VEGF mini-traps and methods of use thereof

Abstract

To provide VEGF mini-trap molecules and methods for treating or preventing angiogenic disorders, such as angiogenic ocular disorders and cancer.SOLUTION: Disclosed is an isolated VEGF mini-trap (e.g., REGN7483F) (e.g., which can be monomeric, homodimeric, or homomultimeric), or a composition thereof, e.g., an aqueous composition. The invention also includes a composition (e.g., an aqueous composition) comprising a VEGF mini-trap (e.g., 2mS, REGN7851, REGN7483F, or REGN7483R) that is the product of a method comprising subjecting the mini-trap to anion exchange chromatography (e.g., in a load buffer at a pH of about 8.3 to 8.6 and / or a conductivity of about REGN7850 / cm), wherein the mini-trap is collected in a flow-through chromatography fraction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 944,635, filed December 6, 2019, which is incorporated herein by reference in its entirety.

[0002] Sequence Listing The Sequence Listing of the present application has been submitted electronically as an ASCII sequence listing with the filename "250298_000141_seqlist.TXT", created on December 2, 2020, and 115,306 bytes in size. This submitted Sequence Listing is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present invention provides VEGF mini-trap molecules, pharmaceutical compositions thereof, and methods for using them, for example, to treat angiogenic eye disorders and cancer. [Background technology]

[0004] Several eye disorders are associated with pathological angiogenesis. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from CNV causes macular edema and the accumulation of submacular fluid, resulting in vision loss. Diabetic macular edema (DME) is another eye disease with a neovascular component. DME is the most common cause of moderate vision loss in patients with diabetes and is a common complication of diabetic retinopathy, a disease affecting the retinal blood vessels. Clinically significant DME occurs when fluid leaks into the center of the macula, the light-sensitive part of the retina that contributes to clear, direct vision. Fluid in the macula can cause severe vision loss or blindness. Yet another eye disorder associated with abnormal angiogenesis is central retinal vein occlusion (CRVO). CRVO is caused by blockage of the central retinal vein, which leads to stagnation of blood and fluid within the retina. The retina may also become ischemic, leading to inappropriate new blood vessel growth, which can lead to further vision loss and more serious complications. The release of vascular endothelial growth factor (VEGF) contributes to increased vascular permeability and inappropriate new blood vessel growth in the eye. Therefore, inhibiting the pro-angiogenic properties of VEGF is an effective strategy for treating neovascular eye disorders. Summary of the Invention [Problem to be solved by the invention]

[0005] Various VEGF inhibitors, including the VEGF trap Eylea (aflibercept), have been approved for the treatment of these ocular disorders. The treatment protocol for delivering the VEGF trap involves intravitreal injection. Such a protocol is painful and inconvenient for patients, psychologically and physically traumatic, and carries the potential for adverse effects, such as infection, with each treatment event. While aflibercept has proven highly effective in treating various neovascular ocular disorders, it requires frequent monthly dosing. A therapeutic VEGF trap treatment that offers comparable efficacy but can be administered less frequently is of great interest. Dosing a higher molar dose of the VEGF minitrap compared to aflibercept would require fewer dosing events while still benefiting from the high therapeutic efficacy of aflibercept. [Means for solving the problem]

[0006] The present invention provides isolated VEGF mini-traps (e.g., REGN7483 F ) (which may be, for example, a monomer, homodimer, or homomultimer) having the following domain structure: and one or more histidines of the VEGF mini-trap are oxidized to 2-oxo-histidine, and / or one or more tryptophans are dioxidized (e.g., to N-formylkynurenine) or oxidized to hydroxytryptophan or dihydroxytryptophan or trihydroxyltryptophan, and / or one or more asparagines thereof are glycosylated, or ((R1D2)-(R2D3)-(R2D4)). a -(MC) b , ((R1D2)-(R2D3)) c -Linker-((R1D2)-(R2D3)) d ;or ((R1D2)-(R2D3)-(R2D4)) e -Linker-((R1D2)-(R2D3)-(R2D4)) fwherein R1D2 is VEGFR1 Ig domain 2; R2D3 is VEGFR2 Ig domain 3; R2D4 is VEGFR2 Ig domain 4; MC is the amino acid sequence: DKTHTCPPC (SEQ ID NO: 22), DKTHTCPPCPPC (SEQ ID NO: 23), DKTHTCPPCPPCPPC (SEQ ID NO: 24), where h is 1, 2, 3, 4, or 5. h (SEQ ID NO:25), DKTHTCPPCPAPELLG (SEQ ID NO:6), DKTHTCPLCPAPELLG (SEQ ID NO:7), DKTHTC (SEQ ID NO:8), or DKTHTCPLCPAP (SEQ ID NO:9), and the linker is a peptide containing about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, and independently, a=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; b=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15;

[0013] In an embodiment of the invention, an isolated VEGF minitrap, or composition thereof, e.g., an aqueous composition, is provided, wherein c=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; d=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; e=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and f=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In an embodiment of the invention, a minitrap (e.g., REGN7483) is provided. F ) is about 90 mg / ml. For example, in an embodiment of the invention, the VEGF mini-trap comprises or consists of an amino acid sequence set forth in a member selected from the group consisting of those set forth in SEQ ID NOs: 10, 11, 12, 13, 26, 27, 28, 29, 30, 32, or 33. In an embodiment of the invention, the mini-trap comprises the domain structure: (i) ((R1D2)-(R2D3)) a -Linker-((R1D2)-(R2D3)) b ; or (ii) ((R1D2)-(R2D3)-(R2D4)) c -Linker-((R1D2)-(R2D3)-(R2D4))d and (i) the R1D2 domains cooperate; (ii) the R2D3 domains cooperate; and / or (iii) the R2D4 domains cooperate to form a VEGF (e.g., VEGF-α) binding domain. In an embodiment of the invention, the linker is (Gly4Ser) n wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In embodiments of the invention, a VEGF minitrap or composition thereof comprises one or more histidines that are oxidized to 2-oxo-histidine, and / or one or more tryptophans that are dioxided, and / or one or more asparagines that are glycosylated. In embodiments of the invention, a composition (e.g., an aqueous composition) comprises a VEGF minitrap wherein 0.1% to 2% of the histidines of the VEGF minitrap are 2-oxo-histidine. In an embodiment of the invention, the composition comprises a VEGF mini-trap, wherein the VEGF mini-trap, which comprises one or more carboxymethylated cysteines and 2-oxo-histidine, is digested with Lys-C and trypsin protease (e.g., S. pyogenes IdeS or a sequence variant thereof) to produce an oligopeptide product, EIGLLTC, which contains approximately 0.006-0.013% 2-oxo-histidine. * EATVNGH * LYK (amino acids 73-89 of SEQ ID NO: 12), QTNTIIDVVLSPSH containing about 0.019-0.028% 2-oxo-histidine * GIELSVGEK (amino acids 97-119 of SEQ ID NO: 12), approximately 0.049-0. ELNVGIDFNWEYPSSKH containing 0.85% 2-oxo-histidine * QHK (amino acids 128-148 of SEQ ID NO: 12), DKTH containing about 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (amino acids 206-221 of SEQ ID NO: 12), TNYLTH containing about 0.010-0.022% 2-oxo-histidine* R (amino acids 90-96 of SEQ ID NO: 12), and / or IIWDSR (amino acids 56-61 of SEQ ID NO: 12) containing about 0.198-0.298% 2-oxo-histidine, wherein H * is histidine which may be oxidized to 2-oxo-histidine, C * is a cysteine ​​which may be carboxymethylated, and optionally one or more tryptophans of the oligopeptide are dioxided; or EIGLLTC containing about 0.0095 or 0.01% 2-oxo-histidine. * EATVNGH * LYK (amino acids 73-89 of SEQ ID NO: 12), QTNTIIDVVLSPSH containing approximately 0.0235 or 0.24% 2-oxo-histidine * GIELSVGEK (amino acids 97-119 of SEQ ID NO: 12), TELNVGIDFNWEYPSSKH containing about 0.067 or 0.07% 2-oxo-histidine * QHK (amino acids 128-148 of SEQ ID NO: 12), DKTH containing about 0.0745 or 0.075% 2-oxo-histidine * TC * PPC * PAPELLG (amino acids 206-221 of SEQ ID NO: 12), TNYLTH containing about 0.016 or 0.02% 2-oxo-histidine * R (amino acids 90-96 of SEQ ID NO: 12), and / or IIWDSR (amino acids 56-61 of SEQ ID NO: 12) containing about 0.248 or 0.25% 2-oxo-histidine, wherein H * is histidine which may be oxidized to 2-oxo-histidine, C * is an optionally carboxymethylated cysteine, and optionally one or more tryptophans of the oligopeptide are dioxided. In an embodiment of the invention, 2-oxo-histidine has the formula: [ka] It is characterized by:

[0007] The present invention relates to VEGF minitraps (e.g., REGN7850, REGN7851, REGN7483, F , or REGN7483 R ), wherein the composition (e.g., an aqueous composition) comprises: (i) a color that is less brownish yellow than the European Color Standard BY2; (ii) a color that is less brown-yellow than the European color standard BY3; (iii) a color less brown-yellow than European color standard BY4; (iv) a color that is less brown-yellow than the European color standard BY5; (v) a color that is less brown-yellow than the European color standard BY6; (vi) a color less brown-yellow than European color standard BY7; (vi) Colors between European color standards BY2 and BY3; (vii) Colors between European color standards BY2 and BY4; (vii) CIEL * a * b * In color space, L is about 70 to 99, a is about -2 to 0, and b is about 20 or lower; (viii) CIEL * a * b * A color in which, in color space, L is about 70 to 99, a is about -2 to 0, and b is about 10 to 31, about 10, about 14, about 12, about 14, about 15, about 18, about 21, about 27, or about 31; (ix) CIEL * a * b * In color space, L * , a * , and b * is roughly the color, which is as shown in one of the rows of Table 9-3 of the specification, or the BY value is approximately as shown in Table 9-3, and in some cases the concentration is also approximately as shown in the table; (x)CIEL* a * b * In color space, L * , a * , and b * is characterized by a color approximately as set forth in any of the rows of Table 17-1 herein, and optionally also at a concentration approximately as set forth in the table; optionally, the concentration of the VEGF mini-trap is about 70-200 mg / ml (e.g., 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / ml); or optionally, the concentration of the VEGF mini-trap is about 70-200 mg / ml (e.g., 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg / ml), but includes compositions characterized by a color when diluted to about 10 or 11 or 10-11 mg / ml. In an embodiment of the invention, the composition comprises a mini-trap of the invention and the color of the composition is determined by the following formula: 0.046 + (0.066 × concentration of mini-trap (mg / ml)) = b * or 0.05 + (0.07 × MiniTrap concentration (mg / ml)) = b * or b * = (0.11 × concentration of minitrap (mg / ml) - 0.56), where L * = approximately 97 to 99 and a = approximately -0.085 to 0.06 (e.g., approximately 0).

[0008] The invention also provides a method for producing a VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483), which comprises subjecting the minitrap to anion exchange chromatography (e.g., in a loading buffer at a pH of about 8.3-8.6 and / or a conductivity of about 2 mS / cm), wherein the minitrap is collected in the flow-through chromatography fraction. F , or REGN7483 RFor example, in an embodiment of the present invention, the method includes: (i) expressing aflibercept or a VEGF mini-trap in a host cell (e.g., Chinese hamster ovary cell) in a chemically defined liquid medium, wherein the aflibercept or the VEGF mini-trap is secreted from the host cell into the medium; (ii) when aflibercept is expressed, proteolytic cleavage of the aflibercept to produce a peptide comprising an Fc domain or a fragment thereof and the VEGF mini-trap, and further removing the Fc domain or a fragment thereof from the VEGF mini-trap; (iii) coupling the VEGF mini-trap to an anion exchange chromatography resin (e.g., a quaternary amine functional group; -O-CH2CHOHCH2OCH2CHOHCH2N + (CH3)3;-N +(CH3)3, or quaternized polyethyleneimine); and (iv) retaining the VEGF mini-trap polypeptide in the chromatography flow-through. In an embodiment of the invention, if aflibercept is expressed, the method further comprises Protein A purification of the aflibercept prior to proteolytic cleavage. In an embodiment of the invention, the proteolytic cleavage is carried out by incubating aflibercept with Streptococcus pyogenes IdeS protease or a variant thereof containing one or more point mutations. In an embodiment, the VEGF mini-trap is applied to an anion exchange chromatography resin that has been equilibrated with a buffer having a pH of about 8.4 or 7.7 and a conductivity of about 2.0 mS / cm, for example, 50 mM Tris pH 8.4±0.1 with a conductivity of 2.0 mS / cm; or an aqueous buffer containing 50 mM Tris, 60 mM NaCl, pH 7.7±0.1. In an embodiment of the present invention, the VEGF mini-trap is applied to the anion exchange resin while it is in an aqueous buffer solution having a pH of about 8.4 or 7.7 and a conductivity of about 2.0 mS / cm, for example, 50 mM Tris pH 8.4±0.1 with a conductivity of 2.0 mS / cm; or 50 mM Tris, 60 mM NaCl, pH 7.7±0.1. After applying the composition, the resin may be washed with an aqueous buffer solution, and the wash solution may be retained. In an embodiment of the present invention, the aflibercept Fc domain or fragment thereof is, after proteolytic cleavage, aflibercept Fc domain or fragment and a VEGF mini-trap are combined. The composition containing Nitrap is applied to a Protein A chromatography resin, and the VEGF minitrap is chromatographed away from the VEGF minitrap composition by retaining it in the flow-through fraction. In embodiments of the invention, the method further comprises adjusting the pH to a more acidic level (e.g., about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2), filtering, depth filtration, ultrafiltration, diafiltration, viral inactivation, cation exchange chromatography, Protein A chromatography purification, and / or hydrophobic interaction chromatography purification (e.g., with phenyl, octyl, or butyl functional groups, and / or running in bind-elute or flow-through mode) on, for example, Phenyl Sepharose FF, Capto Phenyl (GE Healthcare, Uppsala, Sweden), Phenyl 650-M (Tosoh Bioscience, Tokyo, Japan), or Sartobind Phenyl (Sartorius Corporation, NY, USA). In embodiments of the invention, the initial (day 0) cysteine ​​(e.g., cysteine ​​HCl·HO) concentration in the chemically defined liquid medium is about 1.5 mM, and additional cysteine ​​feed is added to the culture medium every two days (e.g., days 2, 4, 6, and 8) at 1.3 mM, 1.7 mM, or 2.1 mM (per volume or culture medium); the chemically defined liquid medium includes EDTA, and / or citric acid, iron, copper, zinc, and nickel; and / or the chemically defined liquid medium includes hypotaurine, taurine, glycine, thioctic acid, and / or vitamin C.

[0009] In an embodiment of the invention, the VEGF minitraps of the invention (e.g., REGN7850, REGN7851, REGN7483) F , or REGN7483 RFor example, the VEGF mini-trap (e.g., expressed in CDM (e.g., in CHO cells) and purified by AEX flow-through chromatography as described herein) has one or more asparagines of the VEGF mini-trap N-glycosylated; one or more serine or threonine of the VEGF mini-trap O-glycosylated; one or more asparagines of the VEGF mini-trap deamidated; one or more aspartate-glycine motifs of the VEGF mini-trap converted to isoaspartate-glycine and / or Asn-Gly; one or more methionines of the VEGF mini-trap oxidized; one or more tryptophans of the VEGF mini-trap converted to N-formylkynurenine; one or more arginines of the VEGF mini-trap converted to Arg3-deoxycyanate; the VEGF minitrap is converted to siglucosone; the C-terminal glycine (or other C-terminal residue) of the VEGF minitrap is absent; one or more non-glycosylated potential glycosylation sites are present in the VEGF minitrap; the VEGF minitrap contains about 40% to about 50% total fucosylated glycans; the VEGF minitrap contains about 30% to about 55% total sialylated glycans; the VEGF minitrap contains about 6% to about 15% mannose-5; the VEGF minitrap contains about 60% to about 79% galactosylated glycans; the VEGF minitrap is xylosylated; the VEGF minitrap is glycosylated at lysine; the VEGF minitrap contains a cystine with a free thiol group; the VEGF minitrap contains a trisulfide bridge; the VEGF minitrap contains an intrachain disulfide bridge; the VEGF minitrap contains disulfide bridges in a parallel orientation; and / or the VEGF minitrap is characterized by comprising a carboxymethylated lysine or arginine; and / or one or more asparagines of the VEGF minitrap are G0-GlcNAc glycosylated; G1-GlcNAc glycosylated; G1S-GlcNAc glycosylated; G0 glycosylated; G1 glycosylated; G1S glycosylated; G2 glycosylated; G2S glycosylated; G2S2 glycosylated; G0F glycosylated; G2F2S glycosylated; G2F2S2 glycosylated; G G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+phosphate glycosylation; Man6+phosphate glycosylation; and / or Man7 glycosylation, e.g., and / or about 30-36% (e.g., about 30, 31, 32, 32-35, 33, 34, 35, or 36%) of asparagine 123 residues and / or about 25-30% (e.g., about 25, 26, 27, 27-30, 28, 29, or 30%) of asparagine 196 residues contain Man5 glycosylation; about 6-8% (e.g., about 6, 7, 8%) of asparagine 36 are glycosylated with Man6-phosphate; and / or about 3-4% (e.g., about 3, 4, or 4.5%) of asparagine 123 are glycosylated with Man7. In embodiments of the invention, a minitrap of the invention (e.g., REGN7483) expressed in CDM (e.g., in CHO cells) and purified by AEX flow-through chromatography as described herein) is used. F ) has high mannose glycosylation at approximately 38% of the asparagine 123 residue and / or high mannose glycosylation at approximately 29% of the asparagine 196 residue.

[0010] The present invention provides VEGF minitraps as provided herein (e.g., REGN7850, REGN7851, REGN7483, F , or REGN7483 R ) or composition (e.g., aqueous composition) and a pharmaceutically acceptable carrier. An injection device (e.g., a pre-filled syringe (PFS), e.g., a sterile PFS) containing the VEGF mini-trap polypeptide, composition, or pharmaceutical formulation is also part of the invention.

[0011] In embodiments of the invention, VEGF minitraps as provided herein (e.g., REGN7850, REGN7851, REGN7483) are used. F , or REGN7483 R ), composition (e.g., aqueous composition), or pharmaceutical preparation is combined with an additional therapeutic agent.

[0012] The present invention also provides VEGF minitraps as provided herein (e.g., REGN7850, REGN7851, REGN7483, F , or REGN7483 R The present invention provides polynucleotides, e.g., DNA, encoding the polypeptides of VEGF minitraps. The present invention also provides vectors comprising the polynucleotides, and host cells (e.g., Chinese hamster ovary (CHO) cells) comprising the VEGF minitraps, polynucleotides, and / or vectors.

[0013] The present invention also provides VEGF minitraps (e.g., REGN7850, REGN7851, REGN7483) described herein. F , or REGN7483 RThe present invention also includes methods for producing a VEGF mini-trap, comprising introducing a polynucleotide encoding the polypeptide of the mini-trap into a host cell (e.g., a CHO cell), culturing the host cell in a medium under conditions in which the polypeptide is expressed, and optionally isolating the polypeptide from the host cell and / or the medium. The product of such a method, or a composition thereof (e.g., an aqueous composition), is also part of the invention.

[0014] The present invention also provides VEGF minitraps (e.g., REGN7850, REGN7851, REGN7483) described herein. F , or REGN7483 R VEGF traps (e.g., aflibercept or conbercept) are cleaved with an enzyme, such as S. pyogenes IdeS or S. pyogenes IdeS, that cleaves immunoglobulin Fc polypeptides after the following sequence: DKTHTCPPCPAPELLG (SEQ ID NO: 20). The present invention also includes methods comprising or consisting essentially of proteolysis of VEGF minitraps with Streptococcus equi subsp. zooepidemicus IdeZ. The products of such methods, or compositions thereof, are also part of the present invention.

[0015] The present invention also provides VEGF minitraps (e.g., REGN7850, REGN7851, REGN7483) described herein. F , or REGN7483 R ) or a composition (e.g., an aqueous composition) or a pharmaceutical formulation thereof to a subject (e.g., a human), comprising introducing the VEGF minitrap, composition, or formulation, and optionally an additional therapeutic agent, into the body of the subject, e.g., by intraocular injection, e.g., by intravitreal injection (e.g., about 100 microliters or less, e.g., about 70 microliters).

[0016] The present invention also relates to a method for treating neovascular ocular disorders (e.g., age-related macular degeneration (wet), age-related macular degeneration (dry), macular edema, macular edema after retinal vein occlusion, retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, postoperative fibrosis in glaucoma, and proliferative vitreous plexus in a subject (e.g., a human) in need thereof. and / or diabetic retinopathy), comprising administering to the patient a therapeutically effective amount (e.g., 0.5 mg, 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg) of a VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483, REGN7484, REGN7485), a VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483), or a VEGF minitrap (e.g., REGN7851, REGN7483 ...1, REGN7483), or a VEGF minitrap (e.g., REGN7851, REGN7483), or a VEGF minitrap (e.g., REGN7851, REGN7483), a VEGF minitrap (e.g., REGN7851, REGN7483), or a F , or REGN7483 R ) or a composition (e.g., an aqueous composition) or a pharmaceutical formulation thereof (e.g., about 100 microliters or less, e.g., about 70 microliters), and optionally an additional therapeutic agent, are injected intraocularly (e.g., intravitreally) into the eye of a subject. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 illustrates the VEGF minitrap molecule (REGN7483F), the product of proteolysis of aflibercept by Streptococcus pyogenes IdeS (FabRICATOR). The homodimeric molecule is depicted with the Ig hinge domain fragment that links each polypeptide together. The VEGFR1 domain, VEGFR2 domain, and hinge domain fragment (MC) are shown. The point within aflibercept where IdeS cleavage occurs is indicated by " / / ". The Fc fragment cleaved from aflibercept is also shown. [Figure 2]

[0023] Figure 1 illustrates a single-chain VEGF minitrap showing the spatial relationship of the domains. The VEGFR1, VEGFR2, and linker domains are shown. The linker shown is (G4S)6 (REGN7080). The present invention includes single-chain VEGF minitraps with (G4S)3; (G4S)9, or (G4S)12 linkers. [Figure 3] Figure 3 (A-C) shows that HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9 cells were treated with increasing concentrations of VEGF110, VEGF121, or VEGF165 (open squares in panels A-C, respectively), resulting in an increase in relative luminescence unit (RLU), reflecting activation of the chimeric VEGF receptor. Neutralization was observed with serial dilutions of REGN3 (filled circles), REGN6824 (filled squares), and REGN7080 (filled triangles) in the presence of 20 pM VEGF110, VEGF121, or VEGF165. [Figure 4] Figure 4 (A-B) shows that HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9 cells were treated with increasing concentrations of VEGF121 or VEGF110 (open squares in panels A-B, respectively), resulting in increased relative luminescence units (RLU), reflecting activation of the chimeric VEGF receptor. Neutralization was observed with serial dilutions of REGN3 (VEGF trap; filled circles), REGN7991 (filled squares), and REGN7992 (open triangles) in the presence of 20 pM VEGF121 or VEGF110. [Figure 5-1]Figure 5 (A-F) shows that treatment of HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9 cells with increasing concentrations of VEGF110 (A-B), VEGF121 (C-D), or VEGF165 (E-F) resulted in an increase in relative luminescence unit (RLU), reflecting activation of the chimeric VEGF receptor. Neutralization was observed in the presence of 20 pM VEGF110, 40 pM VEGF121, or 40 pM VEGF165 at serial dilutions of REGN3 (VEGF trap; small black filled squares); REGN7483F (large black filled squares or gray open squares (separate lot)); REGN7483R (small black filled triangles); REGN112 (open triangles); REGN7850 (gray filled circles); REGN7851 (open circles), or a VEGF control (black open squares). [Figure 5-2] Continued from Figure 5-1. [Figure 5-3] Continued from Figure 5-2. [Figure 6] Figure 1 shows that the REGN6824:REGN110 complex was analyzed by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The relative UV absorbance at 280 nm (right Y-axis) as a function of retention time (X-axis) is shown for each sample, and the molar mass measurements of the separated peaks are also shown (left Y-axis). Peak 1 represents the complex, peak 2 represents REGN6824 alone, and peak 3 represents REGN110 alone. [Figure 7] Figure 1 shows that the REGN7080:REGN110 complex was analyzed by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The relative UV absorbance at 280 nm (right Y-axis) as a function of retention time (X-axis) is shown for each sample, as well as the molar mass measurements of the separated peaks (left Y-axis). Peak 1 represents the complex, peak 2 represents REGN7080 alone, and peak 3 represents REGN110 alone. [Figure 8]Figure 1 shows that the REGN7483F:REGN110 complex was analyzed by particle size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS). The relative UV absorbance at 280 nm (right Y-axis) as a function of retention time (X-axis) is shown for each sample, and molar mass measurements of the separated peaks are also shown (left Y-axis). Peak 1 represents the complex, peak 1a is consistent with a mixture of REGN7483F alone and the REGN110:REGN7483F complex, peak 2 represents REGN7483F alone, and peak 3 represents REGN110 alone. [Figure 9] FIG. 1 shows the surface area of ​​abnormal neovascularization observed in OIR (oxygen-induced retinopathy) model mice after intravitreal administration of control hFc, VEGF trap (aflibercept), single-chain mini-trap REGN7080, or dimeric mini-trap REGN7483F. [Figure 10] Figure 10 (A-B) shows the surface area of ​​abnormal neovascularization observed in an OIR (oxygen-induced retinopathy) mouse model after systemic (ip) administration of the dimeric mini-trap REGN7483F (3 mg / kg, 30 mg / kg, or 100 mg / kg; or 3 mg / kg control hFc) (A). Also shown is a previous study (B) of the surface area (normalized to the hFc control protein) in OIR mice treated systemically (ip) with 2.5 mg / kg, 6.25 mg / kg, 25 mg / kg, or 50 mg / kg aflibercept (VEGF trap). [Figure 11] FIG. 1 shows reduced and non-reduced SDS-PAGE gels of REGN112 (R112), REGN7850 (R7850), and REGN7851 (R7851) molecules (M = molecular weight marker). Dimers and monomers are shown. [Figure 12-1] FIG. 1 shows a diagrammatic summary of VEGF trap and mini-trap constructs. [Figure 12-2] Continued from Figure 12-1. [Figure 12-3] Continued from Figure 12-2. [Figure 13] FIG. 1 illustrates a graphical representation of the CIEL*a*b* color space. [Figure 14-1] Figure 14 (A-D) shows post-translational modifications observed in CDM-expressed and non-CDM-expressed aflibercept (Eylea). The table in (A) shows the site-specific asparagine-linked glycosylation observed in REGN7483F and aflibercept (Eylea). The degree of shading in each box correlates with the indicated degree of glycosylation at the indicated residue. % Manose-rich was calculated by summing Man4, Man5, Man6, and Man7. The table in (B) shows other post-translational modifications, including non-glycosylation at N-linked glycosylation sites, observed in REGN7483F and aflibercept (Eylea). (C) Tables show the site-specific asparagine-linked glycosylation observed in REGN7483F (Minitrap Production 10), REGN7483R, REGN7711, and aflibercept (Eylea). These tables show only glycoforms with levels greater than 1% in any of the samples. (D) shows additional glycan structures. [Figure 14-2] Continued from Figure 14-1. [Figure 14-3] Continued from Figure 14-2. [Figure 14-4] Continued from Figure 14-3. [Figure 14-5] Continued from Figure 14-4. [Figure 14-6] Continued from Figure 14-5. [Figure 14-7] Continued from Figure 14-6. [Figure 15] FIG. 1 shows baseline vascular permeability (leakage / disk area) in each group (aflibercept (doses of 500 μg and 2 mg); REGN7483R (MinitrapR, dose 250.5 μg); REGN7483F (MinitrapF, doses 254.4 μg and 1.4 mg), and placebo). [Figure 16]FIG. 1 shows vascular permeability inhibition (as % of baseline) over time for equimolar doses of aflibercept (500 μg), REGN7483R (minitrap recombinant, 250.5 μg), REGN7483F (minitrap Fabricator, 254.4 μg), and placebo. [Figure 17] FIG. 1 shows vascular permeability inhibition (as % of baseline) over time in high dose aflibercept (2 mg), or REGN7483F (minitrap Fabricator, 1.4 mg); or placebo. [Figure 18] FIG. 1 shows intraocular pressure over time in rabbits in each treatment group (aflibercept (doses of 500 μg and 2 mg); REGN7483R (minitrap recombinant, dose 250.5 μg); REGN7483F (minitrap Fabricator, doses 254.4 μg and 1.4 mg), and placebo). [Figure 19] FIG. 1 shows the pathological vascular regression rate (%) in each group (aflibercept (doses of 500 μg and 2 mg); REGN7483F (Minitrap F, doses of 254.4 μg and 1.4 mg), and placebo). [Figure 20] FIG. 1 shows baseline vascular permeability in aflibercept (500 μg), REGN7483F (minitrap, 213 μg), or placebo groups. [Figure 21] FIG. 1 shows the % inhibition of vascular permeability over time in aflibercept (500 μg), REGN7483F (Minitrap(F), 213 μg), or placebo groups. [Figure 22] FIG. 1 shows a color analysis of the BY color standard in the CIEL*a*b* color space. [Figure 23]FIG. 1 shows an evaluation of the percentage of 2-oxo-histidine (and tryptophan dioxide) in commercially available aflibercept, oligopeptides derived from protease-digested minitrap product 10 purified by AEX chromatography, and oligopeptides derived from protease-digested minitrap product 10 adsorbed and separated from AEX chromatography. [Figure 24] Figure 24 (A-B) shows the effect of incubating various components with aflibercept in fresh CDM on color production (predicted b* values ​​from CIEL*a*b*) (A); and a plot of predicted b* values ​​versus measured values. The B vitamins are thiamine, niacinamide, pantothenic acid, biotin, and pyridoxine. [Figure 25] FIG. 1 shows the effect of metal content and cysteine ​​reduction on color (b* value predicted from CIEL*a*b*). [Figure 26] 26(A-B) show a graphical (A) and tabular summary (B) of the effect on predicted b* values ​​of spiking various antioxidants into spent CDM containing aflibercept drug substance. [Figure 27] FIG. 1 shows the effect of REGN7483 concentration (Minitrap Production 23) on b* values. [Figure 28] FIG. 1 shows the results of an experiment carried out to compare different minitrap preparations and the acidic species present in the fractions obtained when performing strong cation exchange (CEX) chromatography. [Figure 29] Figure 1 shows a strong cation exchange chromatogram performed on minitrap product 23 (before any purification steps, ≦BY3) according to a representative embodiment, and an enriched variant of the desialylated minitrap (dsMT1) was subjected to CEX using a dual salt-pH gradient. [Figure 30]Figure 1 shows imaged capillary isoelectric focusing (iciEF) electropherograms performed according to a representative embodiment for VEGF minitrap preparation 23 (before any purification steps, ≦BY3) subjected to CEX and for an enriched variant of desialylated VEGF minitrap (dsMT1). [Figure 31A] (A) A diagram showing the overall chart of absorbance (at 350 nm) versus time (min) for VEGF mini-trap (MT4) obtained by IdeS (FabRICATOR) cleavage of aflibercept manufactured using a commercial process (non-CDM), and for mini-trap product 10 (M1); (B) A diagram showing the overall chart of absorbance (at 350 nm) versus time (16-30 min) for MT4 and MT1; (C) A diagram showing the overall chart of absorbance (at 350 nm) versus time (30-75 min) for MT4 and MT1. [Figure 31B] (A) A diagram showing the overall chart of absorbance (at 350 nm) versus time (min) for VEGF mini-trap (MT4) obtained by IdeS (FabRICATOR) cleavage of aflibercept manufactured using a commercial process (non-CDM), and for mini-trap product 10 (M1); (B) A diagram showing the overall chart of absorbance (at 350 nm) versus time (16-30 min) for MT4 and MT1; (C) A diagram showing the overall chart of absorbance (at 350 nm) versus time (30-75 min) for MT4 and MT1. [Figure 31C] (A) A diagram showing the overall chart of absorbance (at 350 nm) versus time (min) for VEGF mini-trap (MT4) obtained by IdeS (FabRICATOR) cleavage of aflibercept manufactured using a commercial process (non-CDM), and for mini-trap product 10 (M1); (B) A diagram showing the overall chart of absorbance (at 350 nm) versus time (16-30 min) for MT4 and MT1; (C) A diagram showing the overall chart of absorbance (at 350 nm) versus time (30-75 min) for MT4 and MT1. [Figure 32] Figure 32 (A-B) shows natural log plots of the decay curves of intravitreal (A) VEGF trap REGN3 and (B) VEGF mini-trap REGN7483F in New Zealand White rabbits (rabbits 428, 429, 430, 434, 435, and 436). OD = right eye; OS = left eye. [Figure 33-1] Figure 33 (A-C) shows natural log plots of the decay curves of intravitreal (A) VEGF trap REGN3, (B) VEGF mini-trap REGN7850, and (C) VEGF mini-trap REGN7851 in New Zealand White rabbits (rabbits 472, 473, 475, 476, 477, 431, 432, and 433). OD = right eye; OS = left eye. [Figure 33-2] Continuation of Figure 33-1. [Figure 34] FIG. 10 shows a two-way ANOVA revealing no significant IOP changes between the VEGF trap REGN3 and VEGF mini-trap REGN7483F groups before and 20 minutes after IVT injection. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides a VEGF mini-trap molecule (e.g., REGN7483) that has several advantageous properties and is the result of efforts to overcome significant technical hurdles. F) and compositions thereof. Expression of the minitrap in chemically defined medium (CDM) resulted in a pronounced brown-yellow color. While expression in CDM is a preferred modern method for protein expression (e.g., CDM offers greater reproducibility / consistency than hydrolysate-based media), adding colored substances to the visual organ, the eye, can have negative effects on vision. By analyzing and developing optimized purification methods and host cell growth conditions, we identified a possible cause of coloration (2-oxo-histidine modification) and significantly reduced its presence in the final purified product. In addition, evidence suggests that the minitrap of the present invention has a shorter systemic half-life than aflibercept (Eylea), thereby avoiding certain adverse events associated with intravitreal administration. The cause of this effect is unclear, but it may be due to the higher mannose content of the minitrap compared to aflibercept.

[0019] Accordingly, the present invention encompasses fusion polypeptides capable of binding to vascular endothelial growth factor (VEGF) and therapeutic methods for their use.

[0020] A "variant" of a polypeptide (e.g., of a VEGFR Ig domain) refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a reference amino acid sequence (e.g., any of SEQ ID NOS: 1-5 or 10-13); when the comparison is performed using the BLAST algorithm, the algorithm parameters are selected to obtain the largest match between corresponding sequences over the entire length of the corresponding reference sequences (e.g., threshold: 10; word size: 3; maximum match of query range: 0; BLOSUM62 matrix; gap cost: 11 for presence, 1 for extension; conditional composition score matrix adjustment).

[0021] A variant of a polypeptide (e.g., of a VEGFR Ig domain) can also refer to a polypeptide that contains a reference amino acid sequence except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions, for example, compared to any of SEQ ID NOs: 1-5, 10-13, 26-30, 32, 33, or 36.

[0022] The present invention includes VEGF mini-traps comprising polypeptides whose amino acid sequences are variants of those specifically set forth herein.

[0023] The following references relate to the BLAST algorithm, which is commonly used for sequence analysis: AST ALGORITHMS: Altschul et al. (2005) FEBS J. 272(20):5101-5109; Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403-410; Gish, W. et al. (1993) Nature Genet. 3:266-272; Madden, T.L. et al. (1996) Meth. Enzymol. 266:131-141; Altschul, S.F. et al. (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656; Wootton, JC et al. (1993) Comput. Chem. 17:149-163; Hancock, JM et al. (1994) Comput. Appl. Biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO et al. "A model of evolutionary change in proteins." In Atlas of Protein Sequence and Structure, (1978) Vol. 5, Complement 3. Mo Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, RM et al. "Matrices for detecting distant relationships." In Atlas of Protein Sequence and Structure (1978) Vol. 5, Complement 3., M. O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F. (1991) J. Mol. Biol. Vol. 219: pp. 555-565; States, D. J. et al. (1991) Methods Vol. 3: pp. 66-70; Henikoff, S. et al. (1992) Proc. Natl. Acad. Sci. USA Vol. 89: pp. 10915-10919; Altschul, S. F. et al. (1993) J. Mol. Evol. Vol. 36: pp. 290-300; ALIGNMENT STATISTICS: Karlin, S. et al. (1990) Proc. Natl. Acad.Sci. USA 87:2264-2268; Karlin, S. et al. (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A. et al. (1994) Ann. Prob. 22:2022-2039; and Altschul, S. F., "Evaluating the statistical significance of multiple distinct local alignments." in Theoretical and Computational Methods in Genome Research (ed. S. Suhai) (1997) pp. 1-14, Plenum, NY

[0024] The sequences and domain structures of VEGF, VEGFR1, VEGFR2, and VEGFR3 are known. In an embodiment of the present invention, the VEGF amino acid sequence is set forth in Genbank accession number AH001553; the VEGFR1 amino acid sequence is set forth in Uniprot accession number P17948; the VEGFR2 amino acid sequence is set forth in Uniprot accession number P35968; and / or the VEGFR3 amino acid sequence is set forth in Uniprot accession number P35916. Holash et al., VEGF-Trap: a VEGF blocker with potent antitumor effects, Proc Natl Acad Sci USA. 2002, August 20; 99(17):11393-8.

[0025] VEGF Minitrap The present invention relates to the treatment of ocular diseases such as neovascular ocular disorders and cancer, including VEGF (e.g., VEGF 110 , VEGF 121 , or VEGF 165 The present invention provides a VEGF mini-trap capable of binding to vascular endothelial growth factor (VEGF), which is therapeutically useful for treating or preventing conditions and diseases treatable or preventable by inhibition of VEGF. The term "VEGF" in the context of "VEGF mini-trap" refers to a mini-trap that binds to VEGF and is capable of inhibiting the VEGF-mediated signaling pathway described above. A schematic diagram of the VEGF mini-trap of the present invention is shown in FIG.

[0026] A VEGF mini-trap is a molecule or complex of molecules that binds to VEGF and has one or more sets of VEGF receptor Ig-like domains (or variants thereof) (e.g., VEGFR1 Ig domain 2 and / or VEGFR2 Ig domain 3 and / or 4), with or without a truncated multimerization component (MC), e.g., the MC is a truncated immunoglobulin Fc. Cleavage may be the result of proteolytic digestion of the VEGF trap (e.g., aflibercept or conbercept) or the direct expression of the resulting polypeptide chain with a truncated MC sequence. See the molecular structure illustrated in Figure 1. Figure 1 is an illustration of a VEGF mini-trap molecule that is the product of proteolysis of aflibercept by Streptococcus pyogenes IdeS. A homodimeric molecule with an Ig hinge domain fragment connected by two parallel disulfide bonds is illustrated. The VEGFR1 domain, VEGFR2 domain, and hinge domain fragment (MC) are shown. The point in aflibercept where IdeS cleavage occurs is indicated by " / / ". The Fc fragment cleaved from aflibercept is also indicated. A single, non-dimerized chimeric polypeptide can also be a VEGF mini-trap if it has VEGF-binding activity. The term "VEGF mini-trap" includes a single polypeptide that contains a first set of one or more VEGF receptor Ig domains (or variants thereof), lacks an MC, and is fused to one or more additional sets of one or more VEGF receptor Ig domains (or variants thereof) via a linker (e.g., a peptide linker). The VEGF-binding domains of the VEGF mini-trap of the present invention can be identical to or different from each other. See International Publication No. WO 2005 / 00895.

[0027] For example, in embodiments of the invention, a non-truncated immunoglobulin Fc domain comprises the following amino acid sequence, or amino acids 1-226 thereof: [ka]

[0028] Inhibition of VEGF includes, for example, VEGF (e.g., VEGF 110 , VEGF 121 , and / or VEGF 165 (i.e., antagonism of VEGF binding to a VEGF receptor, e.g., by competing with the VEGF receptor for VEGF-mediated VEGFR activation. Such inhibition can result in inhibition of VEGF-mediated VEGFR activation, e.g., inhibition of luciferase expression in a cell line (e.g., HEK293) expressing on its cell surface a chimeric VEGF receptor (e.g., a homodimer thereof) having a VEGFR extracellular domain fused to an IL18Rα and / or IL18Rβ intracellular domain and harboring an NFkB-luciferase-IRES-eGFP reporter gene, such as the cell line HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ as shown herein.

[0029] The VEGF receptor Ig domain component of the VEGF mini-trap of the invention may comprise: (i) one or more of the immunoglobulin-like (Ig) domains 2 (R1D2) of VEGFR1 (Flt1); (ii) one or more of Ig domain 3 (Flk1D3) (R2D3) of VEGFR2 (Flk1 or KDR); (iii) one or more of Ig domain 4 (Flk1D4) (R2D4) of VEGFR2 (Flk1 or KDR), and / or (iv) one or more of Ig domains 3 (Flt1D3 or R3D3) of VEGFR3 (Flt4).

[0030] The immunoglobulin-like domain of a VEGF receptor may be referred to herein as a VEGFR "Ig" domain. The VEGFR Ig domains referred to herein, such as R1D2 (sometimes referred to herein as VEGFR1(d2)), R2D3 (sometimes referred to herein as VEGFR2(d3)), R2D4 (sometimes referred to herein as VEGFR2(d4)), and R3D3 (sometimes referred to herein as VEGFR3(d3)), are intended to encompass not only complete wild-type Ig domains, but also variants thereof that substantially retain the functional characteristics of the wild-type domain, for example, retain the ability to form a functional VEGF-binding domain when incorporated into a VEGF mini-trap. It will be readily apparent to those skilled in the art that numerous variants of the above Ig domains can be obtained that retain substantially the same functional characteristics as the wild-type domain.

[0031] The present invention provides the following domain structure: ((R1D2)-(R2D3)) a -Linker-((R1D2)-(R2D3)) b ; ((R1D2)-(R2D3)-(R2D4)) c -Linker-((R1D2)-(R2D3)-(R2D4)) d ; ((R1D2)-(R2D3)) e -(MC) g ;or ((R1D2)-(R2D3)-(R2D4)) f -(MC) g Includes; During the ceremony, - R1D2 is VEGF receptor 1 (VEGFR1) Ig domain 2 (D2); - R2D3 is VEGFR2 Ig domain 3; - R2D4 is VEGFR2 Ig domain 4; - MC is a multimerizing component (e.g. IgG1); - the linker is a peptide containing about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, e.g., (GGGS) g and; Independently, a=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; b=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; c=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; d=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; e=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; f=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and g=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 1 5, VEGF mini-trap polypeptides are provided.

[0032] In an embodiment of the invention, R1D2 comprises the following amino acid sequence: [ka] In an embodiment of the invention, R1D2 lacks the N-terminal SDT.

[0033] In an embodiment of the invention, R1D2 comprises the following amino acid sequence: [ka]

[0034] In an embodiment of the invention, R2D3 comprises the following amino acid sequence: [ka]

[0035] In an embodiment of the invention, R2D4 comprises the following amino acid sequence: [ka]

[0036] In an embodiment of the invention, R2D4 comprises the following amino acid sequence: [ka]

[0037] In embodiments of the invention, a multimerization component (MC) for use in a VEGF mini-trap is a peptide, e.g., a truncated Fc immunoglobulin (e.g., IgG1) capable of binding to another multimerization component. In embodiments of the invention, the MC is a truncated Fc immunoglobulin comprising an immunoglobulin hinge region or a fragment thereof. For example, in embodiments of the invention, the MC is a peptide comprising one or more (e.g., 1, 2, 3, 4, 5, or 6) cysteines that can form one or more cysteine ​​bridges with cysteines of another MC, e.g., DKTHTCPPC (SEQ ID NO: 22), DKTHTCPPCPPC (SEQ ID NO: 23), DKTHTCPPCPPCPPC (SEQ ID NO: 24), h is 1, 2, 3, 4, or 5DKTHTC(PPC) h (SEQ ID NO: 25), DKTHTCPPCPAPELLG (SEQ ID NO: 6), DKTHTCPLCPAPELLG (SEQ ID NO: 7), DKTHTC (SEQ ID NO: 8), or DKTHTCPLCPAP (SEQ ID NO: 9).

[0038] The present invention also provides the following domain structure: (i) ((R1D2)-(R2D3)) a -(MC) b ;or (ii) ((R1D2)-(R2D3)-(R2D4)) c -(MC) d Includes; These may be homodimerized with a second of said polypeptides, e.g., by binding between the MCs of each polypeptide, During the ceremony, (i) R1D2 domains cooperate; (ii) the R2D3 domains cooperate; and / or (iii) The R2D4 domain cooperates to Forms a dimeric VEGF-binding domain VEGF mini-trap polypeptides are provided.

[0039] In an embodiment of the invention, the VEGF mini-trap polypeptide comprises or consists of the following amino acid sequence: [ka]

[0040] [ka]

[0041] [ka]

[0042] [ka]

[0043] [ka] or [ka] As discussed, such polypeptides may be multimerized (e.g., dimerized (e.g., homodimerized)), in which case the binding between the polypeptides is mediated by a multimerization component. Such multimers and single polypeptides are part of the present invention.

[0044] In an embodiment of the present invention, REGN7483 FまたはR N36, N68, N123, and / or N196 of REGN7483, REGN7850, or REGN7851 are N-glycosylated. FまたはR , REGN7850, or REGN7851 have intrachain disulfide bridges (i) between C30 and C79 and / or (ii) between C124 and C185.

[0045] In an embodiment of the present invention, REGN7483 FまたはR The interchain disulfide bridges in the hinge region of THTCPPCPAPELLG (amino acids 208-221 of SEQ ID NO: 12) of REGN7850 or REGN7851 are either parallel (between each C211 and between each C214) or crossing (between C211 and C214). In embodiments, the majority of the disulfide bridges are parallel.

[0046] In an embodiment of the present invention, REGN7483 FまたはR , REGN7850, or REGN7851 lack the C-terminal glycine.

[0047] In an embodiment of the present invention, VEGFR1 Ig-like domain 2 of a monomeric VEGF minitrap of the present invention has N-linked glycosylation at N36 and / or N68 and / or an intrachain disulfide bridge between C30 and C79; and / or VEGFR2 Ig-like domain 3 of a monomeric VEGF minitrap of the present invention has N-linked glycosylation at N123 and / or N196 and / or an intrachain disulfide bridge between C124 and C185.

[0048] In an embodiment of the invention, the VEGF mini-trap comprises the following structure: ·(R1D2)1-(R2D3)1-(G4S)3-(R1D2)1-(R2D3)1; ·(R1D2)1-(R2D3)1-(G4S)6-(R1D2)1-(R2D3)1; (R1D2)1-(R2D3)1-(G4S)9-(R1D2)1-(R2D3)1; or (R1D2) 1-(R2D3) 1-(G4S) 12 -(R1D2)1-(R2D3)1. G4S is -Gly-Gly-Gly-Gly-Ser-.

[0049] In an embodiment of the invention, the VEGF mini-trap comprises the following amino acid sequence: (i) [ka]

[0050] (iii) [ka]

[0051] (iv) [ka]

[0052] (v) [ka]

[0053] (vi) [ka] or;

[0054] (vii) [ka]

[0055] As discussed herein, such polypeptides may comprise secondary structures in which VEGFR Ig-like domains associate to form intrachain VEGF-binding domains (see, e.g., Figure 2). In embodiments of the invention, two or more such polypeptides multimerize (e.g., dimerize (e.g., homodimerize)), where the VEGFR Ig domains of each chain associate with the Ig-like domains of another chain to form interchain VEGF-binding domains.

[0056] In certain embodiments of the present invention, the VEGF minitraps of the present invention lack any significant modifications of the amino acid residues of the VEGF minitrap polypeptide (e.g., site-directed chemical modifications such as PEGylation or iodoacetamidation at the N-terminus and / or C-terminus).

[0057] In an embodiment of the invention, the polypeptide is a single chimeric polypeptide (e.g., ((R1D2)-(R2D3)) a -Linker-((R1D2)-(R2D3)) b ;or ((R1D2)-(R2D3)-(R2D4)) c -Linker-((R1D2)-(R2D3)-(R2D4)) d ), or in separate chimeric polypeptides (e.g., homodimers), in which the VEGFR Ig-like domains cooperate to form a VEGF-binding domain. (i) R1D2 domains cooperate; (ii) the R2D3 domains cooperate; and / or (iii) The R2D4 domain cooperates to The VEGFR1, VEGFR2, and linker domains are shown in Figure 2, which illustrates the VEGF-binding domain. The VEGFR1, VEGFR2, and linker domains are shown. The linker shown is (G4S)6. The present invention also provides a method for the preparation of a single-chain VEGF mini-trap that utilizes (G4S)3; (G4S)9, or (G4S) 12 Contains a single-chain VEGF minitrap with a linker.

[0058] Additionally, the present invention provides a complex comprising a VEGF mini-trap as discussed herein complexed with a VEGF polypeptide, or a fragment or fusion thereof. In an embodiment of the present invention, a VEGF (e.g., VEGF 165 ) are homodimerized, and / or the VEGF minitrap is homodimerized into a 2:2 complex (2 VEGF:2 minitrap). The complex may comprise a homodimerized VEGF molecule bound to a homodimerized VEGF minitrap polypeptide. In embodiments of the invention, the complex is present in vitro (e.g., immobilized on a solid substrate) or present in the body of a subject. The present invention also provides a method for the preparation of a VEGF minitrap, e.g., REGN6824, REGN7080, or REGN7483, in a molar ratio as shown in Table 3-3 herein. F VEGF dimers complexed with (e.g., VEGF 165 ) complex compositions.

[0059] IdeS and its variants The present invention includes VEGF minitraps and compositions thereof produced by proteolytic digestion of aflibercept with Streptococcus pyogenes IdeS (FabRICATOR) and its variants. FabRICATOR is commercially available from Genovis, Inc.; Cambridge, Massachusetts; Lund, Sweden.

[0060] In one embodiment, the IdeS polypeptide comprises an amino acid sequence having at least 70% sequence identity over the entire length of the isolated amino acid sequence as set forth in the group consisting of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQ ID NO:52. In one aspect, the isolated amino acid sequence has at least about 80% sequence identity over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has at least about 90% sequence identity over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence has about 100% sequence identity over the entire length of the isolated amino acid sequence. In one aspect, the polypeptide may be capable of cleaving a target protein into fragments. In a particular aspect, the target protein is an IgG. In another particular aspect, the target protein is a fusion protein. In yet another particular aspect, the fragments may comprise a Fab fragment and / or an Fc fragment.

[0061] In one embodiment, the IdeS amino acid sequence comprises the parent amino acid sequence defined by SEQ ID NO: 37, except that the asparagine residues at positions 87, 130, 182, and / or 274 are mutated to amino acids other than asparagine. In one aspect, the mutations can confer increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In another aspect, the mutations can confer a 50% increase in chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In one aspect, the amino acids can be selected from aspartic acid, leucine, and arginine. In a specific aspect, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another specific aspect, the asparagine residue at position 130 is mutated to an arginine residue. In yet another specific aspect, the asparagine residue at position 182 is mutated to a leucine residue. In yet another specific aspect, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another specific embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 130, and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 182, and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 130, 182, and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 130, 182, and 274 are mutated.

[0062] Aflibercept may be cleaved with IdeS immobilized on a solid support, such as chromatography beads. For example, a sample containing aflibercept in a buffered aqueous solution (in cleavage buffer) may be applied to the immobilized IdeS on, for example, a chromatography column. The column may be incubated, for example, at about 18°C, for example, for 30 minutes. The column may then be washed with cleavage buffer. After cleavage, the digestion solution and washing solution may be poured into a plate. The cleaved Fc by-product may be captured by application to a Protein A column, with the mini-trap product being retained in the flow-through. In an embodiment of the invention, the cleavage buffer and / or Protein A column equilibration and wash solution is at pH 7, e.g., 40 mM Tris, 54 mM acetate, pH 7.0±0.1.

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[0079] Protein purification Proteins of interest (e.g., VEGF minitraps (e.g., REGN7850, REGN7851, REGN7483)) produced by methods involving a combination of different purification techniques, including, but not limited to, affinity, ion exchange, mixed-mode, and hydrophobic interaction chromatography. F , or REGN7483 R ), alone or in combination, are contemplated within the scope of the present invention. In an embodiment, the method comprises enzymatically cleaving REGN7483 to produce REGN7483. F This involves purifying aflibercept to produce aflibercept. In these chromatography steps, a mixture of proteins in a sample matrix is ​​separated based on charge, hydrophobicity, or size, or any combination thereof, depending on the particular separation format. Several different chromatography resins are available for each of the techniques mentioned herein, allowing for precise customization of the purification scheme to the specific protein involved. In each separation method, proteins pass through the column at different speeds, achieving increased physical separation when the proteins either pass through the column further or selectively adhere to the separation medium. Proteins are then either (i) differentially eluted using an appropriate elution buffer and / or (ii) collected from the used column, optionally from the flow-through fraction obtained by washing the column with an appropriate equilibration buffer. In some cases, the protein of interest is separated from the impurities (protein variants) when the impurities preferentially adhere to the column and the protein of interest adheres less, i.e., when the protein of interest does not adsorb to the solid phase of a particular column and therefore flows through the column. In some cases, impurities are unable to adsorb to the column and therefore are separated from the protein of interest when they flow through the column.

[0080] The purification method can begin with a separation step after the recombinant protein has been produced using the upstream production methods described herein and / or by alternative production methods conventional in the art. Once a clarified solution or mixture containing the protein of interest, e.g., a VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483), is obtained, separation of the protein of interest from process-related impurities (such as other proteins produced by the cells (e.g., HCPs)) and from product-related substances, such as acidic or basic variants, is performed. In certain non-limiting embodiments, such separation is performed using CEX, AEX, and / or MM (mixed-mode) chromatography. In certain embodiments, a combination of one or more different purification techniques can be used, including affinity, ion exchange, mixed-mode, and / or hydrophobic interaction chromatography. In such additional purification steps, a mixture of components within a sample matrix is ​​separated, for example, based on charge, hydrophobicity, and / or size. Numerous chromatography resins are commercially available for each of the chromatography techniques mentioned herein, allowing for precise customization of purification schemes to the specific proteins involved. Each separation method allows proteins to pass through the column at different rates, either achieving increased physical separation as the proteins pass further through the column or selectively adhering to the separation resin (or medium). The proteins are then differentially eluted using appropriate buffers. In some cases, the protein of interest will be separated from components of the sample matrix if those components specifically adsorb to the column resin and the protein of interest does not; in other cases, the protein of interest will adsorb to the column resin while other components are pushed off the column during wash cycles.

[0081] Primary recovery and viral inactivation In certain embodiments, an initial step in the purification methods disclosed herein involves clarification and primary recovery of the VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483) from the sample matrix. In certain embodiments, primary recovery will include one or more centrifugation steps to separate the protein of interest, e.g., VEGF minitrap (e.g., REGN7850, REGN7851, REGN7483), from host cells and associated cellular debris. Centrifugation of the sample can be performed, for example, but not limited to, at 7,000 x g to approximately 12,750 x g. In large-scale purification situations, such centrifugation can be performed online, for example, at a flow rate set to achieve a turbidity level of 150 NTU in the resulting supernatant. Such supernatant can then be collected for further purification or filtered in-line through one or more depth filters to further clarify the sample.

[0082] In certain exemplary embodiments, primary recovery may involve the use of one or more depth filtration steps to clarify the sample matrix, thereby assisting in the purification of a protein of interest of the present invention (e.g., REGN7850, REGN7851, REGN7483). In other embodiments, primary recovery may involve centrifugation followed by one or more depth filtration steps to further clarify the sample matrix. Non-limiting examples of depth filters that can be used in the context of the present invention include Millistak+X0HC, F0HC, D0HC, A1HC, and B1HC depth filters (EMD Millipore), 3M™ Models 30 / 60ZA, 60 / 90ZA, VR05, and VR07, and delipid depth filters (3M Corp.). Typically, the depth filter is followed by a 0.2 μm filter, such as a Sartorius 0.45 / 0.2 μm Sartopore™ bi-layer or Millipore Express SHR or SHC filter cartridge. Other filters known to those skilled in the art may also be used.

[0083] In certain embodiments, the primary recovery method may be a point for reducing or inactivating viruses that may be present in the sample matrix. For example, any one or more of a variety of methods for reducing / inactivating viruses can be used in the primary recovery stage of purification, including heat inactivation (pasteurization), pH inactivation, buffer / detergent treatment, UV and gamma irradiation, and the addition of certain chemical inactivators such as β-propiolactone or copper phenanthroline, as described in U.S. Pat. No. 4,534,972. In certain exemplary embodiments of the present invention, the sample matrix is ​​exposed to detergent viral inactivation during the primary recovery stage. In other embodiments, the sample matrix may be exposed to low pH inactivation during the primary recovery stage.

[0084] In those embodiments in which viral reduction / inactivation is used, the sample mixture can be adjusted for further purification steps as needed. For example, after low pH viral inactivation, the pH of the sample mixture is typically adjusted to a more neutral pH, e.g., about 4.5 to about 8.5, before continuing the purification method. Additionally, the mixture can be diluted with water for injection (WFI) to obtain the desired conductivity.

[0085] VEGF mini-traps that are the product of a purification process that includes primary recovery, filtration, and / or viral inactivation, e.g., under conditions as discussed herein, as well as compositions comprising the VEGF mini-trap, are part of the present invention.VEGF mini-traps that are the product of a purification process that includes primary recovery, filtration, and / or viral inactivation, e.g., under conditions as discussed herein, of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate the VEGF mini-trap, as well as compositions comprising the VEGF mini-trap, are part of the present invention.

[0086] Affinity chromatography In certain exemplary embodiments, it may be advantageous to subject the sample matrix to affinity chromatography for the purification of a protein of interest. In certain embodiments, the chromatography material can utilize a specific portion of the protein to selectively or specifically bind to the protein of interest. Non-limiting examples of such chromatography materials include Protein A and Protein G. The chromatography material may also include, for example, a protein or a portion thereof capable of binding to the protein of interest. In an embodiment of the present invention, aflibercept, which can be enzymatically cleaved with IdeS, is purified by Protein A or Protein G chromatography. In an embodiment of the present invention, the Fc fragment removed from aflibercept by IdeS cleavage is removed from a sample containing a minitrap by Protein A or Protein G chromatography.

[0087] In certain embodiments, affinity chromatography can involve subjecting the sample matrix to a column containing a suitable Protein A resin. In certain aspects, Protein A resins are useful for affinity purification and isolation of various VEGF minitrap isotypes because they specifically interact with the Fc portion of contaminating molecules if they possess this region (minitraps lacking affinity for Protein A are present in the flow-through fraction). Protein A is a bacterial cell wall protein that binds to mammalian IgG primarily via their Fc region. In its native state, Protein A has five IgG-binding domains and other domains of unknown function. In certain embodiments, the affinity chromatography step involves subjecting the primary recovery sample to a column containing an anti-protein antibody of interest.

[0088] There are several commercial sources of Protein A resin. One suitable resin is GE Protein A. A suitable resin is MabSelect™ manufactured by GE Healthcare. Suitable resins include, but are not limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect Xtra, and rProtein A Sepharose manufactured by GE Healthcare, ProSep HC, ProSep Ultra, and ProSep Ultra Plus manufactured by EMD Millipore, and MapCapture manufactured by Life Technologies. A non-limiting example of a suitable column packed with MabSelect™ is a column approximately 1.0 cm in diameter x approximately 21.6 cm in length (17 mL bed volume). Columns of this size can be used for small-scale purification and can be compared to other columns used for large-scale purification. For example, a 20 cm x 21 cm column with a bed volume of approximately 6.6 L can be used. m columns can be used for larger scale purification. Suitable columns may contain a resin such as MabSelect™ SuRe or a similar resin.

[0089] Before loading the sample, the affinity column may be equilibrated with a suitable buffer. After loading the column, the column may be washed one or more times using a suitable buffer. Once loaded, the column may be eluted using an appropriate elution buffer. For example, glycine-HCl, acetic acid, or citric acid may be used as the elution buffer. The eluate may be monitored using techniques well known to those skilled in the art, such as a UV detector. The eluted fraction of interest may be collected and prepared for further processing.

[0090] In one embodiment, the eluate may be subjected to viral inactivation, for example, by either detergent or low pH. A suitable detergent concentration or pH (and time) can be selected to achieve the desired viral inactivation results. After viral inactivation, the eluate is typically adjusted to a pH and / or conductivity suitable for subsequent purification steps.

[0091] Prior to additional chromatographic polishing steps, the eluate can be subjected to depth filtration to remove turbidity and / or various impurities from the protein of interest. Examples of depth filters include, but are not limited to, Millistak+ XOHC, FOHC, DOHC, AIHC, X0SP, and BIHC pod filters (EMD Millipore), or Zeta Plus 30ZA / 60ZA, 60ZA / 90ZA, delipidated, VR07, and VR05 filters (3M). Emphaze AEX Hybrid Purifier multi-mechanism filters can also be used to clarify the eluate. The eluate pool may need to be adjusted to the appropriate pH and conductivity to achieve the desired impurity removal and product recovery from the depth filtration step. The present invention is not limited to capturing the protein of interest using chromatography.

[0092] Other affinity purification resins are available for VEGF, VEGF 165 , an anti-VEGFR antibody or antigen-binding fragment thereof, an anti-VEGFR1 antibody or antigen-binding fragment thereof, or an anti-VEGFR2 antibody or antigen-binding fragment thereof, capable of binding to the VEGF mini-trap.

[0093] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising affinity purification under conditions such as those discussed herein (e.g., as performed in flow-through mode) are part of the present invention.VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising affinity purification under conditions such as those discussed herein (e.g., as performed in bind-elute mode) of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate the VEGF mini-trap are part of the present invention.

[0094] In an embodiment of the invention, the affinity column is washed with phosphate buffered saline (PBS), for example, Dulbecco's phosphate buffered saline.

[0095] Anion exchange chromatography In certain embodiments, the mini-traps are produced by subjecting the sample matrix to at least one anion exchange separation step. In one aspect, the anion exchange step will occur after affinity chromatography, such as Protein A affinity, as described above. In certain other embodiments, the anion exchange step will occur before affinity chromatography, such as Protein A affinity, as described above. In certain other embodiments, the anion exchange step will occur before affinity chromatography, such as Protein A affinity, as described above. This will be done both pre- and post-affinity.

[0096] The use of anion exchange materials rather than cation exchange materials, such as those discussed in detail herein, is based on the local charge of the protein of interest under suitable conditions. Anion exchange chromatography can be used in combination with other chromatographic procedures.

[0097] In carrying out the separation, the initial protein composition (sample matrix) can be contacted with the anion exchange material by using any of a variety of techniques, for example, by using batch purification techniques or chromatographic techniques.

[0098] For example, in a batch purification situation, an anion exchange material is prepared or equilibrated with a desired starting buffer. Preparation or equilibration results in a slurry of the anion exchange material. A solution of the protein of interest, e.g., VEGF MiniTrap, is contacted with the slurry to allow adsorption of the protein to the anion exchange material. The solution containing acidic species that do not bind to the AEX material is separated from the slurry, for example, by settling the slurry and removing the supernatant. The slurry can be subjected to one or more washing and / or elution steps.

[0099] In the context of chromatographic separation, a chromatographic column is used to house a chromatographic support material (resin or solid phase). A sample matrix containing the protein of interest is loaded onto the specific chromatographic column for separation. The column can then be subjected to one or more washing steps using a suitable buffer. Components of the sample matrix that do not adsorb to the resin will likely flow through the column. Components that do adsorb to the resin can be differentially eluted using an appropriate buffer.

[0100] In certain embodiments, the wash step can be performed using conditions similar to the load conditions in the context of AEX chromatography, or alternatively, by decreasing the pH and / or increasing the ionic strength / conductivity of the wash solution in a stepwise or linear gradient manner. In certain exemplary embodiments, the aqueous salt solutions used in both the loading and wash buffers have a pH at or near the isoelectric point (pI) of the protein of interest. In certain exemplary embodiments, the pH will be about 0-2 units higher or lower than the pI of the protein of interest. In certain exemplary embodiments, the pH will be in the range of 0-0.5 units higher or lower. In certain exemplary embodiments, the pH will be the pI of the protein of interest.

[0101] In an embodiment of the present invention, the AEX chromatography column is washed with (i) a wash buffer of pH 8.40 and 2.00 mS / cm, (ii) a wash buffer of pH 8.00 and 2.50 mS / cm, or (iii) a wash buffer of pH 7.80 and 4.00 mS / cm; after applying a sample containing a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851), the VEGF minitrap is retained in the AEX flow-through fraction. The wash buffer is retained after passing through the column. In an embodiment of the present invention, the wash buffer contains Tris (e.g., 50 mM) and optionally NaCl. In an embodiment of the present invention, the AEX column is pre-equilibrated with NaCl (e.g., 2 M NaCl). In an embodiment of the present invention, the AEX column is equilibrated with the wash buffer.

[0102] In certain non-limiting embodiments, the anionic agent is selected from the group consisting of acetate, chloride, formate, and combinations thereof. In certain non-limiting embodiments, the cationic agent is selected from the group consisting of Tris, arginine, sodium, and combinations thereof. In one embodiment, the buffer is a Tris / formate buffer. Another Exemplary Embodiment In some embodiments, the buffer is selected from the group consisting of pyridine, piperazine, L-histidine, Bis-tris, Bis-Tris propane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propanol), piperazine, 1,3-diaminopropane, and piperidine.

[0103] Packed anion exchange chromatography columns, anion exchange membrane devices, anion exchange monolithic devices, or depth filter media can be operated in either bind-elute mode, flow-through mode, or hybrid mode, in which the product exhibits binding to the chromatographic material but can be washed off the column using a buffer that is the same or substantially similar to the loading buffer. In bind-elute mode, the column or membrane device is first conditioned with a buffer of appropriate ionic strength and pH under conditions that will immobilize a particular protein to the resin-based matrix. For example, during the additional feed load, the protein of interest will adsorb to the resin by electrostatic attraction. After washing the column or membrane device with an equilibration buffer or another buffer with a different pH and / or conductivity, product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion exchange matrix. Changing the pH, and thereby the charge of the solute, is another method for achieving solute elution. The change in conductivity or pH can be a gradient (gradient elution) or a stepwise change (step elution). In flow-through mode, a column or membrane device is run at a selected pH and conductivity; the protein of interest does not bind to the resin or membrane, but acidic species will be retained on the column or will have a different elution profile compared to the protein of interest. In this hybrid strategy, acidic species will bind to (or pass through) the chromatographic material in a manner different from the protein of interest. For example, the protein of interest and certain aggregates and / or fragments of the protein of interest can bind to the chromatographic material, and a wash solution can be applied that preferentially removes the protein of interest. The column is then regenerated before subsequent use.

[0104] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Additional non-limiting examples include: Poros 50PI and Poros 50HQ, rigid polymer beads with a backbone made of cross-linked poly[styrene-divinylbenzene]; Capto Q Impres and Capto DEAE, high-flux agarose beads; Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are polymer-based beads; Fractogel® EMD TMAE Hicap, a synthetic polymer resin with tentacle ion exchangers; Sartobind STIC® PA nano, a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q nano, a strong anion exchange chromatography membrane; CUNO BioCap, a Zeta Plus depth filter media constructed from inorganic filter aid, purified cellulose, and ion exchange resin; and XOHC, a depth filter media constructed from inorganic filter aid, cellulose, and mixed cellulose esters.

[0105] In certain embodiments, the protein load of the mixture containing the protein of interest is adjusted to a total protein load on the column of about 50-500 g / L, or about 75-350 g / L, or about 200-300 g / L. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of about 0.5-50 g / L, about 1-20 g / L, or about 3-10 g / L of material to be loaded onto the column. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of about 37 g / L of material for the column.

[0106] In certain exemplary embodiments, additives such as polyethylene glycol (PEG), surfactants, amino acids, sugars, chaotropic agents, etc. can be added to enhance the performance of the separation to achieve better recovery or product quality.

[0107] The methods of the invention can be used to selectively remove, significantly reduce, or essentially remove, in the case of ion exchange, at least 10% of protein variants in the flow-through fraction while concentrating that in the elution fraction or strip, thereby producing a protein composition that is reduced in or essentially free of protein variants.

[0108] In certain embodiments, a protein variant may include modifications of one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to aspartate-glycine and / or Asn-Gly; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are monohydroxyltryptophan; one or more tryptophans are dihydroxyltryptophan; one or more tryptophans are trihydroxyltryptophan; one or more arginines are converted to Arg3-deoxyglucosone; the C-terminal glycine is absent; and / or one or more non-glycosylated glycosylation sites are present.

[0109] In certain exemplary embodiments, the protein variant of aflibercept or VEGF minitrap may include one or more of the following: (i) oxidized histidine at histidine residues, for example, selected from His86, His110, His145, His209, His95, His19, and / or His203; (ii) oxidized tryptophan residues, for example, selected from tryptophan residues, for example, Trp58 and / or Trp138; (iii) oxidized tyrosine residues, for example, Tyr64; (iv) oxidized phenylalanine residues, for example, selected from Phe44 and / or Phe166; and / or (v) oxidized methionine residues, for example, selected from Met10, Met20, Met163, and / or Met192. Such oxidized histidine is correlated with an undesirable brown-yellow color.

[0110] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising AEX chromatography (e.g., performed in flow-through mode) under conditions such as those discussed herein are part of the present invention. VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising AEX chromatography under conditions such as those discussed herein of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate the VEGF mini-trap are part of the present invention.

[0111] Cation exchange chromatography The compositions of the present invention can be produced by subjecting a composition, such as a primary recovery sample, to at least one cation exchange (CEX) separation step. In certain exemplary embodiments, the CEX step will occur either before or after the AEX described above. Furthermore, CEX steps may occur throughout the purification procedure.

[0112] The use of a cation exchange material rather than an anion exchange material, such as the anion exchange materials discussed herein, is based on the local charge of the protein of interest in a given solution. Thus, it is within the scope of the present invention to use a cation exchange step before an anion exchange step, or to use an anion exchange step before using a cation exchange step. Furthermore, it is within the scope of the present invention to use only a cation exchange step, only an anion exchange step, or any sequential combination of the two (including sequential combinations of one or both ion exchange steps with other chromatographic separation techniques described herein).

[0113] In carrying out the separation, the initial protein mixture can be contacted with the cation exchange material using any of a variety of techniques, such as using batch purification or chromatographic techniques as described above in connection with Protein A or AEX.

[0114] In certain exemplary embodiments, the aqueous salt solution used as both the loading buffer and the wash buffer has a pH lower than the isoelectric point (pI) of the protein of interest. In certain exemplary embodiments, the pH is about 0-5 units lower than the pI of the protein of interest. In certain exemplary embodiments, the pH is in the range of 1-2 units lower. In certain exemplary embodiments, the pH is in the range of 1-1.5 units lower.

[0115] In certain exemplary embodiments, the concentration of anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of about 3.5 to 10.5, or about 4 to 10, or about 4.5 to 9.5, or about 5 to 9, or about 5.5 to 8.5, or about 6 to 8, or about 6.5 to 7.5. In certain exemplary embodiments, the concentration of anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Suitable buffer systems for use in the CEX method include, but are not limited to, Tris formate, Tris acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

[0116] In certain exemplary embodiments, the conductivity and pH of the aqueous salt solution are adjusted by increasing or decreasing the concentration of a cationic agent. In certain exemplary embodiments, the cationic agent is maintained at a concentration ranging from about 20 mM to 500 mM, about 50 mM to 350 mM, about 100 mM to 300 mM, or about 100 mM to 200 mM. In certain non-limiting embodiments, the cationic agent is selected from the group consisting of sodium, Tris, tromethamine, ammonium, arginine, and combinations thereof. In certain non-limiting embodiments, the anionic agent is selected from the group consisting of formate, acetate, citrate, chloride anion, sulfate, phosphate, and combinations thereof.

[0117] Packed cation exchange chromatography columns or cation exchange membrane devices can be operated in either bind-elute mode, flow-through mode, or hybrid mode, in which the product binds to the chromatographic material but can be washed off the column using a buffer that is the same as or substantially similar to the loading buffer. Details of these modes are outlined above.

[0118] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include, but are not limited to: Capto SP ImpRes, a high-flow agarose bead; CM Hyper DF grade, a functionalized hydrogel, ceramic bead coated and impregnated with 250-400 μeq / mL of ionic groups; Eshmuno S, a hydrophilic polyvinyl ether-based matrix with 50-100 μeq / mL ionic capacity; and macroporous highly Nuvia C Prime, a hydrophobic cation exchange medium composed of a cross-linked hydrophilic polymer matrix with 55-75 με / ιηi; Nuvia S, a UNOsphere-based matrix with 90-150 με / ιηI ionic groups; Poros HS, a rigid polymeric bead with a backbone made of cross-linked poly[styrene-divinylbenzene]; Poros XS, a rigid polymeric bead with a backbone made of cross-linked poly[styrene-divinyl-benzene]; Toyo Pearl Giga Cap CM 650M, a polymer-based bead with an ionic capacity of 0.225 meq / mL; and Toyo Pearl Giga Cap CM 650M, a polymer-based bead with an ionic capacity of 0.225 meq / mL. Giga Cap S 650M; Toyo Pearl polymer-based beads MX TRP. Note that CEX chromatography can be used with the MM resins described herein.

[0119] In certain exemplary embodiments, the protein load of the mixture containing the protein of interest (e.g., VEGF minitrap) is adjusted to a total protein load on the column of about 5-150 g / L, or about 10-100 g / L, about 20-80 g / L, about 30-50 g / L, or about 40-50 g / L. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of about 0.5-50 g / L or about 1-20 g / L of the material to be loaded onto the column.

[0120] In certain exemplary embodiments, additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents, etc. may be added to enhance the performance of the separation to achieve better recovery or product quality.

[0121] In certain embodiments, the methods of the invention can be used to selectively remove, significantly reduce, or essentially completely remove variants in a sample matrix, in which case the protein of interest will be essentially present in the flow-through of the CEX step and the oxo-variant will be substantially captured by the column media.

[0122] In an embodiment of the present invention, the CEX column is loaded with a sample containing the VEGF minitrap in a loading buffer at pH 5.0, e.g., 20 mM acetate, pH 5.0. In an embodiment of the present invention, the column is also washed with the loading buffer. Washing can be performed with a pH 7.0 wash buffer, e.g., 10 mM phosphate, pH 7.0. Elution of the VEGF minitrap from the CEX column can be performed with, e.g., 50 mM Tris, 62.5 mM (NH4)2SO4, pH 8.5, e.g., using (NH4)2SO4 at pH 8.5.

[0123] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising CEX chromatography under conditions such as those discussed herein are part of the present invention.VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising CEX chromatography under conditions such as those discussed herein of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate a VEGF mini-trap are part of the present invention.

[0124] Mixed-mode chromatography Mixed-mode ("MM") chromatography can also be used to prepare the compositions of the present invention. MM chromatography, also referred to herein as "multimodal chromatography," is a chromatographic strategy that uses a support containing ligands capable of providing at least two different interactions with the substance to be bound. In certain exemplary embodiments, one of these sites provides an attractive charge-charge interaction between the ligand and the substance of interest, while the other site provides an electron acceptor-donor interaction. and / or hydrophobic and / or hydrophilic interactions. Electron donor-acceptor interactions include hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, and other interactions.

[0125] In certain embodiments, the resin used for mixed-mode separation is Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. Its base matrix is ​​highly cross-linked agarose with a ligand (N-benzyl-N-methylethanolamine) that exhibits different functionalities with respect to interactions, such as ionic interactions, hydrogen bonding, and hydrophobic interactions. In certain aspects, the resin used for mixed-mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrix of PPA-HyperCel and HEA-HyperCel is highly porous cross-linked cellulose. Their ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine offer different selectivities and hydrophobicity options for protein separation. Additional mixed-mode chromatography supports include, but are not limited to, Nuvia C Examples include Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain embodiments, mixed-mode chromatography resins are composed of a ligand coupled directly or via a spacer to an organic or inorganic support, sometimes referred to as a base matrix. The support may be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, and capillaries. In certain embodiments, the support is prepared from natural polymers such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, and cross-linked carbohydrate materials, such as alginate. To achieve high adsorption capacity, the support may be porous, with ligands then coupled to the exterior and pore surfaces. Such natural polymer supports can be prepared according to standard methods, such as inverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2):393-398 (1964)). Alternatively, the support can be prepared from cross-linked synthetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylate esters, methacrylate esters, vinyl esters, and vinylamides. Such synthetic polymers can be produced according to standard methods. See, for example, "Styrene-based polymer supports developed by suspension polymerization" by R. Arshady, Chimica e L'Industria, Vol. 70(9), pp. 70-75 (1988). Porous natural or synthetic polymer supports are also available from commercial sources, such as GE Healthcare, Uppsala, Sweden.

[0126] In certain embodiments, the protein load of the mixture containing the protein of interest is adjusted to a total protein load on the column of about 25-750 g / L, or about 75-500 g / L, or about 100-300 g / L. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of about 1-50 g / L or about 9-25 g / L of material to be loaded onto the column.

[0127] In certain embodiments, additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents, etc. can be added to enhance the performance of the separation to achieve better recovery or product quality.

[0128] The methods of the present invention can be used to selectively remove, significantly reduce, or essentially remove all PTMs such as 2-oxo-histidine that make up proteins in the flow-through fraction while concentrating them in the strip fraction.

[0129] The method for producing the compositions of the present invention can also be carried out in a continuous chromatography mode. In this mode, at least two columns are used (referred to as the "first" and "second" columns). In certain exemplary embodiments, this continuous chromatography mode can then be carried out by subsequently or simultaneously loading (with or without dilution) the elution fraction and / or strip fraction, which may contain higher levels of PTMs such as 2-oxo-histidine constituting proteins, onto the second column, and the two columns can be run in a non-tandem fashion, reducing operational complexity.

[0130] In one embodiment, the media choice for continuous mode may be one of a number of chromatography resins with pendant hydrophobic and anionic exchange functional groups, monolithic media, membrane adsorption media, or depth filtration media.

[0131] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising MM chromatography under conditions such as those discussed herein are part of the present invention.VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising MM chromatography under conditions such as those discussed herein of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate a VEGF mini-trap are part of the present invention.

[0132] Hydrophobic interaction chromatography The compositions of the present invention can also be prepared using hydrophobic interaction chromatography (HIC).

[0133] In carrying out the separation, the sample mixture is contacted with the HIC material, for example, using batch purification techniques or using column or membrane chromatography. It may be desirable to adjust the concentration of the salt buffer prior to HIC purification to achieve the desired protein binding to the resin or membrane.

[0134] While ion exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography exploits the hydrophobic properties of the protein to achieve selective separation. The hydrophobic groups of the protein interact with the hydrophobic groups of the resin or membrane. The more hydrophobic the protein, the stronger its interaction with the column or membrane under suitable conditions. Therefore, HIC can be used to remove process-related impurities (such as HCPs) and product-related materials (such as aggregates and fragments) under suitable conditions.

[0135] Similar to ion-exchange chromatography, HIC columns or membrane devices can be operated in elution mode, flow-through, or hybrid mode, in which the product exhibits binding to the chromatographic material but can be washed off the column using a buffer that is the same as or substantially similar to the loading buffer (details of these modes are outlined herein for AEX purification). Because hydrophobic interactions are strongest at high ionic strength, this form of separation is conveniently performed after a salt elution step, such as that typically used with ion-exchange chromatography. Alternatively, salt can be added to the supplemental feed stream at low salt levels prior to this step. Adsorption of the VEGF minitrap to the HIC column is promoted by high salt concentrations, although the actual concentration can vary over a wide range depending on the nature of the protein of interest, the type of salt, and the particular HIC ligand selected. Various ions either promote hydrophobic interactions (salting-out effect) or disrupt the structure of water, leading to a weakening of hydrophobic interactions (chaotropic effect). The cations can be placed in the so-called solvophobic series depending on whether they have a salting-out effect or not. 2+ ;Ca 2+ ;Mg 2+ ;Li + ;Cs + ;Na + ;K + ;Rb + ;NH4 + The anions are ranked as PO4 in terms of increasing the chaotropic effect. 3- ;SO4 2- ;CH3CO3 - ;Cl - ;Br - ;NO3 - ;ClO4 - ;I - ;SCN - can be ranked.

[0136] In general, Na + , K. + , or NH4 +Sulfate salts effectively promote ligand-protein interactions when using HIC. Salts can be formulated to affect the strength of the interaction according to the following relationship: (NH4)2SO4 > Na2SO4 > NaCl > NH4Cl > NaBr > NaSCN. Generally, salt concentrations of about 0.75 M to about 2 M ammonium sulfate or about 1 to 4 M NaCl are useful.

[0137] HIC media typically comprise a base matrix (e.g., cross-linked agarose or synthetic copolymer materials) to which hydrophobic ligands (e.g., alkyl or aryl groups) are coupled. Suitable HIC media include agarose resins or membranes functionalized with phenyl groups (e.g., Phenyl Sepharose™ from GE Healthcare or phenyl membranes from Sartorius). Many HIC resins are commercially available. Examples include, but are not limited to, Capto Phenyl, low- or high-substituted Phenyl Sepharose™ 6 Fast. Flow, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ Methyl or Macro-Prep™ t-Butyl columns (Bio-Rad, California); WP HI-Propyl (C3)™ (JT Baker, New Jersey); and Toyopearl™ ether, phenyl, or butyl (TosoHaas, Pennsylvania).

[0138] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising HIC chromatography under conditions such as those discussed herein are part of the present invention.VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising HIC chromatography under conditions such as those discussed herein of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate a VEGF mini-trap are part of the present invention.

[0139] Virus filtration Virus filtration is a virus reduction step specific to the purification method. This step is usually performed after the chromatographic polishing step. Virus reduction can be achieved by using a suitable filter, including, but not limited to, Planova 20N™, 50N, or BioEx™ filters manufactured by Asahi Kasei Pharma, Viresolve™ filters manufactured by EMD Millipore, ViroSart CPV filters manufactured by Sartorius, or Ultipor DV20 or DV50™ filters manufactured by Pall Corporation. It will be obvious to those skilled in the art to select a suitable filter to achieve the desired filtration performance.

[0140] VEGF mini-traps and compositions comprising VEGF mini-traps are part of the present invention, for example, the products of a purification process that includes viral filtration under conditions as discussed herein. VEGF mini-traps and compositions comprising VEGF mini-traps are part of the present invention, for example, the products of a purification process that includes viral filtration under conditions as discussed herein of VEGF traps, such as aflibercept, which are subsequently cleaved with IdeS protease to generate VEGF mini-traps. Compositions containing minitraps are part of the present invention.

[0141] Ultrafiltration / Diafiltration In certain embodiments of the present invention, ultrafiltration and diafiltration are used to further concentrate and formulate the protein of interest, e.g., a minitrap. Ultrafiltration is described in detail in the following publications: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, NY, 1996); and Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). One filtration method is tangential flow filtration, as described in the Millipore catalog entitled "Pharmaceutical Process Filtration Catalogue," pp. 177-202 (Bedford, Massachusetts, 1995 / 96). Ultrafiltration is generally considered to mean filtration using filters with pore sizes less than 0.1 μm. The use of filters with such small pore sizes allows for the reduction of sample volume by permeation of sample buffer through the filter membrane pores, while retaining proteins such as the VEGF minitrap on the upper membrane surface.

[0142] Those skilled in the art can select an appropriate membrane filter device for UF / DF operation. Examples of membrane cassettes suitable for the present invention include, but are not limited to, Pellicon 2 or Pellicon 3 cassettes with 10 kD, 30 kD, or 50 kD membranes manufactured by EMD Millipore, Kvick 10 kD, 30 kD, or 50 kD membrane cassettes manufactured by GE Healthcare, and Centramate or Centrasette 10 kD, 30 kD, or 50 kD cassettes manufactured by Pall Corporation.

[0143] VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising UF and / or DF, e.g., under conditions as discussed herein, are part of the present invention. VEGF mini-traps and compositions comprising VEGF mini-traps that are the product of a purification method comprising UF and / or DF, e.g., under conditions as discussed herein, of a VEGF trap, such as aflibercept, that is subsequently cleaved with IdeS protease to generate a VEGF mini-trap are part of the present invention.

[0144] Exemplary Purification Schemes In certain exemplary embodiments, primary recovery can proceed by sequentially removing cells and cell debris (including HCPs) from the production bioreactor harvest using pH reduction, centrifugation, and filtration. In certain embodiments, the invention relates to subjecting the sample mixture from primary recovery to one or more AEX, CEX, and / or MM purification steps. Certain aspects of the invention will include additional purification steps. Examples of additional purification procedures that can be performed before, during, or after ion exchange chromatography include ethanol precipitation, isoelectric focusing, size exclusion chromatography, reverse-phase HPLC, chromatography on silica, chromatography on Heparin Sepharose™, additional anion exchange chromatography and / or additional cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g., using Protein G or Protein A, antibodies, specific substrates, ligands, or antigens as capture reagents). In certain aspects, column temperatures can be independently varied to improve the separation efficiency and / or yield of any particular purification step.

[0145] In certain embodiments, the unbound flow-through and wash fractions may be further fractionated, and a combination of fractions that provides the desired product purity may be pooled.

[0146] In certain exemplary embodiments, the loading and washing steps can be controlled to achieve target product quality and / or yield by in-line, at-line, or offline measurement of product-related impurity / substance levels in either the column eluate or the collection pool, or both. In certain embodiments, the loading concentration can be dynamically controlled by in-line or batch or serial dilution with buffers or other solutions to achieve the required distribution to improve separation efficiency and / or yield.

[0147] An example of such a purification procedure is as follows: The present invention includes a VEGF minitrap that is the product of a process comprising any of the steps of such a purification method. (1) The method for producing aflibercept is (a) expressing aflibercept in CDM; (b) capturing aflibercept using a first chromatographic support, which may comprise affinity capture chromatography; and (c) contacting at least a portion of the aflibercept from step (b) with a second chromatographic support, which may comprise an anion exchange chromatography. may also include: Step (c) can further comprise collecting the flow-through fraction of the mixture that contains the aflibercept that does not bind to the second chromatographic support.Optionally, step (c) can comprise stripping the second chromatographic support and collecting stripping fraction.These steps can be carried out by the methodology mentioned herein and by routine methodology.

[0148] Other additional exemplary embodiments may include (d) contacting at least a portion of the aflibercept of step (c) with a third chromatographic support. In one aspect of such an embodiment, the preparation may include (e) contacting at least a portion of the aflibercept of step (d) with a fourth chromatographic support. In one aspect of this embodiment, the preparation may optionally include subjecting the aflibercept of step (c) to a pH of less than 5.5. In one aspect of this embodiment, the method may optionally include clarifying the solution containing the fusion binding molecule before capturing step (a). In one aspect of this embodiment, the method may optionally include eluting the fusion binding molecule of step (a). In yet another aspect of this embodiment, the method for preparing aflibercept may optionally include collecting the flow-through fraction of step (c). In yet another aspect of this embodiment, the method for preparing aflibercept may optionally include eluting the aflibercept of step (d). In yet another aspect of this embodiment, the method for producing aflibercept may optionally include a step (e) of eluting aflibercept. In one aspect of this embodiment, the first chromatographic support and / or the second chromatographic support and / or the third chromatographic support and / or the fourth chromatographic support may be the same or different and may comprise an affinity chromatographic medium, an ion exchange chromatographic medium, or a hydrophobic interaction chromatographic medium. In a specific aspect of this embodiment, the ion exchange chromatographic medium may be an anion exchange chromatographic medium. In another specific aspect of this embodiment, the ion exchange chromatographic medium may be a cation exchange chromatographic medium. In one aspect of this embodiment, the method for producing aflibercept may optionally include eluting aflibercept in any of the above steps using virus filtration. In one aspect of this embodiment, the manufacturing may optionally include filtering the aflibercept from any of the above steps using an ultrafiltration and / or diafiltration procedure (UF / DF).

[0149] The present invention includes such VEGF minitraps that are the product of a process that includes cleaving aflibercept with IdeS protease.

[0150] (2) The method for producing VEGF mini-traps is (a) expressing aflibercept in CDM; (b) capturing aflibercept using a first chromatographic support, which may comprise affinity capture chromatography; (c) cleaving aflibercept (e.g., with IdeS protease), thereby forming a mixture comprising a VEGF minitrap and an Fc fragment from aflibercept; (d) contacting the mixture with a second chromatographic support, which may be affinity capture chromatography; and (e) contacting at least a portion of the VEGF mini-trap of step (c) with a third chromatographic support, which may comprise an anion exchange chromatography. may also include: Step (d) may optionally further comprise collecting a flow-through fraction of the mixture containing the VEGF mini-trap that does not bind to the second chromatographic support of step (e). Step (e) may optionally comprise collecting a flow-through fraction of the mixture containing the VEGF mini-trap that does not bind to the third chromatographic support. Optionally, step (d) may comprise stripping the third chromatographic support and collecting stripped fractions. These steps can be carried out by routine methodologies as well as the methodologies mentioned herein.

[0151] Other additional exemplary embodiments may include (f) contacting at least a portion of the VEGF mini-trap of step (e) with a fourth chromatographic support. In one aspect of such an embodiment, the manufacturing may include (g) contacting at least a portion of the VEGF mini-trap of step (f) with a fifth chromatographic support. In one aspect of this embodiment, the manufacturing may optionally include subjecting the VEGF mini-trap of step (d) to a pH of less than 5.5. In one aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include clarifying the solution containing the fusion binding molecule prior to the capture step (a). In one aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include eluting the fusion binding molecule of step (a). In yet another aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include collecting the flow-through of step (e). In yet another aspect of this embodiment, the method for producing a VEGF mini-trap may optionally include a step (f) of eluting the VEGF mini-trap. In yet another aspect of this embodiment, the method for producing a VEGF mini-trap may optionally include a step (g) of eluting the VEGF mini-trap. In one aspect of this embodiment, the first chromatographic support and / or the second chromatographic support and / or the third chromatographic support and / or the fourth chromatographic support and / or the fifth chromatographic support may be the same or different and may comprise an affinity chromatographic medium, an ion exchange chromatographic medium, or a hydrophobic interaction chromatographic medium. In a particular aspect of this embodiment, the ion exchange chromatographic medium may be an anion exchange chromatographic medium. In another particular aspect of this embodiment, the ion exchange chromatographic medium is a cation exchange chromatographic medium. In one aspect of this embodiment, the method for producing the VEGF mini-trap may optionally include filtering the VEGF mini-trap from any of the above steps using viral filtration. In one aspect of this embodiment, the production may optionally include filtering the VEGF mini-trap from any of the above steps using ultrafiltration and / or diafiltration procedures (UF / DF).

[0152] The present invention includes the product of such a method, a VEGF mini-trap.

[0153] (3) The method for producing aflibercept is (a) expressing aflibercept in CDM; (b) capturing aflibercept using a first chromatographic support, which may include cation exchange chromatography; and (c) contacting at least a portion of the aflibercept from step (b) with a second chromatographic support, which may comprise an anion exchange chromatography. may also include: Step (c) can further comprise collecting the flow-through fraction of the mixture that contains the aflibercept that does not bind to the second chromatographic support.Optionally, step (c) can comprise stripping the second chromatographic support and collecting stripping fraction.These steps can be carried out by the methodology mentioned herein and by routine methodology.

[0154] Other additional exemplary embodiments may include (d) contacting at least a portion of the aflibercept of step (c) with a third chromatographic support. In one aspect of such an embodiment, the preparation may include (e) contacting at least a portion of the aflibercept of step (d) with a fourth chromatographic support. In one aspect of this embodiment, the preparation may optionally include subjecting the aflibercept of step (c) to a pH of less than 5.5. In one aspect of this embodiment, the method may optionally include clarifying the solution containing the fusion binding molecule before capturing step (a). In one aspect of this embodiment, the method may optionally include eluting the fusion binding molecule of step (a). In yet another aspect of this embodiment, the method for preparing aflibercept may optionally include collecting the flow-through fraction of step (c). In yet another aspect of this embodiment, the method for preparing aflibercept may optionally include eluting the aflibercept of step (d). In yet another aspect of this embodiment, the method for producing aflibercept may optionally comprise the step of eluting aflibercept in step (e).In one aspect of this embodiment, the first chromatography support and / or the second chromatography support and / or the third chromatography support and / or the fourth chromatography support may be the same or different, and may comprise an affinity chromatography medium, an ion exchange chromatography medium, or a hydrophobic interaction chromatography medium.In a specific aspect of this embodiment, the ion exchange chromatography medium may be an anion exchange chromatography medium.In another specific aspect of this embodiment, the ion exchange chromatography medium may be a cation exchange chromatography medium.In one aspect of this embodiment, the method for producing aflibercept may optionally comprise the step of filtering the aflibercept of any of the above steps using virus filtration.In one aspect of this embodiment, the manufacturing may optionally include filtering the aflibercept from any of the above steps using an ultrafiltration and / or diafiltration procedure (UF / DF).

[0155] The present invention includes such VEGF minitraps that are the product of a process that includes cleaving aflibercept with IdeS protease. (4) The method for producing VEGF mini-traps comprises: (a) expressing aflibercept in CDM; (b) capturing aflibercept using a first chromatographic support, which may comprise a cation exchange chromatography; (c) cleaving aflibercept (e.g., with IdeS protease), thereby forming a mixture comprising a VEGF minitrap and an Fc fragment from aflibercept; (d) contacting the mixture with a second chromatographic support, which may be affinity capture chromatography; and (e) contacting at least a portion of the VEGF mini-trap of step (c) with a third chromatographic support, which may comprise an anion exchange chromatography. may also include: Step (d) may optionally further comprise collecting a flow-through fraction of the mixture containing the VEGF mini-trap that does not bind to the second chromatographic support of step (e). Step (e) may optionally comprise collecting a flow-through fraction of the mixture containing the VEGF mini-trap that does not bind to the third chromatographic support. Optionally, step (d) may comprise stripping the third chromatographic support and collecting a stripped fraction. These steps can be carried out by routine methodologies, along with the methodologies mentioned herein.

[0156] Other additional exemplary embodiments may include (f) contacting at least a portion of the VEGF mini-trap of step (e) with a fourth chromatographic support. In one aspect of such an embodiment, the manufacturing may include (g) contacting at least a portion of the VEGF mini-trap of step (f) with a fifth chromatographic support. In one aspect of this embodiment, the manufacturing may optionally include subjecting the VEGF mini-trap of step (d) to a pH of less than 5.5. In one aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include clarifying the solution containing the fusion binding molecule prior to the capture step (a). In one aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include eluting the fusion binding molecule of step (a). In yet another aspect of this embodiment, the method for manufacturing a VEGF mini-trap may optionally include collecting the flow-through of step (e). In yet another aspect of this embodiment, the method for producing a VEGF mini-trap may optionally include a step (f) of eluting the VEGF mini-trap. In yet another aspect of this embodiment, the method for producing a VEGF mini-trap may optionally include a step (g) of eluting the VEGF mini-trap. In one aspect of this embodiment, the first chromatographic support and / or the second chromatographic support and / or the third chromatographic support and / or the fourth chromatographic support and / or the fifth chromatographic support may be the same or different and may comprise an affinity chromatographic medium, an ion exchange chromatographic medium, or a hydrophobic interaction chromatographic medium. In a particular aspect of this embodiment, the ion exchange chromatographic medium may be an anion exchange chromatographic medium. In another particular aspect of this embodiment, the ion exchange chromatographic medium may be a cation exchange chromatographic medium.In one aspect of this embodiment, the method for producing the VEGF mini-trap may optionally include filtering the VEGF mini-trap from any of the above steps using viral filtration. In one aspect of this embodiment, the production may optionally include filtering the VEGF mini-trap from any of the above steps using ultrafiltration and / or diafiltration procedures (UF / DF).

[0157] The present invention includes the product of such a method, a VEGF mini-trap.

[0158] Minitrap Post-translational Modification The VEGF mini-traps of the present invention and compositions thereof can be characterized by a variety of post-translational modifications.

[0159] Oxidizing species 2-oxo-histidine is the result of histidine oxidation and can serve as a marker of protein oxidation. 2-oxo-histidine was detected in VEGF minitraps (e.g., REGN7483) expressed from cells in chemically defined medium (CDM). F ,REGN7483 R This correlates with the presence of a brownish-yellow color in preparations of REGN7850, REGN7851, or REGN7852. Chemically defined cell growth media offer several important advantages to biopharmaceutical manufacturing, including reduced lot-to-lot variability and improved safety, for example, against infectious agents. However, to realize the benefits of these advantages with respect to mini-traps for ophthalmic injection, it is necessary to reduce the brownish-yellow color of mini-trap compositions expressed in CDM. Reducing the 2-oxo-histidine content of mini-traps is a means by which color can be reduced to levels acceptable for intravitreal injection. The present invention provides, in part, methods for reducing 2-oxo-histidine and thus the brownish-yellow color, as well as compositions resulting from such methods.

[0160] A brownish-yellow color is particularly undesirable for any biological product (e.g., VEGF minitrap) to be injected intraocularly. Only traces of 2-oxo-histidine have been observed in commercially available VEGF trap molecules (e.g., Eylea) expressed in chemically undefined media, such as those containing hydrolysates (e.g., soy hydrolysates). Because the eye is a visual organ, introducing colored liquids into the vitreous can have a negative effect on vision. Vision is particularly sensitive to any obstructions within the eye. For example, transparent microdroplets of silicone oil that shed from the syringe wall and are injected intravitreously have been reported to interfere with vision in the form of floaters. Yu et al., Am J Ophthalmol Case Rep. 2018 June;10:142-144.

[0161] Chemically defined medium (CDM) or synthetic medium is a term commonly used in the art and refers to a medium whose chemical composition is known. CDM does not contain hydrolysates, such as soy hydrolysates. Suitable CDMs include Dulbecco's Modified Eagle's Medium (DME), Ham's Nutrient Mixture, EX-CELL Medium, IS CHO-CD Medium, and other CDMs known to those skilled in the art, the use of which is contemplated within the scope of the present invention.

[0162] There are two chemical forms of 2-oxo-histidine (2-oxo-his): The molecular weight is increased by 13.98 Da compared to histidine (13.98 Da type) [ka] or The molecular weight is increased by 15.99 Da compared to histidine (15.99 Da type) [ka] The 13.98 Da form of 2-oxo-histidine may be produced; the 13.98 Da form of 2-oxo-histidine is the major moiety observed in minitraps expressed in CDM. The content of the 13.98 Da form of 2-oxo-histidine in peptides can be assessed spectrophotometrically, as this moiety exhibits an increase in absorbance at 350 nm, whereas the 15.99 Da form does not. The formation of the 13.98 Da form of 2-oxo-histidine in minitraps may be catalyzed by light, whereas the formation of the 15.99 Da form is catalyzed by copper (Cu 2+ The brown-yellow color of the CDM-expressed minitrap does not correlate with the presence of the 15.99 Da form of 2-oxo-histidine.

[0163] Other oxidized amino acids that may lead to brownish yellow color include oxidized tryptophan, methionine, phenylalanine, and / or tyrosine.The method described herein can also be used to reduce the presence of such oxidized amino acids in the VEGF mini-traps discussed herein.Compositions comprising such VEGF mini-traps also form part of the present invention.

[0164] Oxidation of tryptophan can result in a complex mixture of products. The major products may be N-formylkynurenine and kynurenine, along with mono-, di-, and / or tri-oxidation products. Peptides with oxidized Trp modifications are generally classified as kynurenine (KYN), hydroxytryptophan (W), and ketones. ox1 ), and N-formylkynurenine / dihydroxytryptophan (NFK / W ox2 , also known as "double-oxidized Trp"), trihydroxytryptophan (W ox3 , also known as "triply oxidized Trp"), and hydroxykynurenine (KYN ox1 Depending on the formation of these combinations, such as hydroxytryptophan (W, +20 Da), they exhibit mass increases of 4, 16, 32, and 48 Da. ox1) (Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification? J Am Soc Mass Spectrom. July 2010; 21(7): 1114-1117). Tryptophan oxidation, but not methionine or histidine oxidation, has been found to produce color changes in protein products (Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores.dx.doi.org / 10.1021 / ac404218t|Anal.Chem. 2014, 86, 6850-6857). Similar to tryptophan, oxidation of tyrosine primarily yields 3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li, S, C Schoneich, and R.T. Borchardt. 1995, Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48: (pp. 490-500).

[0165] The present invention provides minitraps (e.g., REGN7483) that contain one or more tryptophan residues that have been oxidized (e.g., as discussed herein). F ) and compositions thereof, for example, wherein about 0.1-10% or less (e.g., about 0.1, 0.2, 0.25, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10%) of the tryptophan residues in the composition are oxidized.

[0166] The present invention provides minitrap molecules described herein (e.g., REGN7483) in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) histidines (e.g., selected from H19, H86, H95, H110, H145, H147, H203, and / or H203) have been oxidized to 2-oxo-his. F or REGN7483 R ) and compositions thereof (e.g., aqueous compositions).

[0167] The present invention also provides VEGF mini-traps of the present invention (e.g., REGN7483 F ,REGN7483 R , REGN7850, or REGN7851) (e.g., expressed in CDM in a host cell, e.g., a CHO cell), wherein about 1% or 2% or less, about 0.1% or less, or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1%, or 0.9-1% of the histidines in the composition are 2-oxo-histidine. In such compositions, the mini-trap polypeptides are a heterogeneous population of peptides, each having a varying number of 2-oxo-histidine residues and unoxidized histidine residues. Thus, the percentage of 2-oxo-histidine in a composition refers to the 2-oxo-histidines in all of the mini-trap molecules divided by the total histidines (oxidized + unoxidized) in the mini-trap molecules × 100. In an embodiment of the invention, the composition is characterized by a brown-yellow color profile as described herein (e.g., less dark or clearer than BY3, 4, 5, 6, or 7).

[0168] One method for quantifying the level of 2-oxo-histidine in a composition is to use a VEGF minitrap (e.g., REGN7483). F ,REGN7483 R, REGN7850, or REGN7851) (e.g., expressed in CDM) with a protease (e.g., Lys-C and / or trypsin), and the amount of 2-oxo-histidine in the resulting peptides is analyzed, for example, by mass spectrometry (ms). In an embodiment of the invention, prior to digestion of the minitrap polypeptide, the cysteine ​​sulfhydryl groups are blocked by reaction with iodoacetamide (IDAM); resulting in residues represented by the following chemical structure: [ka] Such modifications protect free thiols from reforming disulfide bridges and prevent scrambling of disulfide bonds. The present invention provides a VEGF minitrap (e.g., REGN7483) comprising a polypeptide containing the following peptides when modified with IDAM, digested with proteases (e.g., Lys-C and trypsin), and analyzed by mass spectrometry: F ) and a composition (e.g., an aqueous composition) comprising: EIGLLTC containing approximately 0.0095% 2-oxo-histidine * EATVNGH * LYK (amino acids 73 to 89 of SEQ ID NO: 12), QTNTIIDVVLSPSH containing approximately 0.0235% 2-oxo-histidine * GIELSVGEK (amino acids 97 to 119 of SEQ ID NO: 12), TELNVGIDFNWEYPSSKH containing approximately 0.067% 2-oxo-histidine * QHK (amino acids 128 to 148 of SEQ ID NO: 12), DKTH containing approximately 0.0745% 2-oxo-histidine * TC * PPC * PAPELLG (amino acids 206-221 of SEQ ID NO: 12), and / or TNYLTH containing approximately 0.016% 2-oxo-histidine * R (amino acids 90-96 of SEQ ID NO: 12), and / or IIW, optionally containing approximately 0.248% tryptophan dioxide* DSR (amino acids 56 to 61 of SEQ ID NO: 12), In the sequence, H * is 2-oxo-histidine, and W * is tryptophan dioxide, and C * is carboxymethylated cysteine; or EIGLLTC containing approximately 0.006-0.013% 2-oxo-histidine * EATVNGH * LYK (amino acids 73 to 89 of SEQ ID NO: 12), QTNTIIDVVLSPSH containing approximately 0.019-0.028% 2-oxo-histidine * GIELSVGEK (amino acids 97 to 119 of SEQ ID NO: 12), TELNVGIDFNWEYPSSKH containing approximately 0.049-0.085% 2-oxo-histidine * QHK (amino acids 128 to 148 of SEQ ID NO: 12), DKTH containing approximately 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (amino acids 206-221 of SEQ ID NO: 12), and / or TNYLTH containing approximately 0.010-0.022% 2-oxo-histidine * R (amino acids 90-96 of SEQ ID NO: 12), and / or IIW, optionally containing approximately 0.198-0.298% tryptophan dioxide * DSR (amino acids 56 to 61 of SEQ ID NO: 12), In the sequence, H * is 2-oxo-histidine, and W * is tryptophan dioxide, and C * is a carboxymethylated cysteine. In an embodiment of the invention, the peptide is deglycosylated, for example with PNGase F.

[0169] brown yellow The brown-yellow color of the polypeptide compositions presented herein can be described in relation to European color standards. See European Pharmacopoeia Chapter 2.2.2. Color Index of Liquids, 8th Edition. EP colors are typically used in the pharmaceutical industry, for example, to assign a color rank to liquid samples to indicate product quality. European Pharmacopoeia Color is a visual liquid color scale used in the pharmaceutical industry. EP 2.2.2. Color Index of Liquids 2 outlines the preparation of 37 separate "reference solutions" belonging to five color families: green-yellow (GY), yellow (Y), brown-yellow (BY), brown (B), and red (R). Of the seven brown-yellow standards (BY standards), BY1 is the darkest standard and BY7 is the lightest. Matching a given sample to a sample of the BY color standards is routine in the art. The composition of the European brown-yellow color standards is set forth in Table A below.

[0170] [Table 1]

[0171] The color test of a liquid is performed by comparing the test solution with a standard color solution. The composition of the standard color solution is selected depending on the hue and intensity of the color of the test solution. Typically, the comparison is performed in a colorless, transparent, neutral glass, flat-bottom tube (e.g., a tube with a diameter of about 12, 15, 16, or 25 mm) that matches the inner diameter and all other aspects as closely as possible. For example, the comparison may be between 2 or 10 mL of the test solution and the standard color solution. For example, the depth of the liquid may be about 15, 25, 40, or 50 mm. The color assigned to the test solution should not be more intense than the standard color. The color comparison is typically performed against a white background in diffuse light (e.g., daylight). Color may be compared along the vertical or horizontal axis of the tube. In an embodiment of the present invention, the VEGF minitrap (e.g., REGN7483) is used. F The color of the composition containing the ) is carried out as described above.

[0172] The color of the BY standard material is CIEL* a * b * It can also be expressed in color space ("CIELAB" or "CIELab" color space). See Table B. CIEL * a * b * In the coordinate system, L * represents the brightness of a color on a scale of 0 to 100, with 0 being the darkest and 100 being the brightest. * represents the redness or greenness of a color (a * Positive values ​​of represent red, and a * (negative values ​​of b represent green). * represents the yellowness or blueness of the sample, and b * Positive values ​​of represent yellow, and b * Negative values ​​of ΔL represent blue. The color difference between the standard or the initial sample in the evaluation is calculated by the change in the individual color components ΔL * , Δa * , and Δb * The composite change or difference in color can be calculated as a simple Euclidean distance in space using the following formula:

number

[0173] [Table 2]

[0174] The present invention provides a VEGF mini-trap (e.g., REGN7483) of the present invention, characterized by having a brownish-yellow color that approximates the color of BY2, BY3, BY4, BY5, BY6, BY7; or is not darker than BY2, not darker than BY3, not darker than BY4, not darker than BY5, not darker than BY6, not darker than BY7; or is between BY2 and BY3, between BY2 and BY4, between BY3 and BY4, between BY3 and BY5, between BY4 and BY5, between BY4 and BY6, between BY5 and BY6, between BY5 and BY7, or between BY6 and BY7. F ,REGN7483 R In one embodiment, compositions (e.g., aqueous compositions) containing REGN7850, REGN7851, or REGN7852 (e.g., expressed in a host cell, e.g., a CHO cell, in CDM) are provided.

[0175] The present invention also provides the following CIEL * a * b * VEGF minitraps of the present invention (e.g., REGN7483) characterized by color in the color space F ,REGN7483 RIn one embodiment, a composition (e.g., an aqueous composition) comprising a REGN7850 or REGN7851 (e.g., expressed in a host cell, e.g., a CHO cell, in CDM): L * = approx. 88.61, a * = approx. 0.53, b * = approximately 31.17; for example, in this case, the minitrap concentration is approximately 169 mg / ml; L * = approx. 89, a * = about 0.5, b * = approximately 31; for example, in this case, the minitrap concentration is approximately 170 mg / ml; L * = approx. 95.01, a * = approx. -1.68, b * = approximately 18.16; for example, in this case, The minitrap concentration is approximately 161 mg / ml; L * = approx. 95, a * = approx. -1.5, b * = about 18; for example, in this case, the minitrap concentration is about 160 mg / ml; L * = approx. 96.1, a * = approx. -1.05, b * = approximately 14.34; for example, in this case, the minitrap concentration is approximately 158 mg / ml; L * = approx. 96, a * = about -1, b * = approximately 14; for example, in this case, the minitrap concentration is approximately 160 mg / ml; L * = approx. 97.18, a * = approx. -0.93, b * = approximately 10.31; for example, in this case, the minitrap concentration is approximately 106 mg / ml; L * = approx. 97, a * = about -1, b * = approximately 10; for example, in this case, the minitrap concentration is approximately 110 mg / ml; L * = approx. 96.06, a * = approx. -1.02, b* = approximately 14.48; for example, in this case, the minitrap concentration is approximately 154 mg / ml; L * = approx. 96, a * = about -1, b * = approximately 14.5; for example, in this case, the minitrap concentration is approximately 150 mg / ml; L * = approximately 96.96, a * = about -0.85, b * = approximately 14.89; for example, in this case, the minitrap concentration is approximately 159 mg / ml; L * = approx. 97, a * = about -1, b * = about 15; for example, in this case, the minitrap concentration is about 160 mg / ml; L * = approx. 97.76, a * = approx. -1.02, b * = approximately 12.16; for example, in this case, the minitrap concentration is approximately 128 mg / ml; L * = approx. 98, a * = about -1, b * = approximately 12; for example, in this case, the minitrap concentration is approximately 130 mg / ml; L * = approx. 95.06, a * = approx. -1.07, b * = approximately 20.87; for example, in this case, the minitrap concentration is approximately 205 mg / ml; L * = approx. 95, a * = about -1, b * = approximately 21; for example, in this case, the minitrap concentration is approximately 205 mg / ml; L * = approx. 96.93, a * = approx. -1.55, b * = approximately 14.02; for example, in this case, the minitrap concentration is approximately 158 mg / ml; L * = approx. 97, a * = approx. -1.5, b * = approximately 14; for example, in this case, the minitrap concentration is approximately 160 mg / ml; L * = approx. 97.36, a * = approx. -0.39, b * = approximately 10.64; for example, in this case, the minitrap concentration is approximately 150 mg / ml; L * = approx. 97, a * = about -0.5, b * = about 11; for example, in this case, the minitrap concentration is about 150 mg / ml; L * = approximately 99.16, a * = about -0.35, b * = approximately 3.41; for example, in this case, the minitrap concentration is approximately 144 mg / ml; L * = approx. 99, a * = about -0.5, b * = approximately 3; for example, in this case, the minitrap concentration is approximately 145 mg / ml; L * = approximately 99.33, a * = approx. -0.19, b * = approximately 2.39; for example, in this case, the minitrap concentration is approximately 79.3 mg / ml; L * = approx. 99, a * = about 0, b * = approximately 2.4; for example, in this case, the minitrap concentration is approximately 79 mg / ml; L * = approx. 97.37, a * = approx. -1.12, b * = approximately 9.58; for example, in this case, the minitrap concentration is approximately 80 mg / ml; L * = approx. 97, a * = about -1, b * = about 9.6; for example, in this case, the minitrap concentration is about 80 mg / ml; L * = approx. 97.1, a * = about -0.85, b * = approximately 9.97; for example, in this case, the minitrap concentration is approximately 154 mg / ml; L * = approx. 97, a * = about -1, b* = approximately 10; for example, in this case the minitrap concentration is It is about 150 mg / ml; L * = approx. 98.04, a * = about -0.67, b * = approximately 6.75; for example, in this case, the minitrap concentration is approximately 100 mg / ml; L * = approx. 98, a * = about -1, b * = approximately 6.8; for example, in this case, the minitrap concentration is approximately 100 mg / ml; L * = approx. 98.5, a * = approx. -0.51, b * = approximately 5.03; for example, in this case, the minitrap concentration is approximately 75 mg / ml; L * = approx. 99, a * = about -0.5, b * = about 5; for example, in this case, the minitrap concentration is about 75 mg / ml; L * = approx. 98.94, a * = about -0.36, b * = approximately 3.58; for example, in this case, the minitrap concentration is approximately 50 mg / ml; L * = approx. 99, a * = about -0.5, b * = approximately 3.6; for example, in this case, the minitrap concentration is approximately 50 mg / ml; L * = approximately 99.47, a * = about -0.13, b * = approximately 1.65; for example, in this case, the minitrap concentration is approximately 25 mg / ml; L * = approx. 99.5, a * = about 0, b * = about 1.7; for example, in this case, the minitrap concentration is about 25 mg / ml; L * = approximately 99.77, a * = approx. -0.02, b *= approximately 0.66; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 100, a * = about 0, b * = approximately 0.7; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approximately 99.9, a * = approx. 0.01, b * = approximately 0.36; for example, in this case, the minitrap concentration is approximately 5 mg / ml; L * = approx. 100, a * = about 0, b * = approximately 0.4; for example, in this case, the minitrap concentration is approximately 5 mg / ml; L * = approximately 99.95, a * = approx. 0.06, b * = approximately 0.08; for example, in this case, the minitrap concentration is approximately 3 mg / ml; L * = approx. 100, a * = approx. 0.1, b * = approximately 0.1; for example, in this case, the minitrap concentration is approximately 3 mg / ml; L * = approx. 98.89, a * = approx. 0.01, b * = approximately 1.05; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 99, a * = about 0, b * = approximately 1.1; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 98.3, a * = approx. -0.03, b * = approximately 0.96; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 98, a * = about 0, b * = approximately 1; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 99.07, a* = about -0.07, b * = approximately 1.33; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 99, a * = about 0, b * = about 1.3; for example, in this case, the minitrap concentration is about 10 mg / ml; L * = approx. 99.42, a * = approx. -0.04, b * = approximately 1.35; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 99, a * = about 0, b * = about 1.4; for example, in this case, the minitrap concentration is about 10 mg / ml; L * = approximately 99.19, a * = approx. -0.09, b * = approximately 1.55; for example, in this case, the minitrap concentration is approximately 10 mg / ml; L * = approx. 99, a * = about 0, b * = about 1.6; for example, in this case, the minitrap concentration is about 10 mg / ml; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 23 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 22 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 21 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 20 or less; L * = approx. 94-100, a *= -3 to 1 or -3 to 0, and b * =about 19 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 18 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 17 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 16 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 15 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 14 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 13 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 12 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 11 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 10 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 9 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b *=about 8 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 7 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 6 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 5 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 4 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 3 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 2 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 1 or less; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 22; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 21; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 20; L * = approx. 94-100, a* = -3 to 1 or -3 to 0, and b * =about 19; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 18; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 17; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 16; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 15; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 14; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 13; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 12; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 11; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 10; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 9; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 8; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 7; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 6; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 5; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 4; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 3; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 2; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 1; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 3~5; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 4~6; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 5~7; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 6~8; L * = approx. 94-100, a *= -3 to 1 or -3 to 0, and b * =about 7~9; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 8~10; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 9~11; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 10~12; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 11~13; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 14~16; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 15~17; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 16~18; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 17~19; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 18~20; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 19~21; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b* =about 20~22; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 21~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 17~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 10~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 5~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 3~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 1~23; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 10~17; L * = approx. 94-100, a * = -3 to 1 or -3 to 0, and b * =about 5~17; L * =70~99, a * = -2 to 0, and b * = 20 or less; and / or L * =70~99, a * = -2 to 0, and b *= 10-31, about 10, about 14, about 12, about 14, about 15, about 18, about 21, about 27, or about 31. In embodiments of the invention, compositions comprising VEGF mini-traps having a color profile as described above have the following concentrations of mini-traps: about 70 mg / ml or higher, 75-200 mg / ml; or 70-205 mg / ml; 10 mg / ml; 11 mg / ml; 12 mg / ml; 13 mg / ml; 14 mg / ml; 15 mg / ml; 16 mg / ml; 17 mg / ml; 18 mg / ml; 19 mg / ml; 20 mg / ml; 21 mg / ml; 22 mg / ml; 23 mg / ml; 24 mg / ml; 25 mg / ml; 26 mg / ml; 27 mg / ml; 28 mg / ml; 29 mg / ml; 30 mg / ml; 31 mg / ml;32mg / ml;33mg / ml;34mg / ml;35mg / ml;36mg / ml;37mg / ml;38mg / ml;39mg / ml;40mg / ml;41mg / ml;42mg / ml;43mg / ml;44mg / ml;45mg / ml;46mg / ml;47mg / ml;48mg / ml; 49mg / ml;50mg / ml;51mg / ml;52mg / ml;53mg / ml;54mg / ml;55mg / ml;56mg / ml;57mg / ml;58mg / ml;59mg / ml;60mg / ml;61mg / ml;62mg / ml;63mg / ml;64mg / ml;65mg / ml;66mg / ml ;67mg / ml;68mg / ml;69mg / ml;70mg / ml;71mg / ml;72mg / ml;73mg / ml;74mg / ml;75mg / ml;76mg / ml;77mg / ml;78mg / ml;79mg / ml;80mg / ml;81mg / ml;82mg / ml;83mg / ml;84mg / ml;85mg / ml;86mg / ml;87mg / ml;88mg / ml;89mg / ml;90mg / ml;91mg / ml;92mg / ml;93mg / ml;94mg / ml;95mg / ml;96mg / ml;97mg / ml;98mg / ml;99mg / ml;100mg / ml;101mg / ml;102mg / ml;103mg / ml;104mg / ml;105mg / ml;106mg / ml;107mg / ml;108mg / ml;109mg / ml;110mg / ml;111mg / ml;112mg / ml;113mg / ml;114mg / ml;115mg / ml;116mg / ml;117mg / ml;118mg / ml;119mg / ml;120mg / ml;121mg / ml;122mg / ml;123mg / ml;124mg / ml;125mg / ml;126mg / ml;127mg / ml;128mg / ml;129mg / ml;130mg / ml;131mg / ml;132mg / ml;133mg / ml;134mg / ml;135mg / ml;136mg / ml;137mg / ml;138mg / ml;139mg / ml;140mg / ml;141mg / ml;142mg / ml;143mg / ml;144mg / ml;145mg / ml;146mg / ml;147mg / ml;148mg / ml;149mg / ml;150mg / ml;151mg / ml;152mg / ml;153mg / ml;154mg / ml;155mg / ml;156mg / ml;157mg / ml;158mg / ml;159mg / ml;160mg / ml;161mg / ml;162mg / ml;163mg / ml;164mg / ml;165mg / ml;166mg / ml;167mg / ml;168mg / ml;169mg / ml;170mg / ml;171mg / ml;172mg / ml;173mg / ml;174mg / ml;175mg / ml;176mg / ml;177mg / ml;178mg / ml;179mg / ml;180mg / ml;181mg / ml;182mg / ml;183mg / ml;184mg / ml;185mg / ml;186mg / ml;187mg / ml;188mg / ml;189mg / ml;190mg / ml;191mg / ml;192mg / ml;193mg / ml;194mg / ml;195mg / ml;196mg / ml;197mg / ml;198mg / ml;199mg / ml;200mg / ml;201mg / ml;202mg / ml;203mg / ml;204mg / ml;or 205mg / ml. ;

[0176] Alternatively, in embodiments of the present invention, the composition has a VEGF mini-trap concentration of about 70 or greater, about 75, about 90, about 106, about 128, about 147, about 154, 158, about 159, about 161, about 169, about 200, about 205, about 75-200, or about 70-205 g / L, but has a color profile as described above when diluted to the following concentrations: for example, about 10 mg / L. ml;11mg / ml;12mg / ml;13mg / ml;14mg / ml;15mg / ml;16mg / ml;17mg / ml;18mg / ml;19mg / ml;20mg / ml;21mg / ml ;22mg / ml;23mg / ml;24mg / ml;25mg / ml;26mg / ml;27mg / ml;28mg / ml;29mg / ml;30mg / ml;31mg / ml;32mg / ml;3 3mg / ml;34mg / ml;35mg / ml;36mg / ml;37mg / ml;38mg / ml;39mg / ml;40mg / ml;41mg / ml;42mg / ml;43mg / ml;44m g / ml;45mg / ml;46mg / ml;47mg / ml;48mg / ml;49mg / ml;50mg / ml;51mg / ml;52mg / ml;53mg / ml;54mg / ml;55mg / ml;56mg / ml;57mg / ml;58mg / ml;59mg / ml;60mg / ml;61mg / ml;62mg / ml;63mg / ml;64mg / ml;65mg / ml;66mg / ml ;67mg / ml;68mg / ml;69mg / ml;70mg / ml;71mg / ml;72mg / ml;73mg / ml;74mg / ml;75mg / ml;76mg / ml;77mg / ml;7 8mg / ml;79mg / ml;80mg / ml;81mg / ml;82mg / ml;83mg / ml;84mg / ml;85mg / ml;86mg / ml;87mg / ml;88mg / ml;89mg / ml;90mg / ml;91mg / ml;92mg / ml;93mg / ml;94mg / ml;95mg / ml;96mg / ml;97mg / ml;98mg / ml;99mg / ml;100mg / ml;101mg / ml;102mg / ml;103mg / ml;104mg / ml;105mg / ml;106mg / ml;107mg / ml;108mg / ml;109mg / ml;110mg / ml;111mg / ml;112mg / ml;113mg / ml;114mg / ml;115mg / ml;116mg / ml;117mg / ml;118mg / ml;119mg / ml;120mg / ml;121mg / ml;122mg / ml;123mg / ml;124mg / ml;125mg / ml;126mg / ml;127mg / ml;128mg / ml;129mg / ml;130mg / ml;131mg / ml;132mg / ml;133mg / ml;134mg / ml;135mg / ml;136mg / ml;137mg / ml;138mg / ml;139mg / ml;140mg / ml;141mg / ml;142mg / ml;143mg / ml;144mg / ml;145mg / ml;146mg / ml;147mg / ml;148mg / ml;149mg / ml;150mg / ml;151mg / ml;152mg / ml;153mg / ml;154mg / ml;155mg / ml;156mg / ml;157mg / ml;158mg / ml;159mg / ml;160mg / ml;161mg / ml;162mg / ml;163mg / ml;164mg / ml;165mg / ml;166mg / ml;167mg / ml;168mg / ml;169mg / ml;170mg / ml;171mg / ml;172mg / ml;173mg / ml;174mg / ml;175mg / ml;176mg / ml;177mg / ml;178mg / ml;179mg / ml;または180mg / ml。

[0177] In embodiments of the present invention, a VEGF minitrap (e.g., REGN7483) expressed in a host cell (e.g., a CHO cell) in, for example, CDM is used. F ) contains about 50 parts per million (ppm) or less of host cell protein.

[0178] In embodiments of the present invention, the color of a composition may be correlated with the concentration of VEGF minitrap (e.g., expressed in CDM) in the composition (e.g., aqueous composition), the correlation being calculated using the following mathematical formula: 0.046 + (0.066 × Minitrap concentration (mg / ml)) = b * For example, in this case, L * = about 97 to 99 and a = about −0.085 to 0.06. In an embodiment of the invention, the formula is as follows: b * = (0.11 × concentration of minitrap (mg / ml) - 0.56). In an embodiment of the invention, the concentration of VEGF minitrap in the composition or pharmaceutical formulation of the invention is about 90, 100, 110, or 120 mg / ml (or any of the concentrations described above), and the CIEL * a * b * It is characterized by a color in a color space.

[0179] The color of a composition containing a VEGF minitrap (e.g., expressed in CDM) also correlates with the pH and conductivity at which AEX chromatographic purification (flow-through mode) is performed. In an embodiment of the present invention, the composition is the product of a method comprising AEX chromatographic purification at a pH of about 8.0 or higher or 8.4 or higher and a conductivity of about 2.0 mS / cm or lower or 4 mS / cm or lower. Thus, in an embodiment of the present invention, the AEX chromatographic conditions are a pH of about 8.1 or higher (e.g., about 8.1-8.4) and / or a conductivity of about 6.5 (e.g., about 2.0, 4.0, or 2-4 mS / cm). In an embodiment of the present invention, the composition is the flow-through from an AEX column and has the above pH (e.g., 8.4) and conductivity (e.g., 2.0 mS / cm). In certain embodiments, the composition is the product of a process that includes AEX chromatography purification and further includes adjusting the composition to a lower pH, for example, about 6.0 (e.g., 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2).

[0180] Thus, the present invention provides a method for producing a VEGF minitrap (e.g., REGN7483) expressed in a chemically defined medium. F ,REGN7483 R , REGN7850, or REGN7851), wherein about 0.1% to 1% of all histidines in the composition are modified to 2-oxo-histidine, and the color of the composition is as discussed herein, e.g., not darker than the European Brown Yellow color standards BY2, BY3, or BY4, and / or CIEL * a * b * In color space, L * =94~100, a * = -3 to 0, and b *= about 3-6; for example, a concentration of about 90, 100, 110, or 120 mg / ml; or any of the concentrations discussed above.

[0181] Acidic and basic species Protein variants can include both acidic and basic species, where acidic species are variants that elute earlier than the main CEX peak or later than the main AEX peak, and basic species are variants that elute later than the main CEX peak or earlier than the main AEX peak.

[0182] The terms "acidic species," "AS," "acidic region," and "AR" refer to protein variants characterized by an overall acidic charge. For example, such acidic species in recombinant protein preparations can be detected by various methods, such as ion exchange, e.g., WCX-10 HPLC (weak cation exchange chromatography) or IEF (isoelectric focusing). Acidic species of VEGF minitraps can include variants, structural variants, and / or fragmentation variants. Exemplary variants include, but are not limited to, deamidated variants, afucosylated variants, oxidized variants, methylglyoxal (MGO) variants, glycated variants, and citrated variants. Exemplary structural variants include, but are not limited to, glycosylated variants and acetonated variants. Exemplary fragmentation variants include any modified protein species derived from the target molecule by peptide chain dissociation, including, but not limited to, enzymatic and / or chemical modifications, including Fc and Fab fragments, fragments lacking Fab, fragments lacking the heavy chain variable domain, C-terminal truncated variants, variants with the N-terminal Asp of the light chain truncated, and variants with N-terminal truncations of the light chain. Other acidic species variants include variants containing unpaired disulfides, host cell proteins and nucleic acids, chromatography materials, and medium components. Generally, acidic species elute earlier than the main peak in CEX or later than the main peak in AEX analysis.

[0183] In embodiments of the present invention, the protein composition may contain more than one type of acidic species variant. For example, but not limited to, the entire acidic species can be divided based on the chromatographic retention time of the peaks that appear. Another example of how the entire acidic species can be divided can be based on the type of variant, i.e., whether it is a variant, a structural variant, or a fragmentation variant.

[0184] The term "acidic species" or "AS" does not refer to process-related impurities. The term "process-related impurities," as used herein, refers to impurities that are present in a composition containing a protein but that are not derived from the protein itself. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatography materials, and media components.

[0185] In some exemplary embodiments of the present invention, the compositions of the present invention may comprise a VEGF minitrap and an acidic species of the VEGF minitrap, wherein the amount of the acidic species in the composition is at most about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% relative to the VEGF minitrap, and ranges within one or more of the above.

[0186] In an exemplary embodiment of the invention, the composition may comprise a VEGF mini-trap and an acidic species of the VEGF mini-trap, and the amount of the acidic species in the composition is about 0% to about 15%, e.g., about 0% to about 15%, about 0.05% to about 15%, about 0.1% to about 15%, about 0.2% to about 15%, about 0.3% to about 15%, about 0.4% to about 15%, about 0.5% to about 15%, about 0.6% to about 15%, about 0.7% to about 15%, about 0.8% to about 15%, about 0.9% to about 15%, about 1% to about 15%, about 1.5% to about 15%, or about 2% to about 25% of the VEGF mini-trap. % to about 15%, about 2% to about 15%, about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5% , about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5%, and ranges within one or more of the above.

[0187] All peaks eluting before the protein of interest can be grouped together as the acidic region, and all peaks eluting after the protein of interest can be grouped together as the basic region. In some exemplary embodiments, acidic species may elute as two or more acidic regions, which can be numbered AR1, AR2, and AR3, etc., based on the particular retention time of the peaks and the ion exchange column used.

[0188] In one exemplary embodiment of the invention, the composition may comprise a VEGF minitrap and an acidic species of the VEGF minitrap, and AR1 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the above, relative to the area of ​​the VEGF minitrap. In another exemplary embodiment, the composition may include a VEGF mini-trap and an acidic species of the VEGF mini-trap, wherein AR1 is present in an amount of about 0.0% to about 10% relative to the area of ​​the anti-VEGF protein. about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the foregoing.

[0189] In one exemplary embodiment of the invention, the composition may comprise a VEGF mini-trap and an acidic species of the VEGF mini-trap, wherein AR2 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and ranges within one or more of the foregoing, relative to the region of the anti-VEGF protein. In another exemplary embodiment, the composition may include a VEGF minitrap and an acidic species of the VEGF minitrap, and AR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the foregoing, relative to the area of ​​the VEGF minitrap.

[0190] Among the chemical degradation pathways that give rise to acidic or basic species, the two most widely observed covalent modifications of proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan, and tyrosine are the amino acids most susceptible to oxidation: Met and Cys due to their sulfur atoms, and His, Trp, and Tyr due to their aromatic rings.

[0191] The terms "basic species," "basic region," and "BR" refer to protein variants characterized by an overall basic charge. For example, such basic species in recombinant protein preparations can be detected by various methods, such as ion exchange, e.g., WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Exemplary variants include, but are not limited to, lysine variants, aspartic acid isomerization, succinimide formation at asparagine, methionine oxidation, amidation, incomplete disulfide bond formation, serine-to-arginine mutations, aglycosylation, fragmentation, and aggregation. Generally, basic species elute later than the main peak in CEX or earlier than the main peak in AEX analysis. (Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies. MAbs. 2012, September 1; Vol. 4(5): 578-585).

[0192] In certain embodiments of the present invention, the protein composition may contain more than one type of basic species variant.For example, but not limited to, the entire basic species can be divided based on the chromatographic retention time of the peaks that appear.Another example of how the entire basic species can be divided can be based on the type of variant, i.e., whether it is a variant, a structural variant, or a fragmentation variant.

[0193] As described for acidic species, the term "basic species" does not include process-related impurities; basic species may be the result of product preparation (referred to herein as "preparation-derived basic species") or storage (referred to herein as "storage-derived basic species").

[0194] In some exemplary embodiments of the invention, the composition may comprise a VEGF minitrap and a basic species of the VEGF minitrap, wherein the amount of basic species in the composition is It may be up to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% relative to Nitrap, and ranges within one or more of the foregoing.

[0195] In other exemplary embodiments of the invention, the composition may comprise a VEGF minitrap and a basic species of the VEGF minitrap, and the amount of the basic species in the composition is about 0% to about 15%, e.g., about 0% to about 15%, about 0.05% to about 15%, about 0.1% to about 15%, about 0.2% to about 15%, about 0.3% to about 15%, about 0.4% to about 15%, about 0.5% to about 15%, about 0.6% to about 15%, about 0.7% to about 15%, about 0.8% to about 15%, about 0.9% to about 15%, about 1% to about 15%, about 1.5% to about 15%, about 2% to about 15%, about 3% to about 15%, about 4% to about 15%, about 5% to about 15%, about 6% to about 15%, about 7% to about 15%, about 8% to about 15%, about 9% to about 15%, about 10% to about 15%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% to about 7.5%, about 0.6% to about 7.5%, about 0.7% to about 7.5%, about 0.8% to about 7.5%, about 0.9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5 %, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0.8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3% to about 5%, about 4% to about 5%, and ranges within one or more of the above.

[0196] In some exemplary embodiments of the invention, the basic species may elute as two or more basic regions, which may be numbered BR1, BR2, and BR3, etc., based on the particular retention time of the peak and the ion exchange used.

[0197] In one exemplary embodiment of the invention, the composition may comprise a VEGF minitrap and a basic species of the VEGF minitrap, wherein BR1 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% relative to the area of ​​the VEGF minitrap, and ranges within one or more of the above. In another exemplary embodiment, the composition may include a VEGF mini-trap and an acidic species of the VEGF mini-trap, wherein BR1 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the foregoing, relative to the region of the anti-VEGF protein.

[0198] In one exemplary embodiment of the invention, the composition comprises a VEGF minitrap and a VEGF The F minitrap may comprise a basic species, and BR2 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0% relative to the area of ​​the VEGF minitrap, and ranges within one or more of the above. In another exemplary embodiment, the composition may include a VEGF mini-trap and an acidic species of the VEGF mini-trap, wherein BR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%, and ranges within one or more of the foregoing, relative to the region of the anti-VEGF protein.

[0199] The levels of protein variants and / or acidic species in chromatography samples produced using the techniques described herein can be analyzed as described in the Examples section. In one specific embodiment, a cIEF method using an iCE3 analyzer (ProteinSimple) equipped with a fluorocarbon-coated capillary cartridge (100 μm × 5 cm) was used. The ampholyte solution consisted of 0.35% methylcellulose (MC), 4% Pharmalyte 3-10 carrier ampholyte, 4% Pharmalyte 5-8 carrier ampholyte, 10 mM L-arginine HCl, 24% formamide, and a mixture of pI markers 5.12 and 9.77 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted to 10 mg / mL with purified water. The sample was mixed with an ampholyte solution and then focused by introducing a potential of 1500 V for 1 minute, followed by a potential of 3000 V for 7 minutes. Images of the focused variants were acquired by directing 280 nm ultraviolet light through the capillary and into the lens of a charge-coupled device digital camera. The images were then analyzed to determine the distribution of the different charge variants.

[0200] Anion exchange (AEX) chromatography In an embodiment of the present invention, the VEGF minitrap (e.g., in AEX equilibration buffer) is loaded onto an AEX resin that has been equilibrated with, for example, a pH 8.4 buffer (e.g., a Tris buffer such as 50 mM Tris), e.g., 50 mM Tris pH 8.4, 2.0 mS / cm (millisiemens per centimeter), and the flow-through fraction is collected. In an embodiment of the present invention, the equilibration buffer is Tris hydrochloride at a pH of about 8.3 to about 8.6. For example, the flow-through fraction may be collected together with the wash fraction from the column. Column washing can be performed, for example, with one or more column volumes (CV) of equilibration buffer (e.g., 2 CV). In an embodiment of the present invention, aflibercept is cleaved with IdeS protease (e.g., derived from Streptococcus pyogenes, e.g., FabRICATOR) prior to AEX chromatography, and the cleaved Fc fragment is removed from the minitrap product using Protein A chromatography. The minitraps are then purified by AEX chromatography (flow-through mode) as discussed.

[0201] Accordingly, the present invention provides a VEGF mini-trap of the present invention (e.g., REGN7483) produced by a method comprising the steps of: F ) a composition comprising: (i) expressing aflibercept in host cells (e.g., Chinese hamster ovary (CHO) cells) grown in CDM (e.g., secreting aflibercept from the host cells into the CDM); (ii) removing aflibercept from the medium and / or the host cells; (iii) optionally purifying the aflibercept by Protein A chromatography; (iii) aflibercept against S. pyogenes IdeS protease (e.g., Fab proteolytic digestion with a PEG-3 PEG-4 ... (iv) applying the minitrap to an AEX chromatography resin (e.g., a column containing the resin) at a rate of, for example, about 50-500 g / L resin; and (v) retaining the mini-trap in the flow-through fraction of the resin; and (vi) Optionally, further purifying the minitrap, for example by hydrophobic interaction chromatography (HIC).

[0202] In an embodiment of the present invention, the AEX resin is Q-sepharose Fast Flow or has the active group: -O-CH2CHOHCH2OCH2CHOHCH2N + (CH3)3 or -N + (CH3)3 or a quaternary amine. In an embodiment of the invention, the resin is POROS 50HQ or contains quaternary polyethyleneimine active groups.

[0203] In an embodiment of the present invention, the conditions for AEX chromatographic purification of VEGF minitrap in flow-through mode are as follows: (1) The AEX column is POROS 50HQ (or an AEX resin with quaternized polyethyleneimine functional groups) equilibrated with a buffer solution of pH 8.30-8.50 with a conductivity of 1.90-2.10 mS / cm; (2) The AEX column is Q Sepharose FF (or -O-CH2CHOHCH2OCH2CHOHCH2N) equilibrated with a pH 7.90-8.10 buffer solution with a conductivity of 2.40-2.60 mS / cm. + (CH3)3 or -N + (CH3)3 or quaternary amine functionalized AEX resin); (3) The AEX column is POROS 50HQ (or an AEX resin with quaternized polyethyleneimine functional groups) equilibrated with a buffer solution of pH 7.90-8.10 with a conductivity of 2.40-2.60 mS / cm; (4) The AEX column is Q Sepharose FF (or -O-CH2CHOHCH2OCH2CHOHCH2N) equilibrated with a pH 7.70-7.90 buffer solution with a conductivity of 3.90-4.10 mS / cm. + (CH3)3 or -N + (CH3)3 or quaternary amine functionalized AEX resin); (5) The AEX column is POROS 50HQ (or an AEX resin with quaternized polyethyleneimine functional groups) equilibrated with a buffer solution of pH 7.70-7.90 with a conductivity of 3.90-4.10 mS / cm; (6) The AEX column is Q Sepharose FF (or -O-CH2CHOHCH2OCH2CHOHCH2N) equilibrated with a pH 7.70±0.1 buffer solution with a conductivity of 9.0±0.1 mS / cm. + (CH3)3 or -N + (CH3)3 or quaternary amine functionalized AEX resin); or (7) The AEX column is POROS 50HQ (or an AEX resin with quaternized polyethyleneimine functional groups) equilibrated with a buffer solution of pH 8.4±0.1 with a conductivity of 2.0±0.1 mS / cm. In an embodiment of the invention, a buffer of pH 8.30-8.50 comprises 50 mM Tris pH 8.4 and 2.0 mS / cm; a buffer of pH 7.90-8.10 comprises 50 mM Tris pH 8.4 and 2.0 mS / cm; pH 7.70-7.90 buffer contains 50 mM Tris, 10 mM acetate, 10 mM NaCl, pH 7.8 and 4.0 mS / cm; pH 7.70+0.1 buffer contains 50 mM Tris, 60 mM NaCl, pH 7.7±0.1; and / or pH 8.4±0.1 buffer contains 50 mM Tris pH 8.4±0.1.

[0204] In an embodiment of the present invention, for example, S. pyogenes IdeS or a variant thereof is used. Aflibercept, which will be proteolytically cleaved to generate the VEGF minitrap, is harvested from the host cells and / or the host cell chemically defined growth medium and then cleaved prior to any AEX chromatographic purification.

[0205] In an embodiment of the present invention, the AEX chromatography column is loaded at a rate of 40 grams of protein per liter of resin.

[0206] In embodiments of the invention, before and / or after AEX chromatography, the VEGF minitrap is purified by additional chromatography (e.g., mixed-mode chromatography, cation exchange chromatography, Protein A chromatography, and / or hydrophobic interaction chromatography (in flow-through or bind-elute mode)) and / or filtration steps (e.g., depth filtration, viral filtration, diafiltration, and / or ultrafiltration).

[0207] Ion exchange chromatography resins have charged functional groups attached to the resin beads that attract biomolecules or surface-exposed patches of opposite charge. Cation exchange resins are negatively charged, while anion exchange resins are positively charged. Ion exchange resins are also classified as "weak" or "strong" exchangers. These terms refer to the degree to which the ionization state of the functional group varies with pH. "Weak" exchangers are ionized only over a limited pH range, while "strong" exchangers do not exhibit a change in ion exchange capacity with changes in pH. Weak exchange resins can gain or lose protons as the buffer pH changes, providing an additional dimension of charge variation and selectivity for binding and elution. Strong exchangers do not vary and remain fully charged over a wide pH range, making separations easier to optimize than weak exchangers. For example, strong anion exchange resins include those with quaternary amine functional groups, e.g., -N + Q Sepharose FF or Capto with -(CH3)3 Q; or POROS 50HQ, which has a quaternary polyethyleneimine functional group. In an embodiment of the present invention, AEX purification can be carried out using, for example, -N + It is carried out using strong or weak anion exchangers with -(CH3)3 or quaternary polyethyleneimine functional groups.

[0208] The present invention also provides VEGF mini-traps of the present invention (e.g., REGN7483 F ,REGN7483 R , REGN7850, or REGN7851), comprising the steps of: (i) culturing host cells containing a polynucleotide encoding the minitrap or aflibercept under conditions such that the minitrap or aflibercept is expressed and, optionally, secreted from the host cells into the growth medium (the host cells can be grown in CDM); and (ii) removing the minitrap or aflibercept from the host cells and / or medium; (iii) optionally, purifying aflibercept, if expressed, by Protein A chromatography; and (iii) if aflibercept is expressed, proteolytically digesting the aflibercept with an IdeS protease (e.g., FabRICATOR) or a variant thereof to generate a minitrap and an Fc fragment; and optionally removing the Fc from the composition by Protein A chromatography, in which the Fc binds to a Protein A resin. The minitraps or compositions thereof (e.g., aqueous compositions) that are the products of such methods are also part of the invention.

[0209] light exposure VEGF minitrap (e.g., REGN7483) expressed in CDM F ,REGN7483 R The brown-yellow color that characterizes REGN7850, REGN7851, and REGN7852 is the anion. The color can be reduced by ion-exchange (AEX) chromatography purification. For example, a minitrap expressed by host cells (e.g., Chinese hamster ovary (CHO) cells) in a chemically defined liquid growth medium can be removed from the growth medium (after removing the host cells) by applying it to an AEX resin (e.g., a strong AEX resin) and retaining the material in the flow-through fraction. In addition, exposure of the minitrap to light has been found to increase its brownish-yellow appearance. Therefore, minimizing light exposure can reduce the color appearance. The present invention includes placing the VEGF minitrap in a colored container (e.g., a brown vial) for storage. In embodiments of the present invention, the purification method (e.g., involving AEX (flow-through) chromatography) and / or expression and / or storage in a CDM can be performed at approximately 240,000, 600,000, 960,000, 1.2 million, or 2.4 million lux. * and / or about 40, 100, 160, 200, or 400 W white light exposure * hours / m 2 This is done while preventing any greater ultraviolet A (UVA) light exposure than

[0210] Cell culture conditions Other methods for reducing the brownish yellow color of the composition containing VEGF minitrap expressed in CDM include adjusting the concentration of various components of culture medium.The presence of cysteine ​​has been shown to correlate with brownish yellow color, especially when iron and zinc are present.For example, reducing the cysteine ​​concentration in CDM and culture supplementation has been shown to reduce brownish yellow color.One way to reduce color or cysteine ​​concentration is to replace cysteine ​​with cystine or cysteine ​​sulfate, and / or reduce the metallic iron and / or zinc and / or nickel and / or copper and / or chelate content in CDM. For example, in embodiments of the invention, the cysteine ​​concentration in the CDM in which the host cells are initially grown (day 0) is about 1.3 to 1.6 (e.g., 1.3, 1.4, 1.5, or 1.6) millimoles per liter, and additional cysteine ​​supplements, e.g., 1.1 to 1.4 (e.g., 1.1, 1.2, 1.3, or 1.4) millimoles per liter of culture, 1.6 to 1.9 (e.g., 1.6, 1.7, 1.8, or 1.9) millimoles per liter of culture, or 2.0 to 2.3 (2.0, 2.1, 2.2, or 2.3) millimoles per liter of culture, are added during culture growth, e.g., every two days, e.g., on days 2, 4, 6, and 8. In an embodiment of the invention, iron (Fe), zinc (Zn), copper (Cu), and nickel (Ni) are included in the initial culture medium along with chelating agents such as ethylenediaminetetraacetic acid (EDTA) and / or citric acid.In an embodiment of the invention, the chelating agents are EDTA present at a concentration of about 38-190 (e.g., 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, or 190) micromolar, and citrate present at a concentration of about 22-110 (e.g., 22, 30, 40, 50, 60, 70, 80, 90, 100, or 110) micromolar; Fe is present at a concentration of about 34-125 (e.g., 34, 40, 50, 60, 70, 75, 80, 90, 100, 120, or 125) micromolar. Zn is present at a concentration of about 3-10 (e.g., 3, 4, 5, 6, 6.5, 7, 8, 8.5, 9, or 10) micromolar; Cu is present at a concentration of about 0.05-0.4 (e.g., 0.05, 0.06, 0.07, 0.08, 0.1, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.3, or 0.4) millimolar; and Ni is present at a concentration of about 0.25-2.0 (e.g., 0.25, 0.5, 0.6, 0.64, 0.65, 0.70, 0.75, 1, 1.5, or 2.0) millimolar. In an embodiment of the invention, the ratio of Fe:Zn:Cu:EDTA:Citrate:Ni is about 441:38:1:500:294:4.

[0211] It has also been shown that including an antioxidant in the CDM in which the VEGF minitrap is expressed causes a reduction in the brown-yellow color. For example, in embodiments of the present invention, the antioxidant is The antioxidants include hypotaurine, taurine, glycine, a combination of hypotaurine, taurine, and glycine; a combination of hypotaurine, taurine, glycine, and glutathione; thioctic acid, and / or vitamin C. Other antioxidants that can be introduced include choline, hydrocortisone, and vitamin E. In an embodiment of the present invention, the initial culture medium has taurine at a concentration of about 10 mM in culture; hypotaurine at a concentration of about 10 mM in culture; glycine at a concentration of about 10 mM in culture; thioctic acid at a concentration of about 0.0024 mM in culture; and / or vitamin C (ascorbic acid) at a concentration of about 0.028 mM in culture. Optionally, the initial culture medium has glutathione at a concentration of about 2 mM in culture; choline chloride at a concentration of about 1.43 mM in culture; hydrocortisone at a concentration of about 0.0014 mM in culture; and / or vitamin E (α-tocopherol) at a concentration of about 0.009 mM in culture.

[0212] With respect to the concentration of culture medium components, the term "cumulative" refers to the total amount or concentration of a particular component or components added over the course of a cell culture to form the CDM, including components added at the beginning of the culture (to the day 0 CDM) and any subsequent additional components ("chemically defined top-up feeds"). Because medium components are metabolized during culture, cultures with the same cumulative amount of a given component will have different absolute levels if that component is added at different times (e.g., not all is present initially, but some is added in a top-up feed).

[0213] In some embodiments of the present invention, modified CDMs are used to produce the VEGF minitraps of the present invention or compositions thereof (e.g., aqueous compositions). Minitraps produced by host cells cultured in modified CDMs and compositions containing such minitraps (e.g., having any of the color characteristics discussed herein) form part of the present invention. Modified CDMs can be obtained by reducing or increasing the cumulative concentration of amino acids in the CDM. Non-limiting examples of such amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (or salts thereof). The cumulative increase or decrease in the amount of such amino acids in the modified CDM relative to the CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the above. Alternatively, the cumulative increase or decrease in the amount of one or more amino acids in the modified CDM relative to the unmodified CDM can be about 5 to about 20%, about 10 to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, and ranges within one or more of the above.

[0214] In some embodiments, the modified CDM can be obtained by reducing the cumulative concentration of cysteine ​​in the CDM. The reduction in the amount of cysteine ​​in the CDM to form the modified CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the foregoing, compared to the unmodified CDM. Alternatively, the cumulative amount of cysteine ​​in the modified CDM may be reduced by about 5 to about 20%, about 10 to about 30%, about 30 to about 40%, about 30 to about 50%, about 40 to about 60%, about 60 to about 70%, about 70 to about 80%, about 80 to about 90%, or about 90 to about 100%, as compared to the CDM, and may range within one or more of the above. In one embodiment, the cumulative amount of cysteine ​​in the modified CDM is less than about 1 mM, less than about 2 mM, less than about 3 mM, less than about 4 mM, less than about 5 mM, less than about 6 mM, less than about 7 mM, less than about 8 mM, less than about 9 mM, or less than about 10 mM.

[0215] In some embodiments, the modified CDM can be obtained by replacing at least a certain percentage of the cumulative cysteines in the CDM with cystine. The replacement can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the above, relative to the CDM. Alternatively, the replacement can be about 5 to about 20%, about 10 to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, and ranges within one or more of the above, relative to the unmodified CDM.

[0216] In some embodiments, the modified CDM can be obtained by replacing at least a certain percentage of the cumulative cysteines in the CDM with cysteine ​​sulfate, which can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the foregoing, relative to the CDM. Alternatively, the replacement may be about 5 to about 20%, about 10 to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, as compared to unmodified CDM, and ranges within one or more of the foregoing.

[0217] In one embodiment, the VEGF mini-trap is produced by a method comprising culturing host cells in a CDM under suitable conditions, wherein the suitable conditions are achieved by reducing the cumulative iron concentration in the CDM to about 50 μM or less. In one embodiment, the VEGF mini-trap is produced by a method comprising culturing host cells in a CDM under suitable conditions, wherein the suitable conditions are achieved by reducing the cumulative copper concentration in the CDM to about 0.1 μM or less. In one embodiment, the VEGF mini-trap is produced by a method comprising culturing host cells in a CDM under suitable conditions, wherein the suitable conditions are achieved by reducing the cumulative zinc concentration in the CDM to about 5 μM or less. Compositions (e.g., aqueous compositions) comprising such mini-traps are part of the invention, e.g., such compositions have color characteristics as set forth herein.

[0218] In some embodiments, the modified CDM can be obtained by reducing or increasing the cumulative concentration of metals in the CDM. Non-limiting examples of metals include iron, copper, manganese, molybdenum, zinc, nickel, calcium, potassium, and sodium. The increase or decrease in the amount of one or more metals in the modified CDM compared to the CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, and ranges within one or more of the above. Alternatively, the increase or decrease in the cumulative amount of one or more metals in the modified CDM compared to the unmodified CDM may be about 5 to about 20%, about 10 to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%, and ranges within one or more of the foregoing.

[0219] In some embodiments, the modified CDM comprises one or more antioxidants. Non-limiting examples of antioxidants include taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, vitamin C, vitamin E, and combinations thereof. In some embodiments, the modified CDM comprises a concentration of about 0.01 mM to about 20 mM, such as 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, or about 0.01 mM to about 10 mM. In some embodiments, the modified CDM contains hypotaurine at about 0.01 mM to about 20 mM, i.e., 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and any range within one or more of the above. In some embodiments, the modified CDM comprises glycine at about 0.01 mM to about 20 mM, i.e., 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and any range within one or more of the above. In some embodiments, the modified CDM comprises thioctic acid at about 0.01 nM to about 5 nM, i.e., about 0.01 nM to about 0.1 nM, about 0.1 nM to about 1 nM, about 1 nM to about 2.5 nM, about 1 nM to about 3 nM, about 1 nM to about 5 nM, and any range within one or more of the above. In some embodiments, the modified CDM comprises glutathione at about 0.01 mM to about 5 mM, i.e., 0.01 mM to about 1 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, and any one or more of the above ranges. In some embodiments, the modified CDM comprises choline chloride at about 0.01 mM to about 5 mM, i.e., 0.01 mM to about 1 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 1 mM to about 5 mM, and any one or more of the above ranges. In some embodiments, the modified CDM comprises about 0.01 nM to about 5 nM hydrocortisone, i.e., about 0.01 nM to about 0.1 nM, about 0.1 nM to about 1 nM, about 1 nM to about 2.5 nM, about 1 nM to about 3 nM, about 1 nM to about 5 nM, and ranges within one or more of the above.In some embodiments, the modified CDM comprises about 1 mM to about 50 mM vitamin C, i.e., about 1 mM to about 5 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 5 mM to 30 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, and any one or more of the above ranges. In some embodiments, the modified CDM comprises about 1 mM to about 50 mM vitamin E, i.e., about 1 mM to about 5 mM, about 5 mM to about 20 mM, about 10 mM to about 30 mM, about 5 mM to 30 mM, about 20 mM to about 50 mM, about 25 mM to about 50 mM, and any one or more of the above ranges.

[0220] Glycosylation For example, VEGF minitraps (e.g., REGN7483, REGN7850, or REGN7851) produced by the methods for modulating their glycosylation and having color characteristics as discussed herein, and compositions thereof (e.g., aqueous compositions), form part of the present invention. Glycosylation can be varied by changing the cumulative concentration of certain components in the CDM in which host cells expressing the minitraps are grown. Based on the cumulative amount of components added to the CDM, the total fucosylation, total galactosylation, total sialylation, and mannose-5% can be varied.

[0221] In an embodiment of the invention, the VEGF minitrap is desialylated.

[0222] In some exemplary embodiments, the method for modulating glycosylation of a VEGF minitrap may include supplementing CDM with uridine. The VEGF minitrap may have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

[0223] In some exemplary embodiments, the method for modulating glycosylation of a VEGF mini-trap may include supplementing the CDM with manganese. The CDM does not contain manganese prior to supplementation. The VEGF minitrap may have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

[0224] In some exemplary embodiments, the method for modulating glycosylation of a VEGF minitrap can include supplementing a CDM with galactose. The CDM as discussed herein does not contain galactose prior to supplementation. The VEGF minitrap can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

[0225] In some exemplary embodiments, the method for modulating glycosylation of a VEGF minitrap can include supplementing a CDM with dexamethasone. The CDM as contemplated herein does not contain dexamethasone prior to supplementation. The VEGF minitrap can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

[0226] In some exemplary embodiments, the method for modulating glycosylation of a VEGF minitrap may include supplementing the CDM with one or more of uridine, manganese, galactose, and dexamethasone. The CDM as contemplated herein does not contain one or more of uridine, manganese, galactose, and dexamethasone prior to supplementation. The anti-VEGF protein may have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

[0227] In an embodiment of the invention, the VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in the composition of the invention: Less than approximately 0.1% is trixylosylated Approximately 1.5% are dixylosylated; Approximately 15% are monoxylosylated; about 0.9% or less than about 1% is modified with xylose-galactose; and / or Approximately 0.7% or less than 1% is modified with xylose-galactose-sialic acid.

[0228] In an embodiment of the invention, the amount of VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in the composition of the invention is · Approximately 8% of 5 arginine residues; less than about 0.1% of arginine 153 residues; and / or Less than approximately 0.1% of the arginine 96 residues are It is modified with 3-deoxyglucosone.

[0229] In an embodiment of the invention, the amount of VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in the composition of the invention is · Approximately 0.1% of 5 arginine residues; Approximately 1.0 or 1.1% of lysine 62 residues; Approximately 0.4% or fewer of the lysine 68 residues; about 0.6% or fewer of lysine 149 residues; and / or Less than approximately 0.1% of lysine 185 residues are It is glycated.

[0230] In embodiments of the invention, in a composition comprising a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851): For example, (R)VTSP NApproximately 98% or more of the 36 asparagine residues corresponding to ITVTLK (underlined) (amino acids 31-42 of SEQ ID NO: 12); For example, (K)GFIIS N approximately 51, 52, 53, 54, or 55% of the asparagine 68 residue corresponding to ATYK (underlined) (amino acids 62-72 of SEQ ID NO: 12); For example, (K)LVL N About 99% or more of the asparagine 123 residues corresponding to CTAR (underlined) (amino acids 119-127 of SEQ ID NO: 12); and / or For example, (K) N Approximately 44, 50, 60, 70, 80, 90, 98, or 99% of the asparagine 196 residues corresponding to STFVR (amino acids 195-201 of SEQ ID NO: 12) are It is N-glycosylated.

[0231] Glycosylation has been shown to have a significant impact on the safety and function of biotherapeutics. Obtaining minitraps with favorable glycosylation profiles would be highly beneficial for their successful use in treating neovascular ocular disorders. VEGF antagonists have generally been shown to have common adverse vascular effects, directly or indirectly attributable to their anti-VEGF effects, including renal vascular damage, often manifested by hypertension, proteinuria, and thrombotic microangiopathy, as well as congestive heart failure. Therefore, any means to reduce the systemic exposure of subjects receiving intravitreally injected minitraps would be beneficial. It is believed that small amounts of intravitreally injected VEGF antagonists may leak into the systemic circulation and exhibit such adverse effects. See, for example, the following references: Avery RL et al., Comparison of Systemic Pharmacokinetics Post Anti-VEGF Intravitreal Injections of Ranibizumab, Bevacizumab, and Aflibercept (abstract). Presented at the 2013 Annual Meeting of the American Society of Retina Specialists (ASRS); Toronto, August 25, 2013; Avery et al., Intravitreal bevacizumab (Avastin) in the treatment of proliferative diabetic retinopathy. Ophthalmology 2006; 113:1695-705; Matsuyama et al., Plasma levels of vascular endothelial growth factor and pigment epithelium-derived factor before and after vitiligo. after intravitreal injection of bevacizumab. Br J Ophthalmol 2010;94:1215-18; and Carneiro et al., Vascular endothelial growth Factor plasma levels before and after treatment of neovascular age-related macular degeneration with bevacizumab or ranibizumab. Acta Ophthalmol 2012;90:e25-30. The in vivo studies presented herein suggest that minitraps have a shorter half-life than aflibercept when administered systemically (see Example 6). One reason for this effect may be the glycosylation profile of minitraps. REGN7483 produced in chemically defined medium F is known to have particularly high levels of high-mannose glycans at N123 and N196. This is higher than the levels observed in IgG antibodies. As discussed further below, Table C and Figure 14 (A and C) show that approximately 30-40% of N123 and N196 residues in the tested compositions were highly mannosylated. For aflibercept, approximately 6-13% of these residues were observed to be highly mannosylated. High-mannose glycans on antibodies have been shown to be associated with rapid systemic clearance and shorter half-lives. See Goetze et al., High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans, Glycobiology 21(7):949-959 (2011). This may be due to binding by mannose receptors, which remove high-mannose-containing pathogens from the blood. A similar mechanism may be responsible for the rapid systemic clearance of MiniTrap.

[0232] In one embodiment of the invention, the glycosylation profile of the VEGF minitrap composition is as follows: about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans. In an embodiment of the invention, the minitrap has Man5 glycosylation at about 32.4% of the asparagine 123 residues and / or about 27.1% of the asparagine 196 residues.

[0233] For example, compositions of the invention can be expressed, e.g., in CHO cells and in CDM, and purified by AEX flow-through chromatography as provided herein; ·Man5 glycosylation on approximately 30-35% of asparagine 123 residues; ·Man5 glycosylation on approximately 25-30% of asparagine 196 residues; Approximately 6-8% of asparagine 36 residues are Man6-phosphate glycosylated; ·Man7 glycosylation on approximately 3-4% of asparagine 123 residues; High-mannose glycosylation of approximately 38% of asparagine 123 residues; and / or Approximately 29% of the asparagine 196 residue is highly mannose glycosylated The VEGF mini-traps of the invention include those (e.g., REGN7483, REGN7850, or REGN7851) comprising:

[0234] The present invention also provides a VEGF minitrap (e.g., REGN7483) comprising Man5 glycosylation at Asn123; Man5 glycosylation at Asn196; Man6-phosphate glycosylation at Asn36; and / or Man7 glycosylation at Asn123. F or REGN7483 R ) is included.

[0235] In embodiments of the invention, the VEGF minitraps of the invention may contain one or more of the glycosylations listed below. Compositions (e.g., aqueous compositions) comprising the minitraps of the invention, e.g., minitrap molecules having such glycosylations at the indicated frequency percentages, are also part of the invention. G0-GlcNAc glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 1.00%), and / or Asn196 (e.g., about 1.40%); G1-GlcNAc glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 4.80%), and / or Asn196 (e.g., about 2.70%); G1S-GlcNAc glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 4.10%), and / or Asn196 (e.g., about 2.20%); G0 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G1 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 6.10%); G1S glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 1.90%); G2 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 11.50%), and / or Asn196 (e.g., about 18.10%); G2S glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 14.50%), and / or Asn196 (e.g., about 18.40%); G2S2 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 1.50%), and / or Asn196 (e.g., about 3.70%); G0F glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G2F2S glycosylation of Asn36 (e.g., about 2.00%), Asn68 (e.g., about 2.00%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G2F2S2 glycosylation of Asn36 (e.g., about 1.60%), Asn68 (e.g., about 0.50%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G1F glycosylation at Asn36 (e.g., about 5.60%), Asn68 (e.g., about 6.10%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G1FS glycosylation at Asn36 (e.g., about 0%), Asn68 (e.g., about 3.80%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G2F glycosylation at Asn36 (e.g., about 20.20%), Asn68 (e.g., about 28.00%), Asn123 (e.g., about 1.80%), and / or Asn196 (e.g., about 2.10%); G2FS glycosylation at Asn36 (e.g., about 35.20%), Asn68 (e.g., about 48.90%), Asn123 (e.g., about 2.80%), and / or Asn196 (e.g., about 2.20%); G2FS2 glycosylation of Asn36 (e.g., about 22.40%), Asn68 (e.g., about 9.10%), Asn123 (e.g., about 0.30%), and / or Asn196 (e.g., about 0.60%); G3FS glycosylation at Asn36 (e.g., about 3.40%), Asn68 (e.g., about 1.60%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G3FS3 glycosylation of Asn36 (e.g., about 1.70%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); G0 of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 3.40%), and / or Asn196 (e.g., about 2.60%) -2GlcNAc glycosylation; Man4 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0.50%), and / or Asn196 (e.g., about 1.60%); Man4_A1G1 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 3.60%), and / or Asn196 (e.g., about 2.10%); Man4_A1G1S1 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 4.60%), and / or Asn196 (e.g., about 3.00%); Man5 glycosylation at Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 32.40%), and / or Asn196 (e.g., about 27.10%); Man5_A1G1 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 4.80%), and / or Asn196 (e.g., about 2.80%); Man5_A1G1S1 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 3.30%), and / or Asn196 (e.g., about 1.50%); Man6 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 1.30%), and / or Asn196 (e.g., about 0%); Man6_G0+ phosphate glycosylation of Asn36 (e.g., about 1.70%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); Man 6+ phosphate glycosylation at Asn36 (e.g., about 6.20%), Asn68 (e.g., about 0%), Asn123 (e.g., about 0%), and / or Asn196 (e.g., about 0%); Man7 glycosylation of Asn36 (e.g., about 0%), Asn68 (e.g., about 0%), Asn123 (e.g., about 3.60%), and / or Asn196 (e.g., about 0%); REGN7483 F or REGN7483 R or REGN7711, or VTSPNITVTLK;KGFIISNATYK;GFIISNATYK;LVLNCTAR;KNSTFVR; or another VEGF minitrap shown herein containing an NSTFVR motif and / or residues N36, N68, N123, or N196, any of the asparagine glycosylation shown in Figure 14 (A or C) (e.g., at approximately the indicated level); REGN7483 F any of the asparagine glycosylation (e.g., at approximately the indicated level) shown in Table C(a or b) herein, whether or not another VEGF minitrap shown herein comprising residues N36, N68, N123, or N196; and / or REGN7483 F or another VEGF minitrap shown herein, any of the glycosylation (eg, at about the indicated level) in Table D herein.

[0236] Compositions (e.g., aqueous compositions) comprising the VEGF mini-traps of the invention may comprise one or more of the glycosylations listed in Table C, e.g., at the frequency percentages shown (e.g., all of the glycosylations at the percentages listed, e.g., ±10% of the percentage number listed). In embodiments of the invention, the VEGF mini-traps of the invention comprise one or more of the glycosylations listed below, e.g., , may be contained in one or more of the residues shown.

[0237] [Table 3]

[0238] [Table 4-1] [Table 4-2]

[0239] The present invention provides VEGF minitraps (e.g., REGN7483) comprising any one or more of the glycosylation percentages shown in Table D below. F ) is included in the composition.

[0240] [Table 5]

[0241] In some exemplary embodiments of the invention, the VEGF mini-trap is at or below about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4% It may have a reduced level of fucosylated glycans of 0.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. A range within one or more of the above values, e.g., 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, or 1-99%.

[0242] In some exemplary embodiments of the invention, the VEGF minitrap may have a level of sialylated glycans reduced by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. A range within one or more of the above values, e.g., 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, or 1-99%.

[0243] In some exemplary embodiments of the invention, the VEGF minitrap may have a level of galactosylated glycans reduced by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. A range within one or more of the above values, e.g., 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 2-51%, 2-52%, 2-53%, 2-54%, 2-55%, 2-56%, 2-57%, 2-58%, 2-59%, 2-60%, 2-61%, 2-62%, 2-63%, 2-64%, 2-65%, 2-66%, 2-67%, 2-68%, 2-69%, 2-70%, 2-71%, 2-72%, 2-73%, 2-74%, 2-75%, 2-76%, 2-77%, 2-78%, 2-79%, 2-80%, 2-81%, 2-82%, 2-83%, 2-84%, 2-85%, 2-86%, 2-87%, 2-88%, 2-89%, 2-90%, 2-91%, 2-92%, 2-93%, 2-94%, 2-95%, 2-96%, 2-97%, 2-98%, 2- %, 2~43%, 2~44%, 2~45%, 2~46%, 2~47%, 2~48%, 2~49%, 2~50%, 3~10%, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44%, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25%, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, or 1-99%.

[0244] In some exemplary embodiments of the invention, the VEGF minitrap may have increased levels of mannosylated glycans by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. A range within one or more of the above values, e.g., 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, or 1-99%.

[0245] Other post-translational modifications (PTMs) In embodiments of the invention, compositions include VEGF minitraps of the invention (e.g., REGN7483, REGN7850, or REGN7851) bearing other PTMs such as free thiols, trisulfide bonds, deamidation, methionine oxidation, and C-terminal amino acid loss.

[0246] In an embodiment of the invention, about 0% of the cysteines in the hinge region of a VEGF mini-trap of the invention (e.g., REGN7483, REGN7850, or REGN7851) in a composition, and / or about 0.3% or fewer of the cysteines in the VEGFR1 domain and / or VEGFR2 domain of a VEGF mini-trap of the invention (e.g., REGN7483, REGN7850, or REGN7851) in a composition, are cysteines of, for example, VEGFR1 cysteines (VEGF1 cysteines). C R, underlined), cysteine ​​of VEGFR2 (LVLN C The underlined amino acids (amino acids 120-127 of SEQ ID NO: 12) corresponding to TAR, and / or the cysteine ​​in the hinge region (THT C PP C PAPELLG (amino acids 208-221 of SEQ ID NO: 12) or THT C PP C PP C (corresponding to amino acids 208-217 of SEQ ID NO: 28, underlined) is a free thiol.

[0247] In an embodiment of the invention, about 4% or fewer of the cysteines in the hinge region of a VEGF mini-trap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention, and / or about 0.1% or fewer of the cysteines in the VEGFR1 domain and / or VEGFR2 domain of a VEGF mini-trap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention, are cysteines that are not cysteines of, for example, VEGFR1 cysteines (ELVIP C R (amino acids 25 to 31 of SEQ ID NO: 12) & EIGLLT C EATVNGHLYK (corresponding to amino acids 73 to 89 of SEQ ID NO: 12, underlined), cysteine ​​of VEGFR2 (LVLN C TAR (amino acids 120-127 of SEQ ID NO: 12) & SDQGLYT C AASSGLMTK(K) (corresponding to amino acids 178 to 195 of SEQ ID NO: 12, underlined), and / or a cysteine ​​in the hinge region (THC PP C PAPELLG&THT C PP C PAPELL(G) (amino acids 208-221 of SEQ ID NO: 12) or THT C PP C P P C &THT C PP C PP C (corresponding to amino acids 208 to 217 of SEQ ID NO: 28, underlined) is trisulfide-bridged.

[0248] In an embodiment of the invention, less than about 0.1% of the cysteines of a VEGF minitrap (eg, REGN7483, REGN7850, or REGN7851) in a composition of the invention are intrachain disulfide and / or trisulfide bonded.

[0249] In an embodiment of the invention, greater than about 99% (eg, about 99.8%) of the disulfide bridges in a VEGF minitrap (eg, REGN7483, REGN7850, or REGN7851) in a composition of the invention are in parallel conformation.

[0250] In an embodiment of the invention, the asparagine 84 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention is selected from the group consisting of: N Approximately 3% of the asparagines corresponding to GHLYK (amino acids 73-89 of SEQ ID NO: 12) (underlined) are deamidated to form succinimides. In embodiments of the invention, approximately 18, 19, 20, 21, or 22% of the asparagines are deamidated to form aspartates / isoaspartates.

[0251] In an embodiment of the invention, the asparagine 99 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention, e.g., QT NLess than about 5% of the asparagines corresponding to TIIDVVLSPSHGIELSVGEK (amino acids 97-119 of SEQ ID NO: 12) (underlined) are deamidated to form succinimides. In embodiments of the invention, less than about 1% of the asparagines are deamidated to form aspartates / isoaspartates.

[0252] In an embodiment of the invention, the methionine 10 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention is removed, e.g., SDTGRPFVE M Approximately 2% or less of the methionines corresponding to YSEIPEIIHMTEGR (amino acids 1-24 of SEQ ID NO: 12) (underlined) are oxidized.

[0253] In an embodiment of the invention, the methionine 20 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention is substituted with methionine 20, e.g., SDTGRPFVEMYSEIPEIIH M Approximately 3% or less of the methionines corresponding to TEGR (amino acids 1-24 of SEQ ID NO: 12) (underlined) are oxidized.

[0254] In an embodiment of the invention, the methionine 163 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention, e.g., TQSGSE M Approximately 2% or less of the methionines corresponding to K (amino acids 157-164 of SEQ ID NO: 12) (underlined) are oxidized.

[0255] In an embodiment of the invention, the methionine 192 residue in a VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in a composition of the invention is replaced with, for example, SDQGLYTCAASSGL M Approximately 4.3% or fewer of the methionines corresponding to TK (amino acids 178-194 of SEQ ID NO: 12) (underlined) are oxidized.

[0256] In an embodiment of the invention, the C-terminal glycine in the VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in the composition of the invention is about 0. .1%, 0.5%, 1%, 1.5%, or 2% are lost / missing.

[0257] In an embodiment of the invention, the amount of VEGF minitrap (e.g., REGN7483, REGN7850, or REGN7851) in the composition of the invention is Approximately 1.5% of the five arginine residues less than about 0.1% of lysine 62 residues; and / or Less than approximately 0.1% of lysine 185 residues are It is carboxymethylated.

[0258] VEGF mini-traps and compositions comprising VEGF mini-traps having any one or more of the following characteristics also form part of the present invention: Asparagine deamidation, for example, at Asn84 (e.g., about 27%), Asn99 (e.g., about 0.5-1.0%), and / or Asn152 (e.g., about 2.5-3.0%). Asp succinimide isomerization at Asp173 (e.g., about 2%). For example, aspartate-glycine isomerization can be achieved by the L-succinamidyl intermediate. ( [ka] ), which is isomerized to isoaspartate-glycine and / or Asn-glycine. See, e.g., Stephenson & Clarke, Succinimide Formation from Aspartyl and Asparaginyl Peptides as a Model for the Spontaneous Degradation of Proteins, J. Biol. Chem. 264(11):6164-6170 (1989). Methionine oxidation, e.g., to methionine sulfoxide and / or methionine sulfone, e.g., at Met10 (e.g., about 5-6%), Met20 (e.g., about 2%), Met163 (e.g., about 7%), and / or Met192 (e.g., about 6-7%). Trp dioxide, for example at Trp58 (for example, about 0.3%), forming, for example, N-formylkynurenine. · For example, Arg3-deoxyglucosone formation at Arg5 (e.g., about 8.1%). · C-terminal glycine loss (e.g., about 7.2%). Non-glycosylated N-linked glycosylation sites, for example, at Asn36 (e.g., about 1.7%), Asn68 (e.g., about 47.3%), Asn123 (e.g., about 0.2%), and / or Asn196 (e.g., about 0.8%).

[0259] Polynucleotides and methods of production Isolated polynucleotides encoding any of the VEGF mini-trap polypeptides described herein form a part of the invention, as do vectors containing the polynucleotides and / or host cells (e.g., Chinese hamster ovary (CHO) cells) containing the polynucleotides, vectors, VEGF mini-traps, and / or polypeptides described herein. Such host cells also form a part of the invention.

[0260] Polynucleotides include DNA and RNA. The present invention relates to, for example, the VEGF mini-trap polypeptides shown herein (e.g., SEQ ID NOs: 10-13). , 26, 27, 28, 30, 32, or 33). Optionally, the polynucleotide is operably linked to a promoter or other expression control sequence. In embodiments of the invention, the polynucleotide of the invention is fused to a secretory signal sequence. Polypeptides encoded by such polynucleotides are also within the scope of the invention.

[0261] The present invention includes a polynucleotide comprising the following nucleotide sequence encoding a precursor VEGF trap, which can be cleaved, for example with an enzyme, to remove the Fc multimerization component and leave behind a hinge sequence that can be joined to another hinge sequence of a similar molecule to create a homodimeric VEGF minitrap:

[0262] REGN7843-VEGF minitrap-hFc DKTHCPPCPAPELLG [ka]

[0263] REGN7850-VEGF minitrap-hFc DKTHCPPCPPC [ka]

[0264] REGN7851-VEGF minitrap-hFc DKTHCPPCPPCPPC [ka]

[0265] Generally, a "promoter" or "promoter sequence" is a DNA regulatory region capable of binding a cellular RNA polymerase (e.g., directly or via other promoter-binding proteins or substances) and initiating transcription of a coding sequence. A promoter may be operably linked to other expression control sequences, including enhancer and repressor sequences, and / or to a polynucleotide of the invention. Promoters that can be used to control gene expression include, but are not limited to, the cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,385,839 and 5,168,062), the SV40 early promoter region (Benoist et al., (1981) Nature 290:304-310), and the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., (1980) Cell 10:101-104). 22:787-797), herpes thymidine kinase promoter (Wagner et al. (1981) Proc. Natl. Acad. Sci. USA 78:1441-1445), regulatory sequences of the metallothionein gene (Brinster et al. (1982) Nature 296:39-42); beta-lactamase promoter (VIIIa-Komaroff et al. (1978) Proc. Natl. Acad. Sci. USA 75:3727-3731) or tac promoter (DeBoer et al. (1983) Proc. Natl. Acad. Sci. USA 80:21-25); prokaryotic expression vectors such as "Useful proteins from recombinant proteins" in Scientific American (1980) 242:74-94 See also "bacteria"; and promoter elements derived from yeast or other fungi, such as the Gal4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, or the alkaline phosphatase promoter.

[0266] A polynucleotide encoding a polypeptide is "operably linked" to a promoter or other expression control sequence when, in a cell or other expression system, this sequence directs RNA polymerase-mediated transcription of the coding sequence to produce RNA, preferably mRNA, which is then spliced ​​(if introns are contained) and optionally translated into the protein encoded by the coding sequence.

[0267] The present invention includes polynucleotides encoding VEGF mini-trap polypeptide chains whose nucleotide sequences are variants of those specifically set forth herein. A "variant" polynucleotide has at least about 70-99.9% (e.g., 70-99.9%) similarity to a reference nucleotide sequence set forth herein (e.g., any of SEQ ID NOS: 14-16). , 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical; in which case the comparison is performed using the BLAST algorithm, whose parameters are selected to maximize matches between corresponding sequences over the entire length of the corresponding reference sequences (e.g., expectation threshold: 10; word size: 28; maximum matches in query range: 0; match / mismatch score: 1, -2; gap cost: linear). In embodiments of the invention, variants of the nucleotide sequences embodied herein contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) point mutations, insertions (e.g., in-frame insertions), or deletions (e.g., in-frame deletions) of one or more nucleotides compared to any of SEQ ID NOs: 14-16. In embodiments of the invention, such mutations may be missense or nonsense mutations. In embodiments of the invention, such variant polynucleotides encode VEGF mini-trap polypeptide chains that retain specific binding to VEGF.

[0268] Eukaryotic host cells, including mammalian cells, and prokaryotic host cells can be used as hosts for expressing VEGF mini-trap polypeptides. Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). Such host cells include, among others, Chinese hamster ovary (CHO) cells, CHO K1, EESYR, NICE, NS0, Sp2 / 0, embryonic kidney cells, and BHK cells. The present invention includes isolated host cells (e.g., CHO cells or any of the types of host cells described above) containing one or more VEGF mini-trap polypeptides (or variants thereof) and / or polynucleotides encoding such polypeptides (e.g., as discussed herein).

[0269] Transformation can be by any known method for introducing polynucleotides into host cells.Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, gene gun injection of DNA into nuclei, and direct microinjection.In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors.Methods for transforming cells are well known in the art.See, for example, U.S. Patent Nos. 4,399,216; 4,912,040; 4,740,461; and 4,959,455.Therefore, the present invention provides a recombinant method for producing VEGF minitraps, comprising: (i) introducing into a host cell one or more polynucleotides encoding a VEGF mini-trap polypeptide (e.g., comprising any one or more nucleotide sequences of SEQ ID NOS: 14-16; or variants thereof), where the polynucleotides are present in a vector; and / or integrated into a host cell chromosome; and / or operably linked to a promoter; (ii) culturing a host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotide; and (iii) optionally isolating the VEGF mini-trap or a chain thereof from the host cell and / or the medium in which the host cell was grown. The method includes: When producing a VEGF minitrap comprising two or more polypeptide chains, the chains can be co-expressed in a single host cell, e.g., intracellularly or on the cell surface. The VEGF mini-traps are coupled to the associated chains such that homodimeric mini-traps are formed at or outside the cell if such chains are secreted. The present invention also includes VEGF mini-traps that are products of the production methods set forth herein and, optionally, the purification methods set forth herein.

[0270] There are several methods for producing recombinant antibodies known in the art. One example of a method for recombinantly producing antibodies is disclosed in U.S. Patent No. 4,816,567. Recombinant VEGF minitraps (e.g., REGN7483) R , REGN7850, or REGN7851) are part of the present invention.

[0271] The present invention also provides a method for producing a VEGF mini-trap (e.g., a homodimeric VEGF mini-trap) as described herein from a VEGF trap (e.g., aflibercept or conbercept), comprising, consisting of, or consisting essentially of proteolysis of the VEGF trap using a protease that cleaves the VEGF trap at the immunoglobulin Fc multimerization component located below (C-terminal to) the Fc hinge domain. For example, proteolysis can be performed with S. pyogenes IdeS (e.g., FabRICATOR protease; Genovis; Cambridge, Massachusetts; Lund, Sweden) or Streptococcus equi subsp. zooepidemicus IdeZ (New England Biolabs; Ipswich, Massachusetts). In embodiments of the present invention, such methods lack any steps involving significant modification of amino acid residues of such VEGF mini-trap polypeptides (e.g., site-directed chemical modification such as PEGylation or iodoacetamidation) and / or disulfide bridge reduction. The VEGF mini-trap product of such a production method is also part of the invention. For example, in an embodiment of the invention, the Fc domain of the VEGF trap has the following amino acid sequence: [ka] In the sequence, the enzyme cleavage site is indicated by " / / ".

[0272] Such methods for producing the VEGF mini-trap may be followed by methods for purifying the VEGF mini-trap from contaminants, such as, for example, Fc fragments (e.g., SEQ ID NO: 19), proteolytic enzymes, or other substances. See, e.g., FIG. 1. In embodiments of the invention, the purification method is performed under conditions that promote the formation of homodimeric VEGF mini-traps (e.g., non-reducing conditions, e.g., in the absence of reducing agents such as dithiothreitol (DTT) or beta-mercaptoethanol). The VEGF mini-trap products of such production and purification methods are also part of the invention. In embodiments of the invention, purification is performed by methods including chromatographic purification.

[0273] In an embodiment of the invention, the protease-cleavable VEGF trap comprises the following amino acid sequence: [ka] or [ka]

[0274] In an embodiment of the present invention, the VEGF trap is aflibercept (marketed as Eylea) or conbercept. See WO 2000 / 75319 or U.S. Patent No. 9,669,069.

[0275] Combinations and Pharmaceutical Formulations The present invention provides a VEGF mini-trap (e.g., REGN7483) in combination with one or more other components. R ,REGN7483 F The present invention also provides compositions comprising VEGF minitraps (REGN7850, REGN7851, or REGN7852), as well as methods for their use and for making such compositions. Pharmaceutical formulations comprising a VEGF minitrap and a pharmaceutically acceptable carrier or excipient are also part of the invention. In embodiments of the invention, the pharmaceutical formulations of the invention have a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2.

[0276] VEGF minitrap (e.g., REGN7483 R ,REGN7483 F To prepare a pharmaceutical formulation of REGN7850, or REGN7851, the Minitrap is mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984); Hardman et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medication s, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In an embodiment of the invention, the pharmaceutical formulation is sterile. Such compositions are part of the present invention.

[0277] The pharmaceutical formulations of the present invention include VEGF minitraps (e.g., REGN7483 R,REGN7483 F , REGN7850, or REGN7851), and a pharmaceutically acceptable carrier comprising, for example, water, a buffer, a preservative, and / or a surfactant.

[0278] The present invention relates to the VEGF minitraps shown herein (e.g., REGN7483 R ,REGN7483 F , REGN7850, or REGN7851) and a pharmaceutically acceptable carrier, wherein, for example, the concentration of the polypeptide is about 40 mg / ml, about 60 mg / ml, about 80 mg / ml; 90 mg / ml; about 100 mg / ml; about 110 mg / ml, about 120 mg / ml, about 133 mg / ml, about 140 mg / ml, about 150 mg / ml, about 200 mg / ml, or about 250 mg / ml.

[0279] The scope of the present invention includes VEGF minitraps (e.g., REGN7483 R ,REGN7483 F , REGN7850, or REGN7851), or a pharmaceutical formulation thereof that includes a pharmaceutically acceptable carrier but is substantially devoid of water.

[0280] In a further embodiment of the invention, the VEGF minitraps disclosed herein (e.g., REGN7483) R ,REGN7483 F Additional therapeutic agents administered to a subject in conjunction with REGN7850, REGN7851, or REGN7852 are administered to the subject in accordance with the Physicians' Desk Reference 2003 (Thomson Healthcare; 57th Edition (November 1, 2002)).

[0281] The present invention relates to VEGF minitraps (e.g., REGN7483 R ,REGN7483 FThe present invention provides a container (e.g., a plastic or glass vial, e.g., with a cap, or a chromatography column, hollow needle, or syringe barrel) containing a pharmaceutical formulation comprising any of the VEGF minitraps or formulations provided herein. The present invention also provides an injection device, e.g., a syringe, pre-filled syringe, or auto-injector, containing the VEGF minitrap or formulation provided herein. In an embodiment of the present invention, the container is colored (e.g., brown) to block light.

[0282] The present invention provides a method for administering a VEGF minitrap (e.g., REGN7483) in combination with one or more additional therapeutic agents. R ,REGN7483 F , REGN7850, or REGN7851). The VEGF minitrap and the additional therapeutic agent may be a single composition or separate compositions. For example, in embodiments of the present invention, the additional therapeutic agent is an Ang-2 inhibitor (e.g., nesbacumab), a Tie-2 receptor activator, an anti-PDGF antibody or antigen-binding fragment thereof, an anti-PDGF receptor or PDGF receptor beta antibody or antigen-binding fragment thereof, and / or an additional VEGF antagonist such as aflibercept, conbercept, bevacizumab, ranibizumab, an anti-VEGF aptamer such as pegaptanib (e.g., pegaptanib sodium), a single chain (e.g., V) such as brolucizumab, or a combination thereof. L -V H ) Anti-VEGF antibody, abyssin parvovirus DAR These include anti-VEGF DARPins such as Pin, bispecific anti-VEGF antibodies such as RG7716 that also bind to ANG2, or soluble human vascular endothelial growth factor receptor-3 (VEGFR-3) containing extracellular domains 1 to 3 expressed as an Fc fusion protein.

[0283] Administration and Treatment The present invention provides a method for treating or preventing cancer (e.g., cancer whose growth and / or metastasis is mediated, at least in part, by VEGF, e.g., VEGF-mediated angiogenesis) or an angiogenic ocular disorder in a subject, comprising administering a therapeutically effective amount of a VEGF minitrap (e.g., REGN7483). R ,REGN7483 F , REGN7850, or REGN7851) to a subject.

[0284] The phrase "neovascular eye disorder," as used herein, means any eye disease caused by or associated with the growth or proliferation of blood vessels or caused by vascular leakage.

[0285] The term "treat" or "treatment" refers to a therapeutic procedure that reverses, stabilizes, or eliminates an undesirable disease or disorder (e.g., an angiogenic eye disorder or cancer), for example, by causing regression, stabilization, or elimination of one or more symptoms or signs of such disease or disorder to any clinically measurable extent, e.g., with respect to an angiogenic eye disorder, by causing a reduction or maintenance of the Diabetic Retinopathy Severity Score (DRSS), by improving or maintaining visual acuity (e.g., with best corrected vision, as measured by an increase in ETDRS letters), by increasing or maintaining visual field, and / or by reducing or maintaining central retinal thickness, and with respect to cancer, by arresting or reversing the proliferation, survival, and / or metastasis of cancer cells in the subject. Typically, the therapeutic procedure is the administration of one or more doses of a therapeutically effective amount of a VEGF minitrap to a subject with the disease or disorder.

[0286] The present invention also provides VEGF minitraps (e.g., REGN7483) described herein. R ,REGN7483 F, REGN7850, or REGN7851) to a subject (e.g., a human), the method comprising introducing into the body of the subject a VEGF minitrap (e.g., about 100 μl or less, e.g., about 50, 70 μl, or 100 μl, e.g., about 0.5 mg, 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, or 20 mg of the polypeptide) and optionally an additional therapeutic agent, for example, by intraocular injection, such as by intravitreal injection.

[0287] The present invention provides methods for treating cancer (e.g., one whose growth and / or metastasis is mediated, at least in part, by VEGF, e.g., VEGF-mediated angiogenesis) or an angiogenic ocular disorder in a subject in need thereof, comprising administering to the body of the subject, e.g., into the eye of the subject, a therapeutically effective amount of a VEGF minitrap (e.g., about 100 μl or less, e.g., 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg) as provided herein and optionally an additional therapeutic agent. In embodiments of the invention, administration is by intravitreal injection. Non-limiting examples of angiogenic ocular disorders that are treatable or preventable using the methods herein include the following: Age-related macular degeneration (e.g., wet or dry), Macular edema, Macular edema after retinal vein occlusion, Retinal vein occlusion (RVO), Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), ·Diabetic macular edema (DME), ·Choroidal neovascularization (CNV), ·iris angiogenesis, Neovascular glaucoma, Postoperative fibrosis in glaucoma Proliferative vitreoretinopathy (PVR), ·Optic disc neovascularization, ·Corneal neovascularization, ·Retinal neovascularization, Vitreous neovascularization, Pannus, ·pterygia, Vascular retinopathy, Diabetic retinopathy in patients with diabetic macular edema; and Diabetic retinopathy (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy, e.g., in a subject not suffering from DME).

[0288] VEGF minitrap (e.g., REGN7483 R ,REGN7483 F The mode of administration of REGN7850, or REGN7851, or compositions thereof, can be varied, including parenteral, non-parenteral, oral, rectal, transmucosal, intestinal, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, intraocular, intravitreal, transdermal, or intra-arterial.

[0289] The present invention relates to VEGF minitraps (e.g., REGN7483 R ,REGN7483 F

[0013] The present invention provides a method for administering an antigen-binding protein (REGN7850, REGN7851, or REGN7852) to a subject, the method comprising introducing the minitrap or a pharmaceutical formulation thereof into the body of the subject. For example, in embodiments of the invention, the method comprises puncturing the body of the subject with, e.g., a syringe needle, and injecting the antigen-binding protein or a pharmaceutical formulation thereof into the body of the subject, e.g., into the eye, vein, artery, muscle tissue, or subcutaneous tissue of the subject.

[0290] In an embodiment of the present invention, the pharmaceutical formulation of the present invention (VEGF minitrap of the present invention (e.g., REGN7483) R ,REGN7483 FIntravitreal injection of VEGF minitrap (including VEGF minitrap, REGN7850, or REGN7851) includes puncturing the eye with a syringe and needle (e.g., a 30 gauge needle) containing the formulation, and injecting the formulation (e.g., equal to or less than about 100 microliters; about 40, 50, 55, 56, 57, 57.1, 58, 60, or 70 microliters) (e.g., an amount sufficient to deliver a therapeutically effective amount as set forth herein, e.g., about 2, 4, 6, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9, 10, or 20 mg of VEGF minitrap) into the vitreous of the eye. Optionally, the method includes administering a local anesthetic (e.g., proparacaine, lidocaine, or tetracaine), an antibiotic (e.g., a fluoroquinolone), an antiseptic (e.g., povidone-iodine), and / or a pupil dilator to the eye being injected. In embodiments of the invention, a sterile field is established around the eye to be injected prior to injection. In embodiments of the invention, after intravitreal injection, the subject is monitored for elevated intraocular pressure, inflammation, and / or blood pressure.

[0291] The term "in combination with" refers to a component, a VEGF minitrap of the invention (e.g., REGN7483 R ,REGN7483 F , REGN7850, or REGN7851) may be formulated in a single composition with another agent, such as an anti-ANG2 agent, for example, for co-delivery, or may be formulated separately in two or more compositions (e.g., The term "combined" refers to a combination of components (e.g., a kit containing each component). Components administered in conjunction with one another may be administered to a subject at a time different from the time the other components are administered; for example, each administration may be spaced apart over a given period of time (e.g., separately or sequentially) rather than simultaneously. Also, separate components administered in conjunction with one another may be administered essentially simultaneously (e.g., exactly simultaneously or separated by a clinically insignificant period) during the same administration session. Furthermore, separate components administered in conjunction with one another can be administered to a subject by the same route or by different routes.

[0292] VEGF minitraps (e.g., REGN7483) for treating or preventing cancer (e.g., mediated at least in part by angiogenesis) or neovascular ocular disorders. R ,REGN7483 FAn effective or therapeutically effective amount of VEGF mini-trap (REGN7850, REGN7851, or REGN7852) refers to an amount of VEGF mini-trap sufficient to cause regression, stabilization, or elimination of cancer or angiogenic eye disorders by, for example, any clinically measurable degree, one or more symptoms or signs of cancer or angiogenic eye disorders, e.g., with respect to angiogenic eye disorders, by causing a reduction or maintenance of the Diabetic Retinopathy Severity Score (DRSS), by improving or maintaining visual acuity (e.g., with best corrected vision, as measured by an increase in ETDRS letters), by increasing or maintaining the visual field, and / or by reducing or maintaining central retinal thickness, and with respect to cancer, by arresting or reversing the proliferation, survival, and / or metastasis of cancer cells in the subject. In embodiments of the present invention, an effective or therapeutically effective amount of a VEGF mini-trap for treating or preventing angiogenic eye disorders is, for example, about 0.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 7.25 mg, 7.7 mg, 7.9 mg, 8.0 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg in about 100 μl or less. The amount may vary depending on the age and size of the subject to be administered, the target disease, condition, and the route of administration, etc. In certain embodiments, the initial dose may be followed by a second or multiple subsequent doses of VEGF minitrap in an amount that may be approximately the same as, less than, or greater than the initial dose, with the subsequent doses spaced apart by at least 1 to 3 days; at least 1 week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0293] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, in need of prevention and / or treatment of, for example, cancer or angiogenic eye disorder. The subject may have cancer or angiogenic eye disorder, or may be predisposed to developing cancer or angiogenic eye disorder.

[0294] Diagnostic Use Also, the VEGF minitrap of the present invention (e.g., REGN7483) R ,REGN7483 F , REGN7850, or REGN7851) can be used to detect and / or measure VEGF or VEGF-expressing cells in a sample, e.g., for diagnostic purposes. For example, a VEGF mini-trap can be used to diagnose a condition or disease characterized by aberrant expression (e.g., overexpression, underexpression, lack of expression, etc.) of VEGF, e.g., to identify tumor cells and / or VEGF-expressing tissues. An exemplary diagnostic assay for VEGF can include, for example, contacting a sample obtained from a patient with a VEGF mini-trap of the invention, where the VEGF mini-trap The VEGF mini-trap is labeled with a detectable label or reporter molecule. The presence of the labeled VEGF mini-trap in a sample indicates the presence of VEGF in the cells and / or tissue. Alternatively, unlabeled VEGF mini-traps may be used in diagnostic applications in combination with a secondary antibody (which has binding affinity for the VEGF mini-trap) that is itself detectably labeled. The detectable label or reporter molecule may be 3 H, 14 C. 32 P, 35 S, or 125The VEGF minitrap may be a radioisotope such as I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase, or luciferase. The presence of a labeled secondary antibody bound to the VEGF minitrap in a sample indicates the presence of VEGF in the cells and / or tissue. For example, in embodiments of the present invention, such a method includes contacting a sample containing cells and / or tissues to be determined for VEGF expression with the VEGF minitrap, and determining that the cells and / or tissues express VEGF if binding of the VEGF minitrap to the cells and / or tissue is observed.

[0295] Conjugates The present invention provides a VEGF minitrap (e.g., REGN7483) that is conjugated to another moiety, e.g., a therapeutic moiety. R ,REGN7483 F As used herein, the term "conjugate" refers to a VEGF trap or minitrap, or a VEGF minitrap that is chemically or biologically linked to an antibody or antigen-binding fragment thereof, a drug, a radioactive agent, a reporter moiety, an enzyme, a peptide, a protein, or a therapeutic agent.

[0296] In certain embodiments, the therapeutic moiety may be a cytotoxin, a chemotherapeutic agent, an immunosuppressant, or a radioisotope. Cytotoxic agents include any agent that is harmful to cells. Examples of suitable cytotoxic agents and chemotherapeutic agents for forming immunoconjugates are known in the art (see, e.g., WO 2005 / 103081).

[0297] The conjugate of VEGF minitrap linked to cytotoxin can be used therapeutically to treat cancer.When the conjugated minitrap binds to tumor tissue, the cytotoxin is localized in the tumor, causing tumor cell death or causing the cessation of growth and / or metastasis.The method of using such conjugated VEGF minitrap is part of the present invention.

[0298] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with respect to numbers used, but some experimental error and deviation should be accounted for. Any formulations presented in these examples are part of the present invention. [Example]

[0299] Recombinant expression of VEGF minitrap The coding region of the recombinant VEGF minitrap was ligated to a signal sequence and cloned into a mammalian expression vector. It was transfected into Chinese hamster ovary (CHO-K1) cells, and after selection with 400 μg / ml hygromycin for 12 days, stably transfected pools were isolated. The stable CHO cell pools were grown in a chemically defined, protein-free medium and used to produce test proteins. The recombinant polypeptide was secreted from the cells into the growth medium, the cells were depth filtered, and the polypeptide was then chromatographically purified from the growth medium and other contaminants. Sequence of the constituent domains of the VEGF mini-trap Human Flt1 (accession number NP_001153392.1) Human Flk1 (accession number NP_002244.1) Human Fc (IGHG1, accession number P01857-1)

[0300] VEGF mini-trap sequence REGN7483 F(FABricator cleaved homodimer minitrap from aflibercept) hFlt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). hFc(D104-G119) [ka]

[0301] REGN7483 R (Homodimer Minitrap, recombinant) hFlt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). hFc(D104-G119) [ka]

[0302] REGN7850 (VEGF minitrap-hFc DKTHCPPCPPC) hFlt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). hFc(D104-G112).PPC [ka]

[0303] REGN7851 (VEGF minitrap-hFc DKTHCPPCPPCPPC) hFlt1 Ig domain 2 (S129-D231), hFLK1 Ig domain 3 (V226-K327), hFc (D104-C112), PPCPPC [ka]

[0304] REGN6824 (hVEGF minitrap-G4Sx3-hVEGF minitrap-mmH) hFlt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). G4Sx3 linker. Flt1 Ig domain 2 (S129-D231), hFLK1 Ig domain 3 (V226-K327), mycmyc6His [ka]

[0305] REGN7080 (VEGF minitrap-G4Sx6-VEGF minitrap mmH) hFlt1 Ig domain 2 (S129-D231) hFLK1 Ig domain 3 (V226-K327) G4Sx6 linker. Flt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). mycmyc6His [ka]

[0306] REGN7991 (hVEGF minitrap-G4Sx9-hVEGF minitrap) hFlt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327). G4Sx9 linker. Flt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327) [ka]

[0307] REGN7992 (hVEGF minitrap-G4Sx12-hVEGF minitrap) hFlt1 Ig domain 2 (S129-D231) hFLK1 Ig domain 3 (V 226-K327). G4Sx12 linker. Flt1 Ig domain 2 (S129-D231). hFLK1 Ig domain 3 (V226-K327) [ka] [Example]

[0308] Proteolytic cleavage of aflibercept VEGF mini-trap molecule REGN7483 F To generate , immobilized IdeS enzyme (FabRICATOR® obtained from Genovis, Cambridge, MA; Lund, Sweden) was used.

[0309] REGN7483 F A column containing FabRICATOR enzyme was used to generate FabRICATOR. Aflibercept (20 mg in 1.0 mL of cleavage buffer) was then added to the column and incubated on the column at 18°C ​​for 30 minutes. After 30 minutes, the column was washed with cleavage buffer (1.0 mL). The digestion mixture and wash solution were combined.

[0310] The mixture was eluted on an analytical proA column (Applied Biosystems™, POROS™ 20 μM Protein A Cartridge 2.1×30 mm, 0.1 mL) (Cat. No. 2-1001-00). Purification can be performed according to the Applied Biosystems™ protocol for POROS™ 20 μM Protein A Cartridge 2.1×30 mm, 0.1 mL (Cat. No. 2-1001-00). [Example]

[0311] Binding kinetics analysis of VEGF minitraps and VEGF on receptor-capturing surfaces VEGF 165 The ability of various VEGF minitrap molecules to bind to VEGF was assessed by surface plasmon resonance (SPR).

[0312] [Table 6]

[0313] VEGF 165 : [ka] The mmh tag is myc-myc-His6.

[0314] Experimental Procedure. Human VEGF binding to various purified VEGF mini-trap constructs. 165 The equilibrium dissociation constant (K D The binding activity (values) was determined using a real-time surface plasmon resonance biosensor using a Biacore3000 instrument. All binding studies were performed at 25°C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween 20, pH 7.4 (HBS-ET). To capture the VEGF minitrap construct, the Biacore sensor surface was first derivatized with a monoclonal mouse anti-VEGFR1 antibody by amine coupling. Binding studies were performed using a human VEGF reagent, human VEGF 165 (Human VEGF 165 ; SEQ ID NO: 31). Different concentrations of VEGF were prepared in HBS-ET running buffer. 165 Reagent (2nM to 62.5pM; human VEGF 165 2-fold serial dilutions) were used to compare the VEGF minitrap conjugates captured by anti-VEGFR1. The tract surface was infused at a flow rate of 90 μL / min for 1.8 min, while VEGF 165 Dissociation of the reagent-bound VEGF minitrap construct was monitored for 60 min in HBS-ET running buffer. a ) and dissociation (k d The binding-dissociation equilibrium constant (K) was determined by fitting the real-time sensorgram to a 1:1 binding model using Scrubber 2.0c curve fitting software. D ) and dissociation half-life (t 1 / 2 ) from the kinetic rate constant:

number

[0315] Human VEGF binding to different VEGF mini-trap constructs at 25°C 165 The binding kinetic parameters for are shown in Tables 1-2 and 1-3.

[0316] [Table 7]

[0317] [Table 8]

[0318] As shown in this example, certain VEGF minitraps of the present invention exhibited binding affinities for the VEGF molecule comparable to full-length aflibercept. [Example]

[0319] VEGFR1 inhibits VEGF in luciferase bioassays 110 , VEGF 121 and VEGF 165 Evaluation of the ability of VEGF minitraps to block activation by VEGF 110 , VEGF 121 , and VEGF 165 The ability of various VEGF mini-traps to inhibit VEGF-mediated VEGFR1 activation in vitro was assessed.

[0320] [Table 9]

[0321] Experimental procedure cell line Two chimeric receptors incorporating the VEGFR1 extracellular domain fused to the cytoplasmic domain of either IL18Rα or IL18Rβ were used to construct the cell line, HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9. The chimeric receptors were transfected into the cell line with an integrated NFκB-luciferase-IRES-eGFP reporter gene. Upon binding to VEGF, extracellular VEGFR1 dimerizes, triggering interaction between the IL18Rα and IL18Rβ intracellular domains, NFκB signaling, and subsequent luciferase production.

[0322] Assay procedure HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9 Cells were seeded at 10,000 cells / well in 96-well white impermeable plates (Nunc, Cat. No. 136101) in OptiMEM (Invitrogen, Cat. No. 31985) containing 0.5% FBS (Seradigm, Cat. No. 1500-500) and incubated overnight at 37°C in 5% CO. The next day, cells were individually treated with 1:3 serial dilutions of VEGF trap or minitrap proteins ranging from 5000 pM to 0.085 pM, followed by a fixed concentration of 20 pM VEGF. 110 (R&D Systems, Catalog No. 298-VS), VEGF 121 (R&D Systems, Catalog No. 4644-VS), or VEGF 165 Ligand protein (R&D Systems, Cat. No. 293-VE) was added and incubated at 37°C, 5% CO for 6 hours. One-Glo luciferase substrate (Promega, Cat. No. E6130) was then added to the cells, and luminescence was measured using a VICTOR™ x5 multilabel plate reader (PerkinElmer, Model 2030-0050). Data were analyzed using a four-parameter logistic equation for an 11-point response curve using GraphPad Prism software to determine EC50 and IC 50 value was determined.

[0323] Summary of results and conclusions. Throughout the experiments, VEGF 110 , VEGF 121 , and VEGF 165 activates HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ clone V3H9 cells and induces EC 50 The values ​​were approximately 11 to 24 pM, approximately 21 to 44 pM, and approximately 28 to 43 pM, respectively (Figures 3 to 5, Table 4-2, Table 4-4, and Table 4-6).

[0324] Single-stranded minitraps with a (G4S)3 linker (REGN6824) or a (G4S)6 linker (REGN7080) reacted with 20 pM VEGF 110 or 20 pM VEGF 121 In the presence of 50 inhibits VEGFR1 signaling at low levels and also inhibits VEGF 165 was partially blocked in the presence of (Figure 3 and Tables 4-2 and 4-3).

[0325] Longer G4S linkers ((G4S)9 or (G4S) 12 Single-stranded mini-traps (REGN7991 and REGN7992) containing β-glucan (GlcNAc) inhibit VEGFR1 signaling with IC values ​​ranging from approximately 24 to approximately 79 pM. 50 The values ​​improved (Figure 4(A-B) and Table 4-4 and Table 4-5).

[0326] Across experiments, VEGF trap (REGN3; aflibercept) had IC values ​​ranging from approximately 9 to 21 pM. 50 inhibited VEGF isoform signaling at low levels.

[0327] REGN7483, a mini-trap cleaved with FabRICATOR F , REGN7483, a recombinant dimeric mini-trap RThe inhibitory activity of REGN7850 and REGN7851 was compared with that of a VEGF trap (aflibercept). F ,REGN7483 R , REGN7850, and REGN7851 inhibit VEGF 110 , VEGF 121 , and VEGF 165 Inhibits VEGFR1 activation mediated by IC 50 Values ​​are the IC observed for full-length VEGF trap 50 The values ​​were similar to those of the VEGF (Figure 5 and Tables 4-6 and 4-7). 110 , VEGF 121 , and VEGF 165 Inhibition of IL-1-mediated VEGFR1 activation by REGN7483 F Approximately 9-12 pM, REGN7483 R IC of approximately 8-19 pM for REGN7850; approximately 12-30 pM for REGN7850, and approximately 15-27 pM for REGN7851 50 The observed values ​​were:

[0328] Data were collected in three different experiments and are presented below.

[0329] Bioassay Experiment 1 [Table 10]

[0330] [Table 11]

[0331] Bioassay Experiment 2 [Table 12]

[0332] [Table 13]

[0333] Bioassay Experiment 3 [Table 14]

[0334] [Table 15]

[0335] [ka]

[0336] This example demonstrates that certain VEGF mini-traps of the present invention exhibit comparable or better efficacy in blocking VEGF-mediated VEGFR1 activity. [Example]

[0337] Size analysis of the in vitro complex formed between VEGF minitraps and VEGF by size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) The stoichiometry of various VEGF minitrap molecules with VEGF was determined.

[0338] [Table 16]

[0339] Experimental procedure Potency determination by size exclusion chromatography with multi-angle light scattering (SEC-MALS) To understand the stoichiometry of different minitrap-VEGF complexes, a series of solutions containing different molar ratios of minitrap and VEGF protein were prepared as shown in Table 5-3 and incubated overnight at 4°C. The complexes tested were as follows: REGN110-REGN7483 F, REGN110-REGN6824, and REGN110-REGN7080. REGN110, REGN7483 F Control samples containing REGN6824 and REGN7080 alone were similarly prepared. The incubated samples were injected into a SEC-MALS system consisting of a miniDAWN Treos MALS device and an Optilab T-rEX (refractometry) (Wyatt Technology Corporation) coupled to a Superose 12 Inc 10 / 300GI column operated by an AKTA microsystem (GE Healthcare Life Sciences). The column running buffer for all samples was 10 mM phosphate, pH 7.0, 500 mM NaCl. 100 μg of BSA (bovine serum albumin, Thermo Scientific) was injected separately as a known molecular weight standard to calibrate the MALS measurements. Size exclusion chromatography data were evaluated by plotting mAU (absorbance at 280 nm) against retention volume (ml) using Unicorn (version 5.20, GE Healthcare Life Sciences). MALS data were evaluated using ASTRA (version 7.0.0.69, Wyatt Technology) by plotting molar mass against volume (ml) and Rayleigh ratio against volume (ml).

[0340] Summary of results and conclusions. SEC-MALS was performed with different versions of MiniTrap (REGN7483 F The molar mass and elution profile of the complexes formed between the VEGF minitrap proteins (REGN6824, REGN7080) and VEGF (REGN110) were evaluated. Table 5-2 presents the theoretical predicted molar mass (calculated from the peptide sequence without including glycosylation), the observed molar mass, and the oligomeric state of the reagents for the VEGF minitrap proteins and REGN110. Table 5-3 shows the weight-average molar mass observed for each peak in the chromatogram for the analyzed complexes.

[0341] REGN110 eluted as a single peak of approximately 42 kDa, consistent with the disulfide-linked homodimer identified in the literature as the major VEGF species (Figures 6-8, peak 3). The minitrap protein migrated as a monomer with a molar mass of approximately 63 kDa, consistent with its theoretical peptide molar mass of 50-51 kDa, plus an additional approximately 12 kDa contributed by eight N-linked glycosylations (Figures 6-8, peak 2). Because FabRICATOR cleavage does not disrupt the hinge disulfide within the native REGN3 Fc domain, REGN7483 F is predicted to be a disulfide-linked homodimer.

[0342] REGN6824 and REGN7080 formed similar complexes with REGN110 under all conditions tested (Table 5-3; Figures 6 and 7). When the single-chain monomer (REGN6824) was combined with a molar equivalent of the VEGF homodimer (REGN110), a peak corresponding to a molar mass of approximately 215 kDa (Figure 6, peak 1) was observed, suggesting a complex consisting of two REGN6824 molecules bound to two REGN110 homodimers. Similar results were observed with REGN7080. When REGN110 or REGN6824 / REGN7080 were present in excess at different molar ratios, the only complex species observed, along with peaks corresponding to excess VEGF or excess MiniTrap, was a 2:2 complex of approximately 215 kDa.

[0343] On the other hand, when combined with equimolar or excess amounts of REGN110, REGN7483 F showed a complex peak of approximately 99 kDa (Figure 8, peak 1). This peak was a single REGN7483 bound to one REGN110 homodimer. F Consistent with a complex consisting of disulfide-linked homodimers. FIn the presence of REGN7483 (Figure 8, peak 1a), the MALS peak has a molar mass of approximately 70 kDa. F +REGN110 complex with excess REGN7483 F therefore, the observed molar mass is the sum of the complex (99 kDa) and REGN7483 F The values ​​represent the average values ​​between the α-glucan and β-glucan alone (63 kDa).

[0344] [Table 17]

[0345] [Table 18] [Example]

[0346] Intravitreal and systemic administration of VEGF trap and dimeric minitrap in a mouse OIR model Mouse pups were placed in a hyperoxic environment (75% O) on postnatal day 6 (P6; postnatal day 6) and returned to room air (21% O) on P11, which caused pathological neovascularization over the next few days. Pups were administered equimolar doses of: VEGF trap (aflibercept) (0.25 μg / eyeball, n=3), Single-stranded minitrap (REGN7080) (0.125 μg / eyeball, n=3), Dimer Minitrap (REGN7483 F ) (0.125 μg / eyeball, n = 3), or · Control protein, hFc (0.125 μg / eyeball, n = 3); administered intravitreally on P13, or 3 mg / kg control protein, hFc, 3mg / kg Dimer Minitrap (REGN7483 F ), 30mg / kg of dimeric minitrap (REGN7483 F );or 100mg / kg Dimer Minitrap (REGN7483 F ) was administered systemically (intraperitoneally) on P12.

[0347] On P16, eyes were harvested. Retinas were dissected, stained with FITC-labeled Griffornia simplicifolia lectin I (Vector Laboratories), and flat-mounted using Prolong Gold (Invitrogen). To measure the area of ​​abnormalities, flat-mounts were imaged using a Nikon 80i with a 4x objective, and the area of ​​retinal neovascularization was quantified using image analysis software (Adobe Photoshop CC2015 Extended).

[0348] Human Fc control, aflibercept (VEGF trap), two [VEGFR1 (d2 The area of ​​abnormal neovascularization (mm ) in mice administered a single-stranded mini-trap with a (G4S)6 linker between the fusion protein [VEGFR2(d3)-VEGFR2(d3) fusion protein] or a dimer mini-trap, which is a FabRICATOR protease cleavage product of aflibercept. 2 ) was evaluated. The performance of the dimeric minitrap was significantly better than that of the single-stranded minitrap and aflibercept in reducing the area of ​​abnormal neovascularization in the mouse retina. See Figure 9.

[0349] When delivered systemically (ip), the dimeric minitrap did not completely inhibit neovascularization, even at 100 mg / kg, and its efficacy was significantly inferior to that of the VEGF trap (aflibercept). See Figure 10A. Previous data using the VEGF trap (aflibercept) showed near-complete inhibition when delivered systemically (ip) at 6.25 mg / kg in an OIR mouse model. See Figure 10B. This suggests that the dimeric minitrap has a shorter half-life than aflibercept when administered systemically. Such a shorter half-life may result in a better safety profile, as any dimeric minitrap leaking from the intravitreal cavity into the blood is cleared relatively quickly. [Example]

[0350] PPC addition to the C-terminus of REGN112 expressed in EESYR CHO cells In this example, various minitraps were evaluated for their ability to form dimers or monomers.

[0351] Recombinant minitraps encoding REGN112, REGN7850, or REGN7851 were cloned into expression plasmids, transfected into CHO cells, and selected for 12 days with 400 μg / ml hygromycin to isolate stably transfected pools. The stable CHO cell pools were grown in chemically defined, protein-free medium and used to produce test proteins. Prior to purification, aliquots (10 μl) of the minitrap-containing medium were loaded onto 4-20% Novex Trys-Glycine (10-well, 1.0 mm minigel) SDS-PAGE gels in 1x Tris-Glycine SDS running buffer under reducing or non-reducing conditions. Proteins were visualized by staining with Coomassie Blue reagent. Monomeric and dimeric species are marked with arrows.

[0352] Visual inspection of SDS-PAGE gels (Figure ​(Figure11)11) suggested that cells expressing REGN112 secreted approximately half of the protein as preformed dimers and the other half as monomers. Addition of one (REGN7850) or two (REGN7851) PPC motifs to the carboxy terminus of REGN112 improved the production of preformed dimers to nearly 100%. [Example]

[0353] Anion exchange chromatography (AEX) to reduce the color of minitraps The AEX set point was optimized during preliminary multivariate characterization studies (negative mode, pH 8.0, 7.0 mS / cm) but did not achieve adequate evacuation of the browner REGN7483 species. A new AEX set point (pH 8.4, 2.0 mS / cm) was evaluated in bind-and-elute mode on three chromatographic resins to determine whether the new set point could achieve further reduction of the brown-colored REGN7483 species. This set point was found to separate the browner REGN3 species from the less brown REGN3 species during previous REGN3 AEX development using Capto Q resin. For the three AEX separations, this set point was used for REGN7483 on Q Sepharose F. F, POROS50HQ, and Capto Q. In the fourth AEX separation, this set point was evaluated for REGN3 on Capto Q. In the fifth AEX separation, the original set point (pH 8.0, 7.0 mS / cm) was evaluated for REGN7483 as a control to determine if the first four AEX separations could achieve further color reduction.

[0354] Design. Five AEX separations were performed in this study, as detailed in Table 8-1. AEX separations 1-4 were performed using the method detailed in Table 8-3, while AEX separation 5 was performed using the method detailed in Table 8-2. All AEX loads originated from similar bioreactors. A 15.7 mL Capto Q column (20.0 cm bed height, 1.0 cm internal diameter), a 14.1 mL POROS50 HQ column (18.0 cm bed height, 1.0 cm internal diameter), and a 16.5 mL Q Sepharose FF column (21.0 cm bed height, 1.0 cm internal diameter) were installed on an AKTA Avant benchtop liquid chromatography controller for this experiment.

[0355] The AEX load pH was adjusted to a target of ±0.05 pH units using 2 M Tris base or 2 M acetic acid. The AEX load conductivity was adjusted to a target of ±0.1 mS / cm using 5 M sodium chloride or RODI (reverse osmosis deionized water). All pooled samples were analyzed for HMW, color, and yield.

[0356] [Table 19]

[0357] [Table 20]

[0358] [Table 21]

[0359] Results. Five AEX separations were performed to determine the optimum resin and set point that would reduce the color of the AEX pool to an acceptable level. All pools were concentrated to 11 g / L and then analyzed for color using the CIELAB color space (L * , a * and b * variable). CIEL *C * h * See Color Scale, Application Notes, 8(11): pp. 1-4 (Hunter Lab; Reston, VA) (2008), and Objective Color Assessment and Quality Control in the Chemical, Pharmaceutical and Cosmetic Industries, Application Report No. 3.9e from Hach Lange GmbH, pp. 1-28, February 2013. The first four AEX separations (1-4) were intended to be evaluated in bind-and-elute mode, but the majority of the product was present in the load and wash blocks (62-94%), i.e., the column was run in negative or flow-through mode.

[0360] The first three separations (1-3) evaluated Capto Q, POROS 50HQ, and Q Sepharose FF resins at pH 8.4 and 2.0 mS / cm set points using REGN7483 as the load material. All three separations demonstrated yields greater than 80% and pool HMW (high molecular weight species content) less than 3.4%. The POROS 50HQ AEX pool exhibited the lowest yellow color of the AEX pools (b * =2.09), Q Sepharose FF AEX pool (b * =2.22) and Capto Q AEX pool products (b * =2.55) followed.

[0361] The fourth AEX separation (4) was performed using REGN3 as the load material while Capto A pH of 8.4 and a 2.0 mS / cm set point were evaluated for Q. At this set point, 61.9% of the yield was found to be collected during the load and wash periods, and 34.0% during the elution period. This AEX pool exhibited minimal yellow color (b *= 1.44). This AEX condition resulted in the least yellow AEX pool, but an increase in yellowness was observed after cleavage with the FabRICATOR enzyme and subsequent removal of the cleaved Fc portion (b in the pre-cleavage pool). * = 3.52, and b in the cleaved and Fc-depleted pools * =4.17). This means that brown is This is likely due to the higher abundance of REGN7483 in the REGN3 molecule rather than the Fc portion. Therefore, removing the Fc, resulting in a doubling of the molar concentration of REGN7483 at a given concentration (g / L) due to enzymatic cleavage, enhances the color. The expected increase in yellow color (Δb * =+0.65) is added to the color of the REGN3 AEX pool product (b * =1.44+0.65=2.09), and after the FabRICATOR unit operation, the REGN7483 AEX pool product had the least yellow color (b * = 2.09). Additionally, the loading and washing yield of 62% is below the development target (>80%), which represents a less desirable set point for AEX separations.

[0362] The fifth AEX separation (5) evaluated previously optimized set points (pH 8.0 and 7.0 mS / cm) on POROS 50HQ resin using REGN7483 as the load material. This AEX separation showed a yield of over 80% and a pool HMW of less than 3.4%, but it also produced the most yellow pool (b * =3.40).

[0363] [Table 22]

[0364] Conclusions. Five AEX separations were performed to evaluate resins (Capto Q, Q Sepharose FF, and POROS 50HQ) and set points (pH 8.0 and 7.0 mS / cm, pH 8.4 and 2.0 mS / cm). Using REGN7483, AEX separations performed on POROS 50HQ with set points of pH 8.4 and 2.0 mS / cm resulted in AEX pools that were less yellow than Q Sepharose FF and Capto Q AEX pools with identical process and load origins. The fourth AEX separation (load origin REGN3, Capto Q resin, pH 8.4 and 2.0 mS / cm set point) was predicted to have a color comparable to the REGN7483 POROS 50HQ AEX pool after the enzymatic cleavage unit run.

[0365] Finally, the fifth AEX separation (load origin REGN7483, POROS 50HQ resin, pH 8.0 and 7.0 mS / cm set point) yielded the most yellow pool. The excessive yellowness is likely due to the relatively low pH (7.9-8.1) and high conductivity (6.5-7.5 mS / cm). These two factors contribute to the degradation of the CDM-expressed REGN7483. F It has been shown to cause higher levels of yellowing in

[0366] Purification of aflibercept (which has a brownish-yellow color) expressed in CDM by protein A chromatography followed by activated charcoal filtration did not result in a significant decrease in the brownish-yellow color (data not shown). [Example]

[0367] Analysis of color and 2-oxo-histidine in AEX-purified minitraps at higher pH and lower conductivity In various minitrap manufacturing lots, the brownish yellow and oxidized 2-oxo-histidine minitraps (REGN7483 F ) The amount of histidine was evaluated in this example.

[0368] Sample preparation. To identify and quantify 2-oxo-histidine post-translational modifications, reduced and alkylated minitrap (REGN7483) was used. F Trypsin mapping of sample lots (10, 23, and 14) was performed. Aliquots (200 μg) of each drug substance lot were denatured in 0.1 M Tris-HCl, pH 7.5, containing 8.0 M urea, reduced with DTT, and then alkylated with iodoacetamide. The denatured, reduced, and alkylated drug substance was first digested with recombinant Lys-C (rLys-C) (enzyme-to-substrate ratio of 1:100 (w / w)) for 30 minutes at 37°C, diluted with 0.1 M Tris-HCl, pH 7.5, to a final urea concentration of 1.8 M, then digested with trypsin at an enzyme-to-substrate ratio of 1:20 (w / w) for 2 hours at 37°C, and then deglycosylated with PNGase F at an enzyme-to-substrate ratio of 1:5 (w / w) for 1 hour at 37°C. The digestion was stopped by using formic acid (FA) to bring the pH below 2.0.

[0369] Minitrap preparations. A 500-liter bioreactor working volume was used to express aflibercept. The aflibercept-containing cell culture was subjected to three filtration steps (depth, polish, and guard), followed by Protein A affinity capture chromatography (bind-and-elute), and further filtration. This material was then enzymatically cleaved using resin-immobilized Streptococcus pyogenes IdeS protease (FabRICATOR, Genovis; Cambridge, MA; Lund, Sweden) to generate the minitrap and a cleaved Fc fragment by-product. The Fc fragment was removed from the reaction by Protein A affinity capture chromatography (minitrap product in the flow-through fraction), followed by a filtration step (minitrap preparations 162, 29, and 30 only). After viral inactivation by low pH hold and filtration steps, the minitraps were purified by anion exchange (AEX) chromatography (flow-through mode) using the parameters presented in Table 9-1.

[0370] [Table 23]

[0371] The purified material was then further purified by hydrophobic interaction chromatography (resin with phenyl ligands) followed by concentration and diafiltration.

[0372] Localization of peptide fragments responsible for increased absorbance at 350 nm. When tryptic peptide maps were compared for minitrap preparation 10 and the VEGF minitrap (obtained by cleavage of aflibercept produced using a commercially available process (non-CDM)), a PTM was observed on minitrap preparation 10 (likely responsible for the strong color of the minitrap preparation 10 sample) (Figure 31(A), showing the absorbance of peptides eluting from 20.0 to 75 minutes). Peptides with distinct UV peaks are highlighted. A magnified view of the chromatogram showing the absorbance of peptides eluting from 16 to 30 minutes is shown in Figure 31(B). There is a striking difference in UV absorbance between minitrap preparation 10 and the VEGF minitrap (obtained by cleavage of aflibercept produced using a commercially available process (non-CDM)). The peptide with the mutation is TNYLTH * R, IIW * DSR and IIIW * It was DSR ( * (Indicates oxidation of residues). Additionally, a magnified view of the chromatogram showing the absorption of peptides eluted between 30 and 75 minutes is shown in Figure 31(C). Peptides with a striking difference in UV absorption between Minitrap Preparation 10 and the VEGF Minitrap (obtained by cleavage of aflibercept produced using a commercial process (non-CDM)) are DKTH * TCPPCPAPELLG, TELNVGIDFNWEYPSSKH * QHK, EIGLLTCEATVNGH * LYK, and QTNTIIDVVLSPSH * It was GIELSVGEK ( * indicates oxidation of residues). Peptide mapping revealed the identity of peptides that differed significantly in abundance between the VEGF minitraps. The relative abundance of peptides identified from peptide mapping analysis is shown in Table 9-2. The amount of 2-oxo-histidine in minitrap preparation 10 was higher than that of the VEGF minitrap (obtained by cleavage of aflibercept produced using a commercial process (non-CDM)), suggesting that the presence of 2-oxo-histidine may be responsible for the intense tan color.

[0373] [Table 24]

[0374] LC-MS Analysis. Aliquots (20 μg) of rLys-C / tryptic peptides from lots 10, 14, and 23 were removed and analyzed by reversed-phase ultra-performance liquid chromatography (UPLC) using a Waters ACQUITY UPLC CSH C18 column (130 Å, 1.7 μm, 2.1 × 150 mm) with online PDA detection (at wavelengths of 280 nm, 320 nm, and 350 nm) and mass spectrometry analysis. Mobile phase A was 0.1% FA in water, and mobile phase B was 0.1% FA in acetonitrile. After sample injection, a gradient was initiated, held at 0.1% B for 5 min, and then linearly increased to 35% B over 75 min for optimal peptide separation. MS and MS / MS experiments were performed on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer, with higher-energy collisional dissociation (HCD) employed to fragment peptides for MS / MS experiments. Identity assignments were based on the experimentally determined accurate masses of a given peptide in the full MS spectrum and the b and y fragment ions in the corresponding HCD MS / MS spectrum. Once extracted ion chromatograms of the 2-oxo-histidine-containing peptide and the corresponding native peptide were generated, peak areas were integrated to determine the identity of REGN7483. F The site-specific percentage of 2-oxo-his present in the sample was calculated.

[0375] Quantification of 2-oxo-histidine in Minitrap preparations 10, 23, and 14. The color of the various Minitrap preparations (in the flow-through fraction of the AEX column) or the material adsorbed and separated from the AEX column is presented in Table 9-3 below, compared to a European brown-yellow standard (BY). The percentage of 2-oxo-histidine in peptides generated from protease digestion, as measured by mass spectrometry, is also shown.

[0376] [Table 25]

[0377] [ka] [+14]: To convert His to 2-oxo-His, an oxygen atom is added to carbon 2, but two hydrogen atoms are lost (one from carbon 2 and another from nitrogen 3), resulting in a net mass increase of approximately +14 over unmodified histidine.

[0378] [ka] [+32]: Tryptophan dioxide leads to the formation of N-formylkynurenine, which has a net mass increase of approximately +32 over unmodified tryptophan.

[0379] Color analysis of Minitrap products 10, 14, 22, 23, 162, 29, 30, and REGN3. CIEL of Minitrap products * a * b * The color analysis is presented in Table 9-4 below.

[0380] [Table 26]

[0381] Aflibercept (manufactured in a chemically undefined medium) and REGN7483 FA series of experiments were performed to assess the percentage of 2-oxo-histidine (and tryptophan dioxide) in the MiniTrap preparation 10. F The material, as well as the material absorbed from the AEX column, was subjected to protease digestion using trypsin and LysC; and PNGase F. The peptides were then applied to a Waters BEH200, 4.6 cm x 150 mm particle size exclusion (SEC) column. Material corresponding to the absorption peak was retained and analyzed by mass spectrometry to determine its identity. The material absorbed from the AEX column was enriched for the presence of 2-oxo-his and tryptophan dioxide species. Furthermore, the levels of 2-oxo-histidine and tryptophan dioxide were very low. See Figure 23 and Table 9-5.

[0382] These data suggest that 2-oxo-his and tryptophan dioxide species have an affinity for AEX resin and that AEX chromatography in flow-through mode is an effective means of removing these species from REGN7483.

[0383] [Table 27]

[0384] A comparison of the acidic species present in the AEX adsorption isolates (Table 9-5) for minitrap preparation 10 (before any purification procedure, BY1), minitrap preparation 23 (before any purification procedure, ≦BY3), minitrap preparation 14 (before any purification procedure, ≦BY3), acidic fraction 1 from minitrap preparation 10 (obtained after the AEX procedure, has a yellowish tint), acidic fraction 2 from minitrap preparation 10 (obtained after the AEX procedure, has a yellowish tint), and the main fraction from minitrap preparation 10 (obtained after the AEX procedure, clear) is shown in Figure 28.

[0385] Strong cation exchange chromatogram (CEX). This method was employed to identify acidic species and other variants present in cell culture harvest samples.

[0386] Strong cation exchange chromatography was performed on a Dionex ProPac WCX-10 analytical column (Dionex, CA). For the sample, the mobile phases used were 10 mM dibasic sodium phosphate pH 7.5 (mobile phase A) and 10 mM dibasic sodium phosphate, 500 mM sodium chloride pH 5.5 (mobile phase B). A binary gradient (94% A, 6% B: 0–20 min; 84% A, 16% B: 20–22 min; 0% A, 100% B: 22–28 min; 94% A, 6% B: 28–34 min) was used with detection at 280 nm. The peak eluting at an earlier relative retention time than the main peak corresponded to the acidic peak.

[0387] A sample from minitrap preparation 23 (before any purification steps, ≦BY3) was subjected to CEX. Desialylation was applied to the sample to reduce the variant complexity of the minitrap preparation. Subsequently, strong cation exchange (CEX) chromatography was applied to enrich for the desialylated minitrap (dsMT1) variants using a dual salt-pH gradient. The procedure resulted in a total of seven fractions (F1-F7, MC is the method control). The tan variant was observed only in the two most acidic protein variants, fractions 1 and 2. This result was supported by a sample generated from adsorptive separation of AEX, which was used to remove most of the tan variants of MT1 and also contained the acidic variants of MT1 (Figure 29).

[0388] Imaging of capillary isoelectric focusing (iciEF) electropherograms. Fractions F1-7 and MC The distribution of variants in the minitrap (derived from post-CEX minitrap production 23) was further evaluated by iCIEF using an iCE280 analyzer (ProteinSimple) equipped with a fluorocarbon-coated capillary cartridge (100 μm × 5 cm). The ampholyte solution consisted of a mixture of 0.35% methylcellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholyte, 4.2% Pharmalyte 8-10.5 carrier ampholyte, 0.2% pi marker 7.40, and 0.15% pi marker 9.77 in purified water. The anolyte was 80 mM phosphoric acid, and the catholyte was 100 mM sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted in purified water, and CpB was added to each diluted sample at a 1:100 enzyme-to-substrate ratio, followed by incubation at 37°C for 20 minutes. The CpB-treated sample was mixed with an ampholyte solution and then focused by introducing a potential of 1500 V for 1 min, followed by a potential of 3000 V for 10 min. Images of the focused ot-PDLl variants were acquired by directing 280 nm ultraviolet light through the capillary and into the lens of a charge-coupled device digital camera. The images were then analyzed to determine the distribution of the various charge variants (Figure 30). [Example]

[0389] REGN7483 F Photostability test In this example, REGN7483 from minitrap production 14 F The photostability of (discussed above) was determined after exposure to different amounts of cool white light or ultraviolet A light. The color and 2-oxo-histidine content of the light-exposed samples were determined.

[0390] [Table 28]

[0391] [Table 29]

[0392] [Table 30]

[0393] REGN7483 F Exposure to cool white light or UVA light correlated with the appearance of oxidized histidine (2-oxo-his). Two 2-oxo-histidines, a 13.98 Da species ( [ka] ), and a 15.99 Da species ( [ka] ) was observed, with the 13.98 Da species dominating in light-stressed minitrap samples. Evidence suggested that the observed brown-yellow color was dependent on the 13.98 Da species but not the 15.99 Da species. The 15.99 Da species is known to be the product of a copper-metal-catalyzed process. Schoneich, J. Pharm. Biomed Anal. 21:1093-1097 (2000). Spiking the minitrap with copper did not cause an obvious color change (data not shown). However, the 13.98 Da species is the product of a light-driven process. Liu et al., Anal. Chem. 86(10:4940-4948 (2014)). [Example]

[0394] Analysis of post-translational modifications (PTMs) by reduced peptide mapping. Mini Trap (REGN7483 F The glycosylation profile of riboflavin (including riboflavin) and aflibercept, as well as the presence of other post-translational modifications, were evaluated in this example.

[0395] Sample preparation: Reductive alkylation minitrap (REGN7483 FTrypsin mapping of Minitrap production 22 and Eylea drug substance lots was performed to identify and quantify post-translational modifications (e.g., site-specific glycosylation, deamidation, and oxidation). Aliquots (1 mg) of each drug substance were denatured in 6.0 M guanidine hydrochloride, reduced with DTT, and alkylated with iodoacetamide at pH 7.5. The modified, reduced, and alkylated drug substances were then desalted and buffer-exchanged into 0.1 M Tris-HCl using a NAP-5 column, followed by trypsin digestion at a 1:20 (w / w) enzyme-to-substance ratio for 2 hours at 37°C. Digestion was halted by adjusting the pH to less than pH 2.0 using TFA.

[0396] LC-MS Analysis. Aliquots (7.6 μg) of tryptic peptides and glycopeptides from each drug substance lot were separated and analyzed by reversed-phase ultra-performance liquid chromatography (UPLC) using a Waters ACQUITY UPLC BEH130 C18 column (1.7 μm, 2.1 × 150 mm), followed by online mass spectrometry analysis to determine peptide and glycopeptide masses and confirm peptide sequences. Mobile phase A was 0.05% TFA in water, and mobile phase B was 0.045% TFA in acetonitrile. After sample injection, a gradient was initiated, held at 0.1% B for 5 minutes, and then linearly increased to 35% B over 75 minutes for optimal peptide separation. MS and MS / MS experiments were performed on a Thermo Scientific Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer, with higher-energy collisional dissociation (HCD) employed to fragment peptides for MS / MS experiments. Peptide and glycopeptide identity assignments were based on the experimentally determined accurate masses of a given peptide or glycopeptide in the full MS spectrum and the b and y fragment ions in the corresponding HCD MS / MS spectrum. For PTM analysis, extracted ion chromatograms of the PTM-containing peptide and the corresponding native peptide were generated, and peak areas were integrated to determine the mass of REGN7483. F and the site-specific percentage (%) of PTMs present in the Eylea samples were calculated.

[0397] Figure 14(A) shows the REGN7483 FThe glycoforms identified at each asparagine glycosylation site on aflibercept (Eylea commercial lot) are presented. Figure 14(A) shows the structures of the glycan residues (GO-GlcNAc; G1-GlcNAc; G1S-GlcNAc; G0; G1; G1S; G2; G2S; G2S2; G0F; G2F2S; G2F2S2; G1F; G1FS; G2F; G2FS; G2FS2; G3FS; G3FS3; G0-2GlcNAc; Man4; Man4_A1G1; Man4_A1G1S1; Man5; Man5_A1G1; Man5_A1G1S1; Man6; Man6_G0+phosphate; Man6+phosphate, and Man7). The nomenclature applied to the various glycan structures has been standardized. Varki et al., Symbol nomenclature for glycan Representation, Proteomics 9:5398-5399 (2009); Harvey et al., Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds.Proteomics 2009, pp. 9, 3796-3801; Kornfeld et al., The synthesis of Complex-type oligosaccharides II characterization of the processing intermediates in the synthesis of the complex oligosaccharide units of the vesicular stomatitis virus G protein. J Biol Chem. 1978, 253, pp. 7771-7778; Varki et al. (Eds.), Essentials of See Glycobiology, 1st Edn., Cold Spring Harbor Laboratory Press, Plainview, NY 1999; Varki et al. (Eds.), Essentials of Glycobiology, 2nd Edn., Cold Spring Harbor Laboratory Press, Plainview, NY 2009; and Dwek, Glycobiology: Moving into the mainstream. Cell 2009, 137, 1175-1176.

[0398] Figure 14(B) shows the REGN7483 F and post-translational modifications other than glycosylation observed in aflibercept.

[0399] Figure 14(C) shows REGN7483 F (Minitrap Manufacturing Product 10), and REGN7483 R The glycosylation profiles of different lots of EGFR, aflibercept, and REGN7711 are presented. [Example]

[0400] Short-term Mini-Trap vascular permeability The eyes of young New Zealand White rabbits were injected with 80 mM DL-α-aminoadipic acid (DL-AAA) solution (80 mcl). Four months later, vascular permeability was assessed by performing fluorescein angiography. The eyes were divided into six groups with similar baseline vascular permeability areas ( FIG. 15 ). Each group was then treated with a single intravitreal injection of one of the following: Group 1: aflibercept, 500 mcg in 50 mcl, n = 6; Group 2: aflibercept, 2 mg in 50 mcl, n = 6; Group 3: Recombinant (R) Minitrap (REGN7483 R ), 250.5 mcg in 50 mcl (equimolar dose to Group 1), n ​​= 6; Group 4: FabRICATOR (F) cleavage minitrap (REGN7483 F ), 254.4 mcg in 50 mcl (equimolar dose to Group 1), n ​​= 6; Group 5: FabRICATOR (F) cleavage minitrap (REGN7483 F ), 1.4 mg in 50 mcl, n=6; Group 6: 50 mcl placebo buffer, n=6

[0401] Ophthalmologic examinations were performed at baseline and at weeks 1, 2, 3, 4, 5, and 6. Each examination included measurements of intraocular pressure (IOP), red-free light (RF) imaging (to determine vascular morphology), fluorescein angiography (FA; to identify vascular leakage), and optical coherence tomography (OCT; to identify vitreous inflammation). Serum (ADA) and plasma (drug levels) were collected at baseline and at weeks 1, 2, and 4.

[0402] Equimolar doses of aflibercept (500 mcg) and minitrap (250.5 or 254.4 mcg) blocked vascular permeability for similar durations (Figure 16). Higher doses of aflibercept or FabRICATOR-cleaving minitrap (REGN7483) blocked vascular permeability for similar durations. F ) blocked vascular permeability for a longer period of time (Figure 17). FabRICATOR cleavage minitrap and recombinant minitrap (REGN7483 R ) did not cause significant changes in intraocular pressure at the doses tested (Figure 18). This resulted in a significant level of pathological vascular regression (Figure 19). [Example]

[0403] Long-term MiniTrap vascular permeability The eyes of young New Zealand White rabbits were injected with 80 mM DL-α-aminoadipic acid (DL-AAA) solution (80 mcl). After 22 months, vascular permeability was assessed by performing fluorescein angiography. The eyes were divided into three groups with similar baseline vascular permeability areas ( FIG. 20 ). After dividing the eyes, they were treated with a single intravitreal injection of one of the following: Group 1: aflibercept, 500 mcg in 50 mcl, n = 4; Group 2: FabRICATOR cleavage minitrap (REGN7483 F ), 213 mcg / eye in 50 mcl, n=4; Group 3: 50 mcl placebo buffer, n=4

[0404] Ophthalmologic examinations were performed at baseline and at weeks 1, 2, 4, 5, 6, 8, 10, and 14. Each examination included measurements of intraocular pressure (IOP), red-free light (RF) imaging (to determine vascular morphology), fluorescein angiography (FA; to determine vascular leakage), and optical coherence tomography (OCT; to identify vitreous inflammation).

[0405] Aflibercept and FabRICATOR cleavage minitrap (REGN7483 F ) blocked vascular permeability. There was no statistically significant difference in the length of block between aflibercept and MiniTrap treatments (Figure 21). [Example]

[0406] Incubation test of fresh chemically defined medium The effect of various components on color when spiked into fresh chemically defined medium (CDM) containing aflibercept (REGN3) was investigated.

[0407] The operating parameters for the incubation test are: 50 mL vented shaker tube with 10 mL working volume Incubation for 7 days, sampling on days 0 and 7 ·Temperature=35.5℃ Adjust to pH 7.35 using 5N HCl or 5N NaOH CO2=6.9% ·Humidity=75% Stirring = 150rpm Component addition (used as DOE) Aflibercept drug substance is spiked into a shaker tube at a concentration of 6g / L. Matrix = Fresh CDM It was.

[0408] The final concentrations reached by adding the components are listed below: Cysteine: 16.6mM Riboflavin: 0.014mM Folic acid: 0.17mM Vitamin B12: 0.014mM Thiamine: 0.18mM Niacinamide: 0.84mM D-pantothenic acid: 0.62mM D-biotin: 0.002mM Pyridoxine: 0.49mM Iron: 0.22mM ·Copper: 0.0071mM Zinc: 0.54mM

[0409] b when each component is added * Value (CIEL * a * b * The effect of cysteine ​​on the color space (color spectrum) is presented in Figure 24 (A-B). Cysteine ​​caused the greatest increase in color. Iron and zinc produced color when incubated with cysteine. Riboflavin and vitamin B12 had no statistically significant effect on color. [Example]

[0410] b when cysteine ​​and metals were reduced * Evaluating the effect on values. The effect of reducing cysteine ​​and metal concentrations on color upon expression of REGN3 was evaluated. Operating parameters for the cell culture assay were: 2L bioreactor ·Temperature: Approx. 35℃ ·pH about 7 Culture medium = CDM as listed below, containing cysteine, plus Fe, Zn, Cu, Ni, EDTA, and citrate Nutritional feed (control): Day 2 = 20 × base CDF Day 4 = 20 × base CDF Day 6 = 13 x base CDF Day 8 = 13 x base CDF * CDF = Chemically Defined Nutrient Feed It was. The following components were added to the cultures on days 2, 4, 6, and 8 as part of the base CDF (whether 20x or 13x): approximately 1-3 micromoles Fe, approximately 6-19 micromoles Zn, approximately 0.1-0.3 micromoles Cu, approximately 8-24 micromoles EDTA, and approximately 1-3 micromoles citrate per liter of culture.

[0411] The bioreactor experimental conditions were as follows: Dissolved oxygen set point = 20.0%, 40.4% (control), or 60.0% Amount of cysteine ​​added per feed * = approximately 1.2-1.3 mmol per L of culture, 1.6-1.7 mmol per L of culture (control), or 2.0-2.1 mmol per L of culture Metals in the starting CDM * = 0.5x, 1x, or 1.5x CDM level - 1x levels are listed below: Fe = 68-83 micromoles per liter of culture Zn = 6-7 micromoles per liter of culture Cu = 0.1-0.2 micromoles per liter of culture EDTA = 76-95 micromoles per liter of culture Citrate = 45-55 micromoles per liter of culture Ni = 0.5 to 1 micromol per liter of culture * Cysteine ​​is fed to the cultures every other day.

[0412] Decreasing the cysteine ​​level to 1.2-1.3 mmol / L of feed reduced color without significantly affecting titer. Decreasing the metal concentration in the medium to 0.5x reduced color but significantly increased titer. VCC (viable cell concentration), viable cell concentration, ... There was minimal effect on survival, ammonia, or osmolality. * The predicted effects of metal content and cysteine ​​on values ​​are presented in FIG. [Example]

[0413] b * Evaluation of the effects of antioxidants on blood sugar levels The effect of antioxidants (taurine, hypotaurine, thioctic acid, glutathione, glycine, and vitamin C) on color was evaluated when spiked into used CDM containing aflibercept (REGN3). The operating parameters for the incubation study were: 50 mL vented shaker tube with 10 mL working volume Incubation for 7 days, sampling on days 0 and 7 ·Temperature=35.5℃ Adjust to pH 7.35 using 5N HCl or 5N NaOH CO2=6.9% ·Humidity=75% Stirring = 150rpm It was.

[0414] The conditions for adding components to the used CDM were as follows: Aflibercept drug substance (purified aflibercept recombinant protein in an aqueous buffered solution containing 5 mM sodium phosphate, 5 mM sodium citrate, and 100 mM sodium chloride, pH 6.2) spiked into shaker tubes at a concentration of 6 g / L. Matrix = spent medium obtained from the MiniTrap 2L Control Bioreactor. Antioxidants were added to reach the following final concentrations: Taurine = 10mM of culture Hypotaurine = 10mM of culture Glycine = 10 mM of culture Thioctic acid = 0.0024 mM of culture Glutathione, reduced form = 2 mM in culture Choline = 1.43 mM of culture Hydrocortisone = 0.0014 mM of culture Vitamin C (ascorbic acid) = 0.028mM of culture Vitamin E (α-tocopherol) = 0.009 mM of culture

[0415] Several antioxidants reduced color formation in spent media: hypotaurine, taurine, and glycine; thioctic acid; and a combination of vitamin C. Glutathione was b * Increased the value.

[0416] [Table 31]

[0417] b * Value (CIEL * a * b * A summary of the predicted effects of various antioxidants on the color space is presented in Figure 26 (A-B). [Example]

[0418] Color assay linearity The mini-traps from Mini-Trap Preparation 23 were diluted from 154 mg / ml to 3.5 mg / ml, and the color of each dilution was measured using a CIEL * a * b * The observed colors were determined in color space. Table 17-1 presents the observed colors.

[0419] [Table 32]

[0420] The color values ​​of the various dilutions were plotted on a graph, and a linear regression analysis of the points was also performed to compare the concentration and b * The relationship between the two was determined and is expressed by the following equation: b * =0.046+(0.066×concentration(mg / ml)); However, L * is about 97 to 99, and a * was approximately 0.06 to 0.85. See Figure 27. [Example]

[0421] Evaluation of color loss when anion exchange chromatography (AEX) is applied to REGN3 Color was measured using two AEX resins (POROS 50HQ and Q Sepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS / cm, pH 8.00 and 2.50 mS / cm, and pH 7.80 and 4.00 mS / cm). The reduction in taste was evaluated for REGN3.

[0422] In this study, five AEX separations were performed as detailed in Table 8-1 using the AEX protocol detailed in Table 18-2. All AEX loads were derived from pilot bioreactor S504-190828 (REGN3 X0SP filtration pool, CCF38105-L8). A 15.7 mL Q Sepharose Fast Flow column (19.5 cm bed height, 1.0 cm internal diameter) and a 14.1 mL POROS 50HQ column (18.0 cm bed height, 1.0 cm internal diameter) were integrated into an AKTA Avant benchtop liquid chromatography controller for this experiment.

[0423] The AEX load pH was adjusted to a target of ±0.05 pH units using 2 M Tris base or 2 M acetic acid. The AEX load conductivity was adjusted to a target of ±0.1 mS / cm using 5 M sodium chloride or RODI. All pooled samples were analyzed for HMW, color, and yield.

[0424] [Table 33]

[0425] [Table 34]

[0426] Five AEX separations were performed on REGN3 to evaluate the effect of resin (Q Sepharose FF, or POROS 50HQ), and pH and conductivity set points (pH 8.40 and 2.00 mS / cm, pH 8.00 and 2.50 mS / cm, or pH 7.80 and 4.00 mS / cm) on color loss. For POROS 50HQ, yields (64.4, 81.9, and 91.4%) and pool HMW levels (1.02, 1.29, and 1.83%) increased as the set points were changed to lower pH and higher conductivity. When the conductivity changes, the color (b *The color values ​​also increased (1.05, 1.33, and 1.55), suggesting that higher pH levels and lower electrical conductivity achieved the greatest color reduction in AEX separations using POROS 50HQ.

[0427] The column equilibration buffer and the buffer used to formulate REG3 when applied to the column were as follows: 50 mM Tris, pH 8.4 and 2.0 mS / cm, 50 mM Tris, 10 mM acetate, pH 8.0 and 2.5 mS / cm; or 50 mM Tris, 10 mM acetate, 10 mM NaCl, pH 7.8 and 4.0 mS / cm

[0428] For Q Sepharose Fast Flow, the yield (49.5 and 77.7%) and pool HMW levels (0.59 and 1.25%) also increased when the set point was changed to lower pH and higher conductivity. * The color values ​​also increased (0.96 and 1.35), suggesting that higher pH levels and lower electrical conductivity achieved the greatest color reduction in AEX separations using Q Sepharose Fast Flow.

[0429] Additionally, Q Sepharose Fast Flow reduced color compared to POROS 50HQ for both resins at the two set points evaluated. At pH 8.00 and 2.50 mS / cm set points, the POROS 50HQ pool had a b of 1.33. * The Q Sepharose Fast Flow pool had a b value of 0.96. * Similarly, at pH 7.80 and a set point of 4.00 mS / cm, the POROS 50HQ pool had a b value of 1.55. * whereas the Q Sepharose Fast Flow pool had a b of 1.35. * had value.

[0430] [Table 35]

[0431] Color reduction was evaluated for REGN3 on two AEX resins (POROS 50HQ and Q Sepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS / cm, pH 8.00 and 2.50 mS / cm, and pH 7.80 and 4.00 mS / cm). For both resins, color reduction was optimal at higher pH and lower conductivity set points. Additionally, Q Sepharose Fast Flow achieved greater color reduction than POROS 50HQ at the two set points evaluated on both resins (pH 8.00 and 2.50 mS / cm, and pH 7.80 and 4.00 mS / cm). [Example]

[0432] Glycosylation and viability studies on aflibercept products obtained using CDM In this example, the generation of host cell lines expressing aflibercept fusion proteins was carried out using CDM1, CDM2 (commercially obtained), and CDM3 (commercially obtained). One series of experiments was carried out using CDM1, 2, and 3 without additional media components. Another series of experiments was carried out using CDM1-3 in which manganese (manganese chloride trihydrate, Sigma, 3.2 mg / L), galactose (Sigma, 8 g / L), and uridine (Sigma, 6 g / L) were added to the feed to modify the galactosylation profile. Finally, a series of experiments was performed using CDMs 1-3, in which manganese (manganese chloride trihydrate, Sigma, 3.2 mg / L), galactose (Sigma, 8 g / L), and uridine (Sigma, 6 g / L) were added to the feed to modify the galactosylation profile, and dexamethasone (Sigma, 12 mg / L) was added to the feed to modify the sialylation profile of the composition. Harvesting using each of the CDMs was prepared by centrifugation followed by 0.45 μm filtration.

[0433] Samples were purified by ProA before N-glycan analysis.

[0434] Titer measurement Aflibercept titers were measured daily using an Agilent (Santa Clara, Calif.) 1200 series HPLC or equivalent operated at low pH and a step elution gradient with detection at 280 nm. Absolute concentrations were assigned to a reference standard calibration curve.

[0435] Viable Cell Density (VCD) and Cell Viability Values Viable cell density (VCD) and cell viability were measured via trypan blue exclusion using a Nova BioProfile Flex automated cell counter (Nova Biomedical, Waltham, MA). Glucose, lactate, offline pH, dissolved oxygen (DO), pCO2 measurement, and osmolality were measured using a Nova BioProfile Flex (Nova Biomedical, Waltham, MA).

[0436] N-glycan oligosaccharide profiling Approximately 15 μg of Protein A-purified samples from CDM1-3 harvests were prepared for N-glycan analysis using the Waters GlycoWorks protocol with the GlycoWorks Rapid Deglycosylation and GlycoWorks RapiFluor-MS Label kits (Waters part numbers 186008939 and 186008091, respectively). N-glycans were removed from the protein by treating the sample with PNGase-F at 50.5°C for 5 minutes, followed by a 5-minute cooldown at 25°C. The released glycans were labeled with RapiFluor-MS fluorescent dye over a 5-minute reaction at room temperature. Proteins were precipitated by adding acetonitrile to the reaction mixture and pelleted to the bottom of the well by centrifugation at 2,204 × g for 10 minutes. The supernatant containing the labeled glycans was collected and analyzed on a UPLC system using hydrophilic interaction liquid chromatography (Waters BEH amide column) with post-column fluorescence detection. After binding to the column, the labeled glycans were separated and eluted using a binary mobile phase gradient consisting of acetonitrile and aqueous 50 mM ammonium formate (pH 4.4). The labeled glycans were detected using a fluorescence detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm. The relative area percentages (%) of N-glycan peaks in the resulting chromatogram were used to characterize the N-glycan distribution: (1) the percentage of N-glycans...

Claims

1. An isolated VEGF mini-trap having the following domain structure: ((R1D2)-(R2D3)) ａ -(MC)c; wherein one or more histidines of the VEGF mini-trap are oxidized to 2-oxo-histidine; and / or one or more tryptophans are dioxided; and / or one or more of the asparagines are glycosylated, or ((R1D2)-(R2D3)-(R2D4)) ａ -(MC) ｂ 、 ((R1D2)-(R2D3)) ｃ -Linker-((R1D2)-(R2D3)) ｄ ;or ((R1D2)-(R2D3)-(R2D4)) ｅ -Linker-((R1D2)-(R2D3)-(R2D4)) ｆ Including; During the ceremony, R1D2 is VEGFR1 Ig domain 2; R2D3 is VEGFR2 Ig domain 3; R2D4 is VEGFR2 Ig domain 4; MC is a fragment of an immunoglobulin hinge region, or the following amino acid sequence: DKTHTCPPC (SEQ ID NO: 22), DKTHTCPPCPPC (SEQ ID NO: 23), DKTHTCPPCPPPCPPC (SEQ ID NO: 24), h is 1, 2, 3, 4, or 5; DKTHTC(PPC) ｈ (SEQ ID NO: 25), DKTHTCPPCPAPELLG (SEQ ID NO: 6), DKTHTCPLCPAPELLG (SEQ ID NO: 7), DKTHTC (SEQ ID NO: 8), or DKTHTCPLCPAP (SEQ ID NO: 9) a multimerization component that is a polypeptide consisting of the linker is a peptide comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids; Independently, a=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; b=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; c=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; d=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; e=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; and f=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. The isolated VEGF mini-trap, or a composition thereof.

2. (i)(R1D2) １ -(R2D3) １ -(MC) １ ; and, (ii)(R1D2) １ -(R2D3) １ -(R2D4) １ -(MC) １ 2. The VEGF mini-trap or composition thereof of claim 1, wherein the VEGF mini-trap is selected from the group consisting of:

3. VEGF mini-trap or composition thereof, 【Chemistry 1】 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 【Transformation 6】 【Transformation 7】 【Transformation 8】 【Chemistry 9】 【Chemistry 10】 and 【Chemistry 11】 VEGF minitrap or composition thereof, comprising the amino acid sequence set forth in a member selected from the group consisting of:

4. Minitraps have the following domain structure: (i) (R1D2) ａ - (R2D3) ｂ -Linker- (R1D2) ｃ - (R2D3) ｄ ;or (ii) (R1D2) ａ - (R2D3) ｂ - (R2D4) ｃ -Linker- (R1D2) ｄ - (R2D3) ｅ - (R2D4) ｆ and (i) R1D2 domains cooperate; (ii) the R2D3 domains cooperate; and / or (iii) The R2D4 domains cooperate to The domain structure has a secondary structure that forms a VEGF-binding domain. The VEGF mini-trap or composition thereof according to any one of claims 1 to 3.

5. The linker is (Gly ４ Ser) ｎ where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15; or MCs are fragments of immunoglobulin hinge regions that form two, three, or four cysteine ​​bridges with another MC.

5. The VEGF mini-trap or composition thereof of claim 1, 3, or 4.

6. The VEGF mini-trap or composition thereof according to any one of claims 1 to 5, wherein the polypeptide is homodimerized.

7. 7. The VEGF mini-trap or composition thereof according to any one of claims 1 to 6, wherein one or more histidines of the VEGF mini-trap are oxidized to 2-oxo-histidine, and / or one or more tryptophans of the VEGF mini-trap are dioxided, and / or one or more asparagines of the VEGF mini-trap are glycosylated.

8. The VEGF minitrap or composition thereof according to any one of claims 1 to 7, wherein the composition comprises the VEGF minitrap, and 0.1% to 2% of the histidines in the VEGF minitrap are 2-oxo-histidine.

9. one or more carboxymethylated cysteines and 2-oxo-histidines The oligopeptide products of Lys-C and trypsin protease digestion of the VEGF mini-trap comprise: EIGLLTC containing about 0.006-0.013% 2-oxo-histidine ＊ EATVNGH ＊ LYK (amino acids 73-89 of SEQ ID NO: 12); QTNTIIDVVLSPSH containing about 0.019-0.028% 2-oxo-histidine ＊ GIELSVGEK (amino acids 97-119 of SEQ ID NO: 12); TELNVGIDFNWEYPSSKH containing about 0.049-0.085% 2-oxo-histidine ＊ QHK (amino acids 128-148 of SEQ ID NO: 12); DKTH containing about 0.057-0.092% 2-oxo-histidine ＊ T.C. ＊ PPC ＊ PAPELLG (amino acids 206-221 of SEQ ID NO: 12), and / or TNYLTH containing about 0.010-0.022% 2-oxo-histidine ＊ R (amino acids 90-96 of SEQ ID NO:12), and Optionally, an IIW containing about 0.198-0.298% 2-oxo-histidine. ＊ DSR (amino acids 56-61 of SEQ ID NO: 12) and in the sequence, H ＊ is histidine which may be oxidized to 2-oxo-histidine, W ＊ is optionally dioxided tryptophan, C ＊ is an optionally carboxymethylated cysteine, The VEGF mini-trap or composition thereof according to any one of claims 1 to 8, which is a composition comprising a VEGF mini-trap polypeptide.

10. The oligopeptide products of digestion of the VEGF mini-trap containing one or more carboxymethylated cysteines and 2-oxo-histidine with Lys-C and trypsin proteases are: EIGLLTC containing approximately 0.0095% 2-oxo-histidine ＊ EATVNGH ＊ LYK (amino acids 73-89 of SEQ ID NO: 12); QTNTIIDVVLSPSH containing about 0.0235% 2-oxo-histidine ＊ GIELSVGEK (amino acids 97-119 of SEQ ID NO: 12); TELNVGIDFNWEYPSSKH containing approximately 0.067% 2-oxo-histidine ＊ QHK (amino acids 128-148 of SEQ ID NO: 12); DKTH containing approximately 0.0745% 2-oxo-histidine ＊ T.C. ＊ PPC ＊ PAPELLG (amino acids 206-221 of SEQ ID NO: 12), and / or TNYLTH containing approximately 0.016% 2-oxo-histidine ＊ R (amino acids 90-96 of SEQ ID NO:12), and Optionally, IIW containing about 0.248% 2-oxo-histidine ＊ DSR (amino acids 56-61 of SEQ ID NO: 12) and in the sequence, H ＊ is histidine which may be oxidized to 2-oxo-histidine, W ＊ is optionally dioxided tryptophan, C ＊ is an optionally carboxymethylated cysteine, The VEGF mini-trap or composition thereof according to any one of claims 1 to 9, which is a composition comprising the VEGF mini-trap.

11. The VEGF mini-trap or composition thereof according to any one of claims 1 to 10, wherein the one or more tryptophans are dioxided.

12. 2-oxo-histidine has the following chemical formula: 【Chemistry 12】 The VEGF mini-trap or composition thereof according to any one of claims 1 to 11, which is a composition characterized by:

13. (i) a color that is less brownish yellow than the European color standard BY2; (ii) a color that is less brownish yellow than the European color standard BY3; (iii) a color that is less brownish yellow than the European color standard BY4; (iv) a color less brownish yellow than the European color standard BY5; (v) a color that is less brownish yellow than the European color standard BY6; (vi) a color that is less brownish yellow than European color standard BY7; (vi) Colors between European color standards BY2 and BY3; (vii) Colors between European color standards BY2 and BY4; (vii) CIE L ＊ a ＊ b ＊ In color space, L ＊ is about 70 to 99, and a ＊ is about −2 to 0, and b ＊ is about 20 or less color; and / or (viii) CIEL ＊ a ＊ b ＊ In color space, L ＊ is about 98-99, and a ＊ is about −1 to 0, and b ＊ is about 5-10, and the mini-trap concentration is 75-100 mg / ml, characterized by color; Optionally, the concentration of the VEGF mini-trap is about 70-200 mg / ml; or Optionally, the concentration of the VEGF mini-trap is about 70-200 mg / ml, but is characterized by said color when diluted to about 10, 11, 10-11, 80, or 90 mg / ml; The VEGF mini-trap or composition thereof according to any one of claims 1 to 12, which is a composition.

14. The color of the composition was determined by the following formula: 0.046 + (0.066 x concentration of Minitrap (mg / ml)) = b ＊ or b ＊ = (0.11 × concentration of minitrap (mg / ml) − 0.56), and L ＊ The VEGF mini-trap or composition thereof according to any one of claims 1 to 12, wherein a is about 97 to 99 and a is about -0.085 to 0.

06.

15. (i) expressing aflibercept or said VEGF mini-trap in a host cell in a chemically defined liquid medium, wherein said aflibercept or said VEGF mini-trap is secreted from the host cell into the medium; (ii) when aflibercept is expressed, proteolytic cleaving aflibercept to produce a peptide comprising an Fc domain or a fragment thereof and said VEGF mini-trap, and further removing the Fc domain or a fragment thereof from the VEGF mini-trap; (iii) applying the VEGF minitrap to an anion exchange chromatography resin; and (iv) retaining the VEGF mini-trap polypeptide in the chromatographic flow-through fraction. VEGF according to any one of claims 1 to 14, which is a composition which is the product of a process comprising: A minitrap or composition thereof.

16. 16. The VEGF minitrap or composition thereof of claim 15, wherein if the aflibercept is expressed, the method further comprises Protein A purifying the aflibercept prior to the proteolytic cleavage.

17. Anion exchange resins are Strong anion exchange resin; quaternary amine functional groups; -O-CH ２ CHOHCH ２ OCH ２ CHOHCH ２ N ＋ (CH ３ ) ３ a functional group; or Quaternized polyethyleneimine functional groups 17. The VEGF mini-trap or composition of claim 15 or 16, which is a composition comprising:

18. The VEGF minitrap or composition of any one of claims 15 to 17, wherein the proteolytic cleavage is carried out by incubating aflibercept with Streptococcus pyogenes IdeS protease, or a variant thereof comprising one or more point mutations.

19. The VEGF mini-trap is (1) The AEX resin contains quaternized polyethyleneimine functional groups and is equilibrated with a buffer solution of pH 8.30-8.50 having a conductivity of 1.90-2.10 mS / cm; (2) AEX resin is —O—CH ２ CHOHCH ２ OCH ２ CHOHCH ２ N ＋ (CH ３ ) ３ or -N ＋ (CH ３ ) ３ or containing a quaternary amine functional group and equilibrated with a buffer solution of pH 7.90-8.10 with a conductivity of 2.40-2.60 mS / cm; (3) the AEX resin contains quaternized polyethyleneimine functional groups and is equilibrated with a buffer solution of pH 7.90-8.10 having a conductivity of 2.40-2.60 mS / cm; (4) AEX resin is —O—CH ２ CHOHCH ２ OCH ２ CHOHCH ２ N ＋ (CH ３ ) ３ or -N ＋ (CH ３ ) ３ or containing a quaternary amine functional group and equilibrated with a buffer solution of pH 7.70-7.90 with a conductivity of 3.90-4.10 mS / cm; (5) The AEX resin contains quaternized polyethyleneimine functional groups and is equilibrated with a buffer solution of pH 7.70-7.90 having a conductivity of 3.90-4.10 mS / cm; (6) AEX resin is —O—CH ２ CHOHCH ２ OCH ２ CHOHCH ２ N ＋ (CH ３ ) ３ or -N ＋ (CH ３ ) ３ or containing a quaternary amine functional group and equilibrated with a buffer solution of pH 7.70±0.1 with a conductivity of 9.0±0.1 mS / cm; and (7) The AEX resin contains quaternized polyethyleneimine functional groups and is equilibrated with a buffer solution of pH 8.4±0.1 having a conductivity of 2.0±0.1 mS / cm; applied to an anion exchange (AEX) chromatography resin under conditions selected from the group consisting of: The buffer solution for condition (1) contained 50 mM Tris, pH 8.4, and 2.0 mS / cm; The buffer for conditions (2)-(3) contained 50 mM Tris, 10 mM acetate, pH 8.0, and 2.5 mS / cm; The buffer for conditions (4)-(5) contained 50 mM Tris, 10 mM acetate, 10 mM NaCl, pH 7.8, and 4.0 mS / cm; The buffer solution under condition (6) contains 50 mM Tris, 60 mM NaCl, pH 7.7±0.1; and / or The buffer solution for condition (7) contained 50 mM Tris, pH 8.4±0.1; The VEGF mini-trap is in a loading buffer, which is an equilibration buffer, before being applied to the resin; and / or After the VEGF mini-trap is applied to the resin, the resin is washed with the aqueous buffer. The VEGF mini-trap or composition of any one of claims 15 to 18, which is a composition.

20. 20. The VEGF minitrap or composition of any one of claims 15 to 19, wherein the Fc domain or fragment thereof is chromatographically removed from the VEGF minitrap after proteolytic cleavage by applying a composition comprising the Fc domain or fragment and the VEGF minitrap to a Protein A chromatography resin and retaining the VEGF minitrap in the flow-through fraction.

21. 21. The VEGF minitrap or composition of any one of claims 15 to 20, wherein the method further comprises adjusting the pH to about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2, filtration, diafiltration, viral inactivation, Protein A chromatography purification, and / or hydrophobic interaction chromatography purification.

22. The VEGF mini-trap or composition of any one of claims 15 to 20, wherein the method further comprises hydrophobic interaction chromatography purification on a resin containing phenyl functional groups.

23. The VEGF minitrap or composition according to any one of claims 15 to 22, wherein the hydrophobic interaction chromatography purification is carried out in bind-elute mode or flow-through mode.

24. The mini-trap is expressed in a host cell present in a chemically defined medium (CDM) or the mini-trap is the product of a process comprising proteolytic cleavage of aflibercept with the enzyme IdeS, wherein the aflibercept is expressed in a host cell present in a liquid chemically defined medium, the host cell being subjected to the following steps: (i) inducing a host cell - approximately 68 micromoles of Fe per liter of culture; - approximately 6 micromoles of Zn per liter of culture; - approximately 0.1 micromoles of Cu per liter of culture; Approximately 76 micromoles of EDTA per liter of culture; approximately 45 micromoles of citrate per liter of culture, and Approximately 0.5 micromoles of Ni per liter of culture, and optionally Approximately 1.2 mmol of cysteine ​​per liter of culture introducing into a CDM comprising: Every two days, - approximately 1.2 mmol of cysteine ​​per liter of culture; Approximately 1 micromole of Fe per liter of culture, - approximately 6 micromoles of Zn per liter of culture; - approximately 0.1 micromoles of Cu per liter of culture; Approximately 8 micromoles of EDTA per liter of culture, and Approximately 1 micromole of citrate per liter of culture adding to the culture optionally, the CDM comprises thioctic acid, vitamin C, and / or a mixture of hypotaurine, taurine, and glycine; Optionally, the CDM is uridine, manganese, galactose, and / or dexamethasone. Including Tazone, 21. The VEGF mini-trap or composition of any one of claims 15 to 20, which is a composition cultured by the method.

25. one or more asparagines of the VEGF mini-trap are N-glycosylated; one or more serines or threonines of the VEGF minitrap are O-glycosylated; - one or more asparagines of the VEGF mini-trap are deamidated; one or more aspartate-glycine motifs of the VEGF minitrap are converted to isoaspartate-glycine and / or Asn-Gly; one or more methionines of the VEGF mini-trap are oxidized; one or more tryptophans of the VEGF minitrap are converted to N-formylkynurenine; One or more arginines of the VEGF minitrap are converted to Arg3-deoxyglucosone; - The C-terminal glycine of the VEGF minitrap is absent; - one or more non-glycosylated glycosylation sites are present in the VEGF minitrap; - VEGF minitraps contain about 40% to about 50% total fucosylated glycans; - VEGF minitraps contain about 30% to about 55% total sialylated glycans; - VEGF minitrap contains about 6% to about 15% mannose-5; - VEGF minitraps contain about 60% to about 79% galactosylated glycans; - VEGF mini-trap is xylosylated; - VEGF mini-traps are glycosylated on lysines; The VEGF mini-trap contains a cystine with a free thiol group; - VEGF minitrap contains a trisulfide bridge; - VEGF minitrap contains intrachain disulfide bridges; the VEGF minitrap contains disulfide bridges in a parallel orientation; and / or - VEGF mini-traps contain carboxymethylated lysine or arginine; 25. The VEGF mini-trap or composition of any one of claims 1 to 24.

26. The one or more asparagines of the VEGF mini-trap may be G0-GlcNAc glycosylation; G1-GlcNAc glycosylation; G1S-GlcNAc glycosylation; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; G2F2S2 glycosylation; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; G2FS2 glycosylation; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; Man4 glycosylation; Man4_A1G1 glycosylation; Man4_A1G1S1 glycosylation; Man5 glycosylation; Man5_A1G1 glycosylation; Man5_A1G1S1 glycosylation; Man6 glycosylation; Man6_G0+phosphoglycosylation; Man6+ phosphate glycosylation; and / or Man7 glycosylation 26. The VEGF mini-trap or composition of any one of claims 1 to 25, comprising:

27. VEGF mini-traps are Man5 glycosylation at approximately 30-35% of the asparagine 123 residue; - Man5 glycosylation at approximately 25-30% of the asparagine 196 residue; Man 6-phosphate glycosylation on approximately 6-8% of asparagine 36 residues; Man7 glycosylation on approximately 3-4% of asparagine 123 residues; - high mannose glycosylation of approximately 38% of asparagine 123 residues; and / or Approximately 29% of asparagine 196 residues are high-mannose glycosylated 27. The VEGF mini-trap or composition of any one of claims 1 to 26, comprising:

28. 28. The VEGF mini-trap or composition of any one of claims 1 to 27, wherein the composition comprises the VEGF mini-trap at a concentration of about 80, 85, 90, 80-90, 100, 105, 110, 115, 120, 125, 130, or 90-120 mg / ml.

29. the composition is aqueous; MiniTrap was expressed in Chinese hamster ovary cells; the pH of the composition is about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2; and / or Mini traps are approximately 240,000, 600,000, 960,000, 1.2 million, or 2.4 million lux. ＊ white light slightly stronger than the standard; and / or about 40, 100, 160, 200, or 400 W ＊ hour / m ２ are not exposed to ultraviolet A (UVA) light even slightly stronger than VEGF mini-trap or composition according to any one of claims 1 to 28.

30. 30. The VEGF minitrap or composition of any one of claims 1 to 29, wherein the VEGF minitrap is a monomer, homodimer, or multimer.

31. A pharmaceutical formulation comprising the VEGF minitrap or composition of any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

32. An injection device comprising a VEGF mini-trap polypeptide, composition or formulation according to any one of claims 1 to 31.

33. 33. The injection device of claim 32, which is a sterile pre-filled syringe.

34. 32. The VEGF mini-trap polypeptide, composition or formulation of any one of claims 1 to 31 in combination with a further therapeutic agent.

35. An isolated polynucleotide encoding the VEGF minitrap of any one of claims 1 to 7.

36. A vector comprising the polynucleotide of claim 35.

37. A host cell comprising the VEGF minitrap, polynucleotide, and / or vector of any one of claims 1 to 7, 35, or 36.

38. 38. The host cell of claim 37, which is a Chinese hamster ovary cell.

39. 8. A method for producing a VEGF mini-trap according to any one of claims 1 to 7, comprising introducing a polynucleotide encoding the polypeptide into a host cell, culturing the host cell in a medium under conditions in which the polypeptide is expressed, and optionally isolating the polypeptide from the host cell and / or the medium.

40. 40. The method of claim 39, wherein the host cell is a Chinese hamster ovary cell.

41. 41. A VEGF mini-trap, the VEGF mini-trap being the product of claim 39 or 40.

42. 8. A method for producing a VEGF mini-trap according to any one of claims 1 to 7, said method essentially consisting of proteolyzing the VEGF trap with an enzyme that cleaves the immunoglobulin Fc polypeptide after the following sequence: DKTHTCPPCPAPELLG (SEQ ID NO: 20).

43. 43. The method of claim 42, wherein the VEGF trap is aflibercept or conbercept.

44. 44. The method of claim 42 or 43, wherein the enzyme is S. pyogenes IdeS or Streptococcus equi subsp. zooepidemicus IdeZ.

45. A method for administering a VEGF minitrap or composition or pharmaceutical formulation described in any one of claims 1 to 31 and 34 to a subject, said method comprising introducing the VEGF minitrap, composition or formulation, and optionally a further therapeutic agent, into the body of the subject.

46. 46. ​​The method of claim 45, wherein the VEGF mini-trap is administered to the subject's body by intraocular injection.

47. 47. The method of claim 45 or 46, wherein the VEGF mini-trap is administered intraocularly to the subject's body by intravitreal injection.

48. 42. A method for treating a neovascular ocular disorder in a subject in need thereof, comprising intraocular injection into the eye of the subject of a therapeutically effective amount of a VEGF minitrap or a composition or pharmaceutical formulation thereof according to any one of claims 1 to 31, 34 or 41, and optionally a further therapeutic agent.

49. 49. The method of any one of claims 45-48, wherein about 0.5 mg, 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg of VEGF mini-trap is intravitreally injected into the subject's eye.

50. Neovascular eye disorders include: - Age-related macular degeneration (wet), - Age-related macular degeneration (dry), -Macular edema, -Macular edema after retinal vein occlusion, - retinal vein occlusion (RVO), - Central retinal vein occlusion (CRVO), Branch retinal vein occlusion (BRVO), ・Diabetic macular edema (DME), ・Choroidal neovascularization (CNV), ・iris angiogenesis, - neovascular glaucoma, - Postoperative fibrosis in glaucoma, - Proliferative vitreoretinopathy (PVR), ・Optic disc neovascularization, ・Corneal neovascularization, ・Retinal neovascularization, - Vitreous neovascularization, Pannus, ・Pterygium, Vascular retinopathy, - diabetic retinopathy in patients with diabetic macular edema; and ・Diabetic retinopathy 50. The method of claim 48 or 49, wherein

51. 51. The method of any one of claims 45 to 50, wherein the VEGF mini-trap is administered in a volume of about 100 microliters or less.

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