second temperature is about 35 °C.PATENTALEXION REF.: 0667 US P123. The method of any one of claims 17-22, wherein the cell density prior to the shifting is about 5 x 106viable cells / mL to about 25 x 106viable cells / mL.

24. The method of claim 23, wherein the cell density prior to the shifting is about 6 x 106viable cells / mL to about 10 x 105viable cells / mL or about 15 x 106viable cells / mL to about 20 x 106viable cells / mL.

25. The method of any one of claims 17-24, wherein the shifting is performed on or about day 7 after the inoculating step (a).

26. The method of any one of claims 1-25, wherein step (d) comprises harvesting the cells at a density from about 10 x 106viable cells / mL to about 30 x 106viable cells / mL.

27. The method of any one of claims 1-26, wherein step (d) is performed on about day 14 after the inoculating step (a).

28. The method of any one of claims 1 -27, further comprising a step of determining cell viability, and wherein step (d) is performed within 24 hours of the cell viability being less than or equal to 75% of total cells, or less than or equal to 80 % of the total cells.

29. The method of any one of claims 1-28, wherein step (d) comprises depth filtration or centrifugation.

30. The method of any one of claims 1 -29, wherein the pH is about 6.9.31 . The method of any one of claims 1 -30, wherein the cells are mammalian cells.

32. The method of claim 31 , wherein the mammalian cells are Chinese Hamster Ovary (CHO) cells.

33. The method of any one of claims 1 -32, wherein the bioreactor has a volume of at least 2 L.

34. The method of claim 33, wherein the volume is at least 1 ,000 L.

35. The method of claim 34, wherein the volume is about 20,000 L.

36. The method of any one of claims 1-35, wherein the recombinant alkaline phosphatase harvested in step (d) comprises the structure of W-sALP-X-Fc-Y-Dn-Z, wherein:W is absent or is an amino acid sequence of at least one amino acid;PATENTALEXION REF.: 0667 US P1X is absent or is an amino acid sequence of at least one amino acid;Y is absent or is an amino acid sequence of at least one amino acid;Z is absent or is an amino acid sequence of at least one amino acid;Fc is a fragment crystallizable region;Dn is a poly-aspartate, poly-glutamate, or combination thereof, wherein n = 5 to 16; and sALP is a soluble alkaline phosphatase.

37. The method of claim 36, wherein the recombinant alkaline phosphatase harvested in step (d) comprises an amino acid sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 2 or 3.

38. The method of claim 37, wherein the recombinant alkaline phosphatase harvested in step (d) comprises the amino acid sequence set forth in SEQ ID NO: 2 or 3.

39. The method of any one of claims 1-38, wherein the recombinant alkaline phosphatase harvested in step (d) comprises the amino acid sequence set forth in SEQ ID NO: 2.

40. The method of claim 39, wherein the recombinant alkaline phosphatase harvested in step (d) is glycosylated at one or more of N123, N254, N413, and N564.

41. The method of claim 40, wherein the glycosylation comprises one or more of sialic acid, fucose, and mannose.

42. The method of claim 41 , wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer.

43. The method of claim 41 or 42, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total fucose content of at least 2.0 mol fucose / mol protein monomer.

44. The method of any one of claims 41-43, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total mannose content of at least 6.0 mol mannose / mol protein monomer.

45. The method of any one of claims 39-44, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a dimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the secondPATENTALEXION REF.: 0667 US P1 polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide.

46. The method of claim 45, wherein the first polypeptide of the recombinant alkaline phosphatase harvested in step (d) comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692.

47. The method of claim 45 or 46, wherein C102 of the first polypeptide and the second polypeptide of the recombinant alkaline phosphatase harvested in step (d) is a free cysteine.

48. The method of claim 47, wherein the recombinant alkaline phosphatase harvested in step (d) comprises the amino acid sequence set forth in SEQ ID NO: 3.

49. The method of claim 48, wherein the recombinant alkaline phosphatase harvested in step (d) is glycosylated at one or more of N123, N213, N254, N286, N413, and N564.

50. The method of claim 49, wherein the glycosylation comprises one or more of sialic acid, fucose, and mannose.

51. The method of claim 50, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total sialic acid content (TSAC) of at least 1.2 mol sialic acid / mol protein monomer.

52. The method of claim 50 or 51 , wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total fucose content of at least 0.6 mol fucose / mol protein monomer.

53. The method of any one of claims 50-52, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a total mannose content of at least 1 .8 mol mannose / mol protein monomer.

54. The method of any one of claims 48-53, wherein the recombinant alkaline phosphatase harvested in step (d) comprises a dimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide.PATENTALEXION REF.: 0667 US P155. The method of claim 54, wherein the first polypeptide of the recombinant alkaline phosphatase harvested in step (d) comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692.

56. The method of claim 1 , wherein step (d) results in a recombinant alkaline phosphatase comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 2, wherein the dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide, wherein the dimer is glycosylated at one or more of N123, N254, N413, and N564, wherein the glycosylation comprises one or more of sialic acid, fucose, and mannose; and wherein the recombinant alkaline phosphatase comprises:(a) a total sialic acid content (TSAC) of at least 4.0 mol sialic acid / mol protein monomer;(b) a total fucose content of at least 2.0 mol fucose / mol protein monomer; and(c) a total mannose content of at least 6.0 mol mannose / mol protein monomer.

57. The method of claim 56, wherein the first polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692.

58. The method of claim 1 , wherein step (d) results in a recombinant alkaline phosphatase comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the dimer is linked by a first disulfide bond between C494 of the first polypeptide and C494 of the second polypeptide and a second disulfide bond between C497 of the first polypeptide and C497 of the second polypeptide, wherein the dimer is glycosylated at one or more of N123, N213, N254, N286, N413, and N564, wherein the glycosylation comprises one or more of sialic acid, fucose, and mannose; and wherein the recombinant alkaline phosphatase comprises:(a) a total sialic acid content (TSAC) of at least 1 .2 mol sialic acid / mol protein monomer;(b) a total fucose content of at least 0.6 mol fucose / mol protein monomer; and(c) a total mannose content of at least 1.8 mol mannose / mol protein monomer.

59. The method of claim 58, wherein the first polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692, and the second polypeptide comprises disulfide bonds between C122 and C184, C472 and C480, C528 and C588, and C634 and C692.PATENTALEXION REF.: 0667 US P160. The method of claim 58 or 59, wherein C102 of the first polypeptide and the second polypeptide is a free cysteine.

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