Method for controlling total sialic acid content (TSAC) during alkaline phosphatase production

Abstract

A method for controlling the total sialic acid content (TSAC) during the production of alkaline phosphatase is provided. [Solution] The subject is a method for producing recombinant alkaline phosphatase, such as asfotase alpha, which provides more precise quality control over the TSAC concentration in the final product by measuring the total sialic acid content (TSAC) concentration during fermentation and adjusting the downstream production steps accordingly. Asfotase alpha is a eukaryotic protein that contains post-translational modifications, such as glycosylation (e.g., sialylation).

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 105,052, filed on October 23, 2020, the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application is electronically filed in ASCII format and includes a sequence listing that is incorporated herein by reference in its entirety. The ASCII copy was created on October 19, 2021, named 0608WO_SL.txt, and is 15,286 bytes in size.

Background Art

[0003] Hypophosphatasia (HPP) is a severe and extremely rare genetic metabolic disorder in which the production of functional tissue - nonspecific alkaline phosphatase (TNSALP) is impaired. It results in the accumulation of unmineralized bone matrix (e.g., rickets, osteomalacia) characterized by hypomineralization of bone and teeth. When growing bones do not mineralize properly, growth disorders can impair the appearance of joints and bones. These consequences can, therefore, affect motor ability, respiratory function, and potentially lead to death. HPP includes perinatal, infantile, juvenile (or pediatric), and adult HPP. Historically, six clinical forms have been defined, including perinatal, benign prenatal, pediatric, juvenile, adult, and dento - localized HPP, mainly based on the age at symptom onset.

[0004] Asfotase alfa (STRENSIQ®, Alexion Pharmaceuticals, Inc.) is an approved, first-in-class targeted enzyme substitution therapy designed to address deficiencies in endogenous TNSALP levels. Asfotase alfa is a soluble fusion glycoprotein consisting of the catalytic domain of human TNSALP, the Fc domain of human immunoglobulin G1, and decaaspartate peptide (e.g., D10) (SEQ ID NO: 2) used as the bone-targeting domain. In vitro, asfotase alfa binds to hydroxyapatite with higher affinity than soluble TNSALP lacking decaaspartate peptide, thereby allowing the TNSALP portion of asfotase alfa to efficiently degrade excess localized inorganic pyrophosphate (PPi) and restore normal mineralization to the bone. Pyrophosphate hydrolysis promotes bone mineralization, and its effect was similar across species evaluated in non-clinical studies. [Overview of the project] [Problems that the invention aims to solve]

[0005] Asfotase alpha is a eukaryotic protein that contains post-translational modifications, such as glycosylation (e.g., sialylation). The production of commercially available therapeutically effective alkaline phosphatases, such as asfotase alpha, involves a multi-step manufacturing process, the conditions of which can significantly affect the final product. During this process, the post-translational modified product may be exposed to glycosidase or other hydrolytic conditions in one or more steps of the manufacturing process that negatively affect the post-translational modifications. For example, the added sialic acid moiety may be lost. These changes can reduce the half-life and enzyme activity of large batch products. Therefore, improved methods for producing alkaline phosphatases are needed to improve quality control of the final protein product and its glycosylation properties. [Means for solving the problem]

[0006] Disclosed herein is a manufacturing process that can be used to improve the quality control of glycosylation in the production of alkaline phosphatases (e.g., asfotase alpha). The method can also be used to maintain, store, regulate and / or improve the enzymatic activity of alkaline phosphatases (e.g., asfotase alpha) produced from recombinant proteins, such as cultured mammalian cells, particularly cultured Chinese hamster ovary (CHO) cells, and especially to maintain, control and / or improve their half-life. Such alkaline phosphatases (e.g., asfotase alpha) are suitable for therapeutic use, for example, in the treatment of conditions associated with reduced alkaline phosphatase protein levels (e.g., HPP) and / or function (e.g., insufficient cleavage of inorganic pyrophosphate (PPi), etc.) in subjects, such as human subjects.

[0007] In one embodiment, the subject is a method for producing recombinant alkaline phosphatase. This method includes the steps of: inoculating cells expressing recombinant alkaline phosphatase (e.g., mammalian cells, e.g., Chinese hamster ovary (CHO) cells) into a bioreactor; obtaining an aqueous medium containing recombinant alkaline phosphatase; obtaining a certain amount from the aqueous medium approximately 6 to 10 days after inoculation, particularly approximately 6 to 8 days (e.g., approximately 6, 7, 8, 9, 10 days, e.g., approximately 7 days); quantifying the total sialic acid content (TSAC) molar concentration per mole of recombinant alkaline phosphatase in the certain amount; collecting the aqueous medium; and finally obtaining a bulk drug substance (BDS) by performing a filtration step (e.g., ultrafiltration, diafiltration, or a combination thereof). This method may further include additional downstream purification steps between the filtration step and the acquisition of BDS (Figure 1).

[0008] A certain amount of the culture medium from the fermentation stage is used to determine the amount of time the filtration pool is maintained. For example, if the certain amount has a TSAC concentration of less than approximately 2.5 mol / mol, the filtration step may be maintained for less than approximately 9 hours. If the certain amount has a TSAC concentration of approximately 2.5 mol / mol to approximately 2.7 mol / mol, the filtration step may be maintained for approximately 10 to 14 hours. If the certain amount has a TSAC concentration of approximately 2.8 mol / mol to approximately 3.0 mol / mol, the filtration step may be maintained for approximately 23 to 27 hours. If the certain amount has a TSAC concentration greater than approximately 3.0 mol / mol, the filtration step may be maintained for approximately 38 to 42 hours.

[0009] In one embodiment, if a certain volume has a TSAC concentration of less than about 2.5 mol / mol, the filtration step may be held for about 7 ± 2 hours or less (e.g., about 5 to 9 hours). If a certain volume has a TSAC concentration of about 2.5 mol / mol to about 2.7 mol / mol, the filtration step may be held for about 18 ± 2 hours or less (e.g., about 16 to 20 hours). If a certain volume has a TSAC concentration greater than about 2.7 mol / mol, the filtration step may be held for about 32 ± 2 hours or less (e.g., about 30 to 34 hours).

[0010] In another embodiment, if a certain volume has a TSAC concentration of about 2.3 mol / mol or less, the filtration step may be held for about 18 ± 4 hours or less (e.g., about 14 to 22 hours). If a certain volume has a TSAC concentration greater than about 2.3 mol / mol to about 3.1 mol / mol (e.g., about 2.4 mol / mol to about 3.1 mol / mol), the filtration step may be held for about 32 ± 4 hours or less (e.g., about 28 to 36 hours). If a certain volume has a TSAC concentration greater than about 3.1 mol / mol (e.g., about 3.2 mol / mol or more), the filtration step may be held for about 44 ± 4 hours or less (e.g., about 40 to 48 hours).

[0011] In another embodiment, if a given volume has a TSAC concentration of less than about 2.4 mol / mol, the filtration step may be held for about 17 ± 3 hours or less (e.g., about 14 to 20 hours). If a given volume has a TSAC concentration of about 2.4 mol / mol to about 3.6 mol / mol, the filtration step may be held for about 31 ± 3 hours or less (e.g., about 28 to 34 hours). If a given volume has a TSAC concentration greater than about 3.6 mol / mol, the filtration step may be held for about 45 ± 3 hours (about 42 to 48 hours). The alkaline phosphatase concentration during the filtration step may be about 3.7 ± 0.4 g / L.

[0012] The alkaline phosphatase concentration during the filtration step may range from approximately 1.8 g / L to approximately 5.0 g / L (e.g., approximately 1.8 to approximately 4.3 g / L, e.g., approximately 2.3 g / L, approximately 3.1 g / L, approximately 3.7 g / L). The TSAC concentration of BDS may range from approximately 1.2 moles to approximately 3.0 moles (e.g., approximately 1.6 moles to approximately 2.4 moles).

[0013] The filtration step may be maintained at a constant temperature, which is any temperature within a defined range. For example, the temperature may be maintained between approximately 15°C and approximately 25°C (e.g., approximately 19°C to approximately 25°C, e.g., approximately 22°C).

[0014] A certain volume can be obtained sterilely from a bioreactor to prevent contamination. This volume may range from approximately 1 mL to approximately 1000 mL (for example, approximately 25 mL to approximately 500 mL, for example, approximately 50 mL to approximately 300 mL, for example, approximately 100 mL or approximately 200 mL).

[0015] Obtaining a specific volume may further include centrifugation of the volume and, optionally, removal of the supernatant from the volume. This step may also include purification of alkaline phosphatase from the supernatant using a chromatography column (e.g., a Protein A column, e.g., a 1 mL HiTrap Protein A column; a 600 μl Protein A Robocolumn; or a MabSelect Sure Protein A solid-phase column). In some embodiments, the alkaline phosphatase may be subjected to buffer exchange. The alkaline phosphatase may also be concentrated, for example, before measuring TSAC analysis. TSAC analysis may include quantifying the TSAC concentration and performing acid hydrolysis to release TSAC.

[0016] After purification, the alkaline phosphatase can be freeze-dried and / or loaded into vials.

[0017] The bioreactor may be of any suitable size for, for example, the commercial-scale production of alkaline phosphatase. For example, the bioreactor may have a volume of at least 2 L, at least 10 L, at least 1,000 L, at least 10,000 L, or at least 20,000 L. The volume may be about 10,000 L or about 20,000 L.

[0018] Any suitable cell medium, such as serum-free medium, may be used. Some suitable examples include EX-CELL® 302 serum-free medium; CD DG44 medium; BD SELECT® medium; SFM4CHO medium; and combinations thereof.

[0019] Alkaline phosphatase is W-sALP-X-Fc-Y-Dn-Z (in the formula, W is either absent or an amino acid sequence of at least one amino acid; X is either absent or an amino acid sequence of at least one amino acid; Y is either absent or 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 crystallizable fragment region; Dn is polyaspartic acid, polyglutamate or a combination thereof, where n = 10 or 16; and sALP is soluble alkaline phosphatase) may include the structure.

[0020] In some embodiments, the recombinant alkaline phosphatase comprises an amino acid sequence having at least 90% (e.g., at least 95%, 97%, 98% or 99%) sequence identity to the sequence shown in SEQ ID NO: 1. For example, the recombinant alkaline phosphatase may comprise or consist of the amino acid sequence shown in SEQ ID NO: 1.

[0021] Definitions As used herein, the terms "about" and "approximately" refer to a range of values that are + / - 10% of the target value when applied to one or more specific cell culture conditions or numerical ranges.

[0022] As used herein, the term "amino acid" generally refers to any of the 20 naturally occurring amino acids used in the form of polypeptides or their amino acid analogs or derivatives. The amino acids of the present disclosure can be provided in the medium for cell culture. The amino acids provided in the medium can be provided as salts or in hydrated form.

[0023] As used herein, the term "batch culture" refers to a method of culturing cells in which all of the components ultimately used to culture the cells, including the medium (see definition of "medium" below), and the cells themselves are provided at the start of the culture process. Batch cultures are typically stopped at some point and the cells and / or components in the medium are collected and optionally purified. In some embodiments, the methods described herein are used in batch cultures.

[0024] As used herein, the term "bioreactor" refers to any vessel used for the growth of a cell culture (e.g., a mammalian cell culture). A bioreactor can be of any size as long as it is useful for culturing cells. Typically, a bioreactor has a volume of at least 1 liter, 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. In some embodiments, the bioreactor has a volume of from 100 liters to 30,000 liters, from 500 liters to 22,000 liters, from 1,000 liters to 22,000 liters, from 2,000 liters to 22,000 liters, from 5,000 liters to 22,000 liters or from 10,000 liters to 22,000 liters. The maximum working volume of a bioreactor can vary by about 1% to 5%, for example 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 adjusted during the culture period. The bioreactor can be constructed from any material suitable for holding a mammalian or other cell culture suspended in a medium under the culture conditions of the present disclosure, including glass, plastic or metal. As used herein, the term "production bioreactor" refers to the final bioreactor used for the production of the polypeptide or protein of interest. The volume of a large-scale cell culture production bioreactor is typically at least 500 liters and can be 1000, 2500, 5000, 8000, 10,000, 12,0000, 20,000 liters or more, or any volume in between. Those skilled in the art will understand and be able to select a bioreactor suitable for use in the practice of the present disclosure.

[0025] As used herein, the term "cell density" refers to the number of cells present in a given volume of medium.

[0026] 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. As used herein, the term also refers to the proportion of cells surviving at a given point in time to the total number of cells (surviving and dead) in culture at that time.

[0027] The terms “culture” and “cell culture” as used herein refer to a population of cells suspended in a culture medium (see the definition of “culture medium” below) under conditions suitable for the survival and / or proliferation of the cell population. As will be obvious to those skilled in the art, these terms as used herein may refer to a combination including a cell population and a culture medium in which the population is suspended.

[0028] 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 point after the start of the culture process. The provided components typically include nutritional supplements for the cells that are depleted during the culture process. Fed-batch culture is typically stopped at some point, and the cells and / or components in the culture medium are collected and optionally purified. Fed-batch culture may be carried out in a corresponding fed-batch bioreactor. In some embodiments, the method includes fed-batch culture.

[0029] The term “fragment,” as used herein, refers to a polypeptide and is defined as any isolated portion of a given polypeptide that is inherent to or characteristic of that polypeptide. The term also, as used herein, refers to any isolated portion of a given polypeptide that retains at least a portion of the activity of the full-length polypeptide. In some embodiments, the percentage of activity retained is at least 10% of the activity of the full-length polypeptide. In various embodiments, the percentage 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 percentage of activity retained is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide. In one embodiment, the percentage of activity retained is 100% of the activity of the full-length polypeptide. The term also, as used herein, 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 extends to at least 4-5 amino acids of the full-length polypeptide. In some embodiments, the sequence elements extend to at least about 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide.

[0030] The term "glycoprotein" or "glycoproteins," as used herein, refers to a protein or polypeptide in which a carbohydrate group (such as sialic acid) is attached to a polypeptide chain.

[0031] The terms “medium,” “media,” “cell medium,” and “culture medium” as used herein refer to a solution containing nutrients that nourish the growth of mammalian cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements that cells require for minimum growth and / or survival. The solutions may also contain components that enhance growth and / or survival beyond the minimum rate, including hormones and growth factors. The solutions may be formulated, for example, to the optimal pH and salt concentration for cell survival and growth. In some embodiments, the medium may be a “limited medium,” i.e., a serum-free medium that does not contain components of proteins, hydrolysates, or unknown compositions. Limited media do not contain animal-derived components, and all components have known chemical structures. In some embodiments, the medium is a non-limited medium containing a basal medium, e.g., a carbon source, water, salts, amino acids, and a nitrogen source (e.g., animal, e.g., beef or enzyme extracts). Various media are commercially available and known to those skilled in the art. In some embodiments, the culture medium is selected from EX-CELL® 302 serum-free medium (Sigam Aldrich, St. Louis, MO), CD DG44 medium (ThermoFisher Scientific, Waltham, MA), BD Select medium (BD Biosciences, San Jose, CA), or a mixture thereof, or a mixture of BD Select medium and SFM4CHO medium (Hyclone®, Logan UT). In some embodiments, the culture medium includes a combination of SFM4CHO medium and BD SELECT® medium. In some embodiments, the culture medium includes a combination of SFM4CHO medium and BD SELECT® medium in a ratio selected from 90 / 10, 80 / 20, 75 / 25, 70 / 30, 60 / 40, or 50 / 50 (including any intermediate ratio between them). In some embodiments, the culture medium includes a combination of SFM4CHO medium and BD SELECT® medium in a ratio of 70 / 30 to 90 / 10. In some embodiments, the culture medium comprises a combination of SFM4CHO medium and BD SELECT® medium in a 75 / 25 ratio.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, hydrolyzate-free medium containing hypoxanthine, thymidine, and L-glutamine, and does not contain PLURONIC® F-68. In some embodiments, alkaline phosphatase (e.g., asfotase alfa) is produced by a process in which the medium is added to a production bioreactor in extra bolus doses. For example, the medium may be added in 1, 2, 3, 4, 5, 6 or more bolus doses. In a particular embodiment, the medium is added in 3 bolus doses. In various embodiments, such extra bolus doses of the culture medium may be added in varying amounts. For example, such bolus doses of the culture medium may be added in amounts of approximately 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 the culture medium. In a particular embodiment, such bolus doses of the culture medium may be added in amounts of approximately 33%, 67%, 100%, or 133% of the original volume. In various embodiments, such addition of extra bolus doses may be made at various points in time during the period of cell proliferation or protein production. For example, a bolus dose may be added on 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 in the process. In a particular embodiment, such a bolus dose of the medium may be added every other day (e.g., (1) on days 3, 5, and 7; (2) on days 4, 6, and 8; or (3) on days 5, 7, and 9). In fact, the frequency, amount, timing, and other parameters of the adjuvant in the bolus dose of the medium can be freely combined according to the above limitations and may be determined by experimental practice.

[0032] The terms "molar osmotic pressure" and "molar osmotic pressure" refer, as used herein, to a measure of the osmotic pressure of solute particles dissolved in an aqueous solution. Solute particles include both ions and non-ionized molecules. Molar osmotic pressure is expressed as the concentration (e.g., osmoles) of osmotically active particles dissolved in 1 kg of solution (1 mOsm / kg of H2O at 38°C is equal to an osmotic pressure of 19 mmHg). In contrast, "molar osmotic pressure" refers to the number of solute particles dissolved in 1 liter of solution. As used herein, the abbreviation "mOsm" means "milliosmoles / kg solution".

[0033] The term “perfusion culture,” as used herein, refers to a method of culturing cells in which additional components are continuously or semi-continuously supplied to the culture after the commencement of the culture process. The supplied components typically include nutritional supplements for the cells that are depleted during the culture process. Some of the cells and / or components in the culture medium are typically collected on a continuous or semi-continuous basis and optionally purified. In some embodiments, nutritional supplements are added under perfusion culture as described herein, for example, continuously over a specified period.

[0034] As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together via peptide bonds. While this term is used to refer to amino acid chains of any length, those skilled in the art will understand that the term is not limited to long chains and may refer to the shortest possible chain containing two amino acids linked together via peptide bonds.

[0035] The term "protein," as used herein, refers to one or more polypeptides that function as a unit of separation. When a single polypeptide is a functional separation unit and does not require permanent physical association with other polypeptides to form that unit, the terms "polypeptide" and "protein" are used interchangeably herein.

[0036] The terms “recombinantly expressed polypeptide” and “recombinant polypeptide,” as used herein, refer to a polypeptide expressed from a host cell that has been genetically engineered to express the polypeptide. Recombinantly expressed polypeptides may typically match or be similar to polypeptides expressed in mammalian host cells. Recombinantly expressed polypeptides may also be exogenous to the host cell, for example, heterologous to peptides typically expressed in the host cell. Alternatively, a recombinantly expressed polypeptide may be a chimeric molecule in that some parts of the polypeptide have an amino acid sequence that matches or is similar to a polypeptide typically expressed in mammalian host cells, while other parts are exogenous to the host cell.

[0037] The term “seeding,” as used herein, refers to the process of providing a cell culture to a bioreactor or another container. Cells may be pre-grown in another bioreactor or container. Alternatively, cells may be frozen and thawed immediately before providing them to the bioreactor or container. This term refers to any number of cells, including single cells. In various embodiments, alkaline phosphatase (e.g., asphotase alpha) is used to provide cells at concentrations of approximately 1.0 × 10⁵ cells / mL, 1.5 × 10⁵ cells / mL, 2.0 × 10⁵ cells / mL, 2.5 × 10⁵ cells / mL, 3.0 × 10⁵ cells / mL, 3.5 × 10⁵ cells / mL, 4.0 × 10⁵ cells / mL, 4.5 × 10⁵ cells / mL, 5.0 × 10⁵ cells / mL, 5.5 × 10⁵ cells / mL, The cells are produced by a process in which they are seeded at densities of 6.0 × 10⁵ cells / mL, 6.5 × 10⁵ cells / mL, 7.0 × 10⁵ cells / mL, 7.5 × 10⁵ cells / mL, 8.0 × 10⁵ cells / mL, 8.5 × 10⁵ cells / mL, 9.0 × 10⁵ cells / mL, 9.5 × 10⁵ cells / mL, 1.0 × 10⁶ cells / mL, 1.5 × 10⁶ cells / mL, 2.0 × 10⁶ cells / mL, or higher densities. In a particular embodiment, in such a process, the cells are seeded at densities of approximately 4.0 × 10⁵ cells / mL, 5.5 × 10⁵ cells / mL, or 8.0 × 10⁵ cells / mL.

[0038] The term “Total Sialic Acid Content” or “TSAC” as used herein refers to the amount of sialic acid (carbohydrate) on a particular protein molecule. It is expressed as TSAC per mole of protein or “mol / mol”. TSAC concentration is measured during the purification process. For example, one method of TSAC quantification involves releasing TSAC from asfotase alpha using acid hydrolysis, and then detecting the released TSAC via electrochemical detection using high-performance anion exchange chromatography (“HPAE-PAD”) with amperometric electrochemical detection.

[0039] As used herein, the term "titer" refers to the total amount of recombinantly expressed polypeptide or protein produced by a cell culture divided by a given volume of culture medium. Titer is typically expressed in milligrams of polypeptide or protein per 1 mL of medium.

[0040] Acronyms used herein include, for example, HCCF: collected clarified culture medium; UF: ultrafiltration; DF: diafiltration; VCD: viable cell density; IVCC: integrated viable cell concentration; TSAC: total sialic acid content; HPAE-PAD: high-performance anion exchange chromatography with amperometric electrochemical detection; SEC: size exclusion chromatography; AEX: anion exchange chromatography; LoC: lab-on-a-chip; and MALDI-TOF: matrix-assisted laser desorption / ionization-time-of-flight type.

[0041] As used herein, the term “hydrophobic interaction chromatography (HIC) column” refers to a column comprising a stationary phase or resin and a mobile phase or liquid phase, on which a protein is separated from impurities, including fragments and aggregates of the target protein, other contaminants such as other proteins or protein fragments and cell flakes, or residual impurities from other purification steps, by hydrophobic interactions between the protein and its hydrophobic groups relative to the stationary phase or resin. The stationary phase or resin comprises a substrate matrix or support to which hydrophobic ligands are bound, such as cross-linked agarose, silica, or synthetic copolymer material. Examples of such stationary phases or resins include agarose, silica, or other synthetic polymers substituted with phenyl, butyl, octyl, hexyl, and other alkyl groups. The column may be of any size including the stationary phase or may be processed in open and batch processes. In some embodiments, recombinant alkaline phosphatases are isolated from cell cultures using HIC.

[0042] As used herein, the term “preparation” refers to a solution containing the protein of interest (e.g., recombinant alkaline phosphatase as described herein) and at least one impurity from a cell culture that produces the protein of interest, and / or a solution used to extract, concentrate and / or purify such protein of interest from the cell culture. For example, a preparation of the protein of interest (e.g., recombinant alkaline phosphatase as described herein) may be prepared by growing cells in a cell culture that produce such protein of interest and homogenizing them in a homogenizing solution. In some embodiments, the preparation is then subjected to one or more purification / isolation processes, such as a chromatography step.

[0043] As used herein, the term “solution” refers to a homogeneous molecular mixture of two or more substances in liquid form. More specifically, in some embodiments, the purified protein, e.g., recombinant alkaline phosphatase or its fusion protein in this disclosure (e.g., asphotase alpha), represents one substance in the solution. The term “buffer” or “buffer solution” refers to a solution that resists changes in pH due to the action of its conjugate acid-base range. Examples of buffers that control pH in the range of approximately pH 5 to approximately pH 7 include hepes, citrates, phosphates, acetates, and other mineral acid or organic acid buffers, and combinations thereof. The cations of the salts include sodium, ammonium, and potassium. As used herein, the term “loading buffer / solution” or “equilibrium buffer / solution” refers to a buffer / solution containing a salt or protein preparation that is mixed with a protein preparation to load the protein preparation onto a chromatography column, e.g., an HIC column. This buffer / solution is also used to equilibrate the column before loading the protein and to wash the column after loading. "Elution buffer / solution" refers to a buffer / solution used to elute proteins from a column. As used herein, the term "solution" refers to either a water-based buffer or a non-buffered solution.

[0044] The term "sialic acid" generally refers to N- or O-substituted derivatives of neuraminic acid, which are monosaccharides having a 9-carbon skeleton. Sialic acid may also specifically refer to N-acetylneuraminic acid compounds, sometimes abbreviated as Neu5Ac or NANA. The presence of sialic acid can affect absorption, serum half-life, and elimination of glycoproteins from serum, as well as the physical, chemical, and immunogenic properties of glycoproteins. In some embodiments of this disclosure, sialic acid associated with alkaline phosphatases, such as asfotase alpha, affects the half-life of the molecule under physiological conditions. In some embodiments, precise and predictable control of the total sialic acid content (TSAC) of asfotase alpha serves as a definitive quality characteristic for recombinant asfotase alpha. In some embodiments, TSAC is 1.2 to 3.0 mol / mol of asfotase alpha monomers. In some embodiments, TSAC is generated in a recombinant protein synthesis process in a bioreactor. In some embodiments, the disclosure provides a method for controlling the total sialic acid content (TSAC) in recombinant proteins containing TSAC through mammalian cell cultures, comprising at least one purification step and at least one chromatography step. In some embodiments, the purification and chromatography steps result in a decrease in glycosidase activity, and therefore an increase in the total sialic acid content of the recombinant protein.

[0045] The term “sialylation” refers to a specific type of glycosylation, such as the addition of one or more sialic acid molecules to a biomolecule, particularly the addition of one or more sialic acid molecules to a protein. In some embodiments of this disclosure, sialylation is carried out by a sialyltransferase enzyme. In some embodiments, the sialyltransferase adds sialic acid to the N or O linked glycans of nascent oligosaccharides and / or glycoproteins. In some embodiments, the sialyltransferase is naturally present in cells producing recombinant alkaline phosphatase. In some embodiments, the sialyltransferase is present in cell culture media and / or nutrient supplements used to culture cells producing recombinant alkaline phosphatase. In some embodiments, the sialyltransferase is recombinantly produced using recombinant protein expression methods known in the art. In some embodiments, the recombinant sialyltransferase, produced separately from the recombinant alkaline phosphatase, is exogenously added to cell cultures, collected clarified culture media (HCCF) and / or filtration pools.

[0046] In some embodiments of this disclosure, the sialic acid group is removed from the glycoprotein by hydrolysis (e.g., “desialylation”). In some embodiments, desialylation is carried out by a glycosidase enzyme. A “glycosidase,” also referred to herein as a “glycoside hydrolase,” is an enzyme that catalyzes the hydrolysis of the bond linking the sugar of a glycoside to an alcohol or another sugar unit. Examples of glycosidases include amylase, xylanase, cellulase, and sialidase. In some embodiments, desialylation is carried out by a sialidase enzyme. In some embodiments, sialidase hydrolyzes the glycosidic bond of the terminal sialic acid residue in glycoproteins, glycolipids, oligosaccharides, colomic acid, and / or synthetic substrates. In some embodiments, sialidase is present in a cell medium that produces recombinant alkaline phosphatase. In some embodiments, sialidase activity is dependent on and / or correlates with the total protein concentration. In some embodiments, sialidase is essentially inactive until it reaches a critically high protein concentration at which point it becomes activated. In some embodiments, sialidase is present in the HCCF or filtration pool of a cell culture producing recombinant alkaline phosphatase. In some embodiments, sialidase removes the sialic acid moiety from the glycosylation site of recombinant alkaline phosphatase, e.g., asfotase alpha, effectively reducing the TSAC of the recombinant alkaline phosphatase. In some embodiments, sialidase is selectively removed from the cell culture, HCCF and / or filtration pool. Sialidase can be selectively removed by one or a combination of, for example, sialidase-specific inhibitors, antibodies, ion exchange and / or affinity chromatography, immunoprecipitation, etc. For an overview of how bioprocess conditions affect the sialic acid content of proteins, see Gramer et al., Biotechnol. Prog. 9(4):366-373 (1993) (the disclosure is incorporated herein by reference in its entirety).In some embodiments, the disclosure provides a method for controlling glycosidase activity in a mammalian cell culture producing recombinant protein, comprising at least one purification and at least one chromatography step. In some embodiments, the purification and chromatography steps result in a decrease in glycosidase activity, and therefore an increase in the total sialic acid content of the recombinant protein.

[0047] The term "Cleared and Cleared Culture Medium," abbreviated as HCCF, refers to the cleared filtrate collected from a cell culture, such as a cell culture in a bioreactor. HCCF typically does not contain cells and cell fragments (e.g., insoluble biomolecules) that may be present in the cell culture. In some embodiments of this disclosure, HCCF is produced through centrifugation, deep filtration, sterile filtration, and / or chromatography. In some embodiments, a cell culture from a bioreactor is first centrifuged and / or filtered, and then subjected to at least one chromatographic step, in order to produce HCCF. In some embodiments, HCCF is concentrated before and / or after at least one chromatographic step. In some embodiments, HCCF is diluted after at least one chromatographic step. In some embodiments, HCCF from a cell culture producing recombinant alkaline phosphatase contains recombinant alkaline phosphatase and contaminant proteins. In some embodiments, the contaminant proteins in HCCF include sialidase enzymes.

[0048] The terms “filtration” and “flow filtration” refer to pressure-driven processes that use membranes to separate components in a solution or suspension based on their size and charge difference. Flow filtration can be conventional flow filtration or “tangential flow filtration,” also known as TFF or cross-flow filtration. TFF is typically used to clarify, concentrate, and purify proteins. During the TFF process, the fluid is pumped tangentially along the surface of at least one membrane. The applied pressure functions to push a portion of the fluid downstream through the membrane as “filtrate.” Particulate matter and macromolecules that are too large to penetrate the membrane pores are retained upstream as “residue.” TFF can be used in various forms, including, for example, microfiltration, ultrafiltration (including viral filtration and high-performance TFF), reverse osmosis, nanofiltration, and diafiltration. In some embodiments of this disclosure, one or more forms of TFF are used in combination for the processing and / or purification of proteins. In some embodiments, ultrafiltration and diafiltration are used in combination for the purification of recombinant alkaline phosphatases. Ultrafiltration and diafiltration are described herein.

[0049] Ultrafiltration (UF) is a purification process used to separate proteins from buffer components in buffer exchange, desalting, or concentration. Depending on the protein being retained, a membrane molecular weight limit ranging from approximately 1 kD to approximately 1000 kD is used. In some embodiments, UF is a TFF process.

[0050] Diafiltration, or DF, is a purification process that washes smaller molecules through a membrane, leaving larger molecules in the holding solution, thus ultimately maintaining their concentration. Typically, DF is used in conjunction with another purification process to increase product yield and / or purity. In DF, a solution (e.g., water or buffer) is introduced into a sample reservoir, while the filtrate is removed from the unit operation. In processes where the desired product is present in the holding solution, diafiltration washes the components from the product pool into the filtrate, thereby exchanging the buffer and reducing the concentration of undesirable species. When the product is present in the filtrate, diafiltration washes it through the membrane into a collection container. In some embodiments, DF is a TFF process.

[0051] The term "filtration pool," sometimes also referred to as "UFDF pool" or simply "UFDF," refers to the total volume of fluid from a filtration process, typically from a combined ultrafiltration / diafiltration (UF / DF) process. In the context of protein purification, UFDF refers to the retained fluid from the ultrafiltration / diafiltration process. [Brief explanation of the drawing]

[0052] [Figure 1] This is a flowchart showing the process for producing recombinant alkaline phosphatases, such as asfotase alpha. [Figure 2] This graph shows the TSAC content of asfotase alpha in the collection of data from multiple batches, the protein A pool step, and the final BDS. [Figure 3] This graph shows the TSAC content of asfotase alfa at 7 days post-vaccination, at the collection step, and at the final BDS for multiple batches. [Modes for carrying out the invention]

[0053] This disclosure provides an improved method for producing recombinant glycoproteins, such as alkaline phosphatases (e.g., asphotase alpha), which provides improved quality control over the TSAC concentration in the final product by measuring the total sialic acid content (TSAC) concentration during fermentation and adjusting downstream production steps according to the TSAC concentration measurement. The method allows for the regulation of the TSAC in the final product by using a dynamic control method that responds to a potentially variable range of TSAC levels from the bioreactor cell culture output. Ultimately, the method provides homogeneous properties of recombinant alkaline phosphatases for commercial production. The method described herein provides a resulting product in which the TSAC of the final product is tightly controlled over a range of input bioreactor aqueous medium TSAC levels.

[0054] Manufacturing method The methods described herein include the steps of: inoculating cells expressing recombinant alkaline phosphatase (e.g., mammalian cells, e.g., Chinese hamster ovary (CHO) cells) into a bioreactor; obtaining an aqueous medium containing recombinant alkaline phosphatase; obtaining a certain volume from the aqueous medium about 6 to 10 days after inoculation, particularly about 6 to 8 days (e.g., about 6, 7, 8, 9, 10 days, e.g., about 7 days); quantifying the total sialic acid content (TSAC) molar concentration per mole of recombinant alkaline phosphatase in the certain volume; collecting the aqueous medium; and performing a filtration step (e.g., ultrafiltration, diafiltration, or a combination thereof) to obtain a bulk drug solution (BDS).

[0055] During fermentation, a certain volume of the culture medium is used to determine the length of time the filtration step is maintained. For example, if the volume has a TSAC concentration of less than approximately 2.5 mol / mol, the filtration step may be maintained for less than approximately 9 hours. If the volume has a TSAC concentration of approximately 2.5 mol / mol to approximately 2.7 mol / mol, the filtration step may be maintained for approximately 10 to 14 hours. If the volume has a TSAC concentration of approximately 2.8 mol / mol to approximately 3.0 mol / mol, the filtration step may be maintained for approximately 23 to 27 hours. If the volume has a TSAC concentration greater than approximately 3.0 mol / mol, the filtration step may be maintained for approximately 38 to 42 hours.

[0056] In one embodiment, if a certain volume has a TSAC concentration of about 2.3 mol / mol or less, the filtration step may be held for about 18 ± 4 hours. If a certain volume has a TSAC concentration of about 2.4 mol / mol to about 3.1 mol / mol, the filtration step may be held for about 32 ± 4 hours. If a certain volume has a TSAC concentration of about 3.2 mol / mol or more, the filtration step may be held for about 44 ± 4 hours.

[0057] In another alternative embodiment, if a given volume has a TSAC concentration of less than about 2.4 mol / mol, the filtration step may be held for about 17 ± 3 hours. If a given volume has a TSAC concentration of about 2.4 mol / mol to about 3.6 mol / mol, the filtration step may be held for about 31 ± 3 hours. If a given volume has a TSAC concentration greater than about 3.6 mol / mol, the filtration step may be held for about 45 ± 3 hours.

[0058] This method allows for the production of BDS with controlled TSAC concentrations in the range of approximately 1.2 mol / mol to approximately 3.0 mol / mol (e.g., approximately 1.6 mol / mol to approximately 2.4 mol / mol). This range of TSAC concentrations provides a commercially viable bulk sample of recombinant alkaline phosphatase that is stable (e.g., therapeutically effective half-life) and enzymatically active for use in human patients.

[0059] The alkaline phosphatase proteins described herein (e.g., asphotase alpha) can be produced by mammalian or other cells, particularly CHO cells, using methods known in the art. Such cells can be grown in culture dishes, flasks, or bioreactors. Specific processes for cell culture and recombinant protein production are known in the art and are described, for example, 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-boil, and continuous reactors. In some embodiments, the alkaline phosphatase proteins are produced in a fed-boil bioreactor.

[0060] Cell culture processes are subject to variability caused by fluctuating physicochemical environments, including, but not limited to, changes in pH, temperature, temperature changes, timing of temperature changes, cell medium composition, cell culture nutritional supplements, lot-to-lot variations in raw materials, media filtration materials, differences in bioreactor size, and gas treatment methods (air, oxygen, and carbon dioxide). As disclosed herein, the yield, relative activity properties, and glycosylation properties of the produced alkaline phosphatase protein may be impaired and can be controlled within a range of specific values ​​by changing one or more of these parameters.

[0061] In recombinant protein production in cell cultures, recombinant genes, along with necessary transcription factors, are first transferred to host cells using known biotechnology techniques. A second gene, which provides a selective advantage to recipient cells, is then introduced selectively. A few days after gene introduction, only cells expressing the selected gene survive in the presence of a potentially applicable selective agent. Two exemplary genes for such selection are dihydrofolate reductase (DHFR) and glutamine synthetase (GS), enzymes involved in nucleotide metabolism. In both cases, selection occurs in the absence of appropriate metabolites (hypoxanthine and thymidine in the case of DHFR, and glutamine in the case of GS), and the proliferation of any non-transformed cells is inhibited. Generally, for efficient recombinant protein expression, it is not important whether the gene encoding the biologic and the selection gene reside on the same plasmid.

[0062] After selection, surviving cells may be introduced as single cells into a second culture vessel, and the culture is expanded to generate a clonal population. Finally, individual clones are evaluated for recombinant protein expression, and the best producers are retained for further culture and analysis. From these candidates, one cell line with suitable growth and productivity characteristics is selected for recombinant protein production. Next, a culture process is developed, determined by the production demand and the requirements of the final product.

[0063] cell Any mammalian or non-mammalian cell type can be cultured to produce polypeptides, and may be used in accordance with this disclosure. Non-limiting examples of mammalian cells that may be used include, for example, Chinese hamster ovary cells + / -DHFR (CHO, Urlaub and Chasin, 1980 Proc. Natl. Acad. Sci. USA, 77:4216); BALB / c mouse myeloma cell line (NSO / 1, ECACC acceptance number: 85110503); human retinoblastoma (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell 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 Examples include CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-I 587); human cervical cancer cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB8065); mouse mammary gland tumors (MMT060562, ATCC CCL 51); TRI cells (Mather et al., 1982, Annals NYAcad. Sci. 383:44-68); MRC5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In certain embodiments, polypeptide and protein culture and expression are performed from Chinese hamster ovary (CHO) cell lines.

[0064] In addition, any number of commercially available and uncommercial hybridoma cell lines expressing polypeptides or proteins may be used in accordance with this disclosure. Those skilled in the art will understand that hybridoma cell lines may have different nutritional requirements and / or require different culture conditions for optimal growth and polypeptide or protein expression, and will be able to modify these conditions as necessary.

[0065] Seeding density In this disclosure, Chinese hamster ovary (CHO) cells are inoculated, i.e., seeded, into a culture medium. Various seeding densities can be used. In some embodiments, seeding densities of 1.0 × 10² cells / mL to 1.0 × 10⁹ cells / mL (e.g., 1.0 × 10³ cells / mL to 1.0 × 10⁸, e.g., 1.0 × 10⁴ cells / mL to 1.0 × 10⁷) can be used. In some embodiments, seeding densities of 1.0 × 10⁵ cells / mL to 1.0 × 10⁶ cells / mL can be used. In some embodiments, seeding densities of 4.0 × 10⁵ cells / mL to 8.0 × 10⁵ cells / mL can be used. In some embodiments, increasing the seeding density may affect the fragmentation of asfotase alpha quality when measured by SEC. In some embodiments, the seeding density is controlled at inoculation to reduce the risk of fragmentation.

[0066] temperature Temperature may have an effect on several parameters, including growth rate, aggregation, fragmentation, and TSAC. In some embodiments, the temperature remains constant when culturing CHO cells in the culture medium. In some embodiments, the temperature when culturing CHO cells in the culture medium is about 30°C to about 40°C, or about 35°C to about 40°C, or about 37°C to about 39°C. In some embodiments, when culturing CHO cells in the culture medium, the temperature is 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. In some embodiments, the temperature remains constant for 40 to 200 hours after inoculation. In some embodiments, the temperature remains constant for 50–150 hours, 60–140 hours, 70–130 hours, 80–120 hours, or 90–110 hours after inoculation. In some embodiments, the temperature remains constant for 80–120 hours after inoculation. In some embodiments, the temperature remains constant for 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 hours after inoculation.

[0067] Temperature change The execution time of cell culture processes, particularly discontinuous processes (e.g., fed-batch processes in bioreactors), is typically limited by the remaining viability of cells, which usually decreases over the execution period. Therefore, extending the time in which cells remain viable is desirable to improve recombinant protein production. Interest in product quality also provides an incentive to minimize the decrease in viable cell density and maintain high cell viability, as cell death can release sialidase into the culture supernatant, potentially reducing the sialic acid content of expressed proteins. Interest in protein purification provides yet another incentive: minimizing the decrease in viable cell density and maintaining high cell viability. Cell debris and dead cell contents in the culture can negatively impact the ability to isolate and / or purify protein products at the end of the culture run. Therefore, maintaining cells viable for longer periods in culture can lead to a decrease in culture medium contamination by cellular proteins and enzymes (e.g., cellular proteases and sialidases), which can result in degradation and ultimately a decline in the quality of desired glycoproteins produced by the cells.

[0068] Many methods can be applied to achieve high cell viability in cell cultures. One involves lowering the culture temperature after initial culture at normal temperature. See, for example, Ressler et al., 1996, Enzyme and Microbial Technology 18:423-427). Generally, mammalian or other cell types capable of expressing the protein of interest are first grown at normal temperature to increase cell number. Such “normal” temperature for each cell type is generally about 37°C (e.g., about 35°C to about 39°C, e.g., 35.0°C, 35.5°C, 36.0°C, 36.5°C, 37.0°C, 37.5°C, 38.0°C, 38.5°C and / or 39.0°C). In a particular embodiment, the temperature for producing asfotase alpha is initially set to about 37°C. When a moderately high cell density is achieved, the culture temperature in the entire cell culture may be altered (e.g., reduced) to promote protein production. In most cases, the decrease in temperature causes cells to shift towards the non-proliferating G1 portion of the cell cycle, which can increase cell density and viability compared to the previous higher temperature environment. Furthermore, lower temperatures may also promote recombinant protein production by increasing the rate of cellular protein production, promoting post-translational modification of proteins (e.g., glycosylation), reducing fragmentation or aggregation of newly produced proteins, promoting protein folding and three-dimensional structure formation (and thus maintaining activity), and / or reducing the degradation of newly produced proteins. In some embodiments, the temperature is reduced by 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, or 10°C. In some embodiments, the temperature is reduced to approximately 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C. In some embodiments, lower temperatures are approximately 30°C to approximately 35°C (e.g., 30.0°C, 30.5°C, 31.0°C, 31.5°C, 32.0°C, 32.5°C, 33.0°C, 33.5°C, 34.0°C, 34.5°C, and / or 35.0°C). In other embodiments, the temperature for producing asfotase alpha is initially set to approximately 35.0°C to approximately 39.0°C, and then changed to approximately 30.0°C to approximately 35.0°C.In one embodiment, the temperature for producing asfotase alpha is initially set to approximately 37.0°C and then changed to approximately 30°C. In another embodiment, the temperature for producing asfotase alpha is initially set to approximately 36.5°C and then changed to approximately 33°C. In yet another embodiment, the temperature for producing asfotase alpha is initially set to approximately 37.0°C and then changed to approximately 33°C. In yet another embodiment, the temperature for producing asfotase alpha is initially set to approximately 36.5°C and then changed to approximately 30°C. In other embodiments, multiple (e.g., two or more) steps of temperature change may be applied.

[0069] Before changing to a different temperature, the time to maintain the culture at a particular temperature can be determined to achieve a sufficient (or desired) cell density while maintaining cell viability and the ability to produce the target protein. In some embodiments, the cell culture is maintained at an initial temperature with a viable cell density of approximately 10⁵ cells / mL to approximately 10⁷ cells / mL (e.g., 1×10⁵, 1.5×10⁵, 2.0×10⁵, 2.5×10⁵, 3.0×10⁵, 3.5×10⁵, 4.0×10⁵, 4.5×10⁵, 5.0×10⁵, 5.5×10⁵, 6.0×10⁵, 6.5×10⁵, 7.0×10⁵, 7.5×10⁵, 8.0×10⁵, 8.5×10⁵) The cells are grown until they reach 9.0×10⁵, 9.5×10⁵, 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, 7.0×10⁶, 7.5×10⁶, 8.0×10⁶, 8.5×10⁶, 9.0×10⁶, 9.5×10⁶, or 1×10⁷ cells / mL or more. In one embodiment, the cell culture is grown at the initial temperature until the viable cell density reaches approximately 2.5 to approximately 3.4×10⁶ cells / mL before being changed to a different temperature. In another embodiment, the cell culture is grown at the initial temperature until the viable cell density reaches approximately 2.5 to approximately 3.2 × 10⁶ cells / mL before being changed to a different temperature. In yet another embodiment, the cell culture is grown at the initial temperature until the viable cell density reaches approximately 2.5 to approximately 2.8 × 10⁶ cells / mL before being changed to a different temperature.

[0070] In some embodiments, the method of the present disclosure provides that the temperature change occurs 50–150 hours, or 60–140 hours, or 70–130 hours, or 80–120 hours, or 90–110 hours after inoculation. In some embodiments, the method of the present disclosure provides that the temperature decreases about 80–150 hours, about 90–100 hours, or about 96 hours after inoculation. In some embodiments, the temperature change occurs 80–120 hours after inoculation. In some embodiments, the temperature change occurs 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, or 110 hours after inoculation. In some embodiments, the temperature after the temperature change is maintained until the CHO cells are collected.

[0071] pH Changes in the pH of the growth medium under cell culture can affect the degradation activity, secretion, and protein production levels of cellular proteins. Most cell lines grow well at approximately pH 7–8. While the variation in optimal pH for cell proliferation is relatively small across various cell lines, the behavior of some normal fibroblast cell lines is best at pH 7.0–7.7, and the behavior of transformed cells is typically best at pH 7.0–7.4 (Eagle, J Cell Physiol 82:1-8, 1973). In some embodiments, the pH of the culture medium for producing asfotase alpha is approximately 6.5 to 7.7 (e.g., 6.50, 6.55, 6.60, 6.65, 6.70, 6.75, 6.80, 6.85, 6.90, 6.95, 7.00, 7.05, 7.10, 7.15, 7.20, 7.25, 7.30, 7.35, 7.39, 7.40, 7.45, 7.50, 7.55, 7.60, 7.65, or 7.70).

[0072] Culture medium In some embodiments, batch culture is used, in which no additional medium is added after inoculation. In some embodiments, fed-batch culture is used, in which medium is added after inoculation in one or more bolus doses. In some embodiments, medium is added after inoculation in two, three, four, five, or six bolus doses.

[0073] In various embodiments, alkaline phosphatase (e.g., asphotase alfa) is produced by a process in which culture medium is added to the production bioreactor in extra bolus doses. For example, the medium may be added in 1, 2, 3, 4, 5, 6 or more bolus doses. In one particular embodiment, the medium is added in 3 bolus doses. In various embodiments, such extra bolus doses of the medium may be added in varying amounts. For example, such bolus doses of culture medium may be added in amounts of approximately 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 a particular embodiment, such bolus doses of culture medium may be added in amounts of approximately 33%, 67%, 100%, or 133% of the original volume. In various embodiments, such addition of extra bolus doses may be made at various points in time during the period of cell proliferation or protein production. For example, a bolus dose may be added on 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 in the process. In a particular embodiment, such a bolus dose of the medium may be added every other day (for example, (1) on days 3, 5, and 7, (2) on days 4, 6, and 8, or (3) on days 5, 7, and 9). In fact, the frequency, amount, timing, and other parameters of the adjuvant in the bolus dose of the medium can be freely combined according to the above limitations and may be determined by experimental practice.

[0074] Various culture media are commercially available. In some embodiments, the culture medium is selected from the group consisting of EX-CELL® 302 serum-free medium, CD DG44 medium, BD Select® medium, SFM4CHO medium, or a combination thereof. In some embodiments, the culture medium includes a commercially available medium, for example, a combination of SFM4CHO medium and BD Select® medium. In some embodiments, the culture medium includes a commercially available medium, for example, a combination of SFM4CHO medium and BD Select® medium, in a ratio selected from 90 / 10, 80 / 20, 75 / 25, 70 / 30, 60 / 40, or 50 / 50.

[0075] Nutritional supplements Various nutritional supplements, also known as “feed media,” are commercially available and known to those skilled in the art. Nutritional supplements include media (different from the culture medium) that are added to cell cultures after inoculation. In some cases, nutritional supplements can be used to substitute nutrients consumed by cells growing under culture. In some embodiments, nutritional supplements are added to optimize the production of a desired protein or the activity of a desired protein. A very large number of nutritional supplements have been developed and are commercially available. While the explicit purpose of nutritional supplements is to enhance aspects of process development, there is no universal nutritional supplement that works for all cells and / or all proteins produced. The selection of a scalable and appropriate cell culture nutritional supplement that can work in combination with a desired cell line, produced protein, and a given basic medium to achieve desired titer and growth characteristics is not routine. Typical methods of screening multiple commercially available nutritional supplements with a specific cell line, specific produced protein, and basic medium combination to identify the optimal supplement may not be successful due to the countless variables present in the cell culture process. In some embodiments, the nutritional supplement is selected from the group consisting of Efficient Feed C+AGT® Supplement (Thermo Fisher Scientific, Waltham, MA), combination of CELL BOOST® 2+CELL BOOST® 4 (GE Healthcare, Sweden), 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 number C1615), or combinations thereof.

[0076] Cell Boost® 7a can be described as a first animal-free (ADCF) nutritional supplement containing one or more amino acids, vitamins, salts, trace elements, poloxamers, and glucose, where the first ADCF nutritional supplement does not contain hypoxanthine, thymidine, insulin, L-glutamine, growth factors, peptides, proteins, hydrolysates, phenol red, and 2-mercaptoethanol. Cell Boost® 7a is a chemically defined supplement. The terms “animal-free” or “ADCF” refer to supplements whose components do not directly originate from animal sources, such as bovine sources. In some embodiments, the nutritional supplement is Cell Boost® 7a.

[0077] Cell Boost® 7b can be described as a second ADCF nutritional supplement containing one or more amino acids, the second ADCF nutritional supplement being deficient in hypoxanthine, thymidine, insulin, L-glutamine, growth factors, peptides, proteins, hydrolysates, phenol red, 2-mercaptoethanol, and poloxamer. Cell Boost® 7b is a chemically defined supplement. In some embodiments, the nutritional supplement is Cell Boost® 7b.

[0078] In some embodiments, a combination of commercially available nutritional supplements is used. The term "nutritional supplement" refers to both a single nutritional supplement and a combination of nutritional supplements. For example, in some embodiments, the combination of nutritional supplements includes a combination of Cell Boost® 7a and Cell Boost® 7b.

[0079] In various embodiments, alkaline phosphatase (e.g., asphotase alfa) is produced by a process in which an excess additive of the nutritional supplement is added to the production bioreactor. In some embodiments, the nutritional supplement is added over a period of time, for example, ranging from 1 minute to 2 hours. In some embodiments, the nutritional supplement is added in a bolus dose. For example, the nutritional supplement may be added in 1, 2, 3, 4, 5, 6 or more bolus doses. In some embodiments, the nutritional supplement is added at three or more different time points, for example, at 2 to 6 different time points. In various embodiments, such an excess bolus dose of the nutritional supplement may be added in varying amounts. For example, such a bolus dose of the nutritional supplement may be added in amounts of about 1% to 20%, 1% to 10%, or 1% to 5% (w / v) of the original volume of the culture medium in the production bioreactor. In one particular embodiment, such a bolus administration of the nutritional supplement may be added in amounts of 1% to 20%, 1% to 10%, or 1% to 5% (w / v) of the original volume.

[0080] In some embodiments, a combination of nutrient supplements is used, in which a first nutrient supplement, such as Cell Boost® 7a, is added to the culture medium at a concentration of 0.5% to 4% (w / v). In some embodiments, a combination of nutrient supplements is used, in which a second nutrient supplement, such as Cell Boost® 7b, is added to the culture medium at a concentration of 0.05% to 0.8% (w / v). In certain embodiments where the combination of nutrient supplements includes Cell Boost® 7a and Cell Boost® 7b, a bolus of the 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.

[0081] In various embodiments, such addition of an additional bolus may be made at various points in time after inoculation. For example, the bolus may be added 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 days after inoculation, or thereafter. In fact, the frequency, amount, timing, and other parameters of bolus addition of nutritional supplements can be freely combined according to the above limitations and may be determined by experimental implementation.

[0082] In some embodiments, the methods disclosed herein further include adding zinc to the culture medium during the preparation of recombinant polypeptides. In some embodiments, zinc may be added to yield a zinc concentration of about 1 to about 300 μM in the culture medium. In one embodiment, zinc may be added to yield a zinc concentration of about 10 to about 200 μM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 μM) in the culture medium. In some embodiments, zinc is added to yield a zinc concentration in the culture medium of about 25 μM to about 150 μM or about 60 μM to about 150 μM. In one embodiment, zinc is added to yield a zinc concentration in the culture medium of about 30, 60, or 90 μM. In some embodiments, zinc is added to the culture medium in bolus doses, continuously, semi-continuously, or in combination thereof. In some embodiments, zinc is added 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and / or 13 days after inoculation.

[0083] collection Previous studies have shown that delays in collection timing are associated with decreased viability and TSAC, suggesting that collection timing may have a potential impact on other CQAs. In various embodiments, alkaline phosphatase (e.g., asfotase alfa) is collected at approximately 200, 210, 220, 230, 240, 250, 260, 264, 270, 280, 288 hours (e.g., 12 days) or after 12 days.

[0084] Downstream processes As used herein, the term “downstream process” generally refers to all or part of the process for the recovery and purification of alkaline phosphatases (e.g., asfotase alpha) produced from a source such as cultured cells or fermented culture medium.

[0085] Generally, downstream processing yields the product from its native state to components of tissues, cells, or fermentation cultures through progressive improvements in purity and concentration. For example, the removal of insoluble materials may be a first step involving the capture of the product as a solute in a liquid free of particulate matter (e.g., separating cells, cell fragments, or other particulate matter from the fermentation culture). Exemplary operations to achieve this include, for example, filtration, centrifugation, sedimentation, siliceous sedimentation, electrostatic precipitation, and gravity sedimentation. Additional operations may include, for example, grinding, homogenization, or leaching to recover the product from solid sources, such as plant and animal tissues. A second step may be a “product isolation” step, which removes components whose properties differ significantly from those of the desired product. In most products, water is the major impurity, and the isolation step is designed to remove most of it, reduce the volume of the material being processed, and concentrate the product. Solvent extraction, adsorption, ultrafiltration, and sedimentation may be used alone or in combination in this step. The next step involves product purification to separate contaminants that closely resemble the product in terms of physical and chemical properties. Possible purification methods include, for example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, mixed-mode chromatography, size exclusion, reverse-phase chromatography, ultrafiltration-diafiltration, crystallization, and fractional precipitation. In some embodiments, the downstream process includes at least one of collection clarification, ultrafiltration, diafiltration, virus inactivation, affinity capture, and a combination thereof. The downstream processes are described herein.

[0086] Measurement of total sialic acid content In some embodiments, the method described herein further includes measuring the total sialic acid content (TSAC) of recombinant alkaline phosphatase from a certain volume removed from the culture medium, for example, on about 6 to about 10 days, particularly on about 6 to about 8 days (e.g., about 6, 7, 8, 9, 10, e.g., 7 days). The certain volume can be obtained sterilely from a bioreactor to prevent contamination. The certain volume may be about 1 mL to about 1000 mL (e.g., about 25 mL to about 500 mL, e.g., about 50 mL to about 300 mL, e.g., about 100 mL or about 200 mL). Obtaining the certain volume may further include centrifugation of the certain volume and / or removal of the supernatant from the certain volume. This step may be performed using a chromatography column (e.g., a Protein A column, a 1 cm Protein A column, e.g., a 1 mL HiTrap Protein A column or a 600 μL Protein A column) This may also include purifying the alkaline phosphatase from the supernatant using Robocolumn. In some embodiments, the alkaline phosphatase may be subjected to buffer exchange. The alkaline phosphatase may also be concentrated, for example, before measuring the TSAC concentration.

[0087] For example, commercial methods for carbohydrate quantification from ThermoFisher are available. Generally, TSAC is released from glycoproteins, such as asfotase alpha, using acid hydrolysis, and the released sugar / TSAC is detected via electrochemical detection using column chromatography such as high-performance anion exchange chromatography (HPAE-PAD) with amperometric electrochemical detection. The resulting levels are quantified per mole against an internal standard and expressed as a function of total moles of protein.

[0088] As described herein, TSAC affects the half-life of recombinant alkaline phosphatase under physiological conditions and therefore functions as a critical quality characteristic for recombinant alkaline phosphatases, such as asfotase alfa. Strict control of the TSAC range is important for reproducibility and cGMP. In some embodiments, TSAC is about 0.8 mol / mol to about 4.0 mol / mol of recombinant alkaline phosphatase. In some embodiments, TSAC is about 0.9 mol / mol to about 3.0 mol / mol of recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.0 mol / mol to about 2.8 mol / mol of recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.2 mol / mol to about 3.0 mol / mol of recombinant alkaline phosphatase. In some embodiments, TSAC is about 1.2 mol / mol to about 2.4 mol / mol of recombinant alkaline phosphatase. In some embodiments, TSAC is a recombinant alkaline phosphatase in concentrations of 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.

[0089] In some embodiments, the TSAC of recombinant alkaline phosphatase decreases during downstream processing. In some embodiments, the TSAC of recombinant alkaline phosphatase decreases as a result of the presence of sialidase enzyme in solutions containing recombinant alkaline phosphatase, such as cell cultures, HCCF, and / or UFDF filtration pools. In some embodiments, sialidase is selectively removed from cell cultures, HCCF, and / or UFDF filtration pools to obtain a TSAC of recombinant alkaline phosphatase of about 0.9 mol / mol to about 3.0 mol / mol. Sialidase can be selectively removed by one or a combination of, for example, sialidase-specific inhibitors, antibodies, ion exchange and / or affinity chromatography, immunoprecipitation, etc.

[0090] In some embodiments, the sialic acid portion is added to the recombinant alkaline phosphatase by a sialyltransferase enzyme present in a solution containing recombinant alkaline phosphatase, such as a cell culture, HCCF, and / or UFDF filtration pool. In some embodiments, recombinant sialyltransferase is added exogenously to the cell culture, HCCF, and / or UFDF filtration pool to obtain a TSAC of about 0.9 to about 3.0 mol / mol of recombinant alkaline phosphatase.

[0091] Measurement of recombinant alkaline phosphatase activity In some embodiments, the method described herein further includes measuring recombinant alkaline phosphatase activity. In some embodiments, the activity is selected from a method selected from at least one of a pNPP-based alkaline phosphatase enzyme assay and an inorganic pyrophosphate (PPi) hydrolysis assay. In some embodiments, at least one of the recombinant alkaline phosphatase Kcat and Km values ​​is increased in the inorganic pyrophosphate (PPi) hydrolysis assay. In some embodiments, the method includes determining the integral viable cell concentration (IVCC).

[0092] The final step may be a process that concludes with product polishing, i.e., packaging the product in a stable, easily transportable, and convenient form. Storage at 2–8°C, freezing at -20–-80°C, crystallization, drying, freeze-drying, and spray-drying are exemplary methods in this final step. Product polishing may further sterilize the product and remove or deactivate trace contaminants (e.g., viruses, endotoxins, metabolic waste products, and pyrogens) that may impair the safety of the product, depending on the product and its intended use.

[0093] Product recovery methods can combine two or more steps discussed herein. For example, in an adsorption fluidized bed (EBA), removal of insoluble matter and product isolation are performed in a single step. For a review of EBA, see Kennedy, Curr Protoc Protein Sci. 2005 Jun; Chapter 8: Unit 8.8. Furthermore, in affinity chromatography, isolation and purification are often performed in a single step.

[0094] For a review of downstream processes for purifying recombinant proteins produced in cultured cells, see Rea, 2008 Solutions for Purification of Fc-fusion Proteins. BioPharm Int. Supplements March 2:20-25. The downstream processes for alkaline phosphatases disclosed herein may include at least one or any combination of the following exemplary steps.

[0095] Collection and clarification process In some embodiments of this method, recombinant alkaline phosphatase is isolated from the cell culture by at least one purification step, e.g., a “collecting” step or a collection clarification step, to form a collection clarified culture medium (HCCF). “Collecting” the cell culture typically refers to the process of collecting the cell culture from a culture vessel, e.g., a bioreactor. In some embodiments, the at least one purification step includes at least one of filtration, centrifugation, and a combination thereof. In some embodiments, the collection clarification step includes centrifugation and / or filtration of the collected cell culture to remove cells and cell fragments (e.g., insoluble biomaterials) and recovery of the product, e.g., recombinant alkaline phosphatase. In some embodiments, cells and cell fragments are removed to obtain a clarified filtrate suitable for chromatography. In some embodiments, the clarified filtrate is known as a collection clarified culture medium or HCCF. In some embodiments, the cell culture is subjected to a combination of centrifugation and deep filtration to produce HCCF. The solutions available in this step may include a recovery buffer (e.g., 50 mM sodium phosphate, 100 mM NaCl, pH 7.50). A suitable recovery buffer composition can be selected by those skilled in the art.

[0096] In some embodiments, HCCF has a total sialic acid content (TSAC) of about 1.9 mol / mol to about 3.1 mol / mol. In some embodiments, HCCF has a total sialic acid content (TSAC) of about 1.9 mol / mol to about 4.3 mol / mol. In some embodiments, HCCF has a total sialic acid content (TSAC) of about 2.2 mol / mol to about 3.6 mol / mol. In some embodiments, HCCF has a total sialic acid content (TSAC) of about 2.2 mol / mol to about 3.4 mol / mol. In some embodiments, the HCCF has about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5 moles / mol of TSAC.

[0097] Ultrafiltration and / or diafiltration after collection In some embodiments of this method, additional purification steps are performed after at least one purification step to form a filtration pool also known as a “UFDF pool” or “UFDF”. In some embodiments, at least one purification step is intended to be concentration and buffer dilution. In some embodiments, at least one purification step includes at least one of collection clarification, filtration, ultrafiltration, diafiltration, virus inactivation, affinity capture and a combination thereof. In some embodiments, at least one purification step includes ultrafiltration (UF) and / or diafiltration (DF). Exemplary steps in the UF process include, for example, washing / storage of the filter membrane before use, flushing after washing / storage, equilibration (e.g., using a buffer containing 50 mM sodium phosphate, 100 mM NaCl, pH 7.50), loading, concentration, diafiltration, dilution / flush / recovery (e.g., using a buffer containing 50 mM sodium phosphate, 100 mM NaCl, pH 7.50), and flushing / washing / storage of the filter membrane after use.

[0098] In some embodiments, after UF / DF, the UFDF is diluted to a protein concentration of about 1.7 g / L to about 5.3 g / L and then maintained at about 13°C to about 27°C for about 0 to about 60 hours before storage and / or further purification. When used herein, "holding" or "maintaining" of UFDF refers to UFDF maintained at the same temperature (e.g., within ± about 1°C or within a specified range, e.g., 19 to 25°C) for a target time, e.g., "holding time" (within ± about 2 hours). Details of "holding" or "maintaining" at a constant temperature may depend on the scale of production and the practical considerations of the production scale. In some embodiments, the UFDF is held to serve as a control point in the recombinant alkaline phosphatase production process. In some embodiments, the UFDF is held to ensure uniform product quality. In some embodiments, the UFDF is held to facilitate downstream processing.

[0099] In some embodiments, the TSAC of recombinant alkaline phosphatase decreases during the UFDF retention period. In some embodiments, the decrease in TSAC correlates with the protein concentration, duration, and / or temperature during the UFDF retention period.

[0100] In some embodiments, the UFDF retention time begins immediately after the completion of diafiltration. In some embodiments, the UFDF retention time begins immediately after the completion of the filtration step. In some embodiments, the UFDF retention time begins immediately after the completion of UF / DF. In some embodiments, the UFDF retention time begins immediately after the completion of recirculation at the end of the UF / DF step. In some embodiments, the UFDF retention time begins immediately after the filtration and transfer of the UF / DF product is completed.

[0101] In some embodiments, UFDF is diluted to achieve a desired protein concentration. In some embodiments, UFDF has a protein concentration of about 1.0 g / L to about 6.0 g / L. In some embodiments, UFDF has a protein concentration of about 1.7 g / L to about 5.3 g / L. In some embodiments, UFDF has a protein concentration of about 1.8 g / L to about 5.0 g / L. In some embodiments, UFDF has a protein concentration of about 2.0 g / L to about 5.0 g / L. In some embodiments, UFDF has a protein concentration of about 1.8 g / L to about 4.3 g / L. In some embodiments, UFDF has a protein concentration of about 2.3 g / L to about 4.3 g / L. In some embodiments, UFDF has a protein concentration of about 3.0 g / L to about 4.5 g / L. In some embodiments, UFDF has a protein concentration of about 3.3 g / L to about 4.1 g / L. In some embodiments, UFDF is approximately 1.0 g / L, approximately 1.1 g / L, approximately 1.2 g / L, approximately 1.3 g / L, approximately 1.4 g / L, approximately 1.5 g / L, approximately 1.6 g / L, approximately 1.7 g / L, approximately 1.8 g / L, approximately 1.9 g / L, 2.0 g / L, approximately 2.1 g / L, approximately 2.2 g / L, approximately 2.3 g / L, approximately 2.4 g / L, approximately 2.5 g / L, approximately 2.6 g / L, approximately 2.7 g / L It has a protein concentration of approximately 2.8 g / L, 2.9 g / L, 3.0 g / L, 3.1 g / L, 3.2 g / L, 3.3 g / L, 3.4 g / L, 3.5 g / L, 3.6 g / L, 3.7 g / L, 3.8 g / L, 3.9 g / L, 4.0 g / L, 4.1 g / L, 4.2 g / L, 4.3 g / L, 4.4 g / L, or 4.5 g / L. In some embodiments, UFDF has a protein concentration of approximately 2.3 g / L. In some embodiments, UFDF has a protein concentration of approximately 3.1 g / L. In some embodiments, UFDF has a protein concentration of approximately 3.7 g / L.

[0102] In some embodiments, the UFDF is held for about 1 hour to about 60 hours. For example, the UFDF may be held for about 1 hour (or less) to about 10 hours (e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours). In some embodiments, the UFDF is held for about 10 hours to about 50 hours. In some embodiments, the UFDF is held for about 12 hours to about 48 hours. In some embodiments, the UFDF is held for about 14 hours to about 42 hours. In some embodiments, the UFDF is held for about 17 hours to about 34 hours. In some embodiments, the UFDF is held for about 19 hours to about 33 hours. In some embodiments, the UFDF is held for about 25 hours to about 38 hours. In some embodiments, the UFDF is held for about 29 hours to about 35 hours. In some embodiments, the UFDF is held for approximately 12, 13, 14, 15, 16, 17, 18, 19, or 20 hours. In some embodiments, the UFDF is held for approximately 29, 30, 31, 32, 33, 34, or 35 hours. In some embodiments, the UFDF is held for approximately 42, 43, 44, 45, 46, 47, or 48 hours. In some embodiments, the UFDF is held for approximately 14 to 20 hours. In some embodiments, the UFDF is held for approximately 28 to 34 hours. In some embodiments, the UFDF is held for approximately 42 to 48 hours.

[0103] As described above, the retention time during the filtration step (e.g., UFDF) depends on the TSAC concentration obtained during cell proliferation (e.g., approximately day 6 to day 10, e.g., approximately day 6 to day 8, e.g., day 7). For example, if a given volume has a TSAC concentration of less than approximately 2.5 mol / mol, the filtration step may be retained for less than approximately 9 hours. If a given volume has a TSAC concentration of approximately 2.5 mol / mol to approximately 2.7 mol / mol, the filtration step may be retained for approximately 10 to 14 hours. If a given volume has a TSAC concentration of approximately 2.8 mol / mol to approximately 3.0 mol / mol, the filtration step may be retained for approximately 23 to 27 hours. If a given volume has a TSAC concentration greater than approximately 3.0 mol / mol, the filtration step may be retained for approximately 38 to 42 hours. In some embodiments, the TSAC concentration of a given volume may be less than approximately 2.5 mol / mol, and the filtration step is retained for less than approximately 9 hours. Alternatively, the TSAC concentration can be approximately 2.5 moles / mol to 2.7 moles / mol, and the filtration step is maintained for approximately 10 to 14 hours.

[0104] In an alternative embodiment, if a given volume has a TSAC concentration of about 2.3 mol / mol or less, the filtration step may be held for about 18 ± 4 hours. If a given volume has a TSAC concentration of about 2.4 mol / mol to about 3.1 mol / mol, the filtration step may be held for about 32 ± 4 hours. If a given volume has a TSAC concentration of about 3.2 mol / mol or more, the filtration step may be held for about 44 ± 4 hours.

[0105] In another alternative embodiment, if a given volume has a TSAC concentration of less than about 2.4 mol / mol, the filtration step may be held for about 17 ± 3 hours. If a given volume has a TSAC concentration of about 2.4 mol / mol to about 3.6 mol / mol, the filtration step may be held for about 31 ± 3 hours. If a given volume has a TSAC concentration greater than about 3.6 mol / mol, the filtration step may be held for about 45 ± 3 hours.

[0106] In some embodiments, UFDF is held at a temperature of approximately 10°C to approximately 30°C. In some embodiments, UFDF is held at a temperature of approximately 13°C to approximately 27°C. In some embodiments, UFDF is held at a temperature of approximately 14°C to approximately 26°C. In some embodiments, UFDF is held at a temperature of approximately 15°C to approximately 26°C. In some embodiments, UFDF is held at a temperature of approximately 15°C to approximately 25°C. In some embodiments, UFDF is held at a temperature of approximately 19°C to approximately 25°C. In some embodiments, UFDF is held at a temperature of approximately 22°C. In some embodiments, UFDF is stored at the end of the holding time until further downstream processing steps are carried out. In some embodiments, UFDF is flash-frozen and then stored at -80°C.

[0107] In some embodiments, at least one additional purification step further comprises a virus inactivation step. In some embodiments, the virus inactivation step comprises a solvent / detergent virus inactivation process for chemically inactivating virus particles. Exemplary solvents / detergents may include 10% polysorbate 80, 3% TNBP, 50 mM sodium phosphate, and 100 mM NaCl.

[0108] chromatography In some embodiments of this method, UFDF is subjected to at least one chromatographic step to obtain partially purified recombinant alkaline phosphatase. In some embodiments, UFDF is subjected to at least one chromatographic step to obtain partially purified recombinant alkaline phosphatase, where the recombinant alkaline phosphatase has a total sialic acid content (TSAC) of about 0.9 mol / mol to about 3.0 mol / mol. In some embodiments, at least one chromatographic step is performed to further purify the product and / or separate impurities / contaminants. In some embodiments, at least one chromatographic step is protein chromatography. In some embodiments, protein chromatography is gel filtration chromatography, ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography, adsorption fluidized bed (EBA), mixed-mode chromatography and / or hydrophobic interaction chromatography (HIC). In some embodiments, protein chromatography is affinity chromatography. In some embodiments, protein chromatography is protein A chromatography. In some embodiments, protein A chromatography captures the product (e.g., alkaline phosphatase, e.g., asphotase alpha). For example, GE The Healthcare Mab Select SuRe Protein A chromatography process may be used. Exemplary buffers and solutions used in Protein A chromatography include, for example, equilibration / washing buffer (e.g., 50 mM sodium phosphate, 100 mM NaCl, pH 7.50), elution buffer (e.g., 50 mM Tris, pH 11.0), exfoliation buffer (e.g., 100 mM sodium citrate, 300 mM NaCl, pH 3.2), flushing buffer, and clean solution (e.g., 0.1 M NaOH).

[0109] In some embodiments, at least one chromatography step includes an additional chromatography and / or purification step. In some embodiments, at least one additional chromatography step includes column chromatography. In some embodiments, column chromatography is gel filtration chromatography, ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography, adsorption fluidized bed (EBA), mixed-mode chromatography and / or hydrophobic interaction chromatography (HIC). In some embodiments, column chromatography includes hydrophobic interaction chromatography (HIC). In some embodiments, butyl Sepharose or CAPTO® butyl agarose columns are used in HIC. Exemplary buffers and solutions used in the CAPTO® butyl agarose HIC process include, for example, additive diluent / pre-equilibrium buffer (e.g., 50 mM sodium phosphate, 1.4 M sodium sulfate, pH 7.50), equilibration buffer / wash buffer / elution buffer (e.g., all containing sodium phosphate and sodium sulfate), strip buffer (e.g., containing sodium phosphate), etc. Examples of buffers and solutions used in the butyl HIC process include, for example, additive diluent / pre-equilibrium buffer (e.g., 10 mM HEPES, 2.0 M ammonium sulfate, pH 7.50), equilibration buffer / wash buffer / elution buffer (e.g., all containing sodium phosphate or HEPES and ammonium sulfate), and strip buffer (e.g., containing sodium phosphate).

[0110] In some embodiments, at least one additional purification step includes additional diafiltration. In some embodiments, at least one additional chromatography and / or purification step includes hydrophobic interaction chromatography and / or at least one additional diafiltration step. In some embodiments, the additional diafiltration step is performed after the hydrophobic interaction chromatography step. In some embodiments, the additional diafiltration step is performed with the intention of product concentration and / or buffer exchange. Exemplary buffers and solutions used in this process include, for example, equilibration buffer (e.g., 20 mM sodium phosphate, 100 mM NaCl, pH 6.75), diafiltration buffer (20 mM sodium phosphate, 100 mM NaCl, pH 6.75), and so on.

[0111] In some embodiments, at least one additional chromatography and / or purification step is performed to obtain recombinant alkaline phosphatase having about 0.5 mol / mol to about 4.0 mol / mol TSAC. In some embodiments, at least one additional chromatography and / or purification step is performed to obtain recombinant alkaline phosphatase having about 0.9 mol / mol to about 3.9 mol / mol TSAC. In some embodiments, at least one additional chromatography and / or purification step is performed to obtain recombinant alkaline phosphatase having about 1.1 mol / mol to about 3.2 mol / mol TSAC. In some embodiments, at least one additional chromatography and / or purification step is performed to obtain recombinant alkaline phosphatase having about 1.4 mol / mol to about 2.6 mol / mol TSAC. In some embodiments, at least one additional chromatography and / or purification step is performed to obtain recombinant alkaline phosphatase having about 1.2 mol / mol to about 3.0 mol / mol TSAC. In some embodiments, approximately 0.8 mol / mol, approximately 0.9 mol / mol, 1.0 mol / mol, approximately 1.1 mol / mol, approximately 1.2 mol / mol, approximately 1.3 mol / mol, approximately 1.4 mol / mol, approximately 1.5 mol / mol, approximately 1.6 mol / mol, approximately 1.7 mol / mol, approximately 1.8 mol / mol, approximately 1.9 mol / mol, approximately 2.0 mol / mol, approximately 2.1 mol / mol, approximately 2.2 mol / mol, approximately 2.3 mol / mol, approximately 2.4 mol / mol, approximately 2.5 mol / mol, approximately 2.6 mol / mol, At least one additional chromatography step is performed to obtain recombinant alkaline phosphatase having TSAC of approximately 2.7 mol / mol, approximately 2.8 mol / mol, approximately 2.9 mol / mol, approximately 3.0 mol / mol, approximately 3.1 mol / mol, approximately 3.2 mol / mol, approximately 3.3 mol / mol, approximately 3.4 mol / mol, approximately 3.5 mol / mol, approximately 3.6 mol / mol, approximately 3.7 mol / mol, approximately 3.8 mol / mol, approximately 3.9 mol / mol, or approximately 4.0 mol / mol.

[0112] Additional downstream processes In some embodiments, additional downstream processes are carried out in addition to at least one purification step, an additional purification step, at least one chromatography step, and / or an additional chromatography step. In some embodiments, the additional downstream processes further purify the product, for example, recombinant alkaline phosphatase.

[0113] In some embodiments, an additional downstream process includes a virus reduction filtration process for further removal of any virus particles. In some embodiments, the virus reduction filtration process is nanofiltration.

[0114] In some embodiments, an additional downstream process includes at least one further chromatographic step. In some embodiments, at least one further chromatographic step is protein chromatography. In some embodiments, protein chromatography is gel filtration chromatography, ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography, adsorption fluidized bed (EBA), mixed-mode chromatography, and / or hydrophobic interaction chromatography (HIC). In some embodiments, the third chromatographic step is mixed-mode chromatography, such as CAPTO® Adhere agarose chromatography. Examples of commercially available mixed-mode materials include resins containing hydrocarbylamine ligands that enable binding at neutral or weakly basic pH through a combination of hydrophobic and electrostatic forces and elution by electrostatic repulsion at low pH (e.g., PPA Hypercel and HEA Hypercel from Pall Corporation, Port Washington, NY) (see Brenac et al., 2008 J Chromatogr A.1177:226-233); resins containing 4-mercapto-ethyl-pyridine ligands that obtain hydrophobic interactions through aromatic residues and promote target protein binding by thiophilic interactions of sulfur atoms (MEP Hypercel, Pall Corporation) (Lees et al., 2009 Bioprocess Int.7:42-48); and resins such as CAPTO® MMC mixed-mode chromatography and CAPTO® adherere agarose chromatography (GE Healthcare, Amersham, UK) that contain ligands with hydrogen bonding groups and aromatic residues near ionic groups that result in salt-tolerant adsorption of proteins at different conductivity levels (Chen et al., 2010 Examples include J Chromatogr A.1217:216-224) and other known chromatographic materials, such as affinity resins with dye ligands, hydroxyapatites, and several ion exchange resins (but not limited to, Amberlite CG50 (Rohm & Haas, Philadelphia, PA) or Lewatit CNP105 (Lanxess, Cologne, DE)). In an exemplary agarose HIC chromatography step, exemplary buffers and solutions used in this process include, for example, a pre-equilibrium buffer (e.g., 0.5 M sodium phosphate, pH 6.00), equilibration / wash buffer (e.g., 20 mM sodium phosphate, 440 mM NaCl, pH 6.50), additive titration buffer (e.g., 20 mM sodium phosphate, 3.2 M NaCl, pH 5.75), pool dilution buffer (e.g., 25 mM sodium phosphate, 150 mM NaCl, pH 7.40), and strip buffer (0.1 M sodium citrate, pH 3.20).

[0115] In some embodiments, an additional downstream process includes a viral filtration step for viral clearance. In some embodiments, the viral filtration step is carried out by size exclusion chromatography. Exemplary buffers and solutions used in this process include, for example, pre-use and post-production flash buffers (e.g., 20 mM sodium phosphate, 100 mM NaCl, pH 6.75).

[0116] In some embodiments, an additional downstream process includes a formulation process. In some embodiments, the formulation process includes at least one further ultrafiltration and / or diafiltration intended for further concentration and / or buffer exchange. Exemplary buffers and solutions used in this process include, for example, a filter flush / equilibrium / diafiltration / recovery buffer (e.g., 25 mM sodium phosphate, 150 mM NaCl, pH 7.40).

[0117] In some embodiments, an additional downstream process includes a bulk filling process. In some embodiments, the bulk filling process includes sterile filtration. An exemplary filter for sterile filtration is a Millipak 60 or equivalent-sized PVDF filter (EMD Millipore, Billerica, MA).

[0118] In some embodiments, as disclosed herein, the steps used to prepare, purify and / or separate alkaline phosphatase from cultured cells further include at least one step selected from the group consisting of a collection and clarification process (or a similar process for removing intact cells and cell fragments from the cell culture), an ultrafiltration (UF) process (or a similar process for concentrating the prepared alkaline phosphatase), a diafiltration (DF) process (or a similar process for modifying or diluting the buffer containing the prepared alkaline phosphatase from the preceding process), a virus inactivation process (or a similar process for inactivating or removing virus particles), an affinity capture process (or one of the chromatographic methods for capturing the prepared alkaline phosphatase and separating it from the rest of the buffer / solution components), a compounding process, and a bulk packing process.In one embodiment, the steps for preparing, purifying and / or separating alkaline phosphatase from cultured cells are as disclosed herein, and include at least: a collection and clarification process (or a similar process for removing intact cells and cell fragments from the cell culture), a post-collection ultrafiltration (UF) process (or a similar process for concentrating the prepared alkaline phosphatase), a post-collection diafiltration (DF) process (or a similar process for modifying or diluting the buffer containing the prepared alkaline phosphatase from the preceding process), a solvent / detergent virus inactivation process (or a similar process for chemically inactivating virus particles), and an intermediate purification process (e.g., hydrophobic interaction chromatography). The process includes either HIC (High Intensity Chromatography) or a chromatographic method for capturing the prepared alkaline phosphatase and separating it from the rest of the buffer / solution components), a post-HIC UF / DF process (or a similar process for concentrating and / or exchanging buffers for the prepared alkaline phosphatase), a virus reduction filtration process (or a similar process for further removing any virus particles or other impurities or contaminants); mixed-mode chromatography (e.g., CAPTO® Adhere agarose chromatography or a similar process for further purifying and / or concentrating the prepared alkaline phosphatase), a compounding process, and a bulk packing process. In one embodiment, the separation step of the method provided herein further includes at least one of collection clarification, ultrafiltration, diafiltration, virus inactivation, affinity capture, HIC chromatography, mixed-mode chromatography, and combinations thereof. Figure 1 is a representative drawing of an embodiment of the process for preparing recombinant alkaline phosphatase, asphotase alpha.

[0119] In some embodiments, the disclosure provides a method for controlling total sialic acid content (TSAC) in recombinant proteins through mammalian cell cultures, comprising at least one purification step and at least one chromatography step. In some embodiments, the disclosure provides a method for controlling glycosidase activity in mammalian cell cultures producing recombinant proteins, comprising at least one purification step and at least one chromatography step. In some embodiments, the at least one purification step comprises at least one of filtration, centrifugation, collection clarification, filtration, ultrafiltration, diafiltration, virus inactivation, affinity capture, and a combination thereof. In some embodiments, the at least one chromatography step comprises protein chromatography. In some embodiments, the protein chromatography is gel filtration chromatography, ion exchange chromatography, reversed-phase chromatography (RP), affinity chromatography, adsorption fluidized bed (EBA), mixed-mode chromatography, and / or hydrophobic interaction chromatography (HIC). In some embodiments, the purification step and the chromatography step are ultrafiltration / diafiltration and protein A chromatography.

[0120] After manufacturing and purification, the process can produce a bulk drug substance (BDS) with a controlled range of sialylation. This method can produce BDS with a controlled TSAC concentration in the range of approximately 1.2 mol / mol to approximately 3.0 mol / mol (e.g., approximately 1.6 mol / mol to approximately 2.4 mol / mol). For example, BDS may have TSAC concentrations of approximately 1.0 mol / mol, 1.1 mol / mol, 1.2 mol / mol, 1.3 mol / mol, 1.4 mol / mol, 1.5 mol / mol, 1.6 mol / mol, 1.7 mol / mol, 1.8 mol / mol, 1.9 mol / mol, 2.0 mol / mol, 2.1 mol / mol, 2.2 mol / mol, 2.3 mol / mol, 2.4 mol / mol, 2.5 mol / mol, 2.6 mol / mol, 2.7 mol / mol, 2.8 mol / mol, 2.9 mol / mol, or 3.0 mol / mol.

[0121] In some embodiments, the BDS is freeze-dried and / or loaded into vials for distribution, for example.

[0122] Alkaline phosphatase (ALP) This disclosure relates to the production of alkaline phosphatase proteins (e.g., asphotase alpha) in recombinant cell culture. Alkaline phosphatase proteins include any polypeptide or molecule containing a polypeptide that has at least some alkaline phosphatase activity. In various embodiments, the alkaline phosphatases disclosed herein include any polypeptide having alkaline phosphatase function, which may include any function of alkaline phosphatases known in the art, such as enzymatic activity against native substrates including phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP).

[0123] In certain embodiments, such alkaline phosphatase proteins may be prepared by the methods disclosed herein, then purified, and used to treat or prevent alkaline phosphatase-related diseases or disorders. For example, such alkaline phosphatase proteins may be administered to subjects having decreased and / or dysfunction of endogenous alkaline phosphatase, or having overexpression (e.g., above normal levels) of alkaline phosphatase substrates. In some embodiments, the alkaline phosphatase proteins in this disclosure are recombinant proteins. In some embodiments, the alkaline phosphatase proteins are fusion proteins. In some embodiments, the alkaline phosphatase proteins in this disclosure specifically target cell types, tissues (e.g., joint, muscle, nerve, or epithelial tissue) or organs (e.g., liver, heart, kidney, muscle, bone, cartilage, ligaments, tendons, etc.). For example, such alkaline phosphatase proteins may comprise full-length alkaline phosphatase (ALP) or fragments of at least one alkaline phosphatase (ALP). In some embodiments, the alkaline phosphatase protein includes a soluble ALP (sALP) linked to a bone target moiety (e.g., a negatively charged peptide as described below). In some embodiments, the alkaline phosphatase protein includes a soluble ALP (sALP) linked to an immunoglobulin moiety (full length or fragment). For example, such an immunoglobulin moiety may include a crystallizable region fragment (Fc). In some embodiments, the alkaline phosphatase protein includes a soluble ALP (sALP) linked to both a bone target moiety and an immunoglobulin moiety (full length or fragment). For a more detailed description of the alkaline phosphatase proteins disclosed herein, see PCT Publications International Publication No. 2005 / 103263 and International Publication No. 2008 / 138131 (the teachings of both of these are incorporated herein by reference in their entirety).

[0124] In some embodiments, the alkaline phosphatase proteins described herein include one of the structures selected from the group consisting of sALP-X, X-sALP, sALP-Y, Y-sALP, sALP-XY, sALP-YX, X-sALP-Y, XY-sALP, Y-sALP-X, and YX-sALP (wherein X comprises a bone targeting moiety as described herein, and Y comprises an immunoglobulin moiety as described herein). In one embodiment, the alkaline phosphatase protein comprises the structure W-sALP-X-Fc-Y-Dn / En-Z (wherein W is an amino acid sequence of absence or at least one amino acid, X is an amino acid sequence of absence or at least one amino acid, Y is an amino acid sequence of absence or at least one amino acid, Z is an amino acid sequence of absence or at least one amino acid, Fc is a crystallizable region fragment, Dn / En is polyaspartic acid, polyglutamic acid or a combination thereof, where n=8 to 20, and sALP is soluble alkaline phosphatase (ALP)). In some embodiments, Dn / En is a polyaspartic acid sequence. For example, Dn may be a polyaspartate sequence, where n is any number from 8 to 20 (including both) (for example, n may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) (SEQ ID NO: 3). In one embodiment, Dn is D10 (SEQ ID NO: 2) or D16 (SEQ ID NO: 4). In some embodiments, Dn / En is a polyglutamate sequence. For example, En may be a polyglutamate sequence, where n is any number from 8 to 20 (including both) (for example, n may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20) (SEQ ID NO: 5). In one embodiment, En is E10 (SEQ ID NO: 6) or E16 (SEQ ID NO: 7).

[0125] For example, such sALP may be fused to the full length or a fragment of an immunoglobulin molecule (e.g., a crystallizable region fragment (Fc)). In some embodiments, the recombinant polypeptide comprises the structure W-sALP-X-Fc-Y-Dn-Z (wherein W is an amino acid sequence of absent or at least one amino acid, X is an amino acid sequence of absent or at least one amino acid, Y is an amino acid sequence of absent or at least one amino acid, Z is an amino acid sequence of absent or at least one amino acid, Fc is a crystallizable region fragment, Dn is polyaspartic acid, polyglutamic acid, or a combination thereof, where n=10 or 16, and the sALP is a soluble alkaline phosphatase). In one embodiment, n=10. In another embodiment, W and Z are absent in the polypeptide. In some embodiments, the Fc comprises a CH2 domain, a CH3 domain, and a hinge region. In some embodiments, the Fc is a constant domain of an immunoglobulin selected from the group consisting of IgG-1, IgG-2, IgG-3, IgG-4, and IgG-4. In one embodiment, the Fc is a constant domain of immunoglobulin IgG-1. In a particular embodiment, the Fc includes the sequence shown in D488-K714 of SEQ ID NO: 1.

[0126] In some embodiments, the alkaline phosphatase disclosed herein comprises the structure W-sALP-X-Fc-Y-Dn-Z (wherein W is an amino acid sequence of absent or at least one amino acid, X is an amino acid sequence of absent or at least one amino acid, Y is an amino acid sequence of absent or at least one amino acid, Z is an amino acid sequence of absent or at least one amino acid, Fc is a crystallizable region fragment, Dn is polyaspartic acid, polyglutamic acid or a combination thereof, where n=10 or 16, and sALP is a soluble alkaline phosphatase). Such sALP is capable of catalyzing the cleavage of at least one of phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP). In various embodiments, the sALP disclosed herein is capable of catalyzing the cleavage of inorganic pyrophosphate (PPi). Such sALP may comprise all the active anchor-type amino acids of alkaline phosphatase (ALP) that lack a C-terminal glycolipid anchor (GPI). Such ALP may be at least one of tissue-nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP), germinal cell alkaline phosphatase (GCALP), and intestinal alkaline phosphatase (IAP), or chimeric or fusion forms or variants thereof disclosed herein. In one particular embodiment, ALP comprises tissue-nonspecific alkaline phosphatase (TNALP). In another embodiment, the sALP disclosed herein is encoded by a polynucleotide encoding a polypeptide containing the sequence shown at L1-S485 of SEQ ID NO: 1. In yet another embodiment, the sALP disclosed herein contains the sequence shown at L1-S485 of SEQ ID NO: 1.

[0127] In one embodiment, the alkaline phosphatase protein comprises the structure TNALP-Fc-D10 (SEQ ID NO: 1). Asparagine (N) residues (e.g., N123, 213, 254, 286, 413, and 564) correspond to expected glycosylation sites. Amino acid residues (L486-K487 and D715-I716) correspond to linkers between sALP and Fc, and between Fc and the D10 (SEQ ID NO: 2) domain, respectively.

[0128] In this embodiment, the polypeptide consists of five parts. The first part (sALP), having amino acids L1-S485, is the soluble portion of a human tissue-nonspecific alkaline phosphatase enzyme with catalytic function. The second part has amino acids L486-K487 as a linker. The third part (Fc), having amino acids D488-K714, is the Fc portion of human immunoglobulin γ1 (IgG1) having hinge, CH2 and CH3 domains. The fourth part has amino acids D715-I716. The fifth part has amino acids D717-D726 (D10 (SEQ ID NO: 2)), which is a bone targeting portion that allows asfotase alpha to bind to the mineral phase of bone. Furthermore, each polypeptide chain has six expected glycosylation sites and 11 cysteine ​​(Cys) residues. Cys102 exists as free cysteine. Each polypeptide chain has four intrachain disulfide bonds between Cys122 and Cys184, Cys472 and Cys480, Cys528 and Cys588, and Cys634 and Cys692. Two polypeptide chains are linked by two interchain disulfide bonds: one between Cys493 on both chains and one between Cys496 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.

[0129] ALP has four known isozymes, namely tissue-nonspecific alkaline phosphatase (TNALP), placental alkaline phosphatase (PALP) (as described, e.g., in Genbank acceptance numbers NP_112603 and NP_001623), germinal cell alkaline phosphatase (GCALP) (as described, e.g., in Genbank acceptance number P10696), and enteric alkaline phosphatase (IAP) (as described, e.g., in Genbank acceptance number NP_001622). These enzymes have very similar three-dimensional structures. Each of their catalytic sites has four metal-binding domains for the metal ions necessary for enzymatic activity, containing two Zn and one Mg. These enzymes catalyze the hydrolysis of phosphate monoesters and also catalyze transphosphorylation 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). The alignment of these isozymes is shown in Figure 30 of International Publication No. 2008 / 138131 (the teachings thereof are incorporated herein by reference in their entirety).

[0130] The alkaline phosphatase proteins in this disclosure may include dimers or polymers of any ALP protein, either alone or in combination. Chimeric ALP proteins or fusion proteins, such as those described in Kiffer-Moreira et al. PLoS One 9:e89374, 2014 (their entire teachings are incorporated herein by reference), may also be constructed.

[0131] In a particular embodiment, the alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide containing the sequence shown in SEQ ID NO: 1. In some embodiments, the alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide containing a sequence having 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 1. In some embodiments, the alkaline phosphatase disclosed herein is encoded by a polynucleotide encoding a polypeptide containing a sequence having 95% or 99% identity to SEQ ID NO: 1. In another embodiment, the alkaline phosphatase disclosed herein contains the sequence shown in SEQ ID NO: 1.

[0132] TNALP As shown above, TNALP is a membrane-bound protein that is immobilized at its C-terminus via a glycolipid (see UniProtKB / Swiss-Prot acceptance number P05186 for human TNALP). This glycolipid anchor (GPI) is added post-translation after the removal of the hydrophobic C-terminus, which serves both as a transient membrane anchor and a signal for GPI addition. Thus, in one embodiment, soluble human TNALP contains a TNALP in which the first amino acid of the hydrophobic C-terminal sequence, i.e., alanine, is replaced with a stop codon. The soluble TNALP thus formed (referred to herein as sTNALP) contains all the amino acids of the native anchor form of TNALP that are necessary for the formation of a catalytic site lacking a GPI membrane anchor. Examples of known TNALPs include human TNALP [Genbank acceptance numbers NP-000469, AAI10910, AAH90861, AAH66116, AAH21289 and AAI26166]; rhesus monkey TNALP [Genbank acceptance number XP-001109717]; rat TNALP [Genbank acceptance number NP_037191]; dog TNALP [Genbank acceptance number AAF64516]; pig TNALP [Genbank acceptance number AAN64273]; mouse TNALP [Genbank acceptance number NP_031457]; cattle TNALP [Genbank acceptance numbers NP_789828, NP_776412, AAM8209 and AAC33858]; and cat TNALP [Genbank acceptance number NP_001036028].

[0133] As used herein, the term “extracellular domain” means any functional extracellular portion of a native protein (e.g., without peptide signaling). Recombinant sTNALP polypeptides retaining the original amino acids 1-501 (18-501 at secretion), 1-502 (18-502 at secretion), 1-504 (18-504 at secretion), or 1-505 (18-505 at secretion) are enzymatically active (see Oda et al., 1999 J. Biochem 126:694-699). This indicates that amino acid residues can be removed from the C-terminus of a native protein without affecting its enzymatic activity. Furthermore, soluble human TNALP may contain one or more amino acid substitutions, and such substitutions do not reduce or at least completely inhibit the enzymatic activity of sTNALP. For example, certain mutations known to cause hypophosphatasia (HPP) are listed in the PCT Public Release International Publication No. 2008 / 138131 and need to be avoided in order to maintain functional sTNALP.

[0134] Negatively charged peptides The alkaline phosphatase proteins of this disclosure may include a target moiety that may specifically target a given cell type, tissue, or organ. In some embodiments, such given cell type, tissue, or organ is bone tissue. Such bone target moiety may include any known polypeptide, polynucleotide, or small molecule compound known in the art. For example, a negatively charged peptide may be used as a bone target moiety. In some embodiments, such a negatively charged peptide may be polyaspartic acid, polyglutamic acid, or a combination thereof (e.g., a polypeptide comprising at least one aspartic acid and at least one glutamic acid, e.g., a negatively charged peptide comprising a combination of aspartic acid and glutamic acid residues). In some embodiments, such negatively charged peptides may be polyaspartic acid having more than 20 aspartic acid molecules, D6 (SEQ ID NO: 8), D7 (SEQ ID NO: 9), D8 (SEQ ID NO: 10), D9 (SEQ ID NO: 11), D10 (SEQ ID NO: 2), D11 (SEQ ID NO: 12), D12 (SEQ ID NO: 13), D13 (SEQ ID NO: 14), D14 (SEQ ID NO: 15), D15 (SEQ ID NO: 16), D16 (SEQ ID NO: 4), D17 (SEQ ID NO: 17), D18 (SEQ ID NO: 18), D19 (SEQ ID NO: 19), D20 (SEQ ID NO: 20), or 20 or more. In some embodiments, such negatively charged peptides may be polyglutamic acids having more than 20 glutamic acids, such as E6 (SEQ ID NO: 21), E7 (SEQ ID NO: 22), E8 (SEQ ID NO: 23), E9 (SEQ ID NO: 24), E10 (SEQ ID NO: 6), E11 (SEQ ID NO: 25), E12 (SEQ ID NO: 26), E13 (SEQ ID NO: 27), E14 (SEQ ID NO: 28), E15 (SEQ ID NO: 29), E16 (SEQ ID NO: 7), E17 (SEQ ID NO: 30), E18 (SEQ ID NO: 31), E19 (SEQ ID NO: 32), E20 (SEQ ID NO: 33), or 20 glutamic acids. In one embodiment, such negatively charged peptides may include at least one selected from the group consisting of D10 (SEQ ID NO: 2) to D16 (SEQ ID NO: 4) or E10 (SEQ ID NO: 6) to E16 (SEQ ID NO: 7).

[0135] Spacer In some embodiments, the alkaline phosphatase proteins of this disclosure include a spacer sequence between the ALP moiety and the target moiety. In one embodiment, such an alkaline phosphatase protein includes a spacer sequence between the ALP (e.g., TNALP) moiety and a negatively charged peptide target moiety. Such a spacer may be any polypeptide, polynucleotide, or small molecule compound. In some embodiments, such a spacer may include a fragment of a crystallizable region (Fc). Useful Fc fragments include Fc fragments of IgG containing a hinge and CH2 and CH3 domains. Such IgG may be IgG-1, IgG-2, IgG-3, IgG-3 and IgG-4, or any combination thereof.

[0136] Without being limited by this theory, assuming that the Fc fragment used in bone-targeting sALP fusion proteins (e.g., asfotase alfa) acts as a spacer, and that the expression of sTNALP-Fc-D10 is higher than that of sTNALP-D10, it is thought that this allows the protein to fold more efficiently. One possible explanation is that the introduction of the Fc fragment mitigates the repulsive force caused by the presence of a highly negatively charged D10 sequence (SEQ ID NO: 2) added to the C-terminus of the sALP sequence exemplified herein. In some embodiments, the alkaline phosphatase proteins described herein include structures selected from the group consisting of sALP-Fc-D10, sALP-D10-Fc, D10-sALP-Fc, D10-Fc-sALP, Fc-sALP-D10, and Fc-D10-sALP. In other embodiments, D10 (SEQ ID NO: 2) in the above structure is replaced with another negatively charged polypeptide (e.g., D8 (SEQ ID NO: 10), D16 (SEQ ID NO: 4), E10 (SEQ ID NO: 6), E8 (SEQ ID NO: 23), E16 (SEQ ID NO: 7), etc.).

[0137] Spacers useful to this disclosure include, for example, Fc-containing polypeptides and hydrophilic and flexible polypeptides that can mitigate the repulsive force caused by the presence of a highly negatively charged bone target sequence (e.g., D10 (SEQ ID NO: 2)) attached to the C-terminus of the sALP sequence.

[0138] dimer / tetramer In specific embodiments, the bone-targeting sALP fusion protein of this disclosure is associated to form a dimer or tetramer.

[0139] In the dimeric configuration, the steric hindrance imposed by the formation of interchain disulfide bonds likely prevents the association of the sALP domain with the smallest catalytically active protein dimer present in normal cells.

[0140] Bone-targeted sALP may optionally contain one or more additional amino acids: 1) downstream from a negatively charged peptide (e.g., a bone labeling agent), and / or 2) between the negatively charged peptide (e.g., a bone labeling agent) and the Fc fragment, and / or 3) between a spacer (e.g., the Fc fragment) and the sALP fragment. This can occur, for example, when exogenous amino acids are introduced at these positions by cloning methods used to construct bone-targeted conjugates. However, the exogenous amino acids must be selected so as not to result in further GPI anchoring signals. The possibility of the designed sequence being cleaved by host cell transamidases is discussed in Ikezawa, 2002 Glycosylphosphatidylinositol (GPI)-anchored proteins. Biol As described in Pharm Bull. 25:409-17, it is predictable.

[0141] This disclosure also includes fusion proteins that are post-translationally modified by glycosylation, acetylation, amidation, sequestering, formylation, γ-carboxyglutamic acid hydroxylation, methylation, phosphorylation, pyrrolidone carboxylic acid, and sulfation, including, for example, those specified herein.

[0142] Asfotase alpha Asfotase alpha is a soluble Fc fusion protein composed of two TNALP-Fc-D10 polypeptides (each having 726 amino acids as shown in SEQ ID NO: 1). Each polypeptide or monomer consists of five parts. The first part (sALP), having amino acids L1-S485, is the soluble portion of a human tissue-nonspecific alkaline phosphatase enzyme with catalytic function. The second part has amino acids L486-K487 as a linker. The third part (Fc), having amino acids D488-K714, is the Fc portion of human immunoglobulin γ1 (IgG1) with hinge, CH2 and CH3 domains. The fourth part has D715-I716 as a linker. The fifth part has amino acids D717-D726 (D10 (SEQ ID NO: 2)), which is a bone targeting portion that enables asfotase alpha to bind to the mineral phase of bone. Furthermore, each polypeptide chain has six expected glycosylation sites and 11 cysteine ​​(Cys) residues. Cys102 exists as free cysteine. Each polypeptide chain has four intrachain disulfide bonds between Cys122 and Cys184, Cys472 and Cys480, Cys528 and Cys588, and Cys634 and Cys692. Two polypeptide chains are linked by two interchain disulfide bonds between Cys493 on both chains and between Cys496 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.

[0143] Asfotase alpha can also be characterized as follows: From the N-terminus to the C-terminus, asfotase alpha contains (1) a soluble catalytic domain of human tissue-nonspecific alkaline phosphatase (TNSALP) (UniProtKB / Swiss-Prot acceptance number P05186), (2) a human immunoglobulin G1 Fc domain (UniProtKB / Swiss-Prot acceptance number P01857), and (3) a decaaspartate peptide (D10 (SEQ ID NO: 2)) used as a bone targeting domain (Nishioka et al. 2006 Mol Genet Metab 88:244-255). The protein associates into a homodimer from two primary protein sequences. This fusion protein has six identified complex N-glycosylation sites. Five of these N-glycosylation sites are located on the sALP domain and one is located on the Fc domain. Another important post-translational modification present on asfotase alpha is the presence of disulfide bridges that stabilize the enzyme and the Fc-domain structure. There are a total of four intramolecular disulfide bridges per monomer and two intramolecular disulfide bridges in the dimer. One cysteine ​​molecule of the alkaline phosphatase domain is free.

[0144] Asfotase alfa is used as an enzyme replacement therapy for the treatment of hypophosphatasia (HPP). In patients with HPP, loss-of-function mutations in the gene encoding TNSALP cause a deficiency in TNSALP enzyme activity, leading 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, restoring a normal skeletal phenotype. For further details regarding asfotase alfa and its therapeutic use, please refer to PCT Publication International Publication Nos. 2005 / 103263 and 2008 / 138131.

[0145] In some embodiments, the method provides an alkaline phosphatase (e.g., asfotase alpha) having improved enzymatic activity compared to alkaline phosphatases prepared by conventional means, as described herein, minimizing the concentration of metal ions having a potentially negative effect on activity, increasing the concentration of metal ions having a potentially positive effect on activity, or both. The activity can be measured by any known method. Such methods include, for example, in vitro and in vivo assays for measuring the enzymatic activity of the prepared alkaline phosphatase (e.g., asfotase alpha) against alkaline phosphatase substrates, such as phosphoethanolamine (PEA), inorganic pyrophosphate (PPi), and pyridoxal 5'-phosphate (PLP).

[0146] In some embodiments, the alkaline phosphatase disclosed herein is encoded by a first polynucleotide that hybridizes to a second polynucleotide under high stringency conditions, compared to a sequence that is fully complementary to a third polynucleotide encoding a polypeptide containing the sequence shown in SEQ ID NO: 1. Such high stringency conditions may include pre-hybridization and hybridization at 68°C in 6×SSC, 5×Denhart reagent, 0.5% SDS, and 100 mg / ml of denatured fragmented salmon sperm DNA, as well as washing with 2×SSC and 0.5% SDS for 10 minutes at room temperature, washing with 2×SSC and 0.1% SDS for 10 minutes at room temperature, and three washes with 0.1×SSC and 0.5% SDS for 5 minutes at 65°C. [Examples]

[0147] Example 1: Asfotase alpha production process Figure 1 shows an exemplary asfotase alpha bulk drug substance (BDS) manufacturing process.

[0148] The asfotase alfa manufacturing process described below details measuring the TSAC content on day 7 of cell culture fermentation in the production bioreactor and using it to determine the retention time in the downstream post-collection ultrafiltration / diafiltration (UF / DF1) step. This added step provides improved quality control and maintains the final TSAC content within the acceptable range already approved for human use. The manufacturing process and target TSAC range provide a final drug product with appropriate enzyme activity, therapeutically effective half-life, and batch-to-batch reproducibility.

[0149] The process of in-process TSAC control Sialic acid is a known form of glycosylation associated with asfotase alpha, which affects the molecular half-life under physiological conditions. Controlling TSAC levels within an acceptable range of 1.2–3.0 moles of sialic acid / asfotase alpha monomer (1 mole / mol) is necessary to provide a final drug product with appropriate potency, therapeutically effective half-life, and batch-to-batch reproducibility. TSAC is generated in the production bioreactor (cell culture medium, CCF, step 2 in Figure 1), and the TSAC in the collected cell culture medium (HCCF, step 3 in Figure 1) is reduced during pool retention after collection with protein A, MabSelect® SuRe® (ProA), and chromatography step (step 5b in Figure 1) (ultrafiltration / diafiltration (UF / DF1) (step 4 in Figure 1).

[0150] UF / DF1 pool retention is a critical in-process control step for BDS TSAC. Preliminary small-scale characterization studies have established that post-collection UF / DF1 retention time, protein concentration, and temperature significantly affect the extent of TSAC reduction during UF / DF1 pool retention.

[0151] TSAC Manufacturing Data Review TSAC was measured at two steps during manufacturing (ProA pooling and BDS release). For a subset of batches, TSAC was also measured at the production bioreactor step (days 7 and 10, referred to as CCF), the collection step (HCCF), and the HIC step. Table 1 summarizes the TSAC data for a 20,000L batch. A review of the manufacturing data, summarized in Table 1 and shown in Figure 2, confirmed that TSAC decreased from the HCCF step (step 3 in Figure 1) to the ProA step (step 5b in Figure 1).

[0152] The TSAC results in BDS were observed to skew towards the lower specification limit, including three out-of-spec (OOS) results (#8, #9, and #11). Upstream process variability, particularly in the production bioreactor, was identified as a verifiable cause of the BDS OOS TSAC. Furthermore, the downstream TSAC control (UF / DF1 retention) of the production bioreactor was not optimally designed to respond to this upstream variability.

[0153] [Table 1]

[0154] Improved TSAC control method An improved TSAC control method has been developed. The improved method involves monitoring TSAC levels in the production bioreactor and adjusting UF / DF1 process parameters accordingly to improve process control and TSAC performance in BDS. Specifically, TSAC measured on day 7 from the production bioreactor sample (referred to as "day 7 TSAC") can provide an estimate of TSAC before UF / DF1 unit operation. Day 7 TSAC is introduced as an in-process control (IPC) for the process, and the relevant processing limit for day 7 TSAC identifies the target UF / DF1 retention time for each batch. Since sample preparation (small-scale protein A purification) and TSAC assay duration currently exclude at-line measurements of actual TSAC before the start of UF / DF1 operation and retention, day 7 TSAC is used as a substitute for TSAC results from the end of cell culture or collection. Protein concentration targets and ranges were adjusted to optimize the UF / DF1 retention method in accordance with TSAC production identified for each batch in the bioreactor. The holding temperature for UF / DF1 was kept constant.

[0155] Table 2 summarizes the changes introduced in UF / DF1 operation to further improve TSAC control. Table 3 summarizes the TSAC processing limits on day 7. Parameters, characteristic ranges, and IPC processing limits were optimized using a predictive model generated from small-scale UF / DF1 characterization tests and additional TSAC data collection from manufacturing-scale batches.

[0156] [Table 2]

[0157] [Table 3]

[0158] The updated TSAC control method improved process control and TSAC performance in BDS compared to the broader range of TSACs generated during the cell culture process in the production bioreactor. The development batch was used to fine-tune the UF / DF1 retention time, demonstrating the feasibility of manufacturing operations with 7-day TSAC in-process control and demonstrating the effectiveness of the modified control method.

[0159] Four initial development batches were successfully carried out up to BDS (Batches 16, 17, 18, and 19; see Table 1 and Figures 2 and 3). Day 7 samples from the production bioreactor were purified using small-scale Protein A chromatography and then tested for TSAC. For three of the four development batches, the UF / DF1 retention time was adjusted based on pre-specified processing limits similar to those specified in Table 3, using the Day 7 TSAC results. One batch (17) was carried out with a fixed retention time target, demonstrating the feasibility of operation with a minimum retention time below the pre-specified range. Three additional batches were manufactured up to BDS using the improved TSAC control method (Batches 20-22). TSAC and UF / DF1 process data for all batches using the improved TSAC control are summarized in Table 1. For all batches, UF / DF1 temperature, protein concentration, and retention time were within the acceptable ranges specified for the improved control method (Table 2). The actual UF / DF1 retention time was within the target range specified for the 7-day processing limit (Table 3), except for the first development batch 16, which was performed using a different 7-day processing limit.

[0160] The TSAC from the production bioreactor (CCF) and after collection (HCCF) (HCCF shown in Figure 2) showed a similar trend in the improved TSAC-controlled batches compared to the preliminary batches, with the day 7 TSAC approximating the TSAC before the UF / DF1 unit operation (HCCF TSAC) (Table 1, Figure 2). The average BDS TSAC for batches 1-15 (n=15) without day 7 TSAC control was 1.3 mol / mol compared to the average BDS TSAC of 2.1 mol / mol (range 1.9-2.4 mol / mol) for batches utilizing day 7 TSAC control (batches 17-22, n=6). The change in BDS TSAC for batches 17-22 was close to the midpoint of the specification range compared to the previous batch, consistent with the UF / DF1 retention conditions performed on those batches using improved TSAC control, demonstrating its effectiveness (Table 1 and Figures 2 and 3). While the TSAC from the production bioreactor (day 7) in these batches resulted in shorter target UF / DF1 retention times, the improved TSAC control method dynamically responds to the range of TSAC output from the production bioreactor. If a higher TSAC output is measured on day 7, the improved method identifies and specifies an appropriate target UF / DF1 retention time, changing the TSAC in the BDS closer to the midpoint of the specification range and avoiding out-of-situation (OOS) results.

[0161] In addition to demonstrating the feasibility and effectiveness of the improved TSAC control method, BDS from batches 17 to 22 meet all criteria for each emission standard, confirming that there are no adverse effects on process performance or other product quality characteristics.

[0162] Other Embodiments All references cited herein are incorporated by reference in their entirety. While the foregoing disclosure is described in some detail through drawings and examples intended to clarify understanding, certain minor changes and modifications will be obvious to those skilled in the art. Therefore, the description and examples should not be construed as limiting the scope of the disclosure.

[0163] The above detailed description and examples are provided for clarity of understanding only. No unnecessary limitations should be inferred from them. This disclosure is not limited to the exact details shown and described, and modifications that are obvious to those skilled in the art are included within this disclosure as defined by the claims.

[0164] Unless otherwise indicated, all figures used in this specification and in the claims to express quantities, molecular weights, etc., of constituents should be understood to be modified in all examples by the term “approximately.” Therefore, unless otherwise indicated, the numerical parameters shown in this specification and in the claims are approximations that may vary depending on the desired properties to be obtained by this disclosure. Each numerical parameter should be interpreted, at least in light of the reported significant figures and by applying the usual rounding techniques, and not in any attempt to limit the doctrine of equivalents in the claims.

[0165] Although the numerical ranges and parameters representing the broad scope of this disclosure are approximate, the numerical ranges shown in the specific examples are reported as accurately as possible. However, all numerical ranges inherently include a range derived from the standard deviation found in each of their test measurements.

[0166] The complete disclosure of all patents, patent applications including provisional patent applications, publications including patent and non-patent publications, and electronically available materials (including, for example, nucleotide sequence registrations in GenBank and RefSeq, and amino acid sequence registrations in SwissProt, PIR, PRF, and PDB, and translations from annotated code regions in GenBank and RefSeq) referenced herein is invoked by reference. The above detailed description and examples are given for clarity of understanding only. No unnecessary limitations should be inferred therefrom. This disclosure is not limited to the exact details shown and described, and modifications that are obvious to those skilled in the art are included within the embodiments defined by the claims. The present invention provides, for example, the following items: (Item 1) A method for producing recombinant alkaline phosphatase, (a) Inoculating a bioreactor with cells expressing recombinant alkaline phosphatase; (b) Obtain an aqueous medium containing the recombinant alkaline phosphatase; (c) Obtain a certain amount from the aqueous culture medium approximately 6 to 10 days after inoculation; (d) To quantify the total sialic acid content (TSAC) molar concentration per mole of recombinant alkaline phosphatase in the aforementioned fixed amount; (e) Collecting the aqueous culture medium; (f) Perform at least one purification step to obtain a bulk drug solution (BDS) Includes, (i) (1) The specified amount contains a TSAC concentration of less than approximately 2.5 moles / mol, and the filtration step is maintained for less than approximately 9 hours; (2) The specified amount contains a TSAC concentration of approximately 2.5 moles / mol to approximately 2.7 moles / mol, and the filtration step is maintained for approximately 10 to 14 hours; (3) The specified amount contains a TSAC concentration of approximately 2.8 mol / mol to approximately 3.0 mol / mol, and the filtration step is maintained for approximately 23 hours to approximately 27 hours; or (4) The specified amount contains a TSAC concentration greater than approximately 3.0 mol / mol, and the filtration step is maintained for approximately 38 hours to approximately 42 hours; or (ii) (1) The specified amount contains a TSAC concentration of less than approximately 2.5 mol / mol, and the filtration step is maintained for approximately 5 to 9 hours; (2) The specified amount contains a TSAC concentration of approximately 2.5 mol / mol to approximately 2.7 mol / mol, and the filtration step is maintained for approximately 16 hours to approximately 20 hours; or (3) The specified amount contains a TSAC concentration greater than approximately 2.7 mol / mol, and the filtration step is maintained for approximately 30 to 34 hours; or (iii) (1) The specified amount contains a TSAC concentration of approximately 2.3 moles / mol or less, and the filtration step is maintained for approximately 14 to 22 hours; (2) The specified amount contains a TSAC concentration of approximately 2.4 moles / mol to approximately 3.1 moles / mol, and the filtration step is maintained for approximately 28 hours to approximately 36 hours; or (3) A method wherein the fixed amount contains a TSAC concentration of about 3.2 moles / mol or more, and the filtration step is maintained for about 40 hours to about 48 hours. (Item 2) The method according to item 1, step (c) comprising obtaining the specified amount from the aqueous medium approximately seven days after inoculation. (Item 3) The filtration step is the method according to item 1 or 2, wherein the filtration step includes ultrafiltration, diafiltration, or a combination thereof. (Item 4) The method according to any one of items 1 to 3, wherein the cells are mammalian cells. (Item 5) The mammalian cells are Chinese hamster ovary (CHO) cells, as described in item 4. (Item 6) The method according to any one of items 1 to 5, wherein the TSAC concentration in the aforementioned fixed amount is less than about 2.5 moles / mol, and the filtration step is maintained for less than about 9 hours. (Item 7) The method according to any one of items 1 to 5, wherein the TSAC concentration in the aforementioned fixed amount is about 2.5 moles / mol to about 2.7 moles / mol, and the filtration step is maintained for about 10 hours to about 14 hours. (Item 8) The method according to any one of items 1 to 7, wherein the alkaline phosphatase concentration during the filtration step is approximately 1.8 g / L to approximately 5.0 g / L. (Item 9) The method according to item 8, wherein the alkaline phosphatase concentration during the filtration step is approximately 1.8 to approximately 4.3 g / L. (Item 10) The method according to item 9, wherein the alkaline phosphatase concentration during the filtration step is approximately 2.3 g / L, approximately 3.1 g / L, or approximately 3.7 g / L. (Item 11) The method according to any one of items 1 to 10, wherein the TSAC concentration of the BDS is approximately 1.2 mol / mol to approximately 3.0 mol / mol. (Item 12) The method according to item 11, wherein the TSAC concentration of the BDS is approximately 1.6 mol / mol to approximately 2.4 mol / mol. (Item 13) The filtration step is performed according to any one of items 1 to 12, wherein the filtration step is maintained at a constant temperature. (Item 14) The method described in item 13, wherein the constant temperature is approximately 15°C to approximately 25°C. (Item 15) The method described in item 14, wherein the constant temperature is approximately 19°C to approximately 25°C. (Item 16) The method according to item 15, wherein the temperature is approximately 22°C. (Item 17) The aforementioned fixed quantity is obtained aseptically by the method described in any one of items 1 to 16. (Item 18) The method according to any one of items 1 to 17, wherein the aforementioned fixed amount is approximately 1 mL to approximately 500 mL. (Item 19) The aforementioned fixed quantity is approximately 50 mL to approximately 300 mL, as described in item 18. (Item 20) The method according to item 19, wherein the aforementioned fixed amount is approximately 100 mL or approximately 200 mL. (Item 21) The method according to any one of items 1 to 20, further comprising step (c) centrifugation of the specified amount. (Item 22) The method according to item 21, further comprising step (c) removing the supernatant from the specified amount. (Item 23) The method according to item 22, further comprising step (c) purifying the alkaline phosphatase from the supernatant using a chromatography column. (Item 24) The chromatography column according to item 23, comprising a Protein A column, a 1 mL HiTrap Protein A column, a 600 μl Protein A Robocolumn, or a MabSelect Sure Protein A solid-phase column. (Item 25) The method described in item 24, wherein the protein A column is a MabSelect Sure protein A column. (Item 26) Step (c) is the method according to any one of items 23 to 25, further comprising performing a buffer exchange. (Item 27) The method according to any one of items 23 to 26, further comprising step (c) concentrating the alkaline phosphatase. (Item 28) The method according to any one of items 1 to 27, wherein step (d) is to carry out acid hydrolysis to release TSAC. (Item 29) The method according to any one of items 1 to 28, further comprising freeze-drying the alkaline phosphatase. (Item 30) The method according to item 29, further comprising loading the alkaline phosphatase into a vial. (Item 31) The bioreactor having a volume of at least 2 L, according to the method of any one of items 1 to 30. (Item 32) The method according to item 31, wherein the volume is at least 10 L. (Item 33) The method according to item 32, wherein the volume is at least 1,000 L. (Item 34) The method according to item 33, wherein the volume is at least 10,000 L. (Item 35) The method according to item 34, wherein the volume is approximately 20,000 L. (Item 36) The culture medium is selected from the group consisting of EX-CELL® 302 serum-free medium; CD DG44 medium; BD Select® medium; SFM4CHO medium; and combinations thereof, as described in any one of items 1 to 34. (Item 37) The recombinant alkaline phosphatase is W-sALP-X-Fc-Y-Dn-Z (wherein the formula, W is either absent or an amino acid sequence of at least one amino acid; X is either absent or an amino acid sequence of at least one amino acid; Y is either absent or an amino acid sequence of at least one amino acid; Z is either absent or an amino acid sequence of at least one amino acid; Fc is a crystallizable region fragment; Dn is polyaspartic acid, polyglutamate, or a combination thereof, where n = 10 or 16; and sALP is a soluble alkaline phosphatase. The method described in any one of items 1 to 36, including the structure. (Item 38) The method according to item 37, wherein the recombinant alkaline phosphatase comprises an amino acid sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 1. (Item 39) The method according to item 37, wherein the recombinant alkaline phosphatase comprises or consists of the amino acid sequence shown in SEQ ID NO: 1.

Claims

[Claim 1] The invention described in the specification.

Images