Method for preparing freeze-dried protein preparations

Abstract

Disclosed herein are methods for preparing lyophilized formulations comprising proteins, such as antibodies or bispecific antibody constructs, that exhibit improved storage stability.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 077,908, filed on September 14, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] The present disclosure provides a method for preparing a lyophilized formulation containing a protein such as an antibody or a bispecific antibody construct, the lyophilized formulation exhibiting improved storage stability.

[0003] Incorporation by Reference Incorporated herein by reference in its entirety is the computer - readable nucleotide / amino acid sequence listing, which was filed simultaneously with this specification and identified as the ASCII (text) file named "55423_Seqlisting.txt", created on September 14, 2020, and having a size of 345,229 bytes.

Background Art

[0004] Protein - based pharmaceuticals such as pharmaceuticals containing antibodies, antibody fragments, and bispecific antibody constructs are becoming increasingly important for the treatment of various diseases and conditions. However, proteins are only marginally stable and are very prone to degradation both chemically and physically. Chemical degradation refers to modifications involving covalent bonds such as deamidation, oxidation, cleavage, clipping / fragmentation, formation of new disulfide bridges, hydrolysis, isomerization, or deglycosylation. Physical degradation includes protein unfolding, unwanted adsorption to surfaces, and aggregation. Addressing these physical and chemical instabilities is one of the most difficult challenges in the development of protein pharmaceuticals (Chi et al., Pharm Res, Vol. 20, No. 9, Sept 2003, pp. 1325 - 1336, Roberts, Trends Biotechnol. 2014 Jul; 32(7):372 - 80).

[0005] Half-life extension antibody constructs (e.g., bispecific T cell engagers (BiTE®) including half-life extension forms such as Fc molecules) should be protected from protein aggregation and / or other degradation phenomena. Protein aggregation of BiTE® molecules is problematic because it can reduce the biological activity and quality (specifications) of therapeutic proteins. Furthermore, aggregation of BiTE® molecules can reduce product yield due to the meticulous purification process required to remove aggregates from the final product. More recently, there has been growing concern and evidence that the presence of aggregated proteins (whether humanized or complete human proteins) can significantly increase the risk of patients developing an immune response to active protein monomers, 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]

[0006] [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 [Overview of the project] [Problems that the invention aims to solve]

[0007] Protein-based pharmaceutical preparations are often freeze-dried and stored in solid form to help preserve the integrity of proteins such as antibodies or bispecific antibody constructs within the preparation during storage. However, many current freeze-drying methods for protein preparations do not yield solid preparations that exhibit suitable stability over time. Therefore, a novel method is needed to produce freeze-dried protein preparations that exhibit improved storage stability. [Means for solving the problem]

[0008] In one embodiment, the present disclosure is a method for preparing a lyophilized formulation, comprising: (a) preparing a lyophilized formulation by cooling a lyophilized chamber containing a liquid formulation comprising a protein, a sugar and a surfactant to a temperature in the range of about -35°C to about -50°C, and holding the chamber at a temperature in the range of about -40°C to about -50°C for a time of about 2 hours to about 24 hours; (b) preparing a primary dried formulation by 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 25 mTorr to about 100 mTorr, and holding the chamber at a temperature in the range of about -30°C to about -20°C The method provides a method comprising the steps of: (c) holding the chamber at a temperature and pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 45 to approximately 60 hours; and (c) heating the chamber to a temperature in the range of approximately 20°C to approximately 35°C to produce a secondary dried formulation, and holding the chamber at a temperature in the range of approximately 20°C to approximately 30°C and pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 5 to approximately 10 hours to produce a lyophilized formulation, wherein the liquid formulation has a pH of approximately 3 to 7 and does not contain mannitol; and the method provides a method that lacks an annealing step.

[0009] In another aspect, the Disclosure provides a lyophilized protein preparation prepared by the method of the Disclosure.

[0010] Further aspects and advantages will become apparent to those skilled in the art by examining the detailed description below. While the methods disclosed herein can take various forms, the following description will give specific examples, with the understanding that this disclosure is illustrative and not intended to limit the invention to any particular embodiment described herein. [Brief explanation of the drawing]

[0011] [Figure 1] This figure compares clipping levels measured by rCE-SDS after one month of storage at 25°C between a liquid formulation containing a 1 mg / mL bispecific antibody construct with the sequences shown in SEQ ID NO: 22 (BiTE A), SEQ ID NO: 77 (BiTE B), SEQ ID NO: 87 (BiTE C), and SEQ ID NO: 97 (BiTE D), and a redissolved (prepared with annealing) lyophilized formulation. The redissolved lyophilized formulation did not show a significant increase in clipping impaired under accelerated stress conditions. [Figure 2] This figure shows a comparison of clipping levels measured by rCE-SDS after one month of storage at 40°C for a 1 mg / mL liquid formulation of BiTE B and a redissolved (prepared with an annealing process) lyophilized formulation. [Figure 3] This figure shows the percentage high molecular weight (HMW) species measured by SE-UHPLC after one month of storage at 40°C for redissolved (prepared with an annealing step) lyophilized formulations containing BiTE B at various protein concentrations. No increase in %HMW was observed under accelerated stress conditions, demonstrating the applicability of the lyophilization cycle to high-concentration protein formulations such as high-concentration antibodies or bispecific antibody construct formulations. [Figure 4]This figure shows the increase in %HMW measured by SE-UHPLC after storage at the freezing temperatures experienced by the protein during the freeze-drying cycle for lyophilized formulations containing 23 mg / mL of BiTE B. "Annealed" samples were stored at -45°C for 48 hours, then at -12°C for 5 hours, then at -45°C for 5 hours, and finally at -25°C for 48 hours. "Non-annealed" samples were stored at -45°C for 58 hours, then at -25°C for 48 hours. [Figure 5] This figure shows a freeze-drying cycle (without an annealing step) for a placebo formulation containing 10 mM glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80) using a drying temperature of -10°C, which did not induce a cake collapse. Cake integrity was acceptable. Some curvature was observed in the cake. [Modes for carrying out the invention]

[0012] This specification discloses a method for preparing lyophilized formulations containing proteins such as antibodies or bispecific antibody constructs (e.g., bispecific antibody constructs with extended half-life) that exhibit improved stability. The lyophilization method disclosed herein is advantageous in that it reduces physical degradation such as aggregation, and chemical degradation such as clipping and deamidation. Furthermore, the lyophilization method disclosed herein can stabilize protein formulations, such as formulations containing antibodies and bispecific antibody constructs, at both low and high concentrations.

[0013] definition As used herein, the term “pharmaceutical composition” refers to a composition suitable for administration to a subject requiring it. The terms “subject,” “individual,” “animal,” or “patient” are used interchangeably herein and refer to any subject, particularly mammalian subjects, for which administration of the pharmaceutical compositions of this disclosure is desirable. Examples of mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc., but humans are preferred. The pharmaceutical formulations of this disclosure are stable and pharmaceutically acceptable, that is, they can exert the desired therapeutic effect without causing any significant undesirable local or systemic effects in the subject to which they are administered. The pharmaceutically acceptable formulations of this disclosure may be sterile. Specifically, the term “pharmaceutically acceptable” may mean that it is approved by a regulatory authority or other generally recognized pharmacopoeia for use in animals (more specifically humans), and is not limited to those approved by a regulatory authority.

[0014] The term “stability” or “stabilization” refers to the stability of the entire pharmaceutical formulation, and more specifically to the stability of the active ingredient itself (e.g., a protein such as a bispecific antibody construct), specifically during formulation, filling, transport, storage, and administration. A “stable formulation” is one in which the proteins within it (e.g., antibodies or bispecific antibody constructs) essentially maintain their physical and / or chemical integrity and biological activity after storage and during 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 enzyme activity, generation of peptide fragments, and shifts in the charge profile, as described in the section on the stability of lyophilized protein formulations below.

[0015] As used herein, the term "aggregation" refers to direct intermolecular attractive forces, such as van der Waals forces or chemical bonds. In particular, aggregation is understood to be the accumulation and clumping of proteins. Aggregates may include amorphous aggregates and oligomers, and are typically called high molecular weight (HMW) species, i.e., molecules with a higher molecular weight than the non-aggregated product molecules.

[0016] As used herein, the term "(protein) aggregate" typically encompasses high molecular weight protein species such as "oligomers" or "multimers," rather than a specific, predetermined species (e.g., monomers). This term is used interchangeably with the terms "high molecular weight" species and "HMW" herein. Protein aggregates can typically differ in size (ranging from small (dimers) to large aggregates (subvisible particles or even visible particles) and in diameters ranging from nanometers to micrometers), morphology (nearly spherical to fibrous), protein structure (natural vs. unnatural / denatured), type of intermolecular bonding (covalent vs. non-covalent), reversibility, and solubility. Soluble aggregates occupy a size range of approximately 1–100 nm, while protein microparticles occupy both subvisible (approximately 0.1–100 nm) and visible (>100 nm) ranges. All of the aforementioned types of protein aggregates are typically encompassed by this term. Therefore, the term "(protein) aggregate" refers to any type of unnatural species in which two or more protein monomers are physically associated or chemically bonded.

[0017] As used herein, the term "low molecular weight (LMW) species" refers to protein fragments such as bispecific antibody constructs.

[0018] method One aspect of the present disclosure provides a method for preparing a lyophilized formulation, which is a method lacking an annealing step. The method includes: (a) cooling a lyophilization chamber containing a liquid formulation having a pH of about 3 to 7, containing a protein, a saccharide, and a surfactant and lacking mannitol, 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 of about 2 hours to about 24 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 25 mTorr to about 100 mTorr to produce a primary dried formulation, 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 25 mTorr to about 100 mTorr for a time of about 45 hours to about 60 hours; and (c) heating the chamber to a temperature in the range of about 20°C to about 35°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 25 mTorr to about 100 mTorr for a time of about 5 hours to about 10 hours to produce a lyophilized formulation. As used herein, the term "temperature" refers to the temperature inside the lyophilization chamber (i.e., the internal temperature of the lyophilization chamber). Similarly, the term "pressure" as used herein refers to the pressure inside the lyophilization chamber (i.e., the internal pressure of the lyophilization chamber).

[0019] Step (a). In step (a), a freeze-drying chamber containing a liquid formulation is cooled to a temperature in the range of approximately -35°C to approximately -50°C (e.g., internal temperature) to produce a freeze-drying formulation, and is held at a temperature in the range of approximately -40°C to approximately -50°C (e.g., internal temperature) for a period of approximately 2 to 24 hours. In some embodiments, cooling is performed to a temperature in the range of approximately -40°C to approximately -50°C (e.g., approximately -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 cases, cooling can be performed to a temperature of approximately -45°C. In some cases, the chamber is cooled at a rate in the range of approximately 0.5°C / min to approximately 1°C / min. In various embodiments, cooling is performed at a rate of approximately 0.5°C / min to approximately 0.8°C / min. In some embodiments, cooling is performed at a rate of approximately 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 cases, cooling is performed at a rate of approximately 0.5°C / min. In some embodiments, the chamber can be held at a temperature of approximately -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, holding is performed at a temperature of approximately -45°C. In some embodiments, the temperature at which the freeze-drying chamber is cooled and the holding temperature are the same. In various embodiments, holding is performed for a period of approximately 2 hours to approximately 5 hours (e.g., approximately 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours). In some cases, holding is performed for approximately 2 hours.

[0020] Step (b). In step (b), the freeze-drying chamber is heated to a temperature in the range of approximately -30°C to approximately -20°C (e.g., internal temperature) and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr (e.g., internal pressure) to produce a primary-dried formulation, which is then held at a temperature in the range of approximately -30°C to approximately -20°C (e.g., internal temperature) and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr (e.g., internal pressure) for a period of approximately 45 to approximately 60 hours. In some embodiments, heating is carried out to temperatures of approximately -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 cases, heating is carried out to a temperature of approximately -25°C. In some cases, heating is carried out at a rate in the range of approximately 0.1°C / min to approximately 1°C / min. In various embodiments, heating is carried out at a rate of approximately 0.1°C / min to approximately 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 cases, heating is carried out at a rate of approximately 0.3°C / min. In various cases, heating is carried out at pressures in the range of approximately 25 mTorr to approximately 75 mTorr, or approximately 50 mTorr to approximately 100 mTorr, or approximately 70 mTorr to approximately 100 mTorr, or approximately 65 mTorr to approximately 75 mTorr. In some cases, heating is carried out at pressures of approximately 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, heating is performed at a pressure of about 70 mTorr. In some embodiments, chamber holding is performed at temperatures of about -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 cases, holding is performed at a temperature of about -25°C. In various embodiments, holding is performed at pressures ranging from about 25 mTorr to about 75 mTorr, or about 50 mTorr to about 100 mTorr, or about 70 mTorr to about 100 mTorr, or about 65 mTorr to about 75 mTorr.In some cases, the holding is performed 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 performed at a pressure of about 70 mTorr. In some embodiments, the temperature at which the lyophilization chamber is heated and the holding temperature are the same temperature. In some embodiments, the pressure at which the lyophilization chamber is heated and the holding pressure are the same pressure. In some embodiments, the temperature at which the lyophilization chamber is being heated and the holding temperature are the same temperature, and also, the pressure at which the lyophilization chamber is being heated and the holding pressure are the same pressure. In some cases, the holding is performed for a time of about 50 hours to about 55 hours (e.g., about 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, or 55 hours). In various cases, the holding is performed for a time of about 52 hours.

[0021] Step (c). In step (c), the chamber is heated to a temperature in the range of approximately 20°C to approximately 35°C (e.g., internal temperature) to produce a secondary dried formulation, and then held at a temperature in the range of approximately 20°C to approximately 30°C (e.g., internal temperature) and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr (e.g., internal pressure) for a period of approximately 5 to 10 hours to produce a lyophilized formulation. In some embodiments, heating is performed up to a temperature of approximately 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 cases, heating is performed up to a temperature of approximately 30°C. In various embodiments, heating is performed at a rate in the range of approximately 0.5°C / min to produce a secondary dried formulation. In some cases, heating is carried out at a rate of approximately 0.05°C / min to approximately 0.5°C / min. In various cases, heating is carried out at a rate of approximately 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, heating is carried out at a rate of approximately 0.1°C / min. In some embodiments, holding is carried out at a temperature of approximately 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 cases, holding is carried out at a temperature of approximately 30°C. In some embodiments, the temperature at which the freeze-drying chamber is heated is the same as the holding temperature. In some embodiments, holding is performed at pressures ranging from approximately 25 mTorr to approximately 75 mTorr, or approximately 50 mTorr to approximately 100 mTorr, or approximately 70 mTorr to approximately 100 mTorr, or approximately 65 mTorr to approximately 75 mTorr. In some cases, holding is performed at pressures of approximately 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, holding is performed at a pressure of approximately 70 mTorr. In some cases, holding is performed for a period of approximately 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours. In various cases, holding is performed for a period of approximately 8 hours.

[0022] Another aspect of the present disclosure provides a method for preparing a lyophilized formulation, comprising an annealing step. The method of this aspect comprises (a) preparing a lyophilized formulation by cooling a lyophilization chamber containing a liquid formulation having a pH of about 3 to 7, containing proteins, sugars, and surfactants, and lacking mannitol, to a temperature in the range of about -35°C to about -50°C, and holding the chamber at a temperature in the range of about -40°C to about -50°C for a period of about 2 to about 24 hours; and (b) preparing a primary dried formulation by 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 25 mTorr to about 100 mTorr, and then... (a) a step of holding the formulation at a temperature in the range of approximately -30°C to approximately -20°C and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 45 hours to approximately 60 hours; and (c) a step of heating the chamber to a temperature in the range of approximately 20°C to approximately 35°C to produce a secondary dried formulation, and holding the chamber at a temperature in the range of approximately 20°C to approximately 30°C and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 5 hours to approximately 10 hours to produce a lyophilized formulation, as described above, but including an annealing step between steps (a) and (b). As used herein, “annealing” refers to the process of cycling the temperature of the formulation (e.g., from low to high temperature and then back to low temperature) to obtain more perfect crystallization. In embodiments, the annealing step may include (i) heating the chamber containing the cryopreserved preparation from step (a) and holding the chamber at the heated temperature for a period of time; and (ii) cooling the chamber containing the cryopreserved preparation back to the temperature of step (a) and holding the chamber containing the cryopreserved preparation at a temperature in the range of approximately -35°C to approximately -50°C for a period of approximately 2 hours to approximately 24 hours.

[0023] Step (i). In step (i), the frozen preparation from step (a) may be heated to a temperature in the range of approximately -20°C to approximately -5°C at a rate ranging from approximately 0.1°C / min to approximately 1°C / min. In some embodiments, heating may be performed to a temperature of approximately -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, or -5°C. In some embodiments, heating may be performed to a temperature in the range of approximately -15°C to approximately -10°C. In various cases, heating may be performed to a temperature of approximately -12°C. In various embodiments, heating is performed at a rate of approximately 0.1°C / min to approximately 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 cases, heating is performed at a rate of approximately 0.5°C / min. The frozen preparation can be held at a temperature in the range of approximately -20°C to approximately -5°C for a period of approximately 2 hours to approximately 10 hours. In some embodiments, holding is performed down to a temperature of approximately -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, or -5°C. In some embodiments, holding is performed at a temperature in the range of approximately -15°C to approximately -10°C. In various cases, holding is performed at a temperature of approximately -12°C. In various embodiments, the holding period is approximately 2 to 5 hours (for example, approximately 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours). In some cases, the holding period is approximately 2 hours.

[0024] Step (ii). In step (ii), cooling can be performed at a rate in the range of about 0.5°C / min to about 1°C / min down to a temperature in the range of about -35°C to about -50°C. In some embodiments, cooling is performed down 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 cases, cooling is performed down to a temperature of about -45°C. In various embodiments, cooling is performed at a rate of about 0.5°C / min to about 0.8°C / min. In some embodiments, 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 cases, cooling is performed at a rate of about 0.5°C / min. In some embodiments, holding can be performed for a period of about 2 to 24 hours at a temperature in the range of about -40°C to about -50°C. In some embodiments, holding is 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, holding is performed at a temperature of about -45°C. In various embodiments, holding is performed for a period of about 2 to 5 hours (e.g., about 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or 5 hours). In some cases, holding is performed for about 2 hours.

[0025] In any embodiment of the method disclosed herein (with or without an annealing step), the method may further include step (d) of cooling the chamber containing the lyophilized formulation from 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 supplying 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 cases, the inert gas is selected from argon, helium, nitrogen, and any combination thereof. In various cases, the inert gas is nitrogen. In embodiments, step (d) may facilitate the capping of the container (e.g., vial) containing the lyophilized formulation. In embodiments, the method further includes storing the lyophilized formulation at a temperature in the range of about 2°C to about 8°C. In embodiments, the method further includes redissolving the lyophilized formulation with water.

[0026] In yet another embodiment, the Disclosure provides lyophilized protein formulations prepared by the methods disclosed herein. In some embodiments, the protein formulations are prepared by the methods disclosed herein, which lack an annealing step. In various embodiments, the methods disclosed herein include an annealing step.

[0027] Freeze-dried protein preparations The lyophilized protein formulations described herein comprise proteins, sugars, surfactants, and optionally buffers, 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 cases, the pH is about 4 to about 6. In some cases, the pH of the formulation is about 4 or about 4.2. In various cases, 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 sugar alcohols. As used herein, “sugar alcohol” refers to a linear polyol in which one hydroxyl group is bonded to each carbon atom. Examples of sugar alcohols as used herein include xylitol, erythritol, mannitol, and sorbitol. In embodiments, the lyophilized formulations do not contain mannitol.

[0028] protein In some embodiments, the protein in the lyophilized formulation is an antigen-binding protein. An "antigen-binding protein" is a protein that contains a domain that binds to a specific target antigen (such as CD3 and / or CDH19, MSLN, DLL3, FLT3, EGFRvll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17). The antigen-binding protein includes a backbone or framework portion that allows the antigen-binding domain to adopt a three-dimensional structure that facilitates the binding of the antigen-binding protein to the antigen.

[0029] In some embodiments, the antigen-binding protein of a lyophilized formulation is an antibody, immunoglobulin, or antigen-binding antibody fragment. In some cases, the antigen-binding protein is an antibody. The term “antibody” refers to an intact antigen-binding immunoglobulin. “Antibody” is a type of antigen-binding protein. An antibody may be an IgA, IgD, IgE, IgG, or IgM antibody containing any one of IgG1, IgG2, IgG3, or IgG4. In various embodiments, an intact antibody contains two full-length heavy chains and two full-length light chains. An antibody has one variable region and one constant region. In IgG form, the one variable region is generally about 100 to 110 or more amino acids and contains three complementarity-determining regions (CDRs) that are primarily involved in antigen recognition and substantially vary among other antibodies that bind to different antigens. A single variable region typically contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J.Mol.Biol.196:901-917; Chothia et al., 1989, Nature 342:877-883), which are located within a framework region (referred to as framework regions 1-4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, cited above). The constant region allows the antibody to recruit cells and molecules of the immune system.

[0030] In some embodiments, the antibody in the formulation is a bispecific antibody, i.e., an antibody that binds to two different targets (e.g., CD3 and a second different target). As used herein, the term “bispecific” refers to an antibody 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, the antibody construct according to this disclosure includes specificity to two different antigens or targets. The term “surface antigen of a target cell” refers to an antigenic structure expressed by a cell and present on its cell surface so that the antibody construct described herein can access it. The target cell surface antigen may be a protein, such as the extracellular portion of a protein, or a carbohydrate structure, such as the carbohydrate structure of a protein, such as a glycoprotein. The target cell surface antigen may be a tumor antigen. This disclosure also includes multispecific antibody constructs, such as triplicate antibody constructs, the latter of which include constructs having three binding domains or more than three (e.g., four, five, or more) specificity.

[0031] The bispecific antibodies and / or antibody constructs understood herein include, but are not limited to, conventional bispecific immunoglobulins (e.g., BsIgG), IgG containing an added antigen-binding domain (e.g., the amino or carboxyl terminus of the light or heavy chain is linked to an additional antigen-binding domain such as a single-domain antibody or a paired antibody variable domain (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 portion), and BsAb conjugates. For example, see Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015), which describes various forms of bispecificity and is incorporated herein by reference. Examples of bispecific constructs include, but are not limited to, diabodies, single-chain diabodies, tandem scFvs, bispecific T cell engagers (BiTE®) (fusion proteins consisting of two single-chain variable fragments (scFvs) linked by a linker), and modified constructs including Fab2 bispecifics and full-length antibodies. For example, all of the following are explicitly incorporated herein: 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; International Publication No. 2009032782 and International Publication No. 2006020258; Zuo et al., 2000, Protein Engineering 13[5]:361-367; U.S. Patent Application Publication No. 20020103345; Shen et al., 2006, J Biol Chem See 281

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

[20] :19665-19672; and Kontermann, 2012 MAbs 4(2):182.

[0032] In some embodiments, the lyophilized formulation described herein is a bispecific antibody 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 T cells, and a third domain optionally comprising a hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain in the order of amino to carboxyl. In some embodiments, each of the first and second binding domains comprises a VH region and a VL region.

[0033] As used herein, the term "binding domain" refers to a domain that (specifically) binds to, interacts with, or recognizes a predetermined target epitope or target site on a target molecule (antigen), such as CDH19, MSLN, DLL3, FLT3, EGFRvll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17 and CD3, respectively.

[0034] The structure and function of the first binding domain (which recognizes, for example, CDH19, MSLN, DLL3, FLT3, EGFRvlll, BCMA, PSMA, CD33, CD19, CD70, CLDN18.2, or MUC17), and the structure and / or function of the second binding domain (which recognizes CD3), are based on the structure and / or function of the antibody, for example, the full-length or full immunoglobulin molecule, and / or derived from the variable heavy chain (VH) domain and / or variable light chain (VL) domain of the antibody or a fragment thereof. In embodiments, the first binding domain is characterized by the presence of three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). In embodiments, the second binding domain also includes the minimum structural requirements of the antibody that enable target binding. In the embodiment, the second binding domain includes at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region). The first and / or second binding domains are assumed to be prepared or obtained by phage display or library screening, rather than by transplanting CDR sequences derived from an existing (monoclonal) antibody into the backbone.

[0035] In some embodiments, the first binding domain that binds to the surface antigen of the target cell and / or the second binding domain that binds to CD3ε are human binding domains. Antibodies and antibody constructs containing at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs that have non-human variable regions and / or constant regions, such as those of rodents (e.g., mice, rats, hamsters, or rabbits). The presence of such rodent-derived proteins may result in rapid clearance of the antibody or antibody construct, or may trigger an immune response by the patient to the antibody or antibody construct. To avoid the use of rodent-derived antibodies or antibody constructs, human or fully human antibodies / antibody constructs can be generated by introducing human antibody function into rodents so that the rodents produce fully human antibodies.

[0036] In some embodiments, the antigen-binding protein comprises a single-chain antibody construct. 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 bound to the N-terminus of the scFv linker, and its C-terminus is bound to the N-terminus of the variable heavy chain (N-vh-linker-vl-C), but the configuration is reversible (N-vl-linker-vh-C). Alternatively, the C-terminus of the variable heavy chain is bound to the N-terminus of the scFv linker, and its C-terminus is bound to the N-terminus of the variable light chain (N-vl-linker-vh-C), but the configuration is reversible (N-vh-linker-vC). Therefore, both orientations of the scFv are specifically included in the description and explanation of the scFv.

[0037] At least two binding and variable domains (VH / VL) of the antibody constructs of this disclosure may or may not contain a peptide linker (spacer peptide). The term “peptide linker” according to this disclosure 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 antibody construct of this disclosure. A peptide linker may also be used to fuse a third domain to other domains of the antibody construct of this disclosure. A characteristic of such peptide linkers is that they do not contain any polymerization activity. Particularly preferred peptide linkers are those described in U.S. Patent No. 4,751,180 and No. 4,935,233 or International Publication No. 88 / 09344, whose disclosures are incorporated herein by reference as a whole. Peptide linkers can also be used to add other domains, modules, or regions (e.g., half-life extension domains) to the bispecific antibody constructs described herein.

[0038] In some embodiments, the third domain includes "Fc" or "Fc region" or "Fc domain," referring to a polypeptide containing the constant region of an antibody that excludes the first constant region immunoglobulin domain. Thus, the "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 mobile hinge N-terminus to these domains. With respect to IgA and IgM, Fc may include the J chain. With respect to IgG, the Fc domain includes the immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3) as well as the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, the bispecific antibody construct is an IgG antibody (including, but not limited to, several subclasses, including IgG1, IgG2, IgG3, and IgG4). The boundaries of the Fc region may vary, but the human IgG heavy chain Fc region is typically defined as encompassing residues C226 or P230 to its carboxyl terminus, and is numbered according to the EU index as described in Kabat. In some embodiments, amino acid modifications are made to the Fc region to alter, for example, binding to one or more FcγR receptors or FcRn receptors.

[0039] In some embodiments, the formulations described herein include a bispecific antibody construct that binds to 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.

[0040] In some embodiments, the first binding domain of the bispecific antibody construct includes one set of 6 CDRs shown in (a) SEQ ID NOs: 24-29, (b) SEQ ID NOs: 34-39, (c) SEQ ID NOs: 78-83, (d) SEQ ID NOs: 10-15, (e) SEQ ID NOs: 46-51, (f) SEQ ID NOs: 88-93, (g) SEQ ID NOs: 67-72, (h) SEQ ID NOs: 56-61, (i) SEQ ID NOs: 112-117, (j) SEQ ID NOs: 100-105, (k) SEQ ID NOs: 148-153, SEQ ID NOs: 157-162, or SEQ ID NOs: 166-171, or SEQ ID NOs: 175-180, (l) SEQ ID NOs: 132-137, or (m) SEQ ID NOs: 123-128.

[0041] In some embodiments, the first binding domain of the bispecific antibody construct includes a VH region containing 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 shown in sequence numbers 30, 40, 84, 16, 17, 52, 94, 73, 62, 118, 154, 163, 172, 181, 106, 138, 143, or 129.

[0042] In some embodiments, the first binding domain of the bispecific antibody construct includes a VL region containing 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 shown in SEQ ID NOs: 31, 41, 85, 18, 19, 53, 95, 74, 63, 119, 155, 164, 173, 182, 107, 139, 144, or 130.

[0043] In some embodiments, the first binding domain is (a) a VH region containing the amino acid sequence shown in SEQ ID NO: 30 and a VL region showing SEQ ID NO: 31; (b) a VH region containing the amino acid sequence shown in SEQ ID NO: 40 and a VL region showing the amino acid sequence shown in SEQ ID NO: 41; (c) a VH region containing the amino acid sequence shown in SEQ ID NO: 84 and a VL region containing the amino acid sequence shown in SEQ ID NO: 85; (d) a VH region containing the amino acid sequence shown in SEQ ID NO: 16 or 17 and a VL region containing the amino acid sequence shown in SEQ ID NO: 18 or 19; (e) a VH region containing the amino acid sequence shown in SEQ ID NO: 52 and a VL region containing the amino acid sequence shown in SEQ ID NO: 53; (f) a VH region containing the amino acid sequence shown in SEQ ID NO: 94 and a VL region containing the amino acid sequence shown in SEQ ID NO: 95; (g) a VH region containing the amino acid sequence shown in SEQ ID NO: 73 and an amino acid sequence shown in SEQ ID NO: 74 (h) A VL region containing a column; (i) A VH region containing the amino acid sequence shown in SEQ ID NO: 62 and a VL region containing the amino acid sequence shown in SEQ ID NO: 63; (j) A VH region containing the amino acid sequence shown in SEQ ID NO: 118 and a VL region containing the amino acid sequence shown in SEQ ID NO: 119; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 154, 163, 172, or 181 and a VL region containing the amino acid sequence shown in SEQ ID NO: 155, 164, 173, or 182; (k) A VH region containing the amino acid sequence shown in SEQ ID NO: 106 and a VL region containing the amino acid sequence shown in SEQ ID NO: 107; (l) A VH region containing the amino acid sequence shown in SEQ ID NO: 138 or 143 and a VL region containing the amino acid sequence shown in SEQ ID NO: 139 or 144; or (m) A VH region containing the amino acid sequence shown in SEQ ID NO: 129 and a VL region containing the amino acid sequence shown in SEQ ID NO: 130.

[0044] In some embodiments, the second binding domain of the bispecific antibody construct contains a set of 6CDRs shown in SEQ ID NOs: 1-6.

[0045] In some embodiments, the second binding domain of the bispecific antibody construct includes a VH region containing 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 shown in SEQ ID NO: 7.

[0046] In some embodiments, the second binding domain of the bispecific antibody construct includes a VL region containing 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 shown in SEQ ID NO: 8.

[0047] In some embodiments, the second binding domain includes (a) a VH region containing the amino acid sequence shown in SEQ ID NO: 7 and a VL region containing the amino acid sequence shown in SEQ ID NO: 8.

[0048] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 87.

[0049] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to MSLN, comprising an anti-MSLN variable light chain domain containing the amino acid sequence of SEQ ID NO: 41 and an anti-MSLN variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 40; a second binding domain comprising an anti-CD3 variable heavy chain domain containing 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 antibody construct includes 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 antibody construct includes the amino acid sequences shown in SEQ ID NO: 43, 44, or 45.

[0050] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 76 or 77.

[0051] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 65 or 66.

[0052] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 33.

[0053] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to BCMA, comprising an anti-BCMA variable light chain domain containing the amino acid sequence of SEQ ID NO: 95 and an anti-BCMA variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 94; a second binding domain comprising an anti-CD3 variable heavy chain domain containing 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 98 or SEQ ID NO: 97.

[0054] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequences shown in SEQ ID NOs: 121, 122, 109, 110, or 111.

[0055] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequences shown in SEQ ID NO: 20, 21, 22, or 23.

[0056] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 55.

[0057] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to 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; 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 156, 165, 174, or 183.

[0058] In some embodiments, the bispecific antibody construct includes a first binding domain that binds to cldn18.2, which includes an anti-cldn18.2 variable light chain domain containing the amino acid sequence of SEQ ID NO: 139 or 144 and an anti-cldn18.2 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 138 or 143; a second binding domain containing an anti-CD3 variable heavy chain domain containing the amino acid sequence of SEQ ID NO: 7; and an anti-CD3 variable light chain domain containing the amino acid sequence of SEQ ID NO: 8. For example, in one embodiment, the bispecific antibody construct includes a first binding domain containing the amino acid sequence of SEQ ID NO: 140 or 145, and a second binding domain containing the amino acid sequence of SEQ ID NO: 9. In some embodiments, the bispecific antibody construct includes the amino acid sequences shown in SEQ ID NO: 141, 142, 146, or 147.

[0059] In some embodiments, the bispecific antibody construct includes 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; 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 antibody construct includes the amino acid sequence shown in SEQ ID NO: 131.

[0060] In some embodiments, the protein of the formulation is an antibody. In various embodiments, the protein of the formulation is a bispecific antibody construct. In some cases, the protein of the formulation is a bispecific antibody construct with extended half-life. Bispecific antibody constructs with extended half-life have already been described herein. In some embodiments, the protein formulations of this disclosure contain the amino acid sequences shown in SEQ ID NOs: 1 to 190. In various embodiments, the protein formulations of the present disclosure include the amino acid sequence shown 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: 55, 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 cases, the protein formulations of the present disclosure include the amino acid sequence shown 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).

[0061] In some embodiments, proteins such as antibodies or bispecific antibody constructs (e.g., HLE bispecific antibody constructs) are present in the liquid formulation (before lyophilization) in amounts 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, proteins are present in the liquid formulation in amounts ranging from about 0.1 mg / mL to about 70 mg / mL. In some cases, proteins are present in the liquid formulation in amounts ranging from approximately 0.5 mg / mL to approximately 30 mg / mL (or approximately 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, or 30 mg / mL).In various cases, the protein is present in liquid formulations in a range of approximately 1 mg / mL to approximately 20 mg / mL (or approximately 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). The protein is present in amounts of mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, 12.5 mg / mL, 13 mg / mL, 13.5 mg / mL, 14 mg / mL, 14.5 mg / mL, 15 mg / mL, 15.5 mg / mL, 16 mg / mL, 16.5 mg / mL, 17 mg / mL, 17.5 mg / mL, 18 mg / mL, 18.5 mg / mL, 19 mg / mL, 19.5 mg / mL, or 20 mg / mL). In some embodiments, the protein is present in the liquid formulation in an amount of approximately 1 mg / mL.

[0062] Sugars The protein formulations of this disclosure contain sugars. In some embodiments, the sugars are monosaccharides or disaccharides. Preferred sugars include, for example, glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof. In some cases, the sugars include sucrose.

[0063] In some embodiments, the liquid formulation (before freeze-drying) contains sugars 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 contains sugars 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 contains sugars 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 contains sugars at concentrations 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 contains sugars at concentrations of about 7% to about 12% w / v. In some embodiments, the liquid formulation contains sugars at a concentration of about 9% w / v. In some embodiments, the sugar is sucrose and is present in the liquid formulation at concentrations ranging from about 6% to about 12% w / v. In some cases, the sugar is sucrose and is present in the liquid formulation at a concentration of about 9% w / v.

[0064] surfactant The protein formulations of this disclosure include surfactants. Preferred surfactants include polysorbates, poloxamers, polyoxyethylenes, or any combination thereof. Surfactants intended 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 includes polysorbate. In some cases, the surfactant is polysorbate 80.

[0065] The protein formulations described herein may comprise one 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 contains 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 contains a surfactant at a concentration of about 0.001% to about 0.5% w / v. In some embodiments, the liquid formulation contains a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some embodiments, the liquid formulation contains a surfactant at a concentration of about 0.001 to about 0.01% w / v. In some embodiments, the liquid formulation contains 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 contains a surfactant at a concentration of about 0.001% to about 0.01% w / v. In some embodiments, the surfactant is polysorbate 80, which is present at a concentration of about 0.01% w / v.

[0066] cushioning agent The protein formulations of this disclosure optionally include a buffer. Suitable buffers include acetate buffers, glutamate buffers, citrate buffers, lactate buffers, succinate buffers, tartarate buffers, fumarate buffers, maleate buffers, histidine buffers, phosphate buffers, 2-(N-morpholino)ethanesulfonic acid buffers, or any combination thereof. In some cases, the buffer may include glutamate.

[0067] Buffers are often used to control the pH of a formulation. In some embodiments, buffers are added at concentrations that maintain the pH of the liquid formulation at approximately 3–7, 4–6, 4–5, or 4.2. The effect of pH on the formulation can be characterized using one or more of several methods, such as accelerated stability testing and calorimetry screening tests (Remmele RLJr., et al., Biochemistry, 38(16):5241-7(1999)).

[0068] 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 concentrations of approximately 0.1 mM to approximately 1000 mM (1 M), or approximately 5 mM to approximately 200 mM, or approximately 5 mM to approximately 100 mM, or approximately 10 mM to approximately 50 mM. A suitable buffer concentration includes concentrations of approximately 200 mM or less. In some embodiments, the buffer in the liquid protein preparation (before freeze-drying) is present at concentrations of approximately 190 mM, 180 mM, 170 mM, 160 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 20 mM, 10 mM, or 5 mM. In some embodiments, the concentration of the buffer 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 buffer 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 and 100 mM. In some embodiments, the concentration of the buffer is between 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM and 50 mM. In some embodiments, the concentration of the buffer is about 10 mM.

[0069] 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.

[0070] Stability of lyophilized protein preparations The methods disclosed herein advantageously yield lyophilized protein formulations that exhibit reduced physical degradation, such as protein aggregation, and reduced chemical degradation, such as clipping and deamidation, after redissolution in liquid. The liquid used to redissolve the lyophilized protein formulation may be any suitable liquid known in the art. In embodiments, the lyophilized protein formulation can be redissolved in water. Furthermore, the lyophilization methods disclosed herein can stabilize protein formulations, such as formulations containing antibodies and bispecific antibody constructs (e.g., bispecific antibody constructs with extended half-lives), at both low and high concentrations.

[0071] The stability of protein formulations, such as those containing antibodies or bispecific antibody constructs (e.g., HLE bispecific antibody constructs), can be quantified by several methods. In some embodiments, the stability of protein formulations is characterized by size exclusion high-performance liquid chromatography (SE-HPLC), size exclusion ultrahigh-performance liquid chromatography (SE-UHPLC), cation exchange high-performance liquid chromatography (CE-HPLC), dynamic light scattering (DLS), ultracentrifugation (AUC), field flow fractionation (FFF), isoelectric focusing, and ion exchange chromatography (IEX). In some embodiments, the stability of protein formulations, such as antibody formulations, is characterized by partial dissociation measured by capillary-sodium decyl sulfate gel electrophoresis (CE-SDS) and / or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In some embodiments, the stability of formulations is evaluated by reduced capillary-sodium decyl sulfate gel electrophoresis (rCE-SDS). The rCE-SDS method separates heavy chain (HC), light chain (LC), non-glycosylated HC (NGHC), and other fine peak species and groups under reducing conditions.

[0072] In some embodiments, the stability of the formulation is characterized by the amount of high molecular weight (HMW) species of the protein, such as the antibody or bispecific antibody construct (e.g., HLE bispecific antibody construct), or by the rate of increase of the amount of HMW species of the protein after storage conditions at various time points. In some embodiments, the amount of HMW species of the protein 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 after redissolution. In some embodiments, the rate of increase of HMW species of the protein 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 after redissolution. In some embodiments, the amount of HMW species of the protein, such as the antibody or bispecific antibody construct (e.g., HLE bispecific antibody construct), in the redissolved lyophilized formulation is measured by SE-UHPLC.

[0073] The stability of proteins such as antibodies or bispecific antibody constructs (e.g., HLE bispecific antibody constructs), and the ability of a formulation to maintain protein stability, can be evaluated over long periods (e.g., several weeks or months). From a formulation perspective, a stable formulation is one in which the proteins within it, such as antibodies or bispecific antibody constructs (e.g., HLE bispecific antibody constructs), essentially maintain their physical and / or chemical integrity and / or biological activity after storage and during processes such as freeze / thaw, mechanical mixing, and lyophilization. Protein stability can be evaluated, for example, by measuring the level and / or rate of high molecular weight (HMW) aggregate formation, the shift in charge profile, and the change in particle size.

[0074] In some embodiments, the relative values ​​of any particular species of protein, such as intact BiTE® molecules or major species, high molecular weight (HMW) species (i.e., aggregates), or low molecular weight (LMW) species (i.e., fragments), are expressed relative to each value of the total product. For example, in some embodiments, less than 2.5% (e.g., 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, or 0.5%) of proteins such as antibodies or bispecific antibody constructs are present as HMW species in the redissolved lyophilized formulation. In some embodiments, the amount of HMW species in the redissolved 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%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the redissolved lyophilized formulation increases by approximately 0.1% to 0.4% (e.g., 0.1%, 0.2%, 0.3%, or 0.4%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of HMW species in the redissolved 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%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of HMW species in the redissolved lyophilized formulation increases by less than 0.5% (e.g., 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of HMW species in the redissolved lyophilized formulation increases by less than 0.5% (e.g., 0.5%, 0.4%, 0.3%, 0.2%, 0.1%) after storage at 40°C for more than one month (e.g., one month, three months, six months, nine months, or twelve months). In some embodiments, the amount of HMW species in the redissolved lyophilized formulation increases by less than 0.5% after storage at 40°C for one month. In some embodiments, the amount of HMW species in the redissolved lyophilized formulation increases by less than 0.3% after storage at 40°C for one month.In some embodiments, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of HMW species in the redissolved 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, after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months), the amount of HMW species in the redissolved lyophilized formulation increases by approximately 0.1% to 0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%). In some embodiments, after storage at 40°C for more than one month (e.g., one, three, six, nine, or twelve months), the amount of HMW species in the redissolved lyophilized formulation increases by approximately 0.1% to 0.5% (e.g., 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%). In some embodiments, the HMW species of the bispecific antibody construct in the redissolved lyophilized formulation are measured by SE-UHPLC.

[0075] In some embodiments, the stability of the formulation is characterized by the amount of low molecular weight (LMW) species of proteins such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs), or by the rate of increase of LMW species of proteins under storage conditions at various time points. 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 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, LMW species of proteins such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs) in the formulation are measured by reduced capillary sodium decyl sulfate gel electrophoresis (rCE-SDS). In some embodiments, LMW species of bispecific antibody constructs in the formulation are measured by size exclusion chromatography (SEC).

[0076] 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 proteins, such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs), are present as low molecular weight (LMW) species in the redissolved lyophilized formulation. In some embodiments, the amount of LMW species in the redissolved 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%) after storage at 4°C for more than one month (e.g., one month, three months, or six months). In some embodiments, the amount of LMW species in the lyophilized formulation redissolved after storage at 4°C for more than one month (e.g., one month, three months, or six months) 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 amount of LMW species in the redissolved 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%, or 0.1%) after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months). In some embodiments, the amount of LMW species in the lyophilized formulation redissolved after storage at 40°C for more than one week (e.g., one week, two weeks, one month, or three months) 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 antibody construct in the redissolved lyophilized formulation is measured by size exclusion chromatography (SEC). In some embodiments, the LMW species of the bispecific antibody construct in the redissolved lyophilized formulation is measured by reductive capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS).

[0077] In some embodiments, the percentage of proteins (i.e., main peak species) such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs) in the redissolved lyophilized formulation is greater than 95% of the total protein content in the formulation.

[0078] In some embodiments, the formulation is stable after storage at approximately 4°C for one month, and the amount of HMW species in the redissolved 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%) during storage for at least one month. In some embodiments, the formulation is stable after storage at 4°C for three months, and the amount of HMW species in the redissolved lyophilized formulation increases by approximately 0.0% to 0.2% (e.g., 0%, 0.1%, or 0.2%) during storage for at least three months. In some embodiments, the formulation is stable after storage at 4°C for at least six months, and the amount of HMW species in the redissolved lyophilized formulation increases by approximately 0.0% to 0.4% (e.g., 0%, 0.1%, 0.2%, 0.3%, or 0.4%) during storage for at least six months. In some embodiments, the HMW species of the bispecific antibody construct in the redissolved lyophilized formulation are measured by SE-UHPLC.

[0079] In some embodiments, the formulation is stable after storage at approximately 4°C for 1 month, 3 months, and 6 months, and the percentage of proteins such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs) is greater than 95% of the total protein content. In some embodiments, the formulation is stable after storage at approximately 4°C for 1 month, 3 months, 6 months, 12 months, and 48 months, and the percentage of proteins such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs) is greater than 96% of the total protein content after redissolution.

[0080] The stability of the formulations described herein is further characterized by changes in the charge distribution, for example, in the amount of charge change peaks of proteins such as antibodies or bispecific antibody constructs (HLE bispecific antibody constructs). 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 the formulation) in the redissolved 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%) 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 formulation) in the redissolved 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., one month, three months, six months, or twelve months). In some embodiments, the amount of major peaks in the redissolved lyophilized formulation decreases by less than 4% (e.g., 4%, 3.5%, 3%, 2.5%, 2%, or 1%) when stored at 4°C for at least one month. In some embodiments, the amount of major peaks in the redissolved lyophilized formulation decreases by less than 6% (e.g., 6%, 5%, 4%, 3.5%, 3%, 2.5%, or 2%) when stored at 4°C for at least three months. In some embodiments, the amount of major peak in the redissolved 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 at 4°C for at least 6 months. In some embodiments, the amount of major peak in the redissolved 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 at 4°C for at least 12 months.

[0081] In some embodiments, the amount of acidic peaks in the redissolved lyophilized formulation increases by less than 30% (e.g., 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 4%, 4%, 3%, 2%, or 1%) 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 relatively high pI) in the redissolved lyophilized formulation increases by less than 15% (e.g., 15%, 10%, 9%, 8%, 7%, 6%, 4%, 4%, 3%, 2%, or 1%) 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 main peak in the redissolved 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 peak in the redissolved 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.

[0082] Protein formulations lyophilized by the method of this disclosure exhibit superior stability compared to corresponding liquid protein formulations. For example, the stability of a protein formulation containing 1 mg / mL of the bispecific antibody construct of this disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80 at pH 4.2, lyophilized by the method of this disclosure using an annealing step, and then redissolved, was determined by reducing capillary decyl sulfate sodium gel electrophoresis (rCE-SDS) to determine the degree of clipping that occurred after 1 month of storage at 25°C and 40°C. See Example 3. As shown in Figures 1 and 2, the redissolved lyophilized formulation showed significantly less clipping than the liquid formulation at both temperatures, demonstrating that protein formulations lyophilized by the method described herein have superior stability compared to corresponding liquid formulations.

[0083] The lyophilization method of this disclosure also advantageously stabilizes both low- and high-concentration protein formulations. For example, a protein formulation of this disclosure containing 1 mg / mL, 5 mg / mL, 13 mg / mL, and 23 mg / mL of the bispecific antibody constructs of this disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80 at pH 4.2 was lyophilized using an annealing step, and then redissolved. The protein formulation was subjected to SEC-UHPLC after 1 month of storage at 40°C, and the degree of aggregation in the formulation was determined by the percentage of high molecular weight species (%HMW). See Example 4. As shown in Figure 3, no increase in %HMW occurred under accelerated stress conditions, demonstrating that the lyophilization method disclosed herein can stabilize formulations containing both low- and high-concentration proteins, such as bispecific antibody constructs.

[0084] Surprisingly, the lyophilization method of this disclosure, which lacks the annealing step, was found to provide superior stability of the protein formulation compared to the lyophilization method that includes the annealing step. For example, a protein formulation containing 15 mg / mL, 20 mg / mL, or 23 mg / mL of the bispecific antibody construct of this disclosure, 10 mM L-glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80 at pH 4.2 was lyophilized with and without the annealing step. The protein formulations were then redissolved and subjected to SE-UHPLC to determine the amount of aggregation in each sample. See Example 5. As shown in Tables 1, 2, Figures 4, and 5, the annealed sample showed significantly more aggregation than the unannealed sample or control sample, as evidenced by the high %HMW.

[0085] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0086] General Procedure Reducing capillary electrophoresis with sodium decyl sulfate (rCE-SDS) separates proteins based on their hydrodynamic size differences under reducing and denaturing conditions. The reducing capillary electrophoresis-sodium decyl sulfate (rCE-SDS) method involves binding protein species to SDS, an anionic detergent, and electrodynamically injecting them into a bare fused silica capillary filled with SDS gel buffer. A voltage is applied across the capillary, under which the SDS-coated proteins are separated by their differences in migration in a hydrophilic polymer-based solution. The proteins are detected by a photodiode array (PDA) detector as they pass through a UV detection window. Purity is assessed by determining the precise peak area percentage of each component. The rCE-SDS method separates heavy chains (HC), light chains (LC), non-glycosylated HC (NGHC), and other fine peak species and groups under reducing conditions. Reduced capillary sodium decyl sulfate gel electrophoresis (rCE-SDS) was performed by incubating the sample in an SDS-MW reduced gel at 79°C for 10 minutes. After incubation, the sample was centrifuged and then electrodynamically injected into a bare 67 cm fused silica capillary with an inner diameter of 50 μm. The effective length of the capillary was 30.2 cm. Separation was performed using a CE-SDS gel (Beckman Coulter, Brea, Calif.) and an effective voltage of 30 kV. Detection was performed at 220 nm by UV absorbance.

[0087] Size exclusion ultrahigh-performance liquid chromatography (SE-UHPLC). SE-UHPLC separates proteins based on their hydrodynamic volume differences. Molecules with higher hydrodynamic volumes elute before molecules with smaller volumes. The sample was loaded onto an SE-UHPLC column (BEH200, 4.6 × 300 mm, (Waters Corporation, 186005226)), separated isocratically, and the eluate was monitored by UV absorbance. Purity was determined by calculating the percentage value of each separated component compared to the total integrated area. The SE-UHPLC settings were as follows: flow rate: 0.4 ml / min, run time: 12 min, UV detection: 280 nm, column temperature: ambient temperature, target protein load: 6 μg, protein-compatible flow cell: 5 mm.

[0088] Protein preparation. The protein preparation was prepared at pH 4.2, containing intact bispecific antibody constructs at concentrations of 1 mg / mL, 5 mg / mL, 13 mg / mL, or 23 mg / mL, 10 mM L-glutamate, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80. This protein preparation was introduced into lyophilized vials (with or without annealing).

[0089] Example 1 Annealing process none Lyophilization of bispecific antibody construct formulations The liquid protein preparation was prepared as described above and introduced into a lyophilization chamber. The chamber was cooled from ambient temperature to -45°C at a rate of 0.5°C / min and held at -45°C for 2 hours. The pressure in the lyophilization chamber was reduced from ambient pressure to 70 mTorr, and the chamber was heated to -25°C at a rate of 0.33°C / min and held at 70 mTorr pressure and -25°C for 52 hours. Subsequently, the chamber was heated to 30°C at a rate of 0.1°C / min and held at 70 mTorr and 30°C for 8 hours. To enable vial capping, the chamber temperature was reduced to 5°C and nitrogen was supplied to the chamber at 500 mTorr. The vials containing the lyophilized protein preparation were removed from the lyophilization chamber and stored at 2-8°C until further processing and analysis.

[0090] The lyophilized protein preparation was redissolved in sterile water for injection.

[0091] Example 2 Annealing process Yes Lyophilization of bispecific antibody construct formulations The liquid protein preparation was prepared as described above and introduced into a lyophilization chamber. The chamber was cooled from ambient temperature to -45°C at a rate of 0.5°C / min and held at -45°C for 2 hours. The chamber temperature was gradually increased to -12°C at a rate of 0.5°C / min and held at -12°C for 5 hours. Then, the chamber was cooled back down to -45°C at a rate of 0.5°C / min and held at -45°C for 2 hours. The chamber pressure was reduced from ambient pressure to 70 mTorr, and the chamber was heated to -25°C at a rate of 0.33°C / min and held at 70 mTorr pressure and -25°C for 52 hours. Then, the chamber was heated to 30°C at a rate of 0.1°C / min and held at 70 mTorr and 30°C for 8 hours. To enable vial capping, the chamber temperature was reduced to 5°C, and nitrogen was supplied to the lyophilization chamber at 500 mTorr. Vials containing the lyophilized protein preparation were removed from the lyophilization chamber and stored at 2–8°C until further processing and analysis.

[0092] The lyophilized protein preparation was redissolved in sterile water for injection.

[0093] Example 3 Comparison of the stability of liquid and lyophilized formulations of bispecific antibody constructs using rCE-SDS. Liquid formulations containing 1 mg / ml of BiTE A, BiTE B, BiTE C, or BiTE D, and lyophilized formulations (prepared with an annealing step), were prepared as described above. The liquid formulations with a protein concentration of 1 mg / mL and the redissolved lyophilized formulations (redissolved in sterile water for injection) were subjected to reduced capillary decyl sulfate sodium gel electrophoresis (rCE-SDS) to determine the degree of clipping that occurred after 1 month of storage at 25°C. As shown in Figure 1, all redissolved lyophilized formulations showed significantly less clipping than the liquid formulations, as evidenced by the lower percentage values ​​(LMS%) for low molecular weight species, indicating that the redissolved lyophilized formulations have superior stability compared to the liquid formulations. Furthermore, the liquid formulations containing 1 mg / ml of BiTE B and the redissolved lyophilized formulations were subjected to rCE-SDS to determine the degree of clipping that occurred after 1 month of storage at 40°C. As shown in Figure 2, the redissolved lyophilized formulation exhibited significantly less clipping than the liquid formulation, as evidenced by the lower LMS%. This indicates that the lyophilized formulation (prepared using the annealing process) has superior stability to the liquid formulation, even at higher temperatures.

[0094] Example 4 The effect of concentration on the stability of lyophilized formulations using SE-UHPLC. Lyophilized formulations containing various concentrations of BiTE B (1 mg / mL, 5 mg / mL, 13 mg / mL, and 23 mg / mL) (prepared with an annealing step) were redissolved after one month of storage at 40°C and subjected to SEC-UHPLC to determine the degree of formulation aggregation. This was determined by the percentage value of high molecular weight species (%HMW). As shown in Figure 3, no increase in %HMW was observed under accelerated stress conditions, demonstrating that the lyophilization cycle can stabilize formulations with both low and high concentrations of the bispecific antibody construct.

[0095] Example 5 Comparison of the stability of lyophilized formulations with and without annealing of bispecific antibody constructs using SE-UHPLC. Annealing of a lyophilized preparation containing 23 mg / mL of BiTE B after storage at freezing temperature. I haven't done it The sample and annealing (prepared according to Example 1) did The amount of aggregation in the samples (prepared according to Example 2) was determined using SE-UHPLC. The annealed samples were stored as follows: 48 hours at 45°C, 5 hours at -12°C, 5 hours at -45°C, and 48 hours at -25°C. The unannealed samples were stored at -46°C for 58 hours, followed by 48 hours at -25°C. As shown in Figure 4, the annealed samples showed significantly more aggregation after redissolution, as evidenced by higher %HMW, compared to the unannealed samples after redissolution, or a control sample containing 10 mM glutamic acid, 9% (w / v) sucrose, and 0.01% (w / v) polysorbate 80 at a dry temperature of -10°C. See Figure 5.

[0096] Annealing of a lyophilized preparation containing 15 mg / ml of BiTE B I haven't done it The sample and annealing (prepared according to Example 1) didThe amount of aggregation in the samples (prepared according to Example 2) was determined using SE-UHPLC before and after freeze-drying. As shown in Table 1 below, the formulations that underwent annealing during the freeze-drying process showed significantly more aggregation after redissolution compared to the samples that did not undergo annealing during the freeze-drying process, as evidenced by the higher %HMW.

[0097] [Table 1]

[0098] Annealing of a lyophilized formulation containing 20 mg / ml of BiTE A, BiTE C, or BiTE E (a bispecific antibody construct having the sequence shown in SEQ ID NO: 122). I haven't done it The sample and annealing (prepared according to Example 1) did The amount of aggregation in the samples (prepared according to Example 2) was determined using SE-UHPLC before and after freeze-drying. As shown in Table 2 below, the formulations that underwent annealing during the redissolved freeze-drying process showed significantly more aggregation, as evidenced by higher %HMW, compared to the samples that did not undergo annealing during the redissolved freeze-drying process.

[0099] [Table 2]

[0100] The above explanation is provided solely for the sake of clarity and should not be interpreted as implying any unnecessary limitations, for modifications that fall within the scope of the present invention would be obvious to those skilled in the art.

[0101] Throughout this specification and the subsequent claims, unless the context requires otherwise, it will be understood that the words “comprise,” and variations such as “comprises,” and “comprising,” mean the inclusion of a specified integer or process, or group of integers or processes, but not the exclusion of other integers or processes, or groups of integers or processes.

[0102] When specifying a range of values, it should be understood that the described characteristics may be individual values ​​found within that range. For example, "pH approximately 4 to approximately 6" is not limited to pH 4, 4.2, 4.6, 5.1, 5.5, etc., and any value between such values. Furthermore, "pH approximately 4 to approximately 6" should not be interpreted as meaning that the pH of the target formulation will fluctuate in 2 pH increments within the pH 4 to pH 6 range during storage, but rather that a value within that range may be selected for the pH of the solution, and the pH will remain buffered around that pH.

[0103] When the term "approximately" is used, it means adding or subtracting 5%, 10%, 15%, or more of the listed number from the listed number. The actual intended variation can be determined from the context.

[0104] Throughout this specification, where a composition is described as comprising components or materials, unless otherwise stated, the composition is also intended to consist essentially of or be composed of any combination of the listed components or materials. Similarly, where a method is described as comprising specific steps, unless otherwise stated, the method is also intended to consist essentially of or be composed of any combination of the listed steps. Inventions disclosed exemplary herein can also be suitably carried out even without elements or steps not specifically disclosed herein.

[0105] The methods disclosed herein and their individual steps can be carried out manually and / or with the help of automation provided by electronic devices. Although the processes have been described in light of specific embodiments, those skilled in the art will readily understand that other means may be used to carry out the actions relating to the Method. For example, the order of the various steps may be changed without departing from the scope or spirit of the Method unless otherwise stated. Also, some of the individual steps may be combined, omitted, or further subdivided into additional steps.

[0106] All patents, publications, and references described herein are incorporated herein by reference in their entirety. In the event of any conflict between this disclosure and the patents, publications, and references incorporated herein, this disclosure shall prevail.

Claims

1. A method for preparing a lyophilized preparation, (a) A step of preparing a frozen formulation by cooling a lyophilization chamber containing a liquid formulation comprising a bispecific antibody construct, a sugar containing a monosaccharide or disaccharide, and a surfactant to a temperature in the range of approximately -35°C to approximately -50°C, and holding the chamber at a temperature in the range of approximately -40°C to approximately -50°C for a period of approximately 2 hours to approximately 24 hours, wherein the surfactant comprises polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, Triton X-100, or a combination thereof; (b) Heating the chamber to a temperature in the range of approximately -30°C to approximately -20°C and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr to produce a primary dried formulation, and holding the chamber at a temperature in the range of approximately -30°C to approximately -20°C and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 45 hours to approximately 60 hours; and (c) A step of producing a lyophilized formulation by heating the chamber to a temperature in the range of approximately 20°C to approximately 35°C, and maintaining the chamber at a temperature in the range of approximately 20°C to approximately 30°C and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr for a period of approximately 5 hours to approximately 10 hours. The liquid formulation contains, has a pH of approximately 3 to 7, and does not contain mannitol; and The above method is a method that lacks an annealing step.

2. The method according to claim 1, wherein the cooling in step (a) is performed to a temperature of approximately -45°C.

3. The method according to claim 1 or 2, wherein the cooling in step (a) is carried out at a rate in the range of about 0.5°C / min to about 1°C / min.

4. The method according to claim 3, wherein the cooling in step (a) is carried out at a rate of about 0.5°C / min.

5. The method according to any one of claims 1 to 4, wherein the holding in step (a) is performed at a temperature of about -45°C.

6. The method according to any one of claims 1 to 5, wherein the holding in step (a) is performed for a period of time of about 2 hours to about 5 hours.

7. The method according to claim 6, wherein the holding in step (a) is performed for about two hours.

8. The method according to any one of claims 1 to 7, wherein the heating in step (b) is carried out to a temperature of about -25°C.

9. The method according to any one of claims 1 to 8, wherein the heating in step (b) is carried out at a rate in the range of about 0.1°C / min to about 1°C / min.

10. The method according to claim 9, wherein the heating in step (b) is carried out at a rate in the range of about 0.1°C / min to about 0.5°C / min.

11. The method according to claim 10, wherein the heating in step (b) is carried out at a rate of about 0.3°C / min.

12. The method according to any one of claims 1 to 11, wherein the heating in step (b) is carried out at a pressure in the range of about 70 mTorr to about 100 mTorr.

13. The method according to claim 12, wherein the heating in step (b) is carried out at a pressure of about 70 mTorr.

14. The method according to any one of claims 1 to 13, wherein the holding in step (b) is performed at a temperature of about -25°C.

15. The method according to any one of claims 1 to 14, wherein the holding in step (b) is performed at a pressure in the range of about 70 mTorr to about 100 mTorr.

16. The method according to claim 15, wherein the holding in step (b) is performed at a pressure of about 70 mTorr.

17. The method according to any one of claims 1 to 16, wherein the holding in step (b) is performed for a period of time of about 50 hours to about 55 hours.

18. The method according to claim 17, wherein the holding in step (b) is performed for approximately 52 hours.

19. The method according to any one of claims 1 to 18, wherein the heating in step (c) is carried out to a temperature of about 30°C.

20. The method according to any one of claims 1 to 19, wherein the heating in step (c) is carried out at a rate up to about 0.5°C / min.

21. The method according to claim 20, wherein the heating in step (c) is carried out at a rate in the range of about 0.05°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 0.1°C / min.

23. The method according to any one of claims 1 to 22, wherein the holding in step (c) is performed at a temperature of about 30°C.

24. The method according to any one of claims 1 to 23, wherein the holding in step (c) is performed at a pressure in the range of about 70 mTorr to about 100 mTorr.

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

26. The method according to any one of claims 1 to 25, wherein the holding in step (c) is performed for about 8 hours.

27. The method according to claim 1, wherein the bispecific antibody construct is a half-life extension (HLE) bispecific antibody construct.

28. The method according to claim 27, wherein the HLE bispecific antibody construct includes the amino acid sequence shown 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: 55, 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.

29. The method according to claim 28, wherein the HLE bispecific antibody construct includes the amino acid sequence shown in SEQ ID NO: 22, SEQ ID NO: 77, SEQ ID NO: 87, or SEQ ID NO:

97.

30. The method according to any one of claims 1 to 29, wherein the bispecific antibody construct is present in the liquid formulation at a concentration in the range of about 0.1 mg / mL to about 100 mg / mL.

31. The method according to claim 32, wherein the bispecific antibody construct is present at a concentration in the range of about 0.1 mg / mL to about 70 mg / mL.

32. The method according to claim 31, wherein the bispecific antibody construct is present at a concentration in the range of about 0.5 mg / mL to about 30 mg / mL.

33. The method according to claim 32, wherein the bispecific antibody construct is present at a concentration in the range of about 1 mg / ml to about 20 mg / mL.

34. The method according to claim 33, wherein the bispecific antibody construct is present at a concentration of about 1 mg / mL.

35. The method according to any one of claims 1 to 34, wherein the liquid formulation in step (a) has a pH of about 4 to 6.

36. The method according to any one of claims 1 to 35, wherein the liquid formulation in step (a) further comprises a buffering agent.

37. The method according to claim 36, wherein the buffer is an acetate buffer, a glutamate buffer, a citrate buffer, a lactic acid buffer, a succinate buffer, a tartaric acid buffer, a fumarate buffer, a maleate buffer, a histidine buffer, a phosphate buffer, a 2-(N-morpholino)ethanesulfonic acid buffer, or any combination thereof.

38. The method according to claim 37, wherein the buffering agent comprises glutamic acid.

39. The method according to any one of claims 36 to 38, wherein the buffer is present in a concentration in the range of about 5 mM to about 200 mM.

40. The method according to claim 39, wherein the buffering agent is present in a concentration in the range of about 10 mM to about 50 mM.

41. The method according to claim 40, wherein the buffering agent is present at a concentration of approximately 10 mM.

42. The method according to any one of claims 1 to 41, wherein the sugars are glucose, galactose, fructose, xylose, sucrose, lactose, maltose, trehalose, or any combination thereof.

43. The method according to claim 42, wherein the sugar is sucrose.

44. The method according to any one of claims 1 to 43, wherein the sugars are present in the liquid formulation at a concentration in the range of about 1 to about 15% (w / v).

45. The method according to claim 44, wherein the sugars are present at a concentration in the range of about 6% to 12% (w / v).

46. The method according to claim 45, wherein the sugars are present at a concentration of about 9% (w / v).

47. The method according to any one of claims 1 to 46, wherein the surfactant is polysorbate 80.

48. The method according to any one of claims 1 to 47, wherein the surfactant is present in the liquid formulation at a concentration in the range of about 0.001% to 0.5% (w / v).

49. The method according to claim 48, wherein the surfactant is present in a concentration in the range of about 0.001% to 0.01% (w / v).

50. The method according to claim 49, wherein the surfactant is present at a concentration of about 0.01% (w / v).

51. The method according to any one of claims 1 to 50, wherein the liquid formulation in step (a) has a pH of about 4 to about 5.

52. The method according to any one of claims 1 to 51, wherein the liquid formulation of step (a) has a pH of about 4.2 and comprises about 10 mM L-glutamic acid, about 9.0% (w / v) sucrose, and about 0.010% (w / v) polysorbate 80.

53. The method according to any one of claims 1 to 52, wherein the freeze-dried formulation shows an increase of 0.5% or less in the percentage value of high molecular weight species after redissolution and storage at 40°C for one month.

54. The method according to claim 53, wherein the freeze-dried formulation shows an increase of 0.3% or less in the percentage value of the high molecular weight species after being redissolved and stored at 40°C for one month.

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