Freeze-drying with controlled nucleation

Abstract

The present invention relates to improved lyophilization methods for therapeutic proteins, such as monoclonal antibodies, using controlled nucleation. The controlled nucleation process is time efficient and yields lyophilized products of high quality and shorter redissolution times. The invention also relates to an efficient freeze-drying process to achieve room temperature stable moderate moisture content in therapeutic proteins.

Description

[0001] This application claims priority to U.S. Application Serial No. 63 / 541,573, filed September 29, 2023, and U.S. Application Serial No. 63 / 681,328, filed August 9, 2024. These applications are incorporated herein by reference in their entirety. Technical Field

[0002] This invention relates to an improved method for lyophilizing therapeutic proteins using controlled nucleation. Background Technology

[0003] Therapeutic proteins include Fc-containing proteins (such as monoclonal antibodies (mAbs)) and receptor Fc fusion proteins (such as trap proteins) used as therapeutic proteins. Lyophilization is a recognized method for long-term storage of therapeutic proteins. Current platform lyophilization methods (i.e., current standard lyophilization) are very time-consuming and often lead to quality problems in lyophilized formulations. This invention provides an improved lyophilization method and improved lyophilized therapeutic products that do not suffer from the disadvantages of conventional platform technologies. This invention also provides room-temperature stable lyophilized therapeutic protein products, methods for their preparation, and methods for obtaining optimal moisture content in the therapeutic products to prepare room-temperature stable lyophilized therapeutic products. Summary of the Invention

[0004] This invention provides a method for lyophilizing a protein product, the method comprising the steps of: (a) cooling a solution containing the protein product to below the freezing point of the solution; (b) pressurizing the cooled solution of step (a) with a gas; (c) releasing the pressure of step (b) to allow nucleation, thereby causing ice nuclei to form in the cooled solution; and (d) lyophilizing the cooled solution of step (c) to form a lyophilized protein product. The gas may be selected from the group consisting of air, helium, nitrogen, or argon. During step (b), the gas may be present at a concentration of 14 to 42 psig, preferably 25 to 30 psig, and more preferably about 28 psig.

[0005] The cooling solution can be stored in a lyophilized bottle. Step (c) can be performed at a temperature between -2°C and -10°C, preferably at about -5°C. This 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 necessary, this temperature can be maintained for more than 1 hour.

[0006] The protein can be an Fc-containing protein, such as an Fc fusion protein, or an antibody. The antibody is preferably a monoclonal antibody.

[0007] When the Fc protein is a monoclonal antibody, the antibody can be reconstituted to 150 mg / ml within approximately 3 minutes, approximately 2 minutes 50 seconds, approximately 2 minutes 40 seconds, approximately 2 minutes 30 seconds, approximately 2 minutes 20 seconds, approximately 2 minutes 10 seconds, approximately 2 minutes, approximately 1 minute 50 seconds, approximately 1 minute 45 seconds, approximately 1 minute 40 seconds or less, and within the range of appendages derived based on the above values.

[0008] Also provided are lyophilized proteins produced by this method. Preferably, the protein can be an Fc-containing protein, such as an antibody and an Fc fusion protein. The invention is further described and discussed below.

[0009] The present invention also provides a method for preparing a room-temperature stable lyophilized therapeutic protein product by providing a target residual moisture content in the lyophilized therapeutic protein product, wherein the method includes the steps of: (a) controlling the shelf temperature (Ts) after ice sublimation without a secondary drying step; and (b) controlling the duration after a primary drying; wherein the target residual moisture content in the lyophilized therapeutic protein product is from about 1% to about 7%. The target residual moisture content in the lyophilized therapeutic protein product can be approximately 2% to 6%, approximately 2% to 5%, approximately 2% to 4%, approximately 2% to 3%, approximately 3% to 7%, approximately 3% to 6%, approximately 3% to 5%, approximately 3% to 4%, approximately 4% to 7%, approximately 4% to 6%, approximately 4% to 5%, approximately 5% to 7%, approximately 5% to 6%, approximately 6% to 7%, approximately 1.0%, approximately 1.5%, approximately 2.0%, 2.5%, approximately 3.0%, approximately 3.5%, approximately 4.0%, approximately 4.5%, approximately 5.0%, approximately 5.5%, approximately 6.0%, approximately 6.5%, or approximately 7.0%, and additional ranges derived from the above values. The stability of the lyophilized therapeutic protein product can be measured by determining the change in %HMW protein aggregates in the product. Determined by size exclusion chromatography-ultra-high performance liquid chromatography (SEC-UPLC), the change in %HMW protein aggregates in a stable lyophilized therapeutic protein product is less than 1.0%. The %HMW protein aggregate variation in the stable lyophilized 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%, and additional sub-ranges derived from the above values. The present invention also provides room-temperature stable lyophilized therapeutic protein products produced by any of the methods disclosed herein. Attached Figure Description

[0010] Figure 1A It is a diagram depicting the freeze-drying process, including the annealing step. Figure 1B and Figure 1CA comparison between conventional and controlled nucleation methodologies for multiple batches of monoclonal antibody formulations is provided. Figure 1B Data from a conventional refrigeration platform is described. Figure 1C The data describes controlled nucleation.

[0011] Figure 2A and Figure 2B Data from controlled nucleation parameter curves of multiple batches of monoclonal antibody formulations are described. Figure 2A For -5℃, Figure 2B For -10℃.

[0012] Figures 3A through 3D depict data from various lyophilization methods for multiple batches of monoclonal antibody formulations. Figure 3A depicts data from lyophilization on a conventional platform. Figure 3B depicts data from controlled nucleation at -5°C. Figure 3C depicts data from controlled nucleation at -10°C. Figure 3D depicts data from lyophilization on a conventional platform with annealing (see the circled portion between 0.0 and 20.0 hours). The arrows in each figure indicate the end of one drying cycle.

[0013] Figure 4 The drying time and freeze-drying time of a conventional platform (see Figure 3A), a controlled nucleation at -5℃ (see Figure 3B), a controlled nucleation at -10℃ (see Figure 3C), and a conventional platform with annealing (see Figure 3D) are described.

[0014] Figure 5A and Figure 5B Size exclusion chromatography (SEC) data depicting the percentage of native mAbs obtained using a buffer containing 150 mg / ml mAb and 5% or 1.5% sucrose. Figure 5A The results for samples stored at 25°C are shown. Figure 5B The results for samples stored at 40°C are shown. Figure 5C and Figure 5D Size exclusion chromatography (SEC) data for formulations F1, F2, F3, and F4 (see formulations in Table 1) were depicted. Figure 5C At 25℃, Figure 5D For 40℃.

[0015] Figure 6A and Figure 6B : Figure 6A The diagram shows the plateau lyophilized mAb formulations in vials: from left to right, plateau drug (DP), low-concentration placebo product (PP), low-concentration drug, and plateau placebo product. See Table 1 for formulations. The arrows in the diagram indicate product shrinkage. Figure 6BThe figure depicts lyophilized mAb formulations in vials: placebo and low-concentration (DP) drugs using lyophilization and annealing at -5°C, controlled nucleation without annealing, and lyophilization with controlled nucleation at -5°C. Arrows in the figure indicate product shrinkage.

[0016] Figure 7 The images show lyophilized mAb formulations in vials prepared using controlled nucleation: from left to right, platform drug product, low-concentration placebo product, platform placebo product, and low-concentration drug product. See Table 1 for formulations. The upward movement was performed at -5°C, and the downward movement at -10°C.

[0017] Figure 8 The diagram shows plateau lyophilized mAb formulations in vials, annealed before lyophilization: from left to right, low-concentration drug (DP), plateau drug, low-concentration placebo product (PP), and plateau placebo product. See Table 1 for formulations. Arrows in the diagram indicate wall products.

[0018] Figure 9 The figure shows platform-based lyophilized mAb formulations using 5% w / v sucrose formulation (left) and 1.5% sucrose formulation (right) in vials, as well as lyophilized mAb formulations prepared via controlled nucleation at -5°C. Arrows in the figure indicate products on the walls used for conventional lyophilization.

[0019] Figures 10A and 10B show scanning electron micrographs (SEM) at the same scale. Figure 10A is from a platform-prepared (conventional) lyophilized formulation, and Figure 10B is from a lyophilized formulation prepared by controlled nucleation at -5°C. Formulations are listed in Table 1.

[0020] Figures 11A and 11B show scanning electron micrographs (SEM) at the same scale. Figure 11A is from a lyophilized formulation prepared by controlled nucleation at -5°C, and Figure 11B is from a lyophilized formulation prepared by controlled nucleation at -10°C. The formulations are shown in Table 1.

[0021] Figures 12A and 12B show scanning electron micrographs (SEM) at the same scale. Figure 12A is from a conventional platform freeze-drying process. Figure 12B is from a conventional platform freeze-drying process with annealing at -5°C.

[0022] Figure 13 This is a graph depicting the effect of annealing on the resistance of the dry product of 5% glycine.

[0023] Figure 14A and Figure 14B It describes the conditions at 5℃, 25℃, and 30℃. Figure 14A ) and at 37℃ and 50℃ ( Figure 14B (A graph showing the moisture content and stability data of the drug preparation.)

[0024] Figure 15A and Figure 15B An efficient freeze-drying process for achieving appropriate moisture content is described. One drying cycle ends when the vapor composition is primarily nitrogen. Pirani gauge pressure ≈ chamber pressure measured via capacitance. Figure 15A This indicates a stop after the ice sublimation process has ended.

[0025] Figure 16 It is the best combination of shelf temperature and the time it takes for ice to sublimate.

[0026] Figure 17 It is a graph depicting the %HMW in stable freeze-dried DP stored at 25°C with moderate moisture content, as determined by SE-UPLC.

[0027] Figure 18 The bar chart depicts data from microfluidic imaging (MFI) analysis of unfiltered particles ≥ 10 µm and its impact on subvisible particles.

[0028] Figure 19 The bar chart depicts data from microfluidic imaging (MFI) analysis of unfiltered particles ≥ 25 µm and its impact on subvisible particles.

[0029] Figures 20A and 20B are graphs showing %HMW aggregates in platform DP (Figure 20A) and low-concentration DP (Figure 20B) as determined by SEC, stored at 25°C and 60% relative humidity (RH) for more than six months.

[0030] Figures 21A and 21B are graphs showing the % main peak (region 2) in platform DP (Figure 21A) and low-concentration DP (Figure 21B) stored for more than six months, as determined by cation exchange chromatography (CEX).

[0031] Figure 22A and Figure 22B This demonstrates the effect of 5% cryoprotectant on 25μm in DP formulation at different temperatures (25℃ and 40℃) and times (t0, 1m and 3m). Figure 22A ) and 10μm ( Figure 22B A diagram illustrating the effect of particles.

[0032] Figure 23A and Figure 23B This demonstrates the effect of 1.5% cryoprotectant on 10 μm of DR formulation at different temperatures (25℃ and 40℃) and times (t0, 1m and 3m). Figure 23A ) and 25 μm ( Figure 23A A diagram illustrating the effect of particles.

[0033] Figure 24 It is a graph depicting the N2 isotherm as a function of relative pressure, measured at 77K.

[0034] Figure 25 It is a graph depicting the logarithmic differential pore size distribution data based on the Hg intrusion volume.

[0035] Figure 26 This is a graph depicting the cumulative pore size distribution data based on Hg intrusion volume. Detailed Implementation

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0037] definition

[0038] In the context of numerical values ​​and ranges, the term "about" refers to a value or range that is approximately or close to the stated value or range, enabling the invention to be practiced, such as the presence of the sought rate, quantity, density, degree, increase, decrease, percentage, value or form, temperature, or time quantity, as is apparent from the teachings contained herein. For example, "about" can mean above or below the stated value, ranging from approximately + / - 10% or more or less, depending on the capability. Thus, the term covers values ​​beyond those simply produced by systematic errors.

[0039] Antibodies (also known as immunoglobulins) are examples of proteins with multiple polypeptide chains and extensive post-translational modifications. A typical immunoglobulin (e.g., IgG) consists of 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 with various polysaccharides at various residues (e.g., asparagine residues), and this glycosylation can vary by species, potentially affecting 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 commonly used as therapeutic biomolecules.

[0040] Antibodies consist of immunoglobulin molecules composed of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain includes a heavy chain variable region (abbreviated as HCVR or VH in this document) and a heavy chain constant region. The heavy chain constant region includes three domains: CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as LCVR or VL in this document) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions scattered with more conserved regions called framework regions (FRs), referred to as complementarity-determining regions (CDRs). Each VH and VL consists of three CDRs and four FRs, with the CDRs and FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs can be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs can be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibody refers to an antibody with at least 10% affinity for its target. -9 M, at least 10 -10 M, at least 10 -11 M or at least 10 -12 The binding affinity of M to antibodies, such as those measured by surface plasmon resonance (e.g., BIACORE™) or solution affinity ELISA.

[0041] 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 may include human antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibody fragments, single-chain antibodies, dimeric antibodies, trisomic or tetrasomic antibodies, Fab fragments or F(ab')2 fragments, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

[0042] The antibody can be an IgG1 antibody. The antibody can be an IgG2 antibody. The antibody can be an IgG3 antibody. The antibody is 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. Derivatives, components, domains, chains, and fragments of the above are also included.

[0043] The phrase "bispecific antibody" refers to antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies typically comprise two distinct heavy chains, each specifically binding to a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), then the affinity of the first heavy chain for the first epitope is typically at least one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa. Epitopes recognized by bispecific antibodies can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding variable sequences of heavy chains recognizing different epitopes of the same antigen can be fused with nucleic acid sequences encoding constant regions of different heavy chains, and such sequences can be expressed in cells expressing immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain (N-terminus to C-terminus), and an immunoglobulin light chain that does not confer antigen-binding specificity but can associate with each heavy chain, or can associate with each heavy chain and bind one or more epitopes bound by the antigen-binding region of the heavy chain, or can associate with each heavy chain such that one or two heavy chains can bind to one or two epitopes.

[0044] The phrase "heavy chain" or "immunoglobulin heavy chain" includes the constant region sequence of the immunoglobulin heavy chain from any organism and, unless otherwise stated, includes the heavy chain variable domain. Unless otherwise stated, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Heavy chain fragments include CDRs, CDRs, and FRs, and combinations thereof. A typical heavy chain has a CH1 domain, a hinge, a CH2 domain, and a CH3 domain (from the N-terminus to the C-terminus) following the variable domain. Functional fragments of the heavy chain include those capable of specifically recognizing antigens (e.g., recognizing antigens with KD in the micromolar, nanomolar, or picomolar range), capable of being expressed and secreted from cells, and containing at least one CDR.

[0045] The phrase "light chain" includes the constant region sequence of immunoglobulin light chains from any organism and, unless otherwise specified, includes human κ and λ light chains. Unless otherwise specified, the light chain variable (VL) domain typically comprises three light chain CDRs and four frame (FR) regions. Typically, a full-length light chain contains a VL domain and a light chain constant domain from the amino terminus to the carboxyl terminus, said VL domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Light chains that can be used in these inventions include, for example, light chains that do not selectively bind to a first or second antigen selectively bound by an antigen-binding protein. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or computer simulations), wherein said light chains substantially do not 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 one or both epitopes bound by the antigen-binding region of an antigen-binding protein.

[0046] The phrase "variable domain" comprises the amino acid sequence of the immunoglobulin light or heavy chain (modified as needed), which, in sequence from the N-terminus to the C-terminus (unless otherwise specified), includes the following amino acid regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. "Variable domain" also comprises the amino acid sequence capable of folding into a typical domain (VH or VL) with a double β-sheet structure, wherein the β-sheet is linked by disulfide bonds between residues of the first and second β-sheets.

[0047] The phrase "complementarity-determining region" or the term "CDR" includes an amino acid sequence encoded by the nucleic acid sequence of an organism's immunoglobulin gene, which typically (i.e., in wild-type animals) appears between two frame regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T cell receptor). For example, a CDR can be encoded by a germline sequence or a rearranged or unrearranged sequence, and can be encoded, for example, by naive or mature B cells or T cells. In some cases (e.g., for CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are discontinuous (e.g., in a nucleic acid sequence that has not yet been rearranged) but are contiguous in the B cell nucleic acid sequence, for example, as a result of splicing or joining said sequences (e.g., VDJ rearrangement to form heavy chain CDR3).

[0048] "Antibody derivatives and fragments" include, but are not limited to: antibody fragments (e.g., Fab, ScFv-Fc, dAB-Fc, haptens and other combinations of heavy and / or light chains), multispecific (e.g., bispecific, IgG-ScFv, IgG-dab, ScFV-Fc-ScFV, trispecific).

[0049] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, Fc-containing antibody derivatives, Fc-containing antibody fragments, Fc fusion proteins, immunoadhesins, and other binding proteins containing at least the 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 participate in complement activation. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that prevent them from binding to any Fc receptors and activating complement, then those CH2 and CH3 regions are non-functional. Fc fusion proteins include, for example, Fc fusions (N-terminus), Fc fusions (C-terminus), mono-Fc fusions, and bispecific Fc fusion proteins.

[0050] "Fc" stands for crystallizable fragment, often referred to as a constant fragment. Antibodies consist of an Fc region composed of two identical protein sequences. IgG has a heavy chain called the γ chain. IgA has a heavy chain called the α chain, and IgM has a heavy chain called the µ chain. IgD has a heavy chain called the σ chain, and IgE has a heavy chain called the ε chain. In nature, the Fc regions of antibodies of a given class and subclass within the same species are identical. Human IgG has four subclasses, each sharing approximately 95% homology. Within each subclass, the Fc sequence is identical. For example, human IgG1 antibodies have the same Fc sequence. Similarly, IgG2 antibodies will have the same Fc sequence; IgG3 antibodies will have the same Fc sequence; and IgG4 antibodies will have the same Fc sequence. Changes in the Fc region result in changes in charge.

[0051] Fc-containing proteins may include modifications in the immunoglobulin domain, including modifications that affect one or more effector functions of the bound protein (e.g., modifications affecting FcyR binding, FcRn binding, and thereby affecting half-life and / or CDC activity). Referring to the EU designations for the immunoglobulin constant region, such modifications include, but are not limited to, the following modifications and combinations thereof: 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, 326, 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.

[0052] For example, and not by means of restriction, the binding protein is an Fc-containing protein that exhibits an enhanced serum half-life (compared to the same Fc-containing protein without the described modification) and has modifications at the following positions: 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T) and 256 (e.g., S / R / Q / E / D or T); or modifications at 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at 250 and / or 428; or modifications at 307 or 308 (e.g., 308F, V308F) and 434. In another example, the modifications may include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V259I) and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254 and 256 modifications (e.g., 252Y, 254T and 256E); 250Q and 428L modifications (e.g., T250Q and M428L); 307 and / or 308 modifications (e.g., 308F or 308P).

[0053] Some recombinant Fc-containing proteins contain receptors or receptor fragments, ligands or ligand fragments that have homologous binding partners in biological systems, and include "receptor Fc fusion proteins," which refer to recombinant molecules containing soluble receptors fused to the Fc domain of immunoglobulins.

[0054] "Fc fusion proteins" comprise parts or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, wherein the two or more proteins do not fuse in their native state. Fc fusion proteins include Fc fusions (N-terminus), Fc fusions (C-terminus), single Fc fusions, and bispecific Fc fusions. The preparation of fusion proteins comprising different portions of antibody-derived peptides (containing Fc domains) has been described: for example, Ashkenazi et al., Proc. Natl. Acad. Sci USA 88: 10535-39 (1991); Byrn et al., Nature 344: 677-70, 1990; and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", Current Protocols in Immunology, Supplement 4, pp. 10.19.1-10.19.11 (1992). "Receptor Fc fusion proteins" include one or more extracellular domains of a receptor coupled to the Fc portion, including, in some embodiments, a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, Fc fusion proteins contain two or more different 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 referred to as "traps," "trap molecules," or "trap proteins." For example, such trap proteins include IL-1 traps (e.g., linasip, which contains an IL-1RAcP ligand-binding region fused to the extracellular region of IL-1R1, which is fused to the Fc region of hlgG1; see U.S. Patent No. 6,927,044), or VEGF traps (e.g., aflibercept, which contains an Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl, which is fused to the Fc region of hlgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0055] Linaserip 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 those containing a peptide fused to an Fc domain. Recombinant Fc-containing proteins are also described in: C. Huang, “Receptor-Fc fusion therapeutics, traps, and MFMETIBODY technology”, 20(6) Curr. Opin. Biotechnol. 692-9 (2009).

[0056] Proteins lacking the Fc moiety, such as recombinant enzymes and microtraps, can also be lyophilized according to the present invention. Microtraps are trap proteins that use a polymerized component (MC) instead of the Fc moiety, as disclosed in U.S. Patent Nos. 7,279,159 and 7,087,411. The derivatives, components, domains, chains, and fragments described above are also included.

[0057] "Freeze-dried cake" or "cake" refers to freeze-dried drugs, including protein drugs in freeze-dried form in containers such as vials.

[0058] "Platform" refers to the currently used methods, including standard methods.

[0059] All numerical limits and ranges described herein include all numbers or values ​​relating to or falling within a range or limit. The ranges and limits disclosed herein explicitly specify and list all integer, decimal, and fractional values ​​defined and covered within such ranges or limits. Therefore, unless otherwise stated herein, descriptions of ranges of values ​​herein are intended only as a separate reference to each individual value falling within the said range, and each individual value is incorporated into this specification as if separately described herein. All referenced numbers or values ​​explicitly state and represent all ranges formed by the referenced numbers or values.

[0060] describe

[0061] Lyophilization of biological products (such as those containing Fc proteins) has long been used for medical purposes. Due to the absence of water and reduced internal molecular dynamics, lyophilized products tend to have a longer shelf life than liquid products. Furthermore, lyophilized products can be reconstituted to a higher protein concentration than the solution before lyophilization. Higher concentrations are useful for intravenous and subcutaneous administration. Subcutaneous administration, in particular, requires biological products with higher concentrations after reconstitution.

[0062] Protein pharmaceutical products are typically stored at 2 to 8°C, and room temperature stability (storage) would be a significant improvement. Depending on the process design, lyophilization is generally a time- and energy-intensive process. A typical lyophilization process comprises three successive stages:

[0063] i. Freezing (which may include annealing or controlled nucleation steps);

[0064] ii. Single-stage drying (which can be designed to improve process efficiency and protein storage stability); and

[0065] iii. Secondary drying (if necessary at this stage).

[0066] The drying stage is typically the longest and is also where process efficiency is crucial. Improving stability and process efficiency are becoming key priorities in achieving patient convenience and saving energy, costs (and the environment). Figure 1A The diagram shows a typical freeze-drying process, including the annealing step.

[0067] Strategies to improve lyophilized pharmaceutical products (DP)

[0068] The following strategies can be used:

[0069] 1. Stabilizers such as sucrose (or trehalose) can be used in lyophilized formulations:

[0070] (a) Sucrose and trehalose are disaccharides and are used as cryoprotectants and lyophilization protectants. Achieving a sufficient stabilizer-to-protein ratio can be challenging for high-concentration protein formulations.

[0071] (b) Formation of an amorphous matrix to stabilize solid proteins, since the stabilizer and protein are in the same phase; and / or

[0072] (c) Using water as an alternative stabilizer will not increase the viscosity / osmotic pressure of the formulation.

[0073] 2. Formulation strategies to improve the efficiency of freeze-drying processes:

[0074] (a) Use crystallization fillers to increase the formulation collapse temperature (Tc). Suitable options include mannitol and glycine (which have high eutectic temperatures, ~33°C); and / or

[0075] (b) Freeze drying can be carried out above Tg' but below Tc to reduce the total ice sublimation time.

[0076] 3. Improve formulation and / or process strategies for lyophilized DP:

[0077] (a) A combination of stabilizers / plasticizers (with small molecular weights, including water molecules) can be used;

[0078] (b) Stable proteins with high water content may require lower strength drying or no secondary drying at all; and / or

[0079] (c) Controlled nucleation and freeze-drying followed by annealing can be used.

[0080] Freeze-drying process design

[0081] The following conditions can be applied:

[0082] 1. Freezing / cooling rate:

[0083] (a) Rapid freezing rate (e.g., 2 °C / min), which is typically not achievable at low temperatures (e.g., <-30 °C); and

[0084] (b) can be 0.5 to 1 °C / min.

[0085] 2. Freezing temperature (simple but crucial):

[0086] (a) Below Tg' to ensure all solutes are in the solid state; and

[0087] (b) Due to product overcooling (non-scalable), the temperature is below -35°C. Overcooling is not a problem if controlled nucleation (CN) technology is used; and

[0088] (c) Usually -40 / -50℃.

[0089] 3. Annealing of fillers or amorphous materials:

[0090] (a) Filler crystallization;

[0091] (b) Annealing at temperatures between Teu and Tg' for 2 to 10 hours. Annealing at higher temperatures requires shorter times; and

[0092] (c) Improve the morphology and drying efficiency of amorphous medicinal cakes

[0093] The experimental design used in this paper is shown in Table 1 and related texts.

[0094] Table 1* .

[0095]

[0096] *20R, each formulation has a 5.3 mL filler. (5.3 mL of lyo DP is reconstituted with 4.56 mL WFI to a 150 mg / mL formulation in a 20 mL Type 1 glass vial). The purpose of this experiment was to evaluate the effect of CN Lyo cycling on the stability of mAb3.

[0097] Freeze-drying cycle type:

[0098] (a) Platform placebo lyophilization cycles (-20°C for 50 hours and 40°C for 12 hours), which is the standard lyophilization method.

[0099] (b) Anneal using a standard lyophilization cycle with annealing (-5°C, 6 hours). Annealing is performed prior to standard lyophilization methods, such as a plateau placebo lyophilization cycle.

[0100] (c) Freeze-drying cycle with controlled nucleation (CN) at -5°C.

[0101] (d) Freeze-drying cycle with controlled nucleation (CN) at -10°C.

[0102] Measurement:

[0103] Typical analytical methods for lyophilized products include visual analysis, size exclusion chromatography (SEC), cation exchange chromatography (CEX), and microfluidic imaging (MFI).

[0104] The freeze-drying process typically involves three stages: (1) freezing, (2) primary drying via sublimation, and (3) secondary drying via desorption. The freezing step is the first part of the freeze-drying process. In conventional platform methods, freezing can occur randomly. See also Figure 1B Conventional platform technologies produce lower freezing temperatures (more supercooling), such as -11°C to -17°C, which leads to significant supercooling and freezing temperature heterogeneity, such as... Figure 1B As shown.

[0105] Controlled nucleation freezing involves less supercooling and has a consistent temperature (e.g., -5°C). Figure 1C Controlled nucleation occurring at -5°C according to the present invention is shown. Figure 1C The arrows in the image point to the same freezing temperature, and... Figure 1B The heterogeneous temperatures shown present a stark contrast. Preferred temperatures are not lower than -10°C, such as approximately -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 derived from the above values.

[0106] There are several methods to achieve controlled nucleation, including (1) inoculation and vial pretreatment, such as using additives / surface roughening, (2) ultrasonic nucleation: short vibration, (3) using ice fog to freeze the moisture in the chamber by introducing cold nitrogen into the chamber, and (4) decompression, but with the risk of product splashing.

[0107] The preferred method according to the invention employs pressure, for example, 14 to 42 psig, preferably 25 to 30 psig, more preferably about 28 psig. The pressure in the freeze-drying chamber can be determined using, for example, a Pirani vacuum gauge. The basic method includes:

[0108] (1) Cool the product to the predetermined freezing temperature (slightly below its freezing temperature).

[0109] (2) Pressurize the freeze-drying chamber with air or an inert gas, such as nitrogen or argon (approximately 28 psig); and

[0110] (3) Rapidly release pressure to induce ice nuclei.

[0111] More specifically, the implementations provided by the present invention may include:

[0112] Load the containers and seal the freeze dryer;

[0113] Cool the partitions and container to the target nucleation temperature;

[0114] Pressurize with air or an inert gas (such as helium, nitrogen, and / or argon);

[0115] Rapid decompression to induce nucleation; and

[0116] Lower the shelf temperature to complete the freezing process.

[0117] Generally, this invention provides an improved freeze-drying method that produces products with improved properties, as described herein. This invention employs controlled nucleation.

[0118] Figure 2A and Figure 2B Data were plotted on controlled nucleation parameter curves from different monoclonal antibody drug formulations. Figure 2A -5℃ Figure 2B The target temperature is -10°C. Controlled nucleation at -5°C ensures ice formation in all vials at the set temperature, with less supercooling compared to controlled nucleation at -10°C. For -5°C, a holding time of approximately 30 minutes during nucleation is sufficient. See also Figure 2A The holding time can 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 longer or shorter, as determined in light of the teachings contained herein.

[0119] Figures 3A through 3D depict data from various lyophilization methods for different monoclonal antibody drug formulations. Figure 3A depicts data from lyophilization on a conventional platform. Figure 3B depicts data from controlled nucleation at -5°C. Figure 3C depicts data from controlled nucleation at -10°C. Figure 3D depicts data from lyophilization on a conventional platform with annealing (see the circled portion between 0.0 and approximately 15 hours). The arrows in each figure indicate the end of one drying cycle.

[0120] The data described in Table 2 below indicate that, compared to conventional platforms, ice sublimation via controlled nucleation (CN) and annealing cycles results in lower product resistance.

[0121] Table 2 .

[0122]

[0123]

[0124] in:

[0125] Rˆp is the area-normalized product resistance (Torr·h·cm). 2 / g), Rp is the resistance (Torr·h / g);

[0126] Ap is the cross-sectional area of ​​the product (cm²) 2 );

[0127] Rp and Rs are the resistances (Torr·h / g) of the drying layer and the stopper to the water vapor sublimation interface, respectively.

[0128] Δm / Δt is the average ice sublimation rate (g / hour per bottle);

[0129] Pice is the equilibrium vapor pressure (Torr) of ice at the sublimation interface temperature;

[0130] PC stands for chamber pressure; and

[0131] TP is the product temperature (K) at the sublimation interface.

[0132] Figure 4 The drying time and freeze-drying time of a conventional platform (see Figure 3A), a controlled nucleation at -5℃ (see Figure 3C), a controlled nucleation at -10℃ (see Figure 3C), and a conventional platform with annealing (see Figure 3D) are described.

[0133] Overall, controlled nucleation significantly shortened the freeze-drying cycle time. Controlled nucleation at -5°C was faster than that at -10°C. Annealing also reduced the drying time per cycle, but added additional cycle time compared to controlled nucleation at -5°C. The total time for controlled nucleation freeze-drying plus one drying cycle at -5°C was approximately 39 hours, while the annealing method took approximately 48 hours.

[0134] Next, the moisture content of the freeze-dried products after conventional platform drying, annealing, controlled nucleation (CN) at -5℃ (twice) and controlled nucleation (twice) at -10℃ was evaluated, and the results are listed in Table 3.

[0135] Table 3 .

[0136]

[0137] *Platform DP = Fill a 20R vial with 5.3 mL of platform formulation (50 mg / mL mAb, 10 mM histidine, 5% sucrose, 0.1% PS20, pH 6.0).

[0138] *Platform PP = 5.3 mL of platform-matched placebo formulation is filled into a 20R vial.

[0139] All methods can produce freeze-dried products with an acceptable moisture content (below 4%). Annealing or controlled nucleation freeze-drying cycles are expected to increase the moisture content. If a lower moisture content is required, more robust secondary drying conditions should be used when using controlled nucleation.

[0140] The reconstitution time of lyophilized drugs prepared using conventional platform, annealing, and controlled nucleation lyophilization methods was then evaluated, and the results are listed in Table 4 below.

[0141] Table 4 .

[0142]

[0143] The fastest reconstitution time was observed at -5°C under controlled nucleation (CN) when reconstituted to a high concentration of antibody (150 mg / ml). Reconstitution time can be less than 3 minutes. More specifically, reconstitution times can be approximately or less than 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. Reconstitution to lower concentrations can be even faster. For example, for a 50 mg / ml antibody, reconstitution times can be approximately or less than 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40 seconds, or less.

[0144] The slowest method is conventional platform lyophilization. A similar but less pronounced trend was observed when reconstituted to 50 g / ml. No significant difference in reconstitution time was observed between very low concentrations of the drug (2 or 6 mg / ml) and the placebo product (both less than 1 minute) (data not shown).

[0145] 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). Figure 5A and Figure 5B SEC data depicting the percentage of native mAb obtained using buffers containing 150 mg / ml mAb and 5% or 1.5% sucrose are presented (see Table 1). Figure 5A It is a formulation intended for storage at 25°C. Figure 5B It is a formulation intended for storage at 40°C. Figure 5C and Figure 5D SEC and high molecular weight (HMW) data for formulations F1, F2, F3 and F4 are depicted (see formulations in Table 1). Figure 5C It is a formulation intended for storage at 25°C. Figure 5D This formulation is intended for storage at 40°C. For formulations containing 1.5% sucrose, compared to conventional lyophilization cycles, CN lyophilized products showed higher protein purity percentages (%) (less HMW formation) (F4 vs. F3). Degradation rates were comparable. For stable formulations containing 5% sucrose, CN (F2) showed slightly better stability compared to conventional (F1).

[0146] Each mAb formulation (5% sucrose and 1.5% sucrose) was lyophilized using standard platform methods and controlled nucleation. The formulations were stored at 25°C and 40°C prior to SEC.

[0147] For storage at 25°C and 40°C, there was almost no difference in molecular weight profiles between conventional and controlled nucleation of the 5% sucrose formulation. In contrast, the 1.5% sucrose formulation degraded over time when stored at both 25°C and 40°C. Using the 1.5% sucrose formulation, controlled nucleation lyophilization showed less degradation than conventional lyophilization at both temperatures.

[0148] In the above comparisons, there was no substantial difference in protein recovery between controlled nucleation lyophilization and conventional lyophilization, as well as storage at both temperatures. Protein recovery rates ranged from 97% to 99%.

[0149] Because more efficient lyophilization processes are needed to obtain more stable drugs, advancements in formulation and process strategies can be applied to improve lyophilized protein drugs.

[0150] This invention proposes that increasing the moisture content of lyophilized drugs can be an effective method to improve DP stability. Water appears to be the most effective plasticizer for reducing the local mobility of mAb molecules, and the optimal moisture content can be achieved through control during the lyophilization process.

[0151] The present invention also provides freeze-dried protein pharmaceuticals that are stable at room temperature and manufactured using an efficient freeze-drying process.

[0152] The invention is further described by way of the following examples, but they do not limit the invention in any way. The order in which the following experiments and / or examples or example steps are performed may be changed or combined, as can be determined by those skilled in the art in light of the teachings and data contained herein.

[0153] Example 1 – Freeze-dried mAb cake in a vial:

[0154] Figure 6A The lyophilized mAb formulations in vials are shown: from left to right, platform drug (DP), low-concentration placebo product (PP), low-concentration drug, and platform placebo product. These formulations are listed in Table 1.

[0155] Three out of the four vials showed product shrinkage at the bottom of the lyophilized mAb, as indicated by the arrows. If visual inspection indicates that the shrinkage is caused by cake collapse or remelting, the vial will be considered unacceptable.

[0156] Figure 7 The lyophilized mAb formulations in vials prepared using controlled nucleation are shown: from left to right: plateau drug, low-concentration placebo product, plateau placebo product, and low-concentration drug. Ascent was performed at -5°C, and descent at -10°C. Formulations are shown in Table 1. Overall, compared with... Figure 6A Compared to conventional lyophilized mAbs, lyophilized mAbs with controlled nucleation did not show significant shrinkage.

[0157] The lyophilized mAb formulation in the vial, such as Figure 6B As shown. Placebo and low-concentration (DP) drugs were lyophilized using annealing at -5°C (bottom row), controlled nucleation without annealing (top row), and controlled nucleation at -5°C (middle row). The arrows in the figure indicate product shrinkage. Although both controlled nucleation and annealing improve the cake structure, annealing may cause other unintended problems, such as:

[0158] (a) Potential products on the wall, and / or

[0159] (b) The crust forms, causing the cake to collapse.

[0160] Moderate product shrinkage is generally acceptable (if it is visually inspected as cake collapse or remelting, then the product is unacceptable).

[0161] Figure 8 The images show lyophilized mAb formulations in vials, annealed before lyophilization: from left to right, low-concentration drug (DP), plateau drug, low-concentration placebo product (PP), and plateau placebo product. See Table 1 for formulations.

[0162] Adding an annealing step during freeze-drying reduces cake shrinkage, but it also causes mAb products to adhere to the bottle walls (see [link]). Figure 8 (The arrow in the image).

[0163] Figure 9 The images show platform-dried high-concentration (150 mg / mL) mAb formulations in vials using a 5% w / v sucrose formulation (left) and a 1.5% sucrose formulation (right), as well as a lyophilized high-concentration (150 mg / mL) mAb formulation prepared by controlled nucleation at -5 °C. The lyophilized mAb cake with controlled nucleation exhibited less breakage than the conventional lyophilized mAb cake. See Table 1 for formulations. Figure 9 The arrows in the text indicate cracks in a conventional freeze-dried mAb cake.

[0164] Improved cake appearance: DP at -5℃ CN showed fewer or no cracks in the freeze-dried cake.

[0165] Product purity: CN shows higher purity (less aggregation) compared to the conventional one (150 mg / mL mAb3 formulation containing 1.5% v / w sucrose).

[0166] No substantial changes in CEX and particulate matter were observed at 25°C and 40°C within three months.

[0167] Example 2 – Electron microscopy examination of lyophilized mAbs:

[0168] The lyophilized mAb samples were examined by scanning electron microscopy (SEM) to assess morphology. 100 µm dimension bars are located in the upper right corner of each figure.

[0169] Figures 10A and 10B show scanning electron micrographs at the same scale. Figure 10A is from a platform lyophilized formulation (50 mg / ml), and Figure 10B is from a lyophilized formulation prepared by controlled nucleation at -5 °C. See Table 1 for formulations.

[0170] Comparison of cake morphology (SEM) between conventional and controlled nucleation freeze-drying cycles:

[0171] Conventional nucleation lyophilization (Fig. 10B) exhibits a larger pore size than conventional lyophilization (Fig. 10A). The larger pore size provided by controlled nucleation results in faster remelting time and lower cake resistance during ice sublimation.

[0172] Figures 11A and 11B show scanning electron micrographs at the same scale. Figure 11A is from a lyophilized platform (50 mg / ml) formulation prepared with controlled nucleation at -5 °C, and Figure 11B is from a lyophilized platform (50 mg / ml) formulation prepared with controlled nucleation at -10 °C. Formulations are listed in Table 1. Controlled nucleation at -5 °C (Figure 11A) provides a larger pore size than controlled nucleation at -10 °C (Figure 11B), resulting in faster reconstitution time and lower cake resistance during ice sublimation. Figure 11A is identical to Figure 10B. Repeated micrographs are provided for comparison.

[0173] Figures 12A and 12B show scanning electron micrographs at the same scale. Figure 12A is from conventional platform lyophilization. Figure 12B is from conventional platform lyophilization with annealing. See the formulations in Table 1. The addition of the annealing step allows conventional lyophilization to provide larger pores. However, as indicated by the 100 μm size bars, the pores in Figures 12A and 12B are smaller than those in Figures 10B and 11A. Similarly, the larger pore size resulting from controlled nucleation at -5°C leads to faster reconstitution times (see Table 4) and less cake resistance during ice sublimation. Furthermore, as... Figure 4 As shown, annealing requires a longer time than controlled nucleation at -5℃.

[0174] SEM was used to reveal the different cake morphologies of the pharmaceutical product produced by controlled nucleation and annealing lyophilization cycles, with a dramatic increase in pore size. SEM micrographs showed that the pore size at -5°C CN was larger than that at -10°C CN (compare Figures 10B and 11B). SEM micrographs further revealed that annealing also significantly increased the pore size (see Figure 12B). The larger pore size resulted in lower cake resistance and faster ice sublimation, thus requiring a shorter drying time per cycle.

[0175] Example 3 – The Effects of Annealing:

[0176] The effect of annealing on the resistance of 5% glycine dry product as follows Figure 13 As shown. The effects of annealing include the following aspects:

[0177] (a) Annealing reduces the resistance of the dried product and the drying time in one pass;

[0178] (b) Annealing increases the ice crystal size and the pore size in the dry cake for ice sublimation;

[0179] (c) The higher the annealing temperature, the more effective it is; and

[0180] (d) The same product resistance can be achieved at a lower temperature for a longer period of time.

[0181] Example 4 – Exemplary antibody for controlled nucleation lyophilization:

[0182] In another respect, the antibody is selected from the group consisting of: anti-programmed cell death 1 antibody (e.g., anti-PD1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203579A1), anti-programmed cell death ligand-1 (e.g., anti-PD-L1 antibody as described in U.S. Patent Application Publication No. US2015 / 0203580A1), anti-Dll4 antibody, anti-angiogenic-2 antibody (e.g., anti-ANG2 antibody as described in U.S. Patent No. 9,402,898), and anti-angiogenic-like 3 antibody (e.g., as described in U.S. Patent No. 9,018,356). Anti-AngPtl3 antibodies, anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Patent No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Patent No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Patent Application Publication No. US2015 / 0313194A1), anti-TNF antibodies, and anti-epidermal growth factor receptor antibodies (e.g., anti-EG antibodies as described in U.S. Patent No. 9,132,192). FR antibodies, or anti-EGFRvIII antibodies as described in U.S. Patent Application Publication No. US2015 / 0259423A1, anti-proprotein convertase subtilisin Kexin-9 antibodies (e.g., anti-PCSK9 antibodies as described in U.S. Patent No. 8,062,640 or U.S. Patent Application Publication No. US2014 / 0044730A1), anti-growth and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies as described in U.S. Patent Nos. 8,871,209 or 9,260,515, also known as anti-myosostatin antibodies), anti-hypertensive antibodies, etc. Glycosin receptors (e.g., anti-GCGR antibodies as described in U.S. 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 U.S. Patent Application Publication No. US2014 / 0271681A1 or U.S. Patent Nos. 8,735,095 or 8,945,559), and anti-interleukin-6 receptor antibodies (e.g., U.S. Patent Nos. 7,582,298, 8,043,617, or 9,173).Anti-IL6R antibody (as described in patent application 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 No. US2014 / 0271658A1 or No. US2014 / 0271642A1), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Application Publication No. US2014 / 0271653A1), anti-differentiation cluster 3 (e.g., as described in U.S. Patent Application Publication 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 No. 880), anti-respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Patent Application Publication No. 880), anti-interleukin 3 (e.g., anti-IL6R antibody as described in U.S. Patent Application Publication 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 No. 880), anti-interleukin 33 (e.g., anti-IL6R antibody as described in U.S. Patent Application Publication No. 880), anti-interleukin 33 (e.g., anti-IL6 Anti-CD3 antibodies, anti-differentiation cluster 20 (e.g., anti-CD20 antibodies as described in US Patent Application Publication No. US2014 / 0088295A1 and US20150266966A1, and US Application No. 62 / 222,605), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation cluster 48 (e.g., anti-CD48 antibodies as described in US Patent Application Publication No. US2014 / 0088295A1 and US20150266966A1, and US Patent No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-differentiation cluster 48 (e.g., anti-CD48 antibodies as described in US Patent No. 9,228,014), anti-Fel d1 antibodies (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 Application Publication No. US2015 / 0337029A1), anti-Ebola virus antibodies (e.g., as described in U.S. Patent Application Publication No. US2016 / 0215040), anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibodies (e.g., as described in U.S. Patent Application Publication No. US2016 / 0017029 and U.S. Patent Nos. 8,309,088 and 9,353,The bispecific antibody described in patent number 176 is an anti-NGF antibody and an anti-activin A antibody. In some embodiments, the bispecific antibody is selected from the group consisting of: anti-CD3 x anti-CD20 bispecific antibodies (such as U.S. Patent Application Publications US2014 / 0088295A1 and US20150266966A1), anti-CD3 x anti-mucin 16 bispecific antibodies (e.g., anti-CD3 x anti-Muc16 bispecific antibody), and anti-CD3 x anti-prostate-specific membrane antigen bispecific antibodies (e.g., anti-CD3 x anti-PSMA bispecific antibody). See also U.S. Patent Publication US2019 / 0285580 A1. It also includes Met x Met antibodies, anti-NPR1 agonist antibodies, LEPR agonist antibodies, BCMA x CD3 antibodies, MUC16 x CD28 antibodies, GITR antibodies, IL-2Rg antibodies, EGFR x CD28 antibodies, factor XI antibodies, antibodies against SARS-CoC-2 variants, Fel d 1 multi-antibody therapy, and Bet v 1 multi-antibody therapy. Derivatives, components, domains, chains, and fragments of the above are also included.

[0183] Cells that produce exemplary antibodies can be cultured according to the present invention. Exemplary antibodies include alikumab, atenvironmentumab, matevironmentumab, oxivironmentumab, oxivironmentumab-ebgn, casciretumab, edevimab, cimiprimab-rwlc (a human IgG4 monoclonal antibody that binds to PD-1), dupilumab (a human monoclonal antibody that binds to the IL-4 Rα(α) subunit and thereby inhibits interleukin 4 (IL-4) and interleukin 13 (IL-13) signaling), everxumab, everxumab-dgnb, fasinumab, fumarazumab, gatoxumab, etagerumab, nesivamab, onitumumab, pazelimab, thalidomab, trogolumab, and linusumab.

[0184] Other exemplary antibodies include rivolizumab-cwvz, abciximab, adalimumab, adalimumab-atto, Ado-trastuzumab, alemtuzumab, atezolizumab, avemumab, baliximab, belimumab, benalizumab, bevacizumab, belotuszumab, bonatetumab, brentuximab, brodatumab, konnatumab, calcimeroxate, pecelizumab, cetuximab, denosumab, denutoximab, durvalumab, ikulizumab, erlotuximab-kxwh, mettansine amorobumabumab, evolocumab, and globulin. Limousinumab, Gusekirumab, Tiimozumab, Idacilumab, Infliximab, Infliximab-abda, Infliximab-dyyb, Ipilimumab, Ixizilumab, Meperizumab, Nexitocilumab, Nivolumab, Otosacoxetine, Otocilumab, Otocilumab, Omalicilumab, Ofamumab, Omalicilumab, Panitumab, Pembrolizumab, Pertuzumab, Ramucirumab, Ralcicilumab, Ralcicilumab, Raxicurumab, Rinusuumab, Rituximab, Secukinumab, Stutuzumab, Tocilizumab, Trastuzumab, Ustenolimab, and Vedolizumab.

[0185] Example 5 – Single-stage drying design: Target product temperature and chamber pressure:

[0186] As a general rule, the temperature of the target product should be within a "safe margin" below the collapse temperature.

[0187] a. The selection of the target product temperature is based on the following factors:

[0188] i. Target Tp = Tc - (safety margin); and

[0189] ii. The target Tp can be significantly higher than Tg' (especially for high-concentration protein drugs).

[0190] b. The chamber pressure selection (calculation) is based on the following factors:

[0191] i. Drive ice sublimation by allowing sufficient vacuum (the lower the chamber pressure, the better);

[0192] ii. By improving heat transfer uniformity (optimal ~150-200 mT); and

[0193] iii. The chamber pressure (Pc) depends on the target product temperature, as shown in the following formula:

[0194]

[0195] c. The shelf temperature and duration can be designed to control the product moisture content:

[0196] i. When a known, appropriate moisture content is required.

[0197] Example 6 – Single-stage drying conditions for high-concentration protein formulations :

[0198] The 150 mg / mL mAb formulation can use a Tc that is 15° to 20° higher than Tg':

[0199] i. The strong vitreous body formed by high concentrations of mAb; and

[0200] ii. Since the formulation is mainly a brittle glass-forming body, the difference between Tc and Tg' was small.

[0201] For high-concentration mAb formulations, lyophilization can be performed at high shelf temperatures, with the product temperature above Tg' (but below Tc).

[0202] i. No collapse of freeze-dried DP cake was observed.

[0203] Table 5 lists the conditions for using several stabilizers.

[0204] Table 5 shows the drying conditions data for protein formulations containing several stabilizers.

[0205] Table 5 .

[0206]

[0207] Example 7 – Secondary Drying Design:

[0208] The design of the secondary drying process takes the following criteria into account:

[0209] a. Chamber pressure

[0210] i. Maintain the same room as the first drying.

[0211] ii. The chamber pressure has little effect on the water desorption rate (equal to or less than 200 mT).

[0212] b. Shelf temperature and heating rate

[0213] i. The "high" temperature required for hydrolysis;

[0214] 1. Below the glass transition temperature,

[0215] ii. Lower shelf temperatures require a longer time;

[0216] iii. Excessive drying may occur at high shelf temperatures (especially if the formulation does not contain sufficient stabilizers); and

[0217] iv. Increase the temperature slowly to ensure the product desorbs before reaching high temperatures;

[0218] 1. This is even more critical for amorphous products.

[0219] c. Duration

[0220] i. Generally, several hours;

[0221] 1. Insufficient secondary drying time may hinder scale-up (uneven moisture content), and

[0222] ii. Avoid excessively long secondary drying times (e.g., > 12 hours), and use the higher permissible temperature for efficiency.

[0223] d. Freeze-dried without secondary drying

[0224] i. When a moderate moisture content is more advantageous than "dryness".

[0225] Example 8 – Water as a plasticizer to stabilize proteins at moderate moisture content:

[0226] The mAb isotonic formulation (150 mg / mL) was subjected to different moisture contents at different storage temperatures. The changes in %HMW aggregates were determined.

[0227] The figure depicts the drug formulation at 5°C, 25°C, and 30°C. Figure 14A ) and 37℃ and 50℃ ( Figure 14B Moisture content and stability under ( ).

[0228] Under storage conditions ranging from 5°C to 50°C, the lowest possible moisture content does not provide optimal stability. In contrast, a moderate moisture content of approximately 4% (about 2% to 5%) is found to be most suitable for room temperature storage. At higher storage temperatures, the optimal moisture content is lower (approximately 2% at 50°C) (see...). Figure 14B It does not have the disadvantage of increasing the viscosity or osmotic pressure of the formulation.

[0229] Example 9 – Achieving an efficient freeze-drying process with appropriate moisture content:

[0230] Experiments were conducted to identify an effective freeze-drying process in order to determine the appropriate moisture content in the pharmaceutical product.

[0231] Figure 15A and Figure 15B Results of an effective freeze-drying process and appropriate moisture content in DP are described. Figure 15A This indicates a stop after the ice sublimation process has ended.

[0232] Use a freeze-drying cycle without a secondary drying step to control the target residual moisture content of the product (e.g., approximately 2% to 4% residual moisture content).

[0233] Two-step freeze-drying cycle: Freezing and drying appear to be the shorter process.

[0234] Moisture content can be controlled by the drying shelf temperature (Ts) and the duration of ice sublimation (without a secondary drying step).

[0235] Example 10 – The optimal combination of shelf temperature and ice sublimation time:

[0236] DP water content (after ice sublimation) is controlled by a combination of shelf temperature and duration after one drying cycle. Figure 16 The optimal combination of shelf temperature and ice sublimation time for achieving the target residual moisture content was depicted. Exponential decay fitting:

[0237]

[0238] The optimal combination of shelf temperature and ice sublimation time is achieved by (a) controlling the DP water content (after ice sublimation) under a combination of shelf temperature and the duration of the first drying cycle. This involves extending the first drying time to achieve the target residual moisture content; and (b) minimizing this extension time to maximize process efficiency, while adjusting the shelf temperature as needed.

[0239] Example 11 – Room temperature stable lyophilized pharmaceutical products with moderate moisture content:

[0240] The isotonic formulation of mAb (150 mg / mL) was stored at room temperature (approximately 25°C) with approximately 0% and approximately 4% moisture content. Changes in %HMW aggregates were determined by SEC-UPLC. Figure 17 The %HMW in stable freeze-dried DP with moderate moisture content at 25 °C was depicted by SE-UPLC.

[0241] Monoclonal Abs with moderate moisture content (about 4%) appear to be more stable when stored at room temperature (about 25°C).

[0242] Lyophilized DP is stable at room temperature with an optimal moisture content (approximately 4% H2O). When DP formulations are lyophilized and stored at a determined optimal moisture content (approximately 4% moisture content), high-concentration (150 mg / mL) isotonic formulations suitable for IV and SC administration are stable at room temperature.

[0243] Example 12 – Separation using size exclusion chromatography (SEC), cation exchange chromatography (CEX), and microfluidic imaging (MFI) Analysis results:

[0244] Microfluidic imaging (MFI) of samples from different freeze-drying cycles. Figure 18 MFI analysis data for unfiltered particles ≥ 10 µm were presented. No substantial effect on subvisible particles was observed.

[0245] Microfluidic imaging (MFI) of samples from different freeze-drying cycles. Figure 19 MFI analysis data for unfiltered particles ≥ 25 µm were presented. No substantial effect on subvisible particles was observed.

[0246] Stability studies were conducted on platform DP (Fig. 20A) and low-concentration DP (Fig. 20B) samples from different lyophilization cycles over a period of more than six months. High molecular weight percentage (%HMW) was determined by SEC. Samples were stored at approximately 25°C and approximately 60% relative humidity (RH). Based on the %HMW aggregates determined by SEC, Figs. 20A and 20B show no significant effect on stability.

[0247] Stability studies were conducted on platform DP (Fig. 21A) and low-concentration DP (Fig. 21B) samples from different lyophilization cycles over a period of more than six months. Samples were stored at 25°C and 60% relative humidity (RH). Based on the % main peak (region 2) determined by cation exchange chromatography (CEX), Figs. 21A and 21B show no significant effect on stability.

[0248] Example 13 – Protein Concentration and Recovery :

[0249] Protein concentrations and percentage recoveries (%recovery) in formulations F1-F4 (see Table 1) stored at 25°C and 40°C for three months were determined using reversed-phase ultra-high performance liquid chromatography (RP-UPLC). No significant changes in protein concentration were observed (i.e., almost complete protein recovery was observed for all formulations F1-F4) (Table 6).

[0250] Table 6 shows the mAb3 protein concentration and % recovery data from RP-UPLC.

[0251] Table 6 .

[0252]

[0253] Example 14 – The effect of cryoprotectants on subvisible particles:

[0254] Samples F1 and F2 of pharmaceutical (DP) formulations containing 5% sucrose from different freeze-drying cycles were analyzed. These samples were stored for one month at T0, 25°C, and 40°C. Figure 22A and Figure 22B This indicates that DP formulation contains 25μm ( Figure 22A ) and 10μm ( Figure 22BThe graph shows the data for particles at different temperatures (25°C and 40°C) and times (t0, 1m, and 3m). Formulation F1 was a plateau cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 5% w / v sucrose, 0.1% w / v PS80, and pH 6.0; F2 was a CN-5°C cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 5% w / v sucrose, 0.1% w / v PS80, and pH 6.0. There was no significant effect on 10 μm and 25 μm particles.

[0255] Samples of pharmaceutical (DP) formulations containing 5% sucrose, F3 and F4, from different freeze-drying cycles, were analyzed after being stored at T0, 25°C, and 40°C for one month. Figure 23A and Figure 23B This graph shows the data for 10 μm and 25 μm particles in the DP formulation at different temperatures (25℃ and 40℃) and times (t0, 1m, and 3m). Formulation F3 was a plateau cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 1.5% w / v sucrose, 0.1% w / v PS80, and pH 6.0; F4 was a CN-5℃ cycle containing 150 mg / mL mAb3, 10 mM L-histidine, 1.5% w / v sucrose, 0.1% w / v PS80, and pH 6.0. There was no significant effect on 10 μm and 25 μm particles.

[0256] The experimental results presented in this paper reveal that controlled nucleation promotes small and consistent supercooling of ice crystals (i.e., the formation of larger ice crystals and thicker pie walls). Larger ice crystals provide lower resistance to the flow of water vapor from the ice sublimation interface, while thicker pie walls improve the pie structure.

[0257] The advantages of controlled nucleation include:

[0258] • Faster drying in one pass and shorter cycles,

[0259] • Reduce reconstitution time

[0260] • Improve the appearance of the pie, and

[0261] • Higher monomer purity after freeze-drying.

[0262] Example 15 – BET (Brunauer, Emmett, and Teller) Surface Area and Hg Porosity of Freeze-Dried Cake Samples analyze:

[0263] The N2 BET surface area and Hg porosity of the freeze-dried cake samples were examined. The BET theory is a popular model used to determine the surface area of ​​exposed surfaces of solid samples at the molecular scale. Nitrogen (N2) is typically used as an adsorbate for BET surface area analysis due to its high affinity for solid surfaces. N2 gas was introduced at low pressure, and the amount adsorbed was determined to calculate the surface area using the BET equation.

[0264] BET analysis showed that the conventional samples had a higher specific surface area than the controlled ice core samples (see Table 7). Additional pore diameter measurements using Hg intrusion volume revealed a significant difference between the conventional and controlled ice samples (see Table 7). Figure 25 The pore diameter of conventional ice is much smaller (see Table 8). The N2 isotherm indicates that the sample has a relatively low surface area. Isotherm data are as follows: Figure 24 As shown in Table 7, the BET surface area results are summarized in Table 7.

[0265] Table 7 Summary of N2 adsorption analysis.

[0266]

[0267] Table 8 Summary of Hg porosity results.

[0268]

[0269] N2 adsorption

[0270] The N2 isotherm was measured on a Micromeritics TriStar II Plus apparatus, with relative pressure (P / P) measured. o The surface area was approximately 0.4 to enable BET surface area analysis. Prior to analysis, the sample was activated on a SmartVacPrep degassing device by dynamic vacuum degassing at ambient temperature.

[0271] N2 BET measurements of standard materials show that when there are at least 50 m in the sample cell... 2 The material can be used for testing when the surface area is 10 m². 2 / g, with an accuracy within approximately 5%; within ~0.5 m 2 At the / g level, the accuracy is within approximately 10%. The repeatability of any given sample depends on the ability to regenerate the sample to the same degree of activation without altering the surface or pore structure.

[0272] Hg porosity determination

[0273] The mercury intrusion experiment was outsourced. The Hg porosity was determined by first filling the void space surrounding the material with Hg into a chamber of known volume at a filling pressure of approximately 0.5 psia. It was assumed that no Hg permeated into the sample at this filling pressure. The Hg pressure was then increased to approximately 60,000 psia to force it into the void space within the sample. The relationship between the intrusion amount and pressure was recorded and converted into a pore size distribution.

[0274] The bulk density obtained by the Hg porosity determination method is usually called Hg density (η). Hg The density of Hg reflects the density of the sample before Hg intrusion, and is therefore similar to the geometric density of the material. Thus, the unit of Hg density is grams of solids / volume of solids + void space (assuming no closed pores). The mercury intrusion volume (MIV), measured by pressing Hg into the sample under pressure, is considered the void space within the sample. Comparing Hg density and MIV values ​​allows one to estimate the total porosity of the material. % porosity can be calculated using the following equation:

[0275]

[0276] The logarithmic differential data of the samples represent unimodal PSD. The pore mode center of sample LT-SS001 CYCLE 3 REPEAT F1 t=0 VIALL19-002020 is approximately 45.2 μm, while that of sample LT-SS001 CYCLE CYCLE 1 F1 t=0 VIALL19-002020 is approximately 21.3 μm. Table 8 provides the values ​​of Hg density (i.e., bulk density at ~0.5 psia), MIV, and calculated porosity. Figure 25 and Figure 26 Logarithmic derivative and cumulative volume PSD plots are provided respectively.

[0277] It should be understood that the descriptions, specific examples, and data are given by way of example and are not intended to limit the invention. Various changes and modifications within the invention, including combinations of the teachings in whole and in part, will become apparent to those skilled in the art based on the discussion, disclosure, and data contained herein, and are therefore considered part of the invention.

Claims

1. A method for freeze-drying a protein product, wherein the method includes the steps of: (a) Cool the solution containing the protein product to below the freezing point of the solution; (b) Pressurize the cooling solution from step (a) with gas; (c) Releasing the pressure from step (b) to allow nucleation, thereby allowing ice nuclei to form in the cooling solution; and (d) Freeze-dry the cooling 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 during step (b), the gas is present at 14 to 42 psig.

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

5. The method according to any one of claims 1 to 4, wherein the cooling solution is in a lyophilized bottle.

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

7. The method according to claim 6, wherein step (c) is performed 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 1 hour or less.

9. The method of 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 of claim 10, wherein the Fc-containing protein is an Fc fusion protein.

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

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

14. The method according to any one of claims 12 to 13, wherein the antibody is reconstituted to 150 mg / ml within about 3 minutes.

15. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 50 seconds.

16. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 40 seconds.

17. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 30 seconds.

18. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 20 seconds.

19. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 10 seconds.

20. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 2 minutes and 5 seconds.

21. The method of claim 14, wherein the antibody is reconstituted to 150 mg / ml in about 1 minute and 45 seconds.

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

23. A method for preparing a room-temperature stable lyophilized therapeutic protein product by providing a target residual moisture content in the lyophilized product, wherein the method comprises the following steps: (a) After ice sublimation, the shelf temperature (Ts) is controlled, eliminating the need for a secondary drying step; and (b) Control the duration of drying after a single drying cycle; The target residual moisture content in the freeze-dried therapeutic protein product is from about 1% to about 7%.

24. The method of 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 of claim 23, wherein the stability of the lyophilized therapeutic protein product is measured by determining the %HMW protein aggregate change in the product.

26. The method of claim 25, wherein the %HMW protein aggregate change in the stable lyophilized therapeutic protein product is less than 1.0%, as determined by SEC-UPLC.

27. The method of claim 25, wherein the %HMW protein aggregate variation in the stable lyophilized 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 room-temperature stable lyophilized therapeutic protein product, produced by the method according to any one of claims 23 to 27.

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