Compositions and methods for minimizing protein loss at low protein concentrations

Abstract

To provide compositions and methods for minimizing protein loss at low protein concentrations.SOLUTION: The present invention relates to the use of low protein concentrations (e.g. by adsorption to a solid surface) To provide compositions and methods for minimizing protein loss. The invention disclosed herein relates generally to the field of compositions comprising proteins, in particular pharmaceutical compositions comprising therapeutic proteins at low protein concentrations. The inventions disclosed herein also relate to methods of administering a composition to a subject in need thereof. The invention disclosed herein is based on the surprising discovery that surfactants can stabilize low concentrations of proteins in liquid compositions and effectively prevent protein loss due to adsorption to solid surfaces, when the surfactant is used at a concentration below its critical micelle concentration.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 926,089, filed October 25, 2019, which is incorporated herein by reference in its entirety.

[0002] Submission of sequence listings recorded in ASCII text files The contents of the following submission, an ASCII text file, are incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: A-2429-WO-PCT_ST25, Data Creation Date: October 23, 2020, Size: 451,608 bytes).

[0003] The invention disclosed herein generally relates to the field of compositions comprising proteins, particularly pharmaceutical compositions comprising therapeutic proteins at low protein concentrations. The invention disclosed herein also relates to methods of administering the compositions to a subject in need thereof. [Background technology]

[0004] Therapeutic proteins are an important type of therapeutic agent for treating patients. Protein molecules are surface-active and potentially adsorb to solid surfaces. Therapeutic proteins in pharmaceutical compositions may be adsorbed to solid surfaces with which they come into contact (e.g., the surface of a container containing the pharmaceutical composition), which may result in protein loss during storage and use. Generally, the concentration of therapeutic proteins in these compositions is high (e.g., 1 mg / mL or higher), so that protein adsorption to solid surfaces does not result in an insufficient amount of drug available for administration to patients. However, when the concentration of protein in a composition is low (e.g., less than 0.1 mg / mL, such as when the composition is diluted before administration to a patient), the risk of protein loss may become more pronounced, which may potentially lead to an insufficient amount of drug available for administration to a patient.

[0005] Surfactants are commonly used in pharmaceutical compositions containing therapeutic proteins, for example, to prevent protein aggregation and stabilize the protein. When the protein is present in a pharmaceutical composition at a low concentration (e.g., 0.1 mg / mL or less), it is unclear whether surfactants can be used to effectively prevent protein loss due to surface adsorption.

[0006] There is a need for protein compositions and methods that minimize protein loss due to adsorption to solid surfaces, especially when the composition contains a protein at a low protein concentration. Summary of the Invention [Means for solving the problem]

[0007] Disclosed herein are compositions comprising proteins at low protein concentrations, as well as methods for administering the compositions to subjects in need thereof. The compositions and methods disclosed herein have the advantage of minimizing or eliminating protein loss due to protein adsorption to solid surfaces, ensuring accurate administration of therapeutic proteins to patients.

[0008] Disclosed herein, in certain embodiments, is an aqueous composition comprising a protein and a surfactant, wherein the protein is present in the composition at a concentration of about 0.001 μg / mL to about 100 μg / mL, and the surfactant is present in the composition at a concentration of at least about 0.25 times the critical micelle concentration (CMC) of the surfactant.

[0009] In certain embodiments, the protein 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 a T cell, and optionally a third domain comprising, in amino to carboxyl order, hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain. In certain embodiments, the second binding domain comprises a polypeptide having the sequence of SEQ ID NO: 201. In certain embodiments, the bispecific antibody construct is present at a concentration of about 0.001 μg / mL to about 50 μg / mL, or about 0.01 μg / mL to about 50 μg / mL, or 0.1 μg / mL to about 50 μg / mL, or 0.1 μg / mL to about 10 μg / mL, or 1 μg / mL to about 10 μg / mL.

[0010] In certain embodiments, the surfactant is polysorbate, poloxamer, or Triton X-100. In certain embodiments, the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or Triton X-100. In certain embodiments, the surfactant is poloxamer 188 or poloxamer 407. In certain embodiments, the surfactant is present at a concentration of about 0.25 to about 20 times the CMC of the surfactant, or about 0.25 to about 10 times the CMC.

[0011] In certain embodiments, the composition further comprises a salt, an amino acid, a sugar or a sugar derivative, or a combination thereof. In certain embodiments, the salt is NaCl. In certain embodiments, the sugar or sugar derivative is a monosaccharide, a disaccharide, a cyclic polysaccharide, or a sugar alcohol. In certain embodiments, the sugar is sucrose, trehalose, mannitol, or sorbitol. In certain embodiments, the amino acid is lysine.

[0012] In certain embodiments, the pH of the composition is from about 3.5 to about 7.5, and in certain embodiments, the pH of the composition is from about 4.2 to about 7.0.

[0013] In certain embodiments, the composition further comprises a buffer or preservative, hi certain embodiments, the buffer is an acetate buffer, a glutamate buffer, a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a maleate buffer, a histidine buffer, or a phosphate buffer.

[0014] In certain embodiments, each of the first and second binding domains of the bispecific antibody construct comprises a VH region and a VL region. In certain embodiments, the bispecific antibody construct is a single-chain antibody construct. In certain embodiments, the bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:176, SEQ ID NO:178, and SEQ ID NO:192. In certain embodiments, the bispecific antibody construct comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192.

[0015] In certain embodiments, the composition is contained in a plastic container such as an IV bag or IV tubing. In certain embodiments, the container is made of a material including polyolefin, polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), or polyurethane. In certain embodiments, the container is made of a material including PVC, wherein the PVC is substantially free of di-2-ethylhexyl phthalate (DEHP) or tri-2-ethylhexyl trimellitate (TOTM).

[0016] Disclosed herein in certain embodiments is a pharmaceutical formulation comprising an aqueous pharmaceutical composition housed in a container, the aqueous pharmaceutical composition comprising: a bispecific antibody construct at a concentration of about 0.001 μg / mL to about 100 μg / mL; and a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant, wherein the surfactant has an HLB value of at least 20. In certain embodiments, the surfactant is poloxamer 188 or poloxamer 407. In certain embodiments, the aqueous pharmaceutical composition comprises the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 50 μg / mL. In certain embodiments, the aqueous pharmaceutical composition comprises the surfactant at a concentration of about 0.25 to about 20 times the CMC of the surfactant, or about 0.25 to about 10 times the CMC.

[0017] In certain embodiments, the aqueous pharmaceutical composition further comprises a salt, a buffering agent, an amino acid, a sugar or sugar derivative, or a combination thereof. In certain embodiments, the aqueous pharmaceutical composition has a pH of about 4.2 to about 7.0.

[0018] In certain embodiments, the container is made from a material including polyolefin, PVC, EVA, or polyurethane (eg, polyester and polyether).

[0019] In certain embodiments, a bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 192. In certain embodiments, a bispecific antibody construct comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192.

[0020] Also disclosed herein is a method of administering a bispecific antibody construct to a patient, the method comprising: preparing an aqueous pharmaceutical composition in a container, the aqueous pharmaceutical composition comprising: the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 100 μg / mL; and a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant; and administering the aqueous pharmaceutical composition to the patient, wherein the bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:176, SEQ ID NO:178, and SEQ ID NO:192. In certain embodiments, the bispecific antibody construct comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192. In certain embodiments, the aqueous pharmaceutical composition comprises the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 50 μg / mL.

[0021] In certain embodiments, the aqueous pharmaceutical composition comprises a surfactant at a concentration of about 0.25 to about 20 times the CMC of the surfactant, or about 0.25 to about 10 times the CMC. In certain embodiments, the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, poloxamer 188, poloxamer 407, or Triton X-100.

[0022] In certain embodiments, the aqueous pharmaceutical composition further comprises one or more selected from salts, buffering agents, amino acids, sugars or sugar derivatives, and preservatives, hi certain embodiments, the aqueous pharmaceutical composition has a pH of about 4.2 to about 7.0.

[0023] In certain embodiments, the container is made from a material including polyolefin, PVC, EVA, polyurethane, hi certain embodiments, the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20, and the container is made from a material including PVC that is substantially free of DEHP or TOTM.

[0024] In certain embodiments, the aqueous pharmaceutical composition is prepared by diluting a first composition comprising a bispecific antibody construct with a suitable aqueous solution. In certain embodiments, the first composition is a liquid composition comprising a bispecific antibody construct. In certain embodiments, the first composition is a liquid composition reconstituted from a lyophilized composition comprising a bispecific antibody construct. In certain embodiments, the suitable solution comprises a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant. In certain embodiments, the aqueous pharmaceutical composition is prepared by adding a suitable aqueous solution to a container, followed by adding an appropriate amount of the first composition to the container.

[0025] In certain embodiments, the patient is a cancer patient. In certain embodiments, the administration is IV administration. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 1. An aqueous composition comprising a bispecific antibody construct at a concentration of about 0.001 μg / mL to about 100 μg / mL and a detergent at a concentration of at least about 0.25 times the critical micelle concentration (CMC) of the detergent, wherein the bispecific antibody construct comprises a first binding domain that binds to a target cell surface antigen, a second binding domain that binds to human CD3 on the surface of a T cell, and optionally a third domain comprising, in amino to carboxyl order, hinge-CH2 domain-CH3 domain-linker-hinge-CH2 domain-CH3 domain, wherein the second binding domain comprises a polypeptide having the sequence of SEQ ID NO: 201. (Item 2) 2. The composition of claim 1, wherein the bispecific antibody construct is present at a concentration of about 0.001 μg / mL to about 50 μg / mL. (Item 3) 3. The composition according to item 1 or 2, wherein the bispecific antibody construct is present at a concentration of from about 0.01 μg / mL to about 50 μg / mL, or from 0.1 μg / mL to about 50 μg / mL, or from 0.1 μg / mL to about 10 μg / mL, or from 1 μg / mL to about 10 μg / mL. (Item 4) 4. The composition according to any one of items 1 to 3, wherein the surfactant is polysorbate, poloxamer, or Triton X-100. (Item 5) 5. The composition according to any one of items 1 to 4, wherein the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or Triton X-100. (Item 6) 5. The composition according to any one of items 1 to 4, wherein the surfactant is poloxamer 188 or poloxamer 407. (Item 7) The surfactant is at a concentration of about 0.25 to about 20 times the CMC of the surfactant, or 7. The composition according to any one of items 1 to 6, wherein the composition is present in a concentration of about 0.25 to about 10 times. (Item 8) 8. The composition according to any one of items 1 to 7, wherein the composition further comprises a salt, an amino acid, a sugar or a sugar derivative, or a combination thereof. (Item 9) 9. The composition according to item 8, wherein the composition further comprises a buffer or a preservative. (Item 10) 10. The composition according to item 8 or 9, wherein the pH of the composition is about 3.5 to about 7.5. (Item 11) Item 11. The composition according to item 10, wherein the pH of the composition is about 4.2 to about 7.0. (Item 12) 12. The composition according to any one of items 8 to 11, wherein the salt is NaCl. (Item 13) 13. The composition according to any one of items 8 to 12, wherein the saccharide or saccharide derivative is a monosaccharide, a disaccharide, a cyclic polysaccharide, or a sugar alcohol. (Item 14) 14. The composition according to any one of items 8 to 13, wherein the sugar is sucrose, trehalose, mannitol, or sorbitol. (Item 15) 15. The composition according to any one of items 8 to 14, wherein the amino acid is lysine. (Item 16) 16. The composition according to any one of items 9 to 15, wherein the buffer is an acetate buffer, a glutamate buffer, a citrate buffer, a succinate buffer, a tartrate buffer, a fumarate buffer, a maleate buffer, a histidine buffer, or a phosphate buffer. (Item 17) 17. The composition of any one of items 1 to 16, wherein each of the first and second binding domains of the bispecific antibody construct comprises a VH region and a VL region. (Item 18) 18. The composition of any one of items 1 to 17, wherein the bispecific antibody construct is a single-chain antibody construct. (Item 19) 19. The composition of any one of Items 1 to 18, wherein the bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 192. (Item 20) 20. The composition according to any one of items 1 to 19, wherein the composition is a pharmaceutical composition. (Item 21) 21. The composition according to any one of items 1 to 20, wherein the composition is contained in a plastic container. (Item 22) 22. The composition according to item 21, wherein the container is made from a material comprising polyolefin, PVC, EVA, or polyurethane. (Item 23) 23. The composition of claim 22, wherein the container is made from a material comprising PVC, wherein the PVC is substantially free of DEHP or TOTM. (Item 24) 24. The method according to any one of items 21 to 23, wherein the container is an IV bag or an IV tube. The composition described above. (Item 25) 1. A pharmaceutical formulation comprising an aqueous pharmaceutical composition contained in a container, the aqueous pharmaceutical composition comprising: a) a bispecific antibody construct at a concentration of 0.001 μg / mL to about 100 μg / mL; b) a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant; wherein the surfactant has an HLB value of at least 20. (Item 26) 26. The pharmaceutical formulation of item 25, wherein the aqueous pharmaceutical composition comprises the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 50 μg / mL. (Item 27) 27. The pharmaceutical formulation according to item 25 or 26, wherein the aqueous pharmaceutical composition comprises the surfactant at a concentration of about 0.25 to about 20 times the CMC of the surfactant, or about 0.25 to about 10 times the CMC. (Item 28) 28. The pharmaceutical formulation according to any one of items 25 to 27, wherein the aqueous pharmaceutical composition further comprises a salt, a buffer, an amino acid, a sugar or a sugar derivative, or a combination thereof. (Item 29) 29. The pharmaceutical formulation according to any one of items 25 to 28, wherein the aqueous pharmaceutical composition has a pH of about 4.2 to about 7.0. (Item 30) 30. The pharmaceutical formulation according to any one of items 25 to 29, wherein the container is made from a material comprising polyolefin, PVC, EVA, or polyurethane. (Item 31) 31. The pharmaceutical formulation according to any one of Aspects 25 to 30, wherein the bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 176, SEQ ID NO: 178, and SEQ ID NO: 192. (Item 32) 1. A method of administering a bispecific antibody construct to a patient, comprising: a) preparing an aqueous pharmaceutical composition in a container, said aqueous pharmaceutical composition comprising the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 100 μg / mL and the surfactant at a concentration of at least about 0.25 times the CMC of the surfactant; b) administering the aqueous pharmaceutical composition to the patient; 10. A method wherein the bispecific antibody construct comprises a polypeptide having an amino acid sequence selected from SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:176, SEQ ID NO:178, and SEQ ID NO:192. (Item 33) 33. The method of claim 32, wherein the aqueous pharmaceutical composition comprises the bispecific antibody construct at a concentration of about 0.001 μg / mL to about 50 μg / mL. (Item 34) Item 34. The aqueous pharmaceutical composition according to Item 32 or 33, wherein the surfactant is contained in a concentration of about 0.25 to about 20 times the CMC of the surfactant, or about 0.25 to about 10 times the CMC. method. (Item 35) 35. The method according to any one of Items 32 to 34, wherein the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, poloxamer 188, poloxamer 407, or Triton X-100. (Item 36) 36. The method according to any one of items 32 to 35, wherein the aqueous pharmaceutical composition further comprises one or more selected from salts, buffering agents, amino acids, sugars, and preservatives. (Item 37) 37. The method according to any one of items 32 to 36, wherein the aqueous pharmaceutical composition has a pH of about 4.2 to about 7.0. (Item 38) 38. The method according to any one of items 32 to 37, wherein the container is made from a material comprising polyolefin, PVC, EVA, or polyurethane. (Item 39) 39. The method of any one of items 32 to 38, wherein the surfactant is polysorbate 80, polysorbate 60, polysorbate 40, or polysorbate 20, and the container is made from a material comprising PVC that is substantially free of DEHP or TOTM. (Item 40) 40. The method of any one of items 32 to 39, wherein the aqueous pharmaceutical composition is prepared by diluting a first composition comprising the bispecific antibody construct with a suitable aqueous solution. (Item 41) 41. The method of claim 40, wherein the first composition is a liquid composition comprising the bispecific antibody construct. (Item 42) 41. The method of claim 40, wherein the first composition is a liquid composition reconstituted from a lyophilized composition comprising the bispecific antibody construct. (Item 43) 43. The method of any one of items 41 to 42, wherein the suitable solution comprises the surfactant at a concentration of at least about 0.25 times the CMC of the surfactant. (Item 44) 44. The method according to any one of items 40 to 43, wherein the aqueous pharmaceutical composition is prepared by adding the suitable aqueous solution to the container, and subsequently adding an appropriate amount of the first composition to the container. (Item 45) 45. The method according to any one of items 32 to 44, wherein the patient is a cancer patient. (Item 46) 46. ​​The method of any one of items 32 to 45, wherein the administration is IV administration. (Item 47) 32. The pharmaceutical formulation according to any one of items 25 to 31, wherein the surfactant is poloxamer 188 or poloxamer 407. (Item 48) 33. The composition of claim 19, the pharmaceutical formulation of claim 31, or the method of claim 32, wherein the bispecific antibody construct comprises a polypeptide having the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 shows a schematic of an assay for measuring protein binding to a solid surface. [Figure 2] FIG. 2 shows proteins binding to a solid surface in the absence of detergent. [Figure 3] FIG. 3 shows that the addition of different surfactants prevents protein binding to solid surfaces. [Figure 4] FIG. 4 shows that adding a surfactant to the solid surface before adding the protein effectively prevents the protein from binding to the surface. [Figure 5] FIG. 5 shows the effect of various surfactants at the same concentration on the leaching of di-2-ethylhexyl phthalate (DEHP) from DEHP-containing PVC. [Figure 6]FIG. 6 shows the effect of various surfactants at the same magnification of CMC on the leaching of DEHP from DEHP-containing PVC. DETAILED DESCRIPTION OF THE INVENTION

[0027] The invention disclosed herein is based on the surprising discovery that surfactants can stabilize low concentrations of proteins in liquid compositions and effectively prevent protein loss due to adsorption to solid surfaces when the surfactants are used at concentrations below their critical micelle concentration.

[0028] Disclosed herein, in some embodiments, is an aqueous composition comprising a protein and a surfactant, wherein the protein is present in the composition at a concentration of about 0.001 μg / mL to about 100 μg / mL, and the surfactant is present in the composition at a concentration of at least about 0.25 times the critical micelle concentration (CMC) of the surfactant.

[0029] The surfactant that can be used in the composition can be any surfactant typically used in pharmaceutical compositions. In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is a polysorbate, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, or polysorbate 85. In some embodiments, the surfactant is polysorbate 20, and in other embodiments, the surfactant is polysorbate 80. In some embodiments, the surfactant is a poloxamer, such as poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407. In some embodiments, the surfactant is Triton X-100. Various surfactants are commercially available (e.g., Tween® 20, Tween® 80, Pluronic F68, and Pluronic F127).

[0030] The surfactant may be present in the composition at a concentration of at least about 0.25 times (0.25x) the critical micelle concentration (CMC) of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.25 to about 20 times the CMC of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.25 to about 15 times, or about 0.25 to about 10 times, or about 0.25 to about 8 times, or about 0.25 to about 6 times, or about 0.25 to about 4 times, or about 0.25 to about 2 times, or about 0.25 to about 1 times the CMC of the surfactant.

[0031] In some embodiments, the surfactant is present in the composition at a concentration of about 0.5 to about 20 times the CMC of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.5 to about 15 times, or about 0.5 to about 10 times, or about 0.5 to about 8 times, or about 0.5 to about 6 times, or about 0.5 to about 4 times, or about 0.5 to about 2 times, or about 0.5 to about 1 times the CMC of the surfactant.

[0032] In some embodiments, the surfactant is present in the composition at a concentration of about 0.25, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 14, about 16, about 18, or about 20 times the CMC of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 1.25, about 2.5, about 3.5, about 4.5, about 5.5, about 6.5, about 7.5, about 8.5, or about 9.5 times the CMC of the surfactant.

[0033] As used herein, the term "about," when used to modify a particular value or range, is understood to mean that there may be a variation in the given value or range within 20 percent, including, for example, within 10 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the stated value or range.

[0034] The critical micelle concentration refers to the concentration of a surfactant above which micelles are formed, and is a characteristic of the surfactant. The CMC value of a surfactant can be measured by experimental methods well known in the art, such as fluorometry, surface tension, conductivity measurement, and dynamic light scattering. See, for example, Norman Scholz, Thomas Behnke, Ute ReschGenger. Journal of Fluorescence 28:465-476 (2018) and Oender Topel, Burcin Acar Cakir, Leyla Budama, Numan Hoda. Journal of Molecular Liquids 177;40-43 (2013). The CMC value of a surfactant can also be measured automatically using an instrument such as the Attention® Sigma 700 or 701.

[0035] In some embodiments, the CMC value for each of the surfactants is listed in Table 1 below.

[0036] [Table 1]

[0037] *For CMC values ​​listed in the tables, see, e.g., le Maire M, Champeil P, Moller JV. 2000. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86-111; Suksiriworapong J, Rungvimolsin T, A-gomol A, Junyaprasert VB, Chantasart D. Development and characterization of lyophilized diazepam-loaded polymeric micelles. 2014. AAPS PharmSciTech. 15(1):52-64; https: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / Sigma / Product_Information_Sheet / 1 / t8532pis.pdf

[0038] The protein may be present in the composition at a concentration of about 0.001 μg / mL to about 100 μg / mL. In some embodiments, the protein is present at a concentration of about 0.001 μg / mL to about 90 μg / mL, or about 0.001 μg / mL to about 80 μg / mL, or about 0.001 μg / mL to about 70 μg / mL, or about 0.001 μg / mL to about 60 μg / mL, or about 0.001 μg / mL to about 50 μg / mL, or about 0.001 μg / mL to about 40 μg / mL. L, or from about 0.001 μg / mL to about 30 μg / mL, or from about 0.001 μg / mL to about 20 μg / mL, or from about 0.001 μg / mL to about 10 μg / mL, or from about 0.001 μg / mL to about 5 μg / mL, or from about 0.001 μg / mL to about 1 μg / mL, or from about 0.001 μg / mL to about 0.01 μg / mL.

[0039] In some embodiments, the protein is present at a concentration of from about 0.01 μg / mL to about 100 μg / mL, or from about 0.01 μg / mL to about 80 μg / mL, or from about 0.01 μg / mL to about 70 μg / mL, or from about 0.01 μg / mL to about 60 μg / mL, or from about 0.01 μg / mL to about 50 μg / mL, or from about 0.01 μg / mL to about 40 μg / mL, or from about 0.01 μg / mL to about 30 μg / mL. μg / mL, or from about 0.01 μg / mL to about 20 μg / mL, or from about 0.01 μg / mL to about 10 μg / mL, or from about 0.01 μg / mL to about 5 μg / mL, or from about 0.01 μg / mL to about 1 μg / mL, or from about 0.01 μg / mL to about 0.1 μg / mL, or from about 0.1 μg / mL to about 1 μg / mL, or from about 0.1 μg / mL to about 51 μg / mL.

[0040] In some embodiments, the protein is present in the composition at a concentration of about 0.001 μg / mL, about 0.005 μg / mL, about 0.01 μg / mL, about 0.05 μg / mL, about 0.1 μg / mL, about 1 μg / mL, about 4 μg / mL, about 8 μg / mL, about 10 μg / mL, about 15 μg / mL, about 20 μg / mL, about 25 μg / mL, about 30 μg / mL, about 35 μg / mL, about 40 μg / mL, about 45 μg / mL, about 50 μg / mL, about 55 μg / mL, about 60 μg / mL, about 65 μg / mL, about 70 μg / mL, about 75 μg / mL, about 80 μg / mL, about 85 μg / mL, about 90 μg / mL, about 95 μg / mL, or about 100 μg / mL.

[0041] In the compositions disclosed herein, any protein concentration or concentration range can be combined with any surfactant concentration or concentration range.

[0042] Any protein can be the protein in the composition, hi some embodiments, the protein in the composition is a therapeutic protein, such as an antigen binding protein or a fusion protein.

[0043] As used herein, the term "antigen-binding protein" refers to a protein that specifically binds to one or more target antigens. Antigen-binding proteins include, but are not limited to, antibodies (e.g., monoclonal antibodies). Antigen-binding proteins are typically proteins that comprise an antigen-binding fragment that specifically binds to an antigen and, optionally, comprise a scaffold or framework portion that enables the antigen-binding fragment to adopt a conformation that promotes binding of the antigen-binding protein to an antigen. An "antigen-binding fragment" refers to a portion of an antibody that lacks at least some of the amino acids present in a full-length heavy and / or light chain, but is still capable of specifically binding to an antigen. Antigen-binding fragments include, but are not limited to, single-chain variable fragments (scFv), nanobodies (e.g., the VH domain of a camelid heavy-chain antibody; VHH fragments, see Cortez-Retamozo et al., Cancer Research, Vol. 64:2853-57, 2004), Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, Fd fragments, and complementarity-determining region (CDR) fragments, and can be derived from any mammalian source, such as human, mouse, rat, rabbit, or camel. Antigen-binding fragments may compete with intact antibodies for binding to the target antigen, and fragments can be produced by modification of intact antibodies (e.g., enzymatic or chemical cleavage) or synthesized de novo using recombinant DNA techniques or peptide synthesis known in the art.

[0044] In some embodiments, the protein is a bispecific antigen-binding protein. As used herein, the term "bispecific" refers to an antigen-binding protein that can specifically bind to two different antigens or targets or epitopes. As used herein, "epitope" refers to any determinant that can be specifically bound by an antigen-binding protein, such as an antibody or fragment thereof. In some embodiments, the bispecific antigen-binding protein comprises a first domain that specifically binds to one antigen or target and a second domain that specifically binds to another antigen or target. In some embodiments, the first domain of the bispecific antigen-binding protein specifically binds to a target cell surface antigen, and the second binding domain of the bispecific antigen-binding protein specifically binds to human CD3, a subunit of the T-cell receptor complex on T cells. In some preferred embodiments, the bispecific antigen-binding protein is a bispecific T-cell engager (BiTE®) antibody construct, e.g., as described in WO2008119567 and WO2017134140.

[0045] As used herein, the term "antibody construct" refers to a molecule whose structure and / or function is based on the structure and / or function of an antibody, e.g., a full-length or complete immunoglobulin molecule, and / or derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or fragment thereof. Thus, the antibody construct is capable of binding to its specific target or antigen. Antibody constructs also include modified fragments of antibodies, such as scFv, di-scFv or bi(s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandabs), tandem di-scFv, tandem tri-scFv, "multibodies," e.g., triabodies or tetrabodies, and single domain antibodies, e.g., nanobodies, or single variable domain antibodies, which contain only one variable domain, which may be a VHH, VH, or VL, that specifically binds to an antigen or epitope independently of other V regions or domains.

[0046] In some embodiments, a bispecific antibody construct comprises a first binding domain and a second binding domain, wherein the first binding domain specifically binds to a first cell surface antigen and the second binding domain specifically binds to human CD3. In some embodiments, the first and second domains of the bispecific antibody construct are "bispecific single-chain antibody constructs," more preferably bispecific "single-chain Fvs" (scFvs). In scFvs, the VL and VH of an antibody are linked as a single protein chain, e.g., by a synthetic linker, where the VL and VH regions pair to form a monovalent molecule; e.g., Huston (See, e.g., J. Am. Chem. Soc. Soc. USA 85:5879-5883). The linker can be a short peptide of about 10 to about 25 amino acids, preferably about 15 to 20 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. This scFv retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. The VH and VL regions are arranged in the order VH-VL or VL-VH. Preferably, the VH region is located at the N-terminus of the linker sequence and the VL region is located at the C-terminus of the linker sequence. In certain embodiments, the first and second domains of the bispecific antibody construct are in a format selected from (scFv)2, scFv-single-domain mAb, diabody, or oligomer, any of these formats.

[0047] In some embodiments, the bispecific antibody construct further comprises a third domain. In some embodiments, the third domain is a single chain Fc (scFc) domain. In some embodiments, the scFc domain is an scFc half-life extension (HLE) domain. In some preferred embodiments, the third domain of the bispecific antibody construct is an HLE domain, which has, in amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3.

[0048] In some embodiments, the first binding domain of the bispecific antibody construct binds to a first cell surface antigen. In some embodiments, the first cell surface antigen is CD70. CD70 (also known as CD27L or TNFSF7) is a type II integral membrane protein whose normal expression is restricted to activated T and B cells, mature dendritic cells, and a subset of medullary thymic epithelial cells.

[0049] In some embodiments, the first cell surface antigen is a tumor antigen. As used herein, the term "tumor antigen" is understood to refer to those antigens presented on tumor cells. These antigens may be presented on the cell surface with an extracellular portion, and often have both transmembrane and cytoplasmic portions of the molecule. These antigens may be presented only by tumor cells and not by normal cells. Tumor antigens may be expressed exclusively on tumor cells or may exhibit tumor-specific mutations compared to normal cells. In this case, they are called tumor-specific antigens. More common antigens are antigens presented by tumor cells and normal cells, and they are called tumor-associated antigens. These tumor-associated antigens may be overexpressed compared to normal cells, or may be accessible for antibody binding in tumor cells due to the less compact structure of tumor tissue compared to normal tissue. In some embodiments, the first binding domain binds to a tumor antigen selected from CD19, CD33, epidermal growth factor receptor variant iii (EGFRvIII), mesothelin (MSLN), cadherin 19 (CDH19), FMS-like tyrosine kinase 3 (FLT3), delta-like ligand 3 (DLL3), placental cadherin (CDH3), B-cell maturation antigen (BCMA), prostate-specific membrane antigen (PSMA), human mucin 17 (MUC17), and claudin-18 isoform 2 (CLDN18.2). In some embodiments, the tumor antigen is a human tumor antigen.

[0050] In some preferred embodiments, the bispecific antibody construct comprises a first domain, a second domain, and optionally a third domain, wherein the first domain binds to CD70, the second domain binds to human CD3, and the third domain (if present) is an HLE domain with, in amino to carboxyl order: hinge-CH2-CH3-linker-hinge-CH2-CH3. In another preferred embodiment, the bispecific antibody construct comprises a first domain, a second domain, and optionally a third domain, wherein the first domain binds to a tumor antigen selected from CD19, CD33, EGFRvIII, MSLN, CDH19, FLT3, DLL3, CDH3, BCMA, PSMA, MUC17, and C:DN18.2, the second domain binds to human CD3, and the third domain (if present) is an HLE domain which, in amino to carboxyl order, is: hinge-CH2-CH3-linker-hinge-CH2-CH3. In a preferred embodiment, the first and second domains are linked together via a peptide linker and to the third domain (if present) via a peptide linker. Preferred peptide linkers are described hereinabove and are characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser, i.e., Gly4Ser, or a polymer thereof, i.e., (Gly4Ser)x, where x is an integer equal to or greater than 1 (e.g., 2, 3, 4, 5, 6, or 7). In some preferred embodiments, the second binding domain comprises a polypeptide having the amino acid sequence of SEQ ID NO:201.

[0051] In some embodiments, the first binding domain specifically binds to CD33. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-15.

[0052] In some embodiments, the first binding domain specifically binds to EGFRvIII. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 16-26.

[0053] In some embodiments, the first binding domain specifically binds to MSLN. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 27-38 and 165.

[0054] In some embodiments, the first binding domain specifically binds to CDH19. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 39-56.

[0055] In some embodiments, the first binding domain specifically binds to DLL3. In some embodiments, the first binding domain comprises a polypeptide having an amino acid sequence of SEQ ID NO: 68-78.

[0056] In some embodiments, the first binding domain specifically binds to CD 19. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 79-88.

[0057] In some embodiments, the first binding domain specifically binds to FLT3. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 57-67.

[0058] In some embodiments, the first binding domain specifically binds to CDH3. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 89-99.

[0059] In some embodiments, the first binding domain specifically binds to BCMA. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 100-110.

[0060] In some embodiments, the first binding domain specifically binds to PSMA. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 111-155, 166, and 167.

[0061] In some embodiments, the first binding domain specifically binds to CD70. In some embodiments, the first binding domain comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 156-164.

[0062] In some embodiments, the second binding domain of the bispecific antibody construct specifically binds to epsilon of human CD3 on the surface of T cells. In some embodiments, the second domain of the bispecific antibody construct specifically binds to an extracellular epitope of the human CD3 epsilon chain. In some embodiments, the second domain of the bispecific antibody construct that specifically binds to human CD3 comprises a VL region comprising a CDR-L1 having the amino acid sequence of SEQ ID NO: 193, a CDR-L2 having the amino acid sequence of SEQ ID NO: 194, and a CDR-L3 having the amino acid sequence of SEQ ID NO: 195, and a VH region comprising a CDR-H1 having the amino acid sequence of SEQ ID NO: 196, a CDR-H2 having the amino acid sequence of SEQ ID NO: 197, and a CDR-H3 having the amino acid sequence of SEQ ID NO: 198.

[0063] In some embodiments, the second domain of the bispecific antibody construct comprises a VH having the amino acid sequence of SEQ ID NO: 199 and a VL having the amino acid sequence of SEQ ID NO: 200. In some embodiments, the second domain of the bispecific antibody construct comprises a polypeptide having the amino acid sequence of SEQ ID NO: 201.

[0064] In some embodiments, the protein is a CD70xCD3 bispecific antibody construct, which comprises a first domain that specifically binds to CD70 and a second domain that specifically binds to CD3. In one embodiment, the first domain specifically binds to CD70 and comprises CDRs as set forth in SEQ ID NOs: 156-161, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In some embodiments, the bispecific antibody construct further comprises an HLE domain (third domain). In one embodiment, the bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 162, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 163. In one embodiment, the CD70xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 164.

[0065] In some embodiments, the protein is a BCMAxCD3 bispecific antibody construct, which comprises a first domain that specifically binds BCMA and a second domain that specifically binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds BCMA and has CDRs as set forth in SEQ ID NOs: 100-105, and the second domain specifically binds CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the BCMAxCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 106, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 107. In one embodiment, the BCMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 108. In one embodiment, the BCMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 109. In another embodiment, the BCMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 110.

[0066] In some embodiments, the bispecific antibody is a CD33xCD3 bispecific antibody construct, which comprises a first domain that specifically binds to CD33 and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody construct further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to CD33 and has CDRs as set forth in SEQ ID NOS: 3-5 and 8-10, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOS: 193-198. In one embodiment, the CD33xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 1 or 2, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 6 or 7. In another embodiment, the CD33xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 11 or 12. In another embodiment, the BCMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 13 or 15.

[0067] In some embodiments, the protein is an EGFRvIIIxCD3 bispecific antibody construct, which comprises a first domain that specifically binds to EGFRvIII and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to EGFRvIII and has CDRs as set forth in SEQ ID NOs: 16-21, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the EGFRvIIIxCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 22, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 23. In one embodiment, the EGFRvIIIxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 24 or 25. In one embodiment, the EGFRvIIIxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:24 or 26.

[0068] In some embodiments, the protein is an MSLNxCD3 bispecific antibody construct, which comprises a first domain that specifically binds to MSLN and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody construct further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to MSLN and has CDRs as set forth in SEQ ID NOs: 27-32, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the MSLNxCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 34. In one embodiment, the MSLNxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 35-38. In one embodiment, the MSLNxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 165.

[0069] In some embodiments, the protein is a CDH19xCD3 bispecific antibody construct, which comprises a first domain that specifically binds CDH19 and a second domain that specifically binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds CDH19 and has CDRs as set forth in SEQ ID NOs: 39-44, and the second domain specifically binds CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the CDH19xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 45 or 52, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 46 or 52. In one embodiment, a CDH19xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 47, 48-50, and 53-56. In one embodiment, a CDH19xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 48.

[0070] In some embodiments, the protein is a DLL3xCD3 bispecific antibody, which comprises a first domain that specifically binds to DLL3 and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to DLL3 and has CDRs as set forth in SEQ ID NOs: 68-73, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the DLL3xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 74, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 75. In one embodiment, the DLL3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NOs: 76-78. In one embodiment, the DLL3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:78.

[0071] In some embodiments, the protein is a FLT3xCD3 bispecific antibody construct, which comprises a first domain that specifically binds to FLT3 and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to FLT3 and has CDRs as set forth in SEQ ID NOs: 57-62, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the FLT3xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 63, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 64. In one embodiment, the FLT3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any of SEQ ID NOs: 65-67. In one embodiment, the FTL3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:67.

[0072] In some embodiments, the protein is a CDH3xCD3 bispecific antibody construct, which comprises a first domain that specifically binds to CDH3 and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody construct further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to CDH3 and has CDRs as set forth in SEQ ID NOs: 89-94, and the second domain specifically binds to CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the CDH3xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 95, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 96. In one embodiment, the CDH3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any of SEQ ID NOs: 97-99. In one embodiment, the CDH3xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:99.

[0073] In some embodiments, the protein is a PSMAxCD3 bispecific antibody construct, which comprises a first domain that specifically binds to PSMA and a second domain that specifically binds to CD3. In some embodiments, the bispecific antibody construct further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds to PSMA and has CDRs as set forth in any of SEQ ID NOS: 111-116, and the second domain specifically binds to CD3 and has CDRs as set forth in any of SEQ ID NOS: 193-198. In one embodiment, the first domain binds to PSMA and has CDRs as set forth in any of SEQ ID NOS: 126-131 and 141-146, and the second domain binds to CD3 and has CDRs as set forth in SEQ ID NOS: 193-198. In one embodiment, a PSMAxCD3 bispecific antibody comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 117, 132, or 147, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 118, 133, or 148. In one embodiment, a PSMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 119-125, 134-140, and 149-155. In one embodiment, a PSMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NOs: 121, 122, 124, 125, 136, 137, 139, 140, 151, 152, 154, and 155. In one embodiment, the PSMAxCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 166 or 167.

[0074] In some embodiments, the protein is a Cldn18.2xCD3 bispecific antibody construct, which comprises a first domain that specifically binds Cldn18.2 and a second domain that specifically binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds Cldn18.2 and has CDRs as set forth in SEQ ID NOs: 168-173, and the second domain specifically binds CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the Cldn18.2xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 174, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 175. In one embodiment, the Cldn18.2xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 176 or 178. In one embodiment, the Cldn18.2xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 178.

[0075] In some embodiments, the protein is a MUC17xCD3 bispecific antibody construct, which comprises a first domain that specifically binds MUC17 and a second domain that specifically binds CD3. In some embodiments, the bispecific antibody further comprises an HLE domain (third domain). In one embodiment, the first domain specifically binds MUC17 and has CDRs as set forth in SEQ ID NOs: 184-189, and the second domain specifically binds CD3 and has CDRs as set forth in SEQ ID NOs: 193-198. In one embodiment, the MUC17xCD3 bispecific antibody construct comprises a VH and a VL, wherein the VH comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 190, and the VL comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 191. In one embodiment, the MUC17xCD3 bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 192.

[0076] In certain embodiments, the bispecific antibody construct comprises a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192.

[0077] The bispecific antibodies disclosed herein can be prepared by methods known in the art, for example, by the methods disclosed in WO 2008 / 119657 and WO 2017 / 134140.

[0078] Further excipients that may be used in the composition In some embodiments, the composition further comprises one or more excipients suitable for pharmaceutical compositions. In some embodiments, the composition further comprises a salt, a buffering agent, a sugar or a sugar derivative, an amino acid, or a preservative, or a combination of two or more of the foregoing. In some embodiments, the composition further comprises a salt, a sugar or a sugar derivative, an amino acid, and, optionally, a preservative. In some embodiments, the composition further comprises a salt, a buffering agent, a sugar or a sugar derivative, and an amino acid. In some embodiments, the composition further comprises a salt, a buffering agent, a sugar or a sugar derivative, an amino acid, and a preservative.

[0079] Exemplary salts that can be used in the compositions include salts suitable for use in pharmaceutical compositions (e.g., NaCl). Exemplary buffers that can be used in the compositions include acetate buffer, glutamate buffer, citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, and phosphate buffer. Exemplary sugars or sugar derivatives include monosaccharides, disaccharides, cyclic polysaccharides, and sugar alcohols, such as sugars (e.g., sucrose and trehalose) and sugar alcohols (e.g., mannitol and sorbitol). Exemplary amino acids include lysine, histidine, arginine, glycine, methionine, and alanine. Exemplary preservatives include benzoates (e.g., benzyl alcohol and sodium benzoate) and sorbates (e.g., potassium sorbate).

[0080] The pH of the composition can range from about 3.5 to 7.5. In some embodiments, the composition has a pH of about 4.0 to about 7.0. In some embodiments, the composition has a pH of about 4.2 to about 7.0, or about 5.0 to about 7.0, or about 5.5 to about 7.0, or about 6.0 to about 7.0, or about 6.5 to about 7.0, or about 4.2 to about 6.5, or about 4.2 to about 6.0, or about 4.2 to about 5.5, or about 4.2 to about 5.0, or about 5.0 to about 6.0, or about 5.0 to about 6.5, or about 5.0 to about 6.0, or about 5.0 to about 5.5, or about 5.5 to about 6.0, or about 5.5 to about 6.5, or about 5.5 to about 6.0, or about 6.0 to about 6.5. In some embodiments, the pH of the composition is about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5.

[0081] The pH of the composition can be achieved by using one or more of the buffers listed above. In some embodiments, the composition comprises a buffer selected from acetate buffer, glutamate buffer, citrate buffer, succinate buffer, tartrate buffer, fumarate buffer, maleate buffer, histidine buffer, or phosphate buffer. In some embodiments, the composition comprises a combination of two buffers selected from the above list of buffers, such as a glutamate buffer and a citrate buffer.

[0082] In some embodiments, the composition is substantially free of additional buffering agents. As used herein, the phrase "substantially free of additional buffering agents" means that the composition does not contain any buffering agents added thereto for the purpose of adjusting and / or maintaining the pH of the composition. For example, saline (0.9% NaCl solution) contains no additional buffering agents and has a pH of about 5.5. The pH of saline is believed to be achieved and maintained in part by atmospheric CO2 dissolved in water. See, e.g., Reddi, B. AJ Int. J. Med. Sci. 10:747-750 (2013). Such compositions may contain residual buffering agents that do not contribute to the buffering capacity of the composition.

[0083] In some embodiments, the composition comprises a protein at any of the concentrations disclosed above and a surfactant at any of the concentrations disclosed above, and further comprises a salt (e.g., NaCl), a sugar (e.g., sucrose), an amino acid (e.g., lysine), and optionally a preservative (e.g., benzyl alcohol). In some embodiments, the composition comprises a protein at any of the concentrations disclosed above and a surfactant at any of the concentrations disclosed above, and further comprises a salt (e.g., NaCl), a sugar (e.g., sucrose), an amino acid (e.g., lysine), a buffer (e.g., glutamate buffer and / or citrate buffer), and optionally a preservative (e.g., benzyl alcohol). The pH of the composition can be any of the pH values ​​disclosed above. In some embodiments, the pH of the composition is about 5.5.

[0084] In some embodiments, the compositions disclosed herein are pharmaceutical compositions. As used herein, the term "pharmaceutical composition" is understood to refer to a formulation comprising a protein (e.g., a bispecific antibody construct) suitable for injection and / or administration to a patient (e.g., a human) in need thereof. More particularly, a pharmaceutical composition is substantially sterile and does not contain any material that is overly toxic or infectious to the recipient.

[0085] Container for the composition In some embodiments, the compositions disclosed herein are contained in a plastic container. Containers that can be used herein include those suitable for pharmaceutical use, such as containers made of materials that are non-toxic and maintain their physical integrity. In some embodiments, the container is an intravenous (IV) administration component. As used herein, "IV administration component" is understood to refer to a container or part of a system that may come into contact with the composition during IV administration. In some embodiments, the container is an IV bag or IV tubing.

[0086] Materials that can be used to manufacture the container include materials typically used to manufacture pharmaceutical containers, such as glass and plastic.

[0087] In some embodiments, the container is a plastic container. In some embodiments, the container is made from a material including polyolefins (e.g., polypropylene (PP) and polyethylene (PE)), polyvinyl chloride (PVC), ethylene vinyl acetate (EVA), or polyurethanes (e.g., polyesters and polyethers). In some embodiments, the container is made from a material including PVC. In some embodiments, the PVC is substantially free of di-2-ethylhexyl phthalate (DEHP) or tri-2-ethylhexyl trimellitate (TOTM). As used herein, the term "substantially free of" is understood to refer to PVC in which DEHP or TOTM is not used and / or detected. DEHP and TOTM are plasticizers and can be used to soften PVC, allowing it to be made into various shapes. In some embodiments, the container is made from a material that does not include PVC. In some embodiments, the container is made from a material including polyolefins, EVA, or polyurethanes (e.g., polyesters and polyethers). In some embodiments, the container is made from a material including PP and / or PE.

[0088] Under certain conditions, DEHP or TOTM contained in certain types of PVC plastics may leach from the plastic in the presence of certain concentrations of certain surfactants (e.g., polysorbates, see Example 3). Without wishing to be bound by any particular theory, it is believed that the more lipophilic the surfactant, the more likely DEHP or TOTM will leach in the presence of the surfactant. The hydrophilic-lipophilic ratio (HLB) of a surfactant is a measure of its degree of hydrophilicity or lipophilicity and can be calculated by methods known in the art. See, for example, Griffin, William C., Calculation of HLB Values ​​of Non-Ionic Surfactants, Journal of the Society of Cosmetic Chemists, 5(4):249-56 (1954). The HLB value can be used to predict surfactant properties; for example, an HLB value >10 indicates that the surfactant is more water-soluble (lipid-insoluble), and an HLB value <10 indicates that the surfactant is more fat-soluble (water-insoluble). Exemplary surfactant HLB values ​​that can be used in the compositions disclosed herein include 17 (polysorbate 20), 15 (polysorbate 80) (https: / / pharmlabs.unc.edu / labs / emulsions / hlb.htm), and 29 (poloxamer 188) (http: / / www.rumapel.com.ar / cosmetica_miscelaneos / ficha_tecnica / Pluracare%20L-%20F%20Grades.pdf). In embodiments in which the container is made from PVC plastic containing DEHP or TOTM, the surfactant in the composition preferably has an HLB value of at least 20 (e.g., poloxamer, see Example 3), more preferably an HLB value of 20-30.

[0089] Containers such as IV components (e.g., IV bags and tubing used for IV administration) made from the above materials are commercially available. For example, various suitable containers are available from manufacturers such as Baxter Healthcare Corporation and B. Braun Medical Inc.

[0090] Pharmaceutical preparations Also disclosed herein is a pharmaceutical formulation comprising the aqueous composition disclosed above. As used herein, "pharmaceutical formulation" is understood to refer to a formulation comprising the aqueous composition disclosed herein in a suitable pharmaceutical container prior to administration to a patient (e.g., a human). In some embodiments, disclosed herein is a pharmaceutical formulation comprising an aqueous pharmaceutical composition in a container, the aqueous pharmaceutical composition comprising: a) a protein at a concentration of about 0.001 μg / mL to about 100 μg / mL; and b) a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant, wherein the protein is not blinatumomab.

[0091] In some embodiments, the surfactant included in the composition is a polysorbate. In some embodiments, the surfactant is a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, and polysorbate 85. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is a poloxamer. In some embodiments, the surfactant is poloxamer 188 or poloxamer 407. In some embodiments, the surfactant is Triton X-100.

[0092] In some embodiments, disclosed herein is a pharmaceutical formulation comprising an aqueous pharmaceutical composition in a container, the aqueous pharmaceutical composition comprising: a) a bispecific antibody construct at a concentration of about 0.001 μg / mL to about 100 μg / mL; and b) a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant, wherein the surfactant is not a poloxamer. In some embodiments, the poloxamer is poloxamer 188 or poloxamer 407.

[0093] In some embodiments, the surfactant is present in the composition at a concentration of about 0.25 to about 20 times the CMC of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration of about 0.25 to about 10 times the CMC of the surfactant. In some embodiments, the surfactant is present in the composition at a concentration within any of the concentration ranges disclosed above. In some embodiments, the surfactant is present in the composition at any of the concentrations disclosed above. The CMC value of the surfactant can be determined by the methods disclosed above. In some embodiments, the CMC values ​​of certain commonly used surfactants are listed in Table 1.

[0094] In some embodiments, the aqueous pharmaceutical composition comprises a protein concentration of about 0.001 μg / mL to about 50 μg / mL. In some embodiments, the composition comprises a protein (e.g., a bispecific antibody construct) at a concentration within any of the ranges disclosed above. In some embodiments, the protein (e.g., a bispecific antibody construct) is present in the composition at any of the concentrations disclosed above. Any of the protein concentrations or concentration ranges disclosed above can be combined with any of the surfactant concentrations or concentration ranges disclosed above.

[0095] As disclosed above, the protein can be a therapeutic protein such as an antigen binding protein or a fusion protein. In some embodiments, the protein is a bispecific antigen binding protein. In some embodiments, the protein is a bispecific antibody construct as disclosed above. In some embodiments, the protein is a CD70xCD3 bispecific antibody construct. In some embodiments, the protein is a BCMAxCD3 bispecific antibody construct. In some embodiments, the protein is a CD33xCD3 bispecific antibody construct. In some embodiments, the protein is an EGFRvIIIxCD3 bispecific antibody construct. In some embodiments, the protein is an MSLNxCD3 bispecific antibody construct. In some embodiments, the protein is a CDH19xCD3 bispecific antibody construct. In some embodiments, the protein is a DLL3xCD3 bispecific antibody construct. In some embodiments, the protein is a FLT3xCD3 bispecific antibody construct. In some embodiments, the protein is a CDH3xCD3 bispecific antibody construct. In some embodiments, the protein is a PSMAxCD3 bispecific antibody construct. In some embodiments, the protein is a Cldn18.2xCD3 bispecific antibody construct. In some embodiments, the protein is a MUC17xCD3 bispecific antibody construct. Each of these bispecific antibody constructs is disclosed above. In certain embodiments, the protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192.

[0096] The pH of the composition can be within any of the pH ranges disclosed above. In some embodiments, the pH of the composition can be any pH disclosed above.

[0097] In some embodiments, the aqueous pharmaceutical composition further comprises one or more excipients suitable for use in pharmaceutical compositions. In some embodiments, the aqueous pharmaceutical composition further comprises a salt, a buffering agent, an amino acid, a sugar or a sugar derivative, a preservative, or a combination thereof. In some embodiments, the aqueous pharmaceutical composition further comprises a salt, an amino acid, a sugar or a sugar derivative, a preservative, or a combination thereof. Exemplary salts, buffering agents, amino acids, sugars or derivatives thereof, and preservatives that can be used in the compositions are disclosed above.

[0098] In some embodiments, the container is a plastic container. In some embodiments, the container is made from a material comprising polyolefins (e.g., PP and PE), PVC, EVA, or polyurethanes (e.g., polyesters and polyethers). In some embodiments, the container is made from a material comprising PVC. In some embodiments, the PVC is substantially free of DEHP or TOTM. In some embodiments, the container is made from a material that is free of PVC.

[0099] In some embodiments, the surfactant included in the composition is a poloxamer, and the container can be made from a material including polyolefin, PVC, EVA, or polyurethane (e.g., polyester and polyether). In some embodiments, the surfactant included in the composition is polysorbate or Triton X-100, and the container can be made from a material including polyolefin, PVC, EVA, or polyurethane (e.g., polyester and polyether) that is substantially free of DEHP or TOTM.

[0100] In some embodiments, the container is an intravenous (IV) administration component, hi some embodiments, the container is an IV bag or IV tubing.

[0101] In some embodiments, the pharmaceutical formulation comprises an aqueous pharmaceutical composition in a container, the aqueous pharmaceutical composition comprising a bispecific antibody construct (e.g., any of the bispecific antibody constructs disclosed herein) and a surfactant (e.g., any of the surfactants disclosed herein), and further comprising a salt (e.g., NaCl), an amino acid (e.g., lysine), a sugar or sugar derivative (e.g., sucrose or mannitol), and optionally a preservative (e.g., benzyl alcohol), and the container is made of a material comprising polyolefin, PVC, EVA, or polyurethane (e.g., polyester and polyether). In some embodiments, the pharmaceutical formulation comprises an aqueous pharmaceutical composition in a container, the aqueous pharmaceutical composition comprising a bispecific antibody construct (e.g., any of the bispecific antibody constructs disclosed herein) and a surfactant (e.g., any of the surfactants disclosed herein), and further comprising a salt (e.g., NaCl), a buffer (e.g., glutamate buffer and / or citrate buffer), an amino acid (e.g., lysine), a sugar or sugar derivative (e.g., sucrose or mannitol), and optionally a preservative (e.g., benzyl alcohol), wherein the container is made of a material including polyolefin, PVC, EVA, or polyurethane (e.g., polyester and polyether). The concentration of the bispecific antibody construct may be any of the concentrations disclosed above for proteins. The concentration of the surfactant may be any of the concentrations disclosed above. The pH of the composition ranges from about 3.5 to about 7.0. In some embodiments, the pH of the composition is about 5.5. In some embodiments, the container is an IV bag.

[0102] Methods of Administering the Composition Also disclosed herein are methods of administering the compositions disclosed herein to a patient. In some embodiments, disclosed herein are methods of administering a protein to a patient, the method comprising: a) preparing an aqueous pharmaceutical composition in a container, wherein the aqueous pharmaceutical composition comprises a protein (e.g., a bispecific antibody construct disclosed herein) at a concentration of about 0.001 μg / mL to about 100 μg / mL and a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant; and b) administering the aqueous pharmaceutical composition to the patient, wherein the protein is not blinatumomab.

[0103] In some embodiments, the aqueous pharmaceutical composition is prepared by diluting a first composition comprising a protein (e.g., a bispecific antibody construct) with a suitable aqueous solution. In some embodiments, the aqueous pharmaceutical composition is prepared by adding a suitable solution to a container, followed by adding an appropriate amount of the first composition to the container, thereby diluting the first composition comprising the protein.

[0104] In some embodiments, the first composition is a liquid composition comprising a protein. In some embodiments, the first composition is a liquid composition reconstituted from a lyophilized composition comprising a protein. In some embodiments, the first composition is a liquid composition reconstituted from a lyophilized composition comprising a protein using sterile water.

[0105] In some embodiments, a suitable aqueous solution used to dilute the first composition comprises a surfactant at a concentration of at least about 0.25 times the CMC of the surfactant. In some embodiments, a suitable aqueous solution used to dilute the first composition comprises a surfactant at a concentration of about 0.25 to about 20 times, or about 0.25 to about 10 times, the CMC of the surfactant. In some embodiments, a suitable aqueous solution used to dilute the first composition comprises a surfactant at a concentration of 0.25, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 times the CMC of the surfactant. In some embodiments, a suitable aqueous solution used to dilute the first composition further comprises NaCl. In some embodiments, a suitable aqueous solution used to dilute the first composition has a pH of about 3.5 to about 7.0, or about 4.0 to about 6.5. In some embodiments, the suitable aqueous solution used to dilute the first composition has a pH of about 5.5. In some embodiments, the method further comprises rinsing the container with the suitable aqueous solution used to dilute the first composition prior to the preparing step.

[0106] In some embodiments, the surfactant in the aqueous pharmaceutical composition is a polysorbate. In some embodiments, the surfactant is a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, and polysorbate 85. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80. In some embodiments, the surfactant is a poloxamer. In some embodiments, the surfactant is poloxamer 188 or poloxamer 407. In some embodiments, the surfactant is Triton X-100.

[0107] In some embodiments, the surfactant in the aqueous pharmaceutical composition is present in the composition at a concentration of about 0.25 to about 20 times the CMC of the surfactant. In some embodiments, the surfactant is present in the aqueous pharmaceutical composition at a concentration of about 0.25 to about 10 times the CMC of the surfactant. In some embodiments, the surfactant is present in the aqueous pharmaceutical composition at a concentration within any of the concentration ranges disclosed above. In some embodiments, the surfactant is present in the aqueous pharmaceutical composition at any of the concentrations disclosed above. The CMC value of a surfactant can be determined by the methods disclosed above. In some embodiments, the CMC values ​​of certain commonly used surfactants are listed in Table 1.

[0108] In some embodiments, the aqueous pharmaceutical composition comprises a protein concentration of about 0.001 μg / mL to about 50 μg / mL. In some embodiments, the aqueous pharmaceutical composition comprises a protein concentration within any of the ranges disclosed above. In some embodiments, the protein is present in the composition at any of the concentrations disclosed above.

[0109] As disclosed above, the protein can be a therapeutic protein such as an antigen binding protein, mAb, or fusion protein. In some embodiments, the protein is a bispecific antigen binding protein. In some embodiments, the protein is a bispecific antibody construct as disclosed above. In some embodiments, the protein is a CD70xCD3 bispecific antibody construct. In some embodiments, the protein is a BCMAxCD3 bispecific antibody construct. In some embodiments, the protein is a CD33xCD3 bispecific antibody construct. In some embodiments, the protein is an EGFRvIIIxCD3 bispecific antibody construct. In some embodiments, the protein is an MSLNxCD3 bispecific antibody construct. In some embodiments, the protein is a CDH19xCD3 bispecific antibody construct. In some embodiments, the protein is a DLL3xCD3 bispecific antibody construct. In some embodiments, the protein is a FLT3xCD3 bispecific antibody construct. In some embodiments, the protein is a CDH3xCD3 bispecific antibody construct. In some embodiments, the protein is a PSMAxCD3 bispecific antibody construct. In some embodiments, the protein is a Cldn18.2xCD3 bispecific antibody construct. In some embodiments, the protein is a MUC17xCD3 bispecific antibody construct. Each of these bispecific antibody constructs is disclosed above. In certain embodiments, the protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 25, SEQ ID NO: 48, SEQ ID NO: 67, SEQ ID NO: 78, SEQ ID NO: 88, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 178, or SEQ ID NO: 192.

[0110] The pH of the composition can be within any of the pH ranges disclosed above. In some embodiments, the pH of the composition can be any pH disclosed above.

[0111] In some embodiments, the aqueous pharmaceutical composition further comprises one or more excipients suitable for use in pharmaceutical compositions. In some embodiments, the aqueous pharmaceutical composition further comprises a salt, a buffering agent, an amino acid, a sugar or a sugar derivative, optionally a preservative, or a combination of two or more of the foregoing. In some embodiments, the aqueous pharmaceutical composition further comprises a salt, an amino acid, a sugar or a sugar derivative, optionally a preservative, or a combination of two or more of the foregoing. Exemplary salts, buffering agents, amino acids, sugars or derivatives thereof, and preservatives that may be used in the compositions are disclosed above.

[0112] In some embodiments, the container is a plastic container or component. In some embodiments, the container is made from a material including polyolefins (e.g., PP and PE), PVC, EVA, or polyurethanes (e.g., polyesters and polyethers). In some embodiments, the container is made from a material including PVC. In some embodiments, the PVC is substantially free of DEHP or TOTM. In some embodiments, the container is made from a material that is PVC-free. In some embodiments, the container is an IV bag or IV tubing.

[0113] In some embodiments, the aqueous pharmaceutical composition is administered to the patient by IV administration. In some embodiments, the patient is a cancer patient. In some embodiments, the patient is human.

[0114] Methods for accessing the binding of proteins to solid surfaces Also disclosed herein are methods for accessing the binding of a protein to a solid surface. In some embodiments, disclosed herein are methods for accessing the binding of a protein to a solid surface, the methods comprising: a) incubating the solid surface with an aqueous solution comprising a protein, wherein the protein is labeled with a fluorophore; b) removing the aqueous solution from the solid surface and rinsing the surface; and c) imaging the solid surface using a confocal microscope.

[0115] Table 2

[0116] Table 3

[0117] Table 4

[0118] Table 5

[0119] Table 6

[0120] Table 7

[0121] Table 8

[0122] Table 9

[0123] Table 10

[0124] Table 11

[0125] Table 12

[0126] Table 13

[0127] Table 14

[0128] Table 15

[0129] Table 16

[0130] Table 17

[0131] Table 18

[0132] Table 19

[0133] Table 20

[0134] Table 21

[0135] Table 22

[0136] Table 23

[0137] Table 24

[0138] Table 25

[0139] Table 26

[0140] Table 27

[0141] Table 28

[0142] Table 29

[0143] Table 30

[0144] Table 31

[0145] Table 32

[0146] Table 33

[0147] Table 34

[0148] Table 35

[0149] Table 36

[0150] Table 37

[0151] Table 38

[0152] Table 39

[0153] Table 40

[0154] Table 41

[0155] Table 42

[0156] Table 43

[0157] [Table 44]

[0158] [Table 45]

[0159] The present invention will be more fully understood by reference to the following examples, which should not, however, be construed as limiting the scope of the invention. [Example]

[0160] Example 1. Methods for measuring binding of proteins to solid surfaces Bispecific antibody constructs were supplied internally, labeled with fluorophores, and purified after the labeling procedure.

[0161] Each measurement chamber is equipped with a plastic coverslip. To measure protein binding to a solid surface, the measurement chambers with plastic coverslips at the bottom were first incubated with a solution containing a fluorophore-protein (e.g., a fluorophore-labeled antibody construct). The sample solution was then aspirated and rinsed from the coverslip, which was then filled with buffer for later imaging with a confocal microscope. The fluorescence intensity measured with the confocal microscope indicates that the bispecific antibody construct has bound to the coverslip. Figure 1 shows the layout of the experimental setup.

[0162] Figure 2 shows the titration of two fluorophore-labeled antibody constructs separately bound to a solid surface (such as a coverslip) in the absence of detergent. The fluorescence intensity of the bound fluorophore-labeled antibody constructs was measured by confocal xy-scanning of the surface.

[0163] Example 2. Treatment of solid surfaces with surfactants to prevent protein binding to the surface Several surfactants were used at different magnifications of their respective CMCs to determine their effectiveness in preventing bispecific antibody constructs from binding to solid surfaces. In this study, a solution containing the surfactant was first incubated with the surface, followed by the addition of a fluorophore-labeled antibody construct. The solution was then aspirated, and the surface was rinsed and filled with buffer for imaging by confocal microscopy. The results are shown in Figure 3. In the figure, the first group of bars represents bispecific antibodies bound to the surface without surfactant. These served as benchmarks to which all subsequent data groups were compared. The second through last groups represent the relative percentages of protein bound to surfaces pretreated with different surfactants at different magnifications of their respective CMCs. The insert shows a magnification of the lower region of the graph. PS80, PS20, P188, P407, and Triton X-100 were investigated.

[0164] The order of adding surfactant and antibody to the surface was tested. For bispecific antibodies 1 and 2, two orders were tested: in the first, the surfactant-containing solution was added to the surface before adding the antibody; in the second, the reverse was done. The results are shown in Figure 4 (antibody 1 on the left, antibody 2 on the right). In both figures, the first group of bars represents bispecific antibody bound to the surface without surfactant. These served as benchmarks to which all subsequent data groups were compared. The second to last groups represent the relative percentage of antibody bound to surfaces pretreated with PS80 at different times its CMC. The surfactant effectively prevented proteins from binding to the surface.

[0165] Example 3. Incompatibility of certain surfactants and plasticizers used in plastic IV components Baxter Viaflex PVC-DEHP pre-filled with saline diluent IV bags were used in the study. The surfactants used in the study were polysorbate 80 (PS80), polysorbate 20 (PS20), poloxamer 188 (P188), poloxamer 407 (P407), and Triton X-100. Different amounts of different surfactants were incubated with the bags at 25°C for 24 or 48 hours. The bags were then sampled and analyzed by reverse-phase ultra-high pressure liquid chromatography (RP-UHPLC) with UV detection. Mobile phases A and B were 0.1% trifluoroacetic acid (TFA) in DI water and 0.1% TFA in acetonitrile. The gradient is listed in Table 3 below. The flow rate was 0.6 mL / min. A standard curve of DEHP was generated under the same conditions for quantification.

[0166] [Table 46]

[0167] PS80, PS20, and P188 were compared at 0.3 wt% in saline in PVC-DEHP IV bags at 25°C for 24 and 48 hours. The results are shown in Figure 5. PS80 and PS20 caused significant leaching of DEHP from the PVC-DEHP IV bags, whereas P188 did not (Figure 5). Saline alone was used as a control.

[0168] PS80, PS20, P188, P407, and Triton X-100 were compared at different magnifications of their respective CMCs by incubating them in saline in PVC-DEHP IV bags at 25°C for 24 hours. The amount of DEHP leached is plotted as a function of CMC magnification in Figure 6. Clearly, polysorbates extract a certain amount of DEHP, whereas poloxamers do not. The amount of DEHP leached also correlates with the amount of surfactant used.

Claims

[Claim 1] The invention described in the specification.

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