Method for preparing buffer solutions for therapeutic protein formulations

Abstract

This disclosure provides materials and methods for preparing a buffer for a therapeutic protein formulation, comprising (a) combining a calcium salt, an organic acid, and a disaccharide at a temperature of 22°C or higher to produce a mixture, and (b) adding sodium hydroxide to the mixture from (a) by bolus addition.

Description

[Technical Field]

[0001] Incorporation by Reference of Electronically Submitted Material The computer-readable nucleotide / amino acid sequence listing submitted concurrently with the present specification is incorporated by reference in its entirety and identified as follows: an XML file named "58404_SeqListing.xml" with a size of 13,970 bytes, created on July 16, 2024.

[0002] Incorporation by Reference The following applications are incorporated herein by reference in their entirety: International Patent Application PCT / U.S. Patent Application Publication No. 2012 / 049331, filed 2 August 2012 (claiming priority to U.S. Provisional Patent Application No. 61 / 515,191, filed 4 August 2011), U.S. Patent Application No. 11 / 410,540, filed 25 April 2006 (U.S. Provisional Patent Application No. 60 / 792,645, filed 17 April 2006, U.S. Provisional Patent Application No. 60 / 782,244, filed 13 March 2006, U.S. Provisional Patent Application No. 60 / 776,847, filed 24 February 2006, and 20 Priority is claimed to U.S. Provisional Patent Application No. 60 / 677,583 filed on 3 May 2005; and U.S. Patent Application No. 11 / 411,003 filed on 25 April 2006 (registered as U.S. Patent No. 7,592,429) (priority is claimed to U.S. Provisional Patent Application No. 60 / 792,645 filed on 17 April 2006, U.S. Provisional Patent Application No. 60 / 782,244 filed on 13 March 2006, U.S. Provisional Patent Application No. 60 / 776,847 filed on 24 February 2006, and U.S. Provisional Patent Application No. 60 / 677,583 filed on 3 May 2005). The following applications are also incorporated herein by reference: U.S. Patent Application No. 12 / 212,327 filed September 17, 2008 (claiming priority to U.S. Provisional Patent Application No. 60 / 973,024 filed September 17, 2007) and U.S. Patent Application No. 12 / 811,171 filed June 29, 2010 (a U.S. national phase application under Section 371 of the U.S. Patent Act to the International Patent Application PCT / U.S. Patent Application Publication No. 08 / 86864 filed December 15, 2008, claiming priority to U.S. Provisional Patent Application No. 61 / 013,917 filed December 14, 2007). [Background technology]

[0003] Therapeutic protein formulations are well known in the art. However, there is a need for the preparation of buffers for such therapeutic protein formulations in a streamlined and accelerated process that also optimizes and equilibrates the solubility of the formulation components. [Overview of the Initiative] [Means for solving the problem]

[0004] In one embodiment, materials and methods for preparing a buffer for a therapeutic protein formulation are described herein, comprising (a) combining a calcium salt, an organic acid, and a disaccharide at a temperature of ≥22°C (22°C or higher) to produce a mixture, and (b) adding sodium hydroxide to the mixture of (a) by bolus addition. In some embodiments, the calcium salt is calcium acetate. In some embodiments, the organic acid is acetic acid, for example, glacial acetic acid. In some embodiments, the disaccharide is sucrose.

[0005] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-restrictive terms unless otherwise specified (i.e., “including but not limited,” allowing for the presence of one or more additional features or components). Various embodiments herein are presented using the term “comprising” under various circumstances, while related embodiments should be understood to be described using the terms “consisting of” or “essentially consisting of.” Note that the terms “a” or “an” refer to one or more; for example, “an immunoglobulin molecule” should be understood to represent one or more immunoglobulin molecules unless otherwise specified by context. Thus, the terms “a” (or “an”), “one or more,” and “at least one” may be used interchangeably herein. Furthermore, “and / or,” when used herein, should be interpreted as a specific disclosure of each of two particular features or components, whether or not they are accompanied by the other feature or component. Therefore, when the term “and / or” is used herein in a phrase such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, when the term “and / or” is used in a phrase such as “A, B, and / or C,” it is intended to include each of the following embodiments: A, B and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0006] When specifying a range of values, it should be understood that the described characteristics may be individual values ​​found within that range. For example, "pH approximately 4 to approximately 6" is not limiting, but could be pH 4, 4.2, 4.6, 5.1, 5.5, and any value between such values. In addition, "pH approximately 4 to approximately 6" should not be interpreted as meaning that the pH of the target formulation will fluctuate within the range of pH 4 to pH 6 in 2 pH increments during storage, but rather as meaning that a value within that range can be selected for the pH of the solution, and that the pH will remain buffered around that pH.

[0007] In any of the scopes described herein, the endpoints of the scope are included within that scope. However, this description also considers the same scopes in which the smaller and / or larger endpoints are excluded. Additional features and variations of the present invention will be apparent to those skilled in the art from the whole of this application, including the drawings and detailed description, and all such features are intended as embodiments of the present invention. Similarly, the features of the present invention described herein can be rearranged to form additional embodiments that are also intended as embodiments of the present invention, whether or not the combination of features is specifically listed above as an embodiment or example of the present invention. Furthermore, only such limitations described herein as essential to the present invention should be considered limitations; variations of the present invention lacking limitations not described herein as essential are also intended as embodiments of the present invention.

[0008] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which this disclosure relates. For example, *Concise Dictionary of Biomedicine and Molecular Biology*, Juo, Pei-Show, 2nd ed., 2002, CRC Press; *The Dictionary of Cell and Molecular Biology*, 3rd ed., 1999, Academic Press; and *Oxford Dictionary of Biochemistry and Molecular Biology*, Revised, 2000, Oxford University Press provide many general dictionaries of the terms used herein.

[0009] All references cited herein are incorporated herein by reference as a whole. [Modes for carrying out the invention]

[0010] This disclosure is based on the discovery of an improved process for preparing buffers for therapeutic protein formulations, which includes a fixed mixing time at a constant mixing rate and a constant temperature, and the addition of a sodium hydroxide bolus as an alternative to NaOH titration, resulting in the preparation of buffers for therapeutic protein formulations. The process is designed to optimize and equilibrate the solubility of different formulation components. Further benefits of the process provided herein include a reduced number of component addition steps, the definition of a fixed mixing rate, a reduction in total mixing time, and the minimization of sample recovery and analysis.

[0011] In one embodiment, a method for preparing a buffer for a therapeutic protein formulation is described herein, comprising (a) combining a calcium salt, an organic acid, and a disaccharide at a temperature of ≥22°C to produce a mixture, and (b) adding sodium hydroxide to the mixture of (a) by bolus addition. In one embodiment, the calcium salt is calcium acetate. In one embodiment, the organic acid is acetic acid, for example, glacial acetic acid. In one embodiment, the disaccharide is sucrose.

[0012] In some embodiments, step (a) includes (i) mixing the calcium salt and organic acid with water (e.g., water for injection or sterile water) for a period of ≤20 minutes (e.g., in the range of 16 to 20 minutes) to produce the mixture of (i), and (ii) adding a disaccharide to the mixture of (i) and mixing for a further ≤20 minutes (e.g., in the range of 16 to 20 minutes) to produce the mixture of (ii). Step (a) is carried out at a temperature of ≥22°C. In one embodiment, steps (i) and (ii) of step (a) are carried out at the same temperature. In another embodiment, steps (i) and (ii) of step (a) are carried out at different temperatures. In some embodiments, step (a) is carried out at a temperature of 22°C to 32°C. In some embodiments, step (a) is carried out at a temperature of 27°C. In some embodiments, the calcium salt is calcium acetate. In some embodiments, the organic acid is acetic acid, e.g., glacial acetic acid. In some embodiments, the disaccharide is sucrose.

[0013] In step (b), sodium hydroxide (NaOH) is added to the mixture of (a) by bolus addition to produce the mixture of (b). In some embodiments, step (b) further includes mixing for a period of ≤4 minutes (e.g., about 2 to about 4 minutes) after the bolus addition of sodium hydroxide. In some embodiments, step (b) includes mixing the mixture for about 2 minutes, about 3 minutes, or about 4 minutes after the bolus addition of sodium hydroxide.

[0014] In some embodiments, step (b) is performed at a temperature of ≥22°C. In some embodiments, step (b) is performed at a temperature of about 22°C to about 32°C. In some embodiments, step (b) is performed at a temperature of 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, or 32°C. In some embodiments, step (b) is performed at a temperature of about 27°C.

[0015] In some embodiments, the mixing speed for step (a) (including steps (i) and (ii)) and step (b) is in the range of about 135 rpm to about 170 rpm. In some embodiments, the mixing speed for steps (a) and (b) is in the range of about 135 rpm to about 155 rpm or about 140 rpm to about 150 rpm. In some embodiments, the mixing speed for steps (a) and (b) is about 145 rpm. In one embodiment, steps (a) and (b) have the same mixing speed. In a further embodiment, steps (i) and (ii) of step (a) have the same mixing speed. In yet another embodiment, steps (i) and (ii) of step (a) and step (b) have the same mixing speed. In some embodiments, the mixing speed is about 135 rpm, or about 140 rpm, or about 145 rpm, or about 150 rpm, or about 155 rpm, or about 160 rpm, or about 165 rpm, or about 170 rpm. In some embodiments, the mixing speed is 135 rpm, 136 rpm, 137 rpm, 138 rpm, 139 rpm, 140 rpm, 141 rpm, 142 rpm, 143 rpm, 144 rpm, 145 rpm, 146 rpm, 147 rpm, 148 rpm, 149 rpm, 150 rpm, 151 rpm, 152 rpm, 153 rpm, 154 rpm, 155 rpm, 156 rpm, 157 rpm, 158 rpm, 159 rpm, 160 rpm, 161 rpm, 162 rpm, 163 rpm, 164 rpm, 165 rpm, 166 rpm, 167 rpm, 168 rpm, 169 rpm, or 170 rpm.

[0016] In some embodiments, the method includes adding water to the mixture of step (b), preferably after mixing. This step may be required to obtain desired concentrations of different components in the (final) buffer. In some embodiments, the method includes adding water to the mixture of step (b) and mixing. In some embodiments, the method includes adding water to the mixture of step (b) and mixing before the cooling step. In some embodiments, the method includes adding water to the mixture of step (b) and mixing for ≤4 minutes (e.g., about 2 to about 4 minutes). In some embodiments, the method includes adding water to the mixture of step (b) and mixing for about 2 minutes, about 3 minutes, or about 4 minutes. In one embodiment, the water is water for injection or sterile water.

[0017] In one embodiment, the mixing of the added water (as referred to in the above section) is carried out at a temperature of ≥22°C. In further embodiments, the mixing of the water is carried out at a temperature of 22°C to 32°C. In some embodiments, the mixing of the water is carried out at a temperature of 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, or 32°C. In some embodiments, the mixing of the water is carried out at a temperature of about 27°C.

[0018] In one embodiment, the mixing of the added water (as mentioned in the two sections above) is carried out at a mixing speed in the range of about 135 rpm to about 155 rpm or about 140 rpm to about 150 rpm. In some embodiments, the mixing speed is about 145 rpm. In some embodiments, the mixing speed is about 135 rpm, or about 140 rpm, or about 145 rpm, or about 150 rpm, or about 155 rpm. In some embodiments, the mixing speed is 135 rpm, 136 rpm, 137 rpm, 138 rpm, 139 rpm, 140 rpm, 141 rpm, 142 rpm, 143 rpm, 144 rpm, 145 rpm, 146 rpm, 147 rpm, 148 rpm, 149 rpm, 150 rpm, 151 rpm, 152 rpm, 153 rpm, 154 rpm, or 155 rpm.

[0019] In some embodiments, the method includes a total mixing time of less than about 60 minutes (e.g., about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 60 minutes) (i.e., mixing time for step (a), for step (b), and for mixing the water added after step (b)). In some embodiments, the method includes a total mixing time in the range of about 36 minutes to about 48 minutes. In some embodiments, the method includes a total mixing time of approximately 36 minutes, or approximately 37 minutes, or approximately 38 minutes, or approximately 39 minutes, or approximately 40 minutes, or approximately 41 minutes, or approximately 42 minutes, or approximately 43 minutes, or approximately 44 minutes, or approximately 45 minutes, or approximately 46 minutes, or approximately 47 minutes, or approximately 48 minutes, or approximately 49 minutes, or approximately 50 minutes, or approximately 51 minutes, or approximately 52 minutes, or approximately 53 minutes, or approximately 54 minutes, or approximately 55 minutes, or approximately 56 minutes, or approximately 57 minutes, or approximately 58 minutes, or approximately 59 minutes, or approximately 60 minutes. In some embodiments, the method includes a total mixing time of approximately 36 to 48 minutes, or approximately 38 to 46 minutes, or approximately 40 to 44 minutes, or approximately 42 minutes.

[0020] In some embodiments, the method includes cooling the buffer prepared by the method disclosed herein (i.e., either after step (b) or after the addition of water to the mixture in step (b)) to a temperature in the range of about 2°C to about 8°C. In some embodiments, the method includes cooling the buffer to a temperature of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, or 8°C. In some embodiments, the method includes cooling the buffer to a temperature of about 5°C.

[0021] In some embodiments, the final concentration of the disaccharide (e.g., sucrose) in the buffer is higher than the final concentration of the calcium salt (e.g., calcium acetate) in the buffer. In some embodiments, the disaccharide (e.g., sucrose) has the maximum final concentration of all components of the buffer (regardless of the concentration of the therapeutic protein that may be added to the final buffer later). In some embodiments, the disaccharide (e.g., sucrose) is added to the mixture in an amount at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, or at least about 30 times greater than the amount of the calcium salt (e.g., calcium acetate) added to the mixture [e.g., expressed in grams per kg of buffer / final buffer].

[0022] The buffer preparation method disclosed herein comprises combining a calcium salt, an organic acid, and a disaccharide, followed by the addition of NaOH. In some embodiments, the calcium salt (e.g., calcium acetate) is added in amounts of about 0.2 g to about 20 g, or about 0.4 g to about 15 g, or about 0.6 g to about 12 g, or about 0.8 g to about 10 g, or about 1 g to about 5 g, or about 1.5 g to about 4 g, or about 1.7 g to about 3 g, or about 2 g per kg of the final buffer. Any range characterized by the above combination of endpoints is also intended.

[0023] In some embodiments, disaccharides (e.g., sucrose) are added in amounts of approximately 6g to 180g, 10g to 150g, 10g to 120g, 20g to 100g, 30g to 90g, 40g to 80g, 50g to 70g, 55g to 65g, 59g, or 60g per kg of final buffer. Any range characterized by the above combinations of endpoints is also intended.

[0024] In some embodiments, the organic acid (e.g., glacial acetic acid) is added in an amount of from about 0.2 g to about 20 g, or from about 0.4 g to about 15 g, or from about 0.6 g to about 12 g, or from about 0.5 g to about 5 g, or from about 0.8 g to about 3 g, or from about 1 g to about 2.5 g, or from about 1.2 g to about 2.2 g, or from about 1.4 g to about 2 g, or about 1.7 g, per 1 kg of the final buffer solution. Any range characterized by a combination of the above endpoints is also contemplated.

[0025] In some embodiments, NaOH is added in an amount of from about 0.2 mL to about 20 mL, or from about 0.4 mL to about 15 mL, or from about 0.6 mL to about 12 mL, or from about 0.5 mL to about 5 mL, or from about 0.8 mL to about 3 mL, or from about 1 mL to about 2.5 mL, or from about 1.2 mL to about 2.2 mL, or from about 1.4 mL to about 2 mL, or about 1.65 mL, per 1 kg of the final buffer solution. Any range characterized by a combination of the above endpoints is also contemplated.

[0026] In some embodiments, the concentration of calcium in the buffer solution prepared by the method described herein ranges from 1 mM to 130 mM. In some embodiments, the concentration of calcium in the buffer solution ranges from 1 mM to 30 mM, alternatively from 5 mM to 20 mM, or alternatively from 10 mM to 15 mM. In some embodiments, calcium is present in the buffer solution at a concentration of at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, or at least 12 mM. In some embodiments, the concentration of calcium in the buffer solution is about 13 mM. In one embodiment, the concentration of calcium in the buffer solution is about 13 mM or less, about 14 mM or less, about 15 mM or less, about 16 mM or less, about 17 mM or less, about 18 mM or less, about 19 mM or less, about 20 mM or less, about 21 mM or less, about 22 mM or less, about 23 mM or less, about 24 mM or less, about 25 mM or less, about 26 mM or less, about 27 mM or less, about 28 mM or less, about 29 mM or less, or about 30 mM or less. Any range characterized by a combination of the above endpoints is contemplated, including but not limited to from about 5 mM to about 30 mM, or from about 10 mM to about 20 mM.

[0027] In some embodiments, the concentration of acetate in the buffer prepared by the method described herein is in the range of about 5 mM to about 100 mM. In some embodiments, the concentration of acetate in the buffer is in the range of about 10 mM to about 90 mM, or about 20 mM to about 80 mM, or about 30 mM to about 70 mM, or about 35 mM to about 75 mM, or about 40 mM to about 70 mM, or about 45 mM to about 65 mM. In some embodiments, the concentration of acetate in the buffer is in the range of about 50 mM to about 60 mM. In some embodiments, the acetate is present in the buffer at a concentration of at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, or at least about 50 mM. In some embodiments, the concentration of acetate in the buffer is about 60 mM or less, about 65 mM or less, about 70 mM or less, about 75 mM or less, about 80 mM or less, about 85 mM or less, about 90 mM or less, about 100 mM or less, about 110 mM or less, about 120 mM or less, or about 130 mM or less. In some embodiments, the concentration of acetate in the buffer is about 55 mM. Any range characterized by the above combinations of endpoints is intended, including but not limited to about 45 mM to about 65 mM.

[0028] In some embodiments, the buffer prepared by the method described herein contains acetate in a total concentration of at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least 55 mM, at least 60 mM, or at least 65 mM. In some embodiments, the concentration of acetate is about 30 mM or less, about 35 mM or less, about 40 mM or less, about 45 mM or less, about 50 mM or less, about 55 mM or less, about 60 mM or less, about 65 mM or less, about 70 mM or less, about 75 mM or less, about 80 mM or less, about 85 mM or less, or about 90 mM or less. Any range characterized by the above combinations of endpoints is intended, including, but not limited to, about 10 mM to about 55 mM, about 20 mM to about 55 mM, about 20 mM to about 40 mM, about 30 mM to about 50 mM, or about 30 mM to about 75 mM. As a non-limiting example, a solution containing 10 mM calcium acetate will have 20 mM acetate anions and 10 mM calcium cations due to the divalent nature of calcium cations, while a solution containing 10 mM sodium acetate will have 10 mM sodium cations and 10 mM acetate anions.

[0029] In some embodiments, the total concentration of ions (cations and anions) in the buffer prepared by the method described herein is at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, or at least about 85 mM. In some embodiments, the total concentration of ions in the buffer is approximately 30 mM or less, approximately 35 mM or less, approximately 40 mM or less, approximately 45 mM or less, approximately 50 mM or less, approximately 55 mM or less, approximately 60 mM or less, approximately 65 mM or less, approximately 70 mM or less, approximately 75 mM or less, approximately 80 mM or less, approximately 85 mM or less, approximately 90 mM or less, approximately 95 mM or less, approximately 100 mM or less, approximately 110 mM or less, approximately 120 mM or less, approximately 130 mM or less, approximately 140 mM or less, approximately 150 mM or less, approximately 160 mM or less, approximately 170 mM or less, approximately 180 mM or less, approximately 190 mM or less, or approximately 200 mM or less. Any range characterized by the above combination of endpoints is intended, including, but not limited to, about 30 mM to about 60 mM, or about 30 mM to about 70 mM, or about 30 mM to about 80 mM, or about 40 mM to about 150 mM, or about 50 mM to about 150 mM. As a non-limiting example, a 10 mM solution of calcium acetate would have a total concentration of 30 mM ions (10 mM cations and 20 mM anions).

[0030] The buffer preparation methods disclosed herein include combining a disaccharide with a calcium salt and an organic acid. Examples of calcium salts, but not limited to, include calcium acetate, calcium carbonate, and calcium chloride. In some embodiments, the calcium salt is calcium acetate.

[0031] The buffer preparation method disclosed herein comprises combining a disaccharide with a calcium salt and an organic acid. Exemplary disaccharides include, but are not limited to, sucrose, trehalose, lactose, and maltose. In some embodiments, the disaccharide is sucrose.

[0032] The buffer preparation methods disclosed herein include combining a disaccharide with a calcium salt and an organic acid. Exemplary organic acids include, but are not limited to, acetic acid, citric acid, glutamic acid, histidine, lactic acid, succinic acid, and aspartic acid. In some embodiments, the organic acid is acetic acid, for example, glacial acetic acid.

[0033] In some embodiments, the buffer prepared by the method described herein contains a disaccharide (which may be sucrose according to one aspect of the invention) in a total concentration of about 0.1% to about 30% w / v, or about 0.5% to about 20% w / v, or about 1% to about 10% w / v. In some embodiments, the disaccharide is present in the buffer at a concentration of about 1% to about 12% w / v. In yet another embodiment, the disaccharide is present in the buffer at a concentration of about 2% to about 10%. In yet another embodiment, the disaccharide is present in the buffer at a concentration of about 3% to about 9%. In yet another embodiment, the disaccharide is present in the buffer at a concentration of about 4% to about 8%. In yet another embodiment, the disaccharide is present in the buffer at a concentration of about 5% to about 7%. In some embodiments, the disaccharide is present in the buffer at a concentration of about 6% w / v. In some embodiments, the disaccharide is present in the buffer at concentrations of at least 0.5, at least 1% w / v, at least 2% w / v, at least 3% w / v, at least 4% w / v, at least 5% w / v, or at least 6% w / v. In some embodiments, the disaccharide is present in the buffer at concentrations of 30% w / v or less, 25% w / v or less, 20% w / v or less, 15% w / v or less, 14% w / v or less, 13% w / v or less, 12% w / v or less, 11% w / v or less, 10% w / v or less, 9% w / v or less, 8% w / v or less, 7% w / v or less, or 6% w / v or less. In some embodiments, the disaccharide is present in the buffer at concentrations of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14%, or about 15% w / v. Any range characterized by the above combination of endpoints is intended. In some embodiments, the disaccharide is sucrose, and the above range and values ​​apply equally to sucrose.

[0034] In some embodiments, the buffer prepared by the method described herein contains 13 mM calcium, 55 mM acetate, and 6% (w / v) sucrose. In one embodiment, the mixture has a pH of 5.2.

[0035] In some embodiments, the pH of the buffer prepared by the method described herein is in the range of about 4 to about 6. In some embodiments, the pH of the buffer is in the range of about 4.5 to about 6 or about 4.8 to about 5.6. In some embodiments, the pH of the buffer is in the range of about 5.0 to about 5.4 or about 5.1 to about 5.3. In some embodiments, the pH of the buffer is 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6. In some embodiments, the pH of the buffer is 5.2.

[0036] Therapeutic protein "Therapeutic proteins" are protein-based drugs. This term may include pharmaceuticals that are genetically modified versions of naturally occurring proteins, such as human proteins. Therapeutic proteins can be used to replace abnormal or deficient proteins in certain diseases. They can also help reduce the effects of disease or chemotherapy by increasing the supply of beneficial proteins to the body. Genetically modified proteins may be very similar to the natural proteins they replace, or they may be enhanced by the addition of sugars or other molecules that extend the duration of protein activity.

[0037] In some embodiments, the therapeutic protein is a signaling protein, cytokine, enzyme or enzyme substitute, CD protein or its extracellular domain, growth factor, growth factor receptor or its extracellular domain, cell adhesion molecule or its extracellular domain, hormone or hormone analog, coagulation factor, coagulation-related protein, colony-stimulating factor, its receptor, or any of the above-mentioned biologically active fragments, analogs, variants, receptors, or receptor fragments.

[0038] In some embodiments, the therapeutic protein is added to the prepared buffer to obtain a concentration of therapeutic protein of at least about 10 mg / ml, at least about 15 mg / ml, at least about 20 mg / ml, at least about 25 mg / ml, at least about 30 mg / ml, at least about 35 mg / ml, at least about 40 mg / ml, at least about 45 mg / ml, at least about 50 mg / ml, at least about 55 mg / ml, at least about 60 mg / ml, at least about 65 mg / ml, at least about 70 mg / ml, at least about 75 mg / ml, at least about 80 mg / ml, at least about 81 mg / ml, at least about 82 mg / ml, at least about 83 mg / ml, at least about 84 mg / ml, at least about 85 mg / ml, at least about 86 mg / ml, at least about 87 mg / ml, at least about 88 mg / ml, at least about 89 mg / ml, or at least about 90 mg / ml. In some embodiments, the therapeutic protein is added to the prepared buffer at concentrations of approximately 90 mg / ml or less, approximately 91 mg / ml or less, approximately 92 mg / ml or less, approximately 93 mg / ml or less, approximately 94 mg / ml or less, approximately 95 mg / ml or less, approximately 96 mg / ml or less, approximately 97 mg / ml or less, approximately 98 mg / ml or less, approximately 99 mg / ml or less, approximately 100 mg / ml or less, approximately 105 mg / ml or less, approximately 110 mg / ml or less, and approximately 115 mg / ml or less. It is added to obtain a therapeutic protein concentration of 1 or less, approximately 120 mg / ml or less, approximately 125 mg / ml or less, approximately 130 mg / ml or less, approximately 135 mg / ml or less, approximately 140 mg / ml or less, approximately 145 mg / ml or less, approximately 150 mg / ml or less, approximately 155 mg / ml or less, approximately 160 mg / ml or less, approximately 165 mg / ml or less, approximately 170 mg / ml or less, approximately 175 mg / ml or less, or approximately 180 mg / ml or less. In some embodiments, the therapeutic protein is added to the prepared buffer to obtain a maximum concentration of therapeutic protein, for example, about 300 mg / ml, about 290 mg / ml, about 280 mg / ml, about 270 mg / ml, about 260 mg / ml, about 250 mg / ml, about 240 mg / ml, about 230 mg / ml, about 220 mg / ml, about 210 mg / ml, about 200 mg / ml, or about 190 mg / ml.Any range characterized by the above-mentioned combination of endpoints is intended, and includes, but is not limited to, approximately 50 mg / ml to approximately 150 mg / ml, approximately 60 mg / ml to approximately 120 mg / ml, approximately 65 mg / ml to approximately 115 mg / ml, approximately 70 mg / ml to approximately 110 mg / ml, approximately 75 mg / ml to approximately 105 mg / ml, approximately 80 mg / ml to approximately 100 mg / ml, or approximately 85 mg / ml to approximately 95 mg / ml. In some embodiments, the therapeutic protein is added to the prepared buffer to obtain a concentration of therapeutic protein of approximately 90 mg / ml.

[0039] In some embodiments, the therapeutic protein is an antigen-binding protein. An "antigen-binding protein" refers to a protein that specifically binds to a particular antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, peptide bodies, antibody fragments, antibody constructs, multispecific (e.g., bispecific or trispecific) antibodies or antibody constructs, BiTE® molecules, and fusion proteins. The term encompasses intact antibodies (including intact bispecific antibodies) containing at least two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and mutants. Antigen-binding proteins also include domain antibodies, such as nanobodies and scFvs.

[0040] In some embodiments, the antigen-binding protein is an antibody. As used herein, the term “antibody” refers to a protein having a conventional immunoglobulin format, comprising heavy and light chains, a variable region and a constant region. Antibodies have a variable region and a constant region. In the IgG format, the variable region is generally about 100 to 110 or more amino acids, contains three complementarity-determining regions (CDRs), and is primarily responsible for antigen recognition, differing substantially from other antibodies that bind to different antigens. The constant region allows the antibody to recruit cells and molecules of the immune system. The variable region consists of the N-terminal regions of each light and heavy chain, while the constant region consists of the C-terminal portions of the heavy and light chains, respectively (Janeway et al., “Structure of the Antibody Molecule and the Immunoglobulin Genes”, Immunobiology: The Immune System in Health and Disease, 4th ed. Elsevier Science Ltd. / Garland Publishing, (1999)).

[0041] The general structure and properties of antibody CDRs have been described in the art. Briefly, in an antibody scaffold, CDRs are embedded within a framework in the variable regions of the heavy and light chains, where they constitute the region responsible for most antigen binding and recognition. The variable region contains at least three heavy or light chain CDRs (see also Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within a framework region (named framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, op. cit.).

[0042] Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. Embodiments of the present invention include all such classes or isotypes of antibodies. The light chain constant region may be, for example, a kappa or lambda type light chain constant region, e.g., a human kappa or lambda type light chain constant region. The heavy chain constant region may be, for example, an alpha, delta, epsilon, gamma, or mu type heavy chain constant region, e.g., a human alpha, delta, epsilon, gamma, or mu type heavy chain constant region. Therefore, in exemplary embodiments, the antibody is an isotype IgA, IgD, IgE, IgG, or IgM antibody, comprising one of IgG1, IgG2, IgG3, or IgG4. An IgG antibody, particularly an IgG2 antibody, represents one preferred embodiment of the present disclosure.

[0043] Antibodies can be monoclonal or polyclonal antibodies. In some embodiments, antibodies contain sequences substantially similar to naturally occurring antibodies produced by mammals, such as mice, rabbits, goats, horses, chickens, hamsters, and humans. In this respect, antibodies can be considered mammalian antibodies, such as mouse antibodies, rabbit antibodies, goat antibodies, horse antibodies, chicken antibodies, hamster antibodies, and human antibodies. In some embodiments, monoclonal antibodies are human antibodies. In some embodiments, monoclonal antibodies are chimeric antibodies or humanized antibodies. The term “chimeric antibody” is used herein to refer to an antibody containing a constant domain from one species and a variable domain from a second species, or more generally, a sequence of amino acids from at least two species. The term “humanized,” when used in reference to antibodies, refers to an antibody having at least a CDR region of a non-human resource that has been manipulated to have a structure and immunological function more similar to a true human antibody than to the original resource antibody. For example, humanization may involve transplanting a CDR from a non-human antibody, such as a mouse antibody, into a human antibody. Humanization may also involve the selection of amino acid substitutions to make non-human sequences appear more human.

[0044] Advantageously, the methods disclosed herein for preparing buffers for therapeutic protein formulations (e.g., antibody formulations) are not limited to the antigen specificity of the antibody. Therefore, an antibody (or antibody fragment or antibody protein product) may have any binding specificity to substantially any antigen. In exemplary embodiments, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor, or any ligand thereof.

[0045] In some embodiments, the antibody is an anti-sclerostin antibody. “Anti-sclerostin antibody” or “sclerostin-binding antibody” refers to an antibody that binds to sclerostin or a portion thereof, as defined in SEQ ID NO: 1. Recombinant human sclerostin / SOST is commercially available, for example, from R&D Systems (Minneapolis, Minn., USA; 2006 catalog number 1406-ST-025). U.S. Patent Nos. 6,395,511 and 6,803,453, and U.S. Patent Publication Nos. 2004 / 0009535 and 2005 / 0106683 (incorporated herein by reference) refer to anti-sclerostin antibodies in general. Examples of anti-sclerostin antibodies suitable for use relating to this disclosure are also described in U.S. Patent Publication No. 2007 / 0110747 and U.S. Patent Publication No. 2007 / 0072797, which are incorporated herein by reference. Suitable anti-sclerostin antibodies, including an antibody designated Ab-5, are further described in U.S. Patent No. 8,715,663 and U.S. Patent No. 7,592,429 (incorporated herein by reference). Additional information regarding materials and methods for producing sclerostin antibodies can be found in U.S. Patent Publication No. 20040158045 (incorporated herein by reference). In some embodiments, the anti-sclerostin antibody is romosozumab, brosozumab, or setrusumab.

[0046] As used herein, "specifically binds" means that an antibody preferentially binds to an antigen compared to other proteins. In some embodiments, "specifically binds" means that an antibody has a higher affinity for an antigen than other proteins.

[0047] In some or any embodiment, the antibody is 1 × 10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11M or less, or 1 × 10 -12 The antibody binds to sclerostin of SEQ ID NO: 1, or its naturally occurring variants, with an affinity (Kd) of M or less. Affinity is determined using various techniques, one example being the affinity ELISA assay. In various embodiments, affinity is determined by the BIAcore assay. In various embodiments, affinity is determined by kinetic methods. In various embodiments, affinity is determined by equilibrium / solution methods. U.S. Patent Application Publication No. 2007 / 0110747 (this disclosure is incorporated herein by reference) includes an additional description of affinity assays suitable for determining the affinity (Kd) of an antibody to sclerostin.

[0048] In various embodiments, the anti-sclerostin antibody comprises at least one CDR sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-H3 having the sequence shown in SEQ ID NO: 2, CDR-H2 having the sequence shown in SEQ ID NO: 3, CDR-H3 having the sequence shown in SEQ ID NO: 4, CDR-L1 having the sequence shown in SEQ ID NO: 5, CDR-L2 having the sequence shown in SEQ ID NO: 6, and CDR-L3 having the sequence shown in SEQ ID NO: 7. The anti-sclerostin antibody may, in various embodiments, contain three, four, five, or six CDRs.

[0049] In a preferred embodiment, the anti-sclerostin antibody comprises the following set of six CDRs: CDR-H1 of SEQ ID NO: 2, CDR-H2 of SEQ ID NO: 3, CDR-H3 of SEQ ID NO: 4, CDR-L1 of SEQ ID NO: 5, CDR-L2 of SEQ ID NO: 6, and CDR-L3 of SEQ ID NO: 7.

[0050] In some or any embodiment, the anti-sclerostin antibody includes a light chain variable region containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 8 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and a heavy chain variable region containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 9 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity). In various embodiments, the sequence difference compared to SEQ ID NO: 8 or 9 lies outside the CDR region in the corresponding sequence. In some or any embodiment, the anti-sclerostin antibody comprises a light chain variable region containing the amino acid sequence described in SEQ ID NO: 8 and a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 9.

[0051] In some or any embodiment, the anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 10 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and all or part of a light chain (e.g., two light chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 11 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0052] In some or any embodiment, the anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 12 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) and all or part of a light chain (e.g., two light chains) containing an amino acid sequence having at least 75% identity to the amino acid sequence described in SEQ ID NO: 13 (e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0053] Other examples of anti-sclerostin antibodies include, but are not limited to, those disclosed in the International Patent Application Publications, International Publication Nos. 2008 / 092894, International Publication Nos. 2008 / 115732, International Publication Nos. 2009 / 056634, International Publication Nos. 2009 / 047356, International Publication Nos. 2010 / 100200, International Publication Nos. 2010 / 100179, International Publication Nos. 2010 / 115932, and International Publication Nos. 2010 / 130830 (each of which is incorporated herein by reference as a whole).

[0054] It is understood by those skilled in the art that several proteins, such as antibodies, can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell line and culture conditions used to express the protein. Such modifications may include variations of glycosylation, methionine oxidation, diketopiperidine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a basic residue at the carboxyl terminus (e.g., lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995).

[0055] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86

[1983] , incorporated by reference as a whole), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0056] The therapeutic protein described above can be added to a buffer prepared by the method described herein to produce a pharmaceutical composition. Such a pharmaceutical composition can be loaded into a container (e.g., a vial or syringe) together with packaging materials and / or materials that provide instructions for the use of such a pharmaceutical composition. Generally, such instructions include specific expressions describing the concentration of the therapeutic protein and the relative amounts of excipient components or diluents (e.g., water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition in certain embodiments. [Examples]

[0057] This example describes a typical method for preparing a buffer solution for a therapeutic protein formulation. Calcium acetate and glacial acetic acid were combined with water for injection (WFI) to produce a first mixture, which was mixed at 145±10 rpm for 16-20 minutes at 27°C±5°C. Next, sucrose was added to produce a second mixture, which was then mixed at 145±10 rpm for a further 16-20 minutes at 27°C±5°C. Then, a bolus amount of sodium hydroxide (NaOH) was added to produce a third mixture, which was then mixed at 145±10 rpm for 2-4 minutes at 27°C±5°C. Finally, water for injection was added and mixed at 145±10 rpm for 2-4 minutes at 27°C±5°C to produce the final buffer solution. The buffer solution was then cooled to 5°C±3°C.

[0058] The buffer solution in this example is prepared by blending 2.012 g / kg of calcium acetate, 1.704 g / kg of glacial acetic acid, 58.708 g / kg of sucrose, and 1.65 mL / kg of sodium hydroxide (all indicated amounts refer to 1 kg of buffer solution). The final buffer solution in this example contains 13 mM calcium, 55 mM acetate, and 6% (w / v) sucrose at pH 5.2. The temperature during the addition and mixing of the components was kept constant at 27°C ± 5°C. Similarly, the mixing rate was kept constant at 145 ± 10 rpm throughout the process.

[0059] The final buffer prepared in this embodiment is suitable for antibodies, particularly IgG isotype antibodies, such as IgG2 antibodies or specifically romosozumab.

[0060] The method described herein offers several advantages compared to standard techniques for preparing buffers for therapeutic protein formulations, which are discussed below.

[0061] The current state of affairs involves preparing buffers at low or room temperature. Preparing buffers at higher temperatures and then cooling them to an acceptable temperature before adding therapeutic proteins is not a standard technique. The buffer preparation temperatures disclosed herein ensure the dissolution of solid excipients during the mixing process. Preparing buffers at higher temperatures requires a special understanding of how different components dissolve in liquid. Calcium acetate and sucrose are the main components affected by buffer preparation at higher temperatures. The solubility of sucrose increases at higher temperatures, while the solubility of calcium acetate decreases. However, sucrose is the largest component per unit volume in the buffer, and it constitutes the largest proportion of its solubility limit; therefore, sucrose becomes the most important excipient in the buffer, aiming to reduce mixing time while ensuring dissolution. An optimal temperature for the solubility of sucrose has now been obtained, while still ensuring the solubility of calcium acetate. In other words, preparing buffer solutions at the temperatures indicated, instead of the generally used lower temperatures, supports the solubility of sucrose in the aqueous buffer solution (i.e., the excipient with the highest final concentration) without affecting the solubility of calcium acetate (which decreases with increasing temperature) in order to properly formulate the buffer solution, thereby reducing the mixing time compared to conventional buffer preparation processes. The average total mixing time was reduced by only 30%, from 60 minutes to 42 minutes.

[0062] Due to the increased temperature, it was also possible to specify a fixed mixing rate for the addition and mixing of all components. It was not necessary to adjust the mixing rate after adding sucrose (e.g., by increasing it) or again after adding NaOH (e.g., by decreasing it).

[0063] Mixing one component in a single step is also a standard technique, but it corresponds to a less efficient process compared to the disclosure. The buffer preparation process described herein combines calcium acetate and glacial acetic acid in a single step, thus reducing the number of excipient addition steps.

[0064] In conventional standard processes, the mixing time after adding individual buffer components varied and depended on the actual dissolution of the components. The newly disclosed process is equilibrated and optimized so as to eliminate the need for visual confirmation (e.g., to control the completion of excipient dissolution) and also allows for the setting of a fixed mixing time.

[0065] Furthermore, adding a bolus of NaOH accelerates the buffer preparation process because it eliminates the need to titrate the NaOH in a multi-step process, thus reducing the mixing time required after NaOH addition (from approximately 20 minutes to approximately 2-4 minutes) while adjusting the buffer pH to the desired value (5.2 in this case) in a single step.

[0066] The method disclosed herein minimizes sample collection and analysis in order to efficiently utilize resources.

Claims

1. (a) Combining calcium salts, organic acids, and disaccharides at a temperature of 22°C or higher to produce a mixture, (b) Add sodium hydroxide to the mixture of (a) by bolus addition. A method for preparing a buffer for a therapeutic protein formulation, including [a specific protein].

2. The method according to claim 1, wherein the method is carried out at a temperature in the range of approximately 22°C to approximately 32°C, preferably at a temperature of approximately 27°C.

3. Step (a) is, (i) Mix the calcium salt and the organic acid with water for about 16 to 20 minutes to produce the mixture in (i), and (ii) Add the disaccharide to the mixture of (i) and mix for a further 16 to 20 minutes. The method according to claim 1 or 2, including the method described in claim 1 or 2.

4. The method according to any one of claims 1 to 3, further comprising step (b) mixing the sodium hydroxide for about 2 to 4 minutes after the addition of the bolus.

5. The method according to claim 3 or 4, wherein the mixing rate for step (a) and / or step (b) is in the range of about 135 rpm to about 155 rpm, preferably the mixing rate for step (a) and / or step (b) is about 145 rpm.

6. The method according to any one of claims 1 to 5, wherein water is added following step (b).

7. The method according to claim 6, further comprising adding the water and then mixing for about 2 to 4 minutes.

8. The method according to claim 7, wherein the total mixing time for step (a), step (b), and the mixing of the added water is less than about 60 minutes, preferably in the range of about 36 minutes to about 48 minutes.

9. The method according to any one of claims 1 to 8, further comprising cooling the buffer solution to a temperature in the range of about 2°C to about 8°C, preferably to a temperature of about 5°C.

10. The method according to any one of claims 1 to 9, wherein the disaccharide is added to the mixture in an amount at least about 5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, or at least about 30 times more than the amount of the calcium salt.

11. The method according to any one of claims 1 to 10, wherein the disaccharide is selected from the group consisting of sucrose, trehalose, lactose, and maltose, and preferably the disaccharide is sucrose.

12. The method according to any one of claims 1 to 11, wherein the calcium salt is selected from the group consisting of calcium acetate, calcium carbonate, and calcium chloride, and preferably the calcium salt is calcium acetate.

13. The method according to any one of claims 1 to 12, wherein the organic acid is selected from the group consisting of acetic acid, citric acid, glutamic acid, histidine, lactic acid, succinic acid, and aspartic acid, and preferably the organic acid is acetic acid.

14. The method according to any one of claims 1 to 13, wherein the pH of the buffer solution is in the range of about 5.0 to about 5.

4.

15. The method according to any one of claims 1 to 14, wherein the buffer solution comprises 13 mM calcium, 55 mM acetate, and 6% (w / v) sucrose at pH 5.2.

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