Freeze drying method

Abstract

The present disclosure provides a method of preparing a lyophilized protein composition, the method comprising: (a) inducing ice nucleation in a liquid protein composition in a vial exposed to a first temperature of about -15°C to about -5°C for about 30 minutes to about 5 hours; (b) exposing the vial to a second temperature of about -25°C to about -50°C for a second period of time; and (c) drying the composition of (b) at a third temperature of about 0°C to about 40°C, resulting in a vial containing the lyophilized protein composition having a fill aspect ratio of about 0.75 or greater. The present disclosure further provides a lyophilized protein composition prepared by the methods described herein.

Description

[Technical field]

[0001] The present disclosure relates to methods for lyophilizing protein compositions and the resulting lyophilized compositions.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 334,785, filed April 26, 2022, which is incorporated herein by reference.

[0003] The computer readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety and is identified as follows: XML file named "57026_Seqlisting.xml" of 97,298 bytes created on April 25, 2023. [Background technology]

[0004] Lyophilization is a manufacturing process that is widely used to increase the stability of pharmaceutical products by removing water content from liquid products by freeze-drying. Lyophilization is a batch process that is typically time-consuming, accounting for more than 50% of drug product processing time. In addition, manufacturing-scale freeze-dryers are limited in terms of capacity. Only a predetermined amount of vials can be loaded in one batch, and the amount of liquid composition that can be dispensed into the vials is limited. Due to these constraints, freeze-dried products are typically expensive. See, for example, Awotwe-Otoo et al., International Journal of Pharmaceutics, 450(2013), 70-78; Esfandiary et al., J.Pharm.Sci., 105(2016), 1427-1433. Efforts to increase the efficiency of the freeze-drying process for biopharmaceutical products are hindered by variability in product attributes (e.g., inconsistent structure of the lyophilized cake) and the possibility of container breakage during the freeze-drying process, resulting in expensive product loss. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Awotwe-Otoo et al.,International Journal of Pharmaceutics,450(2013),70-78 [Non-Patent Document 2] Esfandiary et al.,J.Pharm.Sci.,105(2016),1427-1433 Summary of the Invention [Means for solving the problem]

[0006] The present disclosure provides a method of preparing a lyophilized protein composition, the method comprising: (a) providing a vial containing a liquid protein composition having a packing aspect ratio of about 0.75 or greater; (b) inducing ice nucleation in the vial exposed to a first temperature of about −15° C. to about −5° C. for about 30 minutes to about 5 hours; (c) exposing the vial to a second temperature of about −25° C. to about −50° C. for a second period of time; and (d) drying the composition of (c) at a third temperature of about 0° C. to about 40° C. The present disclosure further provides a lyophilized protein composition prepared by the methods described herein.

[0007] Various embodiments herein are described using the term "comprising" under various circumstances, but it should be understood that related embodiments may also be described using "consisting of" or "consisting essentially of." It is contemplated that an embodiment described as "comprising" a feature includes embodiments "consisting of" or "consisting essentially of" that feature. The term "a" or "an" refers to one or more. Thus, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. The term "or" should be understood to include items alternatively or together unless the context clearly dictates otherwise.

[0008] When describing a range of values, it should be understood that the present disclosure also contemplates each individual value found within that range. For example, "pH of about pH 4 to about pH 6" can be, but is not limited to, pH 4.2, 4.6, 5.2, 5.5, etc., and any value between such values. In any of the ranges described herein, the endpoints of the range are included within the range. However, the description also contemplates the same range in which the lower endpoint and / or the higher endpoint is excluded. When the term "about" is used, it means ±5%, 10%, or more of the recited number. The actual variation intended can be determined from the context.

[0009] Further features and variations of the present invention will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as aspects of the present invention. Similarly, features of the present invention described herein may be recombined into further embodiments, which are also intended as aspects of the present invention, regardless of whether the combination of features is specifically described as an aspect or embodiment of the present invention. It should be understood that the entire document is intended to be related as an integrated disclosure, and all combinations of features described herein are contemplated, even if the combinations of features are not described together in the same sentence, paragraph, or section of this document (even if described in separate sections). Also, only limitations described herein as essential to the present invention should be considered as such, and variations of the present invention lacking limitations not described herein as essential are intended as aspects of the present invention. [Brief description of the drawings]

[0010] [Figure 1]FIG. 1 is a line graph showing the surface temperature profile in a 30R vial as a function of holding time prior to ice nucleation at a shelf temperature of −7° C. A thermocouple was placed at the liquid surface of the vial to monitor the temperature of the top layer of liquid. Temperature is depicted on the Y-axis (° C.), while time (hours) is depicted on the X-axis. The various lines correspond to vials at various positions on the freeze-drying chamber shelves. [Figure 2A] 1 is a cake resistance curve comparison of antibody formulations in 50cc vial (no CIN, no ice nucleation step), 50cc vial (CIN, with ice nucleation step), and 30R (CIN, with ice nucleation step) configurations. Cake resistance (measured as cm2 Torr hr g-1) is depicted on the y-axis, while dry layer thickness (cm) is depicted on the x-axis. [Figure 2B] 1 is a bar graph comparing maximum cake resistance values ​​for the three process conditions described in the Examples. Cake resistance (measured as cm Torr hr g) is listed on the y-axis. [Diagram 3] Scatter plot showing cake moisture in the 30R structure described in the examples as a function of additional retention time in the final drying section, where cake moisture (%) is plotted on the y-axis and additional retention time (hours) when the product is dried is plotted on the x-axis. [Figure 4] 1 is a scatter plot showing the improvement in reconstitution time of protein products lyophilized under the conditions described herein. Reconstitution time (seconds) is listed on the y-axis, while storage time (months) at 40° C. is listed on the x-axis. Circles represent the reconstitution time of lyophilized products lyophilized without a CIN step. Triangles represent samples lyophilized with a CIN step in a 50cc container, while squares represent samples lyophilized with a CIN step in a 30R container. Samples prepared using the methods described herein required reconstitution times of less than 60 seconds. Samples without CIN require longer reconstitution times and may require more than twice the time required for reconstitution of samples prepared using the methods described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure provides a method for preparing a lyophilized protein composition. The method includes providing a vial containing a liquid protein composition having a packing aspect ratio of about 0.75 or more (e.g., 0.8, 0.85, 0.9, or 0.95 or more), inducing ice nucleation in the vial exposed to a first temperature of about -15°C to about -5°C for about 30 minutes to about 5 hours, exposing the vial to a second temperature of about -25°C to about -50°C for a second period of time, and drying the composition at a third temperature of about 0°C to about 40°C. In various embodiments, the packing aspect ratio is about 1 or more (e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 or more). Notably, the process disclosed herein allows for surprisingly high fill volumes (i.e., fill aspect ratios) for a given vial while minimizing the risk of vial breakage and maintaining reasonable drying times. The "fill aspect ratio" is the fill height of the vial (the height of the liquid protein composition in the vial) divided by the internal diameter of the vial (fill aspect ratio = fill height / internal diameter of the vial). Previous methods required a reduction in the amount introduced into the vial (lower fill aspect ratio) to prevent vial breakage and product cake collapse during the lyophilization process. The method described herein allows for higher fill aspect ratios during the lyophilization process, offering a number of potential advantages. For example, for protein therapeutics administered at high doses, fewer vials are required for dosing. Alternatively (or in addition), the process allows for the use of smaller vials compared to other lyophilization methods while maintaining the same fill volume, thereby resulting in more units per batch and therefore increased production. The use of smaller vials also offers the benefit of reduced storage space for manufacturing and clinics. These are just some examples of the benefits of the instant process that are achieved while producing a product cake suitable for biopharmaceutical uses.

[0012] The present disclosure describes various conditions for use in the lyophilization process to produce a lyophilized protein composition. In general, a vial suitable for a pharmaceutical composition (e.g., a glass vial) is filled with a liquid protein composition and exposed to different temperatures and pressures to achieve a lyophilized product. The vial can be of any size or shape suitable for use in a lyophilization process and can be formed from a variety of materials, such as glass, metal, or plastic (e.g., polycarbonate, polystyrene, polypropylene, or polyolefin). For example, the vial can be glass or glass-like and tubular in shape. Molded glass vials are commercially available in a variety of different sizes with different dimensions. Indeed, vials of various sizes are commercially available (e.g., sizes 2R, 4R, 6R, 8R, 10R, 15R, 20R, 25R, 30R, 50R, or 100R). The vial can be configured to include a suitable stopper, such as a commercially available elastomeric stopper available, for example, from Daikyo Seiko, Ltd. or West Pharmaceutical Services, Inc. The steps described herein are carried out in many embodiments in a lyophilization chamber or ice nucleation system. The lyophilization chamber can be carried out according to the manufacturer's instructions suitable for the pharmaceutical composition. In various embodiments of the present disclosure, the lyophilization method does not include an annealing step. "Annealing" refers to the process of cycling the temperature of the formulation (e.g., from low to high and then back to low). Various embodiments of the disclosed method allow for the production of a lyophilized product without such an annealing step.

[0013] The disclosed method includes inducing ice nucleation in a vial (referred to herein as the "ice nucleation step"). Ice nucleation can be initiated using any of a number of methods, including but not limited to ice fog, emergency / rapid depressurization, and vacuum-induced evaporative cooling. Other methods of controlling ice nucleation include, for example, ultrasound, gap freezing, electrical freezing, temperature flash freezing, use of pre-cooling shelves, and mechanical agitation.

[0014] In various aspects of the present disclosure, ice nucleation is induced by ice fog. Ice fog involves "seeding" the supercooled solution in the vial with externally created ice crystals. An ice fog generator is used to generate a suspension of fine ice crystals, which is injected into the freeze dryer chamber. The ice fog crystals act as ice seeds for the supercooled liquid product in the vial. When ice crystals from the fog enter the semi-stoppered vial and contact the surface of the supercooled liquid, ice nucleation occurs instantly in the vial at the designated shelf temperature. This occurs simultaneously in all vials, improving uniformity within the batch. Ice fog systems are available from IMA Life (Tonawanda, NY) and Millrock Technologies (Kingston, NY). Ice fog technology is further described, for example, in Azzarella et al., BioPharm. Int., 29(12) (2017), 36-41.

[0015] In various aspects of the present disclosure, ice nucleation is induced by depressurization. Rapid depressurization generally involves first pressurizing the lyophilizer chamber to 1.5-2 atmospheres (about 20-30 psig) with an inert gas such as nitrogen, and then rapidly releasing the pressure (e.g., 3 seconds or less) to just above atmospheric pressure. The rapid change in pressure induces nucleation in the vial. Rapid depressurization systems are available from SP Scientific (Gardiner, NY) and are further described, for example, in Luoma et al., "Controlled Ice Nucleation Using ControLyo® Pressurization-Depressurization Method", In: Ward K., Matejtschuk P. (eds) Lyophilization of Pharmaceuticals and Biologicals. Methods in Pharmacology and Toxicology. Humana Press, New York, NY, 2019, pp. 57-77.

[0016] Vacuum-induced evaporative cooling generally involves reducing the pressure in the freeze-drying chamber to just above the boiling point of the solution to enhance the evaporative cooling effect at the liquid surface and allow nucleation to occur. Vacuum-induced evaporative cooling systems are available from HOF Sonderanlagenbau GmbH (Lohra, Germany).

[0017] Ice nucleation is induced (e.g., by ice fog) in a vial exposed to a first temperature of about -15°C to about -5°C (e.g., about -15°C to about -6°C or about -15°C to about -7°C or about -10°C to about -7°C, e.g., about -15°C, about -14°C, about -13°C, about -12°C, about -11°C, about -10°C, about -9°C, about -8°C, about -7°C, about -6°C, or about -5°C) for a period of about 30 minutes to about 5 hours. Optionally, the ice nucleation step includes exposing the vial to the first temperature for about 30 minutes to about 2 hours, e.g., about 90 minutes to about 2 hours. In various embodiments, the vial is exposed to a first temperature of about -7°C. Optionally, this step of the method further comprises holding the ice nucleating protein composition at the temperature (optionally at the same temperature) for a post-nucleation period of up to 2 hours. For example, the post-nucleation hold time can be from about 30 minutes to about 90 minutes (e.g., from about 45 minutes to about 75 minutes, e.g., 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, or 75 minutes). In various embodiments, the post-nucleation hold time is about 60 minutes.

[0018] The method further comprises exposing the vial to a second temperature of about -25°C to about -50°C for a second period of time (referred to herein as a "freezing step"). The second temperature can be, for example, about -25°C to about -45°C, about -25°C to about -40°C, about -25°C to about -35°C, about -35°C to about -50°C, about -40°C to about -50°C, or about -45°C to about -50°C (such as about -40°C, about -41°C, about -42°C, about -43°C, about -44°C, about -45°C, about -46°C, about -47°C, about -48°C, about -49°C, or about -50°C). In various embodiments, the freezing step comprises exposing the vial to a second temperature of about -45°C. The second period of time is optionally about 1 hour to about 5 hours, for example, about 2 hours to about 4 hours. The second period of time can be about 60 minutes, about 90 minutes, about 120 minutes, about 150 minutes, about 180 minutes, about 210 minutes, or about 240 minutes (or any range including these endpoints). In various aspects, the second period of time is about 3 hours.

[0019] In various embodiments of the present disclosure, the cooling rate of the vial can be controlled. For example, the first temperature optionally transitions to the second temperature at a rate of about 0.01° C. to about 0.5° C. per minute (e.g., about 0.05° C. to about 0.45° C. per minute, about 0.1° C. to about 0.3° C. per minute, or about 0.15° C. to about 0.25° C. per minute). In various embodiments, the first temperature transitions to the second temperature at a rate of 0.2° C. per minute.

[0020] The method further comprises drying the composition resulting from the freezing step at a third temperature of about 0° C. to about 40° C. (herein referred to as the "drying step"), resulting in a vial containing the lyophilized protein composition having a fill aspect ratio of about 0.75 or greater. In various embodiments, the third temperature utilized in the drying step is about 0° C. to about 35° C., about 0° C. to about 30° C., about 5° C. to about 40° C., about 10° C. to about 40° C., about 15° C. to about 40° C., about 20° C. to about 40° C., about 25° C. to about 40° C., about 30° C. to about 40° C., or about 35° C. to about 40° C. Optionally, the third temperature is about 0° C. to about 25° C., e.g., about 10° C. to about 25° C. (e.g., about 25° C.).

[0021] Optionally, the transition from the freezing step to the drying step involves increasing the temperature at a rate of about 0.2° C. to about 0.7° C. per minute and holding the vial at a temperature of about −40° C. to about −30° C. for about 15 minutes to about 1 hour. For example, the transition can involve increasing the temperature at a rate of about 0.2° C., 0.3° C., 0.4° C., 0.5° C., 0.6° C., or 0.7° C. The transition can occur over a period of, for example, about 15 minutes, 30 minutes, 45 minutes, or 60 minutes.

[0022] In various aspects of the present disclosure, the drying step includes exposing the vial containing the protein composition to an increase in temperature at a rate of about 0.01° C. to about 0.5° C. per minute (e.g., about 0.05° C. to about 0.45° C. per minute, about 0.1° C. to about 0.3° C. per minute, or about 0.15° C. to about 0.25° C. per minute). In various aspects of the present disclosure, the drying step includes (1) holding the vial at a temperature of about -5° C. to about 5° C. for about 8 hours to about 12 hours, and (2) holding the vial at a temperature of about 20° C. to about 30° C. for about 20 hours to about 50 hours. In exemplary embodiments of the present disclosure, drying step 1 includes holding the vial at a temperature of about -5°C to about 0°C, about 0°C to about 5°C, or about -2°C to about 2°C, e.g., about -5°C, about -4°C, about -3°C, about -2°C, about -1°C, about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or any range having these endpoints. In various embodiments, the duration of drying step 1 is about 8 hours to about 10 hours, about 9 hours to about 11 hours, or about 10 hours to about 12 hours, e.g., about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours, or any range having these endpoints. In exemplary aspects of the present disclosure, drying step 2 includes holding the vial at a temperature of about 25° C. to about 30° C., about 20° C. to about 25° C., or about 23° C. to about 27° C., e.g., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., about 26° C., about 27° C., about 28° C., about 29° C., or about 30° C., or any range having these endpoints. The duration of drying step 2 is optionally about 20 hours to about 45 hours, about 20 hours to about 40 hours, about 20 hours to about 35 hours, about 20 hours to about 30 hours, about 25 hours to about 50 hours, about 30 hours to about 50 hours, about 35 hours to about 50 hours, about 40 hours to about 50 hours, or about 45 hours to about 50 hours (e.g., about 20 hours, about 25 hours, about 30 hours, about 35 hours, about 40 hours, about 45 hours, or about 50 hours). Drying step (1) optionally includes holding the vial at a temperature of about 0° C. for about 10 hours. Drying step (2) optionally includes holding the vial at a temperature of about 25° C. for about 40 hours.

[0023] The disclosed method allows for the use of a larger amount of liquid composition in the vial prior to lyophilization. In this regard, the liquid protein composition may fill at least 50% of the vial volume prior to performing the ice nucleation step. The method may include filling at least 50% of the vial volume with the liquid protein composition prior to the ice nucleation step. The liquid protein composition may fill at least 55%, 60%, 65%, or 75% of the vial.

[0024] The methods described herein optionally further comprise reconstituting the lyophilized product with a suitable diluent (e.g., sterile water, saline, etc.). In various aspects, the methods described herein produce lyophilized products that require less reconstitution time (i.e., less time is required to convert the lyophilized cake into a reconstituted liquid composition suitable for administration) than previous lyophilization methods. In this regard, the time required to reconstitute the lyophilized product into a liquid composition is reduced, for example, by at least 25%, at least 30%, at least 40%, at least 45%, or at least 50%, compared to a lyophilized product (having the same protein) that does not include an ice nucleation step (although this is not required).

[0025] The present disclosure further provides a lyophilized protein composition prepared by the methods described herein. The lyophilized product optionally requires a shorter reconstitution time (at least 25%, at least 30%, at least 40%, at least 45%, or at least 50% shorter reconstitution time) compared to, for example, a lyophilized product (having the same protein) that does not include an ice nucleation step. Alternatively or in addition, the maximum cake resistance of the lyophilized product is reduced by at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or at least 85%) compared to a lyophilized product (having the same protein) that does not include one or more of the steps described herein (e.g., an ice nucleation step). Cake resistance is the resistance of the lyophilized product to vapor flow through the drying layer, as further described in the Examples. Cake resistance is typically measured in cm2 Torr h ​​g -1 See, e.g., Jameel et al., AAPS Pharm Sci Tech. 2021 Oct; 22(7): 221.

[0026] The lyophilized protein formulation optionally further comprises a saccharide, a surfactant, and / or a buffering agent. The formulation also optionally has a pH of about 3 to about 7 (or about 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7). In some examples, the pH is about 4 to about 6. In some examples, the pH of the formulation is about 4, or about 4.2. In various examples, the pH of the formulation is about 5. In some embodiments, the pH of the formulation is about 6.

[0027] In some embodiments, the protein of the lyophilized formulation is an antigen-binding protein. An "antigen-binding protein" is a protein that comprises a domain that binds to a designated target antigen (such as HER2 or CD3 and / or DLL3, BCMA, CD33, or STEAP1). An antigen-binding protein comprises a scaffold or framework portion that allows the antigen-binding domain to adopt a conformation that facilitates binding of the antigen-binding protein to an antigen.

[0028] In some embodiments, the antigen-binding protein of the lyophilized formulation is an antibody or immunoglobulin, an antigen-binding antibody fragment, or an antibody protein product that includes an antigen-binding domain in a scaffold, framework, or format that allows the antigen-binding domain to assume a conformation that promotes binding to the antigen. Antibodies or immunoglobulins, antigen-binding antibody fragments, and antibody protein products are also referred to herein as "antibody constructs." In some examples, the antigen-binding protein is an antibody. The term "antibody" refers to an intact antigen-binding immunoglobulin. An "antibody" is one type of antigen-binding protein. The antibody can be an IgA, IgD, IgE, IgG, or IgM antibody, including any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, the intact antibody includes two full-length heavy chains and two full-length light chains. The antibody has one variable region and one constant region. In the IgG format, a variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and varies substantially among other antibodies that bind different antigens. A variable region typically contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; see also Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4 by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). The constant region enables the antibody to recruit cells and molecules of the immune system.

[0029] Antibody structures span a molecular weight range of at least about 12-150 kDa and have been exploited to generate a wide range of alternative formats ranging from monomers (n=1) to dimers (n=2), trimers (n=3), tetramers (n=4), and potentially higher valencies (n), referred to herein as "antibody protein products." Antibody protein products include those based on the complete antibody structure and those that mimic antibody fragments that retain full antigen-binding ability, such as scFvs, disulfide-stabilized scFvs (ds-scFvs), single-chain antibodies (SCAs), single-chain Fabs (scFabs), and minibodies (miniAbs).

[0030] The antibody construct may be "bispecific", i.e., the antibody or antibody protein product binds to two different targets (e.g., CD3 and a second different target). A "bispecific" antibody or antibody-like product generally comprises a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (e.g., a target cell surface antigen) and the second binding domain binds to another antigen or target (e.g., CD3). Thus, the antibody construct optionally comprises specificity for two different antigens or targets. The term "target cell surface antigen" refers to an antigenic structure that is expressed by a cell and present on the cell surface such that it is available to the antibody construct as described herein. This may be a protein, preferably an extracellular portion of a protein, or a carbohydrate structure, preferably a carbohydrate structure of a protein such as a glycoprotein. In various embodiments, it is a tumor antigen. Multispecific antibody constructs are also contemplated, such as trispecific antibody constructs (comprising three binding domains) or constructs with more than three specificities (e.g., four, five, or more).

[0031] In various aspects, the protein is a bispecific antibody construct. Bispecific antibody constructs include, but are not limited to, traditional bispecific immunoglobulins (e.g., BsIgG), IgG with additional antigen-binding domains (e.g., the amino or carboxy terminus of the light or heavy chain is linked to an additional antigen-binding domain, e.g., a single domain antibody or paired antibody variable domains (e.g., Fv or scFv)), BsAb conjugates, and engineered constructs including full-length antibodies. See, for example, Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015) and WO 2015 / 149077, which describe various bispecific formats and are incorporated herein by reference. Examples of bispecific antibody constructs also include, but are not limited to, diabodies, single chain diabodies, tandem scFvs, bispecific T cell engager (BiTE®) formats (fusion proteins consisting of two single chain variable fragments (scFvs) joined by a linker), BsAb fragments (e.g., bispecific single chain antibodies), bispecific fusion proteins (e.g., an antigen binding domain fused to an effector moiety) and Fab2 bispecifics (collectively also referred to as "bispecific antibody protein products").See, e.g., Chames & Baty, 2009, mAbs 1[6]:1-9; and Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-1136; Wu et al., 2007, Nature Biotechnology 25

[11] :1290-1297; Michaelson et al., 2009, mAbs 1[2]:128-141; WO 2009 / 032782 and WO 2006 / 020258; Zuo et al., 2000, Protein Engineering 13[5]:361-367; U.S. Patent Application Publication No. 2002 / 0103345; Shen et al., 2006, J Biol Chem 2006, 1999, 14:111-112, all of which are expressly incorporated herein. 281

[16] :10706-10714; Lu et al., 2005, J Biol Chem 280

[20] :19665-19672; and Kontermann, 2012 MAbs 4(2):182. In addition to the above, the present disclosure contemplates antibody constructs (e.g., bispecific antibody constructs) including, for example, single-chain Fc formats (referred to as scFc), heterogeneous Fc (hetFc or heterodimeric Fc, also referred to as hFc) formats, and half-life extended formats (HLE formats) including fusions of human serum albumin (also referred to as HSA or hALB).

[0032] In various embodiments of the disclosure, the protein in the formulation is an antibody that binds to HER2 (Uniprot Accession No. P04626-1). In various embodiments, the antibody comprises a light chain complementarity determining region 1 (LC CDR1) of SEQ ID NO: 1, a LC CDR2 of SEQ ID NO: 2, a LC CDR3 of SEQ ID NO: 3, a heavy chain (HC) CDR1 of SEQ ID NO: 4, a HC CDR2 of SEQ ID NO: 5, and a HC CDR3 of SEQ ID NO: 6. The anti-HER2 antibody may comprise a light chain variable region (VL) of SEQ ID NO: 7 and / or a heavy chain variable region (VH) of SEQ ID NO: 8, or a sequence at least about 90% identical thereto (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical). The anti-HER2 antibody may comprise a light chain of SEQ ID NO:9 and / or a heavy chain of SEQ ID NO:10, or a sequence that is at least about 90% identical thereto (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical).

[0033] When the protein is a bispecific antibody construct, the first binding domain comprises a set of six CDRs as set forth in (a) SEQ ID NOs: 20-25 (LCDR1-3 and HCDR1-3, respectively, which bind to BCMA (e.g., Uniprot accession no. Q02223)), (b) SEQ ID NOs: 30-35 (LCDR1-3 and HCDR1-3, respectively, which bind to DLL3 (e.g., Uniprot accession no. Q9NYJ7)), or (c) SEQ ID NOs: 41-46 (LCDR1-3 and HCDR1-3, respectively, which bind to CD33 (e.g., Uniprot accession no. P20138)). In some aspects, the first binding domain of the bispecific antibody construct comprises a VL region comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 26, 36, 47, or 48. In some embodiments, the first binding domain of the bispecific antibody construct comprises a VL region comprising an amino acid sequence set forth in SEQ ID NO: 26, 36, 47, or 48. In some aspects, the first binding domain of the bispecific antibody construct comprises a VH region comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 27, 37, 49, or 50. In some embodiments, the first binding domain of the bispecific antibody construct comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 27, 37, 49, or 50. In some aspects, the first binding domain comprises (a) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 27, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 26, (b) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 27, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 36, or (c) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 49 or 50, and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 47 or 48.

[0034] In some embodiments, the second binding domain of the bispecific antibody construct comprises a set of six CDRs set forth in SEQ ID NOs: 11-16 (LCDR1-3 and HCDR1-3, which bind to CD3, respectively (e.g., Uniprot Accession No. P07766)). In some aspects, the second binding domain of the bispecific antibody construct comprises a VL region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 17. In some aspects, the second binding domain of the bispecific antibody construct comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO: 17. In some aspects, the second binding domain of the bispecific antibody construct comprises a VH region comprising an amino acid sequence at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the second binding domain of the bispecific antibody construct comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the second binding domain comprises a VL region comprising an amino acid sequence set forth in SEQ ID NO: 17 and a VH region comprising an amino acid sequence set forth in SEQ ID NO: 18. Anti-CD3 binding domain sequences are also provided in SEQ ID NOs: 54-59 (corresponding to LCDR1-LCDR3 and HCDR1-HCDR3, respectively) and SEQ ID NOs: 60 and 61 (light chain variable region and heavy chain variable region, respectively).

[0035] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to DLL3 comprising an anti-DLL3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 36 and an anti-DLL3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 37, and a second binding domain comprising an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 17 and an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 18. For example, in one embodiment, the bispecific antibody construct comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 38 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the bispecific antibody construct comprises the amino acid sequence set forth in SEQ ID NO: 39 or 40.

[0036] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds BCMA comprising an anti-BCMA variable light chain domain comprising the amino acid sequence of SEQ ID NO: 26 and an anti-BCMA variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 27, and a second binding domain comprising an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 17 and an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 18. For example, in one embodiment, the bispecific antibody construct comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 28 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the bispecific antibody construct comprises the amino acid sequence set forth in SEQ ID NO: 29.

[0037] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to CD33 comprising an anti-CD33 variable light domain comprising the amino acid sequence of SEQ ID NO: 47 or 48 and an anti-CD33 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 49 or 50, and a second binding domain comprising an anti-CD3 variable light domain comprising the amino acid sequence of SEQ ID NO: 17 and an anti-CD3 variable heavy domain comprising the amino acid sequence of SEQ ID NO: 18. For example, in one embodiment, the bispecific antibody construct comprises a first binding domain comprising the amino acid sequences of SEQ ID NO: 47 and 49 or SEQ ID NO: 48 and 50, and a second binding domain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the bispecific antibody construct comprises the amino acid sequence set forth in SEQ ID NO: 52 or 53.

[0038] In some embodiments, the bispecific antibody construct comprises a first binding domain that binds to human STEAP1. The first binding domain may comprise a set of six CDRs set forth in SEQ ID NOs: 66-71 (HCDR1-3 and LCDR1-3, respectively). In some embodiments, the first binding domain of the bispecific antibody construct comprises a VL region that binds to STEAP1 and comprises an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 80. In some embodiments, the STEAP1 binding domain of the bispecific antibody construct comprises a VL that comprises the amino acid sequence set forth in SEQ ID NO: 80. In some aspects, the STEAP1 binding domain of the bispecific antibody construct comprises a VH region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the second binding domain of the bispecific antibody construct comprises a VH comprising an amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the STEAP1 binding domain comprises a VL region comprising an amino acid sequence set forth in SEQ ID NO: 80 and a VH region comprising an amino acid sequence set forth in SEQ ID NO: 81. In this aspect, the bispecific antibody construct may comprise an anti-CD3 binding domain comprising six CDRs comprising the sequences of SEQ ID NOs: 54-59 (corresponding to LCDR1-LCDR3 and HCDR1-HCDR3, respectively) or SEQ ID NOs: 60 and 61 (light chain variable region and heavy chain variable region, respectively). In various aspects, a bispecific antibody construct that binds STEAP1 and CD3 is a construct that includes a polypeptide comprising SEQ ID NO: 89-91 (e.g., a heavy chain comprising SEQ ID NO: 90 and a heavy chain comprising SEQ ID NO: 91 paired with a common light chain of SEQ ID NO: 89). Such bispecific antibodies are further described, for example, in WO 2020 / 010079, which is incorporated herein by reference in its entirety, and is described in particular with respect to bispecific antibody constructs.

[0039] In some aspects of the disclosure, the protein, such an antibody or bispecific antibody construct is present in the liquid formulation (before lyophilization) in an amount ranging from about 0.1 mg / mL to about 100 mg / mL (or about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, or 100 mg / mL). For example, the protein is optionally present in the liquid formulation in an amount ranging from about 0.1 mg / mL to about 70 mg / mL. In some examples, the protein is present in the liquid formulation in an amount ranging from about 0.5 mg / mL to about 30 mg / mL (or about 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, or 30 mg / mL).In various cases, the protein is present in a liquid formulation in a range of about 1 mg / mL to about 20 mg / mL (or about 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 6 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, 15 mg / mL, 15.5 mg / mL, 16 mg / mL, 16.5 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.5 mg / mL, 19 mg / mL, 19.5 mg / mL, or 20 mg / mL). In some embodiments, the protein is present in the liquid formulation in an amount of about 21 mg / mL.

[0040] The protein formulation of the present disclosure optionally comprises a sugar. In some embodiments, the sugar is a monosaccharide or a disaccharide. Suitable sugars include, for example, glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof. In some examples, the sugar comprises trehalose.

[0041] In some aspects, the liquid formulation (before lyophilization) comprises saccharide at a concentration of about 1% to about 15% w / v, or about 4% to about 13% w / v, or about 6% to about 12% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of 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%, or at least 14% w / v. In some embodiments, the liquid formulation comprises saccharide 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%, or about 15% w / v. In some embodiments, the liquid formulation comprises a sugar at a concentration of about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12% w / v. In some embodiments, the liquid formulation comprises a sugar at a concentration of about 7% to about 12% w / v. In some aspects, the liquid formulation comprises a sugar at a concentration of about 9% w / v. In some embodiments, the sugar is sucrose and is present in the liquid formulation at a concentration ranging from about 6% to about 12% w / v.

[0042] The protein formulation of the present disclosure optionally comprises a surfactant. Suitable surfactants include polysorbate, poloxamer, polyoxyethylene, or any combination thereof. Contemplated surfactants include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X-100, polyoxyethylene, PEG3350, PEG4000, and any combination thereof. In some embodiments, the surfactant comprises polysorbate. In some examples, the surfactant is polysorbate 20.

[0043] The protein formulations described herein may, but need not, include a surfactant or a mixture of surfactants. In some embodiments, the liquid formulation (before lyophilization) includes a surfactant at a concentration of about 0.001% to about 5% w / v (or about 0.001% to about 0.5%, or about 0.004 to about 0.5% w / v, or about 0.001 to about 0.01% w / v, or about 0.004 to about 0.01% w / v). In some embodiments, the liquid formulation comprises a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.5% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001 to about 0.01% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001 to about 0.01% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4% to about 0.5% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01% w / v.

[0044] The protein formulation of the present disclosure optionally comprises a buffer.Suitable buffers include acetate buffer, glutamate buffer, citrate buffer, lactate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, phosphate buffer, 2-(N-morpholino)ethanesulfonic acid buffer, or any combination thereof.In some examples, the buffer comprises histidine.

[0045] Buffers are often utilized to control the pH in the formulation. In some embodiments, the buffer is added at a concentration that maintains the pH of the liquid formulation at about 3 to about 7, or about 4 to about 6, about 4 to 5, or about 5 to about 6, or 6 to about 6.5. The effect of pH on the formulation can be characterized using any one or more of several techniques, such as accelerated stability testing and calorimetric screening testing (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)).

[0046] Buffer systems, if present in the protein formulation, are selected to be physiologically compatible and to maintain the desired pH. Buffers may be present in the liquid formulation (before lyophilization) at a concentration of about 0.1 mM to about 1000 mM (1 M), or about 5 mM to about 200 mM, or about 5 mM to about 100 mM, or about 10 mM to about 50 mM. Suitable buffer concentrations include concentrations of about 200 mM or less. In some embodiments, in the liquid formulation (before lyophilization), the buffer is present at a concentration of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM, or about 5 mM. In some embodiments, the concentration of the buffering agent is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffering agent is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM to 100 mM. In some embodiments, the concentration of the buffer is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM to 50 mM.

[0047] In various embodiments, the lyophilized product contains about 420 mg of protein comprising SEQ ID NOs: 9 and 10, about 381.8 mg of α,α-trehalose dihydrate, about 9.5 mg of L-histidine HCl monohydrate, about 6.1 mg of L-histidine, and about 1.7 mg of polysorbate 20. Reconstitution with 20 mL of an appropriate diluent (BWFI or SWFI) results in a solution containing 21 mg / mL of protein, delivering 20 mL (420 mg of protein) at a pH of about 6.

[0048] In a further aspect, a kit is provided that includes the lyophilized protein composition described herein packaged in a manner that facilitates administration to a subject. In one embodiment, the kit includes the lyophilized protein composition described herein packaged in a container, such as a sealed bottle, vessel, single or multiple use vial, pre-filled device (e.g., syringe), or pre-filled injection device, and optionally has a label attached to the container or is included in a package that describes the use of the lyophilized protein composition. In one aspect, the pharmaceutical composition is packaged in a unit dosage form. The kit may include a device suitable for administering the reconstituted protein composition according to a specific route of administration, although this is not required. For example, the present disclosure provides a dual chamber device for delivering the reconstituted protein composition disclosed herein to a subject in need thereof. The dual chamber device is a combination product that contains the lyophilized protein composition disclosed herein and a diluent in two separate chambers of the device. The pre-filled dual chamber device is a combination product that contains a lyophilized drug and a diluent in two separate chambers of the device. Suitable dual chamber devices for use with the present disclosure have been described in the art. See, for example, Ingle R., Fang W. (2021). Int. Journal of Pharmaceutics 597, 12031.

[0049] The formulations described herein are useful as pharmaceutical preparations in the treatment or amelioration of cancer in a subject in need of such treatment or amelioration. The term "subject in need" or "subject in need of treatment" includes subjects already suffering from a disorder as well as subjects in which the disorder is to be prevented. "Subject in need" or "patient" includes human and other mammalian subjects undergoing either prophylactic or therapeutic treatment. "Treatment" does not require complete remission or eradication of the disease, and any improvement in the disease and / or amelioration of symptoms associated with the disease are contemplated. For example, a therapeutic response refers to one or more of the following improvements in the disease: (1) reduction in the number of neoplastic cells; (2) increase in neoplastic cell death; (3) inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth or the appearance of new lesions; (5) delay in disease progression; (6) increase in patient survival; (7) downgrading of the stage of the cancer (e.g., from stage 2 to stage 1) and / or (8) some relief from one or more symptoms associated with the disease or condition. The compositions may also be administered to prevent or delay the onset of disease, for example, to avoid the development or recurrence of tumors or cancer.Disease status is monitored, for example, by clinical examination, X-ray, computed tomography (CT, such as spiral CT), magnetic resonance imaging (MRI), positron emission tomography (PET), ultrasound, endoscopy and laparoscopy, tumor marker levels (e.g., carcinoembryonic antigen (CEA)), cytology, histology, tumor biopsy sampling, and / or counting of circulating tumor cells.These methods are also typically used to diagnose and stage cancer.

[0050] The present disclosure provides a method of treating cancer, comprising administering a therapeutically effective amount of a reconstituted composition based on a lyophilized formulation described herein to a subject in need thereof. In some embodiments, the subject is a human. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is brain cancer, bladder cancer, breast cancer (e.g., triple-negative breast cancer), clear cell renal cancer, cervical cancer, colon and rectal cancer, endometrial cancer, gastric cancer, head and neck squamous cell carcinoma, lip and oral cavity cancer, liver cancer, lung squamous cell carcinoma, melanoma, mesothelioma, non-small cell lung cancer (NSCLC), non-melanoma skin cancer, ovarian cancer, oral cancer, pancreatic cancer, prostate cancer, neuroendocrine prostate cancer, renal cell carcinoma, sarcoma, small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck (SCCHN), or thyroid cancer. In some embodiments, the cancer is acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML). In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL). The present disclosure also provides the use of a reconstituted composition based on a lyophilized formulation in a method for treating cancer, and the use of a lyophilized formulation in the preparation of a medicament for treating cancer.

[0051] Preferably, the pharmaceutical formulation is administered parenterally, for example, intravenously, subcutaneously, intratumorally, or intramuscularly. Parenteral administration can be achieved by injection, such as a bolus injection, or by infusion, such as continuous infusion. Administration can be achieved via a depot for extended release. In some embodiments, the formulation is administered intravenously by an initial bolus followed by continuous infusion to maintain therapeutic circulating levels of the drug product. In some embodiments, the formulation is administered as a single dose. The pharmaceutical formulation can be administered using a medical device. Examples of medical devices for administering pharmaceutical formulations are described in U.S. Pat. Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0052] The following examples illustrate representative features of the present disclosure. From the description of these embodiments, other embodiments of the present invention can be made and / or performed based on the description provided below. The method involves the use of molecular biology techniques described in specialized texts such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001; and Current Protocols in Molecular Biology, Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York. The examples serve only to illustrate the present invention and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0053] This example describes a process for preparing a lyophilized protein composition using the materials and methods described above.

[0054] Previously frozen drug substance was allowed to thaw for 24 hours at room temperature. The drug substance contained an antibody containing heavy and light chains of SEQ ID NOs: 10 and 9, respectively. A target fill volume of 20 mL was accumulated in 50 cc or 30R glass vials, and the fill weight was confirmed at the start of the fill. The filled vials were placed in a tray, manually semi-stoppered, and the tray was loaded onto the freeze dryer shelf. For experiments involving conventional preparation (i.e., loading, equilibration, freezing, primary drying, and secondary drying), the appropriate procedure for the particular freeze dryer used was followed. For freeze drying processes involving induced ice nucleation, Millrock's FreezeBooster equipped freeze dryer was used. The FreezeBooster is an external device that is connected to the main freeze dryer chamber and converts water into ice fog that is then injected into the freeze dryer chamber and ultimately into the semi-stoppered vials. The ice nucleation cycle included a pre-freeze temperature equilibration step to bring the product temperature below the normal freezing point prior to the ice nucleation stage. After nucleation, various freeze ramp rates were used. The drying step was performed in the same manner for runs with and without the ice nucleation step. At the end of each lyophilization run, the chamber pressure was increased to 500 Torr by backfilling with clean, dry nitrogen, and the vials were fully stoppered with a hydraulic ram. The shelf temperature was set at 5°C. After unloading, the vials were manually sealed using a pneumatic crimper and stored at 2-8°C. During the first run, 30R vials were used and parameters applied to 20 mL of drug substance in 50 cc vials. Stability profiles were generated using both vial configurations.

[0055] Hold Time Before Ice Nucleation - When ice fog crystals are introduced into the vial, ice nucleation occurs at the top layer and propagates throughout the vial. The hold time before the nucleation step allows the product to equilibrate at a specific temperature before the ice fog is introduced and helps prevent the ice crystals from melting after nucleation. If the liquid surface temperature is not low enough (supercooling), the ice crystals can melt before the ice propagates throughout the vial. Hold times ranging from 0 to 120 minutes were tested in the 30R configuration. Thermocouples were placed at the liquid surface to monitor the temperature of the top layer as a function of hold time, as shown in Figure 1. For a shelf temperature of -7°C, a liquid surface temperature of 0°C was observed to benefit from hold times greater than 60 minutes. Also, to account for shelf and product temperature variability, a 2 hour hold time was incorporated to ensure that the surface product temperature was below 0°C before the ice fog was injected into the vial. Creation of ice fog by the FreezeBooster device and injection of ice fog into the chamber takes >10 minutes. Therefore, a holding time of 110 min, excluding the time of the ice nucleation step itself, was selected as the pre-ice nucleation holding time for further experiments.

[0056] Ice Nucleation Temperature - Higher ice nucleation temperatures achieve favorable crystal structure and size and reduce cake resistance. However, if the ice nucleation temperature is too high, the ice fog crystals can melt. Temperatures ranging from -10°C to -5°C were evaluated using a 30R structure and batch sizes of >200. The process was evaluated by thermocouple profile and cake appearance. Samples that were successfully nucleated under ice nucleation conditions exhibited a very distinctive dry cake appearance with shiny crystalline features and were easily distinguished from vials that did not undergo the ice nucleation step.

[0057] When the pre-ice nucleation hold time (excluding the ice nucleation step) was >60 minutes, all vials were successfully nucleated at temperatures of -10°C and -7°C. However, when a temperature of -5°C was used, based on the thermocouple profile, one of the thermocouple vials failed to nucleate the particular drug substance used. Also, based on visual inspection, 18 of the 215 vials exhibited a non-ice nucleated type cake appearance. Thus, although -5°C achieved nucleation in many vials, -7°C was selected for further testing as it was the highest temperature at which 100% nucleation was observed.

[0058] Holding time after ice nucleation - Further holding time after ice nucleation (although this is not necessary) may further reduce cake resistance as larger ice crystals are created. However, it is preferred to minimize the holding time after ice nucleation considering that it may affect the quality attributes of the product. Holding times after nucleation from 0 minutes to 60 minutes were tested to evaluate whether there were any differences in product temperature in the sublimation and primary drying profiles. A higher product temperature range (-22.5°C to -20.5°C) was observed during sublimation when the product was not held at the ice nucleation temperature for any further time compared to the product temperature range (-23°C to -21.5°C) when a 60 minute post-nucleation holding step was used. No further impact on product quality and stability was observed. The difference in product temperature range suggests that a holding time after nucleation may be advantageous in at least some circumstances, allowing the formation of larger ice crystal structures and reducing cake resistance. Therefore, a holding time after nucleation of 60 minutes was selected for further testing.

[0059] Ice nucleation conditions have been described as increasing the size of the ice crystal structures and therefore reducing the cake resistance to steam flow compared to processes without a nucleation step. As shown in Figures 2A and 2B, when an ice nucleation step was implemented, a significant reduction in cake resistance was observed compared to the conventional freezing process (Figure 2A). For comparison purposes, the maximum cake resistance values ​​from three drying profiles (50cc no nucleation step, 50cc nucleation step, 30R nucleation step) were used and compared in Figure 2B. The conventional process (9.1cm 2 Torr.hr.g -1 ) compared to the 50cc structure (2.2 cm 2 Torr.hr.g -1 After adding an ice nucleation step at 1.0 cm, a reduction in cake resistance of over 75% was observed. Similarly, very low cake resistance was achieved in the 30R vials that underwent ice nucleation as described herein (1.0 cm). 2 Torr.hr.g -1 ). The difference in cake resistance values ​​for the 50 cc and 30R vials that underwent ice nucleation can be attributed to the difference in product temperature during the sublimation process. The maximum product temperature during sublimation for the 30R structure ranged from -23°C to -21.5°C (collapse temperature above -26.6°C), while the maximum product temperature range for the 50 cc structure ranged from -26.4°C to -24.7°C. Higher product temperatures may result in a higher degree of cake micro-collapse, ultimately reducing the resistance to water vapor flow during the primary drying stage.

[0060] Drying process conditions were also evaluated. A chamber pressure of 150 mTorr was used to ensure sufficient heat transfer during the drying stage. For the 30R vials, an aggressive multi-zone drying process was used by varying the shelf temperature from 0°C to 30°C with 10-hour zones at 5°C to 10°C intervals. Product temperatures ranging from -22.5°C to -21.5°C were observed during sublimation, much higher than the collapse temperature value of -26.6°C. For this product, drying above the collapse temperature and higher product temperatures resulted in shorter drying times without adversely affecting the cake appearance or cake moisture value compared to samples made using a commercial freeze-drying process (without the nucleation step described herein). At the end of the 30°C drying process, the cake moisture value was observed to be 0.3%.

[0061] Subsequent runs were performed to evaluate further reductions in total cycle time. The intermediate primary drying zone was omitted due to a linear ramp rate from 0°C to 25°C at 0.03°C / min. A similar product temperature range (-23°C to -21.5°C) was observed without any obvious effect on cake appearance compared to samples made using a commercial freeze-drying process (without the nucleation step described herein). Pirani pressure gauge data suggests that most of the primary drying was completed during the ramp from 0°C to 25°C, and total drying time was also reduced from 63 hours (multi-step drying) to 54 hours (linear ramp drying). A slight increase in cake moisture value (0.5%) was observed after drying was completed at 25°C compared to multi-step drying ending at 30°C (0.3%). A final drying zone at 25°C was selected and used for both primary and secondary drying purposes.

[0062] Before starting the sublimation of the product, a short (30 min) drying step was added at a shelf temperature of -35°C to remove any additional moisture left by the introduction of ice fog from the chamber. Since a chamber pressure of 150 mTorr was used in the freeze-drying process, a shelf temperature of -35°C (vapor pressure of ice is 168 mTorr) was chosen to avoid a slight increase in temperature after completion of the sublimation of the external ice, and to remove any residual amount of ice from the chamber.

[0063] The removal of residual cake moisture during the secondary drying (SD) step depends on the temperature and time of the SD. After secondary drying at a given temperature and time combination, the cake moisture value reaches a plateau (equilibrium) and then does not decrease significantly with time. To ensure that the cake moisture of the freeze-dried product using the ice nucleation step reaches equilibrium at the end of the last zone of drying, the cake moisture was compared as a function of shelf temperature hold time (time zero is when the Pirani pressure reaches a plateau). As shown in Figure 3, the cake moisture was <0.6% after a 10-hour hold time, and only a slight decrease (0.4%-0.5%) in the cake moisture value was observed after an additional hold time of 15-40 hours. Particularly favorable cake moisture values ​​were achieved with an additional hold time of 10 hours at the end of the last drying zone.

[0064] After developing the process for the 30R vial configuration, the same process was used for 50cc vials containing 20mL of formulation. Product temperatures ranged from -25.6°C to -24.3°C, above the collapse temperature (-26.6°C), and cake appearance and cake moisture range (0.4% to 0.6%) were observed to be comparable to products made using commercial lyophilization process conditions. The combination of an ice nucleation step at 7°C and linear ramp (0°C to 25°C) drying resulted in a total drying time of 35 hours, significantly lower compared to the primary drying time of 100 hours for the commercial lyophilization process at laboratory scale.

[0065] In summary, the inclusion of the ice nucleation step resulted in a reduction in total cycle time for both 50cc and 30R vial configurations compared to the lyophilization process without the step. In addition, the manufacturing throughput (DP vials produced from one batch / total lyophilization cycle time) for both 50cc and 30R was significantly increased compared to the process that did not include the method steps described herein. The increase in throughput was achieved by reducing the total lyophilization cycle time and, in the case of 30R vials, by reducing the vial footprint, i.e., loading more vials per batch.

[0066] The potential impact of the ice nucleation step and drying conditions on the product quality of the drug substance was evaluated by subjecting both the 50cc and 30R versions to accelerated and stressed temperature conditions (25°C / 60RH and 40°C / 75RH). The stability profiles of the stability-indicating assays from these two structures were compared to the stability data of the drug substance lyophilized using the previous conditions (i.e., without the ice nucleation step and less aggressive drying conditions). The attributes of subvisible particle count (HIAC), reconstitution time, cake moisture, high molecular weight (SEC), and acidic / basic peak (CEX) were tested. No negative differences in stability trends were observed between vials processed using the disclosed method and vials of the same drug substance processed using standard lyophilization procedures. Additionally, the process described herein reduced breakage of 30R vials containing 20 mL fills of drug substance. The inclusion of the ice nucleation step allowed for control of the ice morphology, thus reducing lateral stress on the vial surface, helping to reduce vial breakage compared to standard conditions. An additional benefit of the methods disclosed herein was a decrease in reconstitution time for lyophilized protein compositions in both 30R and 50cc vials (see Figure 4). This is due, for example, to the larger pore size of the cake produced by the disclosed process, which facilitates diluent penetration during reconstitution.

[0067] In summary, the process described herein allowed for a substantial reduction in total lyophilization cycle time by approximately 60% and an increase in manufacturing throughput (lyophilizer vial volume divided by lyophilization time) by approximately 150%. Additionally, the process allowed for the use of 20 mL fill volumes in 30R vials, reducing cycle times. The fill aspect ratio for the 30R vials was 1.16 (fill volume 20 mL, fill height 20 mm, vial body diameter 30 mm, vial wall thickness 1.2 mm, vial inner diameter 27.6). The reduction in vial size and short cycle time resulted in an increase in throughput of over 250% compared to previous processes. Product quality and product stability produced using the methods described herein were comparable to products produced using previous lyophilization processes based on 3-month stability data under accelerated and stressed temperature conditions in both 50cc and 30R vials. Further lyophilization runs were performed using buffer alone in 2R (fill volume 2.4 mL, fill height 13 mm, vial body diameter 16 mm, vial wall thickness 1 mm, vial inner diameter 14) and 6R (fill volume 6 mL, fill height 17 mm, vial body diameter 22 mm, vial wall thickness 1 mm, vial inner diameter 20) vials with fill aspect ratios of 0.93 and 0.85, respectively, and no vial breakage was observed.

[0068] All references cited herein, including patents, patent applications, literature publications, and the like, are hereby incorporated by reference in their entirety.

[0069] Although the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that variations of the preferred compounds and methods may be used, and that it is contemplated that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the invention as defined by the following claims.

Claims

1. A method for preparing a freeze-dried protein composition, (a) A step of providing a vial containing a liquid protein composition having a filling aspect ratio of approximately 0.75 or more, (b) A step of inducing ice nucleation in the vial, which has been exposed to a first temperature of approximately -15°C to approximately -7°C for approximately 30 minutes to approximately 5 hours, (c) A step of exposing the vial containing the composition to a second temperature of approximately -50°C to approximately -25°C for a second period of approximately 1 hour to approximately 5 hours, (d) A step of drying the composition of (c) at a third temperature of about 0°C to about 40°C, the step of exposing the vial containing the protein composition to a temperature increase at a rate of about 0.01°C to about 0.5°C per minute. Methods that include...

2. The method according to claim 1, wherein step (b) includes exposing the vial to the first temperature for about 30 minutes to about 2 hours.

3. The method according to claim 2, wherein step (b) includes exposing the vial to the first temperature for about 90 minutes to about 2 hours.

4. The method according to claim 1, wherein the first temperature in step (b) is about -10°C to about -7°C.

5. The method according to claim 4, wherein the first temperature in step (b) is about -7°C.

6. The method according to claim 1, further comprising step (b) holding the ice-nucleated protein composition at the temperature for a period of up to two hours after nucleation.

7. The method according to claim 6, wherein the period after nucleation is approximately 30 minutes to approximately 90 minutes.

8. The method according to claim 7, wherein the period after nucleation is approximately 60 minutes.

9. The method according to claim 1, wherein step (c) includes exposing the vial to a second temperature of about -45°C.

10. The method according to claim 9, wherein the second period is approximately 2 hours to approximately 4 hours.

11. The method according to claim 10, wherein the second period is approximately 3 hours.

12. The method according to claim 1, wherein the first temperature transitions to the second temperature at a rate of approximately 0.01°C to approximately 0.5°C per minute.

13. The method according to claim 12, wherein the first temperature transitions to the second temperature at a rate of approximately 0.2°C per minute.

14. The method according to claim 1, wherein the third temperature in step (d) is about 0°C to about 25°C.

15. The method according to claim 14, wherein the third temperature in step (d) is approximately 10°C to approximately 25°C.

16. The method according to claim 15, wherein the third temperature in step (d) is approximately 25°C.

17. Step (d) is (d1) A step of holding the vial at a temperature of approximately -5°C to approximately 5°C for approximately 8 to 12 hours, (d2) A step of holding the vial at a temperature of approximately 20°C to approximately 30°C for approximately 20 to 50 hours. The method according to claim 1, including the method described in claim 1.

18. The method according to claim 17, wherein step (d1) includes holding the vial at a temperature of about 0°C for about 10 hours.

19. The method according to claim 17, wherein step (d2) includes holding the vial at a temperature of about 25°C for about 40 hours.

20. The method according to claim 1, wherein the transition from step (c) to step (d) includes increasing the temperature at a rate of approximately 0.2°C to approximately 0.7°C per minute and holding the vial at a temperature of approximately -40°C to approximately -30°C for approximately 15 minutes to approximately 1 hour.

21. The method according to claim 1, wherein the liquid protein composition is filled to at least 50% of the vial's volume before carrying out step (b).

22. The method according to claim 1, further comprising filling the vial with the liquid protein composition to at least 50% of its volume before step (b).

23. The method according to claim 1, wherein the method yields a vial containing a freeze-dried protein composition having a filling aspect ratio of about 1 or more.

24. The method according to claim 1, wherein ice nucleation is induced by ice fog or depressurization.

25. The method according to any one of claims 1 to 24, wherein the protein is an antibody comprising a light chain containing the amino acid sequence shown in SEQ ID NO: 9 and a heavy chain containing the amino acid sequence shown in SEQ ID NO:

10.

26. The method according to any one of claims 1 to 24, wherein the protein is a bispecific antibody construct comprising the amino acid sequence shown in SEQ ID NO: 29, 40, 52, or 53.

27. The method according to any one of claims 1 to 24, wherein the protein is an antibody construct.

28. The method according to claim 27, wherein the antibody construct is an antibody or a bispecific antibody construct.

29. The method according to any one of claims 1 to 24, wherein the protein is a bispecific antibody construct comprising a polypeptide having the amino acid sequence shown in SEQ ID NOs. 89 to 91.

30. A freeze-dried protein composition prepared by the method described in any one of claims 1 to 24.

31. The freeze-dried protein composition according to claim 30, wherein the time required to reconstitute the freeze-dried product into a liquid composition is reduced by at least 25% compared to a freeze-dried product that does not involve the ice nucleation step.

32. The freeze-dried protein composition according to claim 30, wherein the maximum cake resistance of the freeze-dried product is reduced by at least 50% compared to a freeze-dried product that does not involve the ice nucleation step.

33. The freeze-dried protein composition according to claim 30, wherein the protein is an antibody construct.

34. The freeze-dried protein composition according to claim 33, wherein the antibody construct is an antibody or a bispecific antibody construct.

35. The lyophilized protein composition according to claim 34, wherein the protein is a bispecific antibody construct comprising a polypeptide having the amino acid sequence shown in SEQ ID NOs. 89 to 91.

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