A rapid method for producing lyophilized protein formulations

Abstract

Disclosed herein is a rapid method for preparing lyophilized formulations comprising proteins, such as antibodies or bispecific antigen-binding molecules, that exhibit improved storage stability.

Description

[Technical field]

[0001] The present disclosure provides a rapid method for preparing lyophilized formulations containing proteins, such as antibodies or bispecific antigen-binding molecules, that exhibit improved storage stability.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety and identified as follows: Filename: I-56846_Seqlisting.txt; Size: 345,286 bytes; Creation Date: May 12, 2022. [Background technology]

[0003] Protein-based drugs, such as drugs containing antibodies, antibody fragments and bispecific antigen-binding molecules, are becoming increasingly important for the treatment of various diseases and conditions. However, proteins are only marginally stable and are highly susceptible to chemical and physical degradation. Chemical degradation refers to covalent modifications such as deamidation, oxidation, cleavage, clipping / fragmentation, formation of new disulfide bridges, hydrolysis, isomerization or deglycosylation. Physical degradation includes protein unfolding, undesirable adsorption to surfaces and aggregation. Addressing these physical and chemical instabilities is one of the most difficult challenges in the development of protein drugs (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325-1336, Roberts, Trends Biotechnol. 2014 Jul; 32(7): 372-80).

[0004] Half-life extended antigen binding molecules (e.g., bispecific T cell engagers (BiTEs) that contain half-life extended modalities such as Fc molecules) need to be particularly protected from protein aggregation and / or other degradation events. Protein aggregation of BiTE molecules is problematic because it can weaken the biological activity and quality (specifications) of the therapeutic protein. Furthermore, aggregation of BiTE molecules can reduce product yields due to the extensive purification steps required to remove aggregates from the final product. More recently, there has also been increasing concern and evidence that the presence of aggregated proteins (even humanized or fully human proteins) can significantly increase the risk that patients will mount an immune response against the active protein monomer, resulting in the formation of neutralizing antibodies and drug resistance or other adverse side effects (Mahler J Pharm Sci. 2009 Sep;98(9):2909-34). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Chi et al.,Pharm Res,Vol.20,No.9,Sept 2003,pp.1325-1336,Roberts,Trends Biotechnol.2014 Jul;32(7):372-80 [Non-Patent Document 2] Mahler J Pharm Sci.2009 Sep;98(9):2909-34 Summary of the Invention [Problem to be solved by the invention]

[0006] Protein-based pharmaceutical formulations are often freeze-dried and stored in solid form to promote the preservation of the integrity of proteins such as antibodies or bispecific antigen-binding molecules in the formulation during storage.However, many current freeze-drying methods for protein formulations do not produce solid formulations that exhibit suitable stability over time and are produced quickly compared to known methods.Therefore, there is a need for a new method for rapidly producing freeze-dried protein formulations that exhibit improved storage stability. [Means for solving the problem]

[0007] In one aspect, the disclosure provides a method for preparing a lyophilized formulation, comprising the steps of: (a) cooling a lyophilization chamber containing a liquid formulation comprising a protein, [saccharide, and surfactant] to a temperature in the range of about -35°C to about -50°C to produce a frozen formulation, and holding the chamber at a temperature in the range of about -40°C to about -50°C for a time period of about 1.0 hour to about 3.0 hours; (b) heating the chamber to a temperature in the range of about -35°C to about -20°C and a pressure in the range of about 75 mTorr to about 125 mTorr to produce a primary dried formulation, and holding the chamber at a temperature in the range of about -35°C to about -20°C and a pressure in the range of about 75 mTorr to about 125 mTorr to produce a primary dried formulation. (c) heating the chamber to a temperature in the range of about 20° C. to about 30° C. to produce a secondary dried formulation, and holding the chamber at a temperature in the range of about 20° C. to about 30° C. and a pressure in the range of about 50 mTorr to about 100 mTorr for a time of about 5 hours to about 12 hours to produce a lyophilized formulation; the liquid formulation has a pH of about 3 to 7 and is free of mannitol; and the method lacks an annealing step.

[0008] In another aspect, the disclosure provides a lyophilized protein formulation prepared by the method of the disclosure.

[0009] Further aspects and advantages will become apparent to those of ordinary skill in the art upon review of the following detailed description. While the methods disclosed herein may take embodiments in a variety of forms, the following description sets forth specific embodiments with the understanding that the disclosure is illustrative and is not intended to limit the invention to the specific embodiments described herein. [Brief description of the drawings]

[0010] [Figure 1] The dried product cake after completion of the cycle is shown and assessed by visual inspection for signs of macroscopic collapse and overall quality. The product cake for BITE B was determined to be acceptable and photographs taken from several angles are shown. [Diagram 2] Figure 1 shows the relative area % values ​​of high molecular weight (HMW) species plotted over time. The data suggests no aggregation instability over the course of the study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Disclosed herein is a method for rapidly preparing lyophilized formulations containing proteins, such as antibodies or bispecific antigen-binding molecules (e.g., extended half-life bispecific antigen-binding molecules), that exhibit improved stability. The accelerated lyophilization method disclosed herein advantageously results in reduced physical degradation, such as aggregation, and reduced chemical degradation, such as reduced clipping and deamidation. Furthermore, the accelerated lyophilization method disclosed herein can stabilize protein formulations, such as those containing antibodies and bispecific antigen-binding molecules, at both low and high concentrations.

[0012] definition As used herein, the term "pharmaceutical formulation" refers to a formulation suitable for administration to a subject in need thereof. The terms "subject", or "individual", or "animal", or "patient" are used interchangeably herein and refer to any subject, particularly a mammalian subject, to which administration of the pharmaceutical formulation of the present invention is desired. Mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, dairy cows, and the like, with humans being preferred. The pharmaceutical formulations of the present disclosure are stable and pharmaceutical acceptable, i.e., capable of exerting the desired therapeutic effect without causing significant undesirable local or systemic effects in the subject to which the pharmaceutical formulation is administered. The pharmaceutical acceptable formulations of the present disclosure may be sterile. In particular, the term "pharmaceutical acceptable" may mean approved by a regulatory agency or other commonly recognized pharmacopoeias for use in animals, more particularly in humans, but is not limited to being approved by a regulatory agency.

[0013] The term "stability" or "stabilization" refers to the stability of the pharmaceutical formulation as a whole, and in particular to the stability of the active ingredient (e.g., a protein such as a bispecific antigen-binding molecule) itself, specifically during formulation, filling, transportation, storage and administration. A "stable formulation" is one in which the protein (e.g., an antibody or bispecific antigen-binding molecule) therein substantially maintains its physical and / or chemical integrity and biological activity during storage and processing (e.g., freeze / thaw, mechanical mixing and lyophilization). Protein stability can be measured by the formation of high molecular weight (HMW) species, loss of enzymatic activity, generation of peptide fragments and shifts in charge profile.

[0014] The term "aggregation" as used herein refers to a direct mutual attraction between molecules, for example, due to van der Waals forces or chemical bonds. In particular, aggregates are understood to be proteins that accumulate and clump together. Aggregates can include amorphous aggregates and oligomers, but are typically referred to as high molecular weight (HMW) species, i.e., molecules that have a higher molecular weight than the product molecule, which is the non-aggregated molecule.

[0015] The term "(protein) aggregates" as used herein generally encompasses high molecular weight protein species such as "oligomers" or "multimers" rather than the desired defined species (e.g., monomers). The term is used interchangeably herein with the terms "high molecular weight" species and "HMW". Protein aggregates may generally differ in size (ranging from small (dimers) to large aggregates (submicroscopic or visible particles) in the nanometer to micrometer diameter range), morphology (nearly spherical to fibrous), protein structure (native vs. non-native / denatured), type of intermolecular bonds (covalent vs. non-covalent), reversibility and solubility. Soluble aggregates occupy the approximate 1-100 nm size range, while protein microparticles occupy the submicroscopic (approximately 0.1-100 nm) and visible (>100 nm) ranges. All of the aforementioned protein aggregate types are generally encompassed by this term. Thus, the term "(protein) aggregate" refers to any type of non-naturally occurring species in which two or more protein monomers are physically associated or chemically linked.

[0016] As used herein, the term "low molecular weight (LMW)" species refers to fragments of proteins such as bispecific antigen-binding molecules.

[0017] As used herein, the term "accelerated lyophilization process" refers to a lyophilization process that is at least 25% faster than known processes that use different temperatures and pressures in a lyophilization device, while maintaining the stability of the lyophilized protein formulations described herein.

[0018] method One aspect of the present disclosure provides a method for rapidly preparing a lyophilized formulation, which does not include an annealing step. The method includes the steps of: (a) cooling a lyophilization chamber containing a liquid formulation having a pH of about 3 to 7, containing a protein, a sugar, and a surfactant, and lacking mannitol, to a temperature in the range of about -40°C to about -50°C to produce a frozen formulation, and holding the chamber at a temperature in the range of about -40°C to about -50°C for a period of about 1 hour to about 3 hours; (b) heating the chamber to a temperature in the range of about -30°C to about -20°C and a pressure in the range of about 75 mTorr to about 125 mTorr to produce a primary dry formulation; (c) preparing a freeze-dried formulation by heating the chamber to a temperature in the range of about 20°C to about 35°C and a pressure in the range of about 50 mTorr to about 100 mTorr for a time period of about 5 hours to about 10 hours; and (b) preparing a freeze-dried formulation by heating the chamber to a temperature in the range of about 20°C to about 30°C and a pressure in the range of about 50 mTorr to about 100 mTorr for a time period of about 5 hours to about 10 hours.

[0019] The term "temperature" as used herein refers to the temperature inside the freeze-drying chamber (i.e., the internal temperature of the freeze-drying chamber "internal temperature"), and the controlled "temperature" of the freeze-dryer during the cycle is precisely measured by the inlet temperature of the silicone oil pumped through the shelf of the chamber. In other words, it is the temperature of the liquid pumped into the metal shelf of the chamber that is in contact with the bottom of the sample vial. Similarly, the term "pressure" as used herein refers to the pressure inside the freeze-drying chamber (i.e., the internal pressure of the freeze-drying chamber "internal pressure").

[0020] Step (a). In step (a), the lyophilization chamber containing the liquid formulation is cooled to a temperature (e.g., internal temperature) in the range of about -35°C to about -50°C to produce a frozen formulation, and is maintained at a temperature (e.g., internal temperature) in the range of about -40°C to about -50°C for a period of about 2 hours to about 24 hours. In some embodiments, cooling is performed to a temperature in the range of about -40°C to about -50°C (e.g., about -40°C, -41°C, -42°C, -43°C, -44°C, -45°C, -46°C, -47°C, -48°C, -49°C, or -50°C). In various examples, cooling is performed to a temperature of about -45°C. In some examples, cooling of the chamber is performed at a rate in the range of about 0.1°C / min to about 1°C / min. In various embodiments, cooling is performed at a rate of about 0.5°C / min to about 0.8°C / min. In some embodiments, the cooling is performed at a rate of about 0.5° C. / min, 0.6° C. / min, 0.7° C. / min, 0.8° C. / min, 0.9° C. / min, or 1° C. / min. In some examples, the cooling is performed at a rate of about 0.5° C. / min. In some embodiments, the holding of the chamber can be performed at a temperature of about −40° C., −41° C., −42° C., −43° C., −44° C., −45° C., −46° C., −47° C., −48° C., −49° C., or 50° C. In some embodiments, the holding is performed at a temperature of about −45° C. In some embodiments, the cooling temperature and the holding temperature of the lyophilization chamber are the same. In various embodiments, the holding is performed for a time period of about 1 hour to about 3 hours (e.g., about 1 hour, 1.5 hours, 2 hours, 2.5 hours, or 3 hours). In some examples, the holding is performed for about 2 hours.

[0021] Step (b): In step (b), the lyophilization chamber is heated to a temperature (e.g., internal temperature) in the range of about -35°C to about -20°C and a pressure (e.g., internal pressure) in the range of about 75mTorr to about 125mTorr to produce a primary dried formulation, and is held at a temperature (e.g., internal temperature) in the range of about -35°C to about -20°C and a pressure (e.g., internal pressure) in the range of about 75mTorr to about 125mTorr for a period of about 12 hours to about 24 hours. In some embodiments, heating is performed to a temperature of about -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, or 20°C. In various examples, heating is performed to a temperature of about -27°C. In some examples, the heating is performed at a rate ranging from about 0.1° C. / min to about 1° C. / min. In various embodiments, the heating is performed at a rate of about 0.1° C. / min to about 0.5° C. / min (e.g., 0.1° C. / min, 0.2° C. / min, 0.3° C. / min, 0.4° C. / min, or 0.5° C. / min). In some examples, the heating is performed at a rate of about 0.3° C. / min. In various examples, the heating is performed at a pressure ranging from about 75 mTorr to about 125 mTorr, or from about 80 mTorr to about 120 mTorr, or from about 85 mTorr to about 115 mTorr, or from about 90 mTorr to about 110 mTorr. In some examples, the heating is performed at a pressure of about 95 mTorr, 96 mTorr, 97 mTorr, 98 mTorr, 99 mTorr, 100 mTorr, 101 mTorr, 102 mTorr, 103 mTorr, 104 mTorr, or 105 mTorr. In various embodiments, the heating is performed at a pressure of about 100 mTorr. In some embodiments, the holding of the chamber is performed at a temperature of about -35°C, -34°C, -33°C, -32°C, -31°C, -30°C, -29°C, -28°C, -27°C, -26°C, -25°C, -24°C, -23°C, -22°C, -21°C, or -20°C. In various embodiments, the holding is performed at a temperature of about -27°C. In various embodiments, the holding is at a pressure ranging from about 75 mTorr to about 125 mTorr, or from about 80 mTorr to about 120 mTorr, or from about 85 mTorr to about 115 mTorr, or from about 90 mTorr to about 110 mTorr.In some examples, the heating is performed at a pressure of about 95 mTorr, 96 mTorr, 97 mTorr, 98 mTorr, 99 mTorr, 100 mTorr, 101 mTorr, 102 mTorr, 103 mTorr, 104 mTorr, or 105 mTorr. In various embodiments, the heating is performed at a pressure of about 100 mTorr. In some embodiments, the temperature to which the lyophilization chamber is heated and the holding temperature are the same. In some embodiments, the pressure to which the lyophilization chamber is heated and the holding pressure are the same. In some embodiments, the temperature to which the lyophilization chamber is heated and the holding temperature are the same, and the pressure to which the lyophilization chamber is heated and the holding pressure are the same. In some examples, the holding is performed for a time period of about 12 hours to about 24 hours (e.g., about 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, or 18 hours). In various examples, the holding is performed for a time period of about 16 to 17 hours, for example, 16.7 hours.

[0022] Step (c). In step (c), the chamber is heated to a temperature (e.g., internal temperature) in the range of about 20° C. to about 35° C. to produce a secondary dried formulation, and is held at a temperature (e.g., internal temperature) in the range of about 20° C. to about 35° C. and a pressure (e.g., internal pressure) in the range of about 50 mTorr to about 100 mTorr for a time period of about 5 hours to about 12 hours to produce a lyophilized formulation. In some embodiments, heating is performed to a temperature of about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., or 35° C. In various examples, heating is performed to a temperature of about 25° C. In various embodiments, heating is performed at a rate in the range of about 0.3 to 0.5° C. / min to produce a secondary dried formulation. In some examples, the heating is performed at a rate of about 0.05° C. / min to about 0.5° C. / min. In various examples, the heating is performed at a rate of about 0.05° C. / min, 0.1° C. / min, 0.15° C. / min, 0.2° C. / min, 0.25° C. / min, 0.3° C. / min, 0.35° C. / min, 0.4° C. / min, 0.45° C. / min, or 0.5° C. / min. In some embodiments, the heating is performed at a rate of about 0.4° C. / min. In some embodiments, the holding is performed at a temperature of about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C. In various examples, the holding is performed at a temperature of about 25° C. In some embodiments, the temperature to which the lyophilization chamber is heated is the same as the holding temperature. In some embodiments, the holding is at a pressure ranging from about 50 mTorr to about 100 mTorr, or from about 70 mTorr to about 100 mTorr, or from about 65 mTorr to about 75 mTorr. In some examples, the holding is at a pressure of about 65 mTorr, 66 mTorr, 67 mTorr, 68 mTorr, 69 mTorr, 70 mTorr, 71 mTorr, 72 mTorr, 73 mTorr, 74 mTorr, or 75 mTorr. In various embodiments, the holding is at a pressure of about 70 mTorr. In some examples, the holding is for a period of about 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.In various examples, the holding is for a period of about 8.1 hours, 8.2 hours, 8.3 hours, 8.4 hours, 8.5 hours, and in one example, the holding is for a period of about 8.3 hours.

[0023] In an embodiment of any method (with or without an annealing step) disclosed herein, the method may further include the step of (d) cooling the chamber containing the lyophilized formulation obtained in step (c) to a temperature in the range of about 1° C. to about 10° C. (or about 2° C. to about 7° C. or about 5° C.) and aerating the lyophilized formulation with an inert gas at a pressure in the range of about 250 mTorr to about 750 mTorr (or about 300 mTorr to about 600 mTorr or about 500 mTorr). In some examples, the inert gas is selected from argon, helium, nitrogen, and any combination thereof. In various examples, the inert gas is nitrogen. In an embodiment, step (d) may facilitate stoppering of a container (e.g., a vial) containing the lyophilized formulation. In an embodiment, the method further includes storing the lyophilized formulation at a temperature in the range of about 2° C. to about 8° C. In an embodiment, the method further includes reconstituting the lyophilized formulation with water.

[0024] In yet another aspect, the disclosure provides a lyophilized protein formulation prepared by the methods disclosed herein. In some embodiments, the protein formulation is prepared by the methods disclosed herein that lack an annealing step.

[0025] Lyophilized Protein Formulations The lyophilized protein formulations described herein comprise a protein, a sugar, a surfactant, and optionally a buffer, and have a pH of about 3 to about 7 (or about 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7). In some examples, the pH is about 4 to about 6. In some examples, the pH of the formulation is about 4 or about 4.2. In various examples, the pH of the formulation is about 5. In some embodiments, the pH of the formulation is about 6. In embodiments, the lyophilized formulations disclosed herein do not contain a sugar alcohol. As used herein, "sugar alcohol" refers to a linear polyol with one hydroxyl group attached to each carbon atom. Examples of sugar alcohols used herein include xylitol, erythritol, mannitol, and sorbitol. In embodiments, the lyophilized formulation does not contain mannitol.

[0026] protein In some embodiments, the protein of the lyophilized formulation is an antigen-binding protein. An "antigen-binding protein" is a protein that comprises a domain that binds to a specific target antigen (such as CD3 and / or CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17). An antigen-binding protein comprises a scaffold or framework portion that allows the antigen-binding domain to adopt a conformation that facilitates binding of the antigen-binding protein to an antigen.

[0027] In some embodiments, the antigen-binding protein of the lyophilized formulation is an antibody or immunoglobulin or an antigen-binding antibody fragment. In some examples, the antigen-binding protein is an antibody. The term "antibody" refers to an intact antigen-binding immunoglobulin. An "antibody" is a type of antigen-binding protein. The antibody can be an IgA, IgD, IgE, IgG, or IgM antibody, including any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, the intact antibody comprises two full-length heavy chains and two full-length light chains. The antibody has one variable region and one constant region. In the IgG format, one variable region is generally about 100-110 or more amino acids, contains three complementarity determining regions (CDRs), is primarily responsible for antigen recognition, and varies substantially among other antibodies that bind different antigens. A variable region usually contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (referred to as framework regions 1 to 4 (FR1, FR2, FR3 and FR4) by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra). The constant regions enable the antibody to recruit cells and molecules of the immune system.

[0028] In some embodiments, the antibody of the formulation is a bispecific antigen-binding molecule, i.e., an antibody that binds to two different targets (e.g., CD3 and a second different target). The term "bispecific" as used herein refers to an antigen-binding molecule or construct that binds to two different target antigens, i.e., it comprises a first binding domain and a second binding domain, where the first binding domain binds to one antigen or target (e.g., a surface antigen of a target cell) and the second binding domain binds to another antigen or target (e.g., CD3). Thus, an antigen-binding molecule according to the present disclosure comprises specificity for two different antigens or targets. The term "target cell surface antigen" refers to an antigenic structure expressed by a cell and present on the cell surface such that it is accessible to an antigen-binding molecule or antigen-binding construct as described herein. The target cell surface antigen may be a protein, such as an extracellular portion of a protein, or a carbohydrate structure, such as a carbohydrate structure of a protein, such as a glycoprotein. The target cell surface antigen may be a tumor antigen. The present disclosure also encompasses multispecific antigen-binding molecules or constructs, such as trispecific antigen-binding molecules or constructs (the latter comprising three binding domains) or constructs with more than three (e.g., four, five...) specificities.

[0029] Bispecific antibodies and / or antigen-binding molecules or constructs as understood herein include, but are not limited to, traditional bispecific immunoglobulins (e.g., BsIgG), IgGs with additional antigen-binding domains (e.g., where the amino or carboxy terminus of the light or heavy chain is linked to an additional antigen-binding domain such as a single domain antibody or paired antibody variable domains (e.g., Fv or scFv)), BsAb fragments (e.g., bispecific single chain antibodies), bispecific fusion proteins (e.g., an antigen-binding domain fused to an effector moiety), and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67(2)Part A:97-106(2015), which is incorporated herein by reference in its entirety. Examples of bispecific constructs include, but are not limited to, diabodies, single chain diabodies, tandem scFvs, Bispecific T Cell Engager (BiTE®) formats (a fusion protein consisting of two single chain variable fragments (scFvs) joined by a linker) and Fab2 bispecifics, as well as engineered constructs including full length antibodies.See, e.g., Chames & Baty, 2009, mAbs 1[6]:1-9; and Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-1136; Wu et al., 2007, Nature Biotechnology 25

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

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

[20] :19665-19672; and Kontermann, 2012 MAbs 4(2):182, which are incorporated by reference in their entireties.

[0030] In some embodiments, the lyophilized formulations described herein comprise a bispecific antigen-binding molecule or construct comprising a first binding domain that binds to a target cell surface antigen, a second binding domain that binds to human CD3 on the surface of a T cell, and optionally a third domain comprising, in amino to carboxyl order, hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain. In some embodiments, each of the first and second binding domains comprises a VH region and a VL region.

[0031] The term "binding domain" as used herein refers to a domain that (specifically) binds to / interacts with / recognizes a given target epitope or a given target site on a target molecule (antigen), such as CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN6, CLDN18.2 or MUC17 and CD3, respectively.

[0032] The structure and function of the first binding domain (e.g. recognizing CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN6, CLDN18.2 or MUC17) and also the structure and / or function of the second binding domain (recognizing CD3) are based on the structure and / or function of an antibody, e.g. a full length or whole immunoglobulin molecule, and / or are derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. In an embodiment, the first binding domain is characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). In an embodiment, the second binding domain also comprises the minimal structural requirements of an antibody to allow target binding. In an embodiment, the second binding domain comprises at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). It is envisaged that the first binding domain and / or the second binding domain are generated or obtained by phage display or library screening methods, rather than grafting CDR sequences from a pre-existing (monoclonal) antibody onto the scaffold.

[0033] In some embodiments, the first binding domain that binds to a surface antigen of a target cell and / or the second binding domain that binds to CD3ε are human binding domains. Antibodies and antigen-binding molecules or constructs that include at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs with non-human variable and / or constant regions, such as from rodents (e.g., mice, rats, hamsters or rabbits). The presence of such rodent-derived proteins may result in rapid clearance of the antibody or antigen-binding molecule or construct, or may generate an immune response against the antibody or antigen-binding molecule or construct by the patient. To avoid the use of rodent-derived antibodies or antigen-binding molecules or constructs, human or fully human antibodies / antigen-binding molecules can be generated by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0034] In some embodiments, the antigen binding protein comprises a single chain antigen binding molecule. The scFv comprises a variable heavy chain, an scFv linker and a variable light chain domain. Optionally, the C-terminus of the variable light chain is attached to the N-terminus of the scFv linker, which is attached to the N-terminus of the variable heavy chain (N-vh-linker-vl-C), although the configuration can be switched (N-vl-linker-vh-C). Alternatively, the C-terminus of the variable heavy chain is attached to the N-terminus of the scFv linker, which is attached to the N-terminus of the variable light chain (N-vl-linker-vh-C), although the configuration can be switched (N-vh-linker-vC). Thus, in depicting and describing scFvs, either orientation of the scFv is specifically included.

[0035] At least two binding domains and variable domain (VH / VL) of the antigen-binding polypeptide of the present disclosure may or may not include a peptide linker (spacer peptide). The term "peptide linker" according to the present invention includes an amino acid sequence that links the amino acid sequences of one (variable and / or binding) domain and the other (variable and / or binding) domain of the antigen-binding molecule of the present disclosure to each other. Peptide linkers can also be used to fuse a third domain to another domain of the antigen-binding molecule of the present disclosure. A characteristic of such peptide linkers is that they do not contain polymerization activity. Suitable peptide linkers include those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88 / 09344, the disclosures of which are incorporated herein by reference in their entirety. Peptide linkers can also be used to add other domains or modules or regions (e.g., half-life extending domains) to the bispecific antigen-binding molecules described herein.

[0036] In some embodiments, the third domain comprises "Fc" or "Fc region" or "Fc domain", which refers to a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. Thus, "Fc domain" refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM and the flexible hinge at the N-terminus of these domains. In IgA and IgM, Fc may include the J chain. In IgG, the Fc domain comprises immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, the bispecific antigen-binding molecule is an IgG antibody (including several subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4). Although the boundaries of the Fc region might vary, the human IgG heavy chain Fc region is usually defined to include residues C226, or P230, from the carboxyl terminus, where numbering is according to the EU index as described in Kabat. In some embodiments, amino acid modifications are made to the Fc region, e.g., to alter binding to one or more FcγR or FcRn receptors.

[0037] In some embodiments, the formulations described herein comprise a bispecific antigen binding molecule that binds human CD3 and human CDH19, or human CD3 and human MSLN, or human CD3 and human DLL3, or human CD3 and human FLT3, or human CD3 and human EGFRvIII, or human CD3 and human BCMA, or human CD3 and PSMA, or human CD3 and human CD33, or human CD3 and human CD19, human CD3 and human CD70, or human CD3 and human MUC17, or human CD3 and human CLDN18.2, or human CD3 and human CLDN6.

[0038] In some embodiments, the first binding domain of the bispecific antigen-binding molecule comprises a set of six CDRs as set forth in (a) SEQ ID NOs: 24 to 29, (b) SEQ ID NOs: 34 to 39, (c) SEQ ID NOs: 78 to 83, (d) SEQ ID NOs: 10 to 15, (e) SEQ ID NOs: 46 to 51, (f) SEQ ID NOs: 88 to 93, (g) SEQ ID NOs: 67 to 72, (h) SEQ ID NOs: 56 to 61, (i) SEQ ID NOs: 112 to 117, (j) SEQ ID NOs: 100 to 105, (k) SEQ ID NOs: 148 to 153, SEQ ID NOs: 157 to 162, or SEQ ID NOs: 166 to 171, or SEQ ID NOs: 175 to 180, (l) SEQ ID NOs: 132 to 137, or (m) SEQ ID NOs: 123 to 128.

[0039] In some embodiments, the first binding domain of the bispecific antigen-binding molecule comprises a VH region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 30, 40, 84, 16, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143 or 129. In some embodiments, the first binding domain of the bispecific antigen-binding molecule comprises a VH region comprising an amino acid sequence set forth in SEQ ID NO: 30, 40, 84, 16, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143 or 129.

[0040] In some embodiments, the first binding domain of the bispecific antigen-binding molecule comprises a VL region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144 or 130. In some embodiments, the first binding domain of the bispecific antigen-binding molecule comprises a VL region comprising an amino acid sequence set forth in SEQ ID NO: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144 or 130.

[0041] In some embodiments, the first binding domain comprises (a) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 30 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 31; (b) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 40 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 41; (c) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 84 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 85; (d) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 16 or 17 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 18 or 19; (e) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 52 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 53; (f) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 94 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 95; (g) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 73 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 74. (h) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 62 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 63; (i) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 118 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 119; (j) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 154, 163, 172 or 181 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 155, 164, 173 or 182; (k) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 106 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 107; (l) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 138 or 143 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 139 or 144; or (m) a VH region comprising the amino acid sequence set forth in SEQ ID NO: 129 and a VL region comprising the amino acid sequence set forth in SEQ ID NO: 130.

[0042] In some embodiments, the second binding domain of the bispecific antigen-binding molecule comprises a set of six CDRs set forth in SEQ ID NOs: 1-6.

[0043] In some embodiments, the second binding domain of the bispecific antigen-binding molecule comprises a VH region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the second binding domain of the bispecific antigen-binding molecule comprises a VH region comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0044] In some embodiments, the second binding domain of the bispecific antigen-binding molecule comprises a VL region comprising an amino acid sequence that is at least 90% identical (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the second binding domain of the bispecific antigen-binding molecule comprises a VL region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0045] In some embodiments, the second binding domain comprises (a) a VH region comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL region comprising the amino acid sequence set forth in SEQ ID NO:8.

[0046] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to CD19 comprising an anti-CD19 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 85 and an anti-CD19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 84, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 86 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 87.

[0047] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to MSLN comprising an anti-MSLN variable light chain domain comprising the amino acid sequence of SEQ ID NO: 41 and an anti-MSLN variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 40, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 42 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 43, 44 or 45.

[0048] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to DLL3 comprising an anti-DLL3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 74 and an anti-DLL3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 73, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 75 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 76 or 77.

[0049] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to FLT3 comprising an anti-FLT3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 63 and an anti-FLT3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 62, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 64 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 65 or 66.

[0050] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to EGFRvIII comprising an anti-EGFRvIII variable light chain domain comprising the amino acid sequence of SEQ ID NO: 31 and an anti-EGFRvIII variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 30, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 32 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0051] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds BCMA comprising an anti-BCMA variable light chain domain comprising the amino acid sequence of SEQ ID NO: 95 and an anti-BCMA variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 94, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 96 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 98 or SEQ ID NO: 97.

[0052] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to PSMA comprising an anti-PSMA variable light chain domain comprising the amino acid sequence of SEQ ID NO: 119 or 107 and an anti-PSMA variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 118 or 106, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 120 or 108 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 121, 122, 109, 110 or 111.

[0053] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to CD33 comprising an anti-CD33 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 18 or 19 and an anti-CD33 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 16 or 17, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 189 or 190 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 20, 21, 22 or 23.

[0054] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to CDH19 comprising an anti-CDH19 variable light chain domain comprising the amino acid sequence of SEQ ID NO:53 and an anti-CDH19 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO:52, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO:7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO:8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO:54 and a second binding domain comprising the amino acid sequence of SEQ ID NO:9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO:55.

[0055] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds MUC17 comprising an anti-MUC17 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 155, 164, 173 or 182 and an anti-MUC17 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 154, 163, 172 or 181, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 156, 165, 174 or 183.

[0056] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to cldn18.2 comprising an anti-cldn18.2 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 139 or 144 and an anti-cldn18.2 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 138 or 143, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antigen binding molecule comprises a first binding domain comprising the amino acid sequence of SEQ ID NO: 140 or 145 and a second binding domain comprising the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 141, 142, 146 or 147.

[0057] In some embodiments, the bispecific antigen binding molecule comprises a first binding domain that binds to CD70 comprising an anti-CD70 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 130 and an anti-CD70 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 129, and a second binding domain comprising an anti-CD3 variable heavy chain domain comprising the amino acid sequence of SEQ ID NO: 7 and an anti-CD3 variable light chain domain comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the bispecific antigen binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 131.

[0058] In some embodiments, the protein of the formulation is an antibody. In various embodiments, the protein of the formulation is a bispecific antigen-binding molecule. In some examples, the protein of the formulation is a half-life extended bispecific antigen-binding molecule. Half-life extended bispecific antigen-binding molecules have been previously described herein. In some embodiments, the protein formulation of the present disclosure comprises an amino acid sequence set forth in SEQ ID NOs: 1-190. In various embodiments, a protein formulation of the disclosure comprises an amino acid sequence set forth in SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:55, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:87, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:131, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:156, SEQ ID NO:165, SEQ ID NO:174, SEQ ID NO:183, SEQ ID NO:184, SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, or SEQ ID NO: 188. In some examples, a protein formulation of the disclosure comprises an amino acid sequence set forth in SEQ ID NO:22 (BiTE A), SEQ ID NO:77 (BiTE B), SEQ ID NO:87 (BiTE C), or SEQ ID NO:97 (BiTE D).

[0059] In some embodiments, a protein such as an antibody or bispecific (e.g., an HLE bispecific antibody construct) is present in the liquid formulation (before lyophilization) in an amount ranging from about 0.1 mg / mL to about 100 mg / mL (or about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, or 100 mg / mL). In various embodiments, the protein is present in the liquid formulation in an amount ranging from about 0.1 mg / mL to about 70 mg / mL. In some examples, the protein is present in the liquid formulation in an amount ranging from about 0.5 mg / mL to about 30 mg / mL (or about 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, or 30 mg / mL).In various examples, the protein is present in a liquid formulation in a range of about 1 mg / mL to about 20 mg / mL (or about 1 mg / mL, 1.5 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 3.5 mg / mL, 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.5 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, 50 mg / mL, 51 mg / mL, 52 mg / mL, 53 mg / mL, 54 mg / mL, 55 mg / mL, 56 mg / mL, 57 mg / mL, 58 mg / mL, 59 mg / mL, 60 mg / mL, 61 mg / mL, 62 mg / mL, 63 mg / mL, 6 In some embodiments, the protein is present in the liquid formulation in an amount of about 1 mg / mL.

[0060] Sugars The protein formulation of the present disclosure comprises a saccharide. In some embodiments, the saccharide is a monosaccharide or a disaccharide. Suitable saccharides include, for example, glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof. In some examples, the saccharide comprises sucrose.

[0061] In some embodiments, the liquid formulation (before lyophilization) comprises saccharide at a concentration of about 1% to about 15% w / v, or about 4% to about 13% w / v, or about 6% to about 12% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, or at least 14% w / v. In some embodiments, the liquid formulation comprises saccharide at a concentration of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% w / v. In some embodiments, the liquid formulation comprises a sugar at a concentration of about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, or about 12% w / v. In some embodiments, the liquid formulation comprises a sugar at a concentration of about 7% to about 12% w / v. In some embodiments, the liquid formulation comprises a sugar at a concentration of about 9% w / v. In some embodiments, the sugar is sucrose and is present in the liquid formulation at a concentration ranging from about 6% to about 12% w / v. In some examples, the sugar is sucrose and is present in the liquid formulation at a concentration of about 9% w / v.

[0062] Surfactants The protein formulation of the present disclosure comprises a surfactant. Suitable surfactants include polysorbate, poloxamer, polyoxyethylene, or any combination thereof. Contemplated surfactants include polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X-100, polyoxyethylene, PEG3350, PEG4000, and any combination thereof. In some embodiments, the surfactant comprises polysorbate. In some examples, the surfactant is polysorbate 80.

[0063] Protein formulations described herein may contain a surfactant or a mixture of surfactants. In some embodiments, the liquid formulation (before lyophilization) contains a surfactant at a concentration of about 0.001% to about 5% w / v (or about 0.001% to about 0.5%, or about 0.004 to about 0.5 w / v%, or about 0.001 to about 0.01 w / v%, or about 0.004 to about 0.01 w / v%). In some embodiments, the liquid formulation comprises a surfactant at a concentration of at least 0.001, at least 0.002, at least 0.003, at least 0.004, at least 0.005, at least 0.007, at least 0.01, at least 0.05, at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, at least 1.5, at least 2.0, at least 2.5, at least 3.0, at least 3.5, at least 4.0, or at least 4.5 w / v%. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.5 w / v%. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.1 w / v%. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01 w / v%. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4% or about 0.5% w / v. In some embodiments, the liquid formulation comprises a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some embodiments, the surfactant is polysorbate 80, and the polysorbate 80 is present at a concentration of about 0.01% w / v.

[0064] Buffer The protein formulation of the present disclosure optionally comprises a buffer. Suitable buffers include acetate buffer, glutamate buffer, citrate buffer, lactate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, phosphate buffer, 2-(N-morpholino)ethanesulfonic acid buffer, or any combination thereof. In some examples, the buffer comprises glutamate.

[0065] Buffers are often used to control the pH of the formulation. In some embodiments, the buffer is added at a concentration that maintains the pH of the formulation at about 3 to about 7, or about 4 to about 6, about 4 to 5, or about 4.2. The effect of pH on the formulation can be characterized using any one or more of several techniques, such as accelerated stability studies and calorimetric screening studies (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)).

[0066] The buffer system present in the protein formulation is selected to be physiologically compatible and to maintain the desired pH. The buffer may be present in the liquid formulation (before lyophilization) at a concentration of about 0.1 mM to about 1000 mM (1 M), or about 5 mM to about 200 mM, or about 5 mM to about 100 mM, or about 10 mM to about 50 mM. Suitable buffer concentrations include concentrations of about 200 mM or less. In some embodiments, in the liquid formulation (before lyophilization), the buffer is present at a concentration of about 190 mM, about 180 mM, about 170 mM, about 160 mM, about 150 mM, about 140 mM, about 130 mM, about 120 mM, about 110 mM, about 100 mM, about 80 mM, about 70 mM, about 60 mM, about 50 mM, about 40 mM, about 30 mM, about 20 mM, about 10 mM, or about 5 mM. In some embodiments, the concentration of the buffering agent is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In some embodiments, the concentration of the buffering agent is between 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM to 100 mM. In some embodiments, the concentration of the buffering agent is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM to 50 mM. In some embodiments, the concentration of the buffering agent is about 10 mM.

[0067] In some embodiments, the liquid protein formulation (before lyophilization) has a pH of about 4.2 and contains about 10 mM L-glutamic acid, about 9.0% (w / v) sucrose, and about 0.01% (w / v) polysorbate 80.

[0068] Stability of Lyophilized Protein Formulations The methods disclosed herein advantageously result in lyophilized protein formulations that exhibit reduced physical degradation, such as aggregation, and reduced chemical degradation, such as clipping and deamidation, of the protein upon reconstitution with a liquid. The liquid used to reconstitute the lyophilized protein formulation may be any suitable liquid known in the art. In embodiments, the lyophilized protein formulation may be reconstituted with water. Furthermore, the lyophilization methods disclosed herein may stabilize protein formulations, such as those containing antibodies and bispecific antigen-binding molecules (e.g., half-life extended bispecific antibody constructs), at both low and high concentrations.

[0069] The stability of a protein formulation, such as a formulation containing an antibody or bispecific antigen-binding molecule (e.g., an HLE bispecific antigen-binding molecule), can be quantified in several ways. In some embodiments, the stability of a protein formulation is characterized by size-exclusion high performance liquid chromatography (SE-HPLC), size-exclusion ultra-high performance liquid chromatography (SE-UHPLC), cation exchange high performance liquid chromatography (CE-HPLC), dynamic light scattering (DLS), analytical ultracentrifugation (AUC), field-flow fractionation (FFF), isoelectric focusing, and ion exchange chromatography (IEX). In some embodiments, the stability of a protein formulation, such as an antibody formulation, is characterized by partial dissociation measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS) and / or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In some embodiments, the stability of the formulation is assessed by reduced capillary electrophoresis sodium dodecyl sulfate (rCE-SDS). The rCE-SDS method separates heavy chain (HC), light chain (LC), non-glycosylated HC (NGHC) and other minor peak species and groups under reducing conditions.

[0070] In some embodiments, the stability of the formulation is characterized by the amount of high molecular weight (HMW) species of a protein, such as an antibody or bispecific antigen binding molecule (e.g., HLE bispecific antigen binding molecule), or the rate of increase in the amount of HMW species of a protein after various storage conditions. In some embodiments, the amount of HMW species of a protein is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months after reconstitution and storage at approximately 4° C. or 40° C. In some embodiments, the rate of increase in HMW species of a protein is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months after reconstitution and storage at approximately 4° C. or 40° C. In some embodiments, the HMW species of a protein, such as an antibody or bispecific antigen binding molecule (e.g., HLE bispecific antigen binding molecule) in a reconstituted lyophilized formulation is measured by SE-UHPLC.

[0071] The stability of a protein, such as an antibody or bispecific antigen-binding molecule (e.g., HLE bispecific antigen-binding molecule), and the ability of a formulation to maintain protein stability can be evaluated over an extended period of time (e.g., weeks or months). In terms of a formulation, a stable formulation is one in which a protein, such as an antibody or bispecific antigen-binding molecule (e.g., HLE bispecific antigen-binding molecule), substantially maintains its physical and / or chemical integrity and / or biological activity during storage and processes such as freezing / thawing, mechanical mixing, and lyophilization. Protein stability can be evaluated, for example, by measuring the level and / or rate of formation of high molecular weight (HMW) aggregates, shifts in charge profile, and changes in particle size.

[0072] In some embodiments, the relative values ​​of any particular species of protein, such as intact BiTE® molecules or major species, or high molecular weight (HMW) species (i.e., aggregates), or low molecular weight (LMW) species (i.e., fragments), are displayed relative to the respective values ​​of the total product. For example, in some embodiments, 2.5% or less (e.g., 2.5%, or 2%, or 1.9%, or 1.8%, or 1.7%, or 1.6%, or 1.5%, or 1.4%, or 1.3%, or 1.2%, or 1.1%, or 1%, or 0.5%) of the protein, such as an antibody or bispecific antigen-binding molecule, is present as HMW species in the reconstituted lyophilized formulation. In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) upon storage at 4° C. for one or more months (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by approximately 0.1%-0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%) upon storage at 4° C. for one or more months (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by less than 0.5% (e.g., 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by less than 0.5% (e.g., 0.5%, 0.4%, 0.3%, 0.2% 0.1%) upon storage at 40° C. for one month or more (e.g., one month, three months, six months, nine months, or twelve months). In some embodiments, the amount of HMW species in a reconstituted lyophilized formulation increases by less than 0.5% upon storage at 40° C. for one month.In some embodiments, the amount of HMW species in the reconstituted lyophilized formulation increases by less than 0.3% upon storage at 40° C. for one month. In some embodiments, the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.1%-0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6% or 0.7%) upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month or three months). In some embodiments, the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.1%-0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4% and 0.5%) upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month or three months). In some embodiments, the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.1% to 0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4% and 0.5%) upon storage at 40° C. for one month or more (e.g., 1 month, 3 months, 6 months, 9 months or 12 months). In some embodiments, the HMW species of the bispecific antigen-binding molecule in the reconstituted lyophilized formulation is measured by SE-UHPLC.

[0073] In some embodiments, the stability of the formulation is characterized by the amount of low molecular weight (LMW) species of a protein, such as an antibody or bispecific antigen binding molecule (HLE bispecific antigen binding molecule), or the rate of increase in the amount of LMW species of a protein under storage conditions at various times. In some embodiments, the amount of LMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4° C. or 40° C. In some embodiments, the rate of increase in LMW species is determined after 1 week, 2 weeks, 1 month, 3 months, 6 months, or 12 months when stored at approximately 4° C. or 40° C. In some embodiments, the LMW species, such as an antibody or bispecific antigen binding molecule (HLE bispecific antigen binding molecule) in the formulation, is measured by capillary electrophoresis sodium dodecyl sulfate method (rCE-SDS). In some embodiments, the LMW species of the bispecific antigen binding molecule in the formulation is measured by size exclusion chromatography (SEC).

[0074] In some embodiments, less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) of the protein, such as an antibody or bispecific antigen binding molecule (HLE bispecific antigen binding molecule), is present as a low molecular weight (LMW) species in the reconstituted lyophilized formulation. In some embodiments, the amount of LMW species in the reconstituted lyophilized formulation increases by less than 2% (e.g., 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) upon storage at 4° C. for one month or more (e.g., one month, three months, or six months). In some embodiments, the amount of LMW species in a reconstituted lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or 0.7%) upon storage at 4° C. for one month or more (e.g., one month, three months, or six months). In some embodiments, the amount of LMW species in a reconstituted lyophilized formulation increases by less than 1% (e.g., 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month, or three months). In some embodiments, upon storage at 40° C. for one week or more (e.g., one week, two weeks, one month, or three months), the amount of LMW species in the reconstituted lyophilized formulation increases by approximately 0.1% to 0.7% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%. or 0.7%). In some embodiments, the LMW species of the bispecific antigen-binding molecule in the reconstituted lyophilized formulation is measured by size exclusion chromatography (SEC). In some embodiments, the LMW species of the bispecific antigen-binding molecule in the reconstituted lyophilized formulation is measured by reduced capillary electrophoresis sodium dodecyl sulfate method (rCE-SDS).

[0075] In some embodiments, the percent of protein, such as antibody or bispecific antigen binding molecule (HLE bispecific antigen binding molecule), in the reconstituted lyophilized formulation (i.e., the main peak species) is greater than 95% of the total protein content in the formulation.

[0076] In some embodiments, the formulation is stable upon storage at about 4° C. for 1 month, and the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.1%-0.7% (e.g., 0.1%, or 0.2%, or 0.3%, or 0.4%, or 0.5%, or 0.6%, or 0.7%) during at least 1 month of storage. In some embodiments, the formulation is stable upon storage at about 4° C. for 3 months, and the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.0%-0.2% (e.g., 0%, or 0.1%, or 0.2%) during at least 3 months of storage. In some embodiments, the formulation is stable upon storage at about 4° C. for 6 months, and the amount of HMW species in the reconstituted lyophilized formulation increases by approximately 0.0%-0.4% (e.g., 0%, or 0.1%, or 0.2%, or 0.3%, or 0.4%) during at least 6 months of storage. In some embodiments, the HMW species of the bispecific antigen-binding molecules in the reconstituted lyophilized formulation are measured by SE-UHPLC.

[0077] In some embodiments, the formulations are stable upon storage at about 4° C. for 1 month, 3 months, and 6 months, and the percentage of protein, such as antibody or bispecific antigen-binding molecule (HLE bispecific antigen-binding molecule), is greater than 95% of the total protein content. In some embodiments, the formulations are stable upon storage at about 4° C. for 1 month, 3 months, 6 months, 12 months, and 48 months, and the percentage of protein, such as antibody or bispecific antigen-binding molecule (HLE bispecific antigen-binding molecule) after reconstitution is greater than 96% of the total protein content.

[0078] The stability of the formulations described herein may also be characterized by a change in charge distribution, e.g., the amount of charge change peaks of a protein, such as an antibody or bispecific antigen-binding molecule (HLE bispecific antigen-binding molecule). For example, in some embodiments, the amount of acidic peaks (e.g., deamidation, which is a charge change with a relatively low isoelectric point (pI)) in a reconstituted lyophilized formulation increases by less than 2% (e.g., 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5% or less) when stored at 4° C. for at least one month (e.g., one month, three months, six months, or twelve months). In some embodiments, the amount of basic peaks (e.g., charge changes with relatively high pI) in the reconstituted lyophilized formulation increases by less than 6% (e.g., 6%, 5%, 4%, 3%, 2% or 1%) when stored at 4° C. for at least one month (e.g., 1 month, 3 months, 6 months or 12 months). In some embodiments, the amount of main peaks in the reconstituted lyophilized formulation decreases by less than 4% (e.g., 4%, 3.5%, 3%, 2.5%, 2%, 1% or less) when stored at 4° C. for at least one month. In some embodiments, the amount of main peaks in the reconstituted lyophilized formulation decreases by less than 6% (e.g., 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored at 4° C. for at least three months. In some embodiments, the amount of the main peak in a reconstituted lyophilized formulation decreases by less than 9% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored for at least 6 months at 4° C. In some embodiments, the amount of the main peak in a reconstituted lyophilized formulation decreases by less than 9% (e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored for at least 12 months at 4° C.

[0079] In some embodiments, the amount of acidic peaks in a reconstituted lyophilized formulation increases by less than 30% (e.g., 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2%, 1% or less) when stored at 40° C. for at least one week (e.g., one week, two weeks, one month or three months). In some embodiments, the amount of basic peaks (e.g., charge changes with a relatively high pI) in a reconstituted lyophilized formulation increases by less than 15% (e.g., 15%, 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2%, 1% or less) when stored at 40° C. for at least one week (e.g., one week, two weeks, one month or three months). In some embodiments, the amount of the main peak in a reconstituted formulation decreases by less than 4% (e.g., 4%, 3.5%, 3%, 2.5%, 2%, 1% or less) when stored for at least 1 month at 4° C. In some embodiments, the amount of the main peak in a reconstituted lyophilized formulation decreases by less than 6% (e.g., 6%, 5%, 4%, 3.5%, 3%, 2.5%, 2% or less) when stored for at least 3 months at 4° C.

[0080] Protein formulations lyophilized by the methods of the present disclosure exhibit superior stability to comparable liquid protein formulations. For example, the stability of a protein formulation containing 1 mg / mL of a bispecific antigen-binding molecule of the present disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80 at pH 4.2, lyophilized according to the present disclosure using an annealing step, and then reconstituted, was determined by reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS) to determine the extent of clipping that occurred after storage at 25°C and 40°C for 1 month.

[0081] The lyophilization method of the present disclosure advantageously stabilizes both low and high concentration protein formulations. For example, protein formulations of the present disclosure containing 1 mg / mL, 5 mg / mL, 13 mg / mL and 23 mg / mL of the bispecific antigen-binding molecule of the present disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose and 0.01% (w / v) polysorbate 80 at pH 4.2, lyophilized using an annealing step, and then reconstituted, were stored at 40° C. for one month and then subjected to SEC-UHPLC to determine the degree of aggregation in the formulations by the percentage of high molecular weight species (HMW%).

[0082] It has been surprisingly found that the freeze-drying method of the present disclosure lacking an annealing step results in superior stability of protein formulations than freeze-drying methods that include an annealing step. For example, protein formulations containing 15 mg / mL, 20 mg / mL or 23 mg / mL of the bispecific antigen-binding molecule of the present disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose and 0.01% (w / v) polysorbate 80 at pH 4.2, freeze-dried with and without an annealing step, were subjected to SE-UHPLC after reconstitution to determine the amount of aggregation in each sample.

[0083] The following examples are offered for illustrative purposes and are not intended to limit the scope of the invention. EXAMPLES

[0084] General Procedure Reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS) separates proteins based on their hydrodynamic size differences under reducing and denaturing conditions. Protein species bind to SDS, an anionic detergent, and are electrokinetically injected into a bare fused silica capillary filled with SDS gel buffer. A voltage is applied across the capillary under which SDS-coated proteins are separated by their differences in migration in a hydrophilic polymer-based solution. Proteins are detected by a photodiode array (PDA) detector as they pass through a UV detection window. Purity is assessed by determining the exact peak area percentages of reach components. The rCE-SDS method separates heavy chain (HC), light chain (LC), nonglycosylated HC (NGHC), and other fine peak species and groups under reducing conditions. Reduced capillary electrophoresis with sodium dodecyl sulfate (rCE-SDS) was performed by incubating the samples in SDS-MW reducing gel at 70 + / - 10 °C for 10 min, followed by 60-80 °C for 10 min, and then returning to room temperature. After incubation, the samples were centrifuged and then electrokinetically injected into a bare 67 cm fused silica capillary with an inner diameter of 50 μm by electrokinetic injection. The effective length of the capillary was 30.2 cm. Separation was performed using CE-SDS gel (Beckman Coulter, Brea, Calif.) and 30 kV effective voltage. Detection was performed by UV absorbance at 220 nm.

[0085] Visual Inspection of the Lyophilized Cake. After completion of the cycle, the dried product cake was evaluated by visual inspection for signs of macroscopic collapse and overall quality. The product cake for BITE B was determined to be acceptable and photographs taken from several angles are shown in Figure 1. An intact visual cake structure serves as a preliminary indication that the cycle parameters allow for adequate drying and homogeneity throughout the sample vial.

[0086] Size Exclusion Ultra High Performance Liquid Chromatography (SE-UHPLC). SE-UHPLC separates proteins based on differences in their hydrodynamic volumes. Molecules with higher hydrodynamic volumes elute before molecules with smaller volumes. Samples were loaded onto an SE-UHPLC column (BEH200, 4.6 x 150 mm, (Waters Corporation, 186005226)) and separated isocratically, and the eluate was monitored by UV absorbance. Purity was determined by calculating the percentage of each separated component compared to the total integrated area. SE-UHPLC settings were as follows: flow rate: 0.4 mL / min, run time: 9 min, UV detection: 220 nm (280 nm is a common wavelength chosen for proteins including antibodies, but most bispecific molecules are detected at 220 nm). Column temperature: ambient, target protein load: 10 μg, protein compatible flow cell: 5 mm. After determining that acceptable moisture content was achievable using this cycle, dried product vials of BiTE B were placed on stability at 5°C, 25°C, and 40°C for a total of 4 weeks. During this period, sample vials were drawn at 2 and 4 weeks and their aggregation was assessed by SE-UHPLC. Samples were reconstituted, mixed by swirling, and injected neat. The relative area % values ​​of high molecular weight (HMW) species are plotted over time in Figure 2, and the data suggests no aggregation instability over the course of the study, supporting the conclusion that no aggregation was introduced by using the proposed cycle.

[0087] Karl Fischer titration (moisture content). To more accurately assess the drying efficiency of the proposed cycle, the moisture content of the product cake was evaluated by Karl Fischer titration. Although the acceptance criteria allows for moisture contents of the order of 5 w / w%, it is highly preferred that the moisture content be less than 2%. The BITE B product cake dried using the cycle of the present invention was compared to cakes previously produced using state-of-the-art BiTE and the moisture content was found to be acceptable and competitive, as shown in Table 1.

[0088] [Table 1]

[0089] Protein formulations were prepared containing intact bispecific antigen-binding molecules at concentrations of 1 mg / mL, 5 mg / mL, 13 mg / mL or 23 mg / mL at pH 4.2, 10 mM L-glutamic acid, 9% (w / v) sucrose, 0.01% (w / v) polysorbate 80. The protein formulations were placed in vials for lyophilization (without annealing step).

[0090] Example 1 Freeze-drying of bispecific antigen-binding molecule formulations without an annealing step Filling vials meeting FIH BiTE platform requirements consisting of Schott ISO 6R glass vials were filled with 1.3 mL of solution and stoppered with D-777 20 mm elastomeric fluoropolymer. For cycle development and analytical control purposes, a formulation buffer designated G42SuT (10 mM glutamic acid, 9.0 w / w% sucrose, 0.01% polysorbate 80, pH 4.2) was used throughout. Sample vials containing BITE B formulations are also formulated in G42SuT at a protein concentration of 1 mg / mL. In the SE-UHPLC method, BITE B is used for system suitability purposes. Standards with 1% and 5% water content are used as standards for Karl Fischer titration.

[0091] Accelerated freeze-drying cycle With a final time of about 32.7 hours, this represents a roughly 67% reduction in time compared to conventional lyophilization cycles. Additionally, this cycle eliminates the optional phase known as annealing, which is believed to promote better drying uniformity but has recently been correlated with product aggregation in lyophilized BiTE-like molecules.

[0092] The liquid protein formulation was prepared as described above and introduced into the lyophilization chamber. The chamber was cooled from a loading temperature of 5°C to -45°C at a rate of 0.5°C / min and held at -45°C for 2 hours. The temperature was then increased at 0.3°C / min and primary drying was carried out at a temperature of approximately -27°C and a chamber pressure of 100 mTorr for 16.7 hours. Secondary drying was carried out at a rate of 0.4°C / min and a pressure of 70 mTorr to 25°C and at a pressure of 70 mTorr for 8.3 hours. To allow stoppering of the vials, the temperature of the chamber was reduced to 5°C and the lyophilization chamber was purged with nitrogen at 500 mTorr. The vials containing the lyophilized protein formulation were removed from the lyophilization chamber and stored at 2-8°C until further processing and analysis. The total cycle time using the cycle of the present invention was 32.7 hours compared to cycle times of over 70 hours (72.7 hours to be exact) using the previous cycle conditions. Table 2 shows the parameters of the accelerated and established freeze-drying cycles.

[0093] [Table 2]

[0094] The above description is given for clarity of understanding only, and no unnecessary limitations should be construed therefrom, since modifications within the scope of the invention may be apparent to those skilled in the art.

[0095] Throughout this specification and the claims which follow, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a specified integer or step or group of integers or steps but not to the exclusion of other integers or steps or groups of integers or steps.

[0096] When describing a range of values, it should be understood that the described characteristic may be any individual value within the range. For example, "a pH of about pH 4 to about pH 6" may be pH 4, 4.2, 4.6, 5.1, 5.5, etc. (but not limited to), and any value between such values. Furthermore, "a pH of about pH 4 to about pH 6" should not be construed to mean that the pH of the formulation of interest will vary by 2 pH units in the range of pH 4 to pH 6 during storage, but rather means that a value within that range may be selected for the pH of the solution, and the pH will be maintained buffered around that pH.

[0097] When the term "about" is used, it means to add or subtract from a recited number 5%, 10%, 15% or more of the recited number. The actual variation intended can be determined from the context.

[0098] Throughout this specification, when a composition is described as comprising components or materials, it is also contemplated that the composition may consist essentially of, or consist of, any combination of the recited components or materials, unless otherwise stated. Similarly, when a method is described as comprising particular steps, it is also contemplated that the method may consist essentially of, or consist of, any combination of the recited steps, unless otherwise stated. The invention illustratively disclosed herein may suitably be practiced in the absence of any element or step not specifically disclosed herein.

[0099] The implementation of the methods disclosed herein and their individual steps can be performed manually and / or with or utilizing automation provided by electronic devices. Although the methods have been described with respect to specific embodiments, those skilled in the art will readily appreciate that other ways of performing the acts associated with the methods may be used. For example, the order of various steps may be altered without departing from the scope or spirit of the method, unless otherwise indicated. In addition, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0100] All patents, publications, and references cited herein are hereby incorporated by reference in their entirety. In the event of a conflict between this disclosure and the incorporated patents, publications, and references, this disclosure shall control.

Claims

Claim 1 A method for preparing a lyophilized preparation, comprising: (a) cooling a lyophilization chamber containing a liquid preparation containing a protein, a saccharide, and a surfactant to a temperature in the range of about -35°C to about -50°C to produce a frozen preparation, and holding the chamber at a temperature in the range of about -40°C to about -50°C for a time period of about 1.5 hours to about 5.0 hours; (b) heating the chamber to a temperature in the range of about -30°C to about -20°C and a pressure in the range of about 75 mTorr to about 125 mTorr to produce a primary dried preparation, and holding the chamber at a temperature in the range of about -30°C to about -20°C and a pressure in the range of about 75 mTorr to about 125 mTorr for a time period of about 12 hours to about 24 hours; (c) heating the chamber to a temperature in the range of about 20°C to about 30°C to produce a secondary dried preparation, and holding the chamber at a temperature in the range of about 20°C to about 30°C and a pressure in the range of about 50 mTorr to about 100 mTorr for a time period of about 5 hours to about 12 hours to produce the lyophilized preparation; wherein the liquid preparation has a pH of about 3 to 7 and does not contain mannitol; and the method lacks an annealing step. Claim 2 The method according to claim 1, wherein the cooling in step (a) is performed to a temperature of about -45°C. Claim 3 The method according to claim 1, wherein the cooling in step (a) is performed at a rate in the range of about 0.3°C / min to about 1°C / min. Claim 4 The method according to claim 3, wherein the cooling in step (a) is performed at a rate of about 0.5°C / min. Claim 5 The method according to claim 1, wherein the holding in step (a) is performed at a temperature of about -45°C. Claim 6 The method according to claim 1, wherein the holding in step (a) is performed for a time period of about 1.5 hours to about 5 hours. Claim 7 The method according to claim 6, wherein the holding in step (a) is performed for about 2 to 3 hours. Claim 8 The method according to claim 1, wherein the heating in step (b) is performed to a temperature of about -25°C to -30°C. Claim 9 The method according to claim 1, wherein the heating in step (b) is performed at a rate in the range of about 0.1°C / min to about 1°C / min. Claim 10 The method according to claim 9, wherein the heating in step (b) is performed at a rate in the range of about 0.1°C / min to about 0.5°C / min. Claim 11 The method according to claim 10, wherein the heating in step (b) is performed at a rate of about 0.3°C / min. Claim 12 The method according to claim 1, wherein the heating in step (b) is carried out at a pressure in the range of about 75 mTorr to about 125 mTorr.

13. The method according to claim 12, wherein the heating in step (b) is carried out at a pressure of about 100 mTorr.

14. The method according to claim 1, wherein the holding in step (b) is carried out at a temperature in the range of about -25 °C to about -30 °C.

15. The method according to claim 1, wherein the holding in step (b) is carried out at a pressure in the range of about 75 mTorr to about 125 mTorr.

16. The method according to claim 15, wherein the holding in step (b) is carried out at a pressure of about 100 mTorr.

17. The method according to claim 1, wherein the holding in step (b) is carried out over a time period of about 10 hours to about 25 hours.

18. The method according to claim 17, wherein the holding in step (b) is carried out over a time period of about 17 hours.

19. The method according to claim 1, wherein the heating in step (c) is carried out up to a temperature of about 25 °C.

20. The method according to claim 1, wherein the heating rate of temperature increase in step (c) occurs at a rate in the range up to about 0.5 °C / min.

21. The method according to claim 20, wherein the heating rate of temperature increase in step (c) occurs at a rate in the range of about 0.1 °C / min to about 0.5 °C / min.

22. The method according to claim 21, wherein the heating in step (c) is carried out at a rate of about 0.4 °C / min.

23. The method according to claim 1, wherein the holding in step (c) is carried out at a temperature of about 25 °C.

24. The method according to claim 1, wherein the holding in step (c) is carried out at a pressure in the range of about 50 mTorr to about 100 mTorr.

25. The method according to claim 24, wherein the holding in step (c) is carried out at a pressure of about 70 mTorr.

26. The method according to claim 1, wherein the holding in step (c) is carried out over a time period of about 8 hours.

27. The method according to claim 1, wherein the protein is an antibody.

28. The method according to claim 1, wherein the protein is a bispecific antigen-binding molecule.

29. The method according to claim 28, wherein the bispecific antigen-binding molecule is a half-life extended (HLE) bispecific antigen-binding molecule.

30. The method according to claim 29, wherein the HLE bispecific antigen-binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 55, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 87, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 131, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 156, SEQ ID NO: 165, SEQ ID NO: 174, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187 or SEQ ID NO:

188.

31. The method according to claim 30, wherein the HLE bispecific antigen-binding molecule comprises the amino acid sequence set forth in SEQ ID NO: 22, SEQ ID NO: 77, SEQ ID NO: 87 or SEQ ID NO:

97.

32. The method according to claim 1, wherein the protein is present in the liquid formulation at a concentration in the range of about 0.1 mg / mL to about 100 mg / mL.

33. The method according to claim 32, wherein the protein is present in a concentration in the range of about 0.1 mg / mL to about 70 mg / mL.

34. The method according to claim 33, wherein the protein is present in a concentration in the range of about 0.5 mg / mL to about 30 mg / mL.

35. The method according to claim 34, wherein the protein is present in a concentration in the range of about 1 mg / mL to about 20 mg / mL.

36. The method according to claim 35, wherein the protein is present at a concentration of about 1 mg / mL.

37. The method according to claim 1, wherein the liquid formulation of step (a) has a pH of about 4 to 6.

38. The method according to claim 1, wherein the liquid formulation of step (a) further comprises a buffer.

39. The method according to claim 38, wherein the buffer is an acetate buffer, a glutamate buffer, a citrate buffer, a lactate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a maleate buffer, a histidine buffer, a phosphate buffer, a 2-(N-morpholino)ethanesulfonic acid buffer, or any combination thereof.

40. The method according to claim 39, wherein the buffer comprises glutamate.

41. The method according to claim 38, wherein the buffer is present at a concentration in the range of about 5 mM to about 200 mM.

42. The method according to claim 41, wherein the buffer is present at a concentration in the range of about 10 mM to about 50 mM.

43. The method according to claim 42, wherein the buffer is present at a concentration of about 10 mM.

44. The method according to claim 1, wherein the saccharide is a monosaccharide or a disaccharide.

45. The method according to claim 44, wherein the saccharide is glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof.

46. The method according to claim 45, wherein the saccharide is sucrose.

47. The method according to claim 1, wherein the saccharide is present at a concentration in the range of about 1% to about 15% (w / v) in the liquid formulation.

48. The method according to claim 47, wherein the saccharide is present at a concentration in the range of about 6% to 12% (w / v).

49. The method according to claim 48, wherein the saccharide is present at a concentration of about 9% (w / v).

50. The method according to claim 1, wherein the surfactant is polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, poloxamer 407, Triton X-100, polyoxyethylene, PEG 3350, PEG 4000, or a combination thereof.

51. The method according to claim 50, wherein the surfactant is polysorbate 80.

52. The method according to claim 1, wherein the surfactant is present at a concentration in the range of about 0.001% to 0.5% (w / v) in the liquid formulation.

53. The method according to claim 52, wherein the surfactant is present at a concentration in the range of about 0.001% to 0.01% (w / v).

54. The method according to claim 53, wherein the surfactant is present at a concentration of about 0.01% (w / v).

55. The method according to claim 1, wherein the liquid formulation of step (a) has a pH of about 4 to about 5.

56. The method according to claim 1, wherein the liquid formulation of step (a) has a pH of about 4.2 and contains about 10 mM L-glutamic acid, about 9.0% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80.

57. The method according to claim 1, wherein the lyophilized formulation shows an increase of 0.5% or less in the proportion of high molecular weight species after storage at 40°C for one month upon reconstitution.

58. The lyophilized formulation according to claim 57, which shows an increase of 0.3% or less in the proportion of high molecular weight species after storage at 40°C for 1 month upon reconstitution.

59. A lyophilized protein formulation prepared by the method according to claim 1.