阅读说明：本技术 制备抗体药物配制品的方法 (Method for producing antibody pharmaceutical formulations ) 是由 A·沙尔玛 B·德朗萨特 于 2019-08-09 设计创作，主要内容包括：本披露提供了用于制备粘度为10cP或更低的抗体药物配制品的材料和方法,所述方法包括在大于30℃的温度下将包含抗原结合蛋白的组合物与包含钙的渗滤缓冲液进行交换。(The present disclosure provides materials and methods for preparing an antibody drug formulation having a viscosity of 10cP or less, the method comprising exchanging a composition comprising an antigen binding protein with a diafiltration buffer comprising calcium at a temperature greater than 30 ℃.)

1. A method of preparing an antibody pharmaceutical formulation having a viscosity of 10cP or less, said method comprising buffer exchanging a composition comprising said antibody with a diafiltration buffer comprising a calcium salt at a temperature of greater than 30 ℃.

2. The method of claim 1, wherein the exchange step is performed by ultrafiltration/diafiltration.

3. The method of claim 1, wherein the composition comprises the antibody at a concentration of at least 90 mg/mL.

4. The method of claim 1, wherein the composition comprises the antibody at a concentration of at least 120 mg/mL.

5. The method of any one of claims 1-4, wherein the exchanging step is performed at a temperature between 30 ℃ and 40 ℃.

6. The method of any one of claims 1-5, wherein the exchanging step is performed at a temperature greater than 35 ℃.

7. The method of any one of claims 1-6, wherein the exchanging step is performed at 37 ℃.

8. The method of claim 1, wherein the calcium salt is calcium acetate.

9. The process of claim wherein said diafiltration buffer comprises at least 20mM calcium acetate.

10. The method of claim 9 wherein the diafiltration buffer comprises about 23mM calcium acetate.

11. The method of any one of claims 1-10, wherein the diafiltration buffer further comprises a polyol.

12. The method of claim 11, wherein the polyol is sucrose.

13. The method of claim 12, wherein diafiltration buffer comprises sucrose present at a concentration of about 1% to about 15%.

14. The method of claim 12, wherein the diafiltration buffer comprises sucrose at a concentration of about 7%.

15. The method of any one of claims 1-14, further comprising the step of filtering the pharmaceutical formulation.

16. The method of any one of claims 1-14, further comprising the step of aliquoting the pharmaceutical formulation into pharmaceutical product forms.

17. The method of any one of claims 1-16, wherein the antibody is lomustizumab, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, viltine-benitumumab, canamumab, cetuximab, certolizumab, daclizumab, denozumab, eculizumab, efavirenzumab, gemtuzumab ozogamicin, golimumab, ibritumomab, infliximab, ipilimumab, moruzumab-CD 3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumumab, tositumomab, trastuzumab, or ustzumab vituzumab.

18. The method of any one of claims 1-17, wherein the pharmaceutical formulation after the exchanging step comprises about 50mM acetate and about 12mM calcium.

Background

Highly concentrated liquid antibody formulations can be used to deliver a dose of therapeutic agent in a smaller volume of carrier. However, highly concentrated protein formulations suffer from several problems, including instability due to particle formation and increased viscosity due to numerous intermolecular interactions by the macromolecular nature of antibodies. High viscosity formulations are also difficult to manufacture, difficult to inhale into a syringe, and difficult to inject. The force of use in handling viscous formulations can lead to excessive foaming, which can lead to denaturation and inactivation of the active biological agent.

Disclosure of Invention

In one aspect, described herein is a method of preparing an antibody pharmaceutical formulation having a viscosity of 10cP or less, the method comprising buffer exchanging a composition comprising the antibody with a diafiltration buffer comprising a calcium salt at a temperature of greater than 30 ℃. In some embodiments, the antibody is lomustizumab (romosozumab), abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, viltine-benitumumab (brentuximab vedotin), canazumab, cetuximab, certolizumab, daclizumab, denozumab, eculizumab, efavirenzumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, moruzumab-CD 3, natalizumab, nivolumab, ofatumumab, omalizumab, palivizumab, rituximab, tositumumab, tositumomab, trastuzumab, or ultuzumab, vituzumab (vedolizumab).

In some embodiments, the exchange step is performed by ultrafiltration/diafiltration. In some embodiments, the composition comprises a concentration of at least 70mg/mL, at least 71mg/mL, at least 72mg/mL, at least 73mg/mL, at least 74mg/mL, at least 75mg/mL, at least 76mg/mL, at least 77mg/mL, at least 78mg/mL, at least 79mg/mL, at least 80mg/mL, at least 81mg/mL, at least 82mg/mL, at least 83mg/mL, at least 84mg/mL, at least 85mg/mL, at least 86mg/mL, at least 87mg/mL, at least 88mg/mL, at least 89mg/mL, at least 90mg/mL, at least 91mg/mL, at least 92mg/mL, at least 93mg/mL, at least 94mg/mL, at least 95mg/mL, at least 96mg/mL, or a combination thereof, At least 97mg/mL, at least 98mg/mL, at least 99mg/mL, at least 100mg/mL, at least 101mg/mL, at least 102mg/mL, at least 103mg/mL, at least 104mg/mL, at least 105mg/mL, at least 106mg/mL, at least 107mg/mL, at least 108mg/mL, at least 109mg/mL, at least 110mg/mL, at least 111mg/mL, at least 112mg/mL, at least 113mg/mL, at least 114mg/mL, at least 115mg/mL, at least 116mg/mL, at least 117mg/mL, at least 118mg/mL, at least 119mg/mL, or at least 120 mg/mL. In some embodiments, the composition comprises the antibody at a concentration ranging from 70mg/mL to 210 mg/mL. In some embodiments, the composition comprises the antibody at a concentration of less than 210 mg/mL. In some embodiments, the exchange step is performed at a temperature between 30 ℃ and 40 ℃ (e.g., 37 ℃) or greater than 35 ℃.

In some embodiments, the calcium salt is calcium acetate.

In some embodiments, the diafiltration buffer comprises at least 20mM (e.g., about 23mM) calcium acetate.

In some embodiments, the diafiltration buffer further comprises a polyol (e.g., sucrose), optionally at a concentration of about 1% to about 15%. In some embodiments, the diafiltration buffer comprises sucrose at a concentration of about 7%.

In some embodiments, the antibody drug formulation after the exchange step comprises about 50mM acetate and about 12mM calcium.

The methods of the present disclosure optionally further comprise the step of filtering the antibody pharmaceutical formulation and/or aliquoting the antibody pharmaceutical formulation into a pharmaceutical product form.

Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to," and permitting the presence of one or more features or components). It should be understood that while various embodiments in the specification are presented using the language "comprising," related embodiments may also be described in various instances using the language "consisting of … …" or "consisting essentially of … …. Unless the context indicates otherwise, it is noted that the term "an" refers to one or more, e.g., "an immunoglobulin molecule" is understood to represent one or more immunoglobulin molecules. Likewise, the terms "a" and "an", "one or more" and "at least one" may be used interchangeably. Further, as used herein, "and/or" should be taken as a specific disclosure of each of the two specified features or components, with or without the other specified feature or component. Thus, the term "and/or" as used in phrases such as "a and/or B" herein is intended to include "a and B," "a or B," "a" (alone) and "B" (alone). Also, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following: 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).

It will also be understood that when a range of values is described, the feature described may be a single value found within the range. For example, "a pH of from about pH 4 to about pH 6" can be, but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, and the like, as well as any value in between these values. In addition, "pH from about pH 4 to about pH 6" should not be construed to mean that the pH of the formulation under consideration changes by 2 pH units over the range of pH 4 to pH 6 during storage, but rather that the pH of the solution may be chosen to be within this range and that the pH remains buffered at about this pH.

In any range described herein, the endpoints of that range are included in the range. However, the description also contemplates the same ranges excluding the lower and/or upper endpoints. Additional features and variations of the present invention will be apparent to those skilled in the art from the present application as a whole, including the figures and detailed description, and all such features are intended as aspects of the invention. Likewise, features of the invention described herein may be recombined into additional embodiments that are also intended as aspects of the invention, whether or not a combination of such features is specifically mentioned above as an aspect or embodiment of the invention. Moreover, only such limitations as described herein as essential to the invention should be viewed as such limitations; variations of the invention that are not described herein as critical are intended as aspects of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, the circumcise Dictionary of Biomedicine and Molecular Biology [ Concise Dictionary of Biomedicine and Molecular Biology ], Juo, Pei-Show, 2 nd edition, 2002, CRC Press [ CRC Press ]; the Dictionary of Cell and Molecular Biology, 3 rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology Dictionary, revised, 2000, Oxford University Press provides the skilled artisan with a comprehensive Dictionary Of many Of the terms used in this disclosure.

Units, prefixes, and symbols are all expressed in their international system of units (SI) accepted form. Numerical ranges include the numbers defining the range. Unless otherwise indicated, amino acid sequences are written from left to right in the amino to carboxyl direction. The headings provided herein are not limitations of the various aspects or aspects of the disclosure, which can be had by reference to the specification as a whole.

All references cited herein are hereby incorporated by reference in their entirety.

Drawings

Figure 1 is a graph showing the effect of various concentrations of calcium acetate on the viscosity of antibody compositions. Viscosity (cp. y axis) was plotted against antibody concentration (mg/mL, X axis).

Figure 2 provides ultrafiltration parameters in the absence of calcium acetate.

Fig. 3 provides ultrafiltration parameters in the presence of calcium acetate.

Fig. 4 is a graph showing the effect of temperature on the viscosity of an antibody composition. Feed pressure (psi, Y-axis) is plotted against retentate concentration (mg/mL, X-axis).

Detailed Description

The present disclosure is based on the following findings: buffer exchange of the antibody-containing composition with a diafiltration buffer containing a calcium salt at a temperature above 30 ℃ may result in an antibody drug formulation with a viscosity of 10cP or less.

The ability to formulate higher concentrations of antibody provides improved patient dosing regimens, smaller injection volumes, a wider range of device options, and improvements in supply chain considerations. Many antibodies are very viscous at higher concentrations, potentially limiting processing and drug delivery options. In particular, high viscosity antibody compositions can be problematic for the final drug substance ultrafiltration/diafiltration process step due to long processing times, high pressures, large membrane areas required for processing, and potentially poor product recovery. As described herein, the combination of elevated temperature and a calcium salt (e.g., calcium acetate) is surprisingly effective in reducing the viscosity of the composition given that the solubility of the calcium salt decreases with increasing temperature.

As used herein, the term "ultrafiltration" or "UF" refers to any technique that passes a solution or suspension through a membrane (e.g., a semi-permeable membrane) to separate a product (e.g., a protein) from other materials in the solution or suspension. For example, ultrafiltration membranes retain molecules larger than the membrane pores, while smaller molecules (such as salts, solvents, and water) pass freely through the membrane. The solution retained by the membrane is referred to as "concentrate" or "retentate", while the solution passing through the membrane is referred to as "filtrate" or "permeate". Ultrafiltration can be used to increase the concentration of macromolecules in a solution or suspension. In one aspect, ultrafiltration is used to increase the concentration of protein in a solution.

Membrane filters of the present disclosure, such as ultrafiltration membranes, can have a pore size of 0.001 to 0.1 microns. In some embodiments, the membrane filter has a pore size of 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085, 0.090, 0.095, or 0.100 microns. In some embodiments, the membrane filters of the present disclosure have a molecular cut-off of 15 kilodaltons (kDa) to 50kDa or higher. For example, in some embodiments, the membrane filter has a molecular cut-off of 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, or 50kDa, or any intermediate value. In some embodiments, the molecular weight cut-off of the membrane is 30 kDa.

As used herein, the term "diafiltration" or "DF" is used to refer to the removal, replacement or reduction of the concentration of salts or solvents from a solution or mixture containing proteins, peptides, nucleic acids or other biomolecules, for example, using ultrafiltration membranes. Diafiltration may or may not result in an increase in the concentration of retained components (including proteins). For example, in continuous diafiltration, solvent is continuously added to the retentate at the same rate as the filtrate is produced. In this case, the volume of the retentate and the concentration of the retained components do not change during the treatment. In discontinuous or sequential dilution diafiltration, on the other hand, the ultrafiltration step is followed by addition of solvent to the retentate side; if the volume of solvent added to the retentate side is not equal to or greater than the volume of filtrate produced, the retained components will have a high concentration. Diafiltration may be used to alter the pH, ionic strength, salt composition, buffer composition or other properties of the macromolecular solution or suspension.

As used herein, the term "ultrafiltration/diafiltration" or "UF/DF" refers to any method, technique, or combination of techniques that accomplish ultrafiltration and/or diafiltration sequentially or simultaneously.

In some embodiments, the viscosity of the antibody-containing composition is measured prior to the buffer exchange step, and the viscosity of the resulting formulation is measured after buffer exchanging the starting composition with a diafiltration buffer comprising a calcium salt at a temperature greater than 30 ℃. Methods of measuring viscosity are well known in the art and include, for example, using a capillary viscometer or a cone and plate rheometer. Any method may be used provided the same method is used to compare the starting composition and the resulting formulation.

As used herein, the term "viscosity" refers to "absolute viscosity". Absolute viscosity (sometimes referred to as dynamic or simple viscosity) is the product of kinematic viscosity and fluid density: absolute viscosity ═ kinematic viscosity x density. Kinematic viscosity of size L²Where L is the length and T is the time. Typically, kinematic viscosity is expressed in centistokes (cSt). SI unit of kinematic viscosity is mm²(ii)/s, which is 1 cSt. Absolute viscosity is expressed in units of centipoise (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s), where 1cP is 1 mPa-s.

Viscosity measurements can be made at storage or application temperatures (e.g., 2 ℃ to 8 ℃ or 25 ℃ (room temperature)). In some embodiments, the resulting pharmaceutical composition has an absolute viscosity of 15cP or less, 14cP or less, 13cP or less, 12cP or less, 11cP or less, 10cP or less, 9cP or less, 8cP or less, 7cP or less, 6cP or less, 5cP or less, or 4cP or less at the storage and/or application temperature.

"diafiltration buffer" is a buffer that does not contain antibodies per se but is used to prepare formulations containing antibodies. The diafiltration buffer contains a calcium salt. Exemplary calcium salts include, but are not limited to, calcium acetate, calcium carbonate, calcium citrate, calcium gluconate, calcium lactate, calcium glutamate, calcium succinate, and calcium chloride. In some embodiments, the calcium salt is present in the diafiltration buffer at a concentration ranging from 5mM to 150 mM. In some embodiments, the calcium salt is present in the diafiltration buffer at a concentration ranging from 10mM to 30 mM. In some embodiments, the calcium salt is present in the diafiltration buffer at a concentration of at least 10mM, at least 11mM, at least 12mM, at least 13mM, at least 14mM, at least 15mM, at least 16mM, at least 17mM, at least 18mM, at least 19mM, at least 20mM, at least 21mM, at least 22mM, at least 23mM, at least 24mM, at least 25mM, at least 26mM, at least 27mM, at least 28mM, at least 29mM, or at least 30 mM. In certain embodiments, the concentration of the calcium salt in the diafiltration buffer is not greater than about 20mM, not greater than about 21mM, not greater than about 22mM, not greater than about 23mM, not greater than about 24mM, not greater than about 25mM, not greater than about 26mM, not greater than about 27mM, not greater than about 28mM, not greater than about 29mM, or not greater than about 30 mM. Any range of combinations having the aforementioned endpoints is contemplated, including, but not limited to, from about 0.5mM to about 30mM, from about 20mM to about 30mM, or from about 20mM to about 25 mM. In some embodiments, the calcium salt is present in the diafiltration buffer at a concentration that reduces the viscosity of the antibody composition produced by the buffer exchange step disclosed herein (compared to the antibody composition prior to buffer exchange with the diafiltration buffer comprising the calcium salt at a temperature greater than 30 ℃) by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or higher, or to a viscosity of 10cP or lower, 9cP or lower, 8cP or lower, 7cP or lower, 6cP or lower, or 5cP or lower.

In all ranges described herein, the concentrations of cations, anions or salts described herein are in relation to the diafiltration buffer. In any range described herein, the endpoints of that range are included in the range. However, the description also contemplates the same ranges excluding the lower and/or upper endpoints.

In some embodiments, the diafiltration buffer described herein comprises a buffer (e.g., an acetate buffer) at a concentration of at least about 5mM, at least about 6mM, at least about 7mM, at least about 8mM, at least about 9mM, at least about 10mM, or at least about 15mM in addition to the calcium salt. In some embodiments, the concentration is no greater than about 10mM, no greater than about 15mM, no greater than about 20mM, no greater than about 25mM, no greater than about 30mM, no greater than about 35mM, no greater than about 40mM, no greater than about 45mM, or no greater than about 50 mM. Any range of combinations having the aforementioned endpoints is contemplated, including, but not limited to, from about 5mM to about 15mM, or from about 5mM to about 10mM, or from about 20mM to about 30mM, or from about 20mM to about 25 mM. The buffer is preferably added to a concentration that maintains the pH at about 5-6 or 5-5.5 or 4.5-5.5. In some embodiments, when the calcium salt in the formulation is calcium acetate, the total concentration of acetate is from about 10mM to about 60mM, or from about 20mM to about 40 mM.

In some aspects, the diafiltration buffer comprises a total concentration of acetate salt of at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, or at least about 50 mM. In some embodiments, the concentration of acetate is no greater than about 30mM, no greater than about 35mM, no greater than about 40mM, no greater than about 45mM, no greater than about 50mM, no greater than about 55mM, no greater than about 60mM, no greater than about 65mM, no greater than about 70mM, no greater than about 75mM, no greater than about 80mM, no greater than about 85mM, or no greater than about 90 mM. Any range having a combination of the foregoing endpoints is contemplated, including, but not limited to: about 10mM to about 50mM, about 20mM to about 40mM, about 30mM to about 50mM, or about 30mM to about 75 mM. By way of non-limiting example, a solution containing 10mM calcium acetate will have 20mM acetate anion and 10mM calcium cation (because calcium cation has divalent properties), while a solution containing 10mM sodium acetate will have 10mM sodium cation and 10mM acetate anion.

In some embodiments, the diafiltration buffer comprises a glutamate buffer or succinate buffer at a concentration of at least about 5mM, at least about 6mM, at least about 7mM, at least about 8mM, at least about 9mM, at least about 10mM, or at least about 15 mM. In some embodiments, the concentration is no greater than about 10mM, no greater than about 15mM, no greater than about 20mM, no greater than about 25mM, no greater than about 30mM, no greater than about 35mM, no greater than about 40mM, no greater than about 45mM, or no greater than about 50 mM. Any range of combinations having the aforementioned endpoints is contemplated, including, but not limited to, from about 5mM to about 15mM, or from about 5mM to about 10mM, or from about 20mM to about 30mM, or from about 20mM to about 25 mM. The buffer is preferably added to a concentration that maintains the pH at about 5-6 or 5-5.5 or 4.5-5.5.

In some embodiments, the total concentration of ions (cations and anions) in the diafiltration buffer is at least about 10mM, at least about 15mM, at least about 20mM, at least about 25mM, at least about 30mM, at least about 35mM, at least about 40mM, at least about 45mM, at least about 50mM, at least about 55mM, at least about 60mM, at least about 65mM, at least about 70mM, at least about 75mM, at least about 80mM, or at least about 85 mM. In some embodiments, the total concentration of ions is no greater than about 30mM, no greater than about 35mM, no greater than about 40mM, no greater than about 45mM, no greater than about 50mM, no greater than about 55mM, no greater than about 60mM, no greater than about 65mM, no greater than about 70mM, no greater than about 75mM, no greater than about 80mM, no greater than about 85mM, no greater than about 90mM, no greater than about 95mM, no greater than about 100mM, no greater than about 110mM, no greater than about 120mM, no greater than about 130mM, no greater than about 140mM, no greater than about 150mM, no greater than about 160mM, no greater than about 170mM, no greater than about 180mM, no greater than about 190mM, or no greater than about 200 mM. Any range having a combination of the foregoing endpoints is contemplated, including, but not limited to: about 30mM to about 60mM, or about 30mM to about 70mM, or about 30mM to about 80mM, or about 40mM to about 150mM, or about 50mM to about 150 mM. By way of non-limiting example, a solution of 10mM calcium acetate will have a total concentration of 30mM ions (10mM cation and 20mM anion).

In various embodiments, the antibody composition is buffer exchanged with a diafiltration buffer at a temperature greater than 30 ℃. In some embodiments, the buffer exchange is performed at a temperature between 30 ℃ and 40 ℃. In some embodiments, the buffer exchange is performed at a temperature greater than 35 ℃. In some embodiments, buffer exchange at 30 ℃,31 ℃,32 ℃, 33 ℃, 34 ℃,35 ℃, 36 ℃, 37 ℃, 38 ℃,39 ℃ or 40 ℃ for. In some embodiments, the buffer exchange is performed at 37 ℃.

The diafiltration buffer described herein optionally comprises at least one polyol. Polyols encompass a class of excipients that includes sugars (e.g., mannitol, sucrose, or sorbitol) and other polyhydric alcohols (e.g., glycerol and propylene glycol). The polymer polyethylene glycol (PEG) is included in this category. Polyols are commonly used as stabilizing excipients and/or isotonics in both liquid and lyophilized parenteral protein formulations. Polyols can protect proteins from physical and chemical degradation pathways.

Exemplary polyols include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, polyols (including, for example, polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, sulfobutyl cyclodextrin (captisol), N-methylpyrrolidine, cellulose, and hyaluronic acid), and sodium chloride (Carpenter et al, develop. biol. standard [ development of biological standardization ]74:225, (1991)).

Additional polyols include, but are not limited to, propylene glycol, glycerol (glycerol), threose, threitol, erythrose, erythritol, ribose, arabinose, arabitol, lyxose, maltitol, sorbitol, sorbose, glucose, mannose, mannitol, levulose, dextrose, maltose, trehalose, fructose, xylitol, inositol, galactose, xylose, fructose, sucrose, 1,2, 6-hexanetriol, and the like. Higher sugars include dextran, propylene glycol or polyethylene glycol. Reducing sugars, such as fructose, maltose or galactose, are more easily oxidized than non-reducing sugars. Further examples of sugar alcohols are glucitol, maltitol, lactitol or isomaltulose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Monoglycosides include compounds obtained by reducing disaccharides (such as lactose, maltose, lactulose, and maltulose).

In some embodiments, the at least one polyol is selected from the group consisting of: monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched glucans, and linear non-branched glucans, or combinations thereof. In some embodiments, the at least one polyol is a disaccharide selected from the group consisting of: sucrose, trehalose, mannitol and sorbitol, or a combination thereof.

In some embodiments, the diafiltration buffer comprises at least one polyol (e.g., sugar) at a concentration of about 0% to about 40% w/v, or about 0% to about 20% w/v, or about 1% to about 15% w/v. In some embodiments, the diafiltration buffer comprises at least one polyol (e.g., sugar) at a concentration of at least 0.5, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, or at least 40% w/v. In some embodiments, the diafiltration buffer comprises at least one polyol (e.g., sugar) at a concentration 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% to about 15% w/v. In some embodiments, the diafiltration buffer comprises at least one polyol (e.g., a sugar) at a concentration of about 1% to about 15% w/v. In yet another embodiment, the diafiltration buffer comprises at least one polyol (e.g., a sugar) at a concentration of about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12%. In some embodiments, the diafiltration buffer comprises at least one polyol (e.g., a sugar) at a concentration of about 9% to about 12% w/v. In some embodiments, the concentration of the at least one polyol (e.g., sugar) in the diafiltration buffer is about 9% w/v. In some embodiments, the at least one polyol is selected from the group consisting of: sucrose, trehalose, mannitol and sorbitol, or a combination thereof. In some embodiments, the polyol is sucrose and is present in the diafiltration buffer at a concentration ranging from about 5% to about 9% w/v.

In some embodiments, the diafiltration buffer comprises 20mM calcium acetate, 7% sucrose. In some embodiments, the pH of the diafiltration buffer ranges from 4 to 6. In some embodiments, the pH of the diafiltration 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 diafiltration buffer is 5.1.

Ultrafiltration/diafiltration

Ultrafiltration/diafiltration (also commonly referred to herein as UF/DF) selectively utilizes permeable (porous) membrane filters to separate the components of solutions and suspensions based on their molecular size. The membrane retains molecules larger than the membrane pores, while smaller molecules that are permeable (e.g., salts, solvents, and water) are free to pass through the membrane. The solution retained by the membrane is referred to as the concentrate or retentate. The solution that passes through the membrane is called the filtrate or permeate. One parameter for selecting a membrane for concentration is its retention characteristics for the sample to be concentrated. As a general rule, the molecular weight cut-off (MWCO) of the membrane should be from 1/3 to 1/6 of the molecular weight of the molecules to be retained. This is to ensure complete retention. The closer the MWCO is to the sample, the greater the risk of some small product loss during concentration. Examples of membranes that can be used with the methods of the present disclosure include Omega^TMPES membranes (30kDa MWCO, i.e., molecules greater than 30kDa are retained by the membrane while molecules less than 30kDa are allowed to pass through the filtrate side of the membrane) (Pall Corp., washington harbor, new york); pelicon^TM30kD regenerated cellulose membranes (Millipore Sigma);GV syringe drive filter unit, PVDF 0.22 μm (Millipore Corp., bellerica, ma);-GP syringe drive filter unit, PES 0.22 mu.m;a 0.22 μm filter unit (millipore, belerica, ma); and a Vivaspin concentrator (MWCO 10kDa, PES; MWCO 3kDa, PES) (Sartorius Corp.), Ergiwood, N.Y.)

UF/DF can take two forms, including a discontinuous mode UF/DF and a continuous mode UF/DF. The method of the present disclosure can be performed according to either mode.

Continuous UF/DF (also known as constant volume UF/DF) involves washing away the original buffer salts (or other low molecular weight species) in the retentate (sample) by adding water or new buffer to the retentate at the same rate as the filtrate is produced. Thus, the retentate volume and product concentration do not change during UF/DF. The amount of salt removed is related to the volume of filtrate produced (relative to the volume of retentate). The resulting filtrate volume is often referred to as the "diafiltration volume". The single Diafiltration Volume (DV) is the volume of retentate at the start of diafiltration. For continuous diafiltration, liquid is added at the same rate as the filtrate is produced. When the volume of filtrate collected was equal to the volume of the starting retentate, 1DV had been processed.

Discontinuous UF/DF involves two different approaches: discontinuous sequential UF/DF and discontinuous UF/DF of decreasing volume. Discontinuous UF/DF by sequential dilution involves first diluting the sample with water to a predetermined volume. The diluted sample was then concentrated back to its original volume by UF. Discontinuous UF/DF by volume reduction involves first concentrating the sample to a predetermined volume and then diluting the sample back to its original volume with water or a replacement buffer. This process is repeated as with continuous UF/DF until the level of unwanted solutes (e.g., ionic excipients) are removed.

UF/DF may be performed according to conventional techniques known in the art using water, such as WFI, as the UF/DF medium (e.g., Industrial Ultrafiltration Design and Application of Diafeitration Processes)Design and diafiltration process applications]Beaton and Klinkowski, J.Separ.Proc.Technol [ J.J.separation technology],4(2)1-10(1983)). Examples of commercially available equipment for carrying out UF/DF include Millipore laboratory scale^TMTFF System (Michlobo corporation), LV Central^TMLaboratory tangential flow systems (pall), UniFlux systems (GE Healthcare), and,UD (Sartorius Stedim Biotech) Inc.),FlexReady TFF (EMD Millipore), Akta^TMReadyflux (GE healthcare group), Allegro^TMDisposable TFF (pall) and stainless steel skids such as the Cogent TFF system.

Depending on the protein in solution, the buffer exchange step with diafiltration buffer can be performed any number of times, where one diafiltration step is equal to one total volume exchange. In one embodiment, the diafiltration process is performed 1,2, 3,4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or as many times as deemed necessary to achieve the desired result. When a volume of diafiltration buffer (which is equal to the starting volume of the antibody composition) has been added to the retentate side, a single round or single step diafiltration is achieved.

In various embodiments, the resulting diafiltration formulation after the exchange step comprises about 50mM acetate and about 12mM calcium.

The methods of the present disclosure also provide a means to concentrate antigen binding proteins at high levels without increasing the viscosity of the resulting diafiltration formulation. The concentration of antigen-binding protein in the aqueous formulation obtained using the methods of the present disclosure may be any amount according to the desired concentration. For example, the concentration of antigen binding protein in a composition prepared according to the methods described herein is at least about 70mg/ml, at least about 71mg/ml, at least about 72mg/ml, at least about 73mg/ml, at least about 74mg/ml, at least about 75mg/ml, at least about 76mg/ml, at least about 77mg/ml, at least about 78mg/ml, at least about 79mg/ml, at least about 80mg/ml, at least about 81mg/ml, at least about 82mg/ml, at least about 83mg/ml, at least about 84mg/ml, at least about 85mg/ml, at least about 86mg/ml, at least about 87mg/ml, at least about 88mg/ml, at least about 89mg/ml, at least about 90mg/ml, at least about 91mg/ml, at least about 92mg/ml, or, At least about 93mg/ml, at least about 94mg/ml, at least about 95mg/ml, at least about 96mg/ml, at least about 97mg/ml, at least about 98mg/ml, at least about 99mg/ml, at least about 100mg/ml, at least about 101mg/ml, at least about 102mg/ml, at least about 103mg/ml, at least about 104mg/ml, at least about 105mg/ml, at least about 106mg/ml, at least about 107mg/ml, at least about 108mg/ml, at least about 109mg/ml, at least about 110mg/ml, at least about 111mg/ml, at least about 112mg/ml, at least about 113mg/ml, at least about 114mg/ml, at least about 115mg/ml, at least about 116mg/ml, at least about 117mg/ml, at least about 118mg/ml, At least about 119mg/ml, at least about 120mg/ml, at least about 121mg/ml, at least about 122mg/ml, at least about 123mg/ml, at least about 124mg/ml, at least about 125mg/ml, at least about 126mg/ml, at least about 127mg/ml, at least about 128mg/ml, at least about 129mg/ml, at least about 130mg/ml, at least about 131mg/ml, at least about 132mg/ml, at least about 133mg/ml, at least about 134mg/ml, at least about 135mg/ml, at least about 136mg/ml, at least about 137mg/ml, at least about 138mg/ml, at least about 139mg/ml, at least about 140mg/ml, at least about 141mg/ml, at least about 142mg/ml, at least about 143mg/ml, at least about, At least about 144mg/ml, at least about 145mg/ml, at least about 146mg/ml, at least about 147mg/ml, at least about 148mg/ml, at least about 149mg/ml, at least about 150mg/ml, at least about 151mg/ml, at least about 152mg/ml, at least about 153mg/ml, at least about 154mg/ml, at least about 155mg/ml, at least about 156mg/ml, at least about 157mg/ml, at least about 158mg/ml, at least about 159mg/ml, or at least about 160mg/ml, and may range up to, for example, about 300mg/ml, about 290mg/ml, about 280mg/ml, about 270mg/ml, about 260mg/ml, about 250mg/ml, about 240mg/ml, about 230mg/ml, about 220mg/ml, about 210mg/ml, about, About 200mg/ml, about 190mg/ml, about 180mg/ml, or about 170 mg/ml. Any range having a combination of the foregoing endpoints is contemplated, including, but not limited to: about 70mg/ml to about 250mg/ml, about 70mg/ml to about 200mg/ml, about 70mg/ml to about 210mg/ml, about 70mg/ml to about 160mg/ml, about 100mg/ml to about 250mg/ml, about 100mg/ml to about 200mg/ml, or about 100mg/ml to about 180 mg/ml.

"antigen binding protein" as used herein means a protein that specifically binds to a specified antigen. Examples of antigen binding proteins include, but are not limited to, antibodies, peptibodies, antibody fragments, antibody constructs, fusion proteins, and antigen receptors, including Chimeric Antigen Receptors (CARs). The term encompasses whole antibodies comprising at least two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutations thereof. Antigen binding proteins also include domain antibodies, such as nanobodies and scfvs, described further below.

In some embodiments, the antigen binding protein is an antibody. The term "antibody" as used herein refers to a protein having the form of a conventional immunoglobulin comprising heavy and light chains and comprising variable and constant regions. Antibodies have variable and constant regions. In the IgG format, the variable region is typically about 100-110 or more amino acids, comprises three Complementarity Determining Regions (CDRs), is primarily responsible for antigen recognition, and is very different from other antibodies that bind different antigens. The constant regions allow the antibody to recruit cells and molecules of the immune system. The variable region is formed by the N-terminal region of each of the light and heavy chains, while the constant region is formed by the C-terminal portion of each of the heavy and light chains. (Janeway et al, "Structure of The Antibody Molecule and Immunoglobulin Gene"; Immunobiology: The Immune System in Health and Disease [ Immune biology: Immune System of Health and Disease ], 4 th edition Elsevier Science Ltd./Garland Publishing [ Elsevierscience GmbH/Karan ], (1999)).

The general structure and properties of antibody CDRs have been described in the art. Briefly, in the framework of an antibody, CDRs are embedded within a framework in the heavy and light chain variable regions, where these CDRs constitute the regions primarily responsible for antigen binding and recognition. The variable region comprises at least three heavy or light chain CDRs (Kabat et al, 1991, Sequences of Proteins of Immunological Interest [ protein Sequences of Immunological Interest ], Public Health Service N.I.H. [ national institutes of Health Public Health Service ], Besserda, Mland; see also Chothia and Lesk,1987, J.mol.biol. [ J. Mol. ]196: 901-917; Chothia et al, 1989, Nature [ Nature ]342:877-883), within the framework regions (framework regions 1-4, i.e., FR1, FR2, FR3 and FR4, designated by Kabat et al, 1991; see also Chothia and Lesk,1987, supra).

Human light chains are divided into kappa and lambda light chains. Heavy chains are divided into μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG 4. IgM has subclasses, including but not limited to IgM1 and IgM 2. Embodiments of the invention include all such classes or isotypes of antibodies. The light chain constant region can be, for example, a kappa or lambda type light chain constant region, such as a human kappa or lambda type light chain constant region. The heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma or mu-type heavy chain constant region, such as a human alpha, delta, epsilon, gamma or mu-type heavy chain constant region. Thus, in exemplary embodiments, the antibody is of isotype IgA, IgD, IgE, IgG, or IgM, including any of IgG1, IgG2, IgG3, or IgG 4. IgG1 antibodies are particularly susceptible to reducing disulfide bonds and therefore represent a preferred embodiment of the present disclosure.

The antibody may be a monoclonal antibody or a polyclonal antibody. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, e.g., a mouse, rabbit, goat, horse, chicken, hamster, human, and the like. In this regard, an antibody can be considered a mammalian antibody, such as a mouse antibody, a rabbit antibody, a goat antibody, a horse antibody, a chicken antibody, a hamster antibody, a human antibody, and the like. In certain aspects, the monoclonal antibody is a human antibody. In certain aspects, the monoclonal antibody is a chimeric antibody or a humanized antibody. The term "chimeric antibody" is used herein to refer to an antibody that contains constant domains from one species and variable domains from a second species, or more generally, amino acid sequence segments from at least two species. The term "humanized" when used in reference to an antibody refers to antibodies having at least CDR regions from a non-human source that are engineered to have a structure and immune function that is more similar to a true human antibody than the original source antibody. For example, humanization may involve grafting CDRs from a non-human antibody (e.g., a mouse antibody) into a human antibody. Humanization may also involve selecting amino acid substitutions to make non-human sequences appear more similar to human sequences.

The methods of the present disclosure are also suitable for obtaining formulations comprising antigen binding proteins, such as antibody fragments (e.g., scFv, Fab, and VHH/VH) that retain intact antigen binding capacity. Both scFv and Fab are widely used fragments that can be readily produced in prokaryotic hosts. Other antibody protein products include disulfide stabilized scFv (ds-scFv), single chain fab (scfab), and dimeric and multimeric antibody formats, such as bifunctional, trifunctional, and tetrafunctional antibodies, or different formats of miniantibodies (miniAb) comprising scFv linked to an oligomerizing domain. The smallest fragments are VHH/VH and single domain Ab (sdab) of camelidae heavy chain Ab. The building blocks most commonly used to generate novel antibody formats are single chain variable (V) domain antibody fragments (scFv), which comprise V domains from heavy and light chains (VH and VL domains) connected by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of the peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are well described in the art. See, e.g., Shimamoto et al, mAbs 4(5): 586-.

In some embodiments, the antigen binding protein is a scFv, Fab VHH/VH, Fv fragment, ds-scFv, scFab, dimeric antibody, multimeric antibody (e.g., bifunctional, trifunctional, tetrafunctional), miniAb, peptibody VHH/VH of a camelid heavy chain antibody, sdAb, bispecific or trispecific antibody, bsig, episg, BsAb fragment, bispecific fusion protein, or BsAb conjugate.

The antigen binding protein may be in monomeric form or in polymeric, oligomeric or polymeric form. In certain embodiments where the antibody comprises two or more different antigen-binding region fragments, the antibody is considered to be bispecific, trispecific, or multispecific, or bivalent, trivalent, or multivalent, depending on the number of different epitopes recognized and bound by the antibody.

Advantageously, these methods are not limited to the antigen specificity of antibodies. Thus, an antibody (or antibody fragment or antibody protein product) has almost any binding specificity for any antigen. In exemplary aspects, the antibody binds to a hormone, growth factor, cytokine, cell surface receptor, or any ligand thereof. In some embodiments, the antibody is lomustizumab, abciximab, adalimumab, alemtuzumab, basiliximab, belimumab, bevacizumab, viltin-present itumumab, canamycin, cetuximab, certolizumab, daclizumab, denozumab, eculizumab, efavirenzumab, gemtuzumab, golimumab, ibritumomab, infliximab, ipilimumab, moruzumab-CD 3, natalizumab, ofatumumab, omalizumab, palivizumab, panitumumab, ranibizumab, rituximab, tositumumab, tositumomab, trastuzumab, ustuzumab, veltuzumab, or a biosimilar of any of the foregoing.

In some embodiments, the antibody is selected from the group consisting of: Moluomamab-CD 3 (Orthoclone under the trade name Orthoclone)Commercially available product), abciximab (under the trade name ofCommercially available product), rituximab (under the trade name rituximab) Commercially available product) (U.S. Pat. No. 5,843,439), basiliximab (under the trade name of Basizumab)Commercially available product), daclizumab (under the trade name daclizumab)Commercially available product), palivizumab (under the trade name ofCommercially available product), infliximab (under the trade nameCommercially available product), trastuzumab (under the trade name trastuzumab)Commercially available product), alemtuzumab (under the trade nameCommercially available product), adalimumab (under the trade name)Commercially available product), tositumomab-I131 (under the trade name tositumomab-I131)Commercially available product), efavirenz (under the trade name of favizumabCommercially available product), cetuximab (under the trade nameCommercially available product), ibritumomab (under the trade name tiumumab)Commercially available product), omalizumab (under the trade name ofCommercially available product), bevacizumab (toTrade nameCommercially available product), natalizumab (under the trade name of natalizumab)Commercially available product), ranibizumab (under the trade nameCommercially available product), panitumumab (under the trade name)Commercially available product), eculizumab (under the trade name of zeleche)Commercially available product), cetuzumab (under the trade nameCommercially available product), golimumab (under the trade name golimumab)Commercially available product), canamumab (under the trade nameCommercially available product), cetuximab (under the trade nameCommercially available product), Ultexanb (under the trade name @)Commercially available product), toslizumab (under the trade name of Tolizumab) Commercially available product), ofatumumab (under the trade name of fagopyrum)Commercially available product), denosumab (under the trade name of denudomab)Commercially available product), belimumab (under the trade name)Commercially available products), rebamipumab, ipilimumab (under the trade name ofCommercially available products), and pertuzumab (under the trade name pertuzumab)Commercially available products).

In some embodiments, the antibody is an anti-sclerostin antibody. An "anti-sclerostin antibody" or "sclerostin-binding antibody" is an antibody or portion thereof that binds to sclerostin of SEQ ID NO: 1. Recombinant human sclerostin/SOST is commercially available from, for example, R & D Systems (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) generally refer to anti-sclerostin antibodies. Examples of sclerostin antibodies suitable for use in the context of the present disclosure are also described in U.S. patent publication nos. 2007/0110747 and 2007/0072797, which are hereby incorporated by reference. Additional information regarding the materials and methods for producing sclerostin antibodies may be found in U.S. patent publication No. 20040158045 (hereby incorporated by reference).

As used herein, "specifically binds" means that the antibody preferentially binds to the antigen and not to other proteins. In some embodiments, "specifically binds" means that the affinity of the antibody for the antigen is higher than for other proteins.

In some or any embodiment, the antibody binds to sclerostin of SEQ ID NO:1, or a naturally occurring variant thereof, at less than or equal to 1x10^-7M, less than or equal to 1x10^-8M, less than or equal to 1x10^-9M, less than or equal to 1x10^-10M, less than or equal to 1x10^-11M, or less than or equal to 1x10^-12The affinity (Kd) of M binds. Affinity is determined using a variety of techniques, one example of which is an affinity ELISA assay. In various embodiments, affinity is determined by BIAcore assay. In various embodiments, affinity is determined by kinetic methods. In various embodiments, affinity is determined by an equilibrium/solution method. U.S. patent publication No. 2007/0110747, the disclosure of which is incorporated herein by reference, contains additional description of an affinity assay suitable for determining the affinity (Kd) of an antibody for sclerostin.

In various aspects, the antibody comprises at least one CDR sequence having at least 75% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identity) to a CDR selected from CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein CDR-H1 has the sequence given in SEQ ID No. 2, CDR-H2 has the sequence given in SEQ ID No. 3, CDR-H3 has the sequence given in SEQ ID No. 4, CDR-L1 has the sequence given in SEQ ID No. 5, CDR-L2 has the sequence given in SEQ ID No. 6, and CDR-L3 has the sequence given in SEQ ID No. 7. In various aspects, the anti-sclerostin antibody comprises two CDRs or six CDRs.

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.

In some or any embodiment, the antibody comprises a light chain variable region comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 8 and a heavy chain variable region comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 9. In various aspects, the difference in sequence compared to SEQ ID NO 8 or 9 is outside the CDR regions in the corresponding sequence. In some or any embodiment, the antibody comprises a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 8 and a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID No. 9.

In some or any embodiment, an anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 11, and all or part of a light chain (e.g., two light chains) comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 10.

In some or any embodiment, an anti-sclerostin antibody comprises all or part of a heavy chain (e.g., two heavy chains) comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID No. 13, and all or part of a light chain (e.g., two light chains) comprising an amino acid sequence at least 75% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO 12.

Examples of other anti-sclerostin antibodies include, but are not limited to, the anti-sclerostin antibodies disclosed in international patent publication nos. WO 2008/092894, WO 2008/115732, WO 2009/056634, WO 2009/047356, WO 2010/100200, WO 2010/100179, WO 2010/115932, and WO 2010/130830, each of which is incorporated herein by reference in its entirety.

One skilled in the art will appreciate that some proteins (e.g., antibodies) may undergo a variety of post-translational modifications. The type and extent of these modifications typically depends on the host cell line used to express the protein and the culture conditions. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation. Frequent modifications are the loss of the carboxy-terminal basic residue (e.g., lysine or arginine) by the action of carboxypeptidases (as described in Harris, RJ. journal of Chromatography 705:129-134, 1995).

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co. [ W.H. Fleminn, san Francisco., pp.79-86 [1983], incorporated by reference in its entirety), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

Pharmaceutical compositions comprising one or more of the antibodies described herein can be placed within a container (e.g., a vial or a syringe) along with packaging materials that provide instructions for using such pharmaceutical compositions. Typically, such instructions will include an express statement describing the antibody concentration, and in certain embodiments, the relative amounts of excipient ingredients or diluents (e.g., water, saline, or PBS) that may be necessary to reconstitute the pharmaceutical composition.

Examples of the invention

This example describes a representative antibody purification process that uses an elevated temperature buffer exchange (e.g., by ultrafiltration and diafiltration (UF/DF)) step to concentrate and buffer the antibody to a diafiltration buffer (pH 5.1) of 20mM calcium acetate, 7% sucrose to produce a final pharmaceutical composition containing the antibody at a concentration of 120 g/L. The unexpected result of this process is the ability to recover high concentrations of protein from over-concentration using both higher temperatures (e.g., 37 ℃) and calcium salts (e.g., calcium acetate). Calcium acetate has the property of decreasing solubility with increasing temperature (see, e.g., Apelblat, A. and Manzurola, E.; J. chem. Thermodynamics [ journal of chemical thermodynamics ],1999,31, 1347-.

This inverse relationship between solubility and temperature suggests that the use of elevated temperatures during buffer exchange (e.g., UF/DF) with formulations containing calcium acetate may lead to abnormal precipitation or other potential negative side effects. In other words, increasing the temperature of the formulation containing calcium acetate appears to decrease the solubility of the buffer salt. Despite calcium acetate (Ca (Ac) in diafiltration buffer₂) Is relatively low, about 20mM Ca (Ac)²However, during UF (especially during the excess concentration step) the local concentration of salts on the ultrafiltration membrane surface can be much higher. These high local Ca (Ac)₂The concentration, together with the elevated temperature and the very high protein concentration, surprisingly enables the ultrafiltration process to provide high yields.

Figure 1 shows the effect of calcium acetate on the viscosity of excess concentrate material. The addition of between 10mM and 23mM calcium significantly reduced the viscosity. This is also shown in the UF/DF parameters of FIGS. 2 and 3, where Cwall (maximum protein concentration at the membrane surface) increased from 186mg/mL to 220mg/mL with the addition of calcium.

Figure 4 shows the effect of temperature on viscosity. For a given UF/DF condition, the temperature increase lowers the viscosity and the resulting feed pressure.

Interestingly, the calcium concentration of the UF pool after over-concentration did not agree with that of the Diafiltration (DF) buffer. Diafiltration was done at a concentration of 55g/L and the buffer was converted to 20mM calcium acetate, 7% sucrose, pH 5.1, over 10 Diafiltration Volumes (DV). Excess concentration increased the protein concentration by a factor of 3.3 (to 180 g/L). If the calcium is concentrated to the same extent, the concentration of 20mM calcium DF buffer will increase to at least 65 mM. Surprisingly, the experimental observations were reversed, where the calcium concentration decreased below the DF buffer concentration upon excessive concentration. As shown in table 1 below, the target 20mM Ca concentration resulted in an excess concentrated calcium level of only 8.2 mM.

Table 1 summary of calcium concentration during various stages of production.

TABLE 2 summary of calcium exclusion during excess concentration.

Antibody concentration (mg/mL)	Actual calcium concentration (mM)
		66.15	14.1
98.66	12.6
		118.59	12.1
135.49	11.7
		153.24	10.7
173.99	10.2

Although the size exclusion effect may alter the composition of the retentate buffer, calcium ions may preferentially coordinate or partition with proteins due to solubility changes caused by elevated temperatures and/or high local protein concentrations during filtration.

This example describes a representative antibody purification process that uses an elevated temperature buffer exchange (e.g., by ultrafiltration and diafiltration (UF/DF)) step to concentrate and buffer exchange with a diafiltration buffer containing a calcium salt to produce a final antibody pharmaceutical composition (e.g., about 120 g/L). Given the solubility impact of calcium acetate at elevated temperatures, it is surprising that high concentrations of protein can be recovered from over-concentration using both higher temperatures (e.g., 37 ℃) and higher concentrations of calcium salts (e.g., calcium acetate).

Sequence listing

<110> Amgen Inc.

<120> Process for preparing antibody pharmaceutical formulation

<130> 31173/53022A

<150> US 62/717,357

<151> 2018-08-10

<160> 13

<170> PatentIn version 3.5

<210> 1

<211> 190

<212> PRT

<213> Intelligent people

<220>

<221> features not yet classified

<223> human sclerostin

<400> 1

Gln Gly Trp Gln Ala Phe Lys Asn Asp Ala Thr Glu Ile Ile Pro Glu

1 5 10 15

Leu Gly Glu Tyr Pro Glu Pro Pro Pro Glu Leu Glu Asn Asn Lys Thr

20 25 30

Met Asn Arg Ala Glu Asn Gly Gly Arg Pro Pro His His Pro Phe Glu

35 40 45

Thr Lys Asp Val Ser Glu Tyr Ser Cys Arg Glu Leu His Phe Thr Arg

50 55 60

Tyr Val Thr Asp Gly Pro Cys Arg Ser Ala Lys Pro Val Thr Glu Leu

65 70 75 80

Val Cys Ser Gly Gln Cys Gly Pro Ala Arg Leu Leu Pro Asn Ala Ile

85 90 95

Gly Arg Gly Lys Trp Trp Arg Pro Ser Gly Pro Asp Phe Arg Cys Ile

100 105 110

Pro Asp Arg Tyr Arg Ala Gln Arg Val Gln Leu Leu Cys Pro Gly Gly

115 120 125

Glu Ala Pro Arg Ala Arg Lys Val Arg Leu Val Ala Ser Cys Lys Cys

130 135 140

Lys Arg Leu Thr Arg Phe His Asn Gln Ser Glu Leu Lys Asp Phe Gly

145 150 155 160

Thr Glu Ala Ala Arg Pro Gln Lys Gly Arg Lys Pro Arg Pro Arg Ala

165 170 175

Arg Ser Ala Lys Ala Asn Gln Ala Glu Leu Glu Asn Ala Tyr

180 185 190

<210> 2

<211> 5

<212> PRT

<213> little mouse (Mus musculus)

<220>

<221> features not yet classified

<223> romo HCDR1

<400> 2

Asp Tyr Asn Met His

1 5

<210> 3

<211> 17

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo HCDR2

<400> 3

Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe Lys

1 5 10 15

Gly

<210> 4

<211> 14

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo HCDR3

<400> 4

Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val

1 5 10

<210> 5

<211> 11

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romoLCDR1

<400> 5

Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn

1 5 10

<210> 6

<211> 7

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo LCDR2

<400> 6

Tyr Thr Ser Arg Leu Leu Ser

1 5

<210> 7

<211> 9

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo LCD3

<400> 7

Gln Gln Gly Asp Thr Leu Pro Tyr Thr

1 5

<210> 8

<211> 123

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo light chain variable region

<400> 8

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 9

<211> 107

<212> PRT

<213> mouse

<220>

<221> features not yet classified

<223> romo heavy chain variable region

<400> 9

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 10

<211> 236

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody sequence

<220>

<221> features not yet classified

<223> romo light chain

<400> 10

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Gly Asp Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 11

<211> 468

<212> PRT

<213> Artificial sequence

<220>

<223> humanized antibody sequence

<220>

<221> features not yet classified

<223> romo heavy chain wild type

<400> 11

Met Asp Trp Thr Trp Arg Ile Leu Phe Leu Val Ala Ala Ala Thr Gly

1 5 10 15

Ala His Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys

20 25 30

Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asp Tyr Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu

50 55 60

Glu Trp Met Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser

85 90 95

Thr Ala Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr

115 120 125

Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser

130 135 140

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

145 150 155 160

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

165 170 175

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

180 185 190

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

195 200 205

Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr

210 215 220

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val

225 230 235 240

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

245 250 255

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

260 265 270

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

275 280 285

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

290 295 300

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

305 310 315 320

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

325 330 335

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

340 345 350

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

355 360 365

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

370 375 380

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

385 390 395 400

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

405 410 415

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

420 425 430

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

435 440 445

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

450 455 460

Ser Pro Gly Lys

465

<210> 12

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<221> features not yet classified

<223> romo light chain wild type, no signal sequence

<400> 12

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asp Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 13

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<223> Synthesis of polypeptide

<220>

<221> features not yet classified

<223> romo heavy chain wild type, no signal sequence

<400> 13

Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Asn Ser Gly Gly Ala Gly Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Gly Tyr Asp Asp Ile Tyr Asp Asp Trp Tyr Phe Asp Val

100 105 110

Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val

195 200 205

Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys

210 215 220

Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val

290 295 300

Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys