Freeze-drying using controlled nucleation

Abstract

This invention relates to an improved lyophilization method for therapeutic proteins, such as monoclonal antibodies, using controlled nucleation. The controlled nucleation method is time-efficient and produces lyophilized products with high quality and short redissolution times. This invention further relates to an efficient lyophilization process for achieving a moderate moisture content in therapeutic proteins that are stable at room temperature.

Description

[Technical Field]

[0001] This application claims priority to U.S. Application No. 63 / 541,573, filed on 29 September 2023, and U.S. Application No. 63 / 681,328, filed on 9 August 2024. These applications are incorporated in their entirety by reference. The present invention relates to an improved freeze-drying method for therapeutic proteins using controlled nucleation. [Background technology]

[0002] Therapeutic proteins include Fc-containing proteins such as monoclonal antibodies (mAbs) and receptor Fc-fusion proteins such as trap proteins, which are used as therapeutic proteins. Lyophilization is a commonly accepted method for the long-term storage of therapeutic proteins. Current platform-type lyophilization methods (i.e., current standard lyophilization methods) are time-consuming and often result in lyophilized preparations with quality issues. This invention provides an improved lyophilization method and an improved lyophilized therapeutic product that does not have the drawbacks of conventional platform technology. This invention further provides a lyophilized therapeutic protein product that is stable at room temperature, a method for producing the same, and a method for obtaining an optimal moisture content in a therapeutic product for producing a lyophilized therapeutic product that is stable at room temperature. [Overview of the Initiative]

[0003] The present invention provides a method for freeze-drying a protein product, the method comprising: (a) cooling a solution containing the protein product to below the freezing point of the solution; (b) pressurizing the solution cooled in step (a) with a gas; (c) releasing the pressure from step (b) to allow nucleation, thereby forming ice nuclei in the cooled solution; and (d) freeze-drying the solution cooled in step (c) to form a freeze-dried protein product. The gas can be selected from the group consisting of air, helium, nitrogen, or argon. During step (b), the gas can be present at 14 to 42 psig, preferably 25 to 30 psig, more preferably about 28 psig.

[0004] The cooled solution can be contained in a lyophilized vial. Step (c) can be carried out at a temperature of -2°C to -10°C, preferably about -5°C. The temperature can be maintained for about 1 hour or less, for example, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 minutes or less. If desired, the temperature can be maintained for more than 1 hour.

[0005] The protein can be an Fc-containing protein, such as an Fc fusion protein, or an antibody. The antibody is preferably a monoclonal antibody.

[0006] If the Fc-containing protein is a monoclonal antibody, the antibody can be redissolved to 150 mg / ml in approximately 3 minutes, 2 minutes 50 seconds, 2 minutes 40 seconds, 2 minutes 30 seconds, 2 minutes 20 seconds, 2 minutes 10 seconds, 2 minutes, 1 minute 50 seconds, 1 minute 45 seconds, 1 minute 40 seconds or less, and additional subranges set based on the above values.

[0007] Lyophilized proteins produced by this method are also provided. Preferably, the protein can be an Fc-containing protein such as an antibody and an Fc fusion protein. The present invention will be further described and discussed below.

[0008] The present invention provides a method for producing a freeze-dried therapeutic protein product that is stable at room temperature by setting a target residual moisture content in the freeze-dried therapeutic protein product that is stable at room temperature, the method further comprising (a) a step of controlling the shelf temperature (Ts) after ice sublimation without performing a secondary drying step, and (b) a step of controlling the duration of time after primary drying, wherein the target residual moisture content in the freeze-dried therapeutic protein product is about 1% to about 7%. The target residual moisture content in lyophilized therapeutic protein products can be approximately 2%–6%, 2%–5%, 2%–4%, 2%–3%, 3%–7%, 3%–6%, 3%–5%, 3%–4%, 4%–7%, 4%–6%, 4%–5%, 5%–7%, 5%–6%, 6%–7%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, or 7.0%, and additional sub-ranges set based on the above values. The stability of lyophilized therapeutic protein products can be measured by evaluating the change in the percentage of high molecular weight protein aggregates in the product. The change in the percentage of high molecular weight protein aggregates in a stable lyophilized therapeutic protein product is less than 1.0% when measured by size exclusion chromatography-ultrahigh performance liquid chromatography (SEC-UPLC). The change in the percentage of high molecular weight protein aggregates in a stable lyophilized therapeutic protein product is approximately 1.0%, approximately 0.9%, approximately 0.8%, approximately 0.7%, approximately 0.6%, approximately 0.5%, approximately 0.4%, approximately 0.3%, approximately 0.2%, approximately 0.1%, or approximately 0%, and additional subranges set based on the above values. The present invention further provides room-temperature stable lyophilized therapeutic protein products prepared by any of the methods disclosed herein. [Brief explanation of the drawing]

[0009] [Figure 1] A is a graph illustrating the freeze-drying process, including the annealing step. B and C show a comparison of conventional and controlled nucleation methods for multiple lots of monoclonal antibody preparations. B illustrates data from a conventional freezing platform. C illustrates data from controlled nucleation. [Figure 2] A and B illustrate data from controlled nucleation parameter profiles for multiple lots of monoclonal antibody preparations. A is at -5°C, and B is at -10°C. [Figure 3] Figures A-D illustrate data from various lyophilization methods for multiple lots of monoclonal antibody preparations. Figure A illustrates data from conventional platform lyophilization. Figure B illustrates data from controlled nucleation at -5°C. Figure C illustrates data from controlled nucleation at -10°C. Figure D illustrates data from conventional platform lyophilization with annealing (see the boxed sections between 0.0 and 20.0 hours). Arrows in each figure indicate the end of primary drying. [Figure 4] The primary drying time and freezing time and primary drying time are illustrated for conventional platform freeze-drying (see Figure 3A), controlled nucleation at -5°C (see Figure 3B), controlled nucleation at -10°C (see Figure 3C), and conventional platform freeze-drying with annealing (see Figure 3D). [Figure 5]Figures A and B illustrate size exclusion chromatography (SEC) data for the percentage of natural mAbs using a buffer containing 150 mg / ml of mAb and 5% or 1.5% sucrose. Figure A shows results from samples stored at 25°C. Figure B shows results from samples stored at 40°C. Figures C and D illustrate size exclusion chromatography (SEC) data for compositions F1, F2, F3, and F4 (see compositions shown in Table 1). Figure C is from 25°C, and Figure D is from 40°C. [Figure 6] A and B: A shows platform-type lyophilized mAb preparations in vials, from left to right: platform-type pharmaceutical product (DP), low-concentration placebo product (PP), low-concentration pharmaceutical formulation, and platform-type placebo product. See the compositions shown in Table 1. The arrows in the figure indicate product shrinkage. B illustrates lyophilized mAb preparations in vials, including placebo pharmaceutical products and low-concentration pharmaceutical products (DP) using lyophilization with annealing at -5°C, controlled nucleation without annealing, and controlled nucleation at -5°C. The arrows in the figure indicate product shrinkage. [Figure 7] The images show lyophilized mAb preparations in vials prepared using controlled nucleation, from left to right: platform-type pharmaceutical product, low-concentration placebo product, platform-type placebo product, and low-concentration pharmaceutical product. See Table 1 for the compositions. The top row was prepared at -5°C, and the bottom row at -10°C. [Figure 8] The image shows platform-type lyophilized mAb preparations in vials that have been annealed prior to lyophilization. From left to right, the products are: low-concentration pharmaceutical product (DP), platform-type pharmaceutical product, low-concentration placebo product (PP), and platform-type placebo product. See Table 1 for the compositions. The arrows in the figure indicate the products on the walls. [Figure 9]The images show a platform-type lyophilized mAb preparation and a lyophilized mAb preparation in a vial prepared using controlled nucleation at -5°C, using a 5% w / v sucrose composition (left) and a 1.5% sucrose composition (right). The arrows in the figure indicate the product on the wall in the case of conventional lyophilization. [Figure 10] A and B show scanning electron microscope (SEM) images at the same scale. A is from a platform-type lyophilized (conventional) preparation, and B is from a lyophilized preparation prepared using controlled nucleation at -5°C. See the compositions shown in Table 1. [Figure 11] Images A and B show scanning electron microscope (SEM) images at the same scale. Image A is from a lyophilized preparation prepared using controlled nucleation at -5°C. Image B is from a lyophilized preparation prepared using controlled nucleation at -10°C. See the compositions shown in Table 1. [Figure 12] Images A and B show scanning electron microscope (SEM) images at the same scale. Image A is from conventional platform freeze-drying. Image B is from conventional platform freeze-drying with annealing at -5°C. [Figure 13] This graph illustrates the data regarding the annealing effect on resistance exhibited by a dried 5% glycine product. [Figure 14] Figures A and B illustrate data regarding moisture content and the stability of the drug composition at 5°C, 25°C, and 30°C, as well as at 37°C and 50°C. [Figure 15] Figures A and B illustrate an efficient freeze-drying process to achieve a moderate moisture content. Primary drying is complete when the vapor composition is predominantly nitrogen. The pressure measured by the Pirani vacuum gauge is approximately equal to the chamber pressure measured by the capacitance meter. Figure A indicates STOP after the completion of ice sublimation. [Figure 16] This graph illustrates the optimal combination of shelf temperature and time after ice sublimation. [Figure 17]This is a graph illustrating the percentage (%) of high molecular weight species measured by SE-UPLC for a stable lyophilized DP having a moderate moisture content and stored at 25°C. [Figure 18] This is a bar graph illustrating data from a Micro-Flow Imaging (MFI) analytical assay for unfiltered particles of 10 μm or larger and the effect on sub-visible particles. [Figure 19] This is a bar graph illustrating data from a Micro-Flow Imaging (MFI) analytical assay for unfiltered particles of 25 μm or larger and the effect on sub-visible particles. [Figure 20] A and B are graphs showing the percentage (%) of high molecular weight aggregates in platform DP (A) and low-concentration DP (B) stored for more than 6 months at a temperature of 25°C under 60% relative humidity (RH), as measured by SEC. [Figure 21] A and B are graphs showing the percentage (%) of the main peak (Region 2) in platform DP (A) and low-concentration DP (B) during 6 months of storage, as measured by cation exchange chromatography (CEX). [Figure 22] This is a graph showing the effect of 5% lyoprotectant on 25 μm particles (A) and 10 μm particles (B) in the DP composition at different temperatures (25°C and 40°C) and different time periods (t0, 1 month and 3 months). [Figure 23] A and B are graphs showing the effect of 1.5% lyoprotectant on 10 μm particles (A) and 25 μm particles (B) in the DR composition at different temperatures (25°C and 40°C) and different time periods (t0, 1 month and 3 months). [Figure 24] This is a graph illustrating the isotherm of N₂ measured at 77K as a function of relative pressure. [Figure 25] This is a graph illustrating logarithmic differential pore size distribution data based on Hg intrusion volume. [Figure 26] This is a graph showing cumulative pore size distribution data based on Hg intrusion volume. MODES FOR CARRYING OUT THE INVENTION

[0010] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0011] definition In the context of numbers and ranges, the term “approximately” refers to a value or range that approximates or is close to the enumerated value or range, as is evident from the teachings contained herein, such that the invention has the presence of, for example, a desired rate, quantity, density, degree, increase, decrease, percentage (%), value, or quantity of form, variant, temperature, or time, so that the invention can function as intended. For example, “approximately” can refer to a value that is either above or below the stated value, depending on its ability to function, and is within or above a range of approximately + / - 10%. Thus, the term encompasses values ​​that exceed the value that would result simply from systematic error.

[0012] Antibodies (also called immunoglobulins) are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. Canonical immunoglobulin proteins (e.g., IgG) contain four polypeptide chains: two light chains and two heavy chains. Each light chain is linked to one heavy chain via a cysteine ​​disulfide bond, and the two heavy chains are linked to each other via two cysteine ​​disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (e.g., asparagine residues) by various polysaccharides, which can vary by species and may affect the antigenicity of therapeutic antibodies. (Butler and Spearman, “The choice of mammalian cell host and possibilities for glycosylation engineering”, Curr. Opin. Biotech. 30:107-112 (2014)). Antibodies are often used as therapeutic biomolecules.

[0013] The antibody comprises an immunoglobulin molecule consisting of four polypeptide chains interconnected by disulfide bonds, namely two heavy (H) chains and two light (L) chains. Each heavy chain includes a heavy chain variable region (hereinafter abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain includes a light chain variable region (hereinafter abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which contain interspersed more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3, and light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody is defined as having at least 10% affinity to the target when measured by surface plasmon resonance, e.g., BIACORE® or solution affinity ELISA. -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 This refers to antibodies that have binding affinity for M.

[0014] Antibodies can be based on all major antibody classes, namely IgG, IgA, IgM, IgD, and IgE. IgG is the preferred class and includes subclasses IgG1 (including IgG1λ and IgG1κ), IgG2, IgG3, and IgG4. Antibodies include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single-chain antibodies, diabodies, triabodies or tetrabodies, Fab fragments or F(ab′)2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

[0015] The antibody can be an IgG1 antibody. The antibody can be an IgG2 antibody. The antibody can be an IgG3 antibody. The antibody can be an IgG4 antibody. The antibody can be a chimeric IgG2 / IgG4 antibody. The antibody can be a chimeric IgG2 / IgG1 antibody. The antibody can be a chimeric IgG2 / IgG1 / IgG4 antibody. The above derivatives, components, domains, chains, and fragments are also included.

[0016] The term "bispecific antibody" refers to an antibody that can selectively bind to two or more epitopes. A bispecific antibody generally contains two distinct heavy chains, each specifically binding to a different epitope on either two different molecules (e.g., antigens) or the same molecule (e.g., the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain to the first epitope is generally at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain to the second epitope, and vice versa. The epitopes recognized by a bispecific antibody can be located on the same target or on different targets (e.g., on the same protein or different proteins). A bispecific antibody can be created, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, a nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused to a nucleic acid sequence encoding a different heavy chain constant region, and such a sequence can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs, then (from N-terminus to C-terminus) a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and can bind to one or more epitopes bound by the heavy chain antigen-binding region, or can associate with each heavy chain and allows one or both heavy chains to bind to one or both epitopes.

[0017] The terms “heavy chain” or “immunoglobulin heavy chain” include the constant region sequence of an immunoglobulin heavy chain from any organism and, unless otherwise specified, the heavy chain variable domain. The heavy chain variable domain includes three heavy chain CDRs and four FR regions unless otherwise specified. Fragments of a heavy chain include CDRs, CDRs, and FRs, as well as combinations thereof. A typical heavy chain has a variable domain followed (from N-terminus to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of a heavy chain are capable of specifically recognizing an antigen (e.g., recognizing an antigen in the micromolar, nanomolar, or picomolar KD range), are capable of being expressed and secreted from cells, and include fragments containing at least one CDR.

[0018] The term “light chain” includes the constant region sequence of an immunoglobulin light chain from any organism, and unless otherwise specified, includes human kappa and lambda light chains. The variable light chain (VL) domain typically includes three light chain CDRs and four framework (FR) regions, unless otherwise specified. Generally, a full-length light chain includes a VL domain containing FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the amino terminus to the carboxyl terminus, and a constant light chain domain. Light chains that can be used in these inventions include, for example, light chains that do not selectively bind to either a first or second antigen selectively bound by an antigen-binding protein. Suitable light chains include those that can be identified by screening for the light chains most commonly used in existing antibody libraries (wet libraries or in silico), and the light chain does not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include those that can bind to one or both epitopes bound by the antigen-binding domain of the antigen-binding protein.

[0019] The term “variable domain” includes an amino acid sequence (as desired, modified) of an immunoglobulin light or heavy chain, including, in order from the N-terminus to the C-terminus (unless otherwise specified), the following amino acid regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. A “variable domain” includes an amino acid sequence that can fold into a canonical domain (VH or VL) having a double beta-sheet structure, where the beta sheets are connected by disulfide bonds between the residues of the first beta-sheet and the residues of the second beta-sheet.

[0020] The term “complementarity-determining region” or “CDR” typically refers to an amino acid sequence encoded by the nucleic acid sequence of an organism’s immunoglobulin gene that appears between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor) in wild-type animals. A CDR can be encoded by, for example, a germline sequence, or by a sequence that has been rearranged or not rearranged, for example, by a naive or mature B cell or T cell. In some situations (e.g., CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not adjacent (e.g., in an unrearranged nucleic acid sequence) but are adjacent in a B cell nucleic acid sequence as a result of, for example, sequence splicing or joining (e.g., VDJ recombination that forms a heavy chain CDR3).

[0021] "Antibody derivatives and fragments" include, but are not limited to, antibody fragments (e.g., Fab, ScFv-Fc, dAB-Fc, semi-antibodies, and other combinations of heavy and / or light chains) and multispecific molecules (e.g., bispecific, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, and triplicate molecules).

[0022] The term "Fc-containing protein" includes antibodies, bispecific antibodies, antibody derivatives containing Fc, antibody fragments containing Fc, Fc fusion proteins, immunoadhesins, and other binding proteins containing at least functional portions of the immunoglobulin CH2 and CH3 regions. "Functional portion" refers to the CH2 and CH3 regions that can bind to Fc receptors (e.g., FcyR, or FcRn (neonatal Fc receptor)) and / or be involved in complement activation. The CH2 and CH3 regions are non-functional if they contain deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any Fc receptor and from activating complement. Examples of Fc fusion proteins include Fc fusion (N-terminal), Fc fusion (C-terminal), single Fc fusion, and bispecific Fc fusion proteins.

[0023] "Fc" is an abbreviation for crystallizable fragment, often referred to as a constant fragment. Antibodies contain an Fc region formed by two identical protein sequences. IgG has a heavy chain known as the γ chain. IgA has a heavy chain known as the α chain, and IgM has a heavy chain known as the μ chain. IgD has a heavy chain known as the σ chain. IgE has a heavy chain known as the ε chain. In nature, the Fc region is identical in all antibodies of a given class and subclass within the same species. Human IgG has four subclasses, sharing approximately 95% homology among them. In each subclass, the Fc sequence is identical. For example, human IgG1 antibodies have the same Fc sequence. Similarly, IgG2 antibodies have the same Fc sequence, IgG3 antibodies have the same Fc sequence, and IgG4 antibodies have the same Fc sequence. Changes in the Fc region result in charge fluctuations.

[0024] Fc-containing proteins may include modifications to their immunoglobulin domains, where the modifications affect one or more effector functions of the binding protein (e.g., modifications affecting FcyR binding, FcRn binding, and therefore half-life, and / or CDC activity). Such modifications refer to the EU numbering of the immunoglobulin constant region, as follows: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 32 Examples of modifications and combinations thereof include 6, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439, but these are linear.

[0025] For example, but not limited to, the binding protein is an Fc-containing protein and exhibits an enhanced serum half-life (compared to the same Fc-containing protein without the listed modifications), and further has modifications at position 250 (e.g., E or Q), position 250 and position 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or position 428 and / or 433 (e.g., L / R / S / I / P / Q or K) and / or position 434 (e.g., H / F or Y), or modifications at position 250 and / or 428, or position 307 or 308 (e.g., 308F, V308F), and modification at position 434. In another example, the modifiers may include 428L (e.g., M428L) and 434S (e.g., N434S) modifiers, 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modifiers, 433K (e.g., H433K) and 434 (e.g., 434Y) modifiers, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifiers, 250Q and 428L modifiers (e.g., T250Q and M428L), and 307 and / or 308 modifiers (e.g., 308F or 308P).

[0026] Some recombinant Fc-containing proteins include receptors or receptor fragments, ligands or ligand fragments that have a congenital binding partner in a biological system, and include "receptor Fc fusion proteins," which refer to recombinant molecules containing a soluble receptor fused to an immunoglobulin Fc domain.

[0027] Fc fusion proteins contain part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, which is not fused in its native state. Examples of Fc fusion proteins include Fc fusion (N-terminus), Fc fusion (C-terminus), mono-Fc fusion, and bispecific Fc fusion. The preparation of fusion proteins containing a specific heterologous polypeptide fused to various parts (including the Fc domain) of an antibody-derived polypeptide has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. Sci USA 88:10535-39 (1991), Byrn et al., Nature 344:677-70, 1990, Byrn et al., Nature 344:677-70, 1990, and Hollenbaugh et al., “Construction of Immunoglobulin Fusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11 (1992). A “receptor Fc fusion protein” comprises one or more extracellular domains of a receptor bound to the Fc portion, and in some embodiments, comprises a hinge region followed by the CH2 and CH3 domains of the immunoglobulin. In some embodiments, the Fc fusion protein contains two or more distinct receptor chains that bind to one or more ligands. Some receptor Fc fusion proteins may contain ligand-binding domains of multiple different receptors. Receptor Fc fusion proteins are also called “traps,” “trap molecules,” or “trap proteins.” For example, such trap proteins include IL-1 traps (e.g., lilonacept, containing an IL-1RAcP ligand-binding domain fused to an IL-1R1 extracellular domain fused to the Fc of hlgGl, see U.S. Patent No. 6,927,044) or VEGF traps (e.g., aflibercept, containing an Ig domain 2 of VEGF receptor Fltl fused to an Ig domain 3 of VEGF receptor Flkl fused to the Fc of hlgG1, see U.S. Patents No. 7,087,411 and 7,279,159).

[0028] Lilonacept and aflibercept are examples of commercially available trap proteins that antagonize IL1R (see U.S. Patent No. 7,927,583) and VEGF (see U.S. Patent No. 7,087,411), respectively. Other recombinant Fc-containing proteins include these recombinant proteins containing peptides fused to the Fc domain. Recombinant Fc-containing proteins are described in C. Huang, “Receptor-Fc fusion therapeutics, traps, and MFMETIBODY technology,” 20(6)Curr.Opin.Biotechnol.692-9(2009).

[0029] Proteins lacking the Fc moiety, such as recombinant enzymes and minitraps, can also be freeze-dried according to the present invention. Minitraps are trap proteins that use a polymerizing component (MC) instead of the Fc moiety, as disclosed in U.S. Patents 7,279,159 and 7,087,411. The above derivatives, components, domains, chains, and fragments are also included.

[0030] "Lyophilized cake" or "cake" refers to a lyophilized drug, including, for example, a protein drug in a lyophilized form within a container such as a vial.

[0031] "Platform" refers to currently used methods, including standard methods.

[0032] All numerical limits and ranges described herein include all numerical values ​​or values ​​immediately before, after, or between the numerical values ​​of the range or limit. The ranges and limits described herein expressly represent and indicate all integers, decimals, and fractional values ​​defined and encompassed by the range or limit. Therefore, the enumeration of value ranges herein is merely intended to serve as a way of individually referring to each individual value within that range unless otherwise indicated herein, and each individual value is incorporated into the specification as if it were individually enumerated herein. All numerical values ​​or values ​​explicitly indicated represent all ranges formed by the enumerated numerical values ​​or values.

[0033] explanation Lyophilization of biological products such as Fc-containing proteins has been used for many years for medical purposes. Lyophilized products tend to have a longer shelf life than liquid products due to the absence of water and reduced internal molecular dynamics. In addition, lyophilized products can be redissolved to higher protein concentrations than the solution before lyophilization. Higher concentrations are useful for intravenous and subcutaneous administration. Furthermore, subcutaneous administration requires higher concentrations of the biological product upon redissolution.

[0034] Protein pharmaceutical products are typically stored at 2-8°C, and room temperature (storage) stability is a significant improvement. The freeze-drying manufacturing process is usually time- and energy-intensive, depending on the process design. A typical freeze-drying process consists of three consecutive stages, namely: i. Freezing stage (which may include annealing or controlled nucleation steps), ii. A primary drying stage (which can be designed to improve process efficiency and protein storage stability), iii. A secondary drying step (if necessary) is included.

[0035] Primary drying is typically the longest stage and is the focus of process efficiency. Improving stability and process efficiency has become a major focus in order to achieve the goals of patient convenience, energy and cost savings (as well as environmental protection). A diagram of a typical freeze-drying process, including the annealing step, is shown in Figure 1A.

[0036] Strategies for improving freeze-dried pharmaceutical products (DP) The following measures can be used. 1. Stabilizers such as sucrose (or trehalose) can be used in the freeze-dried composition as follows: (a) Sucrose and trehalose are disaccharides and are used as cryoprotectants and freeze-drying protectants. In high-concentration protein compositions, it can be difficult to have a sufficient stabilizer ratio to protein. (b) The stabilizer is in the same phase as the protein, forming an amorphous matrix that stabilizes the protein in the solid state, and / or (c) Water is used as an alternative stabilizer, and this stabilizer does not increase the viscosity / osmotic pressure of the composition. 2. The compositional strategies for improving the efficiency of the freeze-drying process are as follows: (a) Use a crystalline filler to increase the decay temperature (Tc) of the composition. Preferred options include mannitol and glycine (with a high eutectic temperature of about 33°C), and / or (b) Freeze-drying can be performed at temperatures greater than Tg' and less than Tc, which can shorten the ice sublimation time. 3. Compositions and / or process strategies for improving freeze-dried DP, as follows: (a) A combination of stabilizers / plasticizers (containing water molecules and having a smaller molecular weight) may be used. (b) Stable proteins with high water content do not require or may not require high-intensity secondary drying, and / or (c) Controlled nucleation and annealing after freeze-drying may be used.

[0037] Freeze-drying process design The following conditions may apply. 1. The freezing / cooling rates are as follows: (a) A fast freezing rate (e.g., 2°C / min), which is usually not achievable at low temperatures (e.g., below -30°C), and (b) The temperature can be set to 0.5 to 1 degree Celsius per minute. 2. The freezing point (simple but extremely important) is as follows: (a) To ensure that all solutes are in a solid state, Tg' is less than, (b) The product is below -35°C due to supercooling (inability to scale up). Supercooling is not a concern when using controlled nucleation (CN) techniques, and (c) Typically, the temperature is -40 / -50°C. 3. Annealing for volume expanders or amorphous agents, as follows: (a) Crystallization of the bulking agent, (b) Having a duration of 2 to 10 hours at a temperature between Teu and Tg'. Annealing at higher temperatures requires a shorter time, and (c) Improve the morphology and drying efficiency of amorphous drug cakes.

[0038] The experimental design used in this specification is as follows, as shown in Table 1 and the relevant text: [Table 1] * Each composition was filled with 5.3 mL using the 20R cycle. (5.3 mL was filled into a 20 mL Type 1 glass vial, and the lyophilized DP was redissolved in 4.56 mL of WFI to obtain a 150 mg / mL composition). The purpose of this experiment was to evaluate the effect of the CN lyophilization cycle on the stability of mAb3 pharmaceutical products.

[0039] Types of freeze-drying cycles (a) Freeze-drying cycle of platform-type placebo (50 hours at -20°C and 12 hours at 40°C). This is a conventional freeze-drying method. (b) Annealing by a conventional freeze-drying cycle with annealing (-5°C for 6 hours). Conventional freeze-drying methods such as a platform-type placebo freeze-drying cycle are used with annealing performed first. (c) Freeze-drying cycle with controlled nucleation (CN) at -5°C. (d) Freeze-drying cycle with controlled nucleation (CN) at -10°C.

[0040] Test method Typical analytical methods for freeze-dried products include appearance, size exclusion chromatography (SEC), cation exchange chromatography (CEX), and microflow imaging (MFI).

[0041] The freeze-drying process typically consists of three stages: (1) freezing, (2) primary drying by sublimation, and (3) secondary drying by desorption. The freezing step is the first part of freeze-drying. In conventional platform-type methods, freezing can occur randomly. See Figure 1B. Conventional platform techniques result in lower freezing temperatures (greater supercooling), such as -11°C to -17°C, which consequently leads to significant supercooling and non-uniformity of freezing temperatures, as shown in Figure 1B.

[0042] Freezing by controlled nucleation results in less supercooling at a constant temperature (e.g., -5°C). Figure 1C shows that controlled nucleation occurs at -5°C according to the present invention. The arrow in Figure 1C points to a constant freezing temperature, in stark contrast to the non-uniform temperature shown in Figure 1B. Temperatures not below -10°C are preferred, for example, about -2°C, -2.5°C, -3°C, -3.5°C, -4°C, -4.5°C, -5°C, -5.5°C, -6°C, -6.5°C, -7°C, -7.5°C, -8°C, -8.5°C, -9°C, -9.5°C, or -10°C, and additional sub-ranges formed based on the above values.

[0043] There are various methods to achieve controlled nucleation, including (1) seeding and vial pretreatment such as the use of additives / surface roughening, (2) ultrasonic nucleation: short-duration vibration, (3) the use of ice mist to freeze moisture in the chamber by introducing cooling nitrogen gas into the chamber, and (4) reduced pressure (however, this carries the risk of product splashing).

[0044] A preferred method according to the present invention uses a pressure of 14 to 42 psig, preferably 25 to 30 psig, and more preferably about 28 psig. The pressure inside the freeze-drying chamber can be measured, for example, by using a Pirani vacuum gauge. Basic methods include the following: (1) Cool the product to its intended freezing point (or a temperature slightly lower than that freezing point). (2) Pressurize the freeze-drying chamber with air or an inert gas, for example, nitrogen or argon (about 28 psig), and (3) Rapidly release pressure to introduce ice nuclei.

[0045] More specifically, the present invention provides embodiments that include the following: Load the container and seal the freeze dryer. To cool the shelves and containers to the target nucleation temperature, Pressurizing with air or an inert gas (e.g., helium, nitrogen, and / or argon), Rapid depressurization to induce nucleation, and Lower the shelf temperature to complete the freezing process.

[0046] Typically, the present invention provides an improved freeze-drying method that yields a product having improved properties, as described herein. The present invention utilizes controlled nucleation.

[0047] Figures 2A and 2B show data from controlled nucleation parameter profiles for the preparation of different monoclonal antibody drug products. Figure 2A is for -5°C, and Figure 2B is for -10°C. Controlled nucleation at -5°C resulted in less supercooling compared to controlled nucleation at -10°C, ensuring ice formation for all vials at the set temperature. At -5°C, a retention time of approximately 30 minutes during nucleation was sufficient. See Figure 2A. The holding time may be 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 minutes, or a longer or shorter time determined in accordance with the teachings contained herein.

[0048] Figures 3A to 3D show data from various lyophilization methods in the preparation of different monoclonal antibody drug products. Figure 3A shows data from conventional platform lyophilization. Figure 3B shows data from controlled nucleation at -5°C. Figure 3C shows data from controlled nucleation at -10°C. Figure 3D shows data from conventional platform lyophilization with annealing (see the circled area between 0.0 hours and approximately 15 hours). Arrows in each figure indicate the end of primary drying.

[0049] Table 2 below shows data demonstrating that the product exhibits lower resistance to ice sublimation from controlled nucleation (CN) and annealing cycles compared to conventional platform-type systems. [Table 2]

number

[0050] Figure 4 shows the primary drying time and freezing time for the conventional platform type (see Figure 3A), controlled nucleation at -5°C (see Figure 3C), controlled nucleation at -10°C (see Figure 3C), and the conventional platform type with annealing (see Figure 3D).

[0051] Overall, controlled nucleation significantly reduced freeze-drying cycle times. Controlled nucleation at -5°C was faster than controlled nucleation at -10°C. Annealing also reduced primary drying time, but it required additional cycle time compared to controlled nucleation at -5°C. For controlled nucleation at -5°C, the freeze-drying and primary drying combined took approximately 39 hours, compared to approximately 48 hours with the annealing method.

[0052] Next, we evaluated the moisture content of freeze-drying using conventional platform-type methods, annealing, controlled nucleation (CN) at -5°C (performed twice), and controlled nucleation at -10°C (performed twice). The results are shown in Table 3. [Table 3] * A platform-type DP=20R vial is filled with 5.3 mL of the platform-type composition (50 mg / mL mAb, 10 mM histidine, 5% sucrose, 0.1% PS20, pH 6.0). **A platform-type PP=20R vial filled with 5.3 mL of a placebo composition compatible with the platform type.**

[0053] All methods produced freeze-dried products with an acceptable moisture content (less than 4%). Increased moisture content was predicted from freeze-drying cycles using annealing or controlled nucleation. When lower moisture content is required, more aggressive secondary drying conditions should be used when employing controlled nucleation.

[0054] Next, the redissolution time of lyophilized pharmaceutical products produced by conventional platform-type, annealing, and controlled nucleation lyophilization methods was evaluated, and the results are shown in Table 4 below. [Table 4]

[0055] When redissolved in a high-concentration antibody drug product (150 mg / mL), the fastest redissolution time was observed at controlled nucleation (CN)-5°C. The redissolution time can be less than 3 minutes. More specifically, the redissolution time can be approximately 2 minutes 50 seconds or less, approximately 2 minutes 40 seconds or less, approximately 2 minutes 30 seconds or less, approximately 2 minutes 20 seconds or less, approximately 2 minutes 10 seconds or less, approximately 2 minutes or less, approximately 1 minute 50 seconds or less, approximately 1 minute 45 seconds or less, and approximately 1 minute 40 seconds or less. Redissolution to lower concentrations is even faster. For example, the redissolution time for a 50 mg / mL antibody drug product may be approximately 59 seconds or less, approximately 58 seconds or less, approximately 57 seconds or less, approximately 56 seconds or less, approximately 55 seconds or less, approximately 54 seconds or less, approximately 53 seconds or less, approximately 52 seconds or less, approximately 51 seconds or less, approximately 50 seconds or less, approximately 49 seconds or less, approximately 48 seconds or less, approximately 47 seconds or less, approximately 46 seconds or less, approximately 45 seconds or less, approximately 44 seconds or less, approximately 43 seconds or less, approximately 42 seconds or less, approximately 41 seconds or less, or 40 seconds or less.

[0056] The slowest method was conventional platform-type lyophilization. Similar, but less pronounced, data trends were observed in redissolution to 50 mg / mL. Very low-concentration pharmaceutical products and placebo products redissolved to 2 mg / mL or 6 mg / mL showed no significant difference in redissolution time (all less than 1 minute) (data not shown).

[0057] The effects of cryoprotectants and the formation of high molecular weight (HMW) aggregates in samples stored at 25°C and 40°C were analyzed using size exclusion chromatography (SEC). Figures 5A and 5B show SEC data for the percentage of native mAb using a buffer containing 150 mg / mL mAb and 5% or 1.5% sucrose (see Table 1). Figure 5A is for compositions stored at 25°C, and Figure 5B is for compositions stored at 40°C. Figures 5C and 5D show SEC and high molecular weight (HMW) data for compositions F1, F2, F3, and F4 (see compositions in Table 1). Figure 5C is for compositions stored at 25°C, and Figure 5D is for compositions stored at 40°C. For compositions containing 1.5% sucrose, a higher percentage of protein purity (%) after lyophilization (less HMW formation) was found compared to conventional lyophilization cycles using CN (F4 vs. F3). The degradation rates were similar. For stable compositions containing 5% sucrose, a slightly superior stability was observed when comparing CN(F2) with the conventional method (F1).

[0058] Each mAb composition (5% sucrose and 1.5% sucrose) was lyophilized using a conventional platform-based method and controlled nucleation. Prior to SEC, the compositions were stored at 25°C and 40°C.

[0059] For storage at both 25°C and 40°C, there was little difference in the molecular weight curves between conventional and controlled nucleation for the 5% sucrose composition. In contrast, the 1.5% sucrose composition showed degradation over time when stored at both 25°C and 40°C. Freeze-drying with controlled nucleation showed less degradation than conventional freeze-drying at both temperatures when using the 1.5% sucrose composition.

[0060] In the above comparison, there was no substantial difference in protein recovery between controlled nucleation-based freeze-drying and conventional freeze-drying, nor between storage at both temperatures. Protein recovery ranged from 97% to 99%.

[0061] As there is a demand for more stable pharmaceutical products using more efficient freeze-drying processes, freeze-dried protein pharmaceutical products can be improved by applying advances in composition and process strategies.

[0062] The present invention provides that increasing the water content of a freeze-dried drug can be an efficient method for improving the stability of DP. Water appears to be the most effective plasticizer for reducing the local mobility of mAb molecules, and the target optimal water content can be achieved by controlling the freeze-drying process.

[0063] The present invention also provides lyophilized protein pharmaceutical products that are stable at room temperature and produced by an efficient freeze-drying process.

[0064] The present invention is further illustrated by the following examples, which do not limit the invention in any way. The following experiments and / or examples, or the order in which the steps of the examples are carried out, may be modified or combined as determined by those skilled in the art in light of the teachings and data contained herein.

[0065] Example 1 - Lyophilized mAb cake in a vial Figure 6A shows the lyophilized mAb preparations in vials, from left to right: platform drug product (DP), low-concentration placebo product (PP), low-concentration drug product, and platform placebo product. The compositions are shown in Table 1.

[0066] In three of the four vials in the lower row, the lyophilized mAbs showed shrinkage of the product at the bottom of the vial, as indicated by the arrows. If visual inspection determines that the shrinkage is due to cake collapse or meltback, the vial will be considered unsuitable.

[0067] Figure 7 shows lyophilized mAb preparations in vials prepared using controlled nucleation, from left to right: platform-type pharmaceutical product, low-concentration placebo product, platform-type placebo product, and low-concentration pharmaceutical product. The top row was prepared at -5°C, and the bottom row at -10°C. See Table 1 for compositions. Overall, lyophilized mAbs using controlled nucleation did not show significant shrinkage compared to conventional lyophilization of mAbs shown in Figure 6A.

[0068] Figure 6B shows lyophilized mAb preparations in vials. The bottom panel shows placebo and low-concentration drug products (DP) using lyophilization with annealing at -5°C, the top panel shows controlled nucleation without annealing, and the middle panel shows controlled nucleation at -5°C. Arrows in the figure indicate product shrinkage. Both controlled nucleation and annealing can improve the cake structure, but annealing... (a) Adhesion of the product to the wall surface, and / or (b) This may result in other unexpected problems, such as skin formation that causes the cake to collapse.

[0069] Moderate product shrinkage is typically acceptable (however, if the product is handled as cake collapse or meltback upon visual inspection, it is unacceptable).

[0070] Figure 8 shows the lyophilized mAb preparations in vials that were annealed prior to lyophilization, from left to right: low-concentration pharmaceutical product (DP), platform pharmaceutical product, low-concentration placebo product (PP), and platform placebo product. See Table 1 for compositions.

[0071] Adding an annealing step to freeze-drying reduced cake shrinkage, but also resulted in the mAb product adhering to the vial walls (see arrow in Figure 8).

[0072] Figure 9 shows a platform-type lyophilized high-concentration (150 mg / mL) mAb preparation, as well as lyophilized high-concentration (150 mg / mL) mAb preparations in vials prepared by controlled nucleation at -5°C using a 5% w / v sucrose composition (left) and a 1.5% sucrose composition (right). The lyophilized mAb cake using controlled nucleation showed fewer cracks than the conventional lyophilized mAb cake. See the compositions in Table 1. The arrows in Figure 9 indicate cracks in the conventional lyophilized mAb cake.

[0073] Improved cake appearance: DP from CN at -5°C resulted in fewer or no cracks in freeze-dried cakes.

[0074] Product purity: CN showed higher purity (less aggregation) compared to the conventional method (for a 150 mg / mL mAb3 composition containing 1.5% v / w sucrose).

[0075] When stored at 25°C and 40°C for 3 months, no substantial changes were observed in cation exchange chromatography (CEX) or in the particles.

[0076] Example 2 - Electron microscopy of freeze-dried mAbs A scanning electron microscope (SEM) was used to evaluate the morphology of freeze-dried mAb samples. A 100 μm scale bar is placed in the upper right corner of each figure.

[0077] Figures 10A and 10B show scanning electron microscope images at the same scale. Figure 10A is from a platform-type lyophilized preparation (50 mg / ml), and Figure 10B is from a lyophilized preparation prepared using controlled nucleation at -5°C. See Table 1 for composition.

[0078] Comparison of cake morphology between conventional freeze-drying cycles and freeze-drying cycles with controlled nucleation (SEM) Conventional nucleation-induced freeze-drying (Figure 10B) resulted in larger pore sizes than conventional freeze-drying (Figure 10A). The larger pore sizes resulting from controlled nucleation consequently lead to faster remelting times and lower cake resistance during ice sublimation.

[0079] Figures 11A and 11B show scanning electron microscope images at the same scale. Figure 11A is from a lyophilized platform (50 mg / ml) preparation prepared using controlled nucleation at -5°C, and Figure 11B is from a lyophilized platform (50 mg / ml) preparation prepared using controlled nucleation at -10°C. See the compositions in Table 1. Controlled nucleation at -5°C (Figure 11A) results in larger pore sizes than controlled nucleation at -10°C (Figure 11B), which in turn results in faster redissolution times and lower cake resistance during ice sublimation. Figure 11A is identical to Figure 10B. The microscope images are reproduced for easier comparison.

[0080] Figures 12A and 12B show scanning electron microscope images at the same scale. Figure 12A is from conventional platform freeze-drying. Figure 12B is from conventional platform freeze-drying with annealing. See the compositions in Table 1. By adding the annealing step, conventional freeze-drying can provide larger pores. However, as the 100 μm scale bar indicates, the pores in both Figures 12A and 12B are smaller than those in Figures 10B and 11A. Again, the larger pore diameter from controlled nucleation at -5°C results in faster redissolution time (see Table 4) and lower cake resistance during ice sublimation. In addition, as shown in Figure 4, annealing takes longer than controlled nucleation at -5°C.

[0081] For pharmaceutical products prepared from freeze-drying cycles involving controlled nucleation and annealing, different cake morphologies with significantly larger pore sizes can be revealed using SEM. SEM images show that CN produced at -5°C exhibits larger pore sizes than CN produced at -10°C (see Figures 10B and 11B). SEM images further reveal that annealing also significantly increased pore size (see Figure 12B). Larger pore sizes result in lower cake resistance and faster ice sublimation, thus leading to shorter primary drying times.

[0082] Example 3 - Effects of Annealing Figure 13 shows the annealing effect on the resistance exhibited by the dried product of 5% glycine. The annealing effect includes the following: (a) Annealing reduces the resistance exhibited by the dried product and the primary drying time. (b) Annealing increases the ice crystal size and pore size in the dried cake for ice sublimation. (c) The higher the annealing temperature, the more effective it is, (d) By applying the process at a lower temperature for a longer period of time, the resistance exhibited by the same product can be achieved.

[0083] Example 4 - Exemplary antibody for lyophilization by controlled nucleation In an additional embodiment, the antibody may be selected from the group consisting of: anti-programmed cell death 1 antibody (e.g., the anti-PD1 antibody described in U.S. Patent Application Publication US2015 / 0203579A1), anti-programmed cell death ligand 1 (e.g., the anti-PD-L1 antibody described in U.S. Patent Application Publication US2015 / 0203580A1), anti-Dll4 antibody, anti-angiopoietin 2 antibody (e.g., the anti-ANG2 antibody described in U.S. Patent No. 9,402,898), anti-angiopoietin-like 3 antibody (e.g., the anti-AngPtl3 antibody described in U.S. Patent No. 9,018,356), Antiplatelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibody described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibody described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibody described in U.S. Patent Publication No. US2015 / 0313194A1), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibody described in U.S. Patent No. 9,132,192, or U.S. Patent Publication No. US2 Anti-EGFRvIII antibody (as described in U.S. Patent No. 015 / 0259423A1), anti-proprotein convertase subtilisin / kexin 9 antibody (e.g., anti-PCSK9 antibody as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-proliferation and differentiation factor-8 antibody (e.g., anti-GDF8 antibody, also known as anti-myostatin antibody as described in U.S. Patent No. 8,871,209 or U.S. Patent No. 9,260,515), anti-glucagon receptor (e.g., U.S. Patent Application Publication No. Anti-GCGR antibodies (as described in US Patent Application Publication No. US2015 / 0337045A1 or US2016 / 0075778A1), anti-VEGF antibodies, anti-IL1R antibodies, interleukin-4 receptor antibodies (e.g., anti-IL4R antibodies as described in US Patent Application Publication No. US2014 / 0271681A1 or US Patent No. 8,735,095 or No. 8,945,559), anti-interleukin-6 receptor antibodies (e.g., US Patent No. 7,582,298, No. 8,043,617 or No. 9,173,Anti-IL6R antibody (as described in Patent No. 880), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL33 antibody as described in U.S. Patent Application Publication US2014 / 0271658A1 or U.S.2014 / 0271642A1), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Application Publication US2014 / 0271653A1), anti-differentiation antigen group 3 (e.g., U.S. Patent Application Publication US Anti-CD3 antibodies described in US Patent Publication No. 2014 / 0088295A1 and US20150266966A1, and US Patent Application No. 62 / 222,605), anti-differentiation antigen group 20 (e.g., anti-CD20 antibodies described in US Patent Publication No. US2014 / 0088295A1 and US20150266966A1, and US Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation antigen group 48 (e.g., anti-CD48 antibodies described in US Patent No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody as described in U.S. Patent Publication No. US2015 / 0337029A1), anti-Ebola virus antibody (e.g., as described in U.S. Patent Publication No. US2016 / 0215040), anti-Zika virus antibody, anti-lymphocyte activator gene 3 antibody (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-neuronal growth factor antibody (e.g., U.S. Patent Publication No. US2016 / 0017029 and U.S. Patent Nos. 8,309,088 and 9,353,Anti-NGF antibodies (as described in Patent Publication No. 176), and anti-activin A antibodies. In some embodiments, the bispecific antibodies are selected from the group consisting of anti-CD3 × anti-CD20 bispecific antibodies (as described in U.S. Patent Publications US2014 / 0088295A1 and US20150266966A1), anti-CD3 × anti-mucin 16 bispecific antibodies (e.g., anti-CD3 × anti-Muc16 bispecific antibodies), and anti-CD3 × anti-prostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3 × anti-PSMA bispecific antibodies). See also U.S. Patent Publication US2019 / 0285580A1. Furthermore, this includes Met×Met antibodies, agonist antibodies against NPR1, LEPR agonist antibodies, BCMA×CD3 antibodies, MUC16×CD28 antibodies, GITR antibodies, IL-2Rg antibodies, EGFR×CD28 antibodies, factor XI antibodies, antibodies against SARS-CoV-2 variants, Fel d 1 multi-antibody therapy, and Bet v 1 multi-antibody therapy. The above derivatives, components, domains, chains, and fragments are also included.

[0084] Cells producing exemplary antibodies can be cultured according to the present invention. Exemplary antibodies include alirocumab, atorutivimab, maftivimab, odesibimab, odesibibumab-ebgn, cacilibimab, imdevimab, semiprimab and semiprimab-rwlc (human IgG4 monoclonal antibodies that bind to PD-1), dupilumab (human monoclonal antibody of the IgG4 subclass that binds to the IL-4R alpha (α) subunit and thereby inhibits interleukin-4 (IL-4) and interleukin-13 (IL-13) signaling), evinacumab, evinacumab-dgnb, facinumab, fianlimab, galetosumab, itepecimab, nesbakumab, odronextamab, pozelimmab, sarilumab, trevoglumab, and reinukumab.

[0085] Additional exemplary antibodies include ravulizumab-cwvz, absiximab, adalimumab, adalimumab-atto, ad-trastuzumab, alemtuzumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotokisumab, blinatumomab, brentuximab vedotin, brodalumab, canakinumab, capromab pendetide, certolizumab pegol, cetuximab, denosumab, dinutuximab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, entansine alirocumab, evolocumab, golimumab, and gsel. Examples include coumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nivolumab, obilutoxaximab, obinutuzumab, ocrelizumab, ofatumumab, oraratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, laxibakumab, reslizumab, linukumab, rituximab, secukinumab, siltuximab, tocilizumab, trastuzumab, ustekinumab, and vedolizumab.

[0086] Example 5 - Primary drying design: Target product temperature and chamber pressure As a general rule, the target product temperature should be lower than the decay temperature by a "safety margin". a. The target product temperature is selected based on the following: i. Target Tp = Tc - (safety margin), and ii. The target Tp can be significantly higher than Tg' (especially in the case of high-concentration protein pharmaceutical products). b. The selection (calculation) of the chamber pressure is based on the following: i. By allowing a vacuum sufficient to drive ice sublimation (the lower the chamber pressure, the better), ii. By improving the uniformity of heat transfer (optimal value is approximately 150-200 mT), and iii. The chamber pressure (Pc) depends on the target product temperature and follows the following formula:

number

[0087] Example 6 - Primary drying conditions for high-concentration protein compositions A 150 mg / mL mAb composition can be used in combination with Tc, and Tc can be 15° to 20°C higher than Tg', with the following advantages: i. A strong glass formed with high-concentration mAb, and ii. The difference between Tc and Tg' is observed to be small because the composition is dominated by a brittle glass-forming agent.

[0088] For high-concentration mAb compositions, freeze-drying can be performed at a high shelf temperature, with the product temperature above Tg' (but below Tc). i. No collapse of the freeze-dried DP cake was observed.

[0089] Table 5 shows the conditions including several stabilizers.

[0090] Table 5 shows data obtained from drying conditions for protein compositions containing several stabilizers. [Table 5]

[0091] Example 7 - Design for secondary drying The following criteria were considered when designing the secondary drying process. a. Chamber pressure i. Maintain the same chamber used for primary drying. ii. Chamber pressure has little effect on the water desorption rate (when it is 200 mT or less). b. Shelf temperature and heating rate i. The "high" temperature required for water to be attached and detached, 1. Below the glass transition temperature, ii. Lower shelf temperatures require a longer time. iii. High shelf temperatures may lead to over-drying (especially if the stabilizers in the composition are insufficient), and iv. Gradual heating to ensure attachment and detachment before reaching the product at high temperatures. 1. This is more serious in amorphous products. c. Duration i. Generally, it is several hours. 1. If the secondary drying time is too short, scaling up may become difficult (due to non-uniform moisture content), and ii. Avoid prolonged secondary drying (e.g., more than 12 hours), and use a higher, more acceptable temperature for efficiency. d. Freeze-drying without secondary drying i. When a moderate moisture content offers advantages over "dryness".

[0092] Example 8 - Water as a plasticizer to stabilize proteins at a moderate moisture content An isotonic composition at mAb (150 mg / mL) was subjected to various moisture content levels at different storage temperatures. The change in the percentage of high molecular weight aggregates was measured.

[0093] Figure 14A shows graphs illustrating the moisture content and stability of the drug composition at 5°C, 25°C, and 30°C, as well as graphs at 37°C and 50°C (Figure 14B).

[0094] Under storage conditions ranging from 5°C to 50°C, the lowest moisture content did not result in the best stability. In contrast, a moderate moisture content of approximately 4% (approximately 2% to 5%) was found to be optimal for room temperature storage. At higher storage temperatures, the optimal moisture content decreased further (approximately 2% at 50°C) (see Figure 14B). No disadvantages were observed due to an increase in the viscosity or osmotic pressure of the composition.

[0095] Example 9 - Efficient freeze-drying process to achieve a moderate moisture content To determine a moderate moisture content in pharmaceutical products, we conducted experiments to elucidate an efficient freeze-drying process.

[0096] Figures 15A and 15B show the results of an efficient freeze-drying process and a moderate moisture content in the freeze-drying process. Figure 15A indicates that the process is STOP (finished) after the completion of ice sublimation.

[0097] To control the target residual moisture content of the product (e.g., approximately 2% to 4%), a freeze-drying cycle without a secondary drying step was used.

[0098] A two-stage freeze-drying cycle: Freezing and drying appeared to be the shorter process.

[0099] The moisture content can be controlled by the shelf temperature (Ts) during drying and the duration after ice sublimation (without a secondary drying step).

[0100] Example 10 - Optimal combination of shelf temperature and time after ice sublimation The moisture content of the DP (after ice sublimation was complete) was controlled by the combination of shelf temperature and duration after primary drying. Figure 16 shows the optimal combination of shelf temperature and time after ice sublimation to achieve the target residual moisture content. Exponential collapse fitting: %H20-30℃=5.59e-0.0388*t+3.36 %H20-20°C=4.72e-0.0528*t+2.42

[0101] (a) The moisture content of DP (after ice sublimation is complete) is controlled by a combination of shelf temperature and duration after primary drying, and the primary drying time is extended to achieve the target residual moisture content; and (b) the extension time is minimized to maximize process efficiency while changing the shelf temperature as needed, thereby obtaining the optimal combination of shelf temperature and time after ice sublimation.

[0102] Example 11 - A lyophilized pharmaceutical product with moderate moisture content that is stable at room temperature. An isotonic composition containing mAb (150 mg / mL) was stored at room temperature (approximately 25°C) with moisture content of approximately 0% and approximately 4%. The change in the percentage of high molecular weight aggregates was measured by SEC-UPLC. Figure 17 shows the percentage of high molecular weight aggregates measured by SE-UPLC in stable freeze-dried DP stored at 25°C with moderate moisture content.

[0103] Monoclonal antibodies appeared to be more stable at a moderate moisture content (approximately 4%) when stored at room temperature (approximately 25°C).

[0104] A lyophilized DP (dihydrotestosterone) with an optimal moisture content (approximately 4% H2O) is stable at room temperature. The high-concentration (150 mg / mL) isotonic composition is suitable for IV and SC administration, and it has been found that the DP composition is stable at room temperature when stored in a lyophilized state with the determined optimal moisture content (approximately 4% moisture content).

[0105] Example 12 - Results of analytical tests using size exclusion chromatography (SEC), cation exchange chromatography (CEX), and microflow imaging (MFI) Microflow imaging (MFI) of samples from different freeze-drying cycles. Figure 18 shows MFI analysis data for unfiltered particles larger than 10 μm. No substantial effect on subvisible particles was observed.

[0106] Microflow imaging (MFI) of samples from different freeze-drying cycles. Figure 19 shows MFI analysis data for unfiltered particles larger than 25 μm. No substantial effect on subvisible particles was observed.

[0107] Stability tests were conducted on platform-type DP (Figure 20A) and low-concentration DP (Figure 20B) samples from different freeze-drying cycles over a storage period exceeding 6 months. The percentage of high molecular weight (%) (%HMW) was measured by SEC. Samples were stored at approximately 25°C under a relative humidity (RH) of approximately 60%. Figures 20A and 20B show that there was no substantial impact on stability based on the percentage of high molecular weight aggregates (%) measured by SEC.

[0108] Stability tests were conducted on platform-type DP (Figure 21A) and low-concentration DP (Figure 21B) samples from different freeze-drying cycles over a storage period exceeding 6 months. Samples were stored at 25°C under 60% relative humidity (RH). Figures 21A and 21B show no substantial impact on stability, based on the percentage of the main peak (region 2) measured by cation exchange chromatography (CEX).

[0109] Example 13 - Protein concentration and protein recovery The protein concentration and percentage of protein recovery (%) in compositions F1-F4 (see Table 1), stored at 25°C and 40°C for 3 months, were measured using reversed-phase ultrahigh-performance liquid chromatography (RP-UPLC). No significant changes in protein concentration were observed (i.e., nearly complete protein recovery was observed in all compositions F1-F4) (Table 6).

[0110] Table 6 shows data from RP-UPLC regarding mAb3 protein concentration and protein recovery rate (%). [Table 6]

[0111] Example 14 - Effect of cryoprotectant on subvisible particles Samples of compositions F1 and F2, pharmaceutical products (DPs) containing 5% sucrose, from different lyophilization cycles were stored at 25°C and 40°C at T0 and 1 month, and analyzed. Figures 22A and 22B are graphs showing data for 25 μm particles (Figure 22A) and 10 μm particles (Figure 22B) in the DP compositions at different temperatures (25°C and 40°C) and time (t0, 1 month, and 3 months). Composition F1 is a platform cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 5% w / v sucrose, and 0.1% w / v PS80, with a pH of 6.0. Composition F2 is a CN-5°C cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 5% w / v sucrose, and 0.1% w / v PS80, with a pH of 6.0. There is no significant effect on 10 μm and 25 μm particles.

[0112] Samples of compositions F3 and F4, which are pharmaceutical products (DPs) containing 5% sucrose, from different lyophilization cycles were stored at 25°C and 40°C at T0 and 1 month, and analyzed. Figures 23A and 23B are graphs showing data for 10 μm and 25 μm particles in the DP compositions at different temperatures (25°C and 40°C) and times (t0, 1 month, and 3 months), respectively. Composition F3 is a platform cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 1.5% w / v sucrose, and 0.1% w / v PS80, with a pH of 6.0. F4 is a CN-5°C cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 1.5% w / v sucrose, and 0.1% w / v PS80, with a pH of 6.0. There is no significant effect on 10 μm and 25 μm particles.

[0113] The experimental results disclosed herein reveal that controlled nucleation promotes small and consistent supercooling for ice crystallization (i.e., the formation of larger ice crystals and thicker cake walls). Larger ice crystals reduce resistance to water vapor flow from the ice sublimation interface, and thicker cake walls improve the cake structure.

[0114] Advantages of controlled nucleation include the following. ·Faster primary drying and shorter cycle times, ·Reduced reconstitution time ·Improved cake appearance, and ·Higher monomer purity after lyophilization

[0115] Example 15 - BET (Brunauer-Emmett-Teller) Surface Area and Hg Porosimetry Analysis in Lyophilized Cake Samples N2 BET surface area test and Hg porosimetry test were performed on the lyophilized cake samples. The BET theory is a common model used to calculate the surface area of the exposed surface of a solid sample at the molecular scale. In general, BET surface area analysis is performed using N2 gas as the adsorbate, since nitrogen (N2) gas has a high affinity for solid surfaces. N2 gas is introduced at low pressure, the adsorption amount is measured, and the surface area is calculated using the BET equation.

[0116] BET analysis shows that samples from the conventional method have a higher specific surface area compared to controlled ice nucleation samples (see Table 7). Additional pore size evaluation measured via Hg intrusion volume showed a clear difference between the conventional method and controlled ice nucleation (see Figure 25). The conventional method exhibits much smaller pore sizes (see Table 8). The N2 isotherm indicates that the sample has a relatively low surface area. The isotherm data is shown in Figure 24. The BET surface area results are summarized in Table 7. [Table 7] [Table 8]

[0117] N2 Sorption Relative pressure (P / P oThe N2 isotherm was measured down to approximately 0.4 using a Micromeritix TriStar II Plus instrument. Prior to analysis, the samples were activated by degassing under dynamic vacuum at ambient temperature using a Smart VacPrep degasser.

[0118] N2BET measurement in standard materials requires at least 50 m of sample cell space. 2 If the material is available for testing, 10m 2 The surface area value per g is approximately within 5%, and about 0.5 m 2 This suggests an accuracy of approximately 10% or less at the / g level. The reproducibility in any given sample depends on the ability to regenerate the sample to the same degree of activation without altering the surface or pore structure.

[0119] Hg porosimetry Mercury penetration experiments were outsourced. Hg porosimetry experiments were performed by loading a sample into a chamber of known volume and first filling the voids around the material with Hg at a packing pressure of approximately 0.5 psia. Here, it is assumed that Hg does not penetrate into the sample at this packing pressure. Subsequently, the Hg pressure was increased to approximately 60,000 psia to allow penetration into the voids inside the sample. The amount of penetration relative to pressure was recorded and converted into a pore size distribution.

[0120] The bulk density from Hg porosimetry is often the Hg density (ρ Hg The mercury penetration volume (MIV) is called the mercury penetration volume (MIV) and reflects the density of the sample prior to the penetration of Hg into the material; therefore, it is similar to the geometric density of the material. Accordingly, the unit of Hg density is the number of grams of solid per volume of solid + voids (assuming no closed pores exist). The mercury penetration volume (MIV) is measured by penetrating Hg into the sample under pressure and is considered to be the voids within the sample. By comparing the Hg density and MIV value, the total porosity of the material can be estimated. The porosity (%) can be calculated from the following formula. Porosity (%)=MIV×ρ Hg ×100

[0121] The logarithmic derivative data for the samples show a unimodal PSD. The pore mode for sample LT-SS001 cycle 3 F1 retest t=0 vial L19-002020 is centered at approximately 45.2 μm, while the pore mode for sample LT-SS001 cycle 1 F1 t=0 vial L19-002020 is centered at approximately 21.3 μm. The values ​​for Hg density (i.e., bulk density at approximately 0.5 psia), MIV, and calculated porosity are shown in Table 8. The logarithmic derivative PSD plot and cumulative volume PSD plot are shown in Figures 25 and 26, respectively.

[0122] It should be understood that these descriptions, examples, and data are provided for illustrative purposes only and are not intended to limit the invention. Various changes and modifications within the invention, including combining all or part of the teachings, will be apparent to those skilled in the art from the discussions, disclosures, and data contained herein, and are therefore also considered part of the invention.

Claims

1. A method for freeze-drying protein products, wherein the method is (a) A step of cooling a solution containing a protein product to below the freezing point of the solution, (b) Step (a) The cooled solution is pressurized with gas, (c) A step in which nucleation is enabled by releasing the pressure of step (b), thereby resulting in the formation of ice nuclei in the cooled solution, A method comprising the steps of (d) freeze-drying the cooled solution from step (c) to form a freeze-dried protein product.

2. The method according to claim 1, wherein the gas is selected from the group consisting of air, helium, nitrogen, or argon.

3. The method according to claim 1 or 2, wherein the gas is present in a quantity of 14 to 42 psig during step (b).

4. The method according to claim 1 or 2, wherein the gas is present at a concentration of 25 to 30 psig during step (b).

5. The method according to any one of claims 1 to 4, wherein the cooled solution is contained in a lyophilization vial.

6. The method according to any one of claims 1 to 5, wherein step (c) is carried out at a temperature of -10°C to -2°C.

7. The method according to claim 6, wherein step (c) is carried out at a temperature of -5°C.

8. The method according to any one of claims 6 to 7, wherein the temperature is maintained for about one hour or less.

9. The method according to claim 8, wherein the temperature is maintained for 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 minutes or less.

10. The method according to any one of claims 1 to 9, wherein the protein is an Fc-containing protein.

11. The method according to claim 10, wherein the Fc-containing protein is an Fc fusion protein.

12. The method according to claim 10, wherein the Fc-containing protein is an antibody.

13. The method according to claim 12, wherein the antibody is a monoclonal antibody.

14. The method according to any one of claims 12 to 13, wherein the antibody can be redissolved to 150 mg / ml in about 3 minutes.

15. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 50 seconds.

16. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 40 seconds.

17. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 30 seconds.

18. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 20 seconds.

19. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 10 seconds.

20. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 2 minutes and 5 seconds.

21. The method according to claim 14, wherein the antibody can be redissolved to 150 mg / ml in about 1 minute and 45 seconds.

22. A freeze-dried protein product prepared by the method described in any one of claims 1 to 21.

23. A method for producing a freeze-dried therapeutic protein product that is stable at room temperature by imparting a target residual moisture content to the freeze-dried therapeutic protein product that is stable at room temperature, wherein the method is (a) A step of controlling the shelf temperature (Ts) after ice sublimation without a secondary drying step, (b) a step of controlling the duration of time after primary drying, A method wherein the target residual moisture content in the freeze-dried therapeutic protein product is approximately 1% to approximately 7%.

24. The method according to claim 23, wherein the target residual moisture content in the freeze-dried therapeutic protein product is about 1.0%, about 1.5%, about 2.0%, 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, or about 7.0%.

25. The method according to claim 23, wherein the stability of the freeze-dried therapeutic protein product is measured by determining the change in the percentage of high molecular weight protein aggregates in the product.

26. The method according to claim 25, wherein the change in the percentage of the high molecular weight protein aggregates in the stable freeze-dried therapeutic protein product is less than 1.0% when measured by SEC-UPLC.

27. The method according to claim 25, wherein the change in the percentage (%) of the high molecular weight protein aggregates in the stable freeze-dried therapeutic protein product is about 1.0%, about 0.9%, about 0.8%, about 0.7%, about 0.6%, about 0.5%, about 0.4%, about 0.3%, about 0.2%, about 0.1%, or about 0%.

28. A freeze-dried therapeutic protein product that is stable at room temperature, prepared by the method described in any one of claims 23 to 27.

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