Affinity chromatography using an elution buffer containing glycine and arginine

Abstract

The present invention provides a method for purifying a protein or polypeptide, in particular by affinity chromatography, such as custom affinity chromatography, followed by subsequent downstream chromatography steps, the method comprising eluting a recombinant protein from an affinity column using an elution buffer, the elution buffer comprising glycine and arginine at a concentration such that the conductivity is below the conductivity limit of the subsequent chromatography resin.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 359,120, filed on July 7, 2022, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] The purification of recombinant proteins and polypeptides has several applications in therapeutics. Typically, protein purification processes include various chromatography steps, including, among others, affinity chromatography, ion - exchange chromatography, mixed - mode chromatography, and / or hydrophobic interaction chromatography. Increasing the yield and maintaining high activity are challenges for the recombinant protein purification process. Adjusting the purification process in multiple steps for efficient and high - yield purification at each step while maintaining compatible elution and pH conditions that work favorably for downstream processing remains a challenge. For example, acidic elution is commonly used in affinity chromatography, but some proteins are not very stable under acidic pH.

Summary of the Invention

Means for Solving the Problems

[0003] The present invention relates, inter alia, to an improved method for purifying a protein or polypeptide by affinity chromatography (e.g., custom affinity chromatography), comprising eluting the polypeptide from an affinity chromatography column using an elution buffer having a pH and conductivity compatible with further downstream purification and processing, thereby minimizing loss of product and product quality. In one aspect, the elution buffer comprises glycine and arginine. The present invention also provides a platform technology for optimizing elution buffers for affinity chromatography (e.g., custom affinity chromatography). In one aspect, the platform technology can provide specific ranges of glycine and arginine concentrations, conductivity, and / or pH of the elution buffer.

[0004] The inventors of the present application have surprisingly and unexpectedly discovered that the affinity resin has an ion exchange effect at the start of elution, causing subsequent changes in conductivity and pH in the affinity column, thereby degrading the quality of the product. For example, when the elution buffer contacts the resin, the resin is acidified and has an anion exchange effect, whereby negatively charged ions bind to the resin and deplete from the solution. The inventors have discovered that multiple affinity resins have an ion exchange effect during elution.

[0005] Furthermore, acidic elution buffers commonly used in affinity chromatography cause denaturation of some proteins, leading to reduced yields and activities. Formation of aggregates resulting in precipitation and reduced yields is another problem. Different strategies exist to raise the elution pH from low pH to a milder pH, including, for example, increasing the salt concentration or adding an organic solvent. However, these have drawbacks, for example, high salt concentrations result in high conductivity, limiting the choice of subsequent purification or polishing steps, while organic solvents denature proteins, are viscous, and pose safety issues.

[0006] Through careful experiments, the inventors of the present application designed an affinity elution buffer that contains, for example, arginine and glycine, has a pH and conductivity compatible with downstream chromatography steps, and increases the stability of proteins. The inventors of the present application obtained, for example, a low-conductivity affinity elution pool that provides flexibility in the selection of downstream chromatography steps. For example, when hydrophobic interaction chromatography (HIC) is used downstream, the conductivity of the elution pool is appropriately increased, for example, by adding salts, so that it is compatible with the HIC column. When the conductivity of the affinity elution pool is high, large-scale dilution is required in some downstream chromatography steps such as ion exchange chromatography, increasing the cost. On the other hand, the higher the conductivity of the elution pool, the more appropriate combinations of arginine buffer and glycine buffer are selected to be compatible with hydrophobic interaction chromatography or mixed-mode chromatography and to be compatible with the next downstream chromatography column. Amino acids such as arginine and glycine are safe excipients, soluble in aqueous solvents, and increase the stability of proteins by promoting preferential interactions between the solvent components and the proteins and through beneficial effects on the surface tension and structure of the aqueous solvent.

[0007] In some embodiments, arginine and glycine are added to the elution buffer to prevent a decrease in conductivity. For example, in an exemplary purification of arylsulfatase A, when a glycine hydrochloride elution buffer is used, the negatively charged chloride ions are depleted, and the remaining protons and zwitterions have no conductivity. The addition of arginine hydrochloride or sodium chloride prevents the conductivity from becoming too low. Without wishing to be bound by any particular theory, it is believed that the resin cannot deplete cations, and the addition of cations to the elution buffer prevents loss of the product. In some embodiments, the addition of cations (e.g., arginine) to the glycine buffer prevents a decrease in conductivity, which is beneficial to the quality of the product.

[0008] In some embodiments, arginine and glycine are added to the elution buffer to prevent a decrease in pH or pH instability. In some embodiments, for example, when purifying arylsulfatase A, if acetate is used as the elution buffer, acetate anions are depleted by the resin, resulting in more acidic radicals and a decrease in pH. The resulting pH instability affects the quality of the product. In some embodiments, the addition of a cation (e.g., arginine) stabilizes the elution pH.

[0009] In one aspect, a method for purifying a polypeptide, comprising loading the polypeptide onto an affinity chromatography column, eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, and subjecting the eluate from the affinity chromatography column to a subsequent chromatography column containing a resin, wherein the conductivity of the elution buffer is below the conductivity limit of the resin in the subsequent chromatography column, is provided.

[0010] In one aspect, a method for purifying a polypeptide as described herein, comprising loading the polypeptide onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, wherein the conductivity of the elution buffer is 30 mS / cm or less, is provided. In some embodiments, the conductivity of the elution buffer is 5, 10, 15, 20, 25, or 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is about 5-10 mS / cm, 6-10 mS / cm, 7-10 mS / cm, 8-10 mS / cm, or 9-10 mS / cm. In some embodiments, the conductivity of the elution buffer is about 5-30 mS / cm, 10-30 mS / cm, 15-30 mS / cm, 20-30 mS / cm, or 25-30 mS / cm.

[0011] In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of 1500 mM or less and arginine at a concentration of 300 mM or less. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 1100 mM, 1200 mM, 1300 mM, 1400 mM or 1500 mM or less and arginine at a concentration of 100 mM, 150 mM, 200 mM, 250 mM, 300 mM or less. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 0 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 1 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 2 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 3 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 4 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 5 < 0.02X + 0.1Y ≤ 30. In some embodiments, methods are provided herein in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 10 < 0.02X + 0.1Y ≤ 30.In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 15 < 0.02X + 0.1Y ≤ 30. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 20 < 0.02X + 0.1Y ≤ 30. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 25 < 0.02X + 0.1Y ≤ 30.

[0012] In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of 500 mM or less and arginine at a concentration of 100 mM or less. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, or 500 mM or less and arginine at a concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM or less. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 0 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 1 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 2 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 3 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 4 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 5 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein are methods in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 6 < 0.02X + 0.1Y ≤ 10.In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 7 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 8 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of X mM and arginine at a concentration of Y mM, where X and Y satisfy the inequality 9 < 0.02X + 0.1Y ≤ 10. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of 500 mM or less and arginine at a concentration of 100 mM or less. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of 10 - 50 mM, 50 - 100 mM, 100 - 150 mM, 150 - 200 mM, 200 - 250 mM, 250 - 300 mM, 300 - 350 mM, 350 - 400 mM, 400 - 450 mM, 450 - 500 mM and arginine at a concentration of 10 - 20 mM, 20 - 30 mM, 30 - 40 mM, 40 - 50 mM, 50 - 60 mM, 60 - 70 mM, 70 - 80 mM, 80 - 90 mM, 90 - 100 mM. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of 300 mM and arginine at a concentration of 40 mM.

[0013] In one aspect, provided herein is a method for purifying a polypeptide, the method comprising loading the polypeptide onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer, wherein the elution buffer contains glycine at a concentration of 500 mM or less and arginine at a concentration of 100 mM or less.

[0014] In one aspect, provided herein is a method for purifying a polypeptide, the method comprising loading a sample onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer, wherein the elution buffer contains glycine and arginine, and glycine and arginine are present in a ratio (molar ratio) of at least 1:2.

[0015] In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.0 to 5.0, and glycine and arginine are present in a ratio (molar ratio) of 1:2 to 40:1.

[0016] In some embodiments, the elution buffer contains arginine at a concentration of about 10 mM to 300 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 10 - 20 mM, 20 - 30 mM, 30 - 40 mM, 40 - 50 mM, 50 - 60 mM, 60 - 70 mM, 70 - 80 mM, 80 - 90 mM, 90 - 100 mM, 100 - 110 mM, 110 - 120 mM, 120 - 130 mM, 130 - 140 mM, 140 - 150 mM, 150 - 160 mM, 160 - 170 mM, 170 - 180 mM, 180 - 190 mM, 190 - 200 mM, 200 - 210 mM, 210 - 220 mM, 220 - 230 mM, 230 - 240 mM, 240 - 250 mM, 250 - 260 mM, 260 - 270 mM, 270 - 280 mM, 280 mM - 290 mM, or 290 - 300 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 5 mM to 100 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 5 - 10 mM, 10 - 15 mM, 15 - 20 mM, 20 - 25 mM, 25 - 30 mM, 30 - 35 mM, 35 - 40 mM, 40 - 45 mM, 45 - 50 mM, 50 - 55 mM, 55 - 60 mM, 60 - 65 mM, 65 - 70 mM, 70 - 75 mM, 75 - 80 mM, 80 - 85 mM, 85 - 90 mM, 90 - 95 mM, or 95 - 100 mM.

[0017] In some embodiments, the elution buffer contains glycine at a concentration of about 50 mM to 1500 mM. In some embodiments, the elution buffer contains glycine at a concentration of about 50 - 100 mM, 100 - 150 mM, 150 - 200 mM, 200 - 250 mM, 250 - 300 mM, 300 - 350 mM, 350 - 400 mM, 400 - 450 mM, 450 - 500 mM, 500 - 550 mM, 550 - 600 mM, 600 - 650 mM, 650 - 700 mM, 700 - 750 mM, 750 - 800 mM, 800 - 850 mM, 850 - 900 mM, 900 mM - 950 mM, 950 - 1000 mM, 1000 - 1100 mM, 1100 - 1200 mM, 1200 - 1300 mM, 1300 - 1400 mM, 1400 - 1500 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 25 mM to 500 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 25 - 50 mM, 50 - 75 mM, 75 - 100 mM, 100 - 125 mM, 125 - 150 mM, 150 - 175 mM, 175 - 200 mM, 200 - 225 mM, 225 - 250 mM, 250 - 275 mM, 275 - 300 mM, 300 - 325 mM, 325 - 350 mM, 350 - 375 mM, 375 - 400 mM, 400 - 425 mM, 425 - 450 mM, 450 mM - 475 mM, or 475 - 500 mM.

[0018] In some embodiments, the method further comprises adjusting the eluate from the affinity chromatography column to a pH of 4.0 - 9.0. In some embodiments, the method further comprises adjusting the eluate from the affinity chromatography column to a pH of 4.0 - 5.0, 5.0 - 6.0, 6.0 - 7.0, 7.0 - 8.0 or 8.0 - 9.0.

[0019] In some embodiments, the elution buffer further comprises a step of inactivating the virus in the eluate from the affinity chromatography column.

[0020] In some embodiments, the eluate of the affinity column contains less than 5% high molecular weight aggregates (HMW). In some embodiments, the eluate of the affinity column contains less than 4% high molecular weight aggregates (HMW). In some embodiments, the eluate of the affinity column contains less than 3% high molecular weight aggregates (HMW). In some embodiments, the eluate of the affinity column contains less than 2.5% high molecular weight aggregates (HMW). In some embodiments, the eluate of the affinity column contains less than 2% high molecular weight aggregates (HMW). In some embodiments, the eluate of the affinity column contains less than 1% high molecular weight aggregates (HMW).

[0021] In some embodiments, the affinity chromatography column uses an antibody or antigen-binding fragment that specifically binds to the polypeptide. In some embodiments, the affinity chromatography column uses an antibody that specifically binds to the polypeptide. In some embodiments, the affinity chromatography column uses an antigen-binding fragment that specifically binds to the polypeptide. In some embodiments, the affinity chromatography column comprises a custom affinity chromatography resin. In some embodiments, the affinity chromatography column comprises an affinity ligand that is screened and selected from a library of ligands for the polypeptide. In some embodiments, the affinity chromatography resin comprises an affinity ligand that is screened and selected from a library of proteins or peptides for the polypeptide. In some embodiments, the affinity chromatography resin comprises an affinity ligand that is screened and selected from a library that comprises more than 100 proteins or peptides.

[0022] In some embodiments, the method further includes adjusting the concentrations of arginine and glycine in the elution buffer based on the conductivity limit of the resin downstream of the polypeptide. In some embodiments, the method further includes subjecting the eluate from the affinity chromatography column to a chromatography step. In some embodiments, the method includes subjecting the eluate from the affinity chromatography column to a chromatography step, and the chromatography step is selected from the group consisting of ion exchange, mixed mode, or hydroxyapatite chromatography. In some embodiments, the chromatography step is mixed mode chromatography. In some embodiments, the chromatography step is hydroxyapatite chromatography. In some embodiments, the chromatography step is ion exchange chromatography. In some embodiments, the ion exchange chromatography is anion exchange chromatography. In some embodiments, the ion exchange chromatography is cation exchange chromatography.

[0023] In one aspect, provided herein is a method comprising determining the conductivity limit of a chromatography step and adjusting the glycine and / or arginine concentration of the affinity chromatography column elution buffer to the conductivity limit. In some embodiments, the conductivity limit is about ≦30 mS / cm. In some embodiments, the conductivity limit is about 30 mS / cm. In some embodiments, the conductivity limit is about 20 mS / cm. In some embodiments, the conductivity limit is about 10 mS / cm.

[0024] In some embodiments, the affinity chromatography column elution buffer has a pH of 3.0 to 5.0. In some embodiments, the affinity chromatography column elution buffer has a pH of 3.0 to 3.5, 3.5 to 4.0, 4.0 to 4.5, or 4.5 to 5.0.

[0025] In some embodiments, the polypeptide is a recombinant protein. In some embodiments, the polypeptide is an enzyme. In some embodiments, the polypeptide retains at least 80% of its specific activity as compared to its specific activity before purification. In some embodiments, the polypeptide retains at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its specific activity as compared to its specific activity before purification. In some embodiments, the polypeptide retains at least 80% - 85%, 85 - 90%, 90 - 95%, 95 - 99% or 100% of its specific activity as compared to its specific activity before purification.

[0026] In some embodiments, the polypeptide is a recombinant protein. In some embodiments, the polypeptide is an enzyme that retains at least 80% of its specific activity as compared to its specific activity before purification. In some embodiments, the polypeptide is an enzyme that retains at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its specific activity as compared to its specific activity before purification. In some embodiments, the polypeptide is an enzyme that retains at least 80% - 85%, 85 - 90%, 90 - 95%, 95 - 99% or 100% of its specific activity as compared to its specific activity before purification. In some embodiments, the polypeptide is recombinant human arylsulfatase A. In some embodiments, the polypeptide is recombinant iduronate 2-sulfatase. In some embodiments, the polypeptide is recombinant alpha-galactosidase A. In some embodiments, the polypeptide is a natural protein. In some embodiments, the polypeptide is a natural human protein.

[0027] In some embodiments, the polypeptide does not include an antibody or Fc fusion protein that binds to protein A or protein G. In some embodiments, the polypeptide does not include a domain that binds to protein A or protein G. In some embodiments, the polypeptide includes an antigen-binding fragment that does not include an Fc region. In some embodiments, the antigen-binding fragment is unable to bind to protein A or protein G. In some embodiments, the Fc region reduces protein A / G binding, e.g., of an IgG3 antibody.

[0028] The affinity chromatography elution buffer can be adjusted to be compatible with downstream chromatography resins by including a cation (e.g., arginine or salt) or zwitterion (e.g., glycine), and by adjusting the concentrations of glycine and arginine in the elution buffer, with the protein yield increasing more greatly at lower pHs with lower acidity. In some embodiments, adjusting the concentrations of glycine and arginine increases protein activity. In some embodiments, changing the concentrations of glycine and arginine reduces protein aggregation. In some embodiments, altering the concentrations of glycine and arginine increases the stability of the protein.

[0029] As described in the Examples section, exemplary protein purification using the specific processes described herein meets the sales purity requirements in the United States and many other countries.

[0030] As used in this application, the terms “about” and “approximately” are used as synonyms. Whether or not there is an about / approximately, any number used in this application is meant to cover any normal variation understood by one of ordinary skill in the relevant art.

[0031] Other features, objects, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description is given by way of illustration only and not limitation, while presenting embodiments of the present invention. Various changes and modifications within the scope of the present invention will be apparent to those skilled in the art from the detailed description.

[0032] The figures described below are constituted together as a whole of the drawings, but are for illustrative purposes only and not for purposes of limitation.

Brief Description of the Drawings

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BEST MODE FOR CARRYING OUT THE INVENTION

[0034] Definitions For the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout this specification.

[0035] Approximately or about: As used herein, when applied to one or more target values, the terms "approximately" or "about" refer to values that are similar to the referenced value being recited. In certain embodiments, the terms "approximately" or "about" refer to a range of values within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or lesser) of the recited reference value, unless otherwise specified or clear from the context (except when such a number exceeds 100% of the possible value).

[0036] Biological activity: As used herein, the phrase "biological activity" refers to a characteristic of any agent that is active in a biological system, particularly in an organism. For example, when administered to an organism, an agent that has a biological effect on that organism is considered to be biologically active. In certain embodiments, when a protein or polypeptide is biologically active, the portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as the "biologically active" portion.

[0037] Chromatography: As used herein, the term "chromatography" refers to a technique for separating mixtures. Typically, a mixture is dissolved in a fluid called the "mobile phase," which passes through a structure that holds another material called the "stationary phase." Column chromatography is a separation technique where the stationary layer is within a tube, i.e., a column.

[0038] Conductivity: As used herein, the term "conductivity" refers to a measure of electrical conduction and indicates the ability of an aqueous solution to conduct an electric current.

[0039] Diluent: As used herein, the term "diluent" refers to a pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) diluting substance useful in the preparation of reconstituted formulations. Typical diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffers (e.g., phosphate buffered saline), sterile saline, Ringer's solution or glucose solution.

[0040] Elution: As used herein, the term "elution" refers to the process of extracting one material from another by washing with a solvent. For example, in ion exchange chromatography, elution is the process of washing the supported resin to remove the captured ions. Elution refers to the dissociation of the binding of the target product, e.g., a recombinant protein, from the resin and its extraction into a solvent or buffer as part of the purification process.

[0041] Eluate: As used herein, the term "eluate" refers to the combination of the mobile phase "carrier" and the analyte material that typically emerges from chromatography as a result of elution.

[0042] Equilibration or Equilibrium: As used herein, the terms "equilibration" or "equilibrium" as related to chromatography generally refer to the process of equilibrating a first liquid (e.g., a buffer) with another liquid in order to achieve a stable and uniform distribution of the liquid components. For example, in some embodiments, a chromatography column can be equilibrated by passing one or more column volumes of a desired liquid (e.g., a buffer) through the column.

[0043] Improvement, Increase, or Decrease: As used herein, the terms "improvement", "increase", or "decrease", or grammatical equivalents thereof, indicate a value relative to a baseline measurement, such as a measurement in the same individual before initiation of the treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of the treatment described herein. A "control individual" is an individual who has the same form of lysosomal storage disease as the individual being treated and is approximately the same age as the individual being treated (to ensure that the stage of the disease is equivalent in the treated individual and the control individual(s)).

[0044] Impurity: As used herein, the term "impurity" refers to a substance within a limited amount of liquid, gas, or solid that is different from the chemical composition of the target material or compound. Impurities are also referred to as contaminants.

[0045] Loading: As used herein, the term "loading" refers to adding a liquid or solid containing a sample to a column in chromatography. In some embodiments, certain components of the sample loaded onto the column are then captured as the loaded sample passes through the column. In some embodiments, certain components of the sample loaded onto the column are not captured by the column or by the "flow" of the column as the loaded sample passes through the column.

[0046] Peak splitting: Usually, after chromatography, a single protein elutes as a single peak. Since different proteins form different peaks, separation between different proteins is achieved. However, when a single protein forms multiple peaks, i.e., peak splitting, it appears as a shoulder peak, or a doublet peak, or a distorted peak with the same baseline. Some factors contributing to peak splitting are hydrophobicity, loading rate, and the tendency of molecules to aggregate. Peak splitting is undesirable in protein purification because the yield and / or purity are affected. When collecting multiple peaks, the purity decreases, and when collecting a single peak, the yield decreases. It is desirable to minimize peak splitting. In some embodiments, arginine in the elution buffer of the present invention minimizes peak splitting.

[0047] Polypeptide: As used herein, "polypeptide" generally refers to at least two series of amino acids linked to each other by peptide bonds. In some embodiments, a polypeptide may contain at least 3 to 5 amino acids, each of which is linked to other amino acids by at least one peptide bond. One of ordinary skill in the art will understand that polypeptides may sometimes contain "non-natural" amino acids or other entities, and nevertheless, they can optionally be incorporated into the polypeptide chain.

[0048] Pool: As used herein, the term "pool" related to chromatography refers to combining one or more fractions of fluid that have passed through a column together. For example, in some embodiments, one or more fractions (e.g., "peak fractions") containing the desired component of a sample separated by chromatography can be combined and "pooled" together to produce a single "pooled" fraction.

[0049] Solubility: As used herein, the term "soluble" refers to the ability of a therapeutic agent to form a homogeneous solution. In some embodiments, the solubility of the therapeutic agent in the solution in which it is administered and thereby transported to the site of action is sufficient to enable delivery of a therapeutically effective amount of the therapeutic agent to the site of action. Several factors can affect the solubility of a therapeutic agent. For example, relevant factors that can affect the solubility of a protein include ionic strength, amino acid sequence, and the presence of other co-solvents or salts (e.g., calcium salts). In some embodiments, the therapeutic agents according to the invention are soluble in their corresponding pharmaceutical compositions.

[0050] Stability: As used herein, the term "stable" refers to the ability of a therapeutic agent (e.g., a recombinant enzyme) to maintain its therapeutic effect (e.g., all or most of its intended biological activity and / or biochemical integrity) over an extended period of time. The stability of a therapeutic agent and the ability of a pharmaceutical composition to maintain the stability of such a therapeutic agent can be evaluated over an extended period of time (e.g., at least 1, 3, 6, 12, 18, 24, 30, 36 months or more). In the context of a formulation, a stable formulation is one in which the therapeutic agent therein essentially retains its physical and / or chemical integrity and biological activity during storage and during processes (such as freezing / thawing, mechanical mixing, and lyophilization). In the case of protein stability, it can be measured by the formation of high molecular weight (HMW) aggregates, loss of enzyme activity, generation of peptide fragments, and shifts in charge profile.

[0051] Virus treatment: As used herein, the term "virus treatment" refers to "virus removal" (e.g., virus filtration) which simply removes viruses from a sample, or "virus inactivation" in which viruses remain in a non-infectious form in the sample. In some embodiments, virus removal can utilize, inter alia, nanofiltration techniques and / or chromatography techniques. In some embodiments, virus inactivation can utilize, inter alia, solvent inactivation, surfactant inactivation, pasteurization, acidic pH inactivation, and / or ultraviolet inactivation.

[0052] The present invention provides an improved method that, among other things, purifies a recombinant protein or polypeptide (e.g., an enzyme for enzyme replacement therapy, such as including arylsulfatase A, iduronate 2-sulfatase, alpha-galactosidase A, etc.) by affinity chromatography, and elutes the polypeptide from an affinity chromatography column using an elution buffer having pH and conductivity characteristics compatible with further downstream purification and processing, thereby minimizing loss of the product and product quality. In one aspect, the elution buffer includes glycine and arginine.

[0053] The present invention is based in part on the surprising and unexpected discovery that the affinity resin has an ion exchange effect at the start of elution, causing subsequent changes in conductivity and pH in the affinity column, thereby degrading the quality of the product. For example, when the elution buffer contacts the resin, the resin becomes acidified and has an anion exchange effect, whereby negatively charged ions bind to the resin and deplete from the solution. The inventors have discovered the ion exchange effect in a plurality of different affinity resins.

[0054] Acidic elution buffers commonly used in affinity chromatography cause denaturation of some proteins, leading to reduced yields and activities. Formation of aggregates that lead to precipitation also leads to reduced yields. The present invention provides an affinity elution buffer that has pH and conductivity characteristics compatible with downstream chromatography steps and leads to increased protein stability, yield, and activity.

[0055] In one aspect, a method for purifying a polypeptide, comprising loading the polypeptide onto an affinity chromatography column, eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, and subjecting the eluate from the affinity chromatography column to a subsequent chromatography column containing a resin, wherein the conductivity of the elution buffer is below the conductivity limit of the resin in the subsequent chromatography column, is provided.

[0056] In one aspect, provided herein is a method for purifying a polypeptide, comprising loading the polypeptide onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, wherein the conductivity of the elution buffer is 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 5, 10, 15, 20, 25, or 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 10 mS / cm or less. In some embodiments, the conductivity of the elution buffer is about 5-10 mS / cm, 6-10 mS / cm, 7-10 mS / cm, 8-10 mS / cm, or 9-10 mS / cm. In some embodiments, the conductivity of the elution buffer is about 5-30 mS / cm, 10-30 mS / cm, 15-30 mS / cm, 20-30 mS / cm, or 25-30 mS / cm.

[0057] Affinity chromatography The purification method described herein may include one or more steps of affinity chromatography (e.g., immunoaffinity chromatography, immobilized metal ion affinity chromatography, and / or immobilized ligand affinity chromatography).

[0058] Briefly described, affinity chromatography is a chromatography technique that relies on highly specific interactions, such as between a receptor and a ligand, an antigen and an antibody, or an enzyme and a substrate. As known to those skilled in the art, the selective molecules used in the affinity chromatography step in the purification method described herein may be based on various properties (such as three-dimensional structure, glycosylation, etc.) of the molecule and can be produced recombinantly (e.g., arylsulfatase A, iduronate 2-sulfatase, alpha-galactosidase A). Exemplary selective molecules (or capture reagents) that can be utilized in the affinity chromatography step include recombinantly purified proteins, protein A, protein G, antibodies, antigen-binding fragments of antibodies (e.g., VHH), such as single-domain VHH antibody fragments derived from camelids, metal ions (e.g., nickel), specific substrates, ligands, or antigens. In some embodiments, an affinity capture ligand, such as a VHH domain affinity ligand, is a low molecular weight 14kD capture ligand that results in increased yield and purity. In some embodiments, the selective molecules suitable for the affinity chromatography step of the present invention utilize recombinantly produced proteins (e.g., anti-human arylsulfatase A antibody). Suitable recombinantly purified protein antibodies (e.g., arylsulfatase A antibody) or antigen-binding fragments of antibodies can be commercially obtained from animals other than humans (e.g., mice, rats, rabbits, chickens, goats, sheep, horses or other suitable animals for generating antibodies against human proteins), or by in vitro display methods (e.g., bacterial or yeast surface display).

[0059] In some embodiments, for example, affinity chromatography resins such as, but not limited to, CaptureSelect®, AVIPure™, or other custom affinity resins are used. For a particular target molecule, an affinity ligand (e.g., a peptide or protein) is screened from a library of ligands of different sizes, shapes, conformations, and contact areas, and once a ligand is identified based on affinity, selectivity, stability, and favorable manufacturing characteristics, the affinity ligand is cross-linked onto the resin to generate a custom affinity resin. In some embodiments, the ligand is a small protein (e.g., CaptureSelect®). In some embodiments, the ligand is a small peptide (e.g., AviPure™). In some embodiments, the custom affinity resin is selected based on criteria including, but not limited to, ligand density, chemical properties, matrix size, porosity, and linker.

[0060] Generally, the recombinant protein produced (e.g., recombinant arylsulfatase A) is captured on a solid or stationary phase or matrix via interaction with a selective molecule, while other unwanted molecules are not captured as they are not bound by the selective molecule(s). The solid matrix is then removed from the mixture, optionally washed, and the molecule of interest can be released from the enclosure by elution. In some embodiments, the affinity column can be eluted by changing the ionic strength via a gradient. For example, salt concentration, pH, pI, and ionic strength can be used to effect separation or form a gradient for separation.

[0061] Provided herein is a method for designing an elution buffer having physical (in terms of conductivity, pH) and chemical (in terms of buffer and dissolved salt composition) properties that enable compatibility with downstream processes and result in a recombinant protein or polypeptide product in high yield, high purity, and high activity for affinity chromatography.

[0062] In some embodiments, the recombinantly produced protein can be produced with a tag to facilitate purification by affinity chromatography. As will be well known to those skilled in the art, protein tags can include, for example, among others, glutathione-S-transferase (GST), hexahistidine (His), maltose binding protein (MBP), and the like. In some embodiments, lectins are used in affinity chromatography to separate components within a sample. For example, certain lectins can specifically bind to specific carbohydrate molecules and can be used to separate glycoproteins from non-glycosylated proteins or to separate one glycoform from another.

[0063] In some embodiments, the elution buffer contains acetate, citrate, guanidine hydrochloride, or glycine hydrochloride. A commonly used elution buffer for protein affinity purification is 0.1 M glycine-HCl (pH 2.5 - 3.0). This buffer effectively dissociates most protein-protein and antibody-antigen binding interactions without permanently affecting the structure of the protein. In some embodiments, an amino acid or amino acid derivative is added to the elution buffer, for example, an amino acid such as glycine, arginine, proline, lysine, histidine, etc., or an amino acid derivative such as an arginine derivative containing acetylarginine or agmatine. In some embodiments, the elution buffer contains arginine and glycine. In some embodiments, the elution buffer contains an arginine derivative and glycine. In some embodiments, the elution buffer contains an arginine derivative and a glycine derivative.

[0064] Arginine and glycine are amino acids added to the elution buffer to prevent a decrease in conductivity. For example, in an exemplary purification of arylsulfatase A, when a glycine hydrochloride elution buffer is used, negatively charged chloride ions are depleted, and the remaining protons and zwitterions have no conductivity. The addition of arginine hydrochloride or sodium chloride prevents the conductivity from becoming too low. Without wishing to be bound by any particular theory, it is believed that the resin cannot deplete cations, and the addition of cations to the elution buffer prevents loss of the product.

[0065] Arginine and glycine are added to the elution buffer to prevent a decrease in pH. In some embodiments, for example, when acetate is used as the elution buffer in purifying arylsulfatase A, acetate anions are depleted by the resin, resulting in more acidic radicals and a decrease in pH. The resulting pH instability affects the quality of the product. The addition of cations (e.g., arginine) or zwitterions (e.g., glycine) stabilizes the elution pH and prevents a decrease in conductivity, thereby benefiting the quality of the product.

[0066] In one aspect, there is provided a method for purifying a polypeptide, comprising loading the polypeptide onto an affinity chromatography column, eluting the polypeptide from the affinity chromatography column using an elution buffer comprising glycine and arginine, and subjecting the eluate from the affinity chromatography column to a subsequent chromatography column comprising a resin, wherein the conductivity of the elution buffer is below the conductivity limit of the resin in the subsequent chromatography column.

[0067] In one aspect, as described herein, a method for purifying a polypeptide, comprising loading the polypeptide onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer comprising glycine and arginine, A method is provided in which the conductivity of the elution buffer is 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 5, 10, 15, 20, 25 or 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 10 mS / cm or less. In some embodiments, the conductivity of the elution buffer is 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, 30 mS / cm or less. In some embodiments, the conductivity of the elution buffer is about 5 - 10 mS / cm, 6 - 10 mS / cm, 7 - 10 mS / cm, 8 - 10 mS / cm, or 9 - 10 mS / cm. In some embodiments, the conductivity of the elution buffer is about 5 - 30 mS / cm, 10 - 30 mS / cm, 15 - 30 mS / cm, 20 - 30 mS / cm, or 25 - 30 mS / cm.

[0068] The concentration of glycine in the elution buffer is selected based on the conductivity and pH that result in a high yield and stability of the protein. For example, in some embodiments, the elution buffer contains glycine at a concentration of 1500 mM or less, and / or arginine at a concentration of 300 mM or less. In some embodiments, the elution buffer contains glycine at a concentration of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1000 mM, 1100 mM, 1200 mM, 1300 mM, 1400 mM or 1500 mM or less, and / or arginine at a concentration of 100 mM, 150 mM, 200 mM, 250 mM, 300 mM or less.

[0069] In some embodiments, the elution buffer contains glycine at a concentration of 500 mM or less, and / or arginine at a concentration of 100 mM or less. In some embodiments, provided herein is a method in which the elution buffer contains glycine at a concentration of 50 mM, 100 mM, 200 mM, 300 mM, 400 mM or 500 mM or less, and / or arginine at a concentration of 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mM or less.

[0070] In one aspect, the present specification provides a method for purifying a polypeptide, which includes loading the polypeptide onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer, wherein the elution buffer contains glycine at a concentration of 500 mM or less and / or arginine at a concentration of 100 mM or less.

[0071] In one aspect, the present specification provides a method for purifying a polypeptide. The method includes loading a sample onto an affinity chromatography column and eluting the polypeptide from the affinity chromatography column using an elution buffer, wherein the elution buffer contains glycine and arginine in a ratio (molar ratio) of at least 1:2. In some embodiments, glycine and arginine are present in a ratio (molar ratio) of 1:2 to 40:1.

[0072] In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.0 to 5.0. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.25 to 4.75. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.0 to 3.5. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.25 to 3.75. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.5 to 4.0. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 3.75 to 4.25. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 4.0 to 4.5. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 4.25 to 4.75. In some embodiments, the elution buffer of the method provided herein contains glycine and arginine at a pH of 4.5 to 5.0.

[0073] In some embodiments, the elution buffer contains arginine at a concentration of about 10 mM to 300 mM. In some embodiments, the elution buffer contains arginine at a concentration of about 10 - 20 mM, 20 - 30 mM, 30 - 40 mM, 40 - 50 mM, 50 - 60 mM, 60 - 70 mM, 70 - 80 mM, 80 - 90 mM, 90 - 100 mM, 100 - 110 mM, 110 - 120 mM, 120 - 130 mM, 130 - 140 mM, 140 - 150 mM, 150 - 160 mM, 160 - 170 mM, 170 - 180 mM, 180 - 190 mM, 190 - 200 mM, 200 - 210 mM, 210 - 220 mM, 220 - 230 mM, 230 - 240 mM, 240 - 250 mM, 250 - 260 mM, 260 - 270 mM, 270 - 280 mM, 280 mM - 290 mM, or 290 - 300 mM.

[0074] In some embodiments, the elution buffer contains glycine at a concentration of about 50 mM to 1500 mM. In some embodiments, the elution buffer contains glycine at a concentration of about 50 - 100 mM, 100 - 150 mM, 150 - 200 mM, 200 - 250 mM, 250 - 300 mM, 300 - 350 mM, 350 - 400 mM, 400 - 450 mM, 450 - 500 mM, 500 - 550 mM, 550 - 600 mM, 600 - 650 mM, 650 - 700 mM, 700 - 750 mM, 750 - 800 mM, 800 - 850 mM, 850 - 900 mM, 900 mM - 950 mM, 950 - 1000 mM, 1000 - 1100 mM, 1100 - 1200 mM, 1200 - 1300 mM, 1300 - 1400 mM, 1400 - 1500 mM.

[0075] In some embodiments, the method further comprises adjusting the eluate from the affinity chromatography column to a pH of 4.0 - 9.0. In some embodiments, the method further comprises adjusting the eluate from the affinity chromatography column to a pH of 4.0 - 5.0, 5.0 - 6.0, 6.0 - 7.0, 7.0 - 8.0 or 8.0 - 9.0.

[0076] In some embodiments, the elution buffer further comprises a step of virus inactivation of the eluate from the affinity chromatography column.

[0077] In some embodiments, the eluate from the affinity column comprises less than 5% high molecular weight aggregates (HMW). In some embodiments, the eluate from the affinity column comprises less than 4% high molecular weight aggregates (HMW). In some embodiments, the eluate from the affinity column comprises less than 3% high molecular weight aggregates (HMW). In some embodiments, the eluate from the affinity column comprises less than 2.5% high molecular weight aggregates (HMW). In some embodiments, the eluate from the affinity column comprises less than 2% high molecular weight aggregates (HMW). In some embodiments, the eluate from the affinity column comprises less than 1% high molecular weight aggregates (HMW).

[0078] In some embodiments, the affinity chromatography column uses an antibody or antigen-binding fragment of an antibody that specifically binds to a polypeptide. In some embodiments, the affinity chromatography column uses the variable domain of a heavy chain antibody that specifically binds to a polypeptide. In some embodiments, the affinity chromatography column binds to a polypeptide utilizing an antibody-antigen binding interaction. For example, in some embodiments, the Capture Select affinity chromatography resin binds to a specific antibody subdomain region and is used to specifically target any therapeutic protein, producing a high yield of pure therapeutic product. The affinity ligand is designed to have a specific affinity, specificity, and elution profile. In some embodiments, gentle elution conditions are used to produce a stable product.

[0079] In some embodiments, the method further includes adjusting the concentrations of arginine and glycine in the elution buffer based on the conductivity limit of the polypeptide resin. In some embodiments, the method further includes subjecting the eluate from the affinity chromatography column to a chromatography step. In some embodiments, the method includes subjecting the eluate from the affinity chromatography column to a chromatography step, the chromatography step being selected from the group consisting of ion exchange, mixed mode, or hydroxyapatite chromatography. In some embodiments, the chromatography step is mixed mode chromatography. In some embodiments, the chromatography step is hydroxyapatite chromatography. In some embodiments, the chromatography step is ion exchange chromatography. In some embodiments, the ion exchange chromatography is anion exchange chromatography. In some embodiments, the ion exchange chromatography is cation exchange chromatography.

[0080] In one aspect, provided herein is a method comprising determining the conductivity limit of a chromatography step and adjusting the glycine and / or arginine concentration of the affinity chromatography column elution buffer to the conductivity limit. In some embodiments, the conductivity limit is about ≦30 mS / cm. In some embodiments, the conductivity limit is about ≦10 mS / cm.

[0081] In some embodiments, the affinity chromatography column elution buffer has a pH of 3.0 to 5.0. In some embodiments, the affinity chromatography column elution buffer has a pH of 3.0 to 3.5, 3.5 to 4.0, 4.0 to 4.5, or 4.5 to 5.0.

[0082] In some embodiments, the polypeptide is a recombinant protein. In some embodiments, the polypeptide is an enzyme. In some embodiments, the polypeptide retains at least 80% of its specific activity compared to its specific activity before purification. In some embodiments, the polypeptide retains at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its specific activity compared to its specific activity before purification. In some embodiments, the polypeptide retains at least 80% - 85%, 85 - 90%, 90 - 95%, 95 - 99% or 100% of its specific activity compared to its specific activity before purification.

[0083] In some embodiments, the polypeptide is a recombinant protein. In some embodiments, the polypeptide is an enzyme that retains at least 80% of its specific activity compared to its specific activity before purification. In some embodiments, the polypeptide retains at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% of its specific activity compared to its specific activity before purification. In some embodiments, the polypeptide is an enzyme that retains at least 80% - 85%, 85 - 90%, 90 - 95%, 95 - 99% or 100% of its specific activity compared to its specific activity before purification. In some embodiments, the polypeptide is a lysosomal enzyme such as glycosidase, protease, and sulfatase. In some embodiments, the polypeptide is recombinant human arylsulfatase A. In some embodiments, the polypeptide is recombinant iduronate 2-sulfatase. In some embodiments, the polypeptide is recombinant alpha-galactosidase A.

[0084] In some embodiments, the polypeptide does not contain an Fc region that binds to protein A or protein G. In some embodiments, the polypeptide does not contain an Fc region.

[0085] The elution buffer for affinity chromatography can be adjusted to be compatible with the downstream chromatography resin by including cations (e.g., arginine or salts) or zwitterions (e.g., glycine), and by adjusting the concentrations of glycine and arginine in the elution buffer. The protein yield increases more significantly at a lower acidity pH. In some embodiments, by adjusting the concentrations of glycine and arginine, the protein activity increases. In some embodiments, by varying the concentrations of glycine and arginine, protein aggregation is reduced. In some embodiments, by changing the concentrations of glycine and arginine, the stability of the protein increases.

[0086] Accordingly, in some embodiments, the present invention provides subjecting the eluate from the affinity chromatography column to one or more subsequent steps of chromatography selected from the group consisting of ion exchange, mixed mode, or hydroxyapatite chromatography.

[0087] In some embodiments, following affinity chromatography, one or more of the following downstream chromatography processes are used.

[0088] Ion exchange chromatography The purification method described herein may include one or more steps of ion exchange chromatography (e.g., anion exchange chromatography and / or cation exchange chromatography).

[0089] As is known to those skilled in the art, ion exchangers (e.g., anion exchangers and / or cation exchangers) may be based on various materials with respect to the matrix and with respect to the charged groups attached. For example, the following matrices may be used, where the materials mentioned may be more or less cross-linked: agarose-based (such as SEPHAROSE (trademark) CL-6B, SEPHAROSE (trademark) Fast Flow and SEPHAROSE (trademark) High Performance), cellulose-based (such as DEAE SEPHACEL (registered trademark)), dextran-based (such as SEPHADEX (registered trademark)), silica-based and synthetic polymer-based.

[0090] Ion exchange resins can be prepared according to known methods. Usually, before loading a sample or composition containing a polypeptide and one or more contaminants onto the resin, an equilibration buffer can be passed through the ion exchange resin to enable the resin to bind its counterion. Conveniently, the equilibration buffer can be the same as the loading buffer, but this is not essential.

[0091] In an optional embodiment of the present invention, the ion exchange resin can be regenerated with a regeneration buffer after elution of the polypeptide, such that the column can be reused. Generally, the salt concentration and / or pH of the regeneration buffer can be such that substantially all contaminants and the polypeptide of interest are eluted from the ion exchange resin. Generally, the regeneration buffer has a very high salt concentration for eluting contaminants and the polypeptide from the ion exchange resin.

[0092] Anion exchange chromatography In some embodiments, the recombinant protein or polypeptide is subjected to anion exchange chromatography, such as the anion exchange chromatography described herein. In some embodiments, a Nuvia Q™ anion exchange filter is used. In the case of an anion exchange resin, the charged group covalently bonded to the matrix can be, for example, diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and / or quaternary ammonium (Q). In some embodiments, the anion exchange resin used is a Q Sepharose column. Anion exchange chromatography can be performed, for example, using Q SEPHAROSE™ Fast Flow, Q SEPHAROSE™ High Performance, Q SEPHAROSE™ XL, CAPTO™ Q, DEAE, TOYOPEARL GIGACAP® Q, FRACTOGEL® TMAE (trimethylaminoethyl, quaternary ammonia resin), ESHMUNO™ Q, NUVIA™ Q, or UNOSPHERE™ Q. Other anion exchangers can be used within the scope of the present invention, including, but not limited to, quaternary amine resins or "Q-resins" (e.g., CAPTOTM-Q, Q-SEPHAROSE®; QAE SEPHADEX®); diethylaminoethane (DEAE) resins (e.g., DEAE-TRISACRYL®, DEAE SEPHAROSE®, benzoylated naphthoylated DEAE, diethylaminoethyl SEPHACEL®); AMBERJET® resins; AMBERLYST® resins; AMBERLITE® resins (e.g., AMBERLITE® IRA-67, AMBERLITE® strongly basic, AMBERLITE® weakly basic), cholestyramine resins, ProPac® resins (e.g., PROPAC® SAX-10, PROPAC® WAX-10, PROPAC® WCX-10); TSK-GEL® resins (e.g., TSKgel DEAE-NPR; TSKgel DEAE-5PW); and ACCLAIM® resins.

[0093] In an embodiment, anion exchange chromatography is performed using FRACTOGEL® TMAE (trimethylaminoethyl, quaternary ammonium resin).

[0094] In some embodiments, a recombinant protein or polypeptide sample (e.g., arylsulfatase A) is subjected to anion exchange chromatography at a temperature of about 23 °C or lower, about 18 °C or lower, or about 16 °C or lower, e.g., about 23 °C, about 20 °C, about 18 °C, or about 16 °C.

[0095] Typical mobile phases for anion exchange chromatography include water, relatively polar solutions such as acetonitrile, organic alcohols such as methanol, ethanol, and isopropanol, or solutions containing 2-(N-morpholino)-ethanesulfonic acid (MES). Thus, in certain embodiments, the mobile phase comprises about 0%, 1%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100% polar solution. In certain embodiments, the mobile phase comprises about 1% to about 100%, about 5% to about 95%, about 10% to about 90%, about 20% to about 80%, about 30% to about 70%, or about 40% to about 60% polar solution at any given point during the separation.

[0096] In certain embodiments, for example, a recombinant protein, such as rASA, is loaded at a binding capacity of about 23 AU / L resin or less, such as about 19 AU / L resin or less, about 15 AU / L resin or less, or about 12 AU / L resin or less, for example, about 12 AU / L resin to about 15 AU / L resin, or about 15 AU / L resin to about 19 AU / L resin. In some embodiments, a sample of a recombinant protein, such as arylsulfatase A, is loaded onto an anion exchange chromatography column at a binding capacity of at least about 4.5 g / L resin (such as at least about 5 g / L resin, 6 g / L resin, 7 g / L resin, 8 g / L resin, 9 g / L resin, 10 g / L resin, 11 g / L resin, 12 g / L resin, 13 g / L resin, 14 g / L resin, or 15 g / L resin). In some embodiments, a sample of a recombinant protein, such as arylsulfatase A, is loaded onto an anion exchange chromatography column at a binding capacity in the range of about 4.5 to 20 g / L resin (such as about 5 to 20 g / L resin; 5 to 19 g / L resin, 5 to 18 g / L resin, 5 to 17 g / L resin, 5 to 16 g / L resin, 5 to 15 g / L resin, 7.5 to 20 g / L resin, 7.5 - 19 g / L resin, 7.5 to 18 g / L resin, 7.5 to 17 g / L resin, 7.5 to 16 g / L resin, 7.5 to 15 g / L resin, 10 to 20 g / L resin, 10 to 19 g / L resin, 10 to 18 g / L resin, 10 to 17 g / L resin, 10 to 16 g / L resin, or 10 to 15 g / L resin).

[0097] An aqueous solution containing a recombinant protein, for example, arylsulfatase A and the contaminant(s), can be loaded onto an anion resin as a mobile phase using a loading buffer having a salt concentration and / or pH such that the polypeptide and the contaminant(s) bind to the anion exchange resin. Next, the resin can be washed with one or more column volumes of the loading buffer, followed by washing with one or more column volumes of a wash buffer. Here, the salt concentration is increasing. Finally, the recombinant protein, for example, arylsulfatase A can be eluted with an elution buffer while increasing the salt concentration. Optionally, elution of the enzyme can also be mediated by gradually or stepwise decreasing the pH. Fractions containing recombinant protein or enzyme (e.g., arylsulfatase A) activity can be collected and combined for further purification.

[0098] In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto an anion exchange chromatography column is performed using a loading buffer. In one embodiment, the loading buffer does not contain sodium chloride. In another embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is from about 1 mM to about 25 mM, such as from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, or from about 5 mM to about 10 mM. In some embodiments, the salt concentration in the mobile phase is a gradient (e.g., linear or non-linear gradient). In some embodiments, the salt concentration in the mobile phase is constant. In some embodiments, the salt concentration in the mobile phase can increase or decrease stepwise. In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto an anion exchange chromatography column is performed at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7.

[0099] In some embodiments, the anion exchange chromatography column is washed with one or more wash buffers. For example, washing of the anion exchange column can include two or more (e.g., first and second) wash steps, each using a different wash buffer. In one embodiment, the wash buffer does not contain sodium chloride. In another embodiment, the wash buffer contains sodium chloride. For example, the sodium chloride concentration in the wash buffer is from about 50 mM to about 200 mM, such as from about 50 mM to about 150 mM, from about 100 mM to about 200 mM, or from about 100 mM to about 150 mM, such as about 80 mM, about 100 mM, about 120 mM, or about 140 mM. In some embodiments, the anion exchange chromatography column is washed at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7.

[0100] In one embodiment, the elution buffer contains sodium phosphate. For example, the sodium phosphate concentration in the elution buffer is from about 20 mM to about 50 mM, such as from about 25 mM to about 45 mM, such as about 30 mM, about 35 mM, or about 40 mM. In another embodiment, the elution buffer does not contain sodium chloride. In yet another embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration in the elution buffer is from about 200 mM to about 300 mM, such as from about 240 mM to about 280 mM. In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from the anion exchange chromatography column is performed at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7.

[0101] In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from an anion exchange chromatography column includes one or more steps of recovering the elution peak. For example, the recovery of the elution peak can start from about 50 mAU on the ascending side to about 50 mAU on the descending side, such as from about 100 mAU on the ascending side to about 50 mAU on the descending side, from about 200 mAU on the ascending side to about 50 mAU on the descending side, from about 50 mAU on the ascending side to about 100 mAU on the descending side, from about 50 mAU on the ascending side to about 200 mAU on the descending side, or from about 100 mAU on the ascending side to about 100 mAU on the descending side, for example, as determined by spectrophotometry, for example, at 280 nM.

[0102] It will be apparent to those skilled in the art that a number of different buffers can be used in the loading, washing, and elution steps. However, generally, the column can be equilibrated with 1 to 10 column washes with a buffer containing 0.05 M MES-Tris (pH 7.0). For convenience, the sample can be loaded in the buffer from a previous step of the purification process, or the sample can be loaded using a loading buffer. The column can be washed with 1 to 10 column volumes of the buffer used for equilibration, followed by washing with a wash buffer containing 0.02 MES-Tris, 0.12 M NaCl (pH 7.0). Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration, loading, and wash buffers described herein for anion exchange chromatography. The sample can be eluted in a buffer containing 0.02 MES-Tris, 0.26 M NaCl (pH 7.0). Alternatively, the sample can be eluted in any other elution buffer described herein for anion exchange chromatography.

[0103] The loading buffer, washing buffer, and elution buffer described in this specification may contain one or more buffering agents. For example, the buffering agent may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffering agent is from about 1 mM to about 500 mM, for example, from about 10 mM to about 250 mM, from about 20 mM to about 100 mM, from about 1 mM to 5 mM, from about 5 mM to and 10 mM, from about 10 mM to 50 mM, or from about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0104] The yield, activity, and purity after anion exchange chromatography can vary. In some embodiments, the activity yield of a recombinant protein, such as arylsulfatase A, is at least about 75%, such as at least about 85%, such as from about 85% to about 99%, or from about 90% to about 99%. In some embodiments, the protein yield (AU or absorbance units), for example, at 280 nM, as determined by spectrophotometry, is from about 10% to 50%, such as from about 20% to about 35%, or from about 25% to about 30%. In some embodiments (e.g., embodiments using the TMAE column described below), the elution pool protein activity yield (AU or absorbance units), for example, at 280 nm, as determined by spectrophotometry, is from about 70% to 400%, such as from about 80% to about 390%, or from about 90% to about 350%, or from about 100% to 150%, exceeding at least 95%. In some embodiments (e.g., embodiments using the TMAE column described below), the log reduction value (LRV) of host cell protein (HCP) is from about 0.5 to about 1.1, such as from about 0.6 to 0.9, or from about 0.7 to 0.8. In some embodiments (e.g., embodiments using the TMAE column described below), the purity, for example, as determined by capillary electrophoresis - SDS PAGE, is at least 75%, such as at least 80%, at least 85%, at least 90% or more. In preferred embodiments, the activity yield, HCP LRV, and purity (determined by capillary electrophoresis - SDS PAGE) after anion exchange chromatography are at least about 90%, at least about 0.6, and at least about 80%, respectively.

[0105] In a preferred embodiment of the present invention, an anion exchange column having a high loading capacity is used. In certain embodiments of the present invention, the column is characterized by a loading range of about 3 to 20 g / L (i.e., about 5 to 15 g / L, about 10 to 15 g / L, about 10 to 20 g / L). In some embodiments, the loading capacity is significantly greater than 4.3 g / L (e.g., about 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, or 20 g / L, or greater). In certain embodiments, the binding capacity of the resin is about 75 to 100 AU / L (e.g., about 75 AU / L, about 80 AU / L, about 85 AU / L, about 90 AU / L, about 95 AU / L). In certain embodiments, the loading capacity is greater than about 80 AU / L. In some embodiments, the high loading capacity column is a TMAE column. In certain embodiments, the column is selected from the group consisting of a Fractogel® TMAE column, a Nuvia Q column, a Q Sepharose Fast Flow column, a Capto Q column, a Q Sepharose XL column, an Eshmuno Q column, a UNOsphere Q column, or a GigaCap Q column.

[0106] In certain embodiments of the present invention, the TMAE column is pre-equilibrated with a buffer containing about 20 mM MES-Tris and 1000 mM NaCl at pH 7.0. In certain embodiments, the column is equilibrated with a buffer containing 50 mM MES-Tris at pH 7.0. In some embodiments, the loading flow rate of the TMAE column is about 75 to 125 cm / hour (i.e., about 75 to 115 cm / hour, about 75 to 110 cm / hour, about 75 to 105 cm / hour, about 75 to 100 cm / hour, about 85 to 115 cm / hour, about 85 to 110 cm / hour, about 85 to 105 cm / hour, about 85 to 100 cm / hour, about 95 to 115 cm / hour, about 95 to 110 cm / hour, about 95 to 105 cm / hour, about 95 to 100 cm / hour, about 100 to 120 cm / hour, about 100 to 115 cm / hour, about 100 to 110 cm / hour, about 100 cm / hour). The loading conditions can be optimized and evaluated by A280 absorbance as described herein.

[0107] In certain embodiments utilizing a TMAE column (e.g., a Fractogel TMAE column), even at a loading capacity exceeding 15 g / L, there is little product loss during loading. The ability to increase the loading capacity while minimizing flow-through loss is a significant improvement in the purification methodology. In certain embodiments of the present invention, the amount of loss of the flow-through product is less than 30% of the loading (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%).

[0108] After loading, in some embodiments, the TMAE column is washed at least once. In certain embodiments, the column is washed twice. The first or second wash buffer may contain an optimal level of sodium chloride. In some embodiments, the amount of sodium chloride that is the first or second wash buffer is about 50 - 150 mM (e.g., about 50 - 140 mM, about 50 - 130 mM, about 50 - 120 mM, about 50 - 110 mM, about 50 - 100 mM, about 50 - 90 mM, about 50 - 80 mM, about 80 - 150 mM, about 80 - 140 mM, about 80 - 130 mM, about 80 - 120 mM, about 80 - 110 mM, 80 - 100 mM, about 80 - 90 mM, about 80 mM, or about 120 mM). In some embodiments, the first wash buffer contains 50 mM MES-Tris at pH 7.0. In some embodiments, the second wash buffer contains 20 mM MES-Tris, 100 mM NaCl at pH 7.0. Further optimization of the wash conditions, particularly the second wash conditions, is encompassed within the embodiments of the present invention. For example, increasing the salt concentration of the second wash can improve the log reduction value (LRV) of host cell protein (HCP) and overall purity, but both activity and A280 yield decrease. As described herein, in order to provide an optimal combination of purity, activity, and yield, specific wash conditions must be balanced with the elution conditions described below.

[0109] In embodiments of the present invention, the recombinant ASA bound to the TMAE column is eluted with an elution buffer. In some embodiments, the amount of sodium chloride in the elution buffer is optimized. In certain embodiments, the amount of sodium chloride in the elution buffer is about 150 - 300 mM (e.g., about 150 - 290 mM, about 150 - 280 mM, about 150 - 270 mM, about 150 - 260 mM, about 150 - 250 mM, about 150 - 240 mM, about 150 - 230 mM, about 150 - 220 mM, about 150 - 210 mM, about 170 - 290 mM, about 170 - 280 mM, about 170 - 270 mM, about 170 - 260 mM, about 170 - 250 mM, about 170 - 240 mM, about 170 - 230 mM, about 170 - 220 mM, about 170 - 210 mM, about 180 - 290 mM, about 180 - 280 mM, about 180 - 270 mM, 180 - 260 mM, about 180 - 250 mM, about 180 - 240 mM, about 180 - 230 mM, about 180 - 220 mM, about 180 - 210 mM, about 180, about 220 or about 260). In a specific example, the elution buffer contains 50 mM MES-Tris and 1 M NaCl at pH 7.0. In some embodiments, the A280 yield after elution is greater than 60% of the loading (e.g., about 60%, about 70%, about 80% or more). Further optimization of the elution conditions is encompassed within embodiments of the present invention. For example, increasing the elution salt concentration (i.e., conductivity) increases the yield but decreases purity and HCP removal. Also, as described above, certain wash conditions need to be balanced with the elution conditions to provide an optimal combination of purity, activity, and yield.

[0110] Cation exchange chromatography In some embodiments, the method further comprises subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography, such as sulfopropyl (SP) cation exchange chromatography, such as those described herein. In some embodiments, the sample of the recombinant protein, arylsulfatase A, is subjected to anion exchange chromatography prior to cation exchange chromatography. In typical embodiments, the cation exchange chromatography comprises sulfopropyl (SP) cation exchange chromatography, but other cation chromatography membranes or resins, such as MUSTANG™ S membranes, S-SEPHAROSE™ resins, or Blue SEPHAROSE™ resins, may be used. In some embodiments, the method further comprises concentrating and / or filtering a sample of a recombinant protein, such as arylsulfatase A, by, for example, ultrafiltration and / or diafiltration, such as tangential flow ultrafiltration. The cation exchange chromatography may be performed at an optimized temperature, for example, as described herein, to enhance target binding and / or reduce impurity binding. For example, the cation exchange chromatography may be performed at about 23°C, 18°C, 16°C, or lower temperatures.

[0111] In one embodiment, cation exchange chromatography includes sulfopropyl (SP) cation exchange chromatography. In another embodiment, cation exchange chromatography is a polishing step. Cation exchange chromatography (e.g., sulfopropyl (SP) cation exchange chromatography) can be performed using, for example, one or more of TOYOPEARL® SP-650, TOYOPEARL® SP-550, TSKGEL® SP-3PW, TSKGEL® SP-5PW, SP SEPHAROSE™ Fast Flow, SP SEPHAROSE™ High Performance, SP SEPHAROSE™ XL, SARTOBIND® S membrane, POROS® HS50, UNOSPHERE™ S, and MACROCAP™ S.

[0112] An aqueous solution containing a recombinant protein, such as arylsulfatase A and contaminants (s), can be loaded onto a cation resin using a loading buffer having a salt concentration and / or pH such that the polypeptide and contaminants bind to the cation exchange resin. Next, the resin can be washed with one or more column volumes of equilibration buffer or loading buffer, and optionally followed by washing with one or more column volumes of wash buffer having an increasing salt concentration. Finally, the recombinant protein, such as arylsulfatase A, can be eluted into an elution buffer. Fractions containing the recombinant protein, such as arylsulfatase A activity, can be collected and combined for further purification.

[0113] In typical embodiments, the NaCl concentration and / or pH of the loading buffer, washing buffer, and / or elution buffer can be optimized, for example, as described herein, to enhance target binding and / or reduce impurity binding. In some embodiments, the NaCl concentration in the loading buffer is about 20 mM, 15 mM, 10 mM, or less. In some embodiments, the loading buffer has a pH of about 4.5, 4.3, 4.0, or less. In some embodiments, the NaCl concentration in the washing buffer is about 20 mM, 15 mM, 10 mM, or less. In some embodiments, the NaCl concentration in the elution buffer is about 55 mM, 50 mM, 45 mM, 40 mM, or less.

[0114] In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography includes loading the sample of the recombinant protein, such as arylsulfatase A, onto a cation chromatography column (e.g., a sulfopropyl (SP) cation exchange column), washing the cation exchange chromatography column, and eluting the recombinant protein, such as arylsulfatase A, from the column. In some embodiments, the column can be equilibrated, for example, three or more times with 5-10 column volumes of 0.01 M NaAc, 0.01 M NaCl, 0.03 M acetic acid (pH 4.2).

[0115] In some embodiments, the sample can be carried in a buffer from a step prior to the purification process, or the sample can be carried using a carrier buffer. In one embodiment, the carrier buffer contains sodium chloride. For example, the sodium chloride concentration of the carrier buffer is from about 1 mM to about 25 mM, such as from about 5 mM to about 20 mM, such as about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In another embodiment, the carrier buffer contains sodium acetate. For example, the sodium acetate concentration of the carrier buffer is from about 10 mM to about 100 mM, such as about 20 mM, about 40 mM, or about 60 mM. In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto a cation exchange chromatography column is performed at a pH of from about 3.0 to about 6.0, such as from about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5. In some embodiments, a sample of a recombinant protein, such as arylsulfatase A, is loaded onto a cation exchange chromatography column at a binding capacity of from about 15 AU / L resin or less, such as from about 14 AU / L resin or less, or from about 12 AU / L resin or less, such as from about 10 AU / L resin to about 14 AU / L resin, or from about 10 AU / L resin to about 12 AU / L resin.

[0116] In some embodiments, the washing of the cation exchange chromatography column is performed using one or more washing buffers. For example, the washing of the cation exchange column can include two or more (e.g., first and second) washing steps, each using a different washing buffer. The column can be washed with 1 to 10 column volumes of the buffer used for equilibration. Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffers described herein for cation exchange chromatography. In one embodiment, the washing buffer contains sodium chloride. For example, the sodium chloride concentration of the washing buffer is from about 1 mM to about 25 mM, such as from about 5 mM to about 20 mM, or from about 10 mM to about 15 mM, such as about 5 mM, about 10 mM, about 15 mM, or about 20 mM. In another embodiment, the washing buffer contains sodium acetate. For example, the sodium acetate concentration of the loading buffer is from about 10 mM to about 100 mM, such as about 20 mM, about 40 mM, or about 60 mM. In some embodiments, the washing of the cation exchange chromatography column is performed at a pH of from about 3.0 to about 6.0, such as from about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5.

[0117] In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from the cation exchange chromatography column is performed using an elution buffer. In one embodiment, the elution buffer contains sodium chloride. For example, the sodium chloride concentration of the elution buffer is from about 25 mM to about 75 mM, such as from about 45 mM to about 60 mM, such as about 45 mM, about 50 mM, about 55 mM, or about 55 mM. In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from the cation exchange chromatography column is performed at a pH of from about 3.0 to about 6.0, such as from about 4.0 to about 5.0, such as about 4.0, about 4.3, or about 4.5. Thus, as one specific example, the sample can be eluted in a buffer containing 0.02 M NaAc, 0.05 M NaCl (pH 4.5). Alternatively, the sample can be eluted in any other elution buffer described herein for cation exchange chromatography.

[0118] In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from a cation exchange chromatography column includes one or more steps of recovering the elution peak. For example, the recovery of the elution peak starts when determined by spectrophotometry, for example, at 280 nM, from about 50 mAU on the ascending side to about 50 mAU on the descending side, such as from about 100 mAU on the ascending side to about 50 mAU on the descending side, from about 200 mAU on the ascending side to about 50 mAU on the descending side, from about 50 mAU on the ascending side to about 100 mAU on the descending side, from about 50 mAU on the ascending side to about 200 mAU on the descending side, or from about 100 mAU on the ascending side to about 100 mAU on the descending side. The recovered elution peaks may be pooled.

[0119] The loading buffer, washing buffer, and elution buffer described herein may contain one or more buffers. For example, the buffer can be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is from about 1 mM to about 500 mM, such as from about 10 mM to about 250 mM, from about 20 mM to about 100 mM, from about 1 mM to 5 mM, from about 5 mM to and 10 mM, from about 10 mM to 50 mM, or from about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0120] In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography is performed at a temperature of about 23°C or lower, about 18°C or lower, or about 16°C or lower, for example, about 23°C, about 20°C, about 18°C, or about 16°C. In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography is performed at about 23°C to about 16°C, for example, about 23°C, about 20°C, about 18°, or about 16°C, and loading a sample of a recombinant protein, such as arylsulfatase A, onto a cation exchange chromatography column is performed at a pH of about 4.5 to about 4.3, for example, about 4.5, about 4.4, or about 4.3. In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography is performed at about 23°C, and loading a sample of a recombinant protein, such as arylsulfatase A, onto a cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting a sample to cation exchange chromatography is performed at about 23°C, and loading a sample of a recombinant protein, such as arylsulfatase A, onto a cation exchange chromatography column is performed at a pH of about 4.3. In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to cation exchange chromatography is performed at about 18°C, and loading a sample of a recombinant protein, such as arylsulfatase A, onto a cation exchange chromatography column is performed at a pH of about 4.5. In some embodiments, subjecting a sample of arylsulfatase A to cation exchange chromatography is performed at about 18°C, and loading a sample of arylsulfatase A onto a cation exchange chromatography column is performed at a pH of about 4.3.

[0121] The yield after cation exchange chromatography can vary. In some embodiments, the active yield of a recombinant protein, such as arylsulfatase A, is at least about 75%, such as at least about 80%, such as from about 80% to about 105%. In some embodiments, the protein yield (in AU or absorbance units) is, for example, from about 65% to 100%, such as from about 70% to about 95%, when determined by spectrophotometry, for example, at 280 nm.

[0122] The purity and activity are significantly improved after cation exchange chromatography. In some embodiments, the host cell protein (HCP) log reduction value (LRV) is from about 1.0 to about 2.5, such as from about 1.5 to about 2.0 or from about 1.7 to about 1.9. In some embodiments, the specific activity of the purified arylsulfatase A is, for example, at least about 50 U / mg to about 140 U / mg, such as at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, when determined by the methods described herein. In some embodiments, the recombinant protein, such as arylsulfatase A, is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. The purity of the recombinant protein, such as arylsulfatase A, can be measured by, for example, one or more of host cell protein (HCP) western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse phase HPLC, and size exclusion HPLC. In certain embodiments, decreasing the salt concentration of the loading buffer and lowering its pH enhances the binding of the recombinant protein, such as the binding of ASA to the cation exchange column, but does not affect the binding of impurities. In other words, the optimal balance of salt concentration and pH as described above can increase the yield after cation exchange chromatography without adversely affecting the purity.

[0123] In some embodiments, the pH of the cation exchange eluate pool can be adjusted. In certain embodiments, the pH is adjusted immediately prior to virus filtration. The cation exchange eluate (e.g., SP eluate) can be adjusted to a pH of about 5.5, about 6.0, about 6.5, or about 7.0 using a pH adjustment buffer containing 0.25 M sodium phosphate, 1.33 M sodium chloride, and 0.34 M sodium citrate (pH 7.0). In certain embodiments, the pH-adjusted SP eluate pool is virus filtered through a Plana 20N filter. In some embodiments, the yield of the pH-adjusted cation exchange eluate after virus filtration, as evaluated by A280 absorbance, is about 90-100%, i.e., about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. In some embodiments, the yield of virus filtration is important because adjusting the pH to about 6.0 allows the octamer of ASA (which is about 20 nm in diameter) to dissociate into the dimer form. Thus, the pore size of the virus filter can be selected to ensure that only the dimer form is filtered (i.e., the octamer form can be retained by the filter or cause blockage of the virus filter). For example, a virus filter with a pore size of 20 nm retains the octamer form of ASA but not the dimer form.

[0124] In some embodiments, mixed-mode chromatography follows ion exchange chromatography.

[0125] Mixed-mode chromatography The purification method described herein may include one or more steps of mixed-mode chromatography. Mixed-mode chromatography is a type of chromatography to which several separation modes are applied and usually decomposes a mixture of different molecules in liquid chromatography. For example, mixed-mode separation may include a combination phase having both ion-exchange and reverse-phase characteristics simultaneously. These stationary phases having multiple interaction types are available from several column manufacturers. In some embodiments, Capto MMC Impres columns are used for mixed-mode chromatography.

[0126] In some embodiments, the method includes subjecting a sample of a recombinant protein, such as arylsulfatase A, to a method comprising mixed-mode chromatography, such as the mixed-mode chromatography described herein, such as ceramic hydroxyapatite (HA) chromatography, such as hydroxyapatite type I or type II chromatography. In some embodiments, the mixed-mode chromatography is performed using one or more of CHT™ ceramic hydroxyapatite type I media, CHT™ ceramic hydroxyapatite type II media, BIO-GEL® HT hydroxyapatite, and BIO-GEL® HTP hydroxyapatite.

[0127] In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to mixed-mode chromatography includes loading the sample of the recombinant protein, such as arylsulfatase A, onto a mixed-mode chromatography column (e.g., HA chromatography), washing the mixed-mode chromatography column, and eluting the recombinant protein, such as arylsulfatase A, from the column. In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to mixed-mode exchange chromatography is performed at a temperature of about 23°C or lower, about 18°C or lower, or about 16°C or lower, such as about 23°C, about 20°C, about 18°C, or about 16°C.

[0128] In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto an anion exchange chromatography column is performed using a loading buffer. In one embodiment, the loading buffer contains sodium phosphate. For example, the sodium phosphate concentration in the loading buffer is from about 1 mM to about 10 mM, such as from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, such as about 1 mM, about 2 mM, or about 5 mM. In another embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration in the loading buffer is from about 100 mM to about 400 mM, such as from about 200 mM to about 300 mM, such as about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0129] In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto a mixed mode chromatography column is performed at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7.

[0130] In some embodiments, mixed-mode chromatography includes ceramic hydroxyapatite (HA) chromatography. Hydroxyapatite (HAP) generally refers to the crystalline form of calcium phosphate. The mechanism of HAP involves non-specific interactions between negatively charged protein carboxyl groups and positively charged calcium ions on the resin, and between positively charged protein amino groups and negatively charged phosphate ions on the resin. By adjusting the pH of the buffer, basic or acidic proteins can be selectively adsorbed onto the column; elution can be achieved by changing the salt concentration of the buffer. Again, it is clear that a number of buffer compositions and combinations of buffers can be used. However, generally, the column can be equilibrated with 1 to 10 column washes with a buffer containing 0.001 M NaPO4, 0.02 M MES-Tris, 0.26 M NaCl (pH 7.0). For convenience, the sample can be carried in the buffer from the previous step of the purification process, or the sample can be carried using a loading buffer. The column can be washed with 1 to 10 column volumes of the buffer used for equilibration, followed by a wash buffer containing 0.005 M NaPO4, 0.02 M MES-Tris, 0.26 M NaCl (pH 7.0). Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration, loading, and wash buffers described herein for mixed-mode chromatography. The sample can be eluted in a buffer containing 0.04 M NaPO4 (pH 7.0). Optionally, the column can be stripped by washing with 1 to 10 column volumes of 0.4 M NaPO4 (pH 12). Alternatively, the sample can be eluted in any other elution buffer described herein for mixed-mode chromatography.

[0131] In some embodiments, washing of the mixed-mode chromatography column is performed using one or more wash buffers. For example, washing of the mixed-mode chromatography column can include two or more (e.g., first and second) washing steps each using a different wash buffer.

[0132] In one embodiment, the washing buffer contains sodium phosphate. For example, the sodium phosphate concentration in the washing buffer is from about 1 mM to about 10 mM, such as from about 1 mM to about 5 mM, from about 5 mM to about 10 mM, for example, about 1 mM, about 5 mM, or about 10 mM. In another embodiment, the washing buffer contains sodium chloride. For example, the sodium chloride concentration in the washing buffer is from about 50 mM to about 600 mM, such as from about 100 mM to about 500 mM, or from about 200 to about 400 mM, for example, about 220 mM, about 240 mM, about 260 mM, or about 280 mM.

[0133] In some embodiments, the washing of the mixed-mode chromatography column is performed at a pH of about 5 to about 9, such as about 6 to about 8, for example, about 7.

[0134] In some embodiments, the elution of the recombinant protein, such as arylsulfatase A, from the mixed-mode chromatography column is performed at a pH of about 5 to about 9, such as about 6 to about 8, for example, about 7. In some embodiments, eluting arylsulfatase A from the mixed-mode chromatography column includes one or more steps of elution peak recovery. For example, the recovery of the elution peak starts from about 50 mAU on the ascending side to about 50 mAU on the descending side, such as from about 100 mAU on the ascending side to about 50 mAU on the descending side, from about 200 mAU on the ascending side to about 50 mAU on the descending side, from about 50 mAU on the ascending side to about 100 mAU on the descending side, from about 50 mAU on the ascending side to about 200 mAU on the descending side, or from about 100 mAU on the ascending side to about 100 mAU on the descending side, for example, as determined by spectrophotometry at, for example, 280 nM.

[0135] The loading buffer, washing buffer, and elution buffer described in this specification may contain one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is from about 1 mM to about 500 mM, for example, from about 10 mM to about 250 mM, from about 20 mM to about 100 mM, from about 1 mM to 5 mM, from about 5 mM to and 10 mM, from about 10 mM to 50 mM, or from about 50 mM to about 100 mM, for example, about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0136] In some embodiments, the purification of a recombinant protein, such as ASA by mixed-mode chromatography, is performed following purification by ion exchange chromatography (e.g., anion exchange chromatography). However, in some embodiments, it is contemplated that these steps can also be performed in the reverse order.

[0137] The yield after mixed-mode chromatography can vary. In some embodiments, the active yield of a recombinant protein, such as arylsulfatase A, is at least about 80%, for example, at least about 90%, for example, from about 80% to about 115%. In some embodiments, the protein yield (AU or absorbance units) is, for example, from about 30% to 80%, for example, from about 35% to about 75%, or from about 50% to about 70% when determined by spectrophotometry, for example, at 280 nm.

[0138] The purity after mixed-mode chromatography is significantly improved. In some embodiments, the specific activity of a purified recombinant protein, such as arylsulfatase A, is, for example, at least about 50 U / mg to about 140 U / mg, such as at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg as determined by the methods described herein. In some embodiments, arylsulfatase A is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. In some embodiments, the purity of arylsulfatase A can be measured by one or more of, for example, host cell protein (HCP) western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse-phase HPLC, and size exclusion HPLC. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is from about 0.3 to about 0.6, such as from about 0.4 to 0.5.

[0139] In some embodiments, hydrophobic interaction chromatography follows mixed-mode chromatography.

[0140] Hydrophobic interaction chromatography (HIC) The purification method described herein can include subjecting a sample of a recombinant protein, such as arylsulfatase A, to hydrophobic interaction chromatography (HIC). In one embodiment, the hydrophobic interaction chromatography includes phenyl chromatography. In some embodiments, HIC is performed using a Capto Phenyl Impres™ column.

[0141] In other embodiments, hydrophobic interaction chromatography includes butyl chromatography or octyl chromatography. In some embodiments, subjecting a sample of arylsulfatase A to HIC is performed at a temperature of about 23°C or less, about 18°C or less, or about 16°C or less, for example, about 23°C, about 20°C, about 18°C, or about 16°C. In some embodiments, a sample of arylsulfatase A is subjected to mixed-mode chromatography prior to HIC.

[0142] Hydrophobic interaction chromatography utilizes the attraction of a given molecule for a polar or nonpolar environment, and with respect to proteins, this tendency is governed by the hydrophobicity or hydrophilicity of the residues on the exposed outer surface of the protein. Thus, proteins are fractionated based on their various degrees of attraction to a hydrophobic matrix, typically an inert support having alkyl linker arms of 2 to 18 carbon chain lengths. The stationary phase consists of small nonpolar groups (butyl, octyl, or phenyl) attached to the backbone of a hydrophilic polymer (e.g., cross-linked Sepharose™, dextran, or agarose). Thus, HIC columns are typically butyl SEPHAROSE™ columns or phenyl SEPHAROSE™ columns, and most typically, phenyl SEPHAROSE™ columns.

[0143] In some embodiments, hydrophobic interaction chromatography includes phenyl chromatography using one or more of Phenyl SEPHAROSE™ High Performance, Phenyl SEPHAROSE™ 6 Fast Flow (low sub), or Phenyl SEPHAROSE™ 6 Fast Flow (high sub).

[0144] In some embodiments, subjecting a sample of a recombinant protein, such as arylsulfatase A, to hydrophobic interaction chromatography involves loading a sample of the recombinant protein, such as arylsulfatase A, onto an HIC column, washing the HIC column, and eluting the recombinant protein, such as arylsulfatase A. Loading, washing, and eluting in HIC generally follow the same principles as described above for ion exchange chromatography, but in many cases, conditions that are nearly opposite to those used in ion exchange chromatography are applied. Thus, the HIC process involves the use of a loading buffer with a high salt concentration, which unfolds the protein to expose hydrophobic sites. The protein is retained by hydrophobic ligands on the column and exposed to a buffer gradient with decreasing salt concentration. As the salt concentration decreases, the protein returns to its native conformation and is ultimately eluted from the column. Alternatively, the protein may be eluted with PEG.

[0145] In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto an HIC column is performed using a loading buffer. In one embodiment, the loading buffer contains sodium chloride. For example, the sodium chloride concentration of the loading buffer is from about 0.5 M to about 2.5 M, such as about 1 M or about 1.5 M. In another embodiment, the loading buffer contains sodium phosphate. For example, the sodium acetate concentration of the loading buffer is from about 10 mM to about 100 mM, such as about 25 mM, about 50 mM, or about 75 mM. In some embodiments, loading a sample of a recombinant protein, such as arylsulfatase A, onto an HIC column is performed at a pH of from about 5 to about 7, such as from about 5.5 to about 6.5, such as about 5.5, about 6.0, or about 6.5. In some embodiments, the sample is loaded onto the HIC column at a binding capacity of from about 12 AU / L resin or less, such as from about 10 AU / L resin or less, about 9 AU / L resin or less, about 7 AU / L resin or less, or about 5 AU / L resin or less, such as from about 5 AU / L resin to about 9 AU / L resin, or from about 5 AU / L resin to about 7 AU / L resin.

[0146] The use of phenyl SEPHAROSE™ as a solid phase in HIC is typical in the present disclosure. Again, it will be readily appreciated that there are numerous different possibilities regarding the exact conditions as well as the buffers and combinations of buffers used in the loading, washing, and elution processes. In typical embodiments, the column can be equilibrated in a buffer containing 0.05 M NaPO4, 1 M NaCl (pH 5.5). For convenience, the sample can be loaded in the buffer from a previous step of the purification process, or the sample can be loaded using a loading buffer.

[0147] In some embodiments, washing the HIC column is performed with one or more washing buffers. For example, washing the HIC column can include two or more (e.g., first and second) washing steps each using a different washing buffer. In some embodiments, the washing buffer contains sodium chloride. For example, the sodium chloride concentration of the washing buffer is from about 100 mM to about 1.5 M, such as from about 250 mM to about 1 M, such as about 250 mM, about 500 mM, about 750 mM, or about 1 M. In another embodiment, the washing buffer contains sodium phosphate. For example, the sodium acetate concentration of the loading buffer is from about 10 mM to about 100 mM, such as about 25 mM, about 50 mM, or about 75 mM. In some embodiments, washing the HIC column is performed at a pH of from about 5 to about 7, such as from about 5.5 to about 6.5, such as about 5.5, about 6.0, or about 6.5. For example, the washing can be performed using 1 - 5 column volumes of 0.02 M MES, 0.05 M NaPO4, 0.5 M NaCl (pH 5.5) following 1 - 2 column washes with the equilibration buffer. Alternatively, the column can be equilibrated, loaded, and washed with any other equilibration, loading, and washing buffers described herein for HIC.

[0148] In some embodiments, eluting the recombinant protein from the HIC column is performed using an elution buffer. In some embodiments, the elution buffer contains sodium chloride. For example, the sodium chloride concentration in the elution buffer is from about 30 mM to about 100 mM, such as from about 45 mM to about 85 mM, such as about 50 mM, about 60 mM, about 70 mM, or about 80 mM. In some embodiments, eluting arylsulfatase A from the HIC column is performed at a pH of from about 5 to about 9, such as from about 6 to about 8, such as about 7. For example, in some embodiments, arylsulfatase A can be eluted using 0.02 M MES-Tris, 0.06 M NaCl (pH 7.0). Alternatively, the sample can be eluted in any other elution buffer described herein for HIC.

[0149] In some embodiments, eluting a recombinant protein, such as arylsulfatase A, from the HIC column includes one or more steps of recovering the elution peak. For example, the recovery of the elution peak starts from about 50 mAU on the rising side to about 50 mAU on the falling side, such as from about 100 mAU on the rising side to about 50 mAU on the falling side, from about 200 mAU on the rising side to about 50 mAU on the falling side, from about 50 mAU on the rising side to about 100 mAU on the falling side, from about 50 mAU on the rising side to about 200 mAU on the falling side, or from about 100 mAU on the rising side to about 100 mAU on the falling side, as determined by spectrophotometry, for example, at 280 nM.

[0150] In some embodiments, purification of the recombinant protein, such as arylsulfatase A by HIC, is carried out following purification by ion exchange chromatography (e.g., anion exchange chromatography) and / or mixed mode chromatography. However, it is contemplated that these steps can also be performed in the reverse order.

[0151] The loading buffer, washing buffer, and elution buffer described in this specification may contain one or more buffers. For example, the buffer may be TRIS, HEPES, MOPS, PIPES, SSC, MES, sodium phosphate, sodium acetate, or a combination thereof. The concentration of the buffer is from about 1 mM to about 500 mM, such as from about 10 mM to about 250 mM, from about 20 mM to about 100 mM, from about 1 mM to 5 mM, from about 5 mM to 10 mM, from about 10 mM to 50 mM, or from about 50 mM to about 100 mM, such as about 1 mM, about 5 mM, about 10 mM, about 20 mM, about 30 mM, about 40 mM, or about 50 mM.

[0152] The yield after HIC can vary. In some embodiments, the active yield of a recombinant protein, such as arylsulfatase A, is at least about 60%, such as at least about 70%, such as from about 70% to about 100%. In some embodiments, the protein yield (in AU or absorbance units) is, for example, determined by spectrophotometry, for example, at 280 nm, from about 45% to 100%, such as from about 50% to about 95%, or from about 55% to about 90%.

[0153] The purity after HIC is significantly improved. In some embodiments, the specific activity of purified arylsulfatase A is, for example, at least about 50 U / mg to about 140 U / mg, such as at least about 70 U / mg, at least about 90 U / mg, at least about 100 U / mg, or at least about 120 U / mg, as determined by the methods described herein.

[0154] In some embodiments, the recombinant protein, such as arylsulfatase A, is purified to at least about 95%, at least about 98%, at least about 99%, at least about 99.5%, at least about 99.6%, at least about 99.7%, at least about 99.8%, or at least about 99.9%. In some embodiments, the purity of arylsulfatase A can be measured by one or more of, for example, host cell protein (HCP) western blot, SDS-PAGE Coomassie staining, SDS-PAGE silver staining, reverse-phase HPLC, and size exclusion HPLC. In some embodiments, the log reduction value (LRV) of host cell protein (HCP) is from about 0.6 to about 1.2, for example, from about 0.7 to 0.95.

[0155] Peptides and proteins The present invention is used for the purification of any protein or polypeptide without limitation, and it is understood that the examples provided herein are merely illustrative. For clarity, based on the mechanism, the present invention is applicable to any affinity purification that depends on protein-protein specific binding. More specifically, this type of binding depends on the interaction of amino acid chains. The affinity ligand (immobilized on the stationary phase) needs to be a protein or peptide, or a derivative thereof. For example, in various embodiments, the protein or polypeptide to be purified can include proteins, polypeptides, viruses, cells, or other particles having one or more protein components. In some embodiments, the polypeptide or protein is a therapeutic protein. In some embodiments, such therapeutic proteins include antibody-based drugs, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, Fc fusion proteins, growth factors, hormones, interferons, interleukins, and thrombolytic agents. In some embodiments, the polypeptide or protein is an antibody. In some embodiments, the polypeptide or protein is an engineered antibody-based molecule or cell including IgG monoclonal antibodies, FAb fragments, Fc fragments, single-chain variable fragments (scFv), heterodimeric IgG-based bispecific antibodies, scFv-based bispecific antibodies, IgG-scFv-based bispecific antibodies, tetravalent scFv-based antibodies, IgG-based immunocytokines, tandem scFv-immunocytokines, Fc fusion proteins, Fc fusion peptides, antibody-drug conjugates, or chimeric antigen receptor (CAR)-T-based antibodies. In some embodiments, the polypeptide or protein is a monoclonal antibody. In some embodiments, the polypeptide or protein is IgG or a derivative thereof. In some embodiments, the polypeptide or protein is IgA or a derivative thereof. In some embodiments, the polypeptide or protein is IgM or a derivative thereof. In some embodiments, the polypeptide or protein is IgE or a derivative thereof.In some embodiments, the polypeptide or protein is IgD or a derivative thereof. In some embodiments, the polypeptide or protein is a bispecific antibody. In some embodiments, the polypeptide or protein is a trispecific antibody. In some embodiments, the polypeptide or protein is an antibody-drug conjugate. In some embodiments, the polypeptide or protein is an antibody fragment (e.g., scFv, Fab, Fc, F(ab’)2). In some embodiments, the polypeptide or protein is an Fc fusion protein. In some embodiments, the polypeptide or protein is an immune cytokine or a derivative thereof. In some embodiments, the polypeptide or protein is a radioimmunoglobulin or a derivative thereof. The present invention also applies to the purification of viruses, cells or other particles expressing such protein components on the surface.

[0156] In some embodiments, the polypeptide or protein is an enzyme. In some embodiments, the polypeptide or protein is a lysosomal enzyme suitable for enzyme replacement therapy as described in Table 1 below. [Table 1-1] [Table 1-2]

[0157] In some embodiments, the polypeptide or protein is a secreted protein. In some embodiments, the polypeptide or protein is, for example, a viral protein for vaccine development. In some embodiments, the polypeptide or protein is a particle having a protein component on its surface. In some embodiments, the polypeptide or protein is a virus or virus-like particle having a protein component on its surface. In some embodiments, the polypeptide or protein is a cell having a protein component on its surface. In some embodiments, the present invention is applied to, for example, T cells or NK cells expressing a CAR antibody.

[0158] In various embodiments, the present invention is applied to any protein or polypeptide that is concerned about high aggregation, low stability, low activity, or low efficacy due to an elution process. For example, (i) highly engineered antibody-based proteins and (ii) recombinant enzymes usually have complex structures and biological functions based on their amino acid chains and glycan(s) structures and tend to have the above concerns.

[0159] In some embodiments, the polypeptide or protein is a recombinant protein comprising at least one active site. In some embodiments, the polypeptide or protein is a recombinant protein comprising at least two active sites. In some embodiments, the polypeptide or protein is a fusion protein comprising at least one active site derived from a biologically active protein. In some embodiments, the polypeptide or protein is a fusion protein comprising at least two active sites derived from a biologically active protein.

[0160] In some embodiments, the polypeptide or protein is a glycoprotein. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising at least one glycosylation site. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising at least two glycosylation sites. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising at least three glycosylation sites. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least one amino acid chain. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least two amino acids. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least three amino acid chains. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least one glycan. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least two glycans. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least three glycans. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least one amino acid chain and at least one biological activity site based on a glycan. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least one amino acid chain and at least two biological activity sites based on glycans. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biological activity site based on at least two amino acid chains and at least one biological activity site based on a glycan.In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a biologically active site based on at least two amino acid chains and a biologically active site based on at least two glycans. In some embodiments, the polypeptide or protein is a recombinant glycoprotein comprising a engineered glycan structure.

[0161] In some embodiments, the polypeptide or protein is a naturally occurring protein. In some embodiments, the polypeptide or protein is a plasma-derived protein including, but not limited to, for example, immunoglobulins, coagulation factors, α1-antitrypsin, fibrin sealants, or albumin.

[0162] Various aspects of the present invention are further described in detail in the following examples. The use of the examples is for illustration only and does not limit the present invention.

Examples

[0163] Example 1. Effect of arginine addition on conductivity in affinity purification of recombinant polypeptides, such as arylsulfatase A, using glycine elution buffer This example shows the effect of arginine addition to prevent a decrease in conductivity in the exemplary elution of a recombinant polypeptide, such as arylsulfatase A, purified from an affinity chromatography column using a glycine elution buffer.

[0164] Briefly stated, the ASA protein was purified using affinity chromatography with an exemplary glycine elution buffer. In some exemplary embodiments, the glycine buffer had a concentration of 325 mM glycine-HCl (pH 3.5).

[0165] Conductivity was measured during elution using standard methods. As shown in Figure 1A, at low pH, most of the glycine exists as zwitterions and has no conductivity. The conductivity curve is shown in Figure 1B. The results showed that when elution was initiated, the conductivity decreased temporarily (circle including enlarged image) and became almost zero at the start of elution. At the start of elution, the conductivity decreased to nearly 0 and the chloride ions were depleted. Without wishing to be bound by any particular theory, it is thought that when the elution buffer contacts the affinity column resin, the resin is acidified and acquires an anion exchange function. Chloride ions (Cl - ) bind to the resin, and without chloride, glycine in solution alone has a conductivity of approximately 0 under neutral pH (the pH transition is delayed compared to the conductivity transition). Since Cl - ions do not affect protons, chloride ions contribute to conductivity regardless of the pH of the elution buffer.

[0166] Arginine was added to the glycine elution buffer so that the elution buffer contained 300 mM glycine-HCl, 30 mM arginine-HCl (pH 3.5). As shown in Figure 1C, the conductivity curve after addition of arginine showed less decrease in conductivity compared to the decrease in conductivity in the absence of arginine. Without wishing to be bound by any particular theory, it is thought that the addition of arginine provides cations that are not depleted by ion exchange, and thus the conductivity is better maintained. Combining arginine with glycine also resolved peak splitting.

[0167] Overall, the results showed that the conductivity decreased at the start of elution of arylsulfatase A from the affinity chromatography column using glycine elution buffer, and this decrease was alleviated by the addition of arginine.

[0168] Example 2. Effect of arginine addition on the yield of arylsulfatase A in affinity purification using glycine elution buffer This example demonstrates the effect of arginine addition on increasing the yield in the exemplary elution of arylsulfatase A purified from an affinity chromatography column using a glycine elution buffer.

[0169] Briefly, the ASA protein was purified using affinity chromatography with an exemplary glycine elution buffer. In some exemplary embodiments, the glycine buffer had a concentration of 325 mM glycine-HCl (pH 3.5).

[0170] Peak splitting was observed. The elution yield was very low when only one peak was recovered; for example, at 50 mM glycine (pH 3.5), the yield was less than 50%. Since both peaks were recovered, an elution yield higher than 70% was observed at 325 mM glycine (pH 3.5). However, when multiple peaks were recovered, the purity of the protein decreased. (Figure 1B).

[0171] By combining arginine and glycine, peak splitting was no longer observed. Instead, a single peak of pure protein was observed, which eluted, for example, with a combination of 300 mM glycine and 30 mM arginine (pH 3.5), and the elution yield was 69% (Figure 1D). With a combination of 10 mM arginine and 400 mM glycine (pH 3.5), the elution yield from the single peak was 70% (Figure 1E).

[0172] At a constant pH of 3.5, the elution yield is optimized by varying the glycine-to-arginine ratio. In an exemplary downstream process following affinity chromatography, Nuvia Q ion exchange chromatography, the conductivity limit of the ion exchange resin is 10 mS / cm. To achieve a conductivity of the arginine-glycine elution buffer below the conductivity limit of the downstream ion exchange resin, an arginine concentration of less than 0.1 M is selected, together with a maximum glycine concentration of 0.5 M. The various combinations listed in Table 2 are screened to determine the concentration with the highest yield.

Table 2

[0173] The results showed that an arginine elution buffer of 100 mM resulted in a yield of approximately 57.7%, while a buffer containing only 325 mM of glycine had a yield of only approximately 70%. Similarly, 400 mM of glycine containing 100 mM of arginine resulted in a yield of approximately 70%. When 40 mM of arginine was added to 300 mM of glycine, the yield increased to 81%.

[0174] Overall, the results showed that adding arginine and glycine at concentrations below the conductivity limit of the downstream resin increased the yield.

[0175] Example 3. Effect of Arginine Addition on the Aryl Sulfatase A Protein Peak in Affinity Chromatography Using a Glycine Elution Buffer This example shows the effect of adding arginine to minimize peak splitting in the exemplary elution of aryl sulfatase A purified from an affinity chromatography column using a glycine elution buffer.

[0176] Briefly, the ASA protein was purified using affinity chromatography with an exemplary glycine elution buffer, such as an elution buffer of 50 mM glycine-HCl, 50 mM NaCl (pH 3.1). The activity of the ASA protein was measured after several elution runs of purification on a single column, for example, after cycles 1, 6, 11, 16, and 21.

[0177] As shown in Figure 1F, the baseline activity level is shown as "load", which is the control group before the purification cycle. Compared to the "load", the purified product groups (for example, replicate tests performed on different individual columns ARM1, ARM4, and ARM3) showed a decrease in enzyme activity.

[0178] In some embodiments, arginine was added to the glycine elution buffer. In some exemplary embodiments, the glycine buffer had a concentration of 300 mM glycine-HCl, 30 mM arginine-HCl (pH 3.5) and had a specific activity of 116 U / mg. In some embodiments, the elution buffer had a concentration of 400 mM glycine-HCl and 10 mM arginine-HCl (pH 3.5) and had a specific activity of 116.5 U / mg. Different ARMs and different cycles showed variability, but typically all ARMs and cycles showed lower activity compared to the load. On average, the activity of the ARM product was lower than the average activity of the load. Considering a particular cycle, the specific activity of any cycle, e.g., cycle 16, showed a reduction in the activity of the product relative to the load. After purification, when eluted with glycine-arginine elution buffer, the protein was found to have an activity level similar to the initial load (Table 3, Figure 1F).

Table 3

[0179] Overall, the results show that adding arginine to the glycine elution buffer results in a product with no perceivable loss of specific activity, i.e., the specific activity of the enzyme in the purified product is comparable to the specific activity of the enzyme in the load, and that the addition of arginine to the glycine elution buffer is beneficial in maintaining the conductivity levels of arylsulfatase A during elution and preventing or minimizing peak splitting by the chromatography step, and thus a single stable protein was successfully eluted in high yield.

[0180] Example 4. Effect of arginine addition on arylsulfatase A protein aggregation in affinity chromatography using glycine elution buffer This example shows the effect of adding arginine on the reduction of arylsulfatase A aggregation in an exemplary elution of arylsulfatase A purified from an affinity chromatography column using a glycine elution buffer.

[0181] In some embodiments, an affinity chromatography buffer containing 50 mM glycine-HCl, 50 mM NaCl (pH 3.1) was used to quantify high molecular weight species as a measure of aggregation (Figure 1G, Table 4). High aggregation is undesirable as it reduces the yield, activity, and quality of the product. [Table 4]

[0182] In some embodiments, arginine was added to the glycine elution buffer. For example, an elution buffer containing 300 mM glycine-HCl, 30 mM arginine-HCl (pH 3.5) had 2.36% HMW species at cycle 17: in some embodiments, 400 mM glycine-HCl, 10 mM arginine-HCl (pH 3.5) had 2.38% HMW species at cycle 18.

[0183] Overall, the results showed that glycine-arginine buffer reduced high molecular weight species formed by aggregation and had a beneficial effect on reducing aggregation. Example 5. Comparison of glycine and acetate elution buffers for affinity chromatography

[0184] This example shows an exemplary elution of arylsulfatase A purified from an affinity chromatography column using an acetate elution buffer for a recombinant polypeptide, e.g., in some embodiments, and shows a comparison of glycine and acetate elution buffers.

[0185] When performing affinity chromatography elution using an acetate buffer, CH3COO - The acetate anion is depleted by the resin due to the anion exchange effect. As shown in Figure 2A, the depletion of the acetate anion is H +Leads to an increase in protons, causing a change in pH. An exemplary acetate elution buffer pH curve showing that the pH decreased and then gradually increased to the pH of the elution buffer is shown in Figure 2B. In contrast, in glycine-HCl buffer, depletion of chloride anions by the resin does not affect the proton concentration.

[0186] Briefly stated, control affinity chromatography runs were performed without loading the protein to prevent pH interference by the protein. Using an exemplary affinity resin for arylsulfatase A, two runs were performed on the same affinity column, one with 142 mM acetate elution buffer and the other with 340 mM glycine elution buffer.

[0187] The results shown in Figure 3A presented a comparison of the elution pH curves of acetate and glycine elution buffers. The acetate buffer produced an unstable pH during elution. The pH decreased to a low level and then slowly rose back to the pH of the elution buffer. A change in the pH range exceeding 0.2 was observed. In some embodiments, the acetate elution pool has a lower pH than the acetate elution buffer itself. Without wishing to be bound by any particular theory, it is contemplated that the change in pH creates a low-pH pocket on the column, which denatures the protein and results in a decrease in the quality of the product.

[0188] Exemplary pH and conductivity curves for two runs are shown in Figure 3B. Run 1 was an exemplary run using 340 mM glycine-HCl (pH 3.4); Run 2 was performed with 142 mM acetate (pH 3.5).

[0189] Exemplary conductivity curves using glycine or acetate buffer are shown in Figure 3C. The elution buffer consisting of only glycine showed a temporary decrease in conductivity (circles in Figure 3C). No decrease in conductivity was observed when using the acetate elution buffer.

[0190] In an exemplary embodiment, the elution of arylsulfatase A using a glycine elution buffer (Figure 3D) and an acetate elution buffer (Figure 3E) is shown. These results showed that the acetate elution buffer resulted in an earlier pH transition than the glycine elution buffer.

[0191] When using the acetate elution buffer, the elution pool of arylsulfatase A was found to have a lower pH than the elution buffer (Figure 3F). Without wishing to be bound by any particular theory, this is presumably due to an increase in proton concentration.

[0192] Overall, the results of this example showed that when using the acetate elution buffer, there is pH instability but no decrease in conductivity. In contrast, the glycine elution buffer causes a temporary decrease in conductivity but does not cause pH instability.

[0193] Example 6. Anion Exchange Effect in Affinity Purification of an Exemplary Recombinant Polypeptide, Such as Alpha-Galactosidase A, Using a Glycine Elution Buffer This example shows that the affinity chromatography of an exemplary recombinant polypeptide using a glycine elution buffer exhibited an anion exchange effect.

[0194] In one embodiment, the alpha-galactosidase A protein was purified using affinity chromatography with an exemplary glycine elution buffer. In some exemplary embodiments, the glycine buffer had a concentration of 0.1 M glycine-HCl (pH 3.0).

[0195] Conductivity was measured during elution using standard methods. As shown in Figure 4, the results showed that when elution was initiated, the conductivity temporarily decreased to nearly zero. Without wishing to be bound by a particular theory, as the elution buffer contacts the affinity column resin, depletion of chloride ions is caused, the resin becomes acidified, and it is thought to acquire an anion exchange function.

[0196] Overall, the results showed a decrease in conductivity at the start of the elution of alpha-galactosidase A from the affinity chromatography column using glycine elution buffer.

[0197] Example 7. Determination of the conductivity limit of the downstream chromatography resin and adjustment of the amino acid concentration of the affinity chromatography elution buffer. This example shows an exemplary process for adjusting the amino acid concentrations of glycine and arginine in the affinity chromatography elution buffer based on the conductivity limit of the downstream resin.

[0198] First, determine the conductivity limit of the next polishing or purification column after affinity purification. Each resin has a conductivity limit for a specific protein denoted by "S". For example, in an exemplary arylsulfatase A purification process, the next step using NuviaQ resin following affinity purification has an experimentally determined conductivity limit of about 10 mS / cm. In an exemplary iduronate 2-sulfatase purification process, hydroxyapatite chromatography with a conductivity limit of 30 mS / cm is used in the next step following affinity purification. In an exemplary alpha-galactosidase A purification, the next step after affinity chromatography is ion exchange chromatography, which has a conductivity limit of 10 mS / cm.

[0199] Thereafter, the maximum glycine and arginine concentrations are used within the conductivity range. Determine the pH of the affinity elution buffer as X. Estimate the loading pH of the next purification column as Y.

[0200] To adjust the pH of the elution pool from the affinity purification step to the final pH near Y, a series of glycine solutions of different concentrations are prepared. The pH is adjusted to X with HCl. Next, for an exemplary arylsulfatase A protein, the pH is adjusted to Y using, for example, Tris-HCl or Tris base. The final conductivity of all solutions is measured. The conductivity of the solutions is plotted against the glycine concentration (Figure 5).

[0201] As shown in Figure 6, the conductivity vs. concentration curves are linear curves for both glycine and arginine. Based on the conductivity limit S, the concentration upper limit of each amino acid is determined from its respective plot. For example, in the case of rhASA, in some embodiments, the maximum limit of glycine is 500 mM and the maximum limit of arginine is 100 mM. After the pH is neutralized, glycine has a very low conductivity, while arginine has a much higher conductivity.

[0202] In some embodiments, for the purification of iduronate 2-sulfatase, the maximum glycine limit is about 1500 mM and the maximum concentration of arginine is about 300 mM. For example, the conductivity of 1 mM glycine is about 30 / 1500 = 0.02, and the conductivity of 1 mM arginine is about 30 / 300 = 0.1. The concentrations of glycine and arginine that satisfy the equation 0.02X + 0.1Y < 30 mS / cm are selected (where X mM represents the glycine concentration and Y mM represents the arginine concentration).

[0203] In some embodiments, for the purification of argasidase alpha, after affinity chromatography using 0.1 M glycine (pH 3.0), ion exchange chromatography is performed at a conductivity limit of about 10 mS / cm. The maximum concentration of glycine is about 500 mM and the maximum concentration of arginine is about 100 mM. The concentrations of glycine and arginine that satisfy the equation 0.02X + 0.1Y < 10 are selected (where X mM represents the glycine concentration and Y mM represents the arginine concentration).

[0204] The concentrations of glycine and arginine are selected such that the total conductivity is below the limit S.

[0205] This example shows steps in adjusting the amino acid concentration of an elution buffer for affinity chromatography in an improved process for purifying a recombinant protein or polypeptide.

[0206] Example 8. Effect of arginine addition on conductivity in the affinity purification of a recombinant polypeptide, e.g., iduronate-2-sulfatase (I2S) using a glycine elution buffer. This example shows the effect of arginine addition on changes in conductivity and protein yield in an exemplary elution of a recombinant polypeptide, e.g., iduronate-2-sulfatase (I2S), purified from an affinity chromatography column using a glycine elution buffer.

[0207] Briefly, the I2S protein was purified using affinity chromatography with an exemplary glycine elution buffer. In various embodiments, a glycine elution buffer containing 50 mM glycine-HCl and 50 mM sodium chloride was titrated with hydrogen chloride to produce elution buffers at various pH ranges, e.g., pH 2.9, pH 3.1, pH 3.5, pH 3.7, and pH 4.1. At pH 4.1, 20 mM histidine-HCl was added as a positively charged agent to provide a buffering effect.

[0208] After affinity chromatography using the glycine elution buffer, the estimated percent yield was measured. The results shown in Table 4 indicated that an increase in the pH of the buffer corresponded to a decrease in the total yield. Chromatograms of the glycine elution buffer at pH 3.1, pH 3.5, and pH 3.7 showed flatter elution peaks and larger dispersion peaks, indicating a low product yield. It was shown that the higher the ratio of the elution peak area to the strip peak area, the higher the yield, but the narrower and sharper the elution peak, the more favorable it was for recovery (Figure 7).

Table 5

[0209] The specific activity of I2S was measured after affinity chromatography using various elution buffers. In some embodiments, arginine was added to the glycine elution buffer such that the elution buffer contained 50 mM glycine-HCl, 50 mM arginine-HCl (pH 3.6). As shown in Table 6, the combination of glycine and arginine had no significant effect on the specific activity of I2S at about pH 3.6.

Table 6

[0210] The elution yield was optimized by changing the ratio of glycine to arginine at an exemplary constant pH of 3.6. The conductivity limit for iduronate-2-sulfatase to enable multiple downstream processing options was 10 mS / cm. Thus, the maximum concentrations of glycine and arginine used were within this conductivity range at pH 3.6.

[0211] As shown in Figure 8, conductivity affected the elution and dispersion peaks on the affinity chromatogram. In this example, a buffer containing 300 mM glycine, 50 mM arginine, and a conductivity of 5.33 mS / cm produced a larger and narrower elution peak than buffers with lower conductivities of 2.87 and 4.26 mS / cm, showing a higher protein yield and, in some embodiments, also an improvement in the quality of the product.

[0212] At an exemplary conductivity of about 5.5 mS / cm, glycine and arginine were combined at different ratios at pH 3.6 and the effect on product yield was measured. As shown in Figure 9A, it is shown that as the ratio of glycine to arginine increases, the elution peak becomes larger and sharper, and the product yield increases.

[0213] Exemplary conductivities of 7.0 - 7.5 mS / cm were used to recombine glycine and arginine at pH 3.6 in different ratios. As shown in Figure 9B, as the ratio of glycine to arginine increased, the elution peak became larger and sharper.

[0214] Overall, the results showed that the addition of arginine to glycine at higher glycine-to-arginine ratios, which are lower than the conductivity limit of the downstream process, increased the protein yield.

[0215] Example 9. Effect of arginine addition on the yield of iduronate-2-sulfatase in affinity purification using a glycine elution buffer. This example shows the effect of arginine addition on the increased yield in an exemplary elution of iduronate-2-sulfatase purified from an affinity chromatography column using a glycine elution buffer.

[0216] Briefly, the I2S protein was purified using affinity chromatography with one exemplary glycine elution buffer. In some embodiments, sodium chloride was added to the glycine buffer such that the buffer contained 50 mM glycine, 50 mM sodium chloride (pH 3.5), and a conductivity of 5.98 mS / cm.

[0217] In some embodiments, arginine was then added to the exemplary glycine buffer at a glycine-to-arginine ratio of 100:3 and a similar conductivity, such that the glycine elution buffer contained 606.4 mM glycine, 18.2 mM arginine (pH 3.6), and a conductivity of 5.70 mS / cm.

[0218] The results in Figure 9C showed that in the glycine elution buffer, a broad elution peak and a large evaporation peak were observed. The addition of arginine was shown to result in a larger and sharper elution peak, a smaller evaporation peak, and an improved product yield. In Figure 9C, the two buffers tested had almost the same conductivity. Under the same conductivity, generally, the lower the pH (rougher pH), the higher the elution yield obtained. By using a combination of glycine and arginine, a higher yield can be achieved under a higher pH (milder pH), which is rare. Compared with pH 3.5, pH 3.6 represents milder conditions and potentially could improve the quality of the product. Even under milder elution conditions, a higher yield could still be achieved. This indicates that based on this approach, both the yield and the quality of the product can be improved.

[0219] Overall, the results showed that adding arginine to the glycine elution buffer increased the product yield even with similar overall conductivity. The results also showed that increasing the glycine concentration can increase the product yield without changing the overall pH and conductivity.

[0220] Example 10. Effect of Arginine Addition on the Specific Activity of Iduronate-2-Sulfatase in Affinity Purification Using Glycine Elution Buffer This example shows the effect of the addition of arginine on the specific activity in an exemplary elution of iduronate-2-sulfatase purified from an affinity chromatography column using a glycine elution buffer.

[0221] Briefly, the I2S protein was purified using affinity chromatography with an exemplary glycine elution buffer, such as 1000 mM glycine, 30 mM arginine (pH 3.6). The specific activity of the purified I2S protein was measured after several elution runs using different elution buffers.

[0222] As shown in Table 7, the baseline activity level is shown as the "assay control", which is a control group using a standard elution buffer. Compared with the "assay control", the purified product using the glycine elution buffer did not show a decrease in enzyme activity.

Table 7

[0223] In some embodiments, arginine was added to the glycine elution buffer. In some embodiments, the glycine buffer had 1000 mM glycine-HCl, 30 mM arginine-HCl (pH 3.6) and had a specific activity of 3.84 U / mg. In some embodiments, the elution buffer contained 50 mM glycine-HCl, 50 mM NaCl (pH 3.1) and had a specific activity of 2.91 U / mg. Different buffers showed different specific activities; however, compared with the "assay control", all the tested buffers showed that there was no increase in specific activity but they were comparable.

[0224] Overall, the results show that the glycine elution buffer with added arginine provides equivalent products rather than an improvement in product quality, that is, the specific activity of the enzyme in the purified product using the glycine buffer with added arginine is comparable or increased compared to the specific activity of the enzyme in the previous standard buffer.

[0225] Example 11. Effect of arginine addition on iduronate-2-sulfatase protein aggregation in affinity chromatography using a glycine elution buffer. This example shows the addition of arginine to reduce iduronate-2-sulfatase aggregation in an exemplary elution of iduronate-2-sulfatase from an affinity chromatography column using a glycine elution buffer.

[0226] Briefly stated, in this example, aggregation of the purified product was measured by measuring the percentage of high molecular weight species after purification by affinity chromatography using various elution buffers. High aggregation is undesirable as it represents a decrease in product quality.

[0227] The results shown in Table 7 indicated that an elution buffer of 50 mM glycine and 50 mM sodium chloride (pH 3.1) contained 7.74% high molecular weight species. In the buffer of 1000 mM glycine and 30 mM arginine (pH 3.6), the high molecular weight species were 0.29% of the total product. [Table 8]

[0228] Overall, the results showed that the addition of arginine at a high glycine to arginine ratio decreased the high molecular weight species formed by aggregation and that it had a beneficial effect on reducing aggregation.

[0229] Example 12. Comparison of glycine and acetate elution buffers for iduronate-2-sulfatase affinity chromatography. This example shows an exemplary elution of a recombinant polypeptide, iduronate-2-sulfatase, purified from an affinity chromatography column using an acetate elution buffer and shows a comparison of acetate and glycine elution buffers.

[0230] Briefly stated, the affinity chromatography was performed using iduronate-2-sulfatase. An acetate elution buffer containing 50 mM sodium acetate, 1.0 M sodium chloride, 20% propylene glycol (pH 4.5) was compared with a glycine elution buffer containing 1 M glycine, 30 mM arginine (pH 3.6).

[0231] The results of Figure 10 showed a comparison of the affinity chromatograms of acetate elution buffer and glycine elution buffer. Similar yields of approximately 90% were recorded for each run. However, the elution peak of glycine was sharper than the acetate elution peak, indicating a higher protein concentration in the product and a smaller pool volume for recovery.

[0232] Furthermore, the conductivity of each buffer was measured. The conductivity of the acetate buffer was 51.0 mS / cm and that of the glycine buffer was 9.4 mS / cm, allowing for different downstream processes. Overall, the results of this example showed that the glycine elution buffer provided both a higher protein concentration and a lower conductivity, providing preferably improved conditions for downstream operations.

Claims

1. A method for purifying a polypeptide, comprising: loading the polypeptide onto an affinity chromatography column; eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine; feeding the eluate from the affinity chromatography column to a subsequent chromatography column containing a resin; wherein the conductivity of the elution buffer is equal to or lower than the conductivity limit of the resin in the subsequent chromatography column.

2. A method for purifying a polypeptide, comprising: loading the polypeptide onto an affinity chromatography column; eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine; wherein the conductivity of the elution buffer is 30 mS / cm or lower.

3. The method according to claim 1, wherein the elution buffer contains glycine at a concentration of 1500 mM or lower and arginine at a concentration of 300 mM or lower.

4. The method according to claim 1, wherein the elution buffer contains glycine at a concentration of 500 mM or lower and arginine at a concentration of 100 mM or lower.

5. A method for purifying a polypeptide, the method comprising: loading the polypeptide onto an affinity chromatography column; eluting the polypeptide from the affinity chromatography column using an elution buffer; wherein the elution buffer contains glycine at a concentration of 500 mM or lower and arginine at a concentration of 100 mM or lower.

6. A method for purifying a polypeptide, the method comprising: loading the sample onto an affinity chromatography column; eluting the polypeptide from the affinity chromatography column using an elution buffer; wherein the elution buffer contains glycine and arginine, and the glycine and arginine are present in a ratio (molar ratio) of at least 1:

2.

7. The method according to claim 6, wherein the elution buffer contains glycine and arginine at a pH of 3.0 to 5.0, and the glycine and arginine are present in a ratio (molar ratio) of 1:2 to 40:

1.

8. The method according to claim 6 or 7, wherein the elution buffer contains arginine at a concentration of about 10 to 300 mM.

9. The method according to any one of claims 6 to 8, wherein the elution buffer contains glycine at a concentration of about 50 to 1500 mM.

10. The method according to any one of the preceding claims, further comprising the step of adjusting the eluate from the affinity chromatography column to pH 4.0 to 9.

0.

11. The method according to any one of the preceding claims, further comprising a step of inactivating the virus in the eluate from the affinity chromatography column.

12. The method according to any one of the preceding claims, wherein the eluate from the affinity column contains less than 5% high molecular weight aggregates (HMW).

13. The method according to any one of the preceding claims, wherein the affinity chromatography column uses an antibody or antigen-binding fragment that specifically binds to the polypeptide.

14. The method according to any one of the preceding claims, further comprising adjusting the concentrations of arginine and glycine in the elution buffer based on the conductivity of the resin for the polypeptide.

15. The method according to any one of claims 5 to 14, further comprising subjecting the eluate from the affinity chromatography column to a chromatography step.

16. The method according to any one of the preceding claims, comprising subjecting the eluate from the affinity chromatography column to a chromatography step, wherein the chromatography step is selected from the group consisting of ion exchange, mixed mode, or hydroxyapatite chromatography.

17. The method according to claim 16, wherein the chromatography step is mixed mode chromatography.

18. The method according to claim 16, wherein the chromatography step is hydroxyapatite chromatography.

19. The method according to claim 16, wherein the chromatography step is ion exchange chromatography.

20. The method according to claim 19, wherein the ion exchange chromatography is anion exchange chromatography.

21. The method according to claim 19, wherein the ion exchange chromatography is cation exchange chromatography.

22. The method according to any one of the preceding claims, comprising determining the conductivity limit of the chromatography step and adjusting the concentration of the glycine and / or arginine in the affinity chromatography column elution buffer to the conductivity limit.

23. The method according to claim 22, wherein the conductivity limit is about ≦ 30 mS / cm.

24. The method according to any one of the preceding claims, wherein the affinity chromatography column elution buffer has a pH of 3.0 to 5.

0.

25. The method according to any one of the preceding claims, wherein the polypeptide is a recombinant protein.

26. The method according to any one of the preceding claims, wherein the polypeptide is an enzyme.

27. The method according to any one of the preceding claims, wherein the polypeptide retains at least 80% of its specific activity compared to its specific activity before purification.

28. The method according to any one of the preceding claims, wherein the polypeptide is recombinant human arylsulfatase A.

29. The method according to any one of claims 1 to 27, wherein the polypeptide is recombinant iduronate 2-sulfatase.

30. The method according to any one of the preceding claims, wherein the polypeptide does not contain a domain that binds to protein A or protein G.

31. The method according to any one of the preceding claims, wherein the affinity chromatography column contains a ligand selected from a library of ligands that specifically bind to the polypeptide.

32. The method according to claim 31, wherein the ligand comprises a complementarity determining region of an antibody.

33. A method for purifying a polypeptide, comprising: (a) immobilizing the polypeptide on an affinity chromatography column, wherein the affinity chromatography column contains a ligand selected from a library of ligands that specifically bind to the polypeptide; (b) eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, wherein the concentration of glycine is X mM, the concentration of arginine is Y mM, the concentrations of glycine and arginine satisfy the inequality 0.02X + 0.1Y ≦ 10, and the pH of the eluate is 3.0 to 5.0; and (c) subjecting the eluate from the affinity chromatography column to ion exchange chromatography.

34. A method for purifying a polypeptide, comprising: (a) immobilizing the polypeptide on an affinity chromatography column, wherein the affinity chromatography column contains a ligand selected from a library of ligands that specifically bind to the polypeptide; (b) eluting the polypeptide from the affinity chromatography column using an elution buffer containing glycine and arginine, wherein the concentration of glycine is X mM, the concentration of arginine is Y mM, the concentrations of glycine and arginine satisfy the inequality 0.02X + 0.1Y ≦ 30, the pH of the elution buffer is 3.0 to 5.0; and (c) subjecting the eluate from the affinity chromatography column to mixed mode chromatography.

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