CN117320700A

CN117320700A - Stable formulations containing anti-MUC 16X anti-CD 3 bispecific antibodies

Info

Publication number: CN117320700A
Application number: CN202280035628.8A
Authority: CN
Inventors: D·卡门; T-C·杨; 徐晓滨; 邱海波; K·格雷厄姆
Original assignee: Regeneron Pharmaceuticals Inc
Current assignee: Regeneron Pharmaceuticals Inc
Priority date: 2021-04-02
Filing date: 2022-04-01
Publication date: 2023-12-29

Abstract

The present invention provides stable liquid pharmaceutical formulations comprising human bispecific antibodies that specifically bind to human MUC16 and human CD 3. In certain embodiments, the formulation contains a buffer, a surfactant, and a sugar in addition to the bispecific antibody. The pharmaceutical formulations of the invention exhibit a considerable degree of antibody stability upon stress and storage.

Description

Stable formulations containing anti-MUC 16X anti-CD 3 bispecific antibodies

Reference to the sequence Listing

The present application incorporates by reference a sequence listing submitted in computer readable form created at 2022, month 4, 1, file name 10820WO01-sequence. Txt, containing 25,436 bytes.

Technical Field

The present disclosure relates to the field of therapeutic antibody formulations. More specifically, the present invention relates to the field of pharmaceutical formulations comprising human bispecific antibodies that specifically bind to human MUC16 and human CD 3.

Background

Therapeutic macromolecules (e.g., antibodies) must be formulated in such a way as to not only render the molecule suitable for administration to a patient, but also to maintain its stability during storage and subsequent use. For example, unless the solution is properly formulated, therapeutic antibodies in the liquid solution are prone to degradation, aggregation, and/or undesirable chemical modification. The stability of an antibody in a liquid formulation depends not only on the kind of excipient used in the formulation, but also on the amount and ratio of excipients relative to each other. In addition, other factors must be considered in addition to stability when preparing liquid antibody formulations. Examples of such additional considerations include the allowable concentration of antibody for a given formulation, as well as the visual quality or appeal of the formulation. Thus, when formulating therapeutic antibodies, great care must be taken to obtain a formulation that remains stable, contains sufficient concentrations of antibody, and has other properties so that the formulation can be conveniently administered to a patient.

Mucin 16 (MUC 16), also known as cancer antigen 125, carbohydrate antigen 125 or CA-125, is an integral membrane glycoprotein that is highly glycosylated with a single transmembrane domain that is highly expressed in ovarian cancer. CD3 is a homodimeric or heterodimeric antigen expressed on T cells associated with the T cell receptor complex (TCR), and is essential for T cell activation.

Bispecific antibodies to human MUC16 and human CD3 are one example of therapeutically relevant macromolecules that require appropriate formulations. Such antibodies are clinically useful, for example, in the treatment of cancers (e.g., MUC16 expressing cancers, ovarian cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer).

Although anti-MUC 16x anti-CD 3 bispecific antibodies are known in the art (see e.g. WO 2018/067331), there remains a need for pharmaceutical formulations comprising anti-MUC 16x anti-CD 3 bispecific antibodies that are sufficiently stable and suitable for administration to a patient.

Disclosure of Invention

Stable liquid pharmaceutical formulations comprising bispecific anti-MUC 16x anti-CD 3 antibodies and one or more excipients are provided, as well as kits, unit dosage forms and containers comprising such formulations, and uses thereof.

In one aspect, the present invention provides a stable liquid pharmaceutical formulation comprising: (a) A bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), and the second antigen-binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), wherein A1-HCDR1, A1-HCDR2 and A1-HCDR3 comprise the amino acid sequences of SEQ ID NOs 7, 8 and 9, and amino acid sequences of LCDR 2-HCDR2 and LCDR2, LCDR2 and HCDR3 comprise the amino acid sequences of SEQ ID NOs 14 and 12, respectively; (b) a buffer comprising sodium acetate; (c) an organic co-solvent comprising polysorbate; and (d) a stabilizer comprising a sugar; wherein the pH of the formulation is 5.0.+ -. 0.5.

In some cases, the antibody concentration is 1 mg/ml.+ -. 0.1mg/ml to 200 mg/ml.+ -. 20mg/ml. In some cases, the antibody concentration is 5 mg/ml.+ -. 0.5mg/ml to 50 mg/ml.+ -. 5mg/ml. In some cases, the antibody concentration is 5 mg/ml.+ -. 0.5mg/ml. In some cases, the antibody concentration is 50 mg/ml.+ -. 5mg/ml. In some cases, the antibody concentration is 150 mg/ml.+ -.15 mg/ml.

In some cases, the acetate buffer concentration is 10 mM+ -1 mM to 50 mM+ -5 mM. In some cases, the acetate buffer concentration is 25 mM+ -2.5 mM to 35 mM+ -3.5 mM. In some cases, the acetate buffer concentration is 30 mM.+ -. 3mM.

In some cases, the polysorbate concentration is 0.01% ± 0.005% to 0.5% ± 0.05% w/v. In some cases, the polysorbate concentration is 0.1% ± 0.05% to 0.3% ± 0.03% w/v. In some cases, the polysorbate concentration is 0.2% ± 0.02% w/v. In some cases, the polysorbate concentration is 0.05% ± 0.01% w/v. In some cases, the polysorbate is polysorbate 20.

In some embodiments, the sugar is sucrose. In some cases, the sucrose concentration is 5% ± 1% to 20% ± 4% w/v. In some cases, the sucrose concentration is 7% ± 0.5% to 12% ± 0.5% w/v. In some cases, the sucrose concentration is 10% + -1% w/v. In some cases, the sucrose concentration is 7% + -0.7% w/v. In some cases, the sucrose concentration is 8% + -0.8% w/v.

In some embodiments, the pharmaceutical formulation comprises: (a) 5 mg/ml.+ -. 0.5mg/ml antibody, (b) 25 mM.+ -. 2mM to 35 mM.+ -. 2mM acetate buffer, (c) 0.1%.+ -. 0.05% to 0.3%.+ -. 0.05% w/v polysorbate, and (d) 5%.+ -. 1% to 15%.+ -. 3% w/v sucrose, pH 5.0.+ -. 0.5.

In some embodiments, the pharmaceutical formulation comprises: (a) 5 mg/ml.+ -. 0.5mg/ml antibody, (b) 30 mM.+ -. 1mM acetate buffer, (c) 0.2%.+ -. 0.02% w/v polysorbate, and (d) 10%.+ -. 1% w/v sucrose, pH 5.0.+ -. 0.3.

In some embodiments, the pharmaceutical formulation comprises: (a) 50mg/ml + -5 mg/ml antibody, (b) 25mM + -2 mM to 35mM + -2 mM acetate buffer, (c) 0.1% + -0.05% to 0.3% + -0.05% w/v polysorbate, and (d) 5% + -1% to 15% + -3% w/v sucrose, at a pH of 5.0 + -0.5.

In some embodiments, the pharmaceutical formulation comprises: (a) 50 mg/ml.+ -. 0.5mg/ml antibody, (b) 30 mM.+ -. 1mM acetate buffer, (c) 0.2%.+ -. 0.02% w/v polysorbate, and (d) 10%.+ -. 1% w/v sucrose, pH 5.0.+ -. 0.3.

In any of these embodiments, the polysorbate may be polysorbate 20.

In any of the various embodiments discussed above, the formulation contains no more than 2.5% High Molecular Weight (HMW) species after 12 months or 24 months of storage at 5 ℃ as determined by size exclusion ultra high performance liquid chromatography (SE-UPLC). In some cases, the formulation contains no more than 3.5% High Molecular Weight (HMW) species after storage for 6 months at 25℃and 60% relative humidity as determined by SE-UPLC. In some cases, the formulation contains no more than 1.5% High Molecular Weight (HMW) species after 12 months of storage at-30℃as determined by SE-UPLC, or no more than 2.0% HMW species after 24 months of storage at-30 ℃. In some cases, the formulation contains no more than 1.5% High Molecular Weight (HMW) species after 12 months of storage at-80℃as determined by SE-UPLC, or no more than 2.0% HMW species after 24 months of storage at-30 ℃.

In one aspect, the present invention provides a stable liquid pharmaceutical formulation reconstituted from a lyophilisate comprising: (a) A bispecific antibody at a concentration of 1mg/ml to 30mg/ml, wherein the bispecific antibody comprises a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a Heavy Chain Variable Region (HCVR), and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a light chain variable region (HCVR), wherein A1-HCDR1, A1-HCDR2 and A1-HCDR3 comprise amino acid sequences of SEQ ID NOs 7, 2-HCDR2 and 3, and HCDR 2-amino acid sequences of SEQ ID NOs 10, 14-HCDR 2 and 3, and amino acid sequences of SEQ ID NOs 10 and 12, respectively; (b) a buffer comprising histidine; (c) an organic co-solvent comprising polysorbate; and (d) a stabilizer comprising a sugar; wherein the pH of the preparation is 6.0+ -0.5.

In some cases, the antibody concentration is 2 mg/ml.+ -. 0.5mg/ml. In some cases, the antibody concentration is 20 mg/ml.+ -. 2mg/ml. In some cases, the histidine buffer concentration is 5 mM+ -1 mM to 15 mM+ -1 mM. In some cases, the histidine buffer concentration is 10 mM.+ -. 1mM. In some cases, polysorbate concentrations are 0.01% to 0.1% w/v. In some cases, the polysorbate concentration is 0.05% ± 0.01% w/v. In some cases, the polysorbate is polysorbate 20. In some embodiments, the sugar is sucrose. In some cases, the sucrose concentration is 8% ± 0.5% to 12% ± 0.5% w/v. In some cases, the sucrose concentration is 10% + -1% w/v.

In any embodiment of this aspect of the invention, the determination is made by SE-UPLC: (a) At least 95% of the antibodies have a native conformation after 12 months, 18 months, 24 months or 36 months of storage at 5 ℃; (b) After 6 months of storage at 25 ℃ and 60% relative humidity, at least 95% of the antibodies have a native conformation; (c) After 3 months of storage at 37 ℃, at least 95% of the antibodies have a native conformation; (d) After 12 months, 18 months, 24 months or 36 months of storage at 5 ℃, the formulation contains no more than 1% High Molecular Weight (HMW) species; (e) After 6 months of storage at 25℃and 60% relative humidity, the formulation contains no more than 1% HMW species; or (f) after 3 months of storage at 37 ℃, the formulation contains no more than 1% HMW species.

In one aspect, the present invention provides a stable liquid pharmaceutical formulation comprising: (a) A bispecific antibody at a concentration of 100mg/ml to 200mg/ml, wherein the bispecific antibody comprises a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a Heavy Chain Variable Region (HCVR), and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a light chain variable region (HCVR), wherein A1-HCDR1, A1-HCDR2 and A1-HCDR3 comprise amino acid sequences of SEQ ID NOs 7, 2-HCDR2 and 3, and HCDR 2-amino acid sequences of SEQ ID NOs 10, 14-HCDR 2 and 3, and amino acid sequences of SEQ ID NOs 10 and 12, respectively; (b) a buffer comprising acetate; (c) a stabilizer comprising a sugar; and (d) a polysorbate-containing surfactant, wherein the pH of the formulation is 5.0±0.5.

In some cases, the antibody concentration is 125mg/ml to 175mg/ml. In some cases, the antibody concentration is 150 mg/ml.+ -.10 mg/ml. In some embodiments, the sugar is sucrose. In some cases, the sucrose concentration is 4% to 12% w/v. In some cases, the sucrose concentration is 8% w/v.+ -. 1% w/v. In some cases, the acetate buffer concentration is 25mM to 35mM. In some cases, the acetate buffer concentration is 30 mM.+ -. 1mM. In some cases, the polysorbate is polysorbate 20. In some cases, polysorbate 20 concentration is 0.01% w/v to 0.1% w/v. Polysorbate 20 concentration was 0.05% w/v.+ -. 0.01% w/v.

In any embodiment of this aspect of the invention, the determination is made by SE-UPLC: (a) After 12 months or 24 months of storage at-30 ℃ or-80 ℃, the formulation contains no more than 2.5% High Molecular Weight (HMW) species; (b) After 6 months of storage at 5 ℃, the formulation contains no more than 4% HMW species; or (c) the formulation contains no more than 6% HMW species after storage at 25℃and 60% relative humidity for 6 months.

In any of the various embodiments discussed above or herein, the formulation contains NO more than 40%, NO more than 39%, NO more than 38%, NO more than 37%, NO more than 36%, or NO more than 35% of a variant of a glycosylated substance, wherein the variant of a glycosylated substance comprises glycosylation at residue 98 of SEQ ID NO:1 or SEQ ID NO:4 or at residue 2 of SEQ ID NO: 9.

In any of the various embodiments discussed above or herein, the first antigen binding domain comprises a HCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 6.

In any of the various embodiments discussed above or herein, the first antigen binding domain comprises a HCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 6.

In any of the various embodiments discussed above or herein, the first antigen binding domain comprises a HCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 6.

In any of the various embodiments discussed above or herein, the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID No. 4 and a LCVR comprising the amino acid sequence of SEQ ID No. 6 and the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID No. 5 and a LCVR comprising the amino acid sequence of SEQ ID No. 6.

In one aspect, the present invention provides a stable pharmaceutical formulation comprising: (a) 5 mg/ml.+ -. 0.5mg/ml of a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 4 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6, and the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 5 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6; (b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2, (c) 0.2% + -0.02% w/v polysorbate 20, and (d) 10% + -1% w/v sucrose.

In one aspect, the present invention provides a stable pharmaceutical formulation comprising: (a) 50 mg/ml.+ -.5 mg/ml of a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 4 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6, and the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 5 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6; (b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2, (c) 0.2% + -0.02% w/v polysorbate 20, and (d) 10% + -1% w/v sucrose.

In one aspect, the present invention provides a stable pharmaceutical formulation comprising: (a) A bispecific antibody of 150mg/ml ± 15mg/ml comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2, (c) 0.05% + -0.01% w/v polysorbate 20, and (d) 8% + -1% w/v sucrose.

In any of the various embodiments discussed above or herein, the antibody comprises a human IgG heavy chain constant region of HCVR attached to each of the first and second antigen binding domains, respectively. In some cases, the heavy chain constant region is of isotype IgG1. In some cases, the heavy chain constant region is of isotype IgG4.

In some embodiments, the heavy chain constant region attached to the HCVR of the first antigen binding domain or the heavy chain constant region attached to the HCVR of the second antigen binding domain, but not both, contains an amino acid modification that reduces binding of protein a relative to an unmodified heavy chain of the same isotype. In some cases, the modification comprises an H435R substitution (EU numbering) in the isotype IgG1 or IgG4 heavy chain. In some cases, the modifications include H435R substitution and Y436F substitution (EU numbering) in the isotype IgG1 or IgG4 heavy chain.

In any of the various embodiments discussed above or herein, the antibody comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 16 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 17. In some embodiments, the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 18 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 19.

In any of the various embodiments discussed above or herein, the antibody comprises a first heavy chain of an HCVR comprising a first antigen binding domain and a second heavy chain of an HCVR comprising a second antigen binding domain, wherein the first heavy chain comprises residues 1-442 of the amino acid sequence of SEQ ID No. 1 and the second heavy chain comprises residues 1-449 of the amino acid sequence of SEQ ID No. 2. In some embodiments, the antibody comprises a common light chain of the LCVR comprising the first and second antigen-binding domains, wherein the common light chain comprises the amino acid sequence of SEQ ID NO. 3.

In any of the various embodiments discussed above or herein, the pharmaceutical formulation may be considered stable by measuring the percent change in "glycosylated species" as determined by cation exchange ultra high performance liquid chromatography (CEX-UPLC) and/or liquid chromatography-mass spectrometry (LC-MS), wherein the percent change in glycosylated species: (i) no more than 1.5% after storage at 5 ℃ for 6 months; (ii) no more than 3% after 12 months of storage at 5 ℃; (iii) No more than 1.5% after 12 months, 18 months or 24 months of storage at-30 ℃; or no more than 1% after 12 months, 18 months or 24 months of storage at-80 ℃.

In one aspect, the invention provides a pharmaceutical composition, wherein the composition comprises a pharmaceutical formulation as discussed above or herein, and the composition is contained in a container.

In some embodiments, the container is a vial. In some cases, the vial is a type 1 clear glass vial of 2ml, 5ml, or 10 ml. In some embodiments, the container is a syringe. In some cases, the injector is low tungsten glass. In some embodiments, the container is a prefilled syringe. In some embodiments, the pharmaceutical composition is contained in an auto-injector.

In one aspect, the invention provides a kit comprising (i) a container containing a composition comprising a pharmaceutical formulation as discussed above or herein, and (ii) instructions for use of the composition.

In some embodiments, the container is a glass vial. In some embodiments, the container is a prefilled syringe. In some embodiments, the container is an automatic injector.

In some embodiments, the instructions describe subcutaneous administration of the composition. In some embodiments, the instructions describe intravenous administration of the composition.

In one aspect, the invention provides a unit dosage form comprising the pharmaceutical formulation discussed above or herein, wherein the antibody is present in an amount of 0.1mg to 500 mg. In some cases, the antibody is present in an amount of 5±1mg to 50±5 mg. In some cases, the antibody is present in an amount of 10±1mg to 200±20 mg. In some embodiments, the antibody is present in an amount of 4mg, 5mg, 10mg, 12.5mg, 40mg, 50mg, 150mg, or 180 mg.

In some embodiments, the unit dosage form is a glass vial, a prefilled syringe, or an auto-injector.

In one aspect, the present invention provides a container containing a composition comprising a pharmaceutical formulation as discussed above or herein. In various embodiments, the container is a glass vial, a prefilled syringe, or an automatic syringe.

In various embodiments, any features or components of the embodiments discussed above or herein may be combined, and such combinations are contemplated as being within the scope of the present disclosure. Any of the specified values discussed above or herein may be combined with another related value discussed above or herein to list a range, the values representing the upper and lower limits of the range, and such ranges and all values falling within such ranges are encompassed within the scope of the disclosure. Each value discussed above or herein may be represented by a 1%, 5%, 10% or 20% change. For example, a concentration of 10mM can be expressed as 10 mM.+ -. 0.1mM (1% change), 10 mM.+ -. 0.5mM (5% change), 10 mM.+ -. 1mM (10% change), or 10 mM.+ -. 2mM (20% change).

Other embodiments will become apparent upon reading the detailed description that follows.

Drawings

FIG. 1 illustrates the relationship between the relative potency of mAb1 and the glycosylation level of HCDR3-Lys 98. Glycosylated and non-glycosylated mAb1 were purified by preparative cation exchange chromatography and the two substances were mixed in different ratios to produce the level of glycosylation. Efficacy data indicate that efficacy is dependent on glycosylation levels.

FIGS. 2A, 2B, 2C and 2D illustrate the effect of pH on glycosylation and High Molecular Weight (HMW) species levels of mAb1 formulated in histidine buffer. The formulations included 2mg/ml mAb1 in 10mM histidine, 10% w/v sucrose and 0.05% w/v polysorbate 20, with different pH values, and incubated for up to 36 months at 5 ℃ or up to 2 months at 25 ℃. Glycosylation levels were monitored by cation exchange chromatography (CEX-UPLC) at 5 ℃ (fig. 2A) or 25 ℃ (fig. 2B), and HMW levels were monitored by size exclusion chromatography (SE-UPLC) at 5 ℃ (fig. 2C) or 25 ℃ (fig. 2D).

FIGS. 3A and 3B illustrate the effect of pH on glycosylation and HMW species levels of mAb1 formulated in acetate buffer. The formulations included 50mg/ml mAb1 in 10mM acetate and 5% w/v sucrose, with different pH values, and incubated at 40℃for 28 days. Glycosylation levels were monitored by CEX-UPLC (FIG. 3A) and HMW levels by SE-UPLC (FIG. 3B).

Figure 4 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on viscosity of mAb1 formulation. These figures represent statistical models of experimental data.

Figure 5 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on the osmotic pressure of mAb1 formulations. These figures represent statistical models of experimental data.

Figure 6 illustrates the effect of mAb1 concentration, sucrose concentration, and arginine concentration on mAb1 formulation stability. These figures represent statistical models of experimental data.

Figures 7A and 7B illustrate the stability of two mAb1 formulations for subcutaneous administration. Both formulations exhibited comparable stability when incubated under the test conditions: at 40 ℃/75% Relative Humidity (RH) for 3 months; at 25 ℃/60% rh for 6 months; and 6 months at 2-8 ℃. As shown, F1 contained 150mg/ml mAb1, 30mM acetate pH 5.0, 8% w/v sucrose and 0.05% w/v polysorbate 80, and F2 contained 150mg/ml mAb1, 30mM acetate pH 5.0, 7% w/v sucrose, 50mM arginine and 0.05% w/v polysorbate 80.

Detailed Description

Before describing the present invention, it is to be understood that this invention is not limited to the particular methodology and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the term "about," when used in reference to a particular recited value or range of values, means that the value may differ from the recited value by no more than 1%. For example, as used herein, the expression "about 100" includes 99 and 101 and all values therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are now described. All patents, applications, and non-patent publications mentioned in this specification are herein incorporated by reference in their entirety.

Pharmaceutical preparation

As used herein, the expression "pharmaceutical formulation" means a combination of at least one active ingredient (e.g., a bispecific anti-MUC 16 x anti-CD 3 antibody, etc., which is capable of exerting a biological effect in a human or non-human animal) and at least one inactive ingredient which, when combined with the active ingredient and/or one or more additional inactive ingredients, is suitable for therapeutic administration to a human or non-human animal. As used herein, the term "formulation" refers to "pharmaceutical formulation" unless explicitly stated otherwise. The present invention provides pharmaceutical formulations comprising at least one therapeutic polypeptide. According to certain embodiments of the invention, the therapeutic polypeptide is a bispecific antibody that specifically binds to human MUC16 and human CD3 or antigen-binding fragments thereof. More specifically, the invention includes, inter alia, pharmaceutical formulations comprising: (i) Human bispecific antibodies that specifically bind human MUC16 and human CD 3; (ii) a buffer comprising acetate; (iii) an organic co-solvent comprising a polysorbate; and (iv) a stabilizer comprising a sugar. Additional components may be included in the formulations of the present invention if such components do not significantly interfere with the stability of the formulation. Specific exemplary components and formulations included in the present invention are described in detail below.

In certain embodiments, the pharmaceutical formulation of the present invention may be a fluid formulation. The expression "fluid formulation" as used herein means a mixture of at least two components that are present predominantly in a fluid state at about 2 ℃ to about 45 ℃. The fluid formulation includes, inter alia, a liquid formulation. The fluid formulation may have a low, medium or high viscosity depending on its particular composition.

Bispecific antibodies that specifically bind to human MUC16 and human CD3

The pharmaceutical formulation of the invention may comprise a human bispecific antibody or antigen-binding fragment thereof that specifically binds human MUC16 and human CD 3.

As used herein, the term "antibody", including "bispecific antibody", is generally intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy chains (H chains) and two light chains (L chains) that are interconnected by disulfide bonds, and multimers thereof (e.g., igM); however, immunoglobulin molecules consisting of heavy chains only (i.e., without light chains) are also encompassed within the definition of the term "antibody". Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL 1). VH and VL regions can also be subdivided into regions of hypervariability (termed Complementarity Determining Regions (CDRs)) interspersed with regions that are more conserved (termed Framework Regions (FR)). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

In certain embodiments of the invention, the anti-MUC 16 x anti-CD 3 bispecific antibodies of the invention are human antibodies. As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo), e.g., in CDRs, particularly CDR 3. However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (such as a mouse) have been implanted onto a human framework sequence. In various embodiments, the anti-MUC 16 x anti-CD 3 bispecific antibody is a human IgG antibody. In various embodiments, the anti-MUC 16 x anti-CD 3 bispecific antibody is a human antibody of isotype IgG1, igG2, igG3, or IgG4 or mixed isotype. In some embodiments, the anti-MUC 16 x anti-CD 3 bispecific antibody is a human IgG1 antibody (i.e., the antibody comprises a human IgG1 heavy chain constant region of HCVR attached to each of the first and second antigen binding domains, respectively). In some embodiments, the anti-MUC 16 x anti-CD 3 bispecific antibody is a human IgG4 antibody (i.e., the antibody comprises a human IgG4 heavy chain constant region of HCVR attached to each of the first antigen binding domain and the second antigen binding domain, respectively; in any of the embodiments discussed above or herein, the anti-MUC 16 x anti-CD 3 bispecific antibody may comprise a human kappa light chain.

In any embodiment, the bispecific antibody may include modifications in one or both heavy chains to facilitate purification of the bispecific antibody (i.e., heterodimer) from homodimeric impurities. In some embodiments, the bispecific antibody comprises a first and a second heavy chain (i.e., the heavy chain of the anti-MUC 16 binding arm and the heavy chain of the anti-CD 3 binding arm) that are identical except for a modification in the CH3 domain of one or the other heavy chain (e.g., both isotypes IgG1 or IgG 4), which modification reduces binding of the bispecific antibody to protein a as compared to an antibody lacking the modification. In some cases, the CH3 domain of the first heavy chain (e.g., the first heavy chain of the anti-MUC 16 binding arm) binds protein a, and the CH3 domain of the second heavy chain (e.g., the second heavy chain of the anti-CD 3 binding arm) contains mutations that reduce or eliminate protein a binding. In some cases, the mutation is an H435R modification (numbered by EU; H95R numbered by IMGT exon). In some cases, the mutations are H435R modification (numbered by EU; H95R numbered by IMGT exon) and Y436F modification (numbered by EU; Y96F numbered by IMGT). Additional modifications that may be found in the second CH3 domain include: in the case of IgG1 CH3 domains, D356E, L358M, N384S, K392N, V397M and V422I by EU numbering (D16E, L18M, N44S, K52N, V57M and V82I by IMGT numbering); and in the case of IgG4CH3 domains, Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU numbering (Q15R, N44S, K52N, V57M, R69K, E79Q and V82I by IMGT numbering).

In any embodiment, the bispecific antibody can comprise a chimeric hinge. The term "chimeric hinge" is intended to include chimeric proteins comprising a first amino acid sequence derived from a hinge region of one Ig molecule and a second amino acid sequence derived from a hinge region of a different class or subclass of Ig molecule. For example, in embodiments, the chimeric hinge comprises a first amino acid sequence or "upper hinge" sequence derived from a human IgG1 hinge region or a human IgG4 hinge region, and a second amino acid sequence or "lower hinge" sequence derived from a human IgG2 hinge region. In certain embodiments, the first or "upper hinge" sequence comprises amino acid residues 216 to 227 (numbering according to EU). In some embodiments, the second or "lower hinge" sequence comprises amino acid residues 228 to 236 (numbering according to EU).

In some embodiments, the presentThe antibodies of the invention may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from recombinant human antibody combinatorial libraries, antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, e.g., taylor et al, (1992) nucleic acids res.20:6287-6295), or antibodies prepared, expressed, produced or isolated by any other means that involves splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when using animals that are transgenic for human Ig sequences, undergo in vivo somatic mutagenesis) and thus V of the recombinant antibodies _H And V _L The amino acid sequence of the region is that which is although derived from human germline V _H And V _L Sequences and related thereto, but may not naturally occur within the germline repertoire of human antibodies in vivo.

As used herein, the term "antigen binding portion" or "antigen binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to human MUC16 or human CD3.

As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated bispecific antibody that specifically binds human MUC16 and human CD3 is substantially free of antibodies that specifically bind antigens other than human MUC16 and human CD 3).

The term "specifically binds" or the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is characterized by at least about 1x10 ^-6 M or greater. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, specific bindingThe isolated antibodies to human MUC16 and human CD3 may have cross-reactivity with other antigens, such as MUC16 or CD3 molecules from other species (orthologs). In the context of the present invention, multispecific (e.g., bispecific) antibodies that bind to human MUC16 and human CD3, as well as one or more additional antigens, are considered to "specifically bind" to human MUC16 and human CD3. Furthermore, the isolated antibodies may be substantially free of other cellular material and/or chemicals.

Exemplary anti-MUC 16x anti-CD 3 bispecific antibodies that may be included in the pharmaceutical formulations of the present invention are listed in WO 2018/067331, the disclosure of which is incorporated by reference in its entirety.

According to certain embodiments of the invention, the anti-MUC 16x anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises heavy chain Complementarity Determining Regions (CDRs) A1-HCDR1, A1-HCDR2 and A1-HCDR3 comprising the amino acid sequences of SEQ ID NO:7, 8 and 9, respectively, and the second antigen binding domain comprises heavy chain CDRs A2-HCDR1, A2-HCDR2 and A2-HCDR3 comprising the amino acid sequences of SEQ ID NO:10, 11 and 12, respectively. According to certain embodiments of the invention, the anti-MUC 16x anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a common (first and second antigen binding domain) light chain complementarity determining regions LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of SEQ ID NOS: 13, 14 and 15, respectively.

In certain embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody or antigen-binding fragment thereof comprises a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises a Heavy Chain Variable Region (HCVR) comprising the amino acid sequence of SEQ ID No. 4 and the second antigen-binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID No. 5. In certain embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a common Light Chain Variable Region (LCVR) comprising the amino acid sequence of SEQ ID NO. 6. In certain embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody or antigen binding fragment thereof comprises a first antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 4/6, and a second antigen binding domain comprising a HCVR/LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO. 5/6. In some embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody comprises the HCVR/LCVR sequence pair described above and a human IgG1 heavy chain constant region. In some embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody comprises the HCVR/LCVR sequence pair described above and a human IgG4 heavy chain constant region. In some embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody comprises the HCVR/LCVR sequence pair described above and a human IgG heavy chain constant region. In some embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody comprises the HCVR/LCVR sequence pair described above and a human IgG1 or IgG4 heavy chain constant region. In some embodiments, the anti-MUC 16x anti-CD 3 bispecific antibody comprises a first heavy chain comprising the amino acid sequence of SEQ ID NO. 1, a second heavy chain comprising the amino acid sequence of SEQ ID NO. 2, and a common light chain comprising the amino acid sequence of SEQ ID NO. 3. The anti-MUC 16x anti-CD 3 bispecific antibody has a first antigen binding domain (which comprises a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6) that specifically binds human MUC16 and a second antigen binding domain (which comprises a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6) that specifically binds human CD3, referred to herein as mAb1. The antibody has a first heavy chain comprising the amino acid sequence of SEQ ID NO. 1 (including a HCVR that specifically binds human MUC 16), a second heavy chain comprising the amino acid sequence of SEQ ID NO. 2 (including a HCVR that specifically binds human CD 3), and a common light chain comprising the amino acid sequence of SEQ ID NO. 3. In some cases, the mature form of the antibody may not include the C-terminal lysine residues of SEQ ID NOS 1 and 2. Thus, in some cases, the anti-MUC 16 binding arm of mAb1 comprises a heavy chain comprising residues 1-442 of SEQ ID NO. 1 and the anti-CD 3 binding arm of mAb1 comprises a heavy chain comprising residues 1-449 of SEQ ID NO. 2.

The amount of antibody or antigen-binding fragment thereof contained in the pharmaceutical formulation of the present invention may vary depending on the particular properties desired for the formulation and the particular situation and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation may contain about 0.1mg/mL to about 500mg/mL of antibody; about 0.5mg/mL to about 400mg/mL of antibody; about 1mg/mL to about 200mg/mL of antibody; about 2mg/mL to about 100mg/mL; about 1mg/mL to about 5mg/mL of antibody; about 10mg/mL to about 30mg/mL of antibody; about 75mg/mL to about 125mg/mL; about 5mg/mL to about 50mg/mL; about 4mg/ml to about 60mg/ml; or about 2mg/mL to about 55mg/mL of antibody. For example, the formulation of the invention may be a liquid formulation comprising about 0.5mg/mL; about 1mg/mL; about 2mg/mL; about 3mg/mL; about 4mg/mL; about 5mg/mL; about 6mg/mL; about 7mg/mL; about 8mg/mL; about 9mg/mL; about 10mg/mL; about 11mg/mL; about 12mg/mL; about 13mg/mL; about 14mg/mL; about 15mg/mL; about 16mg/mL; about 17mg/mL; about 18mg/mL; about 19mg/mL; about 20mg/mL; about 21mg/mL; about 22mg/mL; about 23mg/mL; about 24mg/mL; about 25mg/mL; about 26mg/mL; about 27mg/mL; about 28mg/mL; about 29mg/mL; about 30mg/mL; about 35mg/mL; about 40mg/mL; about 45mg/mL; about 50mg/mL; about 55mg/mL; about 60mg/mL; about 65mg/mL; about 70mg/mL; about 75mg/mL; about 80mg/mL; about 85mg/mL; about 90mg/mL; about 95mg/mL; about 96mg/mL; about 97mg/mL; about 98mg/mL; about 99mg/mL; about 100mg/mL; about 101mg/mL; about 102mg/mL; about 103mg/mL; about 104mg/mL; about 105mg/mL; about 110mg/mL; about 115mg/mL; about 120mg/mL; about 125mg/mL; about 130mg/mL; about 135mg/mL; about 140mg/mL; about 145mg/mL; about 150mg/mL; about 155mg/mL; about 160mg/mL; about 165mg/mL; about 170mg/mL; about 175mg/mL; about 180mg/mL; about 185mg/mL; about 190mg/mL; about 195mg/mL; or about 200mg/mL of an antibody, or antigen-binding fragment thereof, that specifically binds human MUC16 and human CD3. In certain embodiments, the pharmaceutical formulation is a liquid formulation, which may contain 1±0.1mg/mL to 200±20mg/mL of antibody; antibodies ranging from 2±0.2mg/mL to 10±1mg/mL; 1+ -0.5 mg/mL to 30+ -5 mg/mL of antibody; antibodies of 40+ -4 mg/mL to 60+ -6 mg/mL; 1+ -0.1 mg/mL to 3+ -0.3 mg/mL of antibody; 3+ -0.5 mg/mL to 7+ -0.5 mg/mL of antibody; 45+ -1 mg/mL to 55+ -1 mg/mL of antibody; 140+ -5 mg/ml to 160+ -5 mg/ml of antibody; or 175+ -5 mg/mL to 185+ -5 mg/mL of antibody. In some embodiments, the pharmaceutical formulation contains 5.+ -. 0.5mg/mL of antibody. In some embodiments, the pharmaceutical formulation contains 50.+ -.5 mg/mL of antibody. In some embodiments, the pharmaceutical formulation contains 150.+ -.15 mg/ml of antibody. In some embodiments, the pharmaceutical formulation contains 2±0.2mg/ml of antibody. In some embodiments, the pharmaceutical formulation contains 20.+ -.2 mg/ml of antibody. In some embodiments, the pharmaceutical formulation contains 180.+ -.10 mg/ml of antibody.

Bioequivalence

The present invention encompasses antibodies having amino acid sequences different from the exemplary molecules disclosed herein, but retaining the ability to bind human MUC16 and human CD 3. Such variant molecules may comprise one or more amino acid additions, deletions or substitutions when compared to the parent sequence, but exhibit biological activity substantially equivalent to the antibodies discussed herein.

The invention includes antigen binding molecules bioequivalent to any of the exemplary antibodies set forth herein. For example, two antibodies are considered bioequivalent if they are drug equivalents or drug substitutes, i.e., they do not exhibit a significant difference in their rate and extent of absorption when administered in the same molar dose (single or multiple doses) under similar experimental conditions. If certain antibodies are equivalent in the extent of absorption but not in the rate of absorption, they are considered equivalent or drug substitutes, but because such differences in the rate of absorption are intentional and reflected in the label, they may be considered bioequivalent, are not necessary to achieve an effective in vivo drug concentration, for example, over a prolonged period of use, and are not considered clinically significant for the particular drug under study.

In one embodiment, two antibodies are bioequivalent if they have no clinically significant differences in safety, purity, and efficacy.

In one embodiment, two antibodies are bioequivalent if a patient can perform such a switch one or more times without an expected increase in risk of adverse reactions, including clinically significant changes in immunogenicity, or reduced effectiveness, as compared to continuous therapies that do not perform a switch between a reference product and a biologic product.

Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measures include, for example, (a) in vivo tests in humans or other mammals in which the concentration of antibodies or their metabolites in blood, plasma, serum or other biological fluids is measured over time; (b) In vitro tests associated with and reasonably predictive of human bioavailability data; (c) In vivo tests in humans or other mammals, in which appropriate acute pharmacological effects of the antibody (or target thereof) over time are measured; and (d) establishing a clinical trial of a good control of the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein.

Formulation excipients and pH

The pharmaceutical formulations of the present invention comprise one or more excipients. As used herein, the term "excipient" refers to any non-therapeutic agent added to a formulation to provide a desired consistency, viscosity, or stabilizing effect.

In certain embodiments, the pharmaceutical formulations of the present invention comprise one or more carbohydrates, such as one or more sugars. The sugar may be a reducing sugar or a non-reducing sugar. "reducing sugars" include, for example, sugars having ketone or aldehyde groups and contain reactive hemiacetal groups, which allow the sugar to act as a reducing agent. Specific examples of reducing sugars include fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose and maltose. The non-reducing sugar may comprise an anomeric carbon which is an acetal and which is substantially non-reactive with an amino acid or polypeptide to initiate a maillard reaction. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, melezitose, and raffinose. Sugar acids include, for example, sugar acids, gluconate and other polyhydroxy sugars and salts thereof. In some embodiments, the sugar is sucrose. In some cases, a sugar (e.g., sucrose) acts as a heat stabilizer for the anti-MUC 16 x anti-CD 3 bispecific antibody.

The amount of sugar (e.g., sucrose) included in the pharmaceutical formulations of the present invention will vary depending upon the particular situation and the intended purpose of the formulation use. In certain embodiments, the formulation may contain from about 0.1% to about 20% sugar; about 0.5% to about 20% sugar; about 1% to about 20% sugar; about 2% to about 15% sugar; about 5% to about 15% sugar; about 7.5% to about 12.5% sugar; or about 9% to about 11% sugar. For example, a pharmaceutical formulation of the invention may comprise about 0.5%; about 1.0%; about 1.5%; about 2.0%; about 2.5%; about 3.0%; about 3.5%; about 4.0%; about 4.5%; about 5.0%; about 5.5%; about 6.0%; about 6.5%; about 7.0%; about 7.5%; about 8.0%; about 8.5%; about 9.0%; about 9.5%; about 10.0%; about 10.5%; about 11.0%; about 11.5%; about 12.0%; about 12.5%; about 13.0%; about 13.5%; about 14.0%; about 14.5%; about 15%; or about 20% sugar (e.g., sucrose). In some embodiments, the formulation contains about 10% sugar (e.g., sucrose). In some embodiments, the formulation contains about 5% sugar (e.g., sucrose). Each of the percentages mentioned above corresponds to a weight/volume percentage (w/v). In some cases, the formulation contains 5% ± 1% to 20% ± 4% w/v sucrose. In some cases, the formulation contains 5% to 10% w/v sucrose. In some cases, the formulation contains 8% ± 0.5% to 12% ± 0.5% w/v sucrose. In some cases, the formulation contains 10% ± 1% w/v sucrose.

The pharmaceutical formulations of the invention may also comprise one or more organic co-solvents (or interfacial stabilizers), of the type and amount that stabilize the anti-MUC 16 x anti-CD 3 bispecific antibody under rough handling or agitation (such as, for example, rotary shaking). In some embodiments, the organic co-solvent is a surfactant. As used herein, the term "surfactant" means a substance that reduces the surface tension of a fluid dissolved therein and/or reduces the interfacial tension between oil and water. The surfactant may be ionic or nonionic. Exemplary nonionic surfactants that may be included in the formulations of the present invention include, for example, alkyl poly (ethylene oxide), alkyl polyglycosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols (such as cetyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, and cocamide TEA. Specific nonionic surfactants that may be included in the formulations of the present invention include, for example, polysorbates (such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85); poloxamers (such as poloxamer 188 (also known as pluronic F68), poloxamer 407); polyethylene glycol-polypropylene glycol; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate and polyoxyethylene sorbitan monolaurate. In some embodiments, the surfactant is polysorbate 20.

The amount of surfactant included in the pharmaceutical formulations of the present invention may vary depending on the particular nature of the formulation desired and the particular situation and purpose for which the formulation is intended. In certain embodiments, the formulation may contain from about 0.01% to about 1% surfactant; from about 0.01% to about 0.5% surfactant; about 0.1% to about 0.3%; from about 0.15% to about 0.25% surfactant; or about 0.19% to about 0.21% surfactant. For example, the formulation of the present invention may comprise about 0.01%; about 0.02%; about 0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.10%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; or about 0.30% surfactant (e.g., polysorbate 20). In some embodiments, the formulation contains about 0.2% surfactant (e.g., polysorbate 20). In some embodiments, the formulation contains about 0.05% surfactant (e.g., polysorbate 20). Each of the percentages mentioned above corresponds to a weight/volume percentage (w/v). In some cases, the formulation contains 0.01% ± 0.005% to 0.5% ± 0.25% w/v polysorbate 20. In some cases, the formulation contains 0.2% ± 0.05% w/v polysorbate 20. In some cases, the formulation contains 0.2% ± 0.01% w/v polysorbate 20.

The pharmaceutical formulations of the invention may also comprise a buffer or buffer system for maintaining a stable pH and helping to stabilize the anti-MUC 16 x anti-CD 3 bispecific antibody. In some embodiments, the buffer or buffer system comprises at least one buffer having a buffer range that completely or partially overlaps the range of pH 4.5 to 5.5. In certain embodiments, the buffer comprises an acetate buffer (e.g., sodium acetate). In certain embodiments, the buffer (e.g., acetate) is at about 1mM to about 50mM, about 20mM to about 40mM, about 25mM to about 35mM; about 28mM to about 32mM; or about 29mM to about 31 mM. In some embodiments, the buffer (e.g., acetate) is at about 20mM; about 21mM; about 22mM; about 23mM; about 24mM; about 25mM; about 26mM; about 27mM; about 28mM; about 29mM; about 30mM; about 31mM; about 32mM; about 33mM; about 34mM; about 35mM; about 36mM; about 37mM; about 38mM; about 39mM; or about 40 mM. In some cases, the buffer is histidine at about 1mM; about 2mM; about 3mM; about 4mM; about 5mM; about 6mM; about 7mM; about 8mM; about 9mM; about 10mM; about 11mM; about 12mM; about 13mM; about 14mM; about 15mM; about 16mM; about 17mM; about 18mM; about 19mM; or about 20 mM. In some cases, the formulation contains histidine buffer at a concentration of 5 mM+ -1 mM to 15 mM+ -3 mM. In some cases, the formulation contains histidine buffer at a concentration of 10mm±1 mM. In some embodiments, the formulation contains acetate buffer (e.g., at any of the concentrations discussed above or herein). In some embodiments, the formulation contains phosphate buffer (e.g., at any of the concentrations discussed above or herein).

In some embodiments, the pharmaceutical formulation of the present invention may further comprise arginine. In some cases, arginine is present at a concentration of 1 to 100 mM. In some embodiments, arginine is present at a concentration of 25 to 75 mM. In some cases, arginine is present at a concentration of 50mm±5 mM. In one embodiment, the pharmaceutical formulation comprises 30 mM+ -3 mM acetate, pH 5.0+ -0.1, 7% + -0.7% w/v sucrose, 0.05% + -0.01% w/v polysorbate (e.g., polysorbate 20), and 50 mM+ -5 mM arginine. In some cases, the antibody is present at a concentration of 1mg/ml to 200mg/ml or 150 mg/ml.+ -. 10 mg/ml.

During the antibody purification process, it may be necessary or necessary to exchange one buffer for another to obtain the appropriate excipient concentration, antibody concentration, pH, etc. Buffer exchange may be accomplished, for example, by ultrafiltration/diafiltration (UF/DF) using, for example, semi-permeable tangential flow filtration membranes. However, the use of such techniques may result in the Gibbs-Donnan effect (Bolton et al 2011, biotechnol. Prog.27 (1): 140-152). During protein concentration, the accumulation of positive charge on the product side of the membrane is counteracted by preferential movement of cations to the opposite side of the membrane. A potential consequence of this phenomenon is that the final concentration of certain components (e.g., acetate salts, etc.) may be lower than the intended target concentration of these components due to electrostatic repulsion of positively charged antibody proteins by positively charged diafiltration buffer excipients during the UF/DF step. Thus, the present invention includes formulations wherein, due to the Gibbs-Donnan effect, for example, the concentration of acetate is different from the amounts or ranges described herein.

Size exclusion describes the behavior of a highly concentrated sample in which a significant portion of the total volume of the solution is occupied by solutes, particularly macromolecules such as proteins, excluding solvents in that space. This reduces the total volume of solvent available to dissolve other solutes, which can lead to maldistribution on the ultrafiltration membrane. Thus, the present invention includes formulations wherein the concentration of, for example, acetate may vary from the amounts or ranges described herein due to the size exclusion effect.

During the manufacture of the formulations of the present invention, the composition of the formulation may change. These changes may include the concentration of the active ingredient, the concentration of the excipient, and/or the pH of the formulation. The invention includes formulations comprising anti-MUC 16 x anti-CD 3 bispecific antibodies that are stable and remain potent at excipient concentrations varying up to at least 10%. For example, included herein are anti-MUC 16 x anti-CD 3 bispecific antibody formulations, wherein the stability and potency of the formulation is not affected by variations in the concentration of antibodies, sucrose, acetate buffer, and/or polysorbate by ±10% or ±20%.

Stability of pharmaceutical formulations

The pharmaceutical formulations of the present invention exhibit a high level of stability. The term "stable" as used herein with reference to a pharmaceutical formulation means that the antibodies in the pharmaceutical formulation remain acceptably structurally and/or functionally and/or biologically active after storage for a defined amount of time. The formulation may be stable even if the antibodies contained therein do not retain 100% of the structure and/or function and/or biological activity after storage for a defined amount of time. In certain instances, an antibody that retains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure and/or function and/or biological activity after storage for a defined amount of time may be considered "stable".

Stability can be measured, inter alia, by determining the percentage of natural antibodies retained in the formulation after storage for a defined amount of time at a given temperature. The percentage of natural antibodies can be determined by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SE-HPLC ]). The phrase "acceptable degree of stability" as used herein means that at least 90% of the antibody in its native form is detectable in the formulation after storage at a given temperature for a given amount of time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the antibody in its native form can be detected in the formulation after storage for a defined amount of time at a given temperature. The defined amount of time after measuring stability may be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months or more. When evaluating stability, the temperature at which the pharmaceutical formulation may be stored may be any temperature from about-80 ℃ to about 45 ℃, for example, at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ° -8 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 37 ℃, or about 45 ℃. For example, a pharmaceutical formulation is also considered stable if greater than about 90%, 95%, 96% or 97% of the native antibody is detected by SE-HPLC after 3 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96% or 97% of the native antibody is detected by SE-HPLC after 6 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 9 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 12 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 24 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 36 months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 3 months of storage at 25 ℃ (and optionally 60% relative humidity). Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after 6 months of storage at 25 ℃ (and optionally 60% relative humidity). Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% of the native antibody is detected by SE-HPLC after storage for 9 months at 25 ℃ (and optionally 60% relative humidity). Pharmaceutical formulations are also considered stable if greater than about 90%, 95%, 96% or 97% of the native antibody is detected by SE-HPLC after 3 months of storage at 37 ℃. Pharmaceutical formulations are also considered stable if greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% of the native antibody is detected by SE-HPLC after 1 month of storage at 45 ℃ (and optionally 75% relative humidity).

Other methods may be used to evaluate the stability of the formulations of the invention, such as using Differential Scanning Calorimetry (DSC) to determine thermal stability, controlled agitation to determine mechanical stability, and absorbance at about 350nm or about 405nm to determine solution turbidity. For example, if inOD of the formulation after storage at about 5 ℃ to about 25 ℃ for 6 months or longer ₄₀₅ The change in (c) is less than the OD of the formulation at t=0 ₄₀₅ The formulation of the invention may be considered stable if it is about 0.05 (e.g., 0.04, 0.03, 0.02, 0.01, or less).

Measuring the binding affinity of an antibody to its target can also be used to assess stability. For example, a formulation of the invention may be considered stable if the binding affinity of an anti-MUC 16 x anti-CD 3 bispecific antibody comprised in the formulation to human MUC16 and human CD3 is at least 80%, 85%, 90%, 95% or more of the binding affinity of the antibody prior to storage after a defined amount of storage (e.g. 14 days to 9 months) such as-80 ℃, -30 ℃, -20 ℃, 5 ℃, 25 ℃, 37 ℃, 45 ℃, etc. Binding affinity may be determined by any method, such as, for example, ELISA or plasma resonance. Biological activity may be determined by a MUC16 or CD3 activity assay, such as by contacting a cell expressing MUC16 or CD3 with a formulation comprising an anti-MUC 16 x anti-CD 3 bispecific antibody. Binding of antibodies to such cells can be measured directly, such as by FACS analysis.

Stability may be determined, inter alia, by determining the percentage of antibodies that form aggregates (high molecular weight (HMW) species) after a formulation has been stored at a defined temperature for a defined amount of time, wherein stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated antibodies may be determined by, inter alia, size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [ SE-HPLC ] or size exclusion ultra high performance liquid chromatography [ SE-UPLC ]). The phrase "acceptable degree of stability" as used herein means that up to 6% of the antibody in aggregated form is detected in the formulation after storage for a defined amount of time at a given temperature (up to 25 ℃). In certain embodiments, an acceptable degree of stability means that up to about 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody in aggregate form is detectable in the formulation after storage for a defined amount of time at a given temperature. The defined amount of time after measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months or longer. When evaluating stability, the temperature at which the pharmaceutical formulation may be stored may be any temperature from about-80 ℃ to about 45 ℃, for example, at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ° -8 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 37 ℃, or about 45 ℃. For example, a pharmaceutical formulation is considered stable if less than about 3%, 2.75%, 2.5%, 2.25%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the aggregated form of the antibody is detected after twelve months of storage at 5 ℃. In some cases, a pharmaceutical formulation is considered stable if less than about 5%, 4.75%, 4.5%, 4.25%, 4%, 3.75%, 3.5%, 3.25%, 2%, or 1% of the aggregated form of the antibody is detected after six months of storage at 5 ℃. Pharmaceutical formulations are also considered stable if less than about 6%, 5.75%, 5.5%, 5.25%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25% or 0.1% of the aggregated form of the antibody is detected after six months of storage at 25 ℃ and 60% relative humidity. Pharmaceutical formulations are also considered stable if less than about 6%, 5.75%, 5.5%, 5.25%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25% or 0.1% of the aggregated form of the antibody is detected after three months of storage at 37 ℃. Pharmaceutical formulations are also considered stable if less than about 3%, 2.75%, 2.5%, 2.25%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1%, 0.5%, or 0.1% of the aggregated form of the antibody is detected after twelve months of storage at-30 ℃ or-80 ℃.

Stability can be measured, inter alia, by determining the percentage of antibody that remains in the form of glycosylated material. The percentage of "glycosylated species" of an antibody may be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [ CEX-HPLC ] or cation exchange ultra high performance liquid chromatography [ CEX-UPLC ]) and/or by LC-MS. The phrase "acceptable degree of stability" as used herein means that the percentage of antibody in the form of a "glycosylated species" does not change more than a specified amount after storage for a defined amount of time at a defined temperature. In certain embodiments, an acceptable degree of stability means that the percentage of "glycosylated species" does not change by more than 25%, not more than 20%, not more than 15%, not more than 10%, not more than 9%, not more than 8%, not more than 7%, not more than 6%, not more than 5%, not more than 4%, not more than 3%, not more than 2.5%, not more than 2%, not more than 1.5%, or not more than 1% after storage for a defined amount of time at a given temperature. The defined amount of time after measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months or longer. When evaluating stability, the temperature at which the pharmaceutical formulation may be stored may be any temperature from about-80 ℃ to about 45 ℃, for example, at about-80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 4 ° -8 ℃, about 5 ℃, about 25 ℃, or about 45 ℃. For example, a pharmaceutical formulation is considered stable if the percentage of "glycosylated material" does not change by more than 5%, not more than 4%, not more than 3%, not more than 2% or not more than 1.5% after twelve months of storage at-80 ℃ or-30 ℃. In another example, a pharmaceutical formulation is considered stable if the percentage of "glycosylated material" does not change by more than 5%, not more than 4%, not more than 3%, not more than 2% or not more than 1.5% after storage for six months at 5 ℃. In another example, a pharmaceutical formulation is considered stable if the percentage of "glycosylated material" does not change by more than 5%, by more than 4% or by more than 3% after twelve months of storage at 5 ℃. In each case, the measurement may be performed using cation exchange ultra high performance liquid chromatography (CEX-UPLC) and/or by LC-MS.

Reference to stability of a pharmaceutical formulation "after" a specified period of time is intended to mean that the stability parameter (e.g.,% natural form,% HMW species or% acidic form) is measured at or about the end of the specified period of time, and is not intended to mean that the pharmaceutical formulation must thereafter maintain the same degree of stability. For example, reference to a particular stability after 12 months means that the stability measurement is made at the beginning of the study or after about 12 months. Other methods for assessing the stability of antibodies in a formulation are illustrated in the examples below.

As shown in the examples below, the present invention is based in part on the discovery that the combination of the claimed excipients with bispecific anti-MUC 16 x anti-CD 3 antibodies results in stable formulations.

Exemplary formulations

According to one aspect of the invention, a pharmaceutical formulation comprises: (i) A human anti-MUC 16 x anti-CD 3 bispecific antibody that specifically binds human MUC16 and human CD 3; (ii) a buffer comprising acetate (e.g. sodium acetate); (iii) an organic co-solvent comprising a polysorbate; and (iv) a stabilizer comprising a sugar. According to another aspect, a pharmaceutical formulation comprises: (i) A human anti-MUC 16 x anti-CD 3 bispecific antibody that specifically binds human MUC16 and human CD 3; (ii) a buffer comprising acetate; and (iii) a stabilizer comprising a sugar. According to another aspect, a pharmaceutical formulation comprises: (i) A human anti-MUC 16 x anti-CD 3 bispecific antibody that specifically binds human MUC16 and human CD 3; (ii) a buffer comprising histidine; (iii) an organic co-solvent comprising a polysorbate; and (iv) a stabilizer comprising a sugar.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) acetate at a concentration of about 25mM to about 35 mM; (iii) Polysorbate 20 at a concentration of about 0.1% w/v to about 0.3% w/v; and (iv) sucrose at a concentration of about 5% w/v to about 15% w/v, wherein the pH of the formulation is 5.0±0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG1 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 25mM to about 35 mM; (iii) Polysorbate 20 at a concentration of about 0.1% w/v to about 0.3% w/v; and (iv) sucrose at a concentration of about 5% w/v to about 15% w/v, wherein the pH of the formulation is 5.0±0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG4 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 25mM to about 35 mM; (iii) Polysorbate 20 at a concentration of about 0.1% w/v to about 0.3% w/v; and (iv) sucrose at a concentration of about 5% w/v to about 15% w/v, wherein the pH of the formulation is 5.0±0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of about 1mg/ml to about 200mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID No. 1, a second heavy chain comprising the amino acid sequence of SEQ ID No. 2, and a common light chain comprising the amino acid sequence of SEQ ID No. 3; (ii) acetate at a concentration of about 25mM to about 35 mM; (iii) Polysorbate 20 at a concentration of about 0.1% w/v to about 0.3% w/v; and (iv) sucrose at a concentration of about 5% w/v to about 15% w/v, wherein the pH of the formulation is 5.0±0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG1 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG4 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of about 1mg/ml to about 200mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID No. 1, a second heavy chain comprising the amino acid sequence of SEQ ID No. 2, and a common light chain comprising the amino acid sequence of SEQ ID No. 3; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w/v; and (iv) sucrose at a concentration of about 10% w/v, wherein the pH of the formulation is 5.0±0.1.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG1 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w/v; and (iv) sucrose at a concentration of about 10% w/v, wherein the pH of the formulation is 5.0±0.1.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG4 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w/v; and (iv) sucrose at a concentration of about 10% w/v, wherein the pH of the formulation is 5.0±0.1.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of about 1mg/ml to about 200mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID No. 1, a second heavy chain comprising the amino acid sequence of SEQ ID No. 2, and a common light chain comprising the amino acid sequence of SEQ ID No. 3; (ii) histidine at a concentration of about 10 mM; (ii) acetate at a concentration of about 30 mM; (iii) polysorbate 20 at a concentration of about 0.2% w/v; and (iv) sucrose at a concentration of about 10% w/v, wherein the pH of the formulation is 5.0±0.1.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 3mg/ml±1mg/ml to 7mg/ml±1mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 5mg/ml ± 0.5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 40mg/ml±2mg/ml to 60mg/ml±2mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising a HCVR comprising the amino acid sequence of SEQ ID NO:4 and a LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain comprising a HCVR comprising the amino acid sequence of SEQ ID NO:5 and a LCVR comprising the amino acid sequence of SEQ ID NO: 6; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 50mg/ml±5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO 4 and an LCVR comprising the amino acid sequence of SEQ ID NO 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID NO 5 and an LCVR comprising the amino acid sequence of SEQ ID NO 6; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 5mg/ml ± 0.5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG4 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 50mg/ml±5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID NO:4 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, and a second antigen binding domain that comprises an HCVR comprising the amino acid sequence of SEQ ID NO:5 and an LCVR comprising the amino acid sequence of SEQ ID NO:6, wherein the antibody has a heavy chain constant region of isotype IgG4 (optionally wherein one of the two heavy chains has a modification that reduces protein a binding relative to an unmodified heavy chain of the same isotype, and optionally wherein one or both of the two heavy chains has a chimeric hinge); (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 5mg/ml ± 0.5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, wherein the antibody has a heavy chain constant region of isotype IgG4, and wherein one of the two heavy chains has a modification in the CH3 domain (e.g., H435R and Y436F, numbered by EU) that reduces binding to protein a relative to the unmodified IgG4 CH3 domain; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of 50mg/ml ± 0.5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, wherein the antibody has a heavy chain constant region of isotype IgG4, and wherein one of the two heavy chains has a modification in the CH3 domain (e.g., H435R and Y436F, numbered by EU) that reduces binding to protein a relative to the unmodified IgG4 CH3 domain; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of about 5mg/ml±0.5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID No. 1, a second heavy chain comprising the amino acid sequence of SEQ ID No. 2, and a common light chain comprising the amino acid sequence of SEQ ID No. 3; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a concentration of about 50mg/ml±5mg/ml of a human bispecific antibody that specifically binds human MUC16 and human CD3, and comprises a first heavy chain comprising the amino acid sequence of SEQ ID No. 1, a second heavy chain comprising the amino acid sequence of SEQ ID No. 2, and a common light chain comprising the amino acid sequence of SEQ ID No. 3; (ii) acetate at a concentration of about 30mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.2% w/v±0.02% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 5.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 1mg/ml to about 3mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) histidine at a concentration of about 10mM ± 1 mM; (iii) Polysorbate 20 at a concentration of about 0.05% w/v±0.01% w/v; and (iv) sucrose at a concentration of about 10% w/v+ -1% w/v, wherein the pH of the formulation is 6.0+ -0.3.

In some cases, the stable liquid pharmaceutical formulation comprises (i) a human bispecific antibody that specifically binds human MUC16 and human CD3 at a concentration of about 150mg/ml to about 200mg/ml, and comprises a first antigen binding domain that specifically binds human MUC16 comprising an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and a second antigen binding domain comprising an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6; (ii) acetate at a concentration of about 30mM ± 1 mM; and (iii) sucrose at a concentration of about 5% w/v.+ -. 0.5% w/v, wherein the pH of the formulation is 5.0.+ -. 0.3.

In any of these exemplary formulations, "stable" may be defined as: (a) The formulation contains no more than 2.5% High Molecular Weight (HMW) species after storage at 5 ℃ for 12 months as determined by SE-UPLC; (b) The formulation contains no more than 3.5% High Molecular Weight (HMW) species after storage for 6 months at 25 ℃ and 60% relative humidity as determined by SE-UPLC; (c) The formulation contains no more than 1.5% High Molecular Weight (HMW) species after storage at-30 ℃ for 12 months as determined by SE-UPLC; (d) The formulation contains no more than 1.5% High Molecular Weight (HMW) species after storage at-80 ℃ for 12 months as determined by SE-UPLC; (e) At least 95% of the antibodies have a native conformation after 12 months of storage at 5 ℃ as determined by SE-UPLC; (f) At least 95% of the antibodies have a native conformation after storage for 6 months at 25 ℃ and 60% relative humidity as determined by SE-UPLC; (g) At least 95% of the antibodies have a native conformation after 3 months of storage at 37 ℃ as determined by SE-UPLC; (h) The formulation contains no more than 1% High Molecular Weight (HMW) species after storage at 5 ℃ for 12 months as determined by SE-UPLC; (i) The formulation contains no more than 1% HMW species after storage for 6 months at 25℃and 60% relative humidity as determined by SE-UPLC; (j) The formulation contains no more than 1% HMW species after storage at 37 ℃ for 3 months as determined by SE-UPLC; (k) The formulation contains no more than 2% High Molecular Weight (HMW) species after storage at-30deg.C or-80deg.C for 12 months as determined by SE-UPLC; (l) The formulation contains no more than 4% HMW species after storage at 5 ℃ for 6 months as determined by SE-UPLC; or (m) the formulation contains no more than 6% HMW species after storage for 6 months at 25℃and 60% relative humidity as determined by SE-UPLC.

In any of these exemplary formulations, the bispecific antibody may include modifications in one or both heavy chains to facilitate purification of the bispecific antibody (i.e., heterodimer) from homodimeric impurities. In some embodiments, the bispecific antibody comprises a first and a second heavy chain (i.e., the heavy chain of the anti-MUC 16 binding arm and the heavy chain of the anti-CD 3 binding arm) that are identical except for a modification in the CH3 domain of one or the other heavy chain (e.g., both isotypes IgG1 or IgG 4), which modification reduces binding of the bispecific antibody to protein a as compared to an antibody lacking the modification. In some cases, the CH3 domain of the first heavy chain (e.g., the first heavy chain of the anti-MUC 16 binding arm) binds protein a, and the CH3 domain of the second heavy chain (e.g., the second heavy chain of the anti-CD 3 binding arm) contains mutations that reduce or eliminate protein a binding. In some cases, the mutation is an H435R modification (numbered by EU; H95R numbered by IMGT exon). In some cases, the mutations are H435R modification (numbered by EU; H95R numbered by IMGT exon) and Y436F modification (numbered by EU; Y96F numbered by IMGT). Additional modifications that may be found in the second CH3 domain include: in the case of IgG1CH3 domains, D356E, L358M, N384S, K392N, V397M and V422I by EU numbering (D16E, L18M, N44S, K52N, V57M and V82I by IMGT numbering); and in the case of IgG4 CH3 domains, Q355R, N384S, K392N, V397M, R409K, E419Q and V422I by EU numbering (Q15R, N44S, K52N, V57M, R69K, E79Q and V82I by IMGT numbering).

Further non-limiting examples of pharmaceutical formulations encompassed by the present invention are presented elsewhere herein, including the working examples presented below.

Container and method of application

The pharmaceutical formulations of the present invention may be contained in any container suitable for storing medicaments and other therapeutic compositions. For example, the pharmaceutical formulation may be contained in a sealed and sterilized plastic or glass container having a defined volume, such as a vial, ampoule, syringe, cartridge, bottle, or IV bag. Different types of vials may be used to contain the formulations of the present invention, including, for example, clear and opaque (e.g., amber) glass or plastic vials. Likewise, any type of syringe may be used to contain and/or administer the pharmaceutical formulations of the present invention. In some embodiments, the pharmaceutical formulation is contained in a prefilled syringe. In some embodiments, the pharmaceutical formulation is contained in a prefilled needled syringe.

The pharmaceutical formulations of the present invention may be contained in "standard tungsten" syringes or "low tungsten" syringes. As will be appreciated by those of ordinary skill in the art, methods of manufacturing glass syringes generally involve the use of a hot tungsten rod for piercing the glass, thereby creating a hole from which liquid can be drawn and expelled from the syringe. This method results in trace tungsten deposition on the inner surface of the injector. Subsequent cleaning and other processing steps may be used to reduce the amount of tungsten in the injector. As used herein, the term "standard tungsten" means that the injector contains greater than 500 parts per billion (ppb) tungsten. The term "low tungsten" means that the injector contains less than 500ppb tungsten. For example, a low tungsten injector according to the present invention may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10ppb or less tungsten.

Rubber plungers used in syringes and rubber stoppers for closing vial openings may be coated to prevent contamination of the drug contents of the syringe or vial and/or to maintain their stability. Thus, according to certain embodiments, the pharmaceutical formulation of the present invention may be contained within a syringe comprising a coated plunger or within a vial sealed with a coated rubber stopper. For example, the plunger or stopper may be coated with a fluorocarbon film. Examples of coated stoppers and/or plungers suitable for use with vials and syringes containing the pharmaceutical formulations of the invention are described, for example, in U.S. patent 4,997,423;5,908,686;6,286,699;6,645,635; and 7,226,554, the contents of which are incorporated herein by reference in their entirety. Specific exemplary coated rubber stoppers and plungers that may be used in the context of the present invention are available under the trade nameCommercially available from West Pharmaceutical Services, inc (Lionville, PA). />Is an example of a fluorocarbon coating that is used to minimize or prevent adhesion of the drug product to the rubber surface. According to certain embodiments of the present invention, the pharmaceutical formulation may be contained in a low tungsten syringe comprising a fluorocarbon-coated plunger. In some embodiments, the container is a syringe, such as an Ompi EZ-Fill ^TM Syringes or BD Neopaks ^TM A syringe. In some cases, the syringe is a 1mL long glass syringe with a 1mL iWest plunger, a 27G thin-walled needle, and an FM30 needle shield or BD260 needle shield. In some cases, the syringe is a 2.25mL glass syringe with West NovaPure ^TM 1-3mL plunger, 27G thin-walled needle and FM30 needle shield or BD260 needle shield. In various embodiments, the syringe is a 0.5mL, 0.6mL, 0.7mL, 0.8mL, 0.9mL, 1.0mL, 1.1mL, 1.2mL, 1.3mL, 1.4mL, 1.5mL, 1.6mL, 1.7mL, 1.8mL, 1.9mL, 2.0mL, 2.1mL, 2.2mL, 2.3mL, 2.4mL, 2.5mL, 2.6mL, 2.7mL, 2.8mL, 2.9mL, 3.0mL, 3.5mL, 4.0mL, 4.5mL, 5.0mL, 5.5mL, 6.0mL, 6.5mL, 7.0mL, 7.5mL, 8.0mL, 8.5mL, 9.0mL, 9.5mL or 10mL syringe (e.g., a glass syringe).

The pharmaceutical formulations may be administered to the patient by parenteral routes such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, mucosal, nasal, pulmonary, and/or oral administration. Many reusable pen and/or auto-injector delivery devices may be used to subcutaneously deliver the pharmaceutical formulations of the present invention. Examples include, but are not limited to, AUTOPEN ^TM (Owen Mumford,Inc.,Woodstock,UK)、DISETRONIC ^TM Pen (Disetronic Medical Systems, bergdorf, switzerland), HUMALOG MIX 75/25 ^TM Pen and HUMALOG ^TM Pen, HUMALIN 70/30 ^TM Pen (Eli Lilly and Co., indianapolis, ind.), NOVOPEN ^TM I. II and III (Novo Nordisk, copenhagen, denmark), NOVOPEN JUNIOR ^TM (Novo Nordisk,Copenhagen,Denmark)、BD ^TM Pen (Becton Dickinson, franklin Lakes, NJ), OPTIPEN ^TM 、OPTIPEN PRO ^TM 、OPTIPEN STARLET ^TM And OPTICLIK ^TM (sanofi-aventis, frankfurt, germany) and the like. Examples of disposable pen and/or auto-injector delivery devices that may be used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR ^TM Pen (sanofi-aventis), FLEXPEN ^TM (Novo Nordisk), and KWIKPEN ^TM (Eli Lilly)、SURECLICK ^TM Autoinjector (Amgen, thonsand Oaks, calif.), PENLET ^TM (Haselmeier, stuttgart, germany), EPIPEN (Dey, L.P.), and HUMIRA ^TM Pen (Abbott Labs, abbott Park IL), and the like. In some cases, the pharmaceutical formulation is contained in a syringe that is specially adapted for use in an automatic syringe. Subcutaneous injections may be administered using a 20-30 gauge needle or a 25-30 gauge needle. In some cases, the skin may be applied using a 25 gauge needleAnd (5) injecting downwards. In some cases, a 27 gauge needle may be used to administer subcutaneous injections. In some cases, a 29 gauge needle may be used to administer subcutaneous injections.

Another type of delivery device may include a safety system. Such devices can be relatively inexpensive and the safety sleeve can be manually or automatically extended onto the needle once the injection is completed. Examples of security systems may include the ERIS device of West Pharmaceutical, or the UltraSafe device of Becton Dickinson. In addition, the use of a high volume device ("LVD") or a bolus syringe to deliver the pharmaceutical formulation of the invention is also contemplated herein. In some cases, LVD or bolus injectors may be configured to inject a drug into a patient. For example, LVD or bolus syringes may be configured to deliver "large" volumes of drug (typically about 2ml to about 10 ml).

In certain embodiments, the pharmaceutical formulation is administered via IV drip such that the formulation is diluted in an IV bag containing a physiologically acceptable solution. In one embodiment, the pharmaceutical composition is a complex sterile formulation in an iv bag such that a single dose of the drug is diluted into 100mL, 250mL (or other similar amount suitable for intravenous drip delivery) of physiological buffer (e.g., 0.9% saline).

The pharmaceutical formulations of the present invention may also be included in unit dosage forms. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for patients to be treated, each unit containing a predetermined quantity of active compound in association with the required pharmaceutical carrier, diluent or excipient, calculated to produce the desired therapeutic effect. In various embodiments, the unit dosage form is contained in a container as described herein. The actual dosage level of the active ingredient (e.g., anti-MUC 16 x anti-CD 3 bispecific antibody) in the formulations of the present invention may be varied in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without adverse effects on the patient. The dosage level selected will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and past history of the patient being treated, and like factors well known in the medical arts. The term "diluent" as used herein refers to a solution suitable for changing or achieving the exemplary or appropriate concentrations or concentrations described herein.

In various embodiments, the unit dosage form contains an amount of active ingredient (e.g., an anti-MUC 16 x anti-CD 3 bispecific antibody) intended for single use. In various embodiments, the amount of active ingredient in a unit dosage form is from about 0.1mg to about 5000mg, from about 100mg to about 1000mg, from about 100mg to about 500mg, from about 100mg to about 400mg, from about 100mg to about 200mg, from about 40mg to about 60mg, from about 125mg to about 175mg, from about 160mg to about 200mg, from about 1mg to about 250mg, from about 1mg to about 100mg, from about 1mg to about 50mg, from about 1mg to about 25mg, from about 1mg to about 20mg, from about 5mg to about 15mg, or ranges or intervals thereof. Intermediate ranges of the above amounts, for example, from about 2mg to about 100mg or 2mg to 20mg, are also part of the present invention. For example, a range of values using any combination of the above values (or values included within the ranges) as an upper and/or lower limit is intended to be included. In some embodiments, the unit dosage form contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200mg of antibody. In some embodiments, the unit dosage form contains 5mg of antibody. In some embodiments, the unit dosage form contains 12.5mg of antibody. In particular embodiments, the formulation is typically provided as a liquid in a unit dosage form. In some embodiments, the unit dosage form contains 3 to 7mg, or 8 to 12mg, 10 to 15mg, 35 to 45mg, 45 to 55mg, 140 to 160mg, or 170 to 190mg. In some embodiments, unit dosage forms according to the invention are suitable for subcutaneous administration to a patient (e.g., unit dosage forms containing antibodies at a concentration of about 100mg/ml or about 200mg/ml or 150mg/ml + -5 mg/ml).

The invention also includes a method of preparing a unit dosage form. In exemplary embodiments, the method of preparing a pharmaceutical unit dosage form comprises combining any of the formulations of the preceding embodiments in a suitable container, such as those discussed herein.

Therapeutic use of pharmaceutical formulations

The pharmaceutical formulations of the present invention are particularly useful for the treatment, prevention and/or amelioration of any disease or disorder associated with cells expressing human MUC 16. Exemplary, non-limiting diseases and disorders treatable by administration of the pharmaceutical formulations of the present invention include ovarian cancer, breast cancer, pancreatic cancer, and non-small cell lung cancer.

The methods of treatment of the present invention comprise administering to a subject any formulation comprising an anti-MUC 16 x anti-CD 3 bispecific antibody disclosed herein. The subject to whom the pharmaceutical formulation is administered may be, for example, any human or non-human animal in need of such treatment. For example, a subject may be an individual diagnosed with, or considered at risk of suffering from, any of the above-described diseases or disorders. The invention further includes the use of any of the pharmaceutical formulations disclosed herein in the manufacture of a medicament for the treatment of any disease or disorder associated with cells expressing human MUC16, including any of the exemplary diseases, disorders and conditions described above.

In some embodiments, the invention provides kits comprising a pharmaceutical formulation (e.g., a container containing the formulation or unit dosage form) as discussed herein, and a package or label (e.g., a package insert) having instructions for using the pharmaceutical formulation to treat a disease or disorder as discussed herein. In some cases, the instructions provide for the use of a unit dosage form as discussed herein for treating a disease or disorder.

A summary of the sequences cited herein and the corresponding SEQ ID NOs is shown in table 1 below.

Table 1: sequence overview

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees celsius and pressure is at or near atmospheric pressure.

Example 1: development of stable liquid and lyophilized anti-MUC 16 x anti-CD 3 bispecific antibody formulations

Physical stability of a formulation refers to properties such as color, appearance, pH, turbidity and protein concentration. Chemical stability refers to the formation of High Molecular Weight (HMW) species, low Molecular Weight (LMW) species, charge variants, and other chemical modifications of proteins. The physical and chemical stability of mAb1 drug was evaluated using the following assay: visual color and appearance; pH value; turbidity was measured by increasing the Optical Density (OD) at 405 nm; imaging by microfluidics ^TM (MFI) sub-visual particle analysis; concentrating the protein by reverse phase ultra high performance liquid chromatography (RP-UPLC); purity of each drug was assessed using size exclusion ultra high performance liquid chromatography (SE-UPLC) and reduced and non-reduced microchip capillary electrophoresis-sodium dodecyl sulfate (MCESDS); charge variant analysis was determined using cation exchange UPLC (CEX-UPLC) (glycosylated mAb1 was detected by CEX-UPLC) and imaging capillary isoelectric focusing (iCIEF); and bioassay for determining potency (the relative potency of each sample is determined by bioassay and is defined as (IC) ₅₀ Reference sample/IC ₅₀ Sample) ×100%; the measured efficacy of the storage stable samples must be in the range of 50% -150% of the measured efficacy of the reference standard.

Lyophilized formulations of mAb1 for Intravenous (IV) or Subcutaneous (SC) administration were developed. Lyophilized mAb1 drug can be reconstituted with sterile water for injection (WFI) to a concentration of 2mg/mL mAb1 or 20mg/mL mAb1 for IV infusion or SC injection. Formulation development activities included evaluating buffers, pH, organic co-solvents, surfactants, and sucrose (as a heat stabilizer) to determine excipients that enhance protein stability. The results of these studies were used to develop stable lyophilized formulations suitable for reconstitution into liquid form.

Buffer and pH selection

The effect of buffer and pH on mAb1 thermostability was initially examined in liquid formulations by incubating 2mg/mL mAb1 at 45℃for 28 days in a series of buffer systems of different pH. The following pH and buffer systems were studied: acetate (pH 4.5 to 5.5), histidine (pH 5.5 to 6.5) and phosphate (pH 6.5 to 7.5). These assays indicate that HMW species and charge variant formation are the primary degradation pathways. Based on the results of SE-UPLC, as shown in Table 2, the lowest rate of HMW species formation and lowest rate of monomer loss were observed when mAb1 was formulated in histidine buffer at pH between 5.5 and 6.5 or in acetate buffer at pH between 4.5 and 5.5. CEX-UPLC analysis also showed that charge variant spectra were most stable when mAb1 was formulated between pH 5.5 and 6.5 in histidine buffer or between pH 4.5 and 5.5 in acetate buffer, as shown in Table 2. After incubation of the mAb for 128 days at 45 ℃ relative to the starting material, a relative decrease in total acidic species was observed by CEX-UPLC analysis, while the major charge variant peak increased.

A single peak was isolated that contained about 50% of the total acidic charge variant observed in the CEX-UPLC chromatogram. Further analysis identified that the major component of this peak was mAb1 protein, which contains glycosylation of Heavy Chain (HC) Lys98 within CDR3 of the MUC16 binding arm of the bispecific antibody (glycosylation is discussed in detail in the next paragraph). During the initial activities associated with mAb1 formulation development, the stability of the primary charge variants, total acidic species, total basic species, and glycosylated forms of the molecules were monitored. Histidine buffer (pH 6.0) was chosen for clinically viable formulations because of minimal HMW species formation, monomer loss and charge variant formation in this buffer and pH.

Specific mAb 1-derived acidic peaks were observed by CEX-UPLC analysis, comprising approximately 50% of the total mAb 1-derived acidic charge variants. The peak area detected by CEX-UPLC decreased after 28 days incubation at 45 ℃. This peak was identified as containing glycosylated mAb1 variant at HC Lys98 within CDR3 of the MUC16 binding arm of the bispecific antibody. As the level of glycosylated form of mAb1 decreased, an increase in potency was observed by bioassay (see figure 1). Although Lys98 deglycosylation reactions readily occur in solution, the glycosylation level remains unchanged in the lyophilized state when the formulation is stored at 5 deg.c for at least 12 months. Furthermore, the efficacy remained unchanged when the lyophilized formulation was stored at 5 ℃ for up to 12 months. Thus, a lyophilized formulation was chosen as the formulation for mAb 1.

Surfactant/organic co-solvent selection

The effect of the surfactants polysorbate 20 and polysorbate 80 on the stirring stress stability and thermal stability of 5mg/mL mAb1 (10% sucrose in the surfactant-containing formulation) was examined in the liquid formulation. Polysorbate 20 and polysorbate 80 at concentrations of 0-0.1% (w/v) were tested for their ability to stabilize mAb1 (see tables 3 and 4 below). As shown in table 3, both polysorbate 20 and polysorbate 80 were able to stabilize mAb1 against agitation stress when present at levels of 0.01% or higher. When no surfactant was included in the formulation, a 1.9% increase in HMW species was observed. When polysorbate 20 or polysorbate 80 included in the formulation was not less than 0.01%, no increase in HMW species was observed.

Polysorbate 20 and polysorbate 80 at concentrations of 0.05% and 0.05% were selected to examine the effect of heat stress on mAb1 stability. After 28 days incubation at 45 ℃, about 0.5% less HMW species was observed for the formulation containing 0.05% polysorbate 20, and 1.6% more mAb1 monomer was observed, as shown in table 4, compared to the formulation containing 0.05% polysorbate 80. In addition, mAb1 exhibited an improved stability profile in the presence of 0.05% polysorbate 20 as determined by CEX-UPLC analysis after 28 days of incubation at 45 ℃. mAb1 in the presence of 0.05% polysorbate 20 showed (1) about 5% reduction in the formation of acidic species, (2) about 6% reduction in the loss of the major charge variant form, and (3) approximately equivalent change in the level of glycosylated species relative to mAb1 formulated in the presence of 0.05% polysorbate 80 (see table 4). 0.05% polysorbate 20 was chosen as the surfactant for mAb1 pharmaceutical formulations because it was able to stabilize the protein sufficiently against agitation stress.

Heat stabilizer/antifreeze

Stabilizers (such as sucrose) can sometimes be added to liquid and lyophilized antibody formulations to increase thermal stability and stability of the protein to freeze/thaw stress. Sucrose is required to be included in the mAb1 formulation to stabilize the mAb1 response to freeze/thaw stress. In the absence of sucrose, the level of HMW species increased by 0.4% after mAb1 underwent four freeze and thaw cycles (see, table 5). Adding 10% sucrose to the formulation stabilized the mAb1 drug substance to four freeze/thaw cycles (see table 5).

Sucrose is also a necessary cryoprotectant to stabilize mAb1 during lyophilization. After reconstitution of the lyophilized mAb1 formulation, the relative amount of HMW species increased by 0.2% in the absence of sucrose, but did not show significant change when the formulation contained 10% sucrose, as shown in Table 6.

When lyophilized mAb1 was incubated at 50℃for 28 days in the absence of sucrose, the level of HMW species was increased by 5.9% and the level of monomer was decreased by 7.1% as determined by SE-UPLC (see Table 7). Furthermore, after incubation of mAb1 at 50 ℃ in the absence of sucrose, the level of acidic species was reduced by 13.8%, the level of basic species was increased by 14.3% and the level of glycosylated species was reduced by 14.7% as determined by CEX-UPLC (see table 7). However, in the presence of 10% sucrose, no significant change in the level of molecular weight variants or charge variants was observed, as shown in table 7.

10% sucrose has a positive impact on the freeze/thaw stability, thermal stability and stability of mAb1 during lyophilization. During lyophilization, 10% sucrose was also used as bulking agent. Thus, 10% sucrose was chosen as a stabilizer for developing lyophilized mAb1 formulations.

Summary of selected formulation Components

mAb1 exhibited maximum stability when formulated in the presence of 10mM histidine, 0.050% polysorbate 20 and 10% sucrose at pH 6.0. The primary degradation pathway identified during the development of liquid formulations of mAb1 is the formation of HMW species and charge variants. A charge variant of particular interest is the glycosylation of HC-CDR3-Lys 98. It has been demonstrated that there is no change in the level of glycosylated mAb1 after incubation of lyophilized mAb1 under stressed conditions and real-time storage conditions. The information obtained in the liquid formulation development and lyophilization feasibility study forms the basis for developing lyophilized pharmaceutical formulations suitable for clinical use. To be able to reconstitute the lyophilized drug to a concentration of 2mg/mL mAb1 for IV administration, a formulated drug substance containing 2mg/mL mAb1, 10mM histidine pH 6.0, 10% (w/v) sucrose, and 0.05% (w/v) polysorbate 20 was developed.

Table 2: effect of buffer and pH on stability of 2mg/mL mAb1 incubated at 45℃for 28 days

^a Reported as a change in purity relative to the starting material. In all formulations, the starting material (not incubated) contained ≡97.8% of the natural peak (by SE-UPLC) and ≡42.6% of the main peak (by CEX-UPLC).

^b The sample was turbid with no visible particles.

^c CEX, cation exchange was not established due to extensive degradation; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; ND, undetermined; OD, optical density; RP, reverse phase; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 3: effect of surfactant concentration on stability of 5mg/mL mAb1 after stirring (120 min vortex)

^a Reported as a change in purity relative to the starting material; in all formulations, the starting material (not incubated) contained ≡97.7% of the natural peak (by SE-UPLC) and ≡46.8% of the main peak (by CEX-UPLC).

CEX, cation exchange; DS, bulk drug; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; RP, reverse phase; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 4: evaluation of polysorbate 20 and polysorbate 80 concentrations: effect of surfactant concentration on stability of 5mg/mL mAb1 when incubated at 45℃for 28 days

^a Reported as a change in purity relative to the starting material; in all formulations, the starting material (not incubated) contained ≡97.6% of the natural peak (by SE-UPLC) and ≡46.0% of the main peak (by CEX-UPLC).

Table 5: effect of 10% sucrose on stability of 5mg/mL mAb1 after four freezing and thawing cycles

^a In the initial formulation development, a pH of 5.5 was used. The pH is not optimized until after the initial surfactant is selected.

^b Reported as a change in purity relative to the starting material; in all formulations, the starting material (not incubated) contained ≡97.4% of the natural peak (by SE-UPLC) and ≡42.8% of the main peak (by CEX-UPLC).

Table 6: stabilization of mAb1 during lyophilization by addition of 10% sucrose

(immediately after lyophilization was completed, mAb1 was reconstituted and analyzed)

^c Reported as a change in purity relative to the starting material; in both formulations, the starting material (without lyophilization) contained more than or equal to 98.7% of the natural peak (by SE-UPLC) and more than or equal to 45.1% of the main peak (by CEX-UPLC).

Table 7: effect of the presence of 10% sucrose on stability of lyophilized mAb1 DP incubated for 28 days at 50℃

^a Reported as a change in purity relative to the starting material; in both formulationsThe starting material (without lyophilization) contained more than or equal to 98.7% of the natural peak (by SE-UPLC) and more than or equal to 45.1% of the main peak (by CEX-UPLC).

Example 2: storage and stress stability of formulations

Evaluation studies were performed on the storage, accelerated stability and stress stability (agitation) of liquid, lyophilized and reconstituted formulations of mAb1 drug.

Analysis of mAb1 lyophilized drug indicated that mAb1 drug was physically and chemically stable when stored at 5 ℃ for at least 36 months (see tables 8, 10 and 12). No significant change in physical or chemical stability was detected in any of the monitored attributes. Tables 9, 11 and 13 provide the results of analysis of mAb1 lyophilized drug after incubation under accelerated conditions. No significant change in physical or chemical stability of mAb1 was detected in any of the monitored attributes after incubation for 3 months at 37 ℃. Similarly, no significant change in physical or chemical stability was detected in any of the monitored attributes after 6 months of incubation at 25 ℃/60% Relative Humidity (RH). After incubation at 50℃for 3 months, a slight increase in HMW species (0.3%) and alkaline species (7.6% by CEX-UPLC and 4.0% by iciEF) was observed. mAb1 remained potent as determined by bioassay analysis after incubation under accelerated conditions.

Table 8: stability of mAb1 lyophilized drug stored at 5℃was studied

^a CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imagingCapillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

^b The actual storage time of these samples was 4 months

^c Samples were reconstituted to 2mg/mL mAb1 with sterile WFI

Table 9: study of stability of mAb1 lyophilized drug stored under accelerated conditions

^a The actual storage time is 4 months

^b Samples were reconstituted to 2mg/mL mAb1 with sterile WFI

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MFI, microfluidic imaging; NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 10: study of stability of mAb1 lyophilized drug stored at 5℃

^a CEX, cation exchange; DS, bulk drug; FDG, formulation Developing groups; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

^b Samples were reconstituted to 2mg/mL mAb1 with sterile WFI

Table 11: study of stability of mAb1 lyophilized drug stored under accelerated conditions

^a Samples were reconstituted to 2mg/mL mAb1 with sterile WFI

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 12: stability of mAb1 50mg lyophilized drug stored at 5℃was studied

^a CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillaryPerforming tube electrophoresis; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the NA, unavailable; NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

^b The 36 month MFI data was not available due to instrument failure and insufficient backup samples.

^c Samples were reconstituted with sterile WFI to 20mg/mL mAb1

Table 13: study of stability of mAb1 50mg lyophilized drug stored under accelerated conditions

^a CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MFI, microfluidic imaging; NR, not required; OD, optical density; RP, reverse phase; SDS, sodium dodecyl sulfate; SE, size exclusion; UPLC, ultra high performance liquid chromatography

^b Samples were reconstituted with sterile WFI to 20mg/mL mAb1

Additional stability studies were also performed on reconstituted mAb1 drug to evaluate the stability of reconstituted drug incubated at 25 ℃ for 24 hours and under stress (agitation) conditions. Lyophilized drug (2.5 mL of formulated drug substance in 5mL glass bottle) was reconstituted to 2mg/mL mAb1 or 20mg/mL mAb1 (final volume of 2.5 mL) with 2.3mL WFI. The results of these stability studies are shown in tables 14 and 15 below. When incubated at 25℃for 24 hours, a reconstituted mAb1 drug solution of 2mg/mL mAb1 and 20mg/mL mAb1 was found to be physically and chemically stable. No significant change in physical or chemical stability was detected in any of the monitored attributes. These data indicate that the reconstituted drug is stable at room temperature. When stirred (vortexed at ambient temperature) for 60 minutes, the reconstituted mAb1 drug solution of 2mg/mL mAb1 and 20mg/mL mAb1 was also found to be physically and chemically stable. No significant change in physical or chemical stability was detected in any of the monitored attributes.

Table 14: study of stability of mAb1 reconstituted drug for IV administration

Table 15: study of stability of mAb1 reconstituted 20mg/mL drug for IV administration

The results of the mAb1 drug storage and stress stability studies indicate that mAb1 is stable. mAb1 formulations can withstand short exposure times at room temperature without compromising physical or chemical stability. mAb1 formulations were also stable when reconstituted to concentrations of 2mg/mL or 20 mg/mL. Exposure of the reconstituted mAb1 drug to 25 ℃ for up to 24 hours did not compromise protein integrity nor affect agitation of the reconstituted drug.

Tables 16 and 17 below show the reconstituted volumes of mAb1 drug.

Table 16: reconstitution volume, overfill volume and extractable volume of 5mg/ml mAb1 drug reconstituted for IV administration

Formulation components	Reconstitution for IV administration
		mAb1 concentration in FDS (Pre-lyophilization)	2mg/mL
Protein content per bottle	5mg
		mAb1 concentration in the reconstitution DP	2mg/mL
Filling volume	2.5mL
		WFI volume for reconstruction	2.3mL
Final reconstructed volume	2.5mL
		Excess fill volume	0.5mL
Extractable volume	2.0mL

DP, pharmaceutical; FDS, preparing a raw material medicine; IV, veins; WFI, water for injection

Table 17: reconstitution volume, overfill volume and extractable volume of 50mg/mL mAb1 DP reconstituted for IV administration

Formulation components	Reconstitution for IV administration
		mAb1 concentration in FDS (Pre-lyophilization)	20mg/mL
Protein content per bottle	50mg
		mAb1 concentration in the reconstitution DP	20mg/mL
Target filling volume	2.5mL
		WFI volume for reconstruction	2.3mL
Final reconstructed volume	2.5mL
		Excess fill volume	0.5mL
Extractable volume	2.0mL

Stability studies were performed to determine long-term storage, accelerated stability (temperature above storage conditions) and stress stability (40 ℃/75% RH, stirring, freezing and thawing) of mAb1 drug substance (. Gtoreq.180 mg/ml mAb1, 30mM acetate, 5% w/v sucrose pH 5.0) and formulated drug substance (50 mg/ml mAb1, 30mM acetate, 10% w/v sucrose, 0.2% w/v polysorbate 20pH 5.0), as discussed more fully below.

No significant change in physical or chemical stability of mAb1 drug substance was detected when stored for up to 24 months at-80 ℃ and-30 ℃ (see tables 18 and 19). These results indicate that mAb1 drug substance was stable for at least 24 months when stored frozen under storage conditions. The results of the accelerated stability study are shown in tables 20 to 22. After incubation at-20 ℃ for up to 6 months, no significant change was observed in any of the monitored attributes. After incubation at 5 ℃ and 25 ℃/60% rh for up to 6 months, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After incubation at 5℃and 25℃C/60% RH for up to 6 months, an increase in HMW species was observed by SE-UPLC and non-reduced MCE. After 6 months of incubation at 5 ℃ and 25 ℃/60% rh, a decrease in region 1 (acidic species) was observed by CEX-UPLC, while region 2 (main peak) increased due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After 6 months incubation at 25 ℃/60% rh, an increase in region 1 (acidic species) was observed by iCIEF, while region 2 (main peak) decreased, possibly due to deamidation. These results indicate that mAb1 drug substance can be incubated at-20 ℃ for at least 6 months without compromising the physical or chemical stability of the protein. The results of the stress stability study are presented in tables 23, 24 and 17. mAb1 drug substance was physically and chemically stable when stirred (vortexed) for 10 minutes or exposed to 4 freeze/thaw cycles. After incubation at 40 ℃/75% rh for up to 3 months, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After incubation for 3 months at 40 ℃/75% rh, an increase in colour intensity was observed. After incubation at 40℃and 75% RH for up to 3 months, an increase in HMW and LMW species was observed by SE-UPLC and MCE. After incubation at 40 ℃/75% RH, a decrease in region 1 was observed by CEX-UPLC while a decrease in region 2 was observed, due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After 3 months incubation at 40 ℃/75% rh, an increase in region 3 was observed by CEX-UPLC while a decrease in region 2 was observed. The increase in region 3 is due to the increase in the alkaline peak in the high salt elution step required for CEX-UPLC. Results under acceleration and stress conditions indicate that HMW, LMW and charge variants are the primary degradation pathways for mAb1 drug substance.

Table 18: stability of mAb1 drug substance at-80 DEG C

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY2, color intensity is Not greater than the reference solution BY2; not > BY3, color intensity is Not greater than the reference solution BY3; not > I, turbidity is Not greater than the reference suspension I; NR, not required; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 19: stability of mAb1 drug substance at-30 DEG C

Table 20: stability of mAb1 drug substance at-20 DEG C

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CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY3, color intensity is Not greater than the reference solution BY3; not > I, turbidity is Not greater than the reference suspension I; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 21: stability of mAb1 drug substance stored at 5 DEG C

Table 22: stability of mAb1 drug substance stored at 25 ℃/60% RH

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CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY2, color intensity is Not greater than the reference solution BY2; not > BY3, color intensity is Not greater than the reference solution BY3; not > I, turbidity is Not greater than the reference suspension I; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 23: stability of mAb1 drug substance stored at 40 ℃/75% RH

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY1, color intensity is Not greater than the reference solution BY1; not > BY2, color intensity is Not greater than the reference solution BY2; not > BY3, color intensity is Not greater than the reference solution BY3; not > I, turbidity is Not greater than the reference suspension I; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 24: stability of mAb1 drug substance-influence of agitation and freeze/thaw

No significant change in physical or chemical stability of mAb1 formulated drug substance was detected when stored for up to 24 months at-80 ℃ and-30 ℃ (see tables 25 and 26). These results indicate that mAb1 formulated drug substance was stable for at least 24 months when stored frozen under storage conditions. The results of the accelerated stability study are shown in tables 27 to 29. After incubation of mAb1 formulated drug substance at-20 ℃ or 5 ℃ for up to 6 months, no significant change was observed in the monitored attributes. After incubation at 25 ℃/60% rh for up to 6 months, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After incubation at 5℃and 25℃C/60% RH for up to 6 months, an increase in HMW species was observed by SE-UPLC and non-reduced MCE. After 6 months of incubation at 25 ℃/60% rh, a decrease in region 1 (acidic species) was observed by CEX-UPLC, while region 2 (main peak) increased due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After 6 months incubation at 25 ℃/60% rh, an increase in region 1 (acidic species) was observed by iCIEF, while region 2 (main peak) decreased, possibly due to deamidation. These results indicate that mAb1 formulated drug substance can be incubated at-20 ℃ for at least 6 months and at 5 ℃ for 3 months without compromising the physical or chemical stability of the protein. mAb1 formulated drug substance can also withstand short exposure to temperatures of 25 ℃/60% rh. The results of the stress stability study are presented in tables 30 and 31. mAb1 formulated drug substances were physically and chemically stable when stirred (vortexed) for up to 120 minutes or subjected to up to four freeze and thaw cycles (a small increase in particle was observed in MFI after 4 freeze/thaw cycles). After incubation at 40 ℃/75% rh for up to 3 months, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After incubation at 40℃and 75% RH for up to 3 months, an increase in HMW and LMW species was observed by SE-UPLC and MCE. After incubation at 40 ℃/75% RH, a decrease in region 1 was observed by CEX-UPLC while a decrease in region 2 was observed, due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After incubation for 2 months at 40 ℃/75% rh, zone 1 decreased while zone 2 increased. However, after a three month time point, the trend reversed with a clear increase in zone 1 and zone 3 and a decrease in zone 2. The increase in zone 1 may be due to competing deamidation of asparagine or glutamine, while the increase in zone 3 is due to an increase in the alkaline peak eluted in the high salt elution step. Results under acceleration and stress conditions indicate that HMW, LMW and charge variants are the main degradation pathways for mAb1 formulation drug substances. After 4 freeze and thaw cycles, a small increase in 2-10 μm particles was observed by MFI.

Table 25: stability of mAb1 formulated drug substance at-80 DEG C

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY4, color intensity is Not greater than the reference solution BY4; not > BY5, color intensity is Not greater than the reference solution BY5; not > I, turbidity is Not greater than the reference suspension I; NR, not required; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 26: stability of mAb1 formulated drug substance at-30 DEG C

Table 27: stability of mAb1 formulated drug substance stored at-20deg.C

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CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY4, color intensity is Not greater than the reference solution BY4; not > I, turbidity is Not greater than the reference suspension I; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 28: stability of mAb1 formulated drug substance stored at 5 DEG C

Table 29: stability of mAb1 formulated drug substance stored at 25℃/60% RH

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY4, color intensity is Not greater than the reference solution BY4; not > I, turbidity is Not greater than the reference suspension I; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 30: stability of mAb1 formulated drug substance stored at 40 ℃ per 75% RH

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY4, color intensity is Not greater than the reference solution BY4; not > I, turbidity is Not greater than the reference suspension I; not > II, turbidity is no greater than the reference suspension II; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Table 31: stability of mAb1 formulated drug substance

Effects of stirring and freezing/thawing

CEX, cation exchange; HDPE, high density polyethylene; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; not > BY4, color intensity is Not greater than the reference solution BY4; not > I, turbidity is Not greater than the reference suspension I; NR, not required; SE, size exclusion; UPLC, ultra high performance liquid chromatography

Stability studies have been performed to assess the storage and accelerated stability of liquid mAb1 drugs. Liquid medicine for storage and accelerated stability studies was prepared by filling 2.5mL of the formulated drug substance in 6r ISO 1 glass vials. The liquid drug product is incubated under storage, acceleration and stress conditions. No significant change in physical or chemical stability of mAb1 liquid drug was detected when stored at 5 ℃ for up to 12 or 24 months (see tables 32 and 33). These results indicate that mAb1 liquid drug is stable for at least 12 or 24 months under storage conditions. The results of the accelerated stability study are shown in tables 34 and 35. At 25 ℃/60% rh for up to 6 months, a decrease in region 1 was observed by CEX-UPLC while region 2 increased, due to deglycosylation of the MUC16 arm at HC-CDR3-Lys 98. After incubation at 25 ℃/60% rh for up to 6 months, an increase in region 1 was observed by iCIEF while a decrease in region 2 was observed. In other monitoring properties, no significant change was observed. The results of the stress stability study are shown in tables 34 to 37. mAb1 liquid drug was physically and chemically stable when stirred (vortexed) for 120 minutes. After 3 months incubation at 40 ℃/75% RH, an increase in HMW and LMW species was observed by SE-UPLC. After incubation for 3 months at 40 ℃/75% rh, an increase in LMW species was observed by reduced and non-reduced MCE. Due to the different sensitivities of each assay, different trends in charge change were observed by CEX-UPLC and iCIEF. After incubation at 40 ℃/75% rh for up to 3 months, an increase in region 1 was observed by iCIEF, while region 2 decreased, possibly due to deamidation of asparagine or glutamine. After incubation at 40 ℃/75% rh for up to 2 months, a decrease in region 1 was observed by CEX-UPLC while a decrease in region 2 was observed. The clear increase in region 1, while the decrease in region 2 was observed by CEX-UPLC at the 3 month time point, probably due to the competitive deamidation reaction. At 40 ℃/75% rh, a decrease in region 3 was observed by iCIEF at the 3 month time point. After 4 freeze and thaw cycles, an increase in 2-10um particles was observed by MFI. Results under acceleration and stress conditions indicate that HMW, LMW and charge variants are the main degradation pathways for mAb1 liquid drugs.

Table 32: stability of 5.0mg/ml mAb1 liquid drug at 5 ℃

CEX, cation exchange; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MP, main peak; not > BY7, color intensity is Not greater than the reference solution BY7; not > I, turbidity is Not greater than the reference suspension I; NR, not required; eur, european pharmacopoeia; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

Table 33: stability of 50.0mg/ml mAb1 liquid drug at 5 ℃

CEX, cation exchange; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MP, main peak; not > BY5, color intensity is Not greater than the reference solution BY5; not > III, turbidity Not greater than reference suspension III; NR, not required; eur, european pharmacopoeia; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

Table 34: stability of 5.0mg/ml mAb1 liquid drug stored at 25 ℃/60% RH and 40℃/75% RH

CEX, cation exchange; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MP, main peak; not > BY7, color intensity is Not greater than the reference solution BY7; not > I, turbidity is Not greater than the reference suspension I; not > II, turbidity is no greater than the reference suspension II; NR, not required; eur, european pharmacopoeia; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

Table 35: stability of 50.0mg/ml mAb1 liquid drug stored at 25℃/60% RH and 40℃/75% RH

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CEX, cation exchange; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MP, main peak; not > BY4, color intensity is Not greater than the reference solution BY4; not > BY5, color intensity is Not greater than the reference solution BY5; not > III, turbidity Not greater than reference suspension III; NR, not required; eur, european pharmacopoeia; RH, relative humidity; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

Table 36:5.0mg/nl mAb1 liquid drug stability stirring and Effect of freezing/thawing

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Table 37: influence of stability stirring and freezing/thawing of 50.0mg/nl mAb1 liquid drug

CEX, cation exchange; HMW, high molecular weight; a liquid that is substantially free of visible particles; LMW, low molecular weight; MCE, microchip capillary electrophoresis; MP, main peak; not > BY5, color intensity is Not greater than the reference solution BY5; not > II, turbidity is no greater than the reference suspension II; not > III, turbidity Not greater than reference suspension III; NR, not required; eur, european pharmacopoeia; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

Example 3: freeze-drying cycle development

The lyophilization process developed for clinical production consisted of: freezing, primary drying and secondary drying. The lyophilization process is performed using FTS LyoStar ^TM III freeze dryer development, based on the-31.2 ℃ partial freeze-dried block collapse temperature, using a freeze-drying microscope to frozen blending bulk drug determination. During the primary drying process, the product temperature does not exceed the partial freeze-dried block collapse temperature, thereby maintaining the integrity of the freeze-dried block during the freeze-drying cycle. The secondary drying process was developed to ensure that the drug product had a low residual moisture content.

The lyophilization cycle required about 63 hours to produce lyophilized mAb1 drug in a 5mL type 1 glass vial containing 2.5mL 2mg/mL mAb1 formulated drug substance. The lyophilization cycle contained the steps shown in table 38 below.

Table 38: freeze-drying cycle

NA, inapplicable to

Example 4: development of stable liquid anti-MUC 16 x anti-CD 3 bispecific antibody formulations for intravenous administration and formulation stability

Development of Intravenous (IV) formulations, including formulations comprising 5mg/ml mAb1 and 50mg/ml mAb1, was performed to determine a pH that minimizes glycosylation changes of HCDR3-Lys98 in the MUC16 binding arm, a suitable buffer and concentration to maintain pH and overcome the Donnan effect observed during manufacture, a suitable heat stabilizer to maintain the concentration of viscosity and tonicity desired, and a suitable surfactant sufficient to dilute while maintaining the concentration of stable liquid formulation during IV administration.

As discussed herein, glycosylation at HC-CDR3-Lys98 of the MUC16 binding arm has a direct impact on potency. Deglycosylation results in increased potency as measured by bioassay. The main formulation factor affecting the desugarization rate of mAb1 is pH. By having three different pH values: 5.0, 5.5 and 6.0, 2mg/mL mAb1 was incubated at 5℃for 36 months or 25℃for 2 months in 10mM histidine, 10% (w/v) sucrose and 0.05% (w/v) polysorbate 20, and the effect of pH on glycosylation and High Molecular Weight (HMW) species levels in liquid formulations was studied (FIGS. 2A-2D). The main degradation pathways observed under these conditions are the formation of HMW species detected by CEX-UPLC and deglycosylation of HC-CDR3-Lys 98. As the pH of the mAb1 formulation was lowered, the level of HMW species formation and deglycosylation rate decreased.

Since histidine is not a good buffer at pH 5.0, sodium acetate was chosen as the buffer for the liquid formulation of mAb 1. The effect of pH was studied by incubating 50mg/mL mAb1 under heat stress in 10mM acetate containing 5% (w/v) sucrose and five different pH values (4.8, 5.0, 5.2, 5.5 and 5.7). After 28 days of incubation at 40℃the main degradation pathways are the formation of HMW species and deglycosylation of HC-CDR3-Lys 98. Consistent with that observed in histidine buffer, at lower pH levels of HMW species formation and deglycosylation rates were reduced (FIGS. 3A-3B). The pH of 5.0 was chosen as the target for the production of a stable formulation, the normal production variation of which would remain within the range required for stabilizing mAb 1.

The effect of acetate concentration on stability of 150mg/mL mAb1 formulation at pH 5.0 was examined in liquid formulations. Formulations containing acetate buffers ranging from 21 to 40mM were incubated at 45 deg.c for 14 days. The higher temperature allows for rapid detection of protein degradation. Analysis showed that HMW species and charge variant formation was the primary degradation pathway. An increase in acetate concentration resulted in an increase in HMW species, 30mM was selected based on this data. Acetate at a concentration of 30mM can address the Donnan effect observed during manufacture. As a result of the Donnan effect, 30mM acetate was required to maintain the formulation pH at 5.0. The acetate concentration provides improved stability in terms of HMW species formation under the stress conditions compared to formulations with higher concentrations of acetate (table 39). Thus, 30mM was chosen as acetate concentration for mAb1 liquid formulation for IV administration.

Table 39: stability of 150mg/mL mAb1 incubated in acetate buffer pH 5.0 at 45℃for 14 days

^a the results reported at t=0 represent the average of the initial values of all samples in the study

^b If the sample is clear to slightly milky, essentially free of visible particles, and colorless to pale yellow, it is assessed by color and appearance.

^c Compared to t=0 for each formulation

CEX, cation exchange; DS, bulk drug; HMW, high molecular weight; LMW, low molecular weight; OD, optical density; eur, european pharmacopoeia; SE, size exclusion; UPLC, ultra high performance liquid chromatography; USP, united states pharmacopoeia

The effect of sucrose on mAb1 stability was examined under freeze/thaw and heat stress in liquid formulations (150 mg/mL mAb1, 30mM acetate pH 5.0, containing 0% to 13% [ w/v ] sucrose). Higher temperatures allow rapid detection of protein degradation under these stresses. Under freeze/thaw stress, the formation of HMW species is the primary degradation pathway (table 40). The formation of HMW and LMW species and the change in charge variants are the major degradation pathways under thermal stress at 45 deg.c. Formulations with higher sucrose concentrations (. Gtoreq.10%) provided improved stability by reducing the level of HMW and LMW species and the deglycosylation rate of HC-CDR3-Lys98 (Table 41). To maintain the desired viscosity and tonicity of mAb1, 10% (w/v) sucrose was selected as a heat stabilizer for the liquid formulation for IV administration.

Table 40: stability of 150mg/mL mAb1 in 30mM acetate buffer with varying concentrations of sucrose (pH 5.0) under freeze/thaw stress conditions

^c Compared to t=0 for each formulation

Table 41: stability of 150mg/mL mAb1 in 30mM acetate buffer pH 5.0 containing varying concentrations of sucrose at 45℃for 14 days

^b If the sample is clear to slightly milky, essentially free of visible particles, and colorless to pale yellow, then by color andappearance evaluation.

^c Compared to t=0 for each formulation

During the initial development of mAb1 formulations, the need for surfactants was demonstrated. When the formulation was stirred by vortexing, an increase in mAb1 HMW species was observed when no surfactant was present. Surfactant stabilized mAb1 was added to the stirring stress. In the initial development, polysorbate 20 was chosen as the surfactant because polysorbate 20 has improved thermal stability compared to polysorbate 80. Formulations of mAb1 intended for IV administration also require polysorbate 20 in the formulation to stabilize mAb1 when diluted in 0.9% sodium chloride for IV administration.

The effect of polysorbate 20 on mAb1 stability was tested in a liquid formulation (50 mg/mL mAb1, 30mM acetate, 10% [ w/v ] sucrose pH 5.0, containing 0 to 0.25% [ w/v ] polysorbate 20) under agitation and heat stress.

mAb1 was stable in all liquid formulations tested under stirring stress (table 42). The formation of HMW species and charge variants is the primary degradation pathway under heat stress at 45 ℃ (table 43). Increasing the concentration of polysorbate 20 had no meaningful effect on the formation of charge variants, however, as the concentration of polysorbate 20 increased, an increase in the level of HMW-forming species was observed. Since these mAb1 formulations were intended for IV delivery by dilution in 0.9% sodium chloride, the formulations would contain 0.2% (w/v) polysorbate 20. Polysorbate 20 at 0.2% stabilized mAb1 and provided adequate mAb1 stability when diluted for IV administration.

Table 42: stability of 50mg/mL mAb1 in 30mM acetate buffer, 10% (w/v) sucrose pH 5.0 and polysorbate 20 by vortex agitation

^c Compared to t=0 for each formulation

Table 43: stability of 50mg/mL mAb1 in 30mM acetate buffer, 10% (w/v) sucrose pH 5.0 and polysorbate 20 at 45℃for 14 days

^c Compared to t=0 for each formulation

Long term, accelerated and stress stability studies were performed to verify the stability of the liquid formulation. The results indicate that these formulations provide adequate stability.

Example 5: development of stable liquid anti-MUC 16 x anti-CD 3 bispecific antibody formulations for subcutaneous administration and formulation stability

Subcutaneous (SC) formulations were developed, including formulations comprising 150mg/ml mAb1, to determine excipients and maximum concentrations for SC administration and stability.

The buffer, pH and surfactant parameters of the IV formulation (example 4) were determined and applied to the subcutaneous formulation. The concentration of surfactant (polysorbate 20) was also characterized during IV formulation development, and this data was used to select surfactant concentrations suitable for subcutaneous formulations. During the development of the subcutaneous formulation of mAb1, the buffer, pH and surfactant type and concentration were kept constant at the following levels:

pH:5.0. to minimize deglycosylation rate and HMW species formation

Buffer solution: 30mM sodium acetate. Maintaining a pH of 5.0 even though the Donnan effect is observed during processing

And (2) a surfactant: 0.05% (w/v) polysorbate 20. To balance the stability to agitation stress while minimizing the impact on thermal stability

Other factors evaluated against mAb1SC formulation include:

mAb1 concentration: evaluation range is 50-150mg/mL

When stored at 2-8 ℃, mAb1 concentration reached the required clinical dose while maintaining stability for at least 24 months and viscosity below 20cP at 20 ℃, target osmotic pressure of 290-400mOsm/kg.

Sucrose concentration: the evaluation range is 2% -10%

The o-sucrose concentration provides adequate thermal stability while minimizing the effect on osmotic pressure and viscosity

o at least 2% (w/v) sucrose is required to stabilize mAb1 against freeze/thaw stress

Arginine concentration: the evaluation range is 0-100mM

o evaluate arginine's ability to reduce viscosity and evaluate the effect on stability and osmotic pressure

Formulations evaluated for SC administration are shown in table 44 below.

Table 44: testing formulations for mAb1SC administration

Viscosity analysis-figure 4 shows the relationship of mAb1 viscosity to mAb1 concentration, sucrose concentration and arginine concentration. The following observations were made from this study: the main factor contributing to viscosity is mAb1 concentration. In the range of 50-150mg/mL, the viscosity increases exponentially from about 2cP to about 11cP (at 20 ℃); (ii) the viscosity is less dependent on sucrose. At a mAb1 concentration of 150mg/mL, viscosity increased from about 11cP to 13cP (at 20 ℃) when sucrose was changed from 2% (w/v) to 10% (w/v); and (iii) the effect of arginine concentration on viscosity is similar in magnitude to sucrose, but increasing arginine reduces viscosity from about 11cP to 9cP (at 20 ℃).

Osmolarity analysis-figure 5 shows the relationship of mAb1 osmolality with mAb1 concentration, sucrose concentration and arginine concentration. The following observations were made from this study: (i) In the range of 50-150mg/mL, mAb1 concentration contributes negligible to osmotic pressure; (ii) the major factor responsible for osmotic pressure is sucrose. At a mAb1 concentration of 150mg/mL, when sucrose was varied from 2% (w/v) to 10% (w/v), the osmotic pressure increased from about 90mOsm/kg to about 410mOsm/kg; and (iii) increasing arginine concentration also results in increased osmotic pressure. Increasing arginine from 0mM to 100mM increases the osmotic pressure from about 90mOsm/kg to about 300mOsm/kg at 150mg/mL mAb 1.

Stability analysis-figure 6 shows the relationship of stability of mAb1 with mAb1 concentration, sucrose concentration and arginine concentration. In this study, HMW species formation was evaluated at 25 ℃/60% RH and 40 ℃/75% RH. Furthermore, the loss of acidic species and the loss of glycosylated species were evaluated under the same conditions. The following results were observed: (i) At 25 ℃/60% rh and 40 ℃/75% rh, an increase in sucrose concentration results in a decrease in the rate of HMW formation; (ii) Increasing arginine concentration resulted in a decrease in HMW formation rate at 25 ℃/60% rh, but an increase in HMW formation rate at 40 ℃/75% rh; (iii) Increasing sucrose concentration at 25 ℃/60% rh resulted in a decrease in the rate of formation of acidic and glycosylated species. (acidic and glycosylated materials will decrease over time, so a smaller negative rate means less degradation over time); and (iv) increasing the arginine concentration at 25 ℃/60% rh results in a decrease in the rate of glycosylated species formation.

Based on previous development studies (e.g., examples 1 and 2) and the present study, two pilot formulations were selected for further stability, viscosity and osmolarity evaluation. Table 45 below shows the two pilot formulations and the respective viscosities and osmotic pressures. Both formulations met viscosity and osmolality targets at a mAb1 concentration of 150 mg/mL. The two formulations showed comparable stability when incubated for 3 months at 40 ℃/75% rh, 6 months at 25 ℃/60% rh or 6 months at 2 ℃ -8 ℃ (figures 7A and 7B). Both of these formulations showed considerable variation in HMW species and glycosylated species.

Table 45: exemplary mAb1 SC formulations

Based on these data, the two formulations were comparable. Arginine was not selected as an excipient in subcutaneous formulations because it had little improvement in stability or viscosity but resulted in an increase in osmotic pressure. Thus, preferred exemplary subcutaneous formulations are: 150mg/mL mAb1;30mM acetate pH 5.0;8% w/v sucrose; and 0.05% w/v polysorbate 20.

Stability studies were also initiated to assess storage, stress and acceleration stability of mAb1 formulations. Stability studies included Subcutaneous (SC) formulations (150 mg/mL mAb1 in 30mM sodium acetate, 8% (w/v) sucrose, 0.05% (w/v) polysorbate 20, pH 5.0) stored in Schott 6R borosilicate glass vials. The liquid formulation is incubated under storage, stress and acceleration conditions. The stress and acceleration conditions were chosen to mimic the conditions exceeded during drug production and handling and elucidate the degradation pathway of mAb 1. Stability under additional stresses including agitation and freeze/thaw was also evaluated.

The antibody in the formulation (mAb 1) was assessed to be physically and chemically stable when stored at 5 ℃ for at least 6 months (table 46). In the 6R vials, no significant change in stability was detected in any of the monitored attributes at 5 ℃. Table 47 provides the results of analysis of mAb1 formulations after incubation under accelerated and stressed conditions. After 6 months of incubation at 25℃C/60% RH, a 1.7% increase in HMW species was observed by SE-UPLC. Due to the different sensitivities of each assay, different trends in charge change were observed by CEX-UPLC and iCIEF. An increase in region 1 is observed by iCIEF, while a decrease in region 2 is observed. At 25 ℃/60% rh for up to 6 months, a decrease in region 1 was observed by CEX-UPLC while a increase in region 2 was observed, as deglycosylation of HC-CDR3-Lys98 of the MUC16 arm occurred (deglycosylation also occurs under heat stress conditions). The results under accelerated conditions indicate that the liquid formulation is stable under heat stress in a 6R vial.

After 3 months incubation at 40 ℃/75% RH, an increase in HMW and LMW species of 15.9% and 1.1%, respectively, was observed by SE-UPLC. Due to the different sensitivities of each assay, different trends in charge change were observed by CEX-UPLC and iCIEF. After incubation at 40 ℃/75% rh for up to 3 months, an increase in region 1 was observed by iCIEF, while regions 2 and 3 decreased, possibly due to deamidation of asparagine or glutamine. After incubation at 40 ℃/75% rh for up to 2 months, a decrease in region 1 was observed by CEX-UPLC while a decrease in region 2 was observed due to deglycosylation at HC-CDR3-Lys98 of the MUC16 arm (deglycosylation also occurs under accelerated stress conditions). The clear increase in region 1, while the decrease in region 2 was observed by CEX-UPLC at the 3 month time point, probably due to the competitive deamidation reaction. A16.3% increase in region 3 was also observed by CEX-UPLC after 3 months at 40 ℃/75% RH. This increase was determined to consist of oligomeric species of mAb1, mainly including tetrameric, pentameric, hexameric and heptameric species. Results from incubation of mAb1 formulations at 40 ℃/75% rh indicated that the formation of HMW and LMW species and changes in the distribution of charge variants are the primary degradation pathways for mAb1 drugs.

mAb1 formulations were physically and chemically stable when vortexed for 60 or 120 minutes (table 48). No significant change in physical or chemical stability was detected in any of the monitored attributes. mAb1 formulations were also physically and chemically stable when subjected to 4 freeze and thaw cycles (table 48). No significant change in physical or chemical stability was detected in any of the monitored attributes.

Table 46: stability of mAb1 drug at 5℃

^a. The standard of the quality target of the FBP-015-FD FDG platform is adopted, and the project is specifically adjusted. NR means that no test was performed at the set time point.

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; LMW, low molecular weight; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the MP, main peak; SE-UPLC, size exclusion ultra high performance liquid chromatography; NR, not required; LEFVP, a liquid substantially free of visible particles.

Table 47: stability of mAb1 drug incubated at 25℃/60% RH and 40℃C

/>

NR means that no test was performed at the set time point.

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, finished product Isoelectric focusing like capillary; LMW, low molecular weight; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the MP, main peak; SE-UPLC, size exclusion ultra high performance liquid chromatography; NR, not requiring

Table 48: stability of mAb1 drug-stirring and Effect of freezing/thawing

/>

NR means that no test was performed at the set time point.

DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; LMW, low molecular weight; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the MP, main peak; SE-UPLC, size exclusion ultra high performance liquid chromatography; NR, not requiring

Additional stability studies were initiated to determine long term storage, accelerated stability (temperature above storage conditions) and stress stability (40 ℃/75% rh, stirring, freezing and thawing) of mAb1 formulations of 150mg/ml antibody. mAb1 formulations with 150mg/mL antibody were filled into 5mL polycarbonate vials for agitation, freeze/thaw, frozen storage, and accelerated and stressed storage conditions. Polycarbonate vials are representative storage containers for mAb1 formulations (formulated drug substance) produced by GMP factories. The formulations tested contained 150mg/mL of purified mAb1 in an aqueous buffer containing 30mM sodium acetate pH 5.0, 8% (w/v) sucrose and 0.05% (w/v) PS 20.

No significant change in physical or chemical stability of mAb1 formulations was detected when stored for up to 6 months at-80 ℃ and-30 ℃ (tables 49 and 50). These results indicate that mAb1 (150 mg/mL) was stable for at least 6 months when stored frozen under storage conditions.

The results of the accelerated stability study are shown in tables 51A and 51B. After incubation of mAb1 formulation (150 mg/mL antibody) at-20℃for up to 6 months, no significant change in the monitoring properties was observed. After 6 months incubation at 25 ℃/60% rh, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After 6 months of incubation at 5℃and 25℃C/60% RH, an increase in HMW species was observed by SE-UPLC. After 6 months of incubation at 25 ℃/60% rh, a decrease in region 1 (acidic species) was observed by CEX-UPLC, while region 2 (main peak) increased due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After 6 months incubation at 25 ℃/60% rh, an increase in region 1 (acidic species) was observed by iCIEF, while region 2 (main peak) decreased, possibly due to deamidation. These results indicate that mAb1 (150 mg/mL) formulations can be incubated at-20 ℃ for at least 6 months and at 5 ℃ for 3 months without compromising the physical or chemical stability of the protein. mAb1 formulations can also withstand short exposure to temperatures of 25 ℃/60% rh.

The results of the stress stability study are presented in tables 51A and 51B and table 52. mAb1 (150 mg/mL) formulations were physically and chemically stable when stirred (vortexed) for up to 120 minutes or subjected to up to four freeze and thaw cycles. After incubation at 40 ℃/75% rh for up to 3 months, an increase in protein concentration was observed by solohpe, probably due to sample evaporation. After incubation at 40 ℃/75% RH for up to 3 months, increased HMW and LMW species were observed by SE-UPLC. After incubation at 40 ℃/75% RH, a decrease in region 1 was observed by CEX-UPLC while a decrease in region 2 was observed, due to deglycosylation of HC-CDR3-Lys98 of the MUC16 arm. After incubation for 2 months at 40 ℃/75% rh, zone 1 decreased while zone 2 increased. However, after a three month time point, the trend reversed with a clear increase in zone 1 and zone 3 and a decrease in zone 2. The increase in region 1 may be due to competing deamidation of asparagine or glutamine, while the increase in region 3 was determined to consist of oligomeric species of mAb1, mainly including tetrameric, pentameric, hexameric and heptameric species. Results under acceleration and stress conditions indicate that HMW, LMW and charge variants are the primary degradation pathways for mAb1 (150 mg/mL).

Table 49: stability of mAb1 formulated drug substance at-80 DEG C

a. The standard of the quality target of the FBP-015-FD FDG platform is adopted, and the project is specifically adjusted. The percentage of measurement reports of no quality targets is for reference only. The grey box indicates that no test was performed at the set time point.

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; a liquid substantially free of visible particles; LMW, low molecular weight; MFI, microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the MP, main peak; SE-UPLC, size exclusion ultra high performance liquid chromatography.

Table 50: stability of mAb1 formulated drug substance at-30 DEG C

Table 51A: stability of mAb1 formulated drug substance incubated at-20deg.C, 5deg.C

^a By usingThe standard of the quality target of the FBP-015-FD FDG platform is adopted, and the project is specifically adjusted. The percentage of measurement reports of no quality targets is for reference only. The grey box indicates that no test was performed at the set time point.

Table 51B: stability of mAb1 formulated drug substance incubated at 25℃/60% RH and 40℃/75% RH

^a The standard of the quality target of the FBP-015-FD FDG platform is adopted, and the project is specifically adjusted. The percentage of measurement reports of no quality targets is for reference only. The grey box indicates that no test was performed at the set time point.

Table 52: stability of mAb1 formulated drug substance-stirring and influence of freezing/thawing

^a Adopts the standard of the quality target of the FBP-015-FD FDG platform and carries out specific adjustment on the projectAnd (3) finishing. The percentage of measurement reports of no quality targets is for reference only. The grey box indicates that no test was performed at the set time point.

CEX, cation exchange; DS, bulk drug; FDG, formulation development group; HMW, high molecular weight; iCIEF, imaging capillary isoelectric focusing; a liquid substantially free of visible particles; LMW, low molecular weight; microfluidic imaging ^TM The method comprises the steps of carrying out a first treatment on the surface of the MP, main peak; SE-UPLC, size exclusion ultra high performance liquid chromatography.

The scope of the invention is not limited by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

*******

Sequence listing

<110> Ruizhen pharmaceutical Co

<120> Stable formulations containing anti-MUC 16 x anti-CD 3 bispecific antibodies

<130> 10820WO01

<150> 63/170,320

<151> 2021-04-02

<150> 63/313,927

<151> 2022-02-25

<160> 19

<170> patent in version 3.5

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Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

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Ser Tyr Ile Ser Gly Arg Gly Ser Thr Ile Phe Tyr Ala Asp Ser Val

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Lys Gly Arg Ile Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Phe

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Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys

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Val Lys Asp Arg Gly Gly Tyr Ser Pro Tyr Trp Gly Gln Gly Thr Leu

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Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

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Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys

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Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

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Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

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Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

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Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn

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Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro

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Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro

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Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr

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Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn

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Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg

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Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val

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Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser

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Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys

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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu

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Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe

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Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

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Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

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Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly

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Ser Gly Ile Ser Trp Asn Ser Gly Ser Lys Gly Tyr Ala Asp Ser Val

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Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

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Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys

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Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu

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Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro

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Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr

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Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

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Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn

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Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

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Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

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Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

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Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

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Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu

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Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Leu Gly Lys

325

<210> 17

<211> 326

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 17

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 18

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 18

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

<210> 19

<211> 329

<212> PRT

<213> artificial sequence

<220>

<223> Synthesis

<400> 19

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

Claims

1. A stable liquid pharmaceutical formulation comprising:

(a) A bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), and the second antigen-binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) contained in a Heavy Chain Variable Region (HCVR) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Heavy Chain Variable Region (HCVR), wherein A1-HCDR2 and A1-HCDR3 comprise amino acid sequences of SEQ ID NO 7 and 8, A2-HCDR2 and LCDR3, amino acid sequences of SEQ ID NOs 2-1, 1-HCDR2 and 11 and 12, and amino acid sequences of SEQ ID NOs 14 and 12-HCDR 3, respectively;

(b) A buffer comprising sodium acetate;

(c) An organic co-solvent comprising polysorbate; and

(d) A stabilizer comprising a sugar;

wherein the pH of the formulation is 5.0.+ -. 0.5.

2. The pharmaceutical formulation of claim 1, wherein the antibody concentration is 1mg/ml ± 0.1mg/ml to 200mg/ml ± 20mg/ml.

3. The pharmaceutical formulation of claim 2, wherein the antibody concentration is 5mg/ml ± 0.5mg/ml to 50mg/ml ± 5mg/ml.

4. The pharmaceutical formulation of claim 3, wherein the antibody concentration is 5mg/ml ± 0.5mg/ml.

5. The pharmaceutical formulation of claim 3, wherein the antibody concentration is 50mg/ml ± 5mg/ml.

6. The pharmaceutical formulation of any one of claims 1-5, wherein the histidine buffer concentration is 10mM ± 1mM to 50mM ± 5mM.

7. The pharmaceutical formulation of claim 6, wherein the acetate buffer concentration is 25mM ± 2.5mM to 35mM ± 3.5mM.

8. The pharmaceutical formulation of claim 7, wherein the acetate buffer concentration is 30mM ± 3mM.

9. The pharmaceutical formulation of any one of claims 1-8, wherein the polysorbate concentration is 0.01% ± 0.005% to 0.5% ± 0.05% w/v.

10. The pharmaceutical formulation of claim 9, wherein the polysorbate concentration is 0.1% ± 0.05% to 0.3% ± 0.03% w/v.

11. The pharmaceutical formulation of claim 9, wherein the polysorbate concentration is 0.2% ± 0.02% w/v.

12. The pharmaceutical formulation of any one of claims 1-11, wherein the polysorbate is polysorbate 20.

13. The pharmaceutical formulation of any one of claims 1 to 12, wherein the sugar is sucrose.

14. The pharmaceutical formulation of claim 13, wherein the sucrose concentration is 5% ± 1% to 20% ± 4% w/v.

15. The pharmaceutical formulation of claim 14, wherein the sucrose concentration is 7% ± 0.5% to 12% ± 0.5% w/v.

16. The pharmaceutical formulation of claim 15, wherein the sucrose concentration is 10% ± 1% w/v.

17. The pharmaceutical formulation of claim 1, comprising:

(a) 5 mg/ml.+ -. 0.5mg/ml antibody,

(b) Acetate buffer of 25 mM.+ -. 2mM to 35 mM.+ -. 2mM,

(c) 0.1% ± 0.05% to 0.3% ± 0.05% w/v polysorbate, and

(d) 5% + -1% to 15% + -3% w/v sucrose,

the pH was 5.0.+ -. 0.5.

18. The pharmaceutical formulation of claim 17, comprising:

(a) 5 mg/ml.+ -. 0.5mg/ml antibody,

(b) 30 mM.+ -.1 mM acetate buffer,

(c) 0.2% + -0.02% w/v polysorbate, and

(d) 10% + -1% w/v sucrose,

the pH was 5.0.+ -. 0.3.

19. The pharmaceutical formulation of claim 1, comprising:

(a) 50 mg/ml.+ -. 5mg/ml of antibody,

(b) Acetate buffer of 25 mM.+ -. 2mM to 35 mM.+ -. 2mM,

(c) 0.1% ± 0.05% to 0.3% ± 0.05% w/v polysorbate, and

(d) 5% + -1% to 15% + -3% w/v sucrose,

the pH was 5.0.+ -. 0.5.

20. The pharmaceutical formulation of claim 19, comprising:

(a) 50 mg/ml.+ -. 0.5mg/ml antibody,

(b) 30 mM.+ -.1 mM acetate buffer,

(c) 0.2% + -0.02% w/v polysorbate, and

(d) 10% + -1% w/v sucrose,

the pH was 5.0.+ -. 0.3.

21. The pharmaceutical formulation of any one of claims 17-20, wherein the polysorbate is polysorbate 20.

22. The pharmaceutical formulation of any one of claims 1-21, wherein the formulation contains no more than 2.5% High Molecular Weight (HMW) species after 12 months or 24 months of storage at 5 ℃ as determined by SE-UPLC.

23. The pharmaceutical formulation of any one of claims 1-21, wherein the formulation contains no more than 3.5% High Molecular Weight (HMW) species after storage for 6 months at 25 ℃ and 60% relative humidity as determined by SE-UPLC.

24. The pharmaceutical formulation of any one of claims 1-21, wherein the formulation contains no more than 1.5% High Molecular Weight (HMW) species after 12 months of storage at-30 ℃ or no more than 2.0% HMW species after 24 months of storage at-30 ℃ as determined by SE-UPLC.

25. The pharmaceutical formulation of any one of claims 1-21, wherein the formulation contains no more than 1.5% High Molecular Weight (HMW) species after 12 months of storage at-80 ℃ or no more than 2.0% HMW species after 24 months of storage at-30 ℃ as determined by SE-UPLC.

26. A stable liquid pharmaceutical formulation reconstituted from a lyophilisate comprising:

(a) A bispecific antibody at a concentration of 1mg/ml to 30mg/ml, wherein the bispecific antibody comprises a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) and three light chain CDRs (LCDR 2 and LCDR 3) contained in a Heavy Chain Variable Region (HCVR), wherein A1-HCDR1, A1-HCDR2 and LCDR2 comprise amino acid sequences comprising SEQ ID NOs 7, HCDR 2-HCDR2 and HCDR3, amino acid NOs 10-HCDR 2, HCDR2 and amino acid sequences comprising SEQ ID NOs 10, 14-HCDR 2 and 12, HCDR3, and amino acid sequences comprising SEQ ID NOs 10, and 10, respectively;

(b) A buffer comprising histidine;

(c) An organic co-solvent comprising polysorbate; and

(d) A stabilizer comprising a sugar;

wherein the pH of the formulation is 6.0.+ -. 0.5.

27. The pharmaceutical formulation of claim 26, wherein the antibody concentration is 2mg/ml ± 0.5mg/ml or 20mg/ml ± 2mg/ml.

28. The pharmaceutical formulation of claim 27, wherein the histidine buffer concentration is 5mM ± 1mM to 15mM ± 1mM.

29. The pharmaceutical formulation of claim 28, wherein the histidine buffer concentration is 10mM ± 1mM.

30. The pharmaceutical formulation of any one of claims 26-29, wherein the polysorbate concentration is 0.01% to 0.1% w/v.

31. The pharmaceutical formulation of claim 30, wherein the polysorbate concentration is 0.05% ± 0.01% w/v.

32. The pharmaceutical formulation of any one of claims 26-31, wherein the polysorbate is polysorbate 20.

33. The pharmaceutical formulation of any one of claims 26-32, wherein the sugar is sucrose.

34. The pharmaceutical formulation of claim 33, wherein the sucrose concentration is 8% ± 0.5% to 12% ± 0.5% w/v.

35. The pharmaceutical formulation of claim 34, wherein the sucrose concentration is 10% ± 1% w/v.

36. The pharmaceutical formulation of any one of claims 26 to 35, wherein: as determined by SE-UPLC, (a) after 12 months, 18 months, 24 months or 36 months of storage at 5 ℃ at least 95% of said antibodies have a native conformation; (b) At least 95% of the antibodies have a native conformation after 6 months of storage at 25 ℃ and 60% relative humidity; (c) After 3 months of storage at 37 ℃, at least 95% of the antibodies have a native conformation; (d) After 12 months, 18 months, 24 months or 36 months of storage at 5 ℃, the formulation contains no more than 1% High Molecular Weight (HMW) species; (e) After 6 months of storage at 25 ℃ and 60% relative humidity, the formulation contains no more than 1% HMW species; or (f) the formulation contains no more than 1% HMW species after storage at 37℃for 3 months.

37. A stable liquid pharmaceutical formulation comprising:

(a) A bispecific antibody at a concentration of 100mg/ml to 200mg/ml, wherein the bispecific antibody comprises a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises three heavy chain Complementarity Determining Regions (CDRs) contained in a Heavy Chain Variable Region (HCVR) (A1-HCDR 1, A1-HCDR2 and A1-HCDR 3) and three light chain CDRs (LCDR 1, LCDR2 and LCDR 3) contained in a Light Chain Variable Region (LCVR), wherein the second antigen binding domain comprises three heavy chain CDRs (A2-HCDR 1, A2-HCDR2 and A2-HCDR 3) and three light chain CDRs (LCDR 2 and LCDR 3) contained in a Heavy Chain Variable Region (HCVR), wherein A1-HCDR1, A1-HCDR2 and LCDR2 comprise amino acid sequences comprising SEQ ID NOs 7, 1-HCDR2 and HCDR3, amino acid sequences comprising SEQ ID NOs 10 to 12, and 13, respectively;

(b) A buffer comprising acetate;

(c) A stabilizer comprising a sugar; and

(d) A surfactant comprising a polysorbate;

wherein the pH of the formulation is 5.0.+ -. 0.5.

38. The pharmaceutical formulation of claim 37, wherein the antibody concentration is 125mg/ml to 175mg/ml.

39. The pharmaceutical formulation of claim 38, wherein the antibody concentration is 150mg/ml ± 10mg/ml.

40. The pharmaceutical formulation of any one of claims 37-39, wherein the sugar is sucrose.

41. The pharmaceutical formulation of claim 40, wherein the sucrose concentration is 4% to 12% w/v.

42. The pharmaceutical formulation of claim 41, wherein the sucrose concentration is 8% w/v.+ -. 1% w/v.

43. The pharmaceutical formulation of any one of claims 37-42, wherein the acetate buffer concentration is 25mM to 35mM.

44. The pharmaceutical formulation of claim 43, wherein the acetate buffer concentration is 30 mM+ -1 mM.

45. The pharmaceutical formulation of any one of claims 37-44, wherein the polysorbate is polysorbate 20.

46. The pharmaceutical formulation of claim 45, wherein the polysorbate 20 concentration is 0.01% w/v to 0.1% w/v.

47. The pharmaceutical formulation of claim 46, wherein the polysorbate 20 concentration is 0.05% w/v ± 0.01% w/v.

48. The pharmaceutical formulation of any one of claims 37-47, wherein: as determined by SE-UPLC, (a) after 12 months or 24 months of storage at-30℃or-80℃the formulation contains no more than 2.5% High Molecular Weight (HMW) species; (b) After 6 months of storage at 5 ℃, the formulation contains no more than 4% HMW species; or (c) the formulation contains no more than 6% HMW species after storage at 25℃and 60% relative humidity for 6 months.

49. The pharmaceutical formulation of any one of claims 1-48, wherein the formulation contains NO more than 40% of a glycosylation species variant, wherein the glycosylation species variant comprises glycosylation at residue 98 of SEQ ID NO 1 or SEQ ID NO 4 or residue 2 of SEQ ID NO 9.

50. The pharmaceutical formulation of any one of claims 1-49, wherein the first antigen binding domain comprises a HCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 90% identity to the amino acid sequence of SEQ ID No. 6.

51. The pharmaceutical formulation of claim 50, wherein the first antigen binding domain comprises a HCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 95% identity to the amino acid sequence of SEQ ID No. 6.

52. The pharmaceutical formulation of claim 51, wherein the first antigen binding domain comprises a HCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 4 and a LCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises a HCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 5 and a LCVR having at least 99% identity to the amino acid sequence of SEQ ID No. 6.

53. The pharmaceutical formulation of any one of claims 1-52, wherein the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID No. 4 and a LCVR comprising the amino acid sequence of SEQ ID No. 6 and the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID No. 5 and a LCVR comprising the amino acid sequence of SEQ ID No. 6.

54. A stable pharmaceutical formulation comprising:

(a) 5 mg/ml.+ -. 0.5mg/ml of a bispecific antibody comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 4 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6, and the second antigen binding domain comprises a HCVR comprising the amino acid sequence of SEQ ID NO. 5 and a LCVR comprising the amino acid sequence of SEQ ID NO. 6;

(b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2,

(c) 0.2% + -0.02% w/v polysorbate 20, and

(d) 10% + -1% w/v sucrose.

55. A stable pharmaceutical formulation comprising:

(a) 50mg/ml ± 5mg/ml of a bispecific antibody comprising a first antigen-binding domain that specifically binds human MUC16 and a second antigen-binding domain that specifically binds human CD3, wherein the first antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and the second antigen-binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6;

(b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2,

(c) 0.2% + -0.02% w/v polysorbate 20, and

(d) 10% + -1% w/v sucrose.

56. A stable pharmaceutical formulation comprising:

(a) A bispecific antibody of 150mg/ml ± 15mg/ml comprising a first antigen binding domain that specifically binds human MUC16 and a second antigen binding domain that specifically binds human CD3, wherein the first antigen binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 4 and an LCVR comprising the amino acid sequence of SEQ ID No. 6, and the second antigen binding domain comprises an HCVR comprising the amino acid sequence of SEQ ID No. 5 and an LCVR comprising the amino acid sequence of SEQ ID No. 6;

(b) 30 mM+ -1 mM sodium acetate buffer, pH 5.0+ -0.2,

(c) 0.05% + -0.01% w/v polysorbate 20, and

(d) 8% + -1% w/v sucrose.

57. The pharmaceutical formulation of any one of claims 50-56, wherein the antibody comprises a human IgG heavy chain constant region of HCVR attached to each of the first and second antigen binding domains, respectively.

58. The pharmaceutical formulation of claim 57, wherein the heavy chain constant region is of isotype IgG1.

59. The pharmaceutical formulation of claim 57, wherein the heavy chain constant region is of isotype IgG4.

60. The pharmaceutical formulation of any one of claims 57-59, wherein the heavy chain constant region of the HCVR attached to the first antigen binding domain or the heavy chain constant region of the HCVR attached to the second antigen binding domain, but not both, contain amino acid modifications that reduce protein a binding relative to a heavy chain of the same isotype without the modifications.

61. The pharmaceutical formulation of claim 60, wherein the modification comprises an H435R substitution (EU numbering) in the heavy chain of isotype IgG1 or IgG4.

62. The pharmaceutical formulation of claim 60, wherein the modification comprises an H435R substitution and a Y436F substitution (EU numbering) in the heavy chain of isotype IgG1 or IgG4.

63. The pharmaceutical formulation of any one of claims 57-59, wherein the antibody comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of seq id nos: SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18 and SEQ ID NO. 19.

64. The pharmaceutical formulation of claim 63, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 16 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 17.

65. The pharmaceutical formulation of claim 63, wherein the antibody comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 18 and a heavy chain constant region comprising the amino acid sequence of SEQ ID NO. 19.

66. The pharmaceutical formulation of any one of claims 50-56, wherein the antibody comprises a first heavy chain of a HCVR comprising the first antigen binding domain and a second heavy chain of a HCVR comprising the second antigen binding domain, wherein the first heavy chain comprises residues 1-442 of the amino acid sequence of SEQ ID No. 1 and the second heavy chain comprises residues 1-449 of the amino acid sequence of SEQ ID No. 2.

67. The pharmaceutical formulation of claim 66, wherein the antibody comprises a common light chain of LCVR comprising the first antigen binding domain and the second antigen binding domain, wherein the common light chain comprises the amino acid sequence of SEQ ID NO: 3.

68. The pharmaceutical formulation of any one of claims 1 to 67, wherein the percent change in glycosylated species is determined by cation exchange ultra performance liquid chromatography (CEX-UPLC) and/or liquid chromatography-mass spectrometry (LC-MS): (i) no more than 1.5% after storage at 5 ℃ for 6 months; (ii) no more than 3% after 12 months of storage at 5 ℃; (iii) No more than 1.5% after 12 months, 18 months or 24 months of storage at-30 ℃; or no more than 1% after 12 months, 18 months or 24 months of storage at-80 ℃.

69. A pharmaceutical composition, wherein the composition comprises the pharmaceutical formulation of any one of claims 1 to 68, and the composition is contained in a container.

70. The pharmaceutical composition of claim 69, wherein the container is a vial.

71. The pharmaceutical composition of claim 70, wherein the vial is a 2ml, 5ml, or 10ml type 1 clear glass vial.

72. The pharmaceutical composition of claim 69, wherein the container is a syringe.

73. The pharmaceutical composition of claim 72, wherein the syringe is low tungsten glass.

74. The pharmaceutical composition of claim 69, wherein the container is a pre-filled syringe.

75. The pharmaceutical composition of claim 69 contained in an auto-injector.

76. A kit comprising (i) a container containing a composition comprising the pharmaceutical formulation of any one of claims 1 to 68, and (ii) instructions for use of the composition.

77. The kit of claim 76, wherein the container is a glass vial.

78. The kit of claim 76, wherein the container is a prefilled syringe.

79. The kit of claim 76, wherein the container is an automatic injector.

80. The kit of claim 76, wherein the instructions recite subcutaneous administration of the composition.

81. The kit of claim 76, wherein the instructions recite intravenous administration of the composition.

82. A unit dosage form comprising the pharmaceutical formulation of any one of claims 1-68, wherein the antibody is present in an amount of 0.1mg to 500 mg.

83. The unit dosage form of claim 82, wherein the antibody is present in an amount of 1mg to 20 mg.

84. The unit dosage form of claim 82, wherein the antibody is present in an amount of 100mg to 200 mg.

85. The unit dosage form of claim 82, which is a glass vial.

86. The unit dosage form of claim 82, which is a pre-filled syringe.

87. The unit dosage form of claim 82, which is an auto-injector.

88. A container containing a composition comprising the pharmaceutical formulation of any one of claims 1 to 68.

89. The container of claim 88, which is a glass vial.

90. The container of claim 88, which is a prefilled syringe.

91. The container of claim 88, which is an auto-injector.