Anti-VEGF protein composition and method for producing the same

Abstract

To provide a method of producing aflibercept using a cell culture medium.SOLUTION: Provided is a method of producing aflibercept. The method comprises: (a) producing a clarified harvest of cells cultured in a chemically defined medium (CDM); (b) binding aflibercept from the clarified harvest using an affinity chromatography column comprising a polypeptide capable of binding to or interacting with the aflibercept. The polypeptide is an antibody, a fusion protein, a ScFv, or a fragment thereof; (c) eluting the aflibercept of step (b) forming an affinity eluate; optionally, (d) subjecting the eluted aflibercept of (c) to a second chromatography capture step; and (e) collecting a flowthrough fraction. The flowthrough fraction has aflibercept.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 13, 2020, is identified as 070816-02301_SL.txt, and is 134,385 bytes in size.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 065,012, filed August 13, 2020, the contents of which are incorporated herein by reference in their entirety.

[0003] Field The present invention relates generally to anti-VEGF compositions and methods for making same. [Background technology]

[0004] background Protein-based biopharmaceutical compositions have emerged as important research products for the treatment of ophthalmic diseases, cancer, autoimmune diseases, infectious diseases, and other diseases and disorders. Biopharmaceuticals are one of the rapidly growing product segments in the pharmaceutical industry.

[0005] A class of cell-derived dimeric mitogens with selectivity for vascular endothelial cells has been identified and named vascular endothelial growth factors (VEGFs).

[0006] Persistent angiogenesis can cause or exacerbate certain diseases, such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, and neovascular glaucoma. Inhibitors of VEGF activity are useful as therapeutic agents for these diseases, as well as other VEGF-induced pathological angiogenesis and vascular permeability conditions, such as tumor angiogenesis. The angiopoietin and vascular endothelial growth factor (VEGF) family members are the only growth factors believed to be primarily specific for vascular endothelial cells.

[0007] 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 leads to macular edema and the accumulation of fluid under the macula, which can cause vision loss. Diabetic macular edema (DME) is another eye disorder with an angiogenic component. DME is the most widely recognized cause of moderate vision loss in patients with diabetes and is a common complication of diabetic retinopathy, a disease that adversely affects the blood vessels of the retina. Clinically significant DME occurs when fluid leaks into the center of the macula, the light-sensitive part of the retina responsible for clear, direct vision. The presence of fluid in the macula can cause severe vision loss or blindness.

[0008] Various VEGF inhibitors, such as the VEGF trap Eylea (aflibercept), have been approved to treat these eye disorders. Summary of the Invention

[0009] overview The present invention relates to anti-VEGF proteins, including aflibercept, a VEGF trap protein, which is a fusion protein. The present invention also relates to a novel anti-VEGF protein, aflibercept MiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise specified). Disclosed herein are methods for making these anti-VEGF proteins, including manufacturing modes that provide an efficient and effective means of producing the proteins of interest. In one aspect, the present invention is directed to the use of a defined medium (CDM) for producing anti-VEGF proteins. In a particular aspect, the CDM of interest is a CDM that, upon use, produces a protein sample that is tan in color and may contain oxidized species. Furthermore, the present application discloses protein variants of aflibercept and VEGF MiniTrap, along with associated manufacturing methods.

[0010] Aflibercept manufacturing The present disclosure describes the use of cell culture medium to produce aflibercept.In one embodiment, cell culture medium is synthetic medium (" CDM ").CDM is a synthetic preparation that does not contain protein and does not use animal-derived components, and therefore it is often used because there is certainty regarding the composition of the medium.In another embodiment, cell culture medium is soybean hydrolysis medium.

[0011] In one embodiment, a method for producing a recombinant protein comprises: (a) providing a genetically modified host cell to express a recombinant protein of interest; (b) culturing the host cell in CDM under suitable conditions for the cell to express the recombinant protein of interest; and (c) recovering a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a specific aspect, the anti-VEGF protein is selected from the group consisting of aflibercept and recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), aflibercept scFv, and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.

[0012] In one aspect of this embodiment, aflibercept is expressed in suitable host cells, non-limiting examples of which include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2 / 0, embryonic kidney cells, and BHK.

[0013] Suitable CDMs include Dulbecco's Modified Eagle's Medium (DME), Ham's Nutrient Mixture, Excell Medium, and IS CHO-CD Medium. Other CDMs known to those skilled in the art are also contemplated within the scope of the present invention. In certain embodiments, a suitable CDM is CDM1B (Regeneron) or Excell Advanced Medium (SAFC).

[0014] In one embodiment, a clarified harvest sample from a CDM culture containing aflibercept is subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure, e.g., using Protein A. In a further aspect, the eluate from the affinity procedure exhibits a particular color. For example, the eluate may exhibit a yellow-brown color. As explained in more detail below, the color may be determined by either (i) the European color standard "BY," which is a qualitative visual inspection, or (ii) a colorimetric assay (CIE L), which is more quantitative than the BY system. * , a * , b * (or CIELAB). In either case, however, color assessment across multiple samples must be normalized to protein concentration to ensure meaningful investigations. For example, see Example 9 below, Protein A eluate has a "b" of approximately 2.52. * " value, which corresponds approximately to a BY value of BY5 (measured in Protein A eluate at 5 g / L protein concentration). If the color of a Protein A eluate is to be compared with another sample, then the comparison should be made at the same protein concentration. b in the CIELAB color space * The b* values ​​are used to describe the color of a sample and range from blue (-) to yellow (+). A higher b* value of a sample compared to another sample indicates a darker yellow-brown color in the sample compared to the other sample.

[0015] In one embodiment, aflibercept is produced from a host cell genetically modified to express aflibercept using CDM. In one aspect, other species or variants of aflibercept are also produced. These variants include aflibercept isoforms containing one or more oxidized amino acid residues, collectively referred to as oxovariants. The clarified recovered sample produced using CDM, containing aflibercept and its oxovariants, can be subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure, for example, using a protein A column. When a sample extracted from the affinity eluate, which may or may not exhibit a yellow-brown color, is analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more oxidized variants of aflibercept can be detected. Certain amino acid residues of modified aflibercept, including but not limited to histidine and / or tryptophan residues, have been shown to be oxidized. In one aspect, the variants can include oxidation of one or more methionine residues and other residues. See below.

[0016] In another embodiment, the variant can comprise oxidation of one or more tryptophan residues to form N-formylkynurenine. In a further embodiment, the variant can comprise oxidation of one or more tryptophan residues to form mono-hydroxyltryptophan. In a particular embodiment, the protein variant can comprise oxidation of one or more tryptophan residues to form di-hydroxyltryptophan. In a particular embodiment, the protein variant can comprise oxidation of one or more tryptophan residues to form tri-hydroxyltryptophan.

[0017] In another embodiment, the variant may contain one or more modifications selected from the group consisting of: for example, deamidation of one or more asparagines; conversion of one or more aspartates to isoaspartate and / or Asn; oxidation of one or more methionines; oxidation of one or more tryptophans to N-formylkynurenine; oxidation of one or more tryptophans to mono-hydroxyltryptophan; oxidation of one or more tryptophans to di-hydroxyltryptophan; oxidation of one or more tryptophans to tri-hydroxyltryptophan; Arg3-deoxyglucosonation of one or more arginines; removal of a C-terminal glycine; and the presence of one or more unglycosylated glycosites.

[0018] In another embodiment, the present invention relates to a method for producing aflibercept. In one aspect, a clarified recovered sample containing aflibercept and its variants is subjected to a capture step, such as protein A affinity chromatography. Following the affinity step, the affinity eluate can be subjected to ion exchange chromatography. The ion exchange chromatography can be either cation exchange chromatography or anion exchange chromatography. Mixed-mode or multimodal chromatography, as described further below, and other chromatographic procedures are also contemplated within the scope of this embodiment. In a particular aspect, the ion exchange chromatography is anion exchange chromatography (AEX). Suitable conditions for using AEX include, but are not limited to, Tris-HCl at a pH of about 8.3 to about 8.6. After equilibration, for example, with Tris-HCl at a pH of about 8.3 to about 8.6, the sample is loaded onto the AEX column. After loading the column, the column can be washed one or more times, for example, with the equilibration buffer. In certain embodiments, the conditions used can promote differential chromatographic behavior of aflibercept and its oxidation variants, such that the majority of the oxo variants are retained on the stationary phase of the AEX column and can be obtained when the column is stripped, while fractions containing aflibercept without significant oxo variants can be collected in the flow-through fractions. See Example 2 and Figure 11 below. Referring to Figure 11 and Example 2, changes in oxo variants can be observed between different manufacturing processes. For example, this change can be illustrated by the data in the "Tryptophan Oxidation Level (%)" section (specifically the "W138(+16)" column). Here, it can be observed that the oxo variant (specifically oxo-tryptophan) changed from about 0.131% in the load sample to about 0.070% in the flow-through sample after AEX chromatography (AEX Separation 2), indicating a decrease in the oxo variant of aflibercept using AEX.

[0019] Ion exchange can be used to reduce or minimize color. In one aspect of this embodiment, the clarified recovered sample is subjected to capture chromatography using, for example, Protein A affinity chromatography. The affinity column is eluted and the specific BY and / or b * The Protein A eluate has a first color having a specific BY and / or b value. Subsequently, the Protein A eluate is subjected to ion exchange chromatography, such as anion exchange chromatography (AEX). The ion exchange column is washed, and the flow-through is collected and analyzed for specific BY and / or b values ​​assigned thereto. * In certain embodiments, the color value of the first color ("BY" or "b") is * " or ") is different from the second color. In a further embodiment, the first color of the Protein A eluate is B Y and / or b * As reflected by the values, the secondary color of the AEX flow-through has a more tan color when compared to the secondary color of the AEX flow-through. Typically, the tan color of the secondary color after AEX is reduced when compared to the primary color of the Protein A eluate. For example, the use of anion exchange results in a tan color after AEX of b * The tan color observed in the Protein A eluate samples decreased from a value of about 3.06 (color 1) to about 0.96 (color 2). See Example 2, Tables 2-3 below.

[0020] In one aspect of this embodiment, the pH of both the equilibration buffer and the wash buffer for the AEX column can be about 8.30 to about 8.60. In another aspect, the conductivity of both the equilibration buffer and the wash buffer for the AEX column can be about 1.50 to about 3.00 mS / cm.

[0021] In one aspect of this embodiment, the equilibration buffer and wash buffer can be about 50 mM Tris-HCl. In one aspect, the strip buffer contains 2 M sodium chloride or 1 N sodium hydroxide, or both (see Table 2-2).

[0022] This embodiment can include one or more additional steps, in no particular order, such as hydrophobic interaction chromatography (HIC), affinity chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and / or ultrafiltration / diafiltration, and other well-known chromatographic steps.

[0023] In one embodiment, the anti-VEGF protein is glycosylated at one or more asparagines as follows: 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 GlcNAc ... 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. In one embodiment, the anti-VEGF protein can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0024] In one embodiment, the glycosylation profile of the anti-VEGF protein 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 (see Example 6). In one embodiment, the anti-VEGF protein has Man5 glycosylation at about 32.4% asparagine 123 residue and / or about 27.1% asparagine 196 residue.

[0025] In one embodiment, the process may further comprise formulating the drug substance using a pharmaceutically acceptable excipient. In one aspect of this embodiment, the pharmaceutically acceptable excipient may be selected from the following: water, buffers, sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. Other excipients known to those of skill in the art are within the scope of this embodiment.

[0026] In one aspect of this embodiment, the formulation may be suitable for administration to a human subject. In particular, administration may be effected by intravitreal injection. In one aspect, the formulation may have about 40 to about 200 mg / mL of the protein of interest.

[0027] The formulations are intended to treat 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 neovascularization, neovascular glaucoma, post-operative fibrosis of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, It may be used as a method of treating or preventing neovascular eye disorders, which may include diabetic retinopathy in subjects with pannus, pterygium, vascular retinopathy, diabetic macular edema, or diabetic retinopathy (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Score (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects not afflicted with DME).

[0028] VEGF MiniTrap Manufacturing This disclosure describes the production of modified versions of aflibercept, in which the Fc portion has been removed or is absent, termed aflibercept MiniTrap or VEGF MiniTrap, which can be produced in cell culture media including synthetic medium (CDM) or soy hydrolyzed medium.

[0029] In one embodiment, MiniTrap is produced using CDM. In one aspect of MiniTrap production, full-length aflibercept is produced using a suitable host and under suitable conditions, and then further processed to enzymatically remove the Fc portion, resulting in MiniTrap. Alternatively, a gene encoding MiniTrap (e.g., a nucleotide sequence encoding aflibercept without its Fc portion) can be produced using a suitable host cell under suitable conditions.

[0030] In one embodiment, the method for producing MiniTrap involves the production of full-length aflibercept fusion protein, followed by the cleavage of Fc region.In one aspect, this method is involved in producing recombinant protein, such as so-called full-length aflibercept fusion protein (see U.S. Patent No. 7,279,159, the entire teaching of which is incorporated herein by reference), and includes the following steps: (a) provide a genetically modified host cell to express full-length aflibercept; (b) culture the host cell in CDM under suitable conditions, so that the cell expresses full-length aflibercept; (c) recover the preparation of full-length aflibercept produced by this cell; and (d) subject full-length aflibercept to specific enzymatic cleavage, which removes the Fc part of fusion protein.In another aspect, the nucleotide sequence encoding aflibercept minus its Fc part is expressed from a suitable host cell under suitable conditions known to those skilled in the art (see U.S. Patent No. 7,279,159).

[0031] In one aspect of this embodiment, aflibercept is expressed in suitable host cells, non-limiting examples of which include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2 / 0, embryonic kidney cells, and BHK.

[0032] Suitable CDMs include Dulbecco's Modified Eagle's Medium (DME), Ham's Nutrient Mixture, EX-CELL Medium (SAFC), and IS CHO-CD Medium (Irvine). Other CDMs known to those skilled in the art are also contemplated within the scope of the present invention. In certain embodiments, a suitable CDM is CDM1B (Regeneron) or Excell Medium (SAFC).

[0033] In one embodiment, during the manufacture of MiniTrap, samples containing the protein of interest (i.e., aflibercept fusion protein and / or MiniTrap) and its variants (including oxo variants) may exhibit a particular color characteristic, i.e., a tan color. For example, eluate samples from the affinity chromatography step may be purified by BY and / or b * The sample may exhibit a particular tan color measured using the system (see Examples 2 and 9, below). Exemplary sources for the "sample" include affinity chromatography, e.g., Protein A; the sample may be obtained from the flow-through fraction of an ion-exchange chromatography procedure; or the sample may be obtained from a strip of an ion-exchange column. Other sources exist from which samples can be analyzed during the manufacturing process, as are well known to those skilled in the art. As noted above and explained in more detail below, color can be assessed using (i) the European color standard "BY," which performs a qualitative visual inspection, or (ii) a colorimetric assay (CIELAB), which is more quantitative than the BY system. In either case, however, color assessments across multiple samples must be normalized, e.g., using protein concentration, to ensure meaningful inter-sample results.

[0034] In one aspect of this embodiment, the full-length aflibercept fusion protein can be subjected to enzymatic treatment ("cleavage") to generate the VEGF MiniTrap using, for example, proteolytic digestion with a protease or an enzymatically active variant thereof. In one aspect of this embodiment, the protease can be the immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS). In another aspect, the protease can be thrombin trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain, pronase, or a protease from Aspergillus saitoi. In one aspect, the protease can be a cysteine ​​protease. In a particular aspect of this embodiment, the protease can be IdeS. In another aspect, the protease can be a variant of IdeS. Non-limiting examples of variants of IdeS are described below, including polypeptides having an amino acid sequence as set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In one embodiment, the protease can be immobilized on agarose or another suitable matrix.

[0035] In one embodiment, the protein of interest (also its variants) is produced using CDM. In a specific embodiment, the protein of interest comprises aflibercept or MiniTrap. The variants comprise one or more oxidized amino acid residues, collectively referred to as oxovariants. Examples of oxidized residues include, but are not limited to, one or more histidine and / or tryptophan residues, and other oxidized residues can also be detected using LC-MS, such as oxidized methionine, as described below. Subsequent chromatography, such as AEX, can be used to isolate these oxovariants from the protein of interest in a given sample, as described herein.

[0036] In one embodiment, the variant may comprise oxidation of one or more tryptophan residues to form N-formylkynurenine. In a further embodiment, the variant may comprise oxidation of one or more tryptophan residues to form mono-hydroxyltryptophan. In a particular embodiment, the protein variant may comprise oxidation of one or more tryptophan residues to form di-hydroxyltryptophan. In a particular embodiment, the protein variant may comprise oxidation of one or more tryptophan residues to form tri-hydroxyltryptophan.

[0037] In another embodiment, the oxo variant can contain one or more modifications selected from the group consisting of: deamidation of one or more asparagine residues; conversion of one or more aspartic acids to isoaspartate and / or Asn; oxidation of one or more methionine residues; oxidation of one or more tryptophan residues to form N-formylkynurenine; oxidation of one or more tryptophan residues to form mono-hydroxyltryptophan; oxidation of one or more tryptophan residues to form di-hydroxyltryptophan; oxidation of one or more tryptophan residues to form tri-hydroxyltryptophan; Arg3-deoxyglucosonation of one or more arginine residues; removal of the C-terminal glycine; and the presence of one or more unglycosylated glycosites.

[0038] In one embodiment, the method for producing MiniTrap protein comprises (a) capturing a full-length aflibercept fusion protein on a first chromatographic platform, and (b) cleaving aflibercept to form a MiniTrap protein, i.e., aflibercept without its Fc domain. In one embodiment, the first chromatographic support comprises an affinity chromatography medium, an ion exchange chromatography medium, or a hydrophobic interaction chromatography medium. In a specific embodiment, the first chromatographic platform comprises an affinity chromatography platform, such as Protein A. In a further embodiment, the protein from capture step (a) is eluted from the first chromatographic platform before cleavage step (b). In yet a further embodiment, cleavage step (b) is followed by a second capture step. In a specific embodiment, this second capture step can be facilitated by affinity chromatography, such as Protein A affinity chromatography. The flow-through from this second capture step (including MiniTrap) has a first color, e.g., tan, and is characterized by a specific BY and / or b *The resulting oxidized MiniTrap protein is measured to have a value of 0.01 (see, e.g., Example 9 below). In addition, LC-MS analysis of this second capture flow-through demonstrates the presence of oxo variants in which one or more residues of MiniTrap are oxidized (see, e.g., Example 9 below).

[0039] In a further embodiment, the second capture flow-through can be subjected to ion exchange chromatography, such as AEX. The AEX column can be washed with a suitable buffer, and the AEX flow-through fraction, essentially containing MiniTrap, can be collected. The AEX flow-through fraction can be used to identify specific BY and / or b * In a further embodiment, the first color (flow-through from the second capture step) and the second color (flow-through from the ion exchange procedure) may have a second color that is a tan color having a value of BY and / or b * In one embodiment, the second color is a different color as measured by either the BY and / or b system after AEX. * When comparing to the first color using either value, a reduction in tan color is demonstrated.

[0040] In another embodiment, the cleavage activity of step (b) can be carried out using a chromatographic column, where the cleavage activity is, for example, an enzymatic activity, attached or immobilized to a column matrix. The column used in step (b) can contain one or more of the proteases mentioned above and described more fully below.

[0041] In one embodiment, the ion exchange chromatography procedure can include an anion exchange (AEX) chromatography medium. In another embodiment, the ion exchange chromatography medium can include a cation exchange (CEX) chromatography medium. Suitable conditions for using AEX include, but are not limited to, Tris-HCl at a pH of about 8.3 to about 8.6. For example, after equilibration using Tris-HCl at a pH of about 8.3 to about 8.6, the sample is loaded onto the AEX column. After loading the column, the column can be washed one or more times, for example, with the equilibration buffer. In certain embodiments, the conditions used can promote differential chromatographic behavior of MiniTrap and its oxo variants using AEX, such that the oxo variants are substantially retained on the AEX column and can be collected when the column is stripped, while the MiniTrap is substantially present in the flow-through fraction (see Example 9, below).

[0042] In one example, samples from different stages of production were analyzed for color and the presence of oxo variants. Referring to Example 9, the affinity flow-through pool (flow-through from the second Protein A affinity step) had a 1b chromatin ratio of about 1.58. * This second affinity flow-through was subjected to AEX. The AEX flow-through had a second affinity of approximately 0.50 (see Table 9-3). * The strip samples were obtained by stripping the AEX column and the third b value of about 6.10 was obtained, which indicates that the yellow-brown color was significantly reduced after using AEX. * Values ​​were observed which indicated that the strip sample had a more tan color when compared to either the load or the flow-through.

[0043] Referring again to Example 9, oxo variant analysis was also performed. The samples analyzed were the affinity flow-through pool (second Protein A affinity eluate), the AEX flow-through, and the AEX strip. Referring to Tables 9-5 and 9-6, variations in oxo variants can be observed between different manufacturing processes. For example, this variation can be illustrated by the data in the "Tryptophan Oxidation Level (%)" section (specifically the "W58(+16)" column). Here, it can be observed that the oxo variant (specifically oxo-tryptophan) went from approximately 0.055% in the load sample to approximately 0.038% in the flow-through sample after AEX chromatography, indicating a decrease in oxo variants after AEX. The AEX strip was analyzed, and the proportion of oxotryptophan species was found to be approximately 0.089%. Comparing this strip value to the load (and flow-through) revealed that the majority of this oxo variant was retained on the AEX column.

[0044] This embodiment can include one or more additional steps, in no particular order, such as hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and / or ultrafiltration / diafiltration.

[0045] One embodiment of the present invention relates to a method for regenerating a chromatography column containing a resin. In one aspect of this embodiment, the resin has an immobilized hydrolytic agent. In yet another aspect of this embodiment, the resin comprises an immobilized protease enzyme. In yet another aspect of this embodiment, the resin is a FabRICATOR® resin or a variant of the resin. In one aspect of this embodiment, the method for regenerating a column containing a resin improves the reaction efficiency of the resin.

[0046] In one aspect of this embodiment, the method for regenerating a column containing a resin comprises incubating the column resin with acetic acid. In one aspect, the concentration of acetic acid used is about 0.1 M to about 2 M. In one aspect, the concentration of acetic acid is about 0.5 M. In one aspect, the resin is incubated for at least about 10 minutes. In another aspect, the resin is incubated for at least about 30 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 50 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 100 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 200 minutes. In yet another aspect of this embodiment, the resin is incubated for at least about 300 minutes.

[0047] Optionally, the column resin is further incubated with guanidine hydrochloride (Gu-HCl). In one embodiment, the column resin is regenerated using Gu-HCl without acetic acid. The concentration of Gu-HCl used is from about 1N to about 10N. In another embodiment, the concentration of Gu-HCl is about 6N. In a further embodiment, the column resin may be incubated with the regenerant (acetic acid, Gu-HCl) for at least about 10 minutes. In yet another embodiment, the resin is incubated for at least about 30 minutes. In yet another embodiment, the resin is incubated for at least about 50 minutes. In yet another embodiment, the resin is incubated for at least about 100 minutes.

[0048] In one embodiment, the column containing the resin is stored in ethanol. In one aspect, the column is stored in ethanol, the percentage of ethanol being about 5% v / v to about 20% v / v. In a particular aspect, the column is stored using 20% ​​v / v ethanol.

[0049] In one embodiment, the process may further comprise formulating the VEGF MiniTrap using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient may be selected from the following: water, buffers, sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. Other excipients known to those skilled in the art are within the scope of this embodiment.

[0050] The formulations of the present invention are suitable for administration to human subjects. In one aspect of this embodiment, administration can be by intravitreal injection. In one aspect, the formulation can have about 40 to about 200 mg / mL of the protein of interest. In a specific aspect, the protein of interest is either aflibercept or aflibercept MiniTrap.

[0051] The formulations are intended to treat 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 neovascularization, neovascular glaucoma, postoperative fibrosis of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, and vitreous neovascularization. The present invention may be used in methods for treating or preventing neovascular eye disorders, which may include diabetic retinopathy in subjects with diabetic macular edema, pannus, pterygium, vascular retinopathy, or diabetic retinopathy (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Score (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects not afflicted with DME).

[0052] IdeS variants The present disclosure describes the use of IdeS (FabRICATOR) (SEQ ID NO: 1) or other polypeptides that are IdeS variants (SEQ ID NOs: 2-16) to produce VEGF MiniTrap. IdeS (SEQ ID NO: 1) contains asparagine residues at positions 87, 130, 182, and / or 274 (denoted "N" in bold and italics in SEQ ID NO: 1 below). * "). The asparagines at these positions can be mutated to amino acids other than asparagine to form IdeS variants (the mutated amino acid(s) are shown as italicized and underlined amino acid(s): TIFF0007744391000001.tif115165TIFF0007744391000002.tif209165TIFF00077443910 00003.tif216165TIFF0007744391000004.tif201165TIFF0007744391000005.tif165165

[0053] In one embodiment, the polypeptide has an isolated amino acid sequence comprising 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:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. 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 aspect, the target protein is a fusion protein. In yet another aspect, the fragments may comprise a Fab fragment and / or an Fc fragment.

[0054] SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16.

[0055] The present disclosure also includes an isolated nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising 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:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In one embodiment, the isolated amino acid sequence comprises at least about 80% sequence identity over the entire length of the isolated amino acid sequence. In another embodiment, the isolated amino acid sequence comprises at least about 90% sequence identity over the entire length of the isolated amino acid sequence. In another embodiment, the isolated amino acid sequence comprises about 100% sequence identity over the entire length of the isolated amino acid sequence. In one embodiment, the polypeptide may be capable of cleaving a target protein into fragments. In a particular embodiment, the target protein is an IgG. In another particular embodiment, the target protein is a fusion protein. In yet another particular embodiment, the fragments may comprise a Fab fragment and / or an Fc fragment.

[0056] The present disclosure also includes a vector comprising a nucleic acid encoding a polypeptide having an isolated amino acid sequence comprising at least 70% sequence identity over its entire length to an isolated amino acid sequence as set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In one embodiment, the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. In one embodiment, the vector is a plasmid. In one embodiment, the isolated amino acid sequence comprises at least about 80% sequence identity over its entire length. In another embodiment, the isolated amino acid sequence comprises at least about 90% sequence identity over its entire length. In another embodiment, the isolated amino acid sequence comprises about 100% sequence identity over its entire length. In one embodiment, the polypeptide may be capable of cleaving a target protein into fragments. In a particular embodiment, the target protein is an IgG. In another embodiment, the target protein is a fusion protein. In yet another embodiment, the fragment may comprise a Fab fragment and / or an Fc fragment.

[0057] In one embodiment, the isolated amino acid sequence may comprise the parent amino acid sequence defined by SEQ ID NO: 1 with the asparagine residues at positions 87, 130, 182, and / or 274 mutated to an amino acid other than asparagine. In one embodiment, the mutations may result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In another embodiment, the mutations may result in a 50% increase in chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In one embodiment, the amino acid may be selected from aspartic acid, leucine, and arginine. In a particular embodiment, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another embodiment, the asparagine residue at position 130 is mutated to an arginine residue. In yet another embodiment, the asparagine residue at position 182 is mutated to a leucine residue. In yet another embodiment, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 87, 130 and 182 are mutated. In yet another embodiment, the asparagine residues at positions 87, 182 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 130, 182 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 87, 130, 182 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 87, 130, 182 and 274 are mutated.

[0058] In a related embodiment, the disclosure includes an isolated nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising the parent amino acid sequence defined by SEQ ID NO: 1, with asparagine residues at positions 87, 130, 182, and / or 274 mutated to an amino acid other than asparagine. See supra. The mutations may result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence.

[0059] In a further related embodiment, the present disclosure includes a vector. The vector comprises a nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence comprising the parent amino acid sequence defined by SEQ ID NO: 1, with asparagine residues at positions 87, 130, 182, and / or 274 mutated to an amino acid other than asparagine. See supra. The mutations can result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In one embodiment, the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. In one embodiment, the vector can be a plasmid.

[0060] Affinity-based production The present disclosure also provides methods for reducing host cell proteins and other unwanted proteins and nucleic acids during production of anti-VEGF proteins using affinity chromatography.

[0061] In one embodiment, a method for producing a recombinant protein includes (a) providing a genetically modified host cell to express a recombinant protein of interest, (b) culturing the host cell under suitable conditions such that the cell expresses the recombinant protein of interest, and (c) recovering a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins.

[0062] In one aspect of this embodiment, the recombinant protein of interest is expressed in a suitable host cell, non-limiting examples of which include, but are not limited to, CHO, CHOK1, EESYR®, NICE®, NS0, Sp2 / 0, embryonic kidney cells, and BHK.

[0063] In one aspect of this embodiment, the recombinant protein of interest is cultured in a CDM. Suitable CDMs include Dulbecco's Modified Eagle's Medium (DME), Ham's Nutrient Mixture, Excell's Medium, and IS CHO-CD Medium, and CDM1B. Other CDMs known to those skilled in the art are also contemplated as being within the scope of the present invention.

[0064] The production preparation may contain at least one contaminant, including one or more host cell proteins in addition to the recombinant protein of interest. The at least one contaminant may be derived from the cell substrate, the cell culture, or a downstream process.

[0065] In one embodiment, the present invention relates to a method for producing an anti-VEGF protein from a biological sample using affinity chromatography. In certain aspects, the methods disclosed herein can be used to separate an anti-VEGF protein, at least in part, from one or more host cell proteins and nucleic acids (e.g., DNA) formed during the culture production process of the anti-VEGF protein.

[0066] In one embodiment, the method may include subjecting a biological sample containing an anti-VEGF protein, along with associated contaminants, to affinity chromatography under suitable conditions. In certain embodiments, the affinity chromatography may include a material capable of selectively or specifically binding ("capturing") the anti-VEGF protein. Non-limiting examples of such chromatographic materials include chromatographic materials comprising proteins such as Protein A and Protein G, capable of binding to the anti-VEGF protein, and chromatographic materials comprising Fc-binding proteins. In certain embodiments, the protein capable of binding to or interacting with the anti-VEGF protein may be an antibody, a fusion protein, or a fragment thereof. Non-limiting examples of such materials capable of selectively or specifically binding to the anti-VEGF protein are described in Example 7.

[0067] In one aspect of this embodiment, the method can include subjecting a biological sample containing an anti-VEGF protein and one or more host cell proteins / contaminants to affinity chromatography under suitable conditions, wherein the stationary phase of the affinity chromatography comprises a protein capable of selectively or specifically binding to the anti-VEGF protein. In certain aspects, the protein can be an antibody, a fusion protein, an scFv, or an antibody fragment. In certain aspects, the protein can be a VEGF. 165 , VEGF 121For example, VEGF may be a form of VEGF from another species, such as rabbit. For example, as exemplified in Table 7-1 and Table 7-10, VEGF may be used as a protein capable of selectively or specifically binding to or interacting with an anti-VEGF protein. 165 The use of these proteins has led to the successful production of MT5 (an anti-VEGF protein), aflibercept, and an anti-VEGF scFv fragment. In another specific embodiment, the protein can be one or more of the proteins having the amino acid sequences set forth in SEQ ID NOs: 73-80. Table 7-1 also discloses the successful production of anti-VEGF protein (MT5) using proteins having the amino acid sequences set forth in SEQ ID NOs: 73-80 as proteins capable of selectively or specifically binding to MT5.

[0068] In one aspect of this embodiment, the method can include subjecting a biological sample containing an anti-VEGF protein and one or more host cell proteins / contaminants to affinity chromatography under suitable conditions, where the stationary phase of the affinity chromatography comprises a protein capable of selectively or specifically binding to or interacting with the anti-VEGF protein, which can be selected from aflibercept, a VEGF MiniTrap, or an anti-VEGF antibody. In certain embodiments, the VEGF MiniTrap can further be derived from a VEGF receptor component, which can be formed by recombinant expression of the VEGF MiniTrap in a host cell. By performing this method, the amount of one or more host cell proteins in a sample can be reduced. For example, Figures 35A and 35B show a significant reduction in all host cell proteins in a sample containing MT5 (an anti-VEGF protein) when five different affinity chromatography columns are used. The five different affinity chromatography columns can be used to detect different proteins capable of selectively or specifically binding to MT5, including: (i) VEGF 165(SEQ ID NO: 72), (ii) mAb1 (murine anti-VEGFR1 mAb human IgG1 where SEQ ID NO: 73 is the heavy chain and SEQ ID NO: 74 is the light chain), (iii) mAb2 (murine anti-VEGFR1 mAb human IgG1 where SEQ ID NO: 75 is the heavy chain and SEQ ID NO: 76 is the light chain), (iv) mAb3 (murine anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO: 77 is the heavy chain and SEQ ID NO: 78 is the light chain), and (v) mAb4 (murine anti-VEGFR1 mAb mouse IgG1 where SEQ ID NO: 79 is the heavy chain and SEQ ID NO: 80 is the light chain). As seen in Figures 35A and 35B, the eluates from each of the affinity-based manufacturing processes had host cell protein reduced from over 7000 ppm to about 25 ppm and about 55 ppm, respectively.

[0069] Suitable conditions for affinity chromatography include, but are not limited to, equilibrating an affinity chromatography column using an equilibration buffer. For example, equilibration using Tris-HCl at a pH of about 8.3 to about 8.6 is followed by loading the affinity chromatography column with a biological sample. After loading the column, the column can be washed one or more times using an equilibration buffer, such as Dulbecco's phosphate-buffered saline (DPBS). Other washes, including washes using different buffers, can be used before eluting the column. Column elution can be affected by the type, pH, and conductivity of the buffer, and other elution conditions known to those skilled in the art can be applied. After elution using one or more elution buffers, such as glycine at a pH of about 2.0 to about 3.0, the eluted fraction can be neutralized by adding a neutralization buffer, such as 1 M Tris at pH 7.5.

[0070] In one aspect of this embodiment, the pH of both the wash buffer and the equilibration buffer can be about 7.0 to about 8.6. In one aspect of this embodiment, the wash buffer can be DPBS. In one aspect, the elution buffer can comprise a 100 mM glycine buffer having a pH of about 2.5. In another aspect, the elution buffer can be a buffer having a pH of about 2.0 to about 3.0. In one aspect, the neutralization buffer can comprise 1 M Tris having a pH of about 7.5.

[0071] In one aspect of this embodiment, the method may further comprise washing the column with a wash buffer. In one aspect of this embodiment, the method may further comprise eluting the column with an elution buffer to obtain an elution fraction. In certain aspects, the amount of host cell protein in the elution fraction is significantly reduced, e.g., by about 70%, about 80%, about 90%, about 95%, about 98%, or about 99%, compared to the amount of host cell protein in the biological sample.

[0072] This embodiment can include one or more additional steps, in no particular order, such as hydrophobic interaction chromatography, affinity-based chromatography, multimodal chromatography, viral inactivation (e.g., using low pH), viral filtration, and / or ultrafiltration / diafiltration.

[0073] In one embodiment, the glycosylation profile of the anti-VEGF protein composition is the following: 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.

[0074] In one aspect of this embodiment, the anti-VEGF protein has Man5 glycosylation at about 32.4% of asparagine 123 residues and / or about 27.1% of asparagine 196 residues. In certain embodiments, the anti-VEGF protein can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0075] In one embodiment, the method may further comprise formulating the drug substance using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient may be selected from the following: water, buffers, sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. Other excipients known to those skilled in the art are within the scope of this embodiment.

[0076] In one aspect of this embodiment, the formulation may be suitable for administration to a human subject. In one aspect of this embodiment, administration may be by intravitreal injection. In one aspect, the formulation may have about 40 to about 200 mg / mL of the protein of interest. In particular aspects, the protein of interest may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0077] The formulations are intended to treat 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 neovascularization, neovascular glaucoma, postoperative fibrosis of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, and vitreous neovascularization. The present invention may be used in methods for treating or preventing neovascular eye disorders, which may include diabetic retinopathy in subjects with diabetic macular edema, pannus, pterygium, vascular retinopathy, or diabetic retinopathy (e.g., non-proliferative diabetic retinopathy (e.g., characterized by a Diabetic Retinopathy Severity Score (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects not afflicted with DME).

[0078] Synthesis of oxo species One embodiment of the present invention relates to one or more methods for synthesizing oxidized protein species using light. In one aspect of this embodiment, the protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is aflibercept. In another aspect, the anti-VEGF protein is VEGF MiniTrap, including recombinant VEGF MiniTrap. In yet another aspect of this embodiment, the anti-VEGF protein is a single-chain variable fragment (scFv).

[0079] In one aspect of this embodiment, the sample contains a protein of interest, such as an aflibercept fusion protein with minimal or no oxo variants. The sample is subjected to photostress to synthesize oxidized species of aflibercept. In a specific aspect, the sample is subjected to photostress using cool white light. In another specific aspect, the sample is subjected to photostress using ultraviolet light.

[0080] In a particular aspect of this embodiment, a sample containing aflibercept or another anti-VEGF protein is exposed to cool white light for about 30 hours to about 300 hours, resulting in about a 1.5 to about a 50-fold increase in modified oligopeptides. These peptides are enzymatically digested and analyzed. DKTH * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), TNYLTH * R (SEQ ID NO: 21), SDTGRPFVEMYSEIPEIIH * MTEGR (SEQ ID NO: 22), VH * EKDK (SEQ ID NO: 23), SDTGRPFVEM * YSEIPEIIHMTEGR (SEQ ID NO: 64), SDTGRPFVEMYSEIPEIIHM* TEGR (SEQ ID NO: 65), TQSGSEM * K (SEQ ID NO: 66), SDQGLYTC * AASSGLM * TK (SEQ ID NO: 67), IIW * DSR (SEQ ID NO: 28), RIIW*DSR (SEQ ID NO: 115), IIW * DSRK (SEQ ID NO: 114), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO: 70) F * LSTLTIDGVTR (SEQ ID NO: 32) and one or more from the group consisting of: * is histidine and is oxidized to 2-oxo-histidine, and C * is a cysteine ​​and is carboxymethylated, and M * is oxidized methionine, and W * is oxidized tryptophan, and Y * is oxidized tyrosine, and F * is oxidized phenylalanine. Digestion can be carried out with the proteases mentioned above, for example with trypsin. The oligopeptides can be analyzed using mass spectrometry.

[0081] In a particular aspect of this embodiment, a sample containing aflibercept or another anti-VEGF protein is exposed to ultraviolet light for about 4 hours to about 40 hours, resulting in about a 1.5 to about 25 fold increase in modified oligopeptide products (obtained upon digestion), wherein the sample is DKTH * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), TNYLTH *R (SEQ ID NO: 21), SDTGRPFVEMYSEIPEIIH * MTEGR (SEQ ID NO: 22), VH * EKDK (SEQ ID NO: 23), SDTGRPFVEM * YSEIPEIIHMTEGR (SEQ ID NO: 64), SDTGRPFVEMYSEIPEIIHM * TEGR (SEQ ID NO: 65), TQSGSEM * K (SEQ ID NO: 66), SDQGLYTC * AASSGLM * TK (SEQ ID NO: 67), IIW * DSR (SEQ ID NO: 28), RIIW*DSR (SEQ ID NO: 115), IIW * DSRK (SEQ ID NO: 114), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO: 70) F * LSTLTIDGVTR (SEQ ID NO: 32) wherein H * is histidine and is oxidized to 2-oxo-histidine, and C * is a cysteine ​​and is carboxymethylated, and M * is oxidized methionine, and W * is oxidized tryptophan, and Y * is oxidized tyrosine, and F * is oxidized phenylalanine. Digestion can be carried out with the proteases mentioned above, for example with trypsin. The oligopeptides can be analyzed using mass spectrometry.

[0082] How to Minimize Tanning The present disclosure provides methods for reducing tan coloration during production of aflibercept, MiniTrap, or equivalents produced in CDM.

[0083] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest in CDM under suitable conditions, followed by recovering a preparation comprising the recombinant protein of interest. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a particular aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in U.S. Patent No. 7,279,159, which is incorporated herein by reference in its entirety), scFv, and other anti-VEGF proteins. In one aspect, the method produces a preparation of the recombinant protein of interest, the color of which can be measured by the European BY method or the CIELAB method (b * ) In addition, the presence of oxo variants can be analyzed using, for example, LC-MS.

[0084] In one aspect of this embodiment, the alleviation conditions include increasing or decreasing the cumulative concentration of one or more medium components, such as amino acids, metals, or antioxidants, including salts and precursors. This corresponds to a reduction in color and protein variants of aflibercept and VEGF MiniTrap. Non-limiting examples of 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. In certain aspects, reducing cysteine ​​can be effective in reducing the yellow-brown color of the preparation. Cysteine ​​concentration can also affect oxovariants.

[0085] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions, and recovering a preparation of the protein of interest produced by the cells, where the suitable conditions are achieved in part by reducing the cumulative concentration of cysteine ​​in the CDM to about 10 mM or less. Examples of suitable media include, but are not limited to, CDM1B, Excell, or equivalents. As used herein, the term "cumulative amount" refers to the total amount of a particular component added to a bioreactor throughout the course of cell culture to form a CDM, including the amount added at the beginning of the culture (CDM on day 0) and the amount of the component added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or inoculum prior to production in the bioreactor (i.e., prior to CDM on day 0) is also included. The cumulative amount is not affected by loss of the component over time during culture (e.g., due to metabolic or chemical degradation). Thus, for example, if a component is added to two cultures at different times (e.g., in one culture, all of the components are added at the beginning and in another culture, the components are added over time), two cultures with the same cumulative amount of the component may have different absolute levels. The cumulative amount is also not affected by synthesizing the component in situ (e.g., by metabolic or chemical conversion) over time during the culture. Thus, two cultures with the same cumulative amount of a given component may have different absolute levels if, for example, the component is synthesized in situ in one of the two cultures during the bioconversion process. The cumulative amount may be expressed in units such as grams or moles of the component.

[0086] As used herein, the term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the start of a production batch, including additions to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the start of a production batch and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onwards is 2 g / L (i.e., 4 grams divided by 2 liters). If, on day 4, an additional liter of liquid not containing component X is added to the bioreactor, the cumulative concentration will remain at 2 g / L. If, on day 5, some amount of liquid is lost from the bioreactor (e.g., due to evaporation), the cumulative concentration will remain at 2 g / L. The cumulative concentration may be expressed in units such as grams / liter or moles / liter, for example.

[0087] In one aspect of this embodiment, the method comprises culturing host cells expressing a recombinant protein of interest in a CDM under suitable conditions, and recovering a preparation of the protein produced by the cells, wherein the suitable conditions are achieved by reducing the ratio of cumulative cysteine ​​concentration to cumulative total amino acid concentration from about 1:10 to 1:29.

[0088] In one embodiment, the method comprises (i) culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions, and (ii) recovering a preparation of the recombinant protein of interest produced by the cells, wherein the suitable conditions are achieved by reducing the cumulative iron concentration in the CDM to less than about 55.0 μM. In one aspect of this embodiment, the preparation obtained by this method exhibits a reduced tan color compared to preparations obtained by methods in which the cumulative iron concentration in the CDM is greater than about 55.0 μM.

[0089] In one embodiment, this method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions.This method further comprises recovering a preparation of the recombinant protein of interest produced by the cells, and the suitable conditions are obtained by reducing the cumulative copper concentration in the CDM to about 0.8 μM or less.In one aspect of this embodiment, the preparation obtained by this method exhibits a reduced yellow-brown color compared to the preparation obtained by a method in which the cumulative copper concentration in the CDM is greater than about 0.8 μM.

[0090] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions, and recovering a preparation of the recombinant protein of interest produced by the cells, wherein the suitable conditions are achieved by reducing the cumulative nickel concentration in the CDM to about 0.40 μM or less. In one aspect of this embodiment, the preparation obtained by this method exhibits a reduced tan color compared to preparations obtained by methods in which the cumulative nickel concentration in the CDM is greater than about 0.40 μM.

[0091] In one embodiment, this method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under suitable conditions.This method further comprises recovering a preparation of the recombinant protein of interest produced by the cells, and the suitable conditions are obtained by reducing the cumulative zinc concentration in the CDM to about 56 μM or less.In one aspect of this embodiment, the preparation obtained by this method exhibits a reduced yellow-brown color compared to the preparation obtained by a method in which the cumulative zinc concentration in the CDM is greater than about 56 μM.

[0092] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions. The method further includes recovering a preparation of the recombinant protein of interest produced by the cells, the suitable conditions being achieved by the presence of an antioxidant in the CDM at a cumulative concentration of about 0.001 mM to about 10 mM for a single antioxidant, or at a cumulative concentration of about 30 mM or less when multiple antioxidants are added to the CDM. In one aspect of this embodiment, the preparation obtained by this method exhibits a reduced tan color compared to a preparation obtained by a method in which no antioxidant is present in the CDM at a cumulative concentration of less than about 0.01 mM or greater than about 100 mM. Non-limiting examples of antioxidants can be taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, vitamin C, vitamin E, a chelating agent, catalase, S-carboxymethyl-L-cysteine, and combinations thereof. Non-limiting examples of chelating agents include aurintricarboxylic acid (ATA), deferoxamine (DFO), EDTA, and citric acid.

[0093] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions. The method further includes recovering a preparation of the recombinant protein of interest produced by the cells, the suitable conditions including a CDM having a cumulative iron concentration in the CDM of less than about 55 μM, a cumulative copper concentration in the CDM of about 0.8 μM or less, a cumulative nickel concentration in the CDM of about 0.40 μM or less, a cumulative zinc concentration in the CDM of about 56 μM or less, a cumulative cysteine ​​concentration in the CDM of less than 10 mM, and / or a concentration of antioxidants in the CDM of about 0.001 mM to about 10 mM for a single antioxidant, and a cumulative concentration of about 30 mM or less if multiple antioxidants are added to the CDM.

[0094] In one aspect of this embodiment, the preparation obtained by using suitable conditions results in a reduction of aflibercept and VEGF MiniTrap protein variants to a desired amount of aflibercept and VEGF MiniTrap protein variants (referred to as a "target value" for aflibercept and VEGF MiniTrap protein variants). In a further aspect of this embodiment, the preparation obtained by using suitable conditions results in a reduction of aflibercept and VEGF MiniTrap protein variants to a desired level when the preparation of aflibercept and VEGF MiniTrap protein variants is normalized to a concentration of 5 g / L or 10 g / L. * value or BY value (respectively "target b * In a further aspect of this embodiment, the target b * The value (or target BY value) and / or target value of the variant is obtainable in a preparation if the titer is increased or not significantly decreased.

[0095] [The present invention 1001] 1. A method for producing aflibercept, comprising: (a) producing a clarified harvest of cells cultured in a defined medium (CDM); (b) binding aflibercept from the clarified harvest using an affinity chromatography column comprising a polypeptide capable of binding to or interacting with aflibercept, wherein the polypeptide is an antibody, a fusion protein, an ScFv, or a fragment thereof; (c) eluting the aflibercept of step (b), forming an affinity eluate; Optionally, (d) subjecting the eluted aflibercept of (c) to a second chromatographic capture step; (e) collecting the flow-through fraction, wherein the flow-through fraction comprises aflibercept; A method comprising: [The present invention 1002] 1001. The method of claim 1001, wherein the polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80. [The present invention 1003] 1001. The method of claim 1001, further comprising the step of (b) equilibrating said affinity chromatography column using an equilibration buffer. [The present invention 1004] 1004. The method of claim 1003, wherein said equilibration buffer is Dulbecco's phosphate buffered saline or Tris-HCl. [The present invention 1005] 1005. The method of claim 1004, wherein said equilibration buffer has a pH of about 8.3 to about 8.6. [The present invention 1006] 1001. The method of claim 1001, further comprising the step of washing the column (d) with an equilibration buffer to obtain one or more flow-through fractions. [The present invention 1007] 1006. The method of claim 10, wherein said equilibration buffer is Dulbecco's phosphate buffered saline and has a pH of about 7.0 to about 8.6. [The present invention 1008] 1001. The method of claim 1001, further comprising the step of subjecting the column of (b) to an elution buffer to obtain one or more eluted fractions. [The present invention 1009] 1008. The method of claim 10, wherein said elution buffer comprises a 100 mM glycine buffer having a pH of about 2.5. [The present invention 1010] 1008. The method of claim 10, wherein the pH of said elution buffer is about 2.0 to about 3.5. [The present invention 1011] 1008. The method of claim 8, further comprising the step of neutralizing said elution fraction by the addition of a neutralization buffer. [The present invention 1012] 1011. The method of claim 1011, wherein said neutralization buffer is Tris-HCl. [The present invention 1013] The method of claim 1001, wherein the amount of host cell protein in (c) is significantly reduced by about 90%, about 95%, about 98%, or about 99% compared to the amount of host cell protein in the clarified harvest. [The present invention 1014] 1001. The method of claim 1001, wherein said second capture chromatography in (d) comprises anion exchange chromatography (AEX). [The present invention 1015] The method of claim 10, wherein the conductivity of both the equilibration buffer and the wash buffer for the AEX column can be from about 1.50 to about 3.0 mS / cm. [The present invention 1016] 1014. The method of claim 1014, wherein the aflibercept from said one or more flow-through fractions of (e) comprises less than 20% total acidic species of aflibercept, wherein the acidic species correspond to a peak eluting earlier than the main peak in a cation exchange chromatography (CEX) chromatogram of aflibercept, wherein the chromatogram is generated using a first mobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 5.7) and a second mobile phase of 40 mM sodium phosphate, 100 mM sodium chloride (pH 9.0) (mobile phase B), and wherein the chromatogram is generated using detection at 280 nm. [The present invention 1017] 1. A method for producing aflibercept, comprising: (a) providing a host cell genetically modified to express aflibercept; (b) culturing the host cells under conditions suitable for expression of the aflibercept; (c) recovering a preparation comprising aflibercept and at least one impurity produced by the host cell; (d) subjecting the preparation to affinity chromatography under suitable conditions, wherein the affinity chromatography comprises a polypeptide capable of binding to or interacting with the aflibercept; A method comprising: [The present invention 1018] 1017. The method of claim 1017, wherein said polypeptide capable of binding to aflibercept is an antibody, a fusion protein, an ScFv, or a fragment thereof. [The present invention 1019] 1017. The method of claim 1017, wherein the polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80. [The present invention 1020] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 72. [The present invention 1021] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 73. [The present invention 1022] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 74. [The present invention 1023] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 75. [The present invention 1024] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 76. [The present invention 1025] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 77. [The present invention 1026] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 78. [The present invention 1027] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 79. [The present invention 1028] 1019. The method of claim 1019, wherein said polypeptide capable of binding to or interacting with aflibercept comprises an isolated amino acid sequence selected from SEQ ID NO: 80. [The present invention 1029] 1017. The method of claim 1017, wherein the amount of host cell protein in said elution fraction is significantly reduced by about 90%, about 95%, about 98%, or about 99% compared to the amount of host cell protein in (c). These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not limitation. Many substitutions, modifications, additions, or rearrangements may be made within the scope of the invention. [Brief explanation of the drawings]

[0096] [Figure 1] 1 shows a VEGF MiniTrap generated using an exemplary embodiment, which contains VEGFR1 (SEQ ID NO: 34), VEGFR2 (SEQ ID NO: 36, hinge domain fragment (SEQ ID NO: 60)), and an Fc fragment (SEQ ID NO: 113) cleaved from aflibercept (SEQ ID NO: 55). [Figure 2] 1 shows a proposed mechanism for histidine oxidation to 2-oxo-histidine (14 Da). [Figure 3] 1 shows a proposed mechanism for histidine oxidation to 2-oxo-histidine (16 Da). [Figure 4] 1 shows a proposed mechanism for the oxidation of tryptophan to N-formylkynurenine and kynurenine. [Figure 5] 1 shows an exemplary embodiment for the production of aflibercept. [Figure 6]1 shows an exemplary embodiment for the production of VEGF MiniTrap. [Figure 7] 1 shows an exemplary embodiment for the production of aflibercept. [Figure 8] 1 shows an exemplary embodiment for the production of VEGF MiniTrap. [Figure 9] 1 shows a chart of calculated b* values ​​versus calculated BY standards as an exemplary embodiment. [Figure 10] 1 shows the results of an experiment performed to assess the rate of 2-oxo-histidine and tryptophan oxidation (underlined residues represent oxidation) in oligopeptides from protease-digested AEX load and flow-through, including fragments of reduced and alkylated aflibercept (SEQ ID NO: 55), including, in order of appearance, SEQ ID NOs: 114, 115, 21, 115, 28, 28, 20, 18, 17, 116, 117, and 19, respectively. [Figure 11] Shown are the relative abundances of peptides identified from peptide mapping analysis performed using oligopeptides from protease-digested AEX load and flow-through (underlined portions represent oxidation of residues in the peptide sequence), including fragments of aflibercept (SEQ ID NO: 55), including SEQ ID NOs: 22, 18, 21, 19, 20, 118, 119, 28, and 29, respectively, in order of appearance. [Figure 12A] A full chromatogram chart of absorbance versus time (min) for MT4 and MT1 at 350 nm is shown. [Figure 12B] Shown is an enlarged view of a chromatogram chart of absorbance versus time (16-30 min) for MT4 and MT1 at 350 nm, containing SEQ ID NOs: 21, 28, and 28, respectively, in order of appearance. [Figure 12C] Shown is an enlarged view of a chromatogram chart of absorbance versus time (30-75 min) for MT4 and MT1 at 350 nm, containing SEQ ID NOs: 17, 20, 18, and 19, respectively, in order of appearance. [Figure 13]The results of an experiment conducted to evaluate the proportion of 2-oxo-histidine (and tryptophan dioxide) in oligopeptides from protease-digested MT1 treated by AEX chromatography and in oligopeptides from protease-digested MT1 stripped from AEX chromatography, including SEQ ID NOs: 21, 28, 17, 20, 18, and 19, respectively, in order of appearance, are shown. [Figure 14] Shown are the results of experiments conducted to compare the acidic species present in different production lots of MT1, including SEQ ID NOs: 21, 28, 28, 17, 20, 18, and 19, respectively, in order of appearance, with the acidic acid fractions obtained when performing strong cation exchange (CEX) chromatography. [Figure 15] An exemplary method is shown for enriching acidic species and other variants present in a cell culture harvest sample using strong cation exchange chromatography. [Figure 16] 1 shows fractions from a strong cation exchange chromatography run, according to an exemplary embodiment. [Figure 17] 1 shows strong cation exchange chromatograms performed according to exemplary embodiments for MT1 production (before any production steps, BY3 or below) subjected to CEX and for enriched variants of desialylated MiniTrap (dsMT1) using a dual salt-pH gradient. [Figure 18A] 1 shows a 3D chromatogram for an unfractionated parent control performed by strong cation exchange chromatography according to an exemplary embodiment. [Figure 18B] 1 shows a 3D chromatogram for MT1, fraction 1, illustrating some of the tailing characteristics for an experiment performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18C] 1 shows a 3D chromatogram for a characterization of MT1, fraction 2, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18D]1 shows a 3D chromatogram for a characterization of MT1, fraction 3, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18E] 1 shows a 3D chromatogram for a characterization of MT1, fraction 4, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18F] 1 shows a 3D chromatogram for the characterization of MT1, fraction 5, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18G] 1 shows a 3D chromatogram for a characterization of MT1, fraction 6, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 18H] 1 shows a 3D chromatogram for a characterization of MT1, fraction 7, performed by strong cation exchange chromatography, according to an exemplary embodiment. [Figure 19] 1 shows an electropherogram of an imaged capillary isoelectric focusing (icIEF) performed according to an exemplary embodiment for MT1 production. [Figure 20] 1 shows the results of a study correlating the occurrence of oxidized amino acid residues with exposure of MT1 to cool white light or UVA light, including SEQ ID NOs: 114, 114, 115, 21, 115, 28, 28, 28, 17, 83, 20, 18, 29, 29, 19, and 22, respectively, in order of appearance. [Figure 21] 1 shows 3D SEC-PDA (size-exclusion chromatography coupled with photodiode array detection) chromatograms of CWL-stressed MT1 with absorbance at about 350 nm (e.g., see circle highlighting about 350 nm) according to an exemplary embodiment, where A shows the chromatogram at T=0, B shows the chromatogram at 0.5×ICH, C shows the chromatogram at 2.0×ICH, and D depicts MT1 in a vial (normalized to 80 mg / mL) stressed by CWL at different time intervals. [Figure 22]3D SEC-PDA chromatograms are shown for UVA-stressed MT1 with absorbance at approximately 350 nm (e.g., see circle highlighting approximately 350 nm) according to an exemplary embodiment, where A shows the chromatogram at T=0, B shows the chromatogram at 0.5×ICH, C shows the chromatogram at 2.0×ICH, and D depicts MT1 in a vial (normalized to 80 mg / mL) stressed with UVA for different time intervals. [Figure 23A] A320 / 280 absorbance ratios quantified from SEC-PDA chromatograms for samples stressed using CWL (top panel) are shown. [Figure 23B] FIG. 10 is a chart of A320 / 280 absorbance ratios for size variants in a sample stressed using CWL (bottom panel), which sample has been stressed according to an exemplary embodiment. [Figure 24A] A320 / 280 absorbance ratios quantified from SEC-PDA chromatograms for samples stressed using UVA (top panel) are shown. [Figure 24B] 1 is a chart of A320 / 280 absorbance ratios for size variants in a sample stressed using UVA (bottom panel), the sample being stressed according to an exemplary embodiment. [Figure 25A] Figure 1 shows estimated estimates of the effect that incubation of various ingredients with aflibercept has on color (b* predicted by CIE L*, a*, b values). [Figure 25B] A plot of the predicted b-values ​​versus the measured values ​​is shown. [Figure 26A] 1 shows the effect of low-cysteine ​​and low-metal CDM on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E). [Figure 26B] 1 shows the effect of low-cysteine ​​and low-metal CDM on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E). [Figure 26C] 1 shows the effect of low-cysteine ​​and low-metal CDM on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E). [Figure 26D] 1 shows the effect of low-cysteine ​​and low-metal CDM on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E). [Figure 26E] 1 shows the effect of low-cysteine ​​and low-metal CDM on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmolality (E). [Figure 27] 1 is a chart showing predicted profiles of harvest color (as determined as b* value at day 13) with respect to increasing / decreasing concentrations of metals and cysteine, according to an exemplary embodiment. [Figure 28A] 1 shows the effect of incubation of various ingredients with aflibercept in consumed CDM on the production of color (b* predicted by CIE L*, a*, b values). [Figure 28B] A plot of the estimated predicted effect on b-values ​​is shown. [Figure 28C] Figure 1 shows the estimated effect of incubation of various components with aflibercept in CDM on color (b* predicted by CIE L*, a*, b values) production in shake flask cultures. [Figure 28D] 1 shows the effect of incubation of hypotaurine and deferoxamine mesylate (DFO) with aflibercept in consumed CDM on the production of color (CIE L*, a*, b* predicted by "b" values). [Figure 28E] 1 shows the effect of incubation of each of the various components with aflibercept from shake flask cultures on the production of color (CIE L*, a*, b* predicted by "b" values). [Figure 29] 1 is a chart showing the effect of addition of uridine, manganese, galactose, and dexamethasone in CDM on the titer of aflibercept produced. [Figure 30] 1 is a chart showing the effect of adding uridine, manganese, galactose, and dexamethasone in CDM on the viability of cells expressing produced aflibercept. [Figure 31] 1 is a chart showing the effect of adding uridine, manganese, galactose, and dexamethasone in CDM on the viable cell number of cells expressing aflibercept produced. [Figure 32] 1 is a chart showing a standard curve of absorbance versus host cell protein concentration (ng / mL) prepared using a standard host cell protein solution from Cygnus 3G (F550). [Figure 33] 1 is an image of an SDS-PAGE analysis performed using non-reducing SDS-PAGE sample buffer. [Figure 34] 1 is an image of an SDS-PAGE analysis performed using reducing SDS-PAGE sample buffer. [Figure 35A] 1 is a chart of total host cell proteins detected in the elution fractions from each of affinity chromatography columns 1-3 containing the loading solution, VEGF165, mAb1, and mAb2. [Figure 35B] 1 is a chart of total host cell proteins detected in the elution fractions from affinity chromatography columns 1, 2, 4, and 5, respectively, containing the loading solution, VEGF165, mAb1, mAb3, and mAb4. [Figure 36A] SEC profile of VEGF MiniTrap before affinity chromatography purification. [Figure 36B] 1 shows the SEC profile of VEGF MiniTrap after affinity chromatography purification. [Figure 37] 1 shows a cartoon representation of kinetic testing of VEGF MiniTrap against VEGF 165. In this case, the VEGF MiniTrap constructs tested were derived before and after affinity chromatography purification according to some exemplary embodiments. [Figure 38] 1 shows SPR sensorgrams from a kinetic study of VEGF MiniTrap against VEGF165, where the VEGF MiniTrap constructs tested were derived before and after affinity chromatography purification according to some exemplary embodiments. [Figure 39] 1 is a chart of total host cell proteins detected in elution fractions from an affinity chromatography column run repeatedly for columns containing loading solution, VEGF165, mAb1, and mAb2. [Figure 40] 1 shows the structure of VEGF MiniTrap MT1 (SEQ ID NO: 46), according to an exemplary embodiment. [Figure 41] 1 shows the structure of VEGF MiniTrap MT6 (SEQ ID NO: 51), according to an exemplary embodiment. [Figure 42] Total ion chromatograms (TICs) of relative absorbance versus time (min) of native SEC-MS analysis of MT1, MT5, and MT6, as well as a zoom of the low molecular weight region from the TICs, are shown. [Figure 43] Deconvoluted mass spectra of the main peaks for MT1 and MT5 are shown to confirm the identity of the MiniTrap dimers along with elucidation of some PTMs, with the N-terminal amino acids (SEQ ID NO: 120) indicated. [Figure 44] Deconvoluted mass spectrum of the main peak for MT6 is shown to confirm the identity of single-stranded MiniTrap along with elucidation of some PTMs. [Figure 45A] A chart of relative absorbance versus time (min) is shown for low molecular weight impurities in MT1. [Figure 45B] 1 shows the mass spectrum of low molecular weight impurities in MT1. [Figure 46] Shown is the relative absorbance versus time (min) for MT1, indicating the absence of the FabRICATOR enzyme used to cleave aflibercept into MT1. [Figure 47]Relative absorbance versus time (min) is shown for low molecular weight impurities in MT5. [Figure 48] Relative absorbance versus time (min) is shown for low molecular weight impurities in MT6. [Figure 49A] 1 shows a chart of absorbance versus time (min) obtained by performing HILIC-UV / MS on VEGF MiniTrap MT6, showing the elution of the main peak at 21 min and O-glycans at approximately 21.5 min. [Figure 49B] The mass spectrum obtained by performing HILIC-UV / MS on VEGF MiniTrap MT6, which shows a main peak at 47985.8 Da, is shown. [Figure 49C] 1 shows the mass spectrum of O-glycans of VEGF MiniTrap MT6 obtained by performing HILIC-UV / MS. [Figure 50] Image of a VEGF MiniTrap dimer, where the disulfide bridges in the hinge region of VEGF MiniTrap (SEQ ID NOs: 83, 123, 83, and 123) can be parallel or crossover. [Figure 51] Figure 1 shows the relative abundance of the distribution of glycans observed at Asn36 in MT1, MT5 and MT6. The figure discloses SEQ ID NO: 121. [Figure 52] Figure 1 shows the relative abundance of the distribution of glycans observed at Asn68 in MT1, MT5, and MT6. The figure discloses SEQ ID NOs: 101 and 30, respectively, in order of appearance. [Figure 53] Figure 1 shows the relative abundance of the distribution of glycans observed at Asn123 in MT1, MT5, and MT6. The figure discloses SEQ ID NO: 82. [Figure 54] Figure 1 shows the relative abundance of the distribution of glycans observed at Asn196 in MT1, MT5, and MT6. The figure discloses SEQ ID NOs: 103 and 122, respectively, in order of appearance. [Figure 55]1 shows released N-linked glycan analysis by hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection and mass spectrometry analysis (maximum detection limit and stacking). [Figure 56] HILIC-FLR chromatograms for MT1, MT5, and MT6 are shown. [Figure 57] 1 shows released N-linked glycan analysis by HILIC coupled with fluorescence detection and mass spectrometry analysis (maximum detection limit, stacking, and normalization). [Figure 58A] 1 is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6. [Figure 58B] 1 is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6. [Figure 58C] 1 is a table of detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5 and MT6. [Figure 59] 1 shows an exemplary production procedure for manufacturing MiniTrap, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0097] Detailed Description Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a highly orchestrated process that is important for proper embryonic and postnatal vascular development. Abnormal or pathological angiogenesis is a hallmark of cancer and several retinal diseases. In this case, upregulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) leads to increased endothelial proliferation, changes in vascular morphology, and increased vascular permeability. High levels of VEGF have been found in the vitreous humor and retinal vessels of patients with various ocular diseases. Blocking VEGF activity is also an optimal therapy for treating DME, wet AMD, CNV, retinal vein occlusion, and other ocular diseases in which abnormal angiogenesis underlies the etiology.

[0098] As used herein, aflibercept is one such anti-VEGF protein containing a fully human amino acid sequence, including the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2, expressed as an inline fusion with human IgG1 (Fc). Aflibercept binds to all forms of VEGF-A (VEGF), but also binds to PlGF and VEGF-B. Several other homodimers, VEGF MiniTrap, are produced as enzymatic cleavage products from aflibercept or as direct recombinant expression from host cell lines. An example of such a VEGF MiniTrap is shown in Figure 1. In this figure, the terminal lysine is indicated (k); some culture processes remove this terminal lysine, while others do not. Figure 1 illustrates a process in which the terminal lysine remains. In general, aflibercept encompasses both cases where the terminal lysine is present and cases where it is absent.

[0099] As shown herein, the present invention partially discloses the production of anti-VEGF proteins using CDM (Example 1). Analysis of solutions containing aflibercept produced using specific CDMs showed certain color characteristics, such as a strong yellow-brown color. The color intensity of the solutions varied depending on the CDM used. Not all of the investigated CDMs produced samples with a distinct yellow-brown color after normalizing the solutions to a concentration of 5 g / L.

[0100] In injectable therapeutic solutions, a color such as tan can be an undesirable characteristic. It can be an important parameter used to determine whether a drug product meets the required level of purification and quality for a particular treatment. Colors such as tan observed along the manufacturing pathway of a biopharmaceutical can result from chemical modifications of the biopharmaceutical, degradation products of formulation excipients, or degradation products formed by the reaction of the biopharmaceutical with formulation excipients. However, such information can be useful in understanding the cause of the color change. This can also aid in the design of short-term and long-term storage conditions to prevent modifications that promote such color change.

[0101] The present inventors have observed that the use of AEX during the production of anti-VEGF protein solutions minimizes the yellow-brown coloration. In addition, the present inventors have discovered that the yellow-brown coloration can be reduced by modifying the cell culture used to produce recombinant proteins such as aflibercept or modified aflibercept, such as MiniTrap.

[0102] The present invention encompasses anti-VEGF proteins and their production using CDM. Additionally, the present invention is based on identifying and optimizing upstream and downstream process technologies for protein production.

[0103] As provided herein, some of the examples set forth below describe the production of anti-VEGF proteins (Example 1), the production of oxidized species of anti-VEGF proteins (Example 4), a method for reducing oxidized species of anti-VEGF proteins by optimizing the culture medium (Example 5), and a method for reducing oxidized species of anti-VEGF proteins by optimizing the manufacturing process (Example 2).

[0104] Although several recent patent applications and issued patents purport to describe various aflibercept species and methods for making them, none describe or suggest the anti-VEGF compositions described herein and methods for making them. See, for example, U.S. Patent Application No. 16 / 566,847 to Coherus Biosciences Inc., U.S. Patent No. 10,646,546 to Sam Chun Dang Pharm. Co., Ltd., U.S. Patent No. 10,576,128 to Formycon AG, International Application No. PCT / US2020 / 015659 to Amgen Inc., and U.S. Patent Nos. 8,956,830, 9,217,168, 9,487,810, 9,663,810, 9,926,583, and 10,144,944 to Momenta Pharmaceuticals, Inc.

[0105] I. Explanation of Selected Terms Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein that are known to those skilled in the art can be used in the practice of specific embodiments described herein. All publications mentioned are incorporated herein by reference in their entirety.

[0106] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variation as understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included.

[0107] As used herein, the term "neovascular eye disorder" means any disease of the eye caused by or associated with the growth or proliferation of blood vessels or vascular leakage.

[0108] As used herein, the terms "chemically defined medium" or "chemically defined media" (both abbreviated as "CDM") refer to a synthetic growth medium in which the identity and concentration of all components are defined. Synthetic media do not contain microorganisms, yeast, animal or plant extracts, animal serum, or plasma, although individual plant- or animal-derived components (e.g., proteins, polypeptides, etc.) may be added. Synthetic media may contain inorganic salts such as phosphate, sulfate, and equivalents required to support growth. The carbon source is defined and is usually a sugar, such as glucose, lactose, galactose, and equivalents, or other compounds, such as glycerol, lactate, acetate, and equivalents. Certain synthetic media also use phosphate as a buffer, while other buffers, such as sodium bicarbonate, HEPES, citric acid, triethanolamine, and equivalents, may be used. Examples of commercially available synthetic media include, but are not limited to, various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham's Nutrient Mix (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various EX-CELL media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD media (FUJIFILM Irvine Scientific), combinations thereof, and equivalents thereof. Methods for preparing synthetic media are known in the art, for example, U.S. Patent Nos. 6,171,825 and 6,936,441, WO 2007 / 077217, and U.S. Patent Application Publication Nos. 2008 / 0009040 and 2007 / 0212770, the entire teachings of which are incorporated herein by reference.

[0109] As used herein, the term "cumulative amount" refers to the total amount of a particular component added to a bioreactor over the course of cell culture to form a CDM, including the amount added at the beginning of the culture (CDM on Day 0) and the amount of the component added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or inoculum prior to production in the bioreactor (i.e., prior to CDM on Day 0) is also included. The cumulative amount is not affected by loss of the component (e.g., due to metabolic or chemical degradation) over time during culture. Thus, for example, if a component is added to two cultures at different times (e.g., in one culture, all of the components are added initially, and in another culture, the components are added sequentially), even if the two cultures have the same cumulative amount of the component, they may have different absolute levels. The cumulative amount is also not affected by in situ synthesis of the component (e.g., due to metabolic or chemical conversion) over time during culture. Thus, for example, two cultures with the same cumulative amount of a given component may have different absolute levels if the component is synthesized in situ in one of the cultures during the bioconversion process, where the cumulative amount can be expressed, for example, in grams or moles of the component.

[0110] As used herein, the term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the beginning of a production batch, including additions to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the beginning of a production batch and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onwards is 2 g / L (i.e., 4 grams divided by 2 liters). If, on day 4, an additional liter of liquid not containing component X is added to the bioreactor, the cumulative concentration will remain at 2 g / L. If, on day 5, some amount of liquid is lost from the bioreactor (e.g., due to evaporation), the cumulative concentration will remain at 2 g / L. The cumulative concentration may be expressed in units such as grams / liter or moles / liter, for example.

[0111] As used herein, the term "formulation" refers to a protein of interest formulated with one or more pharmaceutically acceptable vehicles. In one aspect, the protein of interest is aflibercept and / or MiniTrap. In some exemplary embodiments, the amount of the protein of interest in the formulation may range from about 0.01 mg / mL to about 600 mg / mL. In some specific embodiments, the amount of the protein of interest in the formulation may range from about 0.01 mg / mL, about 0.02 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0. ... mL, approximately 0.6 mg / mL, approximately 0.7 mg / mL, approximately 0.8 mg / mL, approximately 0.9 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 5 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 110 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, approximately 140 mg / mL, approximately 150 mg / mL, approximately 160 mg / mL, approximately 170 mg / mL, approximately 180 mg / mL, approximately 190 m The pH of the composition may be about 200 mg / mL, about 225 mg / mL, about 250 mg / mL, about 275 mg / mL, about 300 mg / mL, about 325 mg / mL, about 350 mg / mL, about 375 mg / mL, about 400 mg / mL, about 425 mg / mL, about 450 mg / mL, about 475 mg / mL, about 500 mg / mL, about 525 mg / mL, about 550 mg / mL, about 575 mg / mL, or about 600 mg / mL. In some exemplary embodiments, the pH of the composition may be greater than about 5.0. In one exemplary embodiment, the pH may be greater than about 5.0, greater than about 5.5, greater than about 6, greater than about 6.5, greater than about 7, greater than about 7.5, greater than about 8, or greater than about 8.5.

[0112] As used herein, the term "database" refers to a bioinformatics tool that provides the possibility to search uninterpreted MS-MS spectra against all possible sequences in one or more databases. Non-limiting examples of such tools include Mascot (http: / / www.matrixscience.com), Spectrum Mill (http: / / www.chem.agilent.com), PLGS (http: / / www.waters.com), PEAKS (http: / / www.bioinformaticssolutions.com), Proteinpilot (http: / / download.appliedbiosystems.com / / proteinpilot), Phenyx (http: / / www.phenyx-ms.com), Sorcerer (http: / / www.sagenresearch.com), OMSSA (http: / / www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (http: / / www.thegpm.org / TANDEM / ), ProteinProspector (http: / / www.http: / / prospector.ucsf. edu / prospector / mshome.htm), Byonic (https: / / www.proteinmetrics.com / products / byonic), or Sequest (http: / / fields.scripps.edu / sequest).

[0113] As used herein, the term "ultrafiltration" or "UF" may include a membrane filtration process similar to reverse osmosis, which uses hydrostatic pressure to force water through a semipermeable membrane. Ultrafiltration is described in detail in Leos J. Zeman & Andrew L. Zydney, Microfiltration and Ultrafiltration: Principles and Applications (1996), the entire teachings of which are incorporated herein. Filters with pore sizes smaller than 0.1 μm can be used for ultrafiltration. The use of filters with such small pore sizes allows the sample volume to be reduced by allowing the sample buffer to permeate through the filter, while proteins are retained behind the filter.

[0114] As used herein, "diafiltration" or "DF" can include methods that use an ultrafilter to remove and exchange salts, sugars, and non-aqueous solvents, separate them from bound species, remove low molecular weight materials, and / or cause a rapid change in the ionic and / or pH environment. Microsolutes are most efficiently removed by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultrafiltration rate. In this way, microspecies are washed out of the solution at a constant rate. In certain exemplary embodiments of the invention, a diafiltration process can be used to exchange various buffers used in connection with the invention, e.g., prior to chromatography or other manufacturing steps, and to remove impurities from protein preparations. As used herein, the term "downstream process technology" refers to one or more techniques used after an upstream process technology to produce a protein. Downstream processing techniques include, for example, purification of protein products using affinity chromatography, including, for example, Protein A affinity chromatography and affinity chromatography using a solid phase bearing a well-defined molecule such as VEGF that can interact with its cognate, such as VEGF receptor (VEGF R); ion exchange chromatography, such as anion exchange chromatography or cation exchange chromatography; hydrophobic interaction chromatography; or displacement chromatography.

[0115] The phrase "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector encoding a protein of interest has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur over successive generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In one aspect, eukaryotic cells include protist cells, fungal cells, plant cells, and animal cells. In further aspects, host cells include eukaryotic cells, such as plant cells and / or animal cells. The cells may be mammalian cells, fish cells, insect cells, amphibian cells, or avian cells. In particular aspects, the host cells are mammalian cells. A variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other depositories, as well as from commercial suppliers. Cells that can be used in the process of the present invention include MK2.7 cells; PER-C6 cells; Chinese hamster ovary cells (CHO), such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO 01 / 92337 A2), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12. CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); SV40 (COS cells, COS-7, ATCC CRL-1651) transformed monkey kidney CV1 cells;HEK293 cells and Sp2 / 0 cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2 / 0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells, and retinal epithelial cells), and established cell lines and their cell lines (e.g., human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned to grow in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2; canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB8065); mouse mammary carcinoma cells (MMT 060562, ATCC CCL-51); buffalo rat hepatocytes (BRL3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR5 cells; FS4 cells;PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or their derivatives), fibroblasts from any tissue or organ (heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), Lymphoid tissues (lymph glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit551 cells, Detroit510 cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587;VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN 205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac muntac) cells, SIRC cells, C;II Cells, and Jensen cells, or derivatives thereof) cell lines) or any other cell type known to those of skill in the art.

[0116] As used herein, the term "host cell proteins" (HCPs) includes proteins derived from host cells and may be unrelated to the desired protein of interest. Host cell proteins may be process-related impurities that may originate from the manufacturing process, which may include three major categories: cell matrix-derived, cell culture-derived, and downstream-derived. Cell matrix-derived impurities include, but are not limited to, proteins derived from the host organism and nucleic acids (host cell genome, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antimicrobials, serum, and other media components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biological treatment reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachable materials.

[0117] In some exemplary embodiments, the host cell protein can have a pI in the range of about 4.5 to about 9.0. In exemplary embodiments, the pI can be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0.

[0118] As used herein, the term "hydrolytic agent" refers to any one or combination of a number of different agents capable of performing enzymatic digestion of proteins. Non-limiting examples of hydrolytic agents capable of performing enzymatic digestion include protease from Aspergillus niger, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysC endoproteinase (Lys-C), endoproteinase Asp-N (Asp-N), endoproteinase Arg-C (Arg-C), endoproteinase Glu-C (Glu-C), or outer membrane protein T (OmpT), immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or biologically active fragments, homologs thereof, or combinations thereof. Non-limiting examples of hydrolysis agents that can perform non-enzymatic digestion include high temperature, microwaves, ultrasound, high pressure, infrared radiation, solvents (non-limiting examples include ethanol and acetonitrile), immobilized enzyme digestion (IMER), enzymes immobilized on magnetic particles, and on-chip immobilized enzymes. For a recent review describing available techniques for protein digestion, see Switzer et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzer, Martin Giera & Wilfried Maniessen, Protein Digestion: An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013), the entire teachings of which are incorporated herein). One or a combination of hydrolysis agents can cleave the peptide bonds of proteins or polypeptides in a sequence-specific manner, generating a predictable collection of shorter peptides. The ratio of hydrolysis agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of the protein.An inappropriately high enzyme-to-substrate ratio can result in a correspondingly fast digestion rate, which may not allow sufficient time for peptide analysis by the mass spectrometer, resulting in poor sequence coverage. On the other hand, a low E / S ratio can require a long digestion time, thereby increasing data acquisition time. The enzyme-to-substrate ratio can range from about 1:0.5 to about 1:200. As used herein, the term "digestion" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to perform digestion of proteins in biological samples using appropriate hydrolytic agents, such as enzymatic digestion or non-enzymatic digestion. One widely accepted method for digesting proteins in a sample involves the use of proteases. Many proteases are available, each with unique characteristics in terms of specificity, efficiency, and optimal digestion conditions. Proteases, which refer to both endopeptidases and exopeptidases, are classified based on their ability to cleave at non-terminal or terminal amino acids within peptides. Alternatively, proteases can also refer to six different classes, classified based on their mechanism of catalysis: aspartic proteases, glutamine proteases, and metalloproteases, cysteine ​​proteases, serine proteases, and threonine proteases. The terms "protease" and "peptidase" are used interchangeably to refer to enzymes that hydrolyze peptide bonds.

[0119] The term "in association with" indicates that components, such as the anti-VEGF composition of the present invention, together with another agent, such as anti-ANG2, can be formulated into a single composition for simultaneous delivery, or can be separately formulated into two or more compositions (e.g., a kit containing each component). Components administered in association with each other can be administered to a subject at a different time than when the other components are administered. For example, each administration can be given non-simultaneously (e.g., separately or sequentially) spaced apart over a given period of time. Separate components administered in association with each other can also be administered essentially simultaneously (e.g., at the exact same time or separated by a non-clinically significant period) during the same administration session. Furthermore, separate components administered in association with each other can be administered to a subject by the same route or by different routes; for example, a composition of aflibercept is administered in association with another agent, such as anti-ANG2, and the aflibercept composition contains less than about 15% of its variants.

[0120] As used herein, the term "liquid chromatography" refers to a process by which a fluid-borne biological / chemical mixture can be separated into its components as a result of differential partitioning of the components as they flow through (or into) a stationary fluid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, mixed-mode chromatography, hydrophobic chromatography, or mixed-mode chromatography.

[0121] As used herein, "affinity chromatography" can include any method of separating two substances based on their affinity for a chromatographic material. This can include subjecting a substance to a column containing a suitable affinity chromatography medium. Non-limiting examples of such chromatographic media include, but are not limited to, protein A resin, protein G resin, affinity supports containing an antigen to which a binding molecule (e.g., an antibody) has been raised, a protein capable of binding to a protein of interest, and affinity supports containing an Fc-binding protein. In one embodiment, the affinity column can be equilibrated with a suitable buffer before loading the sample. An example of a suitable buffer is Tris / NaCl buffer (pH approximately 7.0-8.0). Those skilled in the art can develop suitable buffers without undue burden. After this equilibration, the sample can be loaded onto the column. After loading the column, the column can be washed one or more times, for example, with the equilibration buffer. Other washes, including washes using a different buffer, can be used before eluting the column. The affinity column can then be eluted using an appropriate elution buffer. An example of a suitable elution buffer is an acetic acid / NaCl buffer (pH approximately 2.0-3.5). Furthermore, those skilled in the art can develop suitable elution buffers without undue burden. The eluate is monitored using techniques well known to those skilled in the art, including ultraviolet light, e.g., absorbance at 280 nm, which can be used particularly when the sample of interest contains aromatic rings (e.g., proteins with aromatic amino acids such as tryptophan).

[0122] As used herein, "ion exchange chromatography" can refer to any method of separation that separates two substances based on the difference in their respective ionic charges, either collectively or locally on a molecule of interest and / or on a chromatographic material, or on a specific region of the molecule of interest and / or on a chromatographic material. Therefore, it can use either a cation or anion exchange material. Ion exchange chromatography separates molecules based on the difference between the local charge of the molecule of interest and the local charge of the chromatographic material. Packed columns or ion exchange membrane devices for ion exchange chromatography can be operated in bind-elute mode, flow-through mode, or hybrid mode. After washing the column or membrane device with an equilibration buffer or another buffer, product recovery can be achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer, which competes with the solute for the charged sites of the ion exchange matrix. Changing the pH, thereby altering the charge of the solute, can be another way to achieve solute elution. The change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution). Anionic or cationic substituents may be attached to the matrix to form anionic or cationic supports for chromatography. Non-limiting examples of anionic exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Cellulose ion exchange media or supports can include DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™, which are available from Whatman Ltd., Maidstone, Kent, UK. SEPHADEX®-based ion exchangers and SEPHADEX® cross-linked ion exchangers are also known.For example, DEAE-SEPHADEX®, QAE-SEPHADEX®, CM-SEPHADEX®, and SP-SEPHADEX®, and DEAE-SEPHAROSE®, Q-SEPHAROSE®, CM-SEPHAROSE®, S-SEPHAROSE®, and SEPHAROSE® Fast Flow, and Capto™ S are all available from GE Healthcare. Additionally, both DEAE and CM derivatized ethylene glycol-methacrylate copolymers, such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or M, are available from Toso Haas Co. (Philadelphia, Pa.), Nuvia S and UNOSphere™ S are available from BioRad, (Hercules, Calif.), and Eshmuno® S is available from EMD Millipore (MA).

[0123] As used herein, the term "hydrophobic interaction chromatography resin" can include a solid phase that can be covalently modified with phenyl, octyl, butyl, or equivalents. Hydrophobic interaction chromatography uses properties such as hydrophobicity to separate molecules from one another. In this type of chromatography, hydrophobic groups such as phenyl, octyl, hexyl, or butyl can form the stationary phase of the column. Molecules such as proteins, peptides, and the like can pass through an HIC (hydrophobic interaction chromatography) column that has one or more hydrophobic regions or hydrophobic pockets on its surface and interact with the hydrophobic groups that make up the HIC stationary phase. Examples of HIC resins or supports include Phenyl Sepharose FF, Capto Phenyl (GE Healthcare, Uppsala, Sweden), Phenyl 650-M (Tosoh Bioscience, Tokyo, Japan), and Sartobind Phenyl (Sartorius Corporation, New York, USA).

[0124] As used herein, the terms "mixed-mode chromatography" or "multimodal chromatography" (both "MMC") include chromatographic methods in which solutes interact with a stationary phase through multiple interaction modes or mechanisms. MMC can be used as an alternative or complementary tool to traditional reversed-phase (RP), ion-exchange (IEX), and normal-phase (NP) chromatography. Unlike RP, NP, and IEX chromatography, in which hydrophobic, hydrophilic, and ionic interactions are the dominant interaction modes, mixed-mode chromatography can use a combination of two or more of these interaction modes. Mixed-mode chromatography media can offer unique selectivity that single-mode chromatography cannot reproduce. Mixed-mode chromatography can also offer potential cost savings, extended column lifetime, and operational flexibility compared to affinity-based methods. In some exemplary embodiments, mixed-mode chromatography media, sometimes referred to as a base matrix, can be composed of mixed-mode ligands attached directly or via a spacer to an organic or inorganic support. The support may be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, etc. In some exemplary embodiments, the support may be prepared from natural polymers such as cross-linked carbohydrate materials, such as agarose, agPV, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc. To achieve high adsorption capacity, the support may be porous, and ligands may then be attached to the outer surface and pore surfaces. Such natural polymer supports may be prepared according to standard methods, such as inverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teachings of which are incorporated herein).Alternatively, the support can be prepared from synthetic polymers, such as crosslinked synthetic polymers, e.g., styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, and the like. Such synthetic polymers can be prepared according to standard methods, e.g., "Styrene-based polymer supports developed by suspension polymerization" (R. Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teachings of which are incorporated herein). Porous natural or synthetic polymer supports are also available from manufacturers such as GE Healthcare (Uppsala, Sweden).

[0125] As used herein, the term "mass spectrometer" includes an instrument capable of identifying specific molecular species and measuring their exact mass. This term is meant to include any molecular detector in which a polypeptide or peptide can be characterized. A mass spectrometer may include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create gas-phase ions. Analyte atoms, molecules, or clusters can be transferred to the gas phase and ionized either simultaneously (in electrospray ionization) or through a separation process. The choice of ion source depends on the application. In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" includes techniques that obtain structural information on sample molecules by using multiple steps, such as mass selection and mass separation. A prerequisite is that the sample molecules be transferred to the gas phase and ionized to form fragments in a predictable and controllable manner after the initial mass selection step. Multi-stage MS / MS or MS n First, the precursor ion (MS 2 ) is selected and isolated, and fragmented to generate the first fragment ion (MS 3) is separated and fragmented to give a second fragment (MS 4 Tandem MS is performed as long as significant information can be obtained or fragment ion signals can be detected, such as by separating fragments of a given ion. Tandem MS has been successfully performed using many types of analyzers in combination. The choice of analyzer combination for a particular application is determined by many different factors, such as sensitivity, selectivity, and speed, as well as size, cost, and availability. The two main categories of tandem MS techniques are spatial tandem and temporal tandem, although hybrids that spatially couple temporal tandem analyzers or couple spatial tandem analyzers also exist. Spatial tandem mass spectrometers comprise an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function can be designed to select ions in one section of the instrument, dissociate them in an intermediate region, and then send the product ions to another analyzer for m / z separation and data acquisition. In temporal tandem, mass spectrometer ions generated in the ion source can be trapped, isolated, fragmented, and separated by m / z within the same physical device. Peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications. These can be used for protein characterization by correlating experimental and theoretical MS / MS data (the latter generated from potential peptides in protein sequence databases). Characterization can include, but is not limited to, amino acid sequencing of protein fragments, protein sequence determination, de novo protein sequence determination, post-translational modification location or identification, or comparative analysis, or a combination thereof.

[0126] As used herein, "Mini-Trap" or "MiniTrap" or "MiniTrap-binding molecule" refers to a molecule capable of binding to a VEGF molecule. Such MiniTrap molecules may include (i) chimeric polypeptides and (ii) multimeric (e.g., dimeric) molecules comprising two or more polypeptides non-covalently linked, e.g., by one or more disulfide bridges. MiniTrap molecules may be produced by chemical modification, enzymatic activity, or recombinant production.

[0127] As used herein, a "VEGF MiniTrap" or "VEGF MiniTrap-binding molecule" can refer to a molecule or complex of molecules that bind to VEGF, and that have one or more sets of VEGF receptor Ig-like domains (or variants thereof) (e.g., VEGFR1 Ig domain 2 and / or VEGFR2 Ig domains 3 and / or 4) and a modified multimerization component (MC), or no modified multimerization component, where the MC is a modified immunoglobulin Fc. This modification can be the result of proteolytic digestion of a VEGF trap (e.g., aflibercept or conbercept) or the direct expression of the resulting polypeptide chain with a shortened MC sequence. (See the molecular structure shown in Figure 1.) Figure 1 shows a VEGF MiniTrap molecule, which is the product of proteolysis of aflibercept with Streptococcus pyogenes IdeS. The homodimeric molecule is shown as having two parallel Ig hinge domain fragments connected by disulfide bonds. 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 shown. Such a single, non-dimerized chimeric polypeptide can also be a VEGF MiniTrap if it has VEGF binding activity. The term "VEGF MiniTrap" includes a single polypeptide comprising a first set of one or more VEGF receptor Ig domains (or variants thereof), lacking an MC, but fused with a linker (e.g., a peptide linker) to one or more additional sets of one or more VEGF receptor Ig domains (or variants thereof). The VEGF binding domains in the VEGF MiniTrap of the present invention may be identical or different from one another (see WO 2005 / 00895, the entire teachings of which are incorporated herein).

[0128] For example, in one embodiment of the present invention, the unmodified immunoglobulin Fc domain comprises the amino acid sequence or amino acids 1-226 thereof: TIFF0007744391000006.tif26165 (SEQ ID NO: 33, in which X1 is L or P and X2 is A or T).

[0129] Inhibition of VEGF includes, for example, VEGF (e.g., VEGF 110 , VEGF 121 , and / or VEGF 165 Such inhibition can include, for example, antagonism of VEGF binding to a VEGF receptor by competing with the VEGF receptor for IL18Rα and / or IL18Rβ binding. Such inhibition can inhibit VEGF activation of a VEGFR, such as by inhibiting luciferase expression in a cell line (e.g., HEK293) expressing a chimeric VEGF receptor (e.g., a homodimer thereof) having a VEGFR extracellular domain fused to the IL18Rα and / or IL18Rβ intracellular domain on the cell surface and an NFkB-luciferase-IRES-eGFP reporter gene, e.g., the cell line HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ as described herein.

[0130] The VEGF receptor Ig domain component of the VEGF MiniTrap of the present invention is (i) one or more immunoglobulin-like (Ig) domains 2 (R1D2) of VEGFR1 (Flt1); (ii) one or more Ig domains 3 (Flk1D3)(R2D3) of VEGFR2 (Flk1 or KDR); (iii) one or more Ig domains 4 (Flk1D4) (R2D4) of VEGFR2 (Flt1 or KDR), and / or (iv) one or more Ig domains 3 of VEGFR3 (Flt4) (FltD3 or R3D3) may include:

[0131] The immunoglobulin-like domains of VEGF receptors may be referred to herein as VEGFR "Ig" domains. References to VEGFR Ig domains herein, such as R1D2 (which may be referred to herein as VEGFR1(d2)), R2D3 (which may be referred to herein as VEGFR2(d3)), R2D4 (which may be referred to herein as VEGFR2(d4)), and R3D3 (which may be 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 properties of the wild-type domains, e.g., retain the ability to form a functional VEGF-binding domain when incorporated into a VEGF MiniTrap. 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 properties as the wild-type domains.

[0132] The present invention provides a VEGF MiniTrap polypeptide comprising 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 ; ((R1D2)-(R2D3)-(R2D4)) f -(MC) g ; 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 multimerization component (e.g. derived from IgG1, e.g. an IgG hinge domain or a fragment thereof); - the linker is a peptide containing about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, such as (GGGS) g (SEQ ID NO: 104); 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 15.

[0133] In one embodiment of the invention, R1D2 has the amino acid sequence: TIFF0007744391000007.tif11165 (SEQ ID NO: 34). In one embodiment, R1D2 lacks the N-terminal SDT.

[0134] In one embodiment of the invention, R1D2 has the amino acid sequence: Contains TIFF0007744391000008.tif11165 (SEQ ID NO: 35).

[0135] In one embodiment of the invention, R2D3 has the amino acid sequence: Contains TIFF0007744391000009.tif11164 (SEQ ID NO: 36).

[0136] In one embodiment of the invention, R2D4 has the amino acid sequence: Contains TIFF0007744391000010.tif12164 (SEQ ID NO: 37).

[0137] In one embodiment of the invention, R2D4 has the amino acid sequence: Contains TIFF0007744391000011.tif11164 (SEQ ID NO: 38).

[0138] In one embodiment of the present invention, the multimerization component (MC) for use in the VEGF MiniTrap is a peptide, e.g., a modified Fc immunoglobulin (e.g., derived from IgG1) capable of binding to another multimerization component. In one aspect, the MC is a modified Fc immunoglobulin comprising an immunoglobulin hinge region. For example, in one embodiment of the present invention, the MC is a peptide containing one or more (e.g., 1, 2, 3, 4, 5, or 6) cysteines capable of forming one or more cysteine ​​bridges with cysteines in another MC, e.g., DKTHTCPPC (SEQ ID NO: 39), DKTHTCPPCPPC (SEQ ID NO: 40), DKTHTCPPCPPCPPC (SEQ ID NO: 41), DKTHTC(PPC) h (wherein h is 1, 2, 3, 4, or 5) (SEQ ID NO: 105), DKTHTCPPCPAPELLG (SEQ ID NO: 60), DKTHTCPLCPAPELLG (SEQ ID NO: 43), DKTHTC (SEQ ID NO: 44), or DKTHTCPLCPAP (SEQ ID NO: 45).

[0139] The present invention also provides a VEGF MiniTrap polypeptide comprising the following domain structure: (i)(R1D2) a -(R2D3) b -(MC) c or (ii) (R1D2) a -(R2D3) b -(R2D4) c -(MC) d ; They can homodimerize with the second polypeptide, for example, by binding between the MC of each polypeptide. in this case, (i) the R1D2 domains are aligned with each other, (ii) the R2D3 domains are aligned with each other; and / or (iii) the R2D4 domains are aligned with each other, Forms a dimeric VEGF-binding domain.

[0140] In one embodiment of the present invention, the VEGF MiniTrap polypeptide has the amino acid sequence: TIFF0007744391000012.tif165166TIFF0007744391000013.tif55165. As mentioned, such polypeptides can be multimerized (e.g., dimerized (e.g., homodimerized)), where binding between the polypeptides is mediated via a multimerization component.

[0141] In one embodiment of the present invention, the VEGFR1 Ig-like domain 2 of the 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 the VEGFR2 Ig-like domain 3 of the 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.

[0142] In one embodiment of the present invention, the VEGF MiniTrap has the structure: (R1D2)1-(R2D3)1-(G4S)3-(R1D2)1-(R2D3)1 ("(G4S)3": disclosed as SEQ ID NO: 107); (R1D2)1-(R2D3)1-(G4S)6-(R1D2)1-(R2D3)1 ("(G4S)6": disclosed as SEQ ID NO: 108); (R1D2)1-(R2D3)1-(G4S)9-(R1D2)1-(R2D3)1 ("(G4S)9": disclosed as SEQ ID NO: 109); or (R1D2) 1-(R2D3) 1-(G4S) 12-(R1D2)1-(R2D3)1("(G4S) 12 ": disclosed as SEQ ID NO: 110) G4S is -Gly-Gly-Gly-Gly-Ser- (SEQ ID NO: 111).

[0143] In one embodiment of the present invention, the VEGF MiniTrap has the amino acid sequence: TIFF0007744391000014.tif25165TIFF0007744391000015.tif213166TIFF0007744391000016.tif52165 (where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). As described herein, these polypeptides can comprise secondary structure, where similar VEGFR Ig domains combine to form intrachain VEGF-binding domains (e.g., FIG. 2). In one embodiment of the invention, two or more of these polypeptides multimerize (e.g., dimerize (e.g., homodimerize)), where a VEGFR Ig domain from each chain combines with a similar Ig domain from another chain to form an intrachain VEGF-binding domain.

[0144] In certain embodiments of the present invention, the VEGF MiniTrap of the present invention lacks any significant modification of the amino acid residues of the VEGF MiniTrap polypeptide (e.g., directed chemical modification at the N-terminus and / or C-terminus, such as PEGylation or iodoacetamidation).

[0145] In one embodiment of the invention, the polypeptide comprises a secondary structure, in which case a single chimeric polypeptide (e.g., (R1D2) a -(R2D3) b -Linker-(R1D2) c -(R2D3) d ;or (R1D2) a -(R2D3) b -(R2D4) c -Linker-(R1D2) d -(R2D3) e-(R2D4) f ) or similar VEGFR Ig domains in separate chimeric polypeptides (e.g., homodimers) align to form a VEGF-binding domain. For example, in this case: (i) the R1D2 domains are aligned with each other, (ii) the R2D3 domains are aligned with each other; and / or (iii) the R2D4 domains are aligned with each other, The VEGF-binding domain is formed. Figure 2 shows a single-chain VEGF MiniTrap showing such a domain arrangement. The VEGFR1, VEGFR2, and linker domains are shown. The linker shown is (G4S)6 (SEQ ID NO: 108). The present invention also provides a method for the preparation of (G4S)3 (SEQ ID NO: 107); (G4S)9 (SEQ ID NO: 109); or (G4S) 12 (SEQ ID NO: 110) single chain VEGF MiniTrap with linker.

[0146] In addition, the present invention also provides a complex comprising a VEGF MiniTrap as described herein complexed with a VEGF polypeptide, or a fragment thereof, or a fusion thereof. 165 ) is homodimerized, and / or VEGF MiniTrap is homodimerized in a 2:2 complex (2VEGF:2MiniTrap), and / or VEGF MiniTrap is homodimerized in a 1:1 complex. The complex includes a homodimerized VEGF molecule bound to a homodimerized VEGF MiniTrap polypeptide. In one embodiment of the invention, the complex is in vitro (e.g., immobilized on a solid substrate) or present in the body of a subject. The present invention also relates to the preparation of a VEGF dimer (e.g., VEGF) complexed with a VEGF MiniTrap. 165 ) complex compositions.

[0147] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer having covalently linked amide bonds. Examples of proteins of interest include, but are not limited to, aflibercept and MiniTrap. Proteins comprise one or more amino acid polymer chains, commonly known in the art as "polypeptides." "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof linked via peptide bonds, related naturally occurring structural variants, and non-naturally occurring synthetic analogs thereof. "Synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Various solid-phase peptide synthesis methods are known to those skilled in the art. A protein can comprise one or more polypeptides to form a single functional biomolecule. In another exemplary embodiment, proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. The protein of interest may include biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In certain embodiments, the protein of interest is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap). Proteins may be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells).For a recent review describing biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teachings of which are incorporated herein). In some exemplary embodiments, the proteins comprise modifications, additions, and other covalently attached moieties. Such modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose-binding protein (MBP), chitin-binding protein (CBP), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes, and their equivalents. Proteins can be classified based on their composition and solubility, and thus include simple proteins, such as globular and fibrous proteins; complex proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary and secondary derived proteins.

[0148] In some exemplary embodiments, the protein of interest may be a recombinant protein, an antibody, a bispecific antibody, a multispecific antibody, an antibody fragment, a monoclonal antibody, a fusion protein, an scFv, and combinations thereof.

[0149] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried in a recombinant expression vector incorporated into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be a fusion protein. In certain aspects, the recombinant protein is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap). In certain exemplary embodiments, the recombinant protein may be an antibody, such as a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule may be a full-length antibody (e.g., IgG1), or alternatively, the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment).

[0150] As used herein, the term "antibody" includes immunoglobulin molecules comprising four polypeptide chains (two heavy (H) chains and two light (L) chains interconnected by disulfide bonds), as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain (CL1). The VH and VL regions are further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In different embodiments of the present invention, the FRs (or antigen-binding portions thereof) of the anti-big-ET-1 antibody may be identical to the human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence may be defined based on a side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and their equivalents include any naturally occurring, enzymatically obtainable, synthetic, or genetically modified polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be derived from whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized.The DNA can be sequenced and manipulated chemically or using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add, or delete amino acids.

[0151] As used herein, "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity-determining region (CDR) regions, as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an scFv protein is a recombinant single-chain polypeptide molecule in which an immunoglobulin light chain variable region and a heavy chain variable region are connected by a peptide linker. In some exemplary embodiments, an antibody fragment contains sufficient amino acid sequence of a parent antibody to be a fragment that binds to the same antigen as the parent antibody, and in some exemplary embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and competes with the parent antibody for binding to the antigen. Antibody fragments can be produced by any means. For example, antibody fragments can be enzymatically or chemically produced by fragmentation of an intact antibody and / or recombinantly produced from a gene encoding a partial antibody sequence. Alternatively or additionally, antibody fragments can be wholly or partially synthetically produced. Antibody fragments can optionally include single-chain antibody fragments. Alternatively or additionally, antibody fragments can include multiple chains linked together, for example, by disulfide bonds. Antibody fragments can optionally include multimolecular complexes. Functional antibody fragments typically contain at least about 50 amino acids, more typically at least about 200 amino acids.

[0152] The term "bispecific antibody" includes antibodies capable of selectively binding two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, with each heavy chain specifically binding to a different epitope, either on two different molecules (e.g., multiple antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least 1-2, 1-3, or 1-4 orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody can be on the same target or different targets (e.g., the same protein or different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences can be expressed in cells that express immunoglobulin light chains.

[0153] A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain; an immunoglobulin light chain, none of which provides antigen-binding specificity but which is capable of binding to each heavy chain; or an immunoglobulin light chain capable of binding to each heavy chain and to one or more epitopes bound by the heavy chain antigen-binding region; or an immunoglobulin light chain capable of binding to each heavy chain and which may be capable of binding one or both epitopes of one or both heavy chains. BsAbs can be divided into two major classes: those with an Fc region (IgG-like) and those lacking an Fc region; the latter are usually smaller than IgG and Fc-containing IgG-like bispecific molecules. IgG-like bsAbs can have different formats, such as, but not limited to, triomabs, knobs-into-holes IgG (kih IgG), crossmab, orth-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual action Fab (DAF), IgG single chain Fv (IgG-scFv), or κλ-body. Different non-IgG-like formats include tandem scFvs, diabody formats, single-chain diabodies, tandem diabodies (TandAbs), dual affinity retargeting molecules (DARTs), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachings of which are incorporated herein).Methods for producing bsAbs include, but are not limited to, quadroma technology based on somatic cell fusion of two different hybridoma cell lines, chemical conjugation involving chemical cross-linkers, and genetic approaches utilizing recombinant DNA technology. Examples of bsAbs include those described in U.S. Patent No. 12 / 823838 filed June 25, 2010, U.S. Patent No. 13 / 488628 filed June 5, 2012, U.S. Patent No. 14 / 031075 filed September 19, 2013, U.S. Patent No. 14 / 808171 filed July 24, 2015, U.S. Patent No. 15 / 713574 filed September 22, 2017, and U.S. Patent No. 15 / 713575 filed September 22, 2017. Examples of suitable homodimeric impurities include those disclosed in patent applications such as U.S. Patent No. 15 / 713,569, U.S. Patent No. 15 / 386,453, filed December 21, 2016, U.S. Patent No. 15 / 386,443, filed December 21, 2016, U.S. Patent No. 15 / 22,343, filed July 29, 2016, and U.S. Patent No. 15,814,095, filed November 15, 2017, which are incorporated herein by reference. Low levels of homodimeric impurities can be present at multiple steps during the production of bispecific antibodies. Detection of such homodimeric impurities can be challenging when performed using intact mass spectrometry, due to their low abundance and the fact that these impurities co-elute with the major species when performed using conventional liquid chromatography methods.

[0154] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Such molecules typically bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecifics, can also be processed by the systems and methods disclosed herein.

[0155] As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available and known in the art. Monoclonal antibodies useful in the present disclosure may be prepared using a variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0156] In some exemplary embodiments, the protein of interest may have a pI ranging from about 4.5 to about 9.0. In one particular exemplary embodiment, the pI may be about 4.5, about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9.0. In some exemplary embodiments, the composition may contain two or more types of proteins of interest.

[0157] In some exemplary embodiments, the protein of interest may be produced from mammalian cells. The mammalian cells may be of human origin, or of non-human origin, such as primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their strains (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells and PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLaS3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells, and the like.VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, G H3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK-cells, PK-15 cells or derivatives thereof), any Fibroblasts from tissues or organs (heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph glands, pharyngeal tonsils, tonsils, bone marrow and blood), spleen, and fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit551 cells, Detroit510 cells, etc.) cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F The present invention may include, but is not limited to, mouse L cells, including, but not limited to, mouse L cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDMiC3 cells, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, LM line (mouse L) cells, L-MTK' (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntac cells, SIRC cells, Cn cells, and Jensen cells, Sp2 / 0, NS0, NS1 cells, or derivatives thereof.

[0158] As used herein, the term "protein alkylating agent" refers to an agent used to alkylate specific free amino acid residues in proteins. Non-limiting examples of protein alkylating agents include iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methyl methanethiosulfonate (MMTS), and 4-vinylpyridine, or a combination thereof.

[0159] As used herein, "protein denaturation" may refer to a process that changes the three-dimensional shape of a molecule from its native state. Protein denaturation can be carried out using a protein denaturant. Non-limiting examples of protein denaturants include heat, high or low pH, exposure to a reducing agent such as DTT (see below), or a chaotropic agent. Several chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of a system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic interactions. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauroylsarcosine, urea, and salts thereof.

[0160] As used herein, the term "protein reducing agent" refers to an agent used to reduce disulfide bridges in proteins. Non-limiting examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Ellman's reagent, hydroxylamine hydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or a combination thereof.

[0161] As used herein, the term "variant" of a polypeptide (e.g., a variant of a VEGFR Ig domain) refers to a polypeptide containing an amino acid sequence that is at least about 70-99.9% (e.g., 70, 71, 72, 74, 75, 76, 77, 78, 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 or naturally occurring amino acid sequence of a protein of interest. Sequence comparison can be performed, for example, using the BLAST algorithm, where the parameters of the algorithm are selected to maximize matches between the respective sequences over the entire length of the respective reference sequences (e.g., assuming threshold: 10, word size: 3, query width: maximum match with 0, BLOSUM62 matrix, gap cost: 11 for presence, 1 for extension, and conditional composition score matrix adjustment). A variant of a polypeptide (e.g., a variant of a VEGFR Ig domain) also refers to a polypeptide comprising 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, for example, missense substitutions (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. The following references relate to the BLAST algorithm, which is often used for sequence analysis: BLAST 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, J.C., et al. al.,(1993)Comput.Chem.17:149-163;Hancock,JMet al.,(1994)Comput.Appl.Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M.O., et al., 「A model of evolutionary change in proteins.」 In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R.M., et al., 「Matrices for detecting distant relationships」 In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; 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 (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.the entire teachings of which are incorporated herein.

[0162] Some variants may be covalent modifications that polypeptides undergo either during their ribosomal synthesis (co-translational modification) or after their ribosomal synthesis (post-translational modification, "PTM"). PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at the site of specific characteristic protein sequences (e.g., signature sequences) within the protein backbone. Hundreds of PTMs have been documented, and these modifications consistently affect some aspect of protein structure or function (Walsh, G. "Proteins" (2014) 2nd Edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853, the entire teachings of which are incorporated herein). In certain exemplary embodiments, a protein composition may contain two or more types of protein variants of a protein of interest.

[0163] Protein variants in the case of aflibercept (and proteins sharing structural characteristics of aflibercept, e.g., one or more heavy or light chain regions of aflibercept) can include, but are not limited to, oxidation variants, which can result from oxidation of one or more amino acid residues, for example, at histidine, cysteine, methionine, tryptophan, phenylalanine, and / or tyrosine residues; deamidation variants, which can result from deamidation at asparagine residues and / or deoxyglucosylated arginine residues.

[0164] With respect to aflibercept (and proteins that share structural characteristics of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), oxidation variants include oxidation of histidine residues at His86, His110, His145, His209, His95, His19, and / or His203 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept); Trp58 and / or Trp138 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept). oxidation of a tryptophan residue at Tyr64 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of a tyrosine residue at Tyr64 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of a phenylalanine residue at Phe44 and / or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); and / or oxidation of a methionine residue at Met10, Met20, Met163 and / or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept).

[0165] With respect to aflibercept (and proteins that share structural characteristics of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), deamidated variants can include deamidation of the asparagine residues at Asn84 and / or Asn99 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept).

[0166] With respect to aflibercept (and proteins that share structural characteristics of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), a deoxyglucosonation variant can include 3-deoxyglucosonation of the arginine residue at Arg5 (or the equivalent residue position on a protein that shares certain structural characteristics of aflibercept).

[0167] Protein variants may include both acidic and basic species, where acidic species are typically variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while basic species are variants that elute later than the main peak from CEX or earlier than the main peak from AEX.

[0168] As used herein, the terms "acidic species," "AS," "acidic region," and "AR" refer to protein variants characterized by an overall acidic charge. For example, in recombinant protein preparations, such acidic species 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 antibodies may include variants, structural variants, and / or fragmentation variants. Exemplary variants may include, but are not limited to, deamidated variants, defucosylated 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, enzymatic and / or chemical modification, including, but not limited to, Fc and Fab fragments, fragments lacking the Fab, and fragments lacking the heavy chain variable domain; C-terminal truncated variants; variants with the N-terminal Asp in the light chain removed; and variants with N-terminal truncation of the light chain. Other acidic species variants include variants containing unpaired disulfides, host cell proteins and nucleic acids, chromatographic materials, and medium components. Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17).

[0169] In certain embodiments, a protein composition may contain two or more types of acidic species variants. For example, but not limited to, the total acidic species may be classified based on the chromatographic retention time at which the peak appears. Another example in which the total acidic species may be classified may be based on the type of variant (variant, structural variant, or fragmentation variant).

[0170] The term "acidic species" or "AS" does not refer to process-related impurities. As used herein, the term "process-related impurities" refers to impurities that are present in a composition containing a protein but are not themselves derived from the protein. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatography materials, and media components.

[0171] In an exemplary embodiment, the amount of acidic species in the anti-VEGF composition relative to the protein of interest can be up to about 20%, 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%, including within one or more of the above ranges. Exemplary anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0172] Among the chemical degradation pathways involving acidic or basic species, the two most commonly observed covalent modifications occurring in proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan, and tyrosine are the amino acids most susceptible to oxidation: Met and Cys because of their sulfur atoms, and His, Trip, and Tyr because of their aromatic rings.

[0173] As used herein, the terms "oxidized species," "OS," or "oxidized variant" refer to variants of proteins formed by oxidation. Such acidic species can also be detected by various methods, such as ion exchange, e.g., WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Oxidized variants can result from oxidation occurring at histidine, cysteine, methionine, tryptophan, phenylalanine, and / or tyrosine residues. In particular, with respect to aflibercept (and proteins that share structural characteristics of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), oxidation variants include oxidation of histidine residues at His86, His110, His145, His209, His95, His19, and / or His203 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept); oxidation of Trp58 and / or Trp138 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept); oxidation of a tryptophan residue at Tyr64 (or an equivalent position on a protein that shares certain structural characteristics of aflibercept); oxidation of a tyrosine residue at Phe44 and / or Phe166 (or an equivalent residue position on a protein that shares certain structural characteristics of aflibercept); and / or oxidation of a methionine residue at Met10, Met20, Met163 and / or Met192 (or an equivalent residue position on a protein that shares certain structural characteristics of aflibercept).

[0174] In an exemplary embodiment, the amount of oxidized species in the anti-VEGF composition relative to the protein of interest can 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%, including within one or more of the above ranges. Exemplary anti-VEGF compositions are described below in Section III. In one embodiment, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept or MiniTrap.

[0175] Cysteine ​​residues can undergo spontaneous oxidation to form either intra- or intermolecular disulfide bonds or unimolecular by-products such as sulfenic acids.

[0176] Histidine residues are also highly susceptible to oxidation via reaction with their imidazole ring, which can in turn generate additional hydroxyl species (Li, S, C Schoneich, and R.T. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500, the entire teachings of which are incorporated herein). A proposed mechanism for histidine oxidation is highlighted in Figures 2 and 3. Detailed mechanistic studies are available in Anal. Chem. 2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. 21 (2000) 1093-1097, the entire teachings of which are incorporated herein.

[0177] Oxidation of methionine can lead to the formation of methionine sulfoxide (Li, S, C Schoneich, and R.T. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500). Various possible oxidation mechanisms of methionine residues have been described in the literature (Brot, N., Weissbach, H. 1982. The biochemistry of methionine sulfoxide residues in proteins. Trends Biochem. Sci. 7:137-139, the entire teachings of which are incorporated herein).

[0178] The oxidation of tryptophan can result in a complex mixture of products. The primary products can be N-formylkynurenine and kynurenine, with mono-, di-, and / or tri-oxidation products (Figure 4). Peptides with oxidized Trp modifications are generally classified as kynurenine (KYN), hydroxytryptophan (W), and ketones. OX1 ), and N-formylkynurenine / dihydroxytryptophan (NFK / W OX2 , also called "double-oxidized Trp"), trihydroxytryptophan (W OX3 , also called "triply oxidized Trp"), and hydroxykynurenine (KYN OX1 The formation of these combinations, such as hydroxytryptophan (W, +20 Da), results in mass increases of 4 Da, 16 Da, 32 Da, 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. 2010 Jul;21(7):1114-1117, the entire teachings of which are incorporated herein.) Tryptophan oxidation, but not methionine and histidine oxidation, was found to produce a color change in the protein product (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). Like tryptophan, tyrosine oxidation initially produces 3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li, S, C Schoneich, and RT Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500).

[0179] As used herein, the terms "basic species," "basic region," and "BR" refer to variants of proteins, e.g., antibodies or antigen-binding portions thereof, characterized by an overall basic charge relative to the primary charge variant species present in the protein. For example, in recombinant protein preparations, such basic species 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 mutation, glycosylation, fragmentation, and aggregation. Generally, basic species elute later than the main peak during CEX or earlier than the main peak during AEX analysis. (Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, MAbs. 2012 Sep 1;4(5):578-585. doi:10.4161 / mabs.21328, the entire teachings of which are incorporated herein.)

[0180] In certain embodiments, a protein composition may contain two or more types of basic species variants. For example, but not limited to, the total basic species may be separated based on the chromatographic retention time at which the peaks appear. Another example of how the total basic species may be separated may be based on the type of variant (variant, structural variant, or fragmentation variant).

[0181] As mentioned 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 as a result of storage (referred to herein as "storage-derived basic species").

[0182] In an exemplary embodiment, the amount of basic species in the anti-VEGF composition relative to the protein of interest can 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%, including within one or more of the above ranges. Exemplary anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0183] As used herein, a "sample matrix" or "biological sample" can be obtained from any step in a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), or any step in downstream processing (Drug Substance (DS) or Drug Product (DP) including the final formulated product). In some other specific exemplary embodiments, the biological sample can be selected from any step in downstream processing, such as clarification, chromatographic purification, viral inactivation, or filtration. In some specific exemplary embodiments, the drug product can be selected from a drug product manufactured in the clinic, transported, stored, or handled.

[0184] 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 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.

[0185] As used herein with respect to protein formulations, the term "stable" refers to a protein of interest in a formulation that can retain an acceptable degree of chemical structure or biological function after storage under exemplary conditions defined herein. A formulation can be stable even if the protein of interest contained therein does not maintain 100% of its chemical structure or biological function after storage for a specified period of time. Under certain circumstances, retention of about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of protein structure or function after storage for a specified period of time can be considered "stable."

[0186] 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) to any clinically measurable extent, for example, by causing regression, stabilization, or elimination of one or more symptoms or signs of such disease or disorder (e.g., with respect to an angiogenic eye disorder, by causing a reduction or maintenance of the Diabetic Retinopathy Severity Score (DRSS), improving or maintaining vision (e.g., in terms of best-corrected visual acuity, e.g., as measured by an increase in letters on the ETDRS visual acuity test), increasing or maintaining visual field, and / or reducing or maintaining central retinal thickness; with respect to cancer, by arresting or regressing the proliferation, survival time, and / or metastasis of cancer cells in the subject). Typically, a therapeutic measure is the administration of a therapeutically effective amount of VEGF MiniTrap to a subject with a disease or disorder in one or more doses.

[0187] As used herein, the term "upstream processing technologies" in the context of protein preparation refers to the operations involved in producing and harvesting a protein from cells during or after cell culture of a protein of interest. As used herein, the term "cell culture" refers to methods and techniques for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as for optimizing the production and harvest of the protein of interest. For example, once an expression vector is introduced into a suitable host cell, the host cell can be maintained under conditions suitable for expression of the relevant nucleotide coding sequence and the harvest and production of the desired recombinant protein.

[0188] When using the cell culture techniques of the present invention, the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In embodiments in which the protein of interest is produced intracellularly, particulate debris (e.g., resulting from homogenization), either host cells or lysed cells, can be removed by various means, including, but not limited to, centrifugation or ultrafiltration. If the protein of interest is secreted into the medium, the supernatant from such expression systems can first be concentrated using a commercially available protein concentration filter, for example, using an Amicon™ Millipore Pellicon™ ultrafiltration unit. In one embodiment, the protein of interest can be recovered by centrifugation followed by depth filtration and then affinity capture chromatography.

[0189] As used herein, a "VEGF antagonist" is any protein or peptide that binds to or interacts with VEGF. Typically, such binding or interaction inhibits VEGF from binding to its receptors (VEGFR1 and VEGFR2) and / or inhibits the biological signaling of VEGF and its activity. VEGF antagonists include molecules that interfere with the interaction of VEGF with a native VEGF receptor, such as molecules that bind to VEGF or a VEGF receptor, and molecules that prevent or otherwise interfere with the interaction of VEGF with a VEGF receptor. Certain exemplary VEGF antagonists include anti-VEGF antibodies (e.g., ranibizumab [LUCENTIS®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, and their equivalents), and VEGF receptor-based chimeric molecules or VEGF inhibitory fusion proteins ("VEGF-Trap" or "VEGF"), such as aflibercept, dib-aflibercept, and a protein having an amino acid sequence having SEQ ID NO: 60. Other examples of VEGF-Traps include ALT-L9, M710, FYB203, and CHS-2020. Additional examples of VEGF-Traps can be found in U.S. Patent Nos. 7,070,959, 7,306,799, 7,374,757, 7,374,758, 7,531,173, 7,608,261, 5,952,199, 6,100,071, 6,383,486, 6,897,294, and 7,771,721, each of which is specifically incorporated herein by reference in its entirety.

[0190] VEGF receptor-based chimeric molecules include chimeric polypeptides comprising two or more immunoglobulin (Ig)-like domains of VEGF receptors, such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), which may also comprise a multimerization domain (e.g., an Fc domain that promotes multimerization (e.g., dimerization) of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is a molecule designated VEGFR1R2-FcΔC1(a) (also known as aflibercept and sold under the trade name EYLEA®). In certain exemplary embodiments, aflibercept is It contains the amino acid sequence set forth as TIFF0007744391000017.tif43165 (SEQ ID NO: 55).

[0191] As used herein, "virus filtration" may include filtration using a suitable filter, including, but not limited to, Planova 20N™, Planova 50N, or BioEx™ manufactured by Asahi Kasei Pharma, Viresolve™ filters manufactured by EMD Millipore, ViroSart CPV manufactured by Sartorius, and Ultipor DV20 or Ultipor DV50™ manufactured by Pall Corporation. Selecting a suitable filter to obtain the desired filtration performance will be apparent to one skilled in the art.

[0192] II. Color Judgment As used herein, the color observed during the production of a recombinant protein, particularly an anti-VEGF protein, can be measured by a variety of methods, including, but not limited to, the Iodine Color Index, Hazen Color Index, Gardner Color Index, Lovibond Color Index, Saybolt Color Index, Mineral Oil Color Index, European Pharmacopoeia Color Index, United States Pharmacopoeia Color Index, CIE L * , a * , b *(or CIELAB), Klett color scale, Hess-Ives color scale, yellowness index, ADMI color scale, and ASBC and EBC Breweries color scales. Details on such scales can be found in Application Report No. 3.9e by Lange, the entire teachings of which are incorporated herein.

[0193] Visual color matching based on the standards of the European Pharmacopoeia (Ph Eur) (European color standards, European Pharmacopoeia. Chapter 2.2.2. Degree of coloration of liquids. 8 th ed. EP, the entire teachings of which are incorporated herein) can involve the preparation of color reference liquids as described in the European Pharmacopoeia (EP 2.2.2. Degree of Coloration of Liquids 2): using three parent solutions, red (cobalt(II) chloride), yellow (iron(III) chloride), and blue (copper(II) sulfate), and 1% hydrochloric acid, five reference solutions are prepared: yellow (Y), green-yellow (GY), tan (BY), brown (B), and red (R) hues. These five reference solutions are used in turn to prepare a total of 37 reference solutions (Y1-Y7, GY1-GY7, BY1-BY7, B1-B9, and R1-R7). Each reference liquid is clearly defined in the CIE-Lab color space, e.g., by lightness, hue, and saturation. Of the seven tan standards (BY standards), BY1 is the darkest standard and BY7 is the lightest dark. Matching of a given sample with a BY color standard is typically performed under diffused sunlight. The composition of the European tan standards is set forth in Table 1 below.

[0194] Table 1. Composition of the European Tan Standard TIFF0007744391000018.tif51166 Tan standard solution (BY): 10.8 g / L FeCl3.6H2O, 6.0 g / L CoCl2.6H2O, and 2.5 g / L CuSO4.5H2O

[0195] The color of a liquid is examined by comparing the test liquid with a color standard solution. The composition of the color standard solution is selected depending on the hue and intensity of the color of the test liquid. The comparison is typically performed in a colorless, clear, neutral glass, flat-bottomed test tube (e.g., approximately 12 mm, 15 mm, 16 mm, or 25 mm diameter test tube) that matches the inner diameter and all other dimensions as closely as possible. For example, the comparison can be between 2 and 10 mL of test liquid and the color standard solution. The liquid depth can be, for example, approximately 15 mm, 25 mm, 40 mm, or 50 mm. The color assigned to the test liquid should not be darker than the standard color. Color comparisons are typically performed against a white background in diffused light (e.g., sunlight). Colors can be compared down the vertical or horizontal axis of the test tube.

[0196] In contrast to EP color measurement, United States Pharmacopoeia Monograph 1061, Color - Instrumental Measurement is based on the CIE L * , a * , b * (or CIELAB) color measurement to accurately and objectively quantify color. A total of 20 color reference solutions (identified sequentially by the letters A through T) are defined by the United States Pharmacopeia. The measured color of a sample is automatically correlated with the color reference solution. This is done by selecting the color reference solution that is closest to the sample (i.e., the one with the smallest color difference ΔE * This means that the reference solution having ΔL is displayed. * value, Δa * value and Δb * The value is the L * value, a * value, b * values ​​and the L of the USP solution displayed * value, a * value, b * It shows the quantitative difference between the CIE L * , a * , b * In the coordinate system, L * represents the brightness of the color on a scale of 0 to 100 (0 being the darkest and 100 being the brightest), and a * represents the colors red and green (a * Positive values ​​of represent red, while a *Negative values ​​of represent green), b * represents the yellow or blue color of the sample (b * Positive values ​​of b represent yellow, while * (Negative values ​​of ΔL represent blue.) The color difference from the standard or the first sample in the evaluation is calculated by the ΔL * , Δa * and Δb * The composite change, or color difference, can be represented by a change in TIFF0007744391000019.tif5128 and can be calculated as a simple Euclidean distance in space. CIE L * , a * , b * Color coordinates can be generated, for example, using a Hunter Labs UltrascanPro (Hunter Associates Laboratory, Reston, Virginia) or on a BYK Gardner LCS IV (BYK-Gardner, Columbia, Maryland). For the Hunter Labs UltraScanPro, a Didymium Filter Test can be performed to calibrate the wavelength. The instrument can be standardized on a TTRAN with a 0.780-inch port insert and DIW before use. This uses a light trap and a black plate to establish the top (L=100) and bottom (L=0) of the photometric scale. See Pack et al., "Modernization of Physical Appearance and Solution Color Tests Using Quantitative Tristimulus Colorimetry: Advantages, Harmonization, and Validation Strategies," J. Pharmaceutical Sci. 104:3299-3313 (2015), the entire teachings of which are incorporated herein. The BY standard color is also known as the CIE L * , a * , b * It can be expressed under a color space ("CIELAB" or "CIELab" color space). See Table 2.

[0197] (Table 2) CIE L * , a * , b * Characterization of the European tan standard in color space TIFF0007744391000020.tif63170 ^ Reported by Pack et al. ~ For each BY color standard, the L * value and b * Values ​​are measured experimentally.

[0198] To enable high-throughput screening of color assays, a spectrophotometric assay (CIELAB) is a more suitable quantitative measurement than BY color standards. The surrogate assay was further optimized as described in the Examples section.

[0199] For any sample evaluated for color, the protein concentration of the test sample must be normalized to the protein concentration in the sample for comparison, e.g., 5 g / L, 10 g / L, and the like.

[0200] III. Anti-VEGF composition There are at least five members of the VEGF family of proteins that regulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). Anti-VEGF compositions can include VEGF antagonists, which specifically interact with one or more members of the VEGF family of proteins and inhibit one or more of their biological activities, such as, for example, mitogenic activity, angiogenic activity, and / or vascular permeability activity.

[0201] In one embodiment, the method for producing an anti-VEGF protein comprises: (a) providing a host cell that has been genetically modified to express an anti-VEGF protein; (b) culturing the host cell in CDM under conditions suitable for the cell to express the anti-VEGF protein; and (c) recovering the preparation of the anti-VEGF protein produced by the cell. In one aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0202] The present inventors discovered that producing an anti-VEGF protein (e.g., aflibercept) in a specific CDM produced a biological sample with a distinct color. Distinct color characteristics were observed during different manufacturing steps, as well as in the final formulation containing the anti-VEGF protein. As observed in Example 9, for the production of VEGF MiniTrap, culturing cells in CDM produced an anti-VEGF protein (e.g., aflibercept) with a strong yellow-brown color. The post-harvest affinity capture step also produced an eluate with a specific color, such as yellow-brown. Further manufacturing steps using AEX also produced a yellow-brown color, but the intensity was reduced.

[0203] As described in more detail below, color can be assessed using either (i) the European color standard "BY," which is a qualitative visual test, or (ii) CIELAB, a colorimetric assay that is more quantitative than the BY system. In either case, however, color assessments across samples were normalized to protein concentration to ensure meaningful studies / comparisons. For example, referring to Example 9, and particularly Table 9-2, the Protein A eluate had a "b" of approximately 2.52. *" value, which corresponds to an approximate BY value of BY5 (measured at 5 g / L protein concentration in Protein A eluate). If the color of a Protein A eluate is to be compared with another sample, then the comparison must be made using the same protein concentration. Therefore, a b value of approximately 0.74 is used for Protein A eluate. * When compared to the AEX pool, which has a high chromatographic value (as measured at a protein concentration of 5 g / L in the Protein A eluate), this production method shows a substantial reduction in the yellow-brown color of samples from the Protein A eluate relative to the AEX pool after AEX chromatography.

[0204] The compositions of the present invention may be characterized by a tan color as described herein, for example, a darkness / intensity equivalent to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), having a b of 17-23, 10-17, 5-10, 3-5, or 1-3. * The composition has a value of about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the clarified harvest or as a Protein A eluate of the clarified harvest.

[0205] In one embodiment, the compositions of the invention produced using CDM produce biological samples with a distinct tan color, which samples: (i) A yellow-brown color similar to the European color standard BY2; (ii) a tan color similar to the European color standard BY3; (iii) a tan color similar to the European color standard BY4; (iv) a tan color similar to the European color standard BY5; (v) European color standard between BY2 and BY3; (vi) European color standard between BY3 and BY4; (vii) European color standard between BY4 and BY5 The composition may be characterized by standard color characterization as recognized by NIH 2004 / 010222. The composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the clarified harvest Protein A eluate.

[0206] In another embodiment, the compositions of the present invention produced using CDM produce biological samples with different tan colors, which range from 100 to 1200 colors on the CIELAB scale: (i) About 22-23 b * Yellow-brown color comparable to the value; (ii) about 16-17 b * Yellow-brown color comparable to the value; (iii) 9-10 b * Yellow-brown color comparable to the value; (iv) 4-5 b * Yellow-brown color comparable to the value; (v) 2-3 b * Yellow-brown color comparable to the value; (vi) b between 17 and 23 * value; (vii) b between 10 and 17 * value; (viii) b between 5 and 10 * value; (ix) b between 3 and 5 * value; or (x) b between 1 and 3 * value The composition may be characterized by standard color characterization as recognized by NIH 2004 / 010222. The composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the clarified harvest Protein A eluate.

[0207] In one embodiment, compositions of the invention produced using CDM may contain other species or variants of anti-VEGF proteins. These variants include anti-VEGF protein isoforms that contain one or more oxidized amino acid residues, collectively referred to as oxovariants. Enzymatic digestion of such compositions containing anti-VEGF proteins and their oxovariants can be performed as follows: EIGLLTC, containing approximately 0.004-0.013% 2-oxo-histidine * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing approximately 0.006-0.028% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH, containing approximately 0.049-0.085% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing approximately 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing approximately 0.008-0.022% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 0.185 to 0.298% tryptophan dioxide; or EIGLLTC, containing approximately 0.008% 2-oxo-histidine * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing approximately 0.02% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), Contains approximately 0.06% 2-oxo-histidine, TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), DKTH, containing approximately 0.07% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing approximately 0.01% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing approximately 0.23% di-oxotryptophan may contain one or more of H* is histidine that can be oxidized to 2-oxo-histidine, and C * is a cysteine ​​that can be carboxymethylated. In certain embodiments, the anti-VEGF protein is aflibercept. In another embodiment, the anti-VEGF protein is VEGF MiniTrap.

[0208] In an exemplary embodiment of the present invention, a composition of the present invention comprises an anti-VEGF protein, wherein about 1% or less, about 0.1% or less, or about 0.1-1%, about 0.2-1%, about 0.3-1%, about 0.4-1%, about 0.5-1%, about 0.6-1%, about 0.7-1%, about 0.8-1%, or about 0.9-1% of the histidine residues in the anti-VEGF protein are 2-oxo-histidine. In such a composition, there may be a heterogeneous population of anti-VEGF protein variants having varying amounts of 2-oxo-histidine residues and non-oxidized histidine residues. Therefore, the percentage of 2-oxo-histidine anti-VEGF protein in a composition refers to the site-specific 2-oxo-histidine in the anti-VEGF molecule divided by the total site-specific histidines in the anti-VEGF protein (oxidized plus non-oxidized) molecule, multiplied by 100. One method of quantifying the level of 2-oxo-histidine in a composition is to digest the polypeptide with a protease (e.g., Lys-C and / or trypsin) and analyze the amount of 2-oxo-histidine in the resulting peptides, for example, by mass spectrometry (ms).

[0209] Prior to digestion of the anti-VEGF protein, the sulfhydryl groups of the cysteines were blocked by reaction with iodoacetamide (IAM), resulting in the following chemical structure: TIFF0007744391000021.tif17128. Such modification prevents free thiols from reforming disulfide bridges and prevents scrambling of disulfide bonds. The present invention includes compositions (e.g., aqueous compositions) comprising anti-VEGF proteins and variants thereof, which, when modified with IAM, digested with proteases (e.g., Lys-C and trypsin), and analyzed by mass spectrometry, yield the following peptides: EIGLLTC, containing approximately 0.004-0.013% 2-oxo-histidine * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing approximately 0.006-0.028% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH, containing approximately 0.049-0.085% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing approximately 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing approximately 0.008-0.022% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 0.185 to 0.298% tryptophan dioxide; or EIGLLTC, containing approximately 0.008% 2-oxo-histidine * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing approximately 0.02% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), Contains approximately 0.06% 2-oxo-histidine, TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), DKTH, containing approximately 0.07% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing approximately 0.01% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing approximately 0.23% di-oxotryptophan where H * is 2-oxo-histidine, and C * is a carboxymethylated cysteine. In one embodiment of the invention, the peptide is deglycosylated with PNGase F.

[0210] The present invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of all histidines in the anti-VEGF protein are modified to 2-oxo-histidine, and further wherein the color of the composition is, for example, equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), alternatively, a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * b characterized by using * The composition has a concentration of about 5 g / L or about 10 g / L of anti-VEGF protein. The composition can be obtained either as a clarified harvest or as a sample derived from a Protein A eluate of the clarified harvest. Such a composition can be obtained from a clarified harvest when the harvested material is subjected to a capture chromatography procedure. In one embodiment, the capture step is an affinity chromatography procedure, for example, using a Protein A affinity column. When the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more variants can be detected.

[0211] The present invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophans of the anti-VEGF protein are modified to kynurenines, and further wherein the color of the composition is equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or has a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * As characterized by b * The composition has a value of about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from a clarified harvest or a Protein A eluate of a clarified harvest. Such a composition can be obtained from a clarified harvest when subjected to a capture chromatography procedure. The capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. When the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0212] The present invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of the total tryptophans of the anti-VEGF protein are modified to mono-hydroxyltryptophan, and further wherein the color of the composition is equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or has a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * characterized by b *The composition has a value of about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from a clarified harvest or a Protein A eluate of a clarified harvest. Such a composition can be obtained from a clarified harvest when subjected to a capture chromatography procedure. The capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. When a sample extracted from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0213] The present invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of the total tryptophans of the anti-VEGF protein are modified to di-hydroxyltryptophan, and further wherein the color is equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or has a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * b characterized by using * The composition has a concentration of about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from a clarified harvest or a Protein A eluate of a clarified harvest. Such a composition can be obtained from a clarified harvest made using a CDM containing anti-VEGF protein and its oxo variants that is subjected to a capture chromatography procedure. The capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. When the sample extracted from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0214] The present invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of the total tryptophans of the anti-VEGF protein are modified to tri-hydroxyltryptophan, and further wherein the color of the composition is equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or has a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * characterized by b * The composition has a value of about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from a clarified harvest or a Protein A eluate of a clarified harvest. Such a composition can be obtained using capture chromatography. The capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. When the affinity-extracted sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0215] In one embodiment, the compositions of the present invention may comprise an anti-VEGF protein, wherein the anti-VEGF protein may contain modifications of one or more residues, such as: one or more asparagines are deamidated; one or more aspartic acids are converted to isoaspartate and / or Asn; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenines; one or more tryptophans are mono-hydroxyltryptophans; one or more tryptophans are di-hydroxyltryptophans; one or more tryptophans are tri-hydroxyltryptophans; one or more arginines are converted to Arg3-deoxyglucosone; the C-terminal glycine is absent; and / or one or more unglycosylated glycosites are present.

[0216] Such compositions can be obtained, for example, from clarified harvests made using CDM containing anti-VEGF proteins and variants thereof that are subjected to a capture chromatography procedure, such as an affinity chromatography procedure using a Protein A column. When the sample extracted from the affinity procedure is analyzed, for example, using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0217] In an exemplary embodiment, a composition of the invention may comprise an anti-VEGF protein sharing structural characteristics of aflibercept that may be oxidized at one or more of the following: His86, His110, His145, His209, His95, His19, and / or His203 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept), Trp58 and / or Trp138 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept), Tyr64 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept), Phe44 and / or Phe166 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept), and / or Met10, Met20, Met163, and / or Met192 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept). Such composition can be obtained from the clarified collection of CDM that contains aflibercept and its oxo variants, which is subjected to capture chromatography procedure.This capture step can be, for example, an affinity chromatography procedure using a protein A column.When the sample extracted from the affinity step is analyzed, for example, by liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

[0218] In one embodiment, a composition of the invention can comprise a VEGF MiniTrap having the amino acid sequence of SEQ ID NO: 46, which can be oxidized at His86, His110, His145, His209, His95, His19, and / or His203; Trp58 and / or Trp138; Tyr64; Phe44 and / or Phe166; and / or Met10, Met20, Met163, and / or Met192. Such a composition can be obtained from a clarified harvest made using a CDM containing VEGF MiniTrap and its oxo variants, which is subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure, for example, using a Protein A column, and one or more of these variants can be detected when analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0219] In some exemplary embodiments, compositions of the invention may include anti-VEGF proteins and variants thereof (including oxovariants), wherein the amount of protein variant in the composition may be up to about 20%, 19%, 18%, 17%, 16%, 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%, or within one or more of the above ranges. Such compositions can be obtained from clarified harvests made using CDMs containing anti-VEGF proteins and variants thereof that are subjected to a capture chromatography procedure. The capture step can be, for example, an affinity chromatography procedure using a Protein A column, and when analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected. In one embodiment, the color of such compositions is, for example, equivalent in darkness / intensity to the European Tan Standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * characterized by b * value, and the composition comprises about 5 g / L or about 10 g / L of anti-VEGF protein.

[0220] In other exemplary embodiments, the compositions of the invention comprise an anti-VEGF protein and its variants, and the amount of protein variant in the composition can be from about 0% to about 20%, for example, from about 0% to about 20%, from about 0.05% to about 20%, from about 0.1% to about 20%, from about 0.2% to about 20%, from about 0.3% to about 20%, from about 0.4% to about 20%, from about 0.5% to about 20%, from about 0.6% to about 20%, from about 0.7% to about 20%, from about 0.8% to about 20%, from about 0.9% to about 20%, from about 1% to about 20%, from about 1.5% to about 20%, from about 2 ... % to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, 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 1 0%, 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% The composition may be within one or more of the above ranges. Such compositions may be obtained by performing capture chromatography on the collected sample. This capture step is, for example, an affinity chromatography procedure using a Protein A column.One or more of these variants can be detected when a sample is analyzed using liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the color of such a composition is, for example, equivalent in darkness / intensity to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * , a * , b * characterized by b * value, and the composition comprises about 5 g / L or about 10 g / L of anti-VEGF protein.

[0221] In one embodiment, a composition of the invention can include an anti-VEGF protein including its acidic species, wherein the amount of acidic species in the composition can be about 20%, 19%, 18%, 17%, 16%, 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%, or within one or more ranges thereof. As mentioned above, such acidic species can be detected by various methods, such as ion exchange, e.g., WCX (WCX-10 HPLC, weak cation exchange chromatography), or IEF (isoelectric focusing). Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17). Compositions containing acidic species can be obtained from biological materials, such as harvested or affinity purified materials using ion exchange chromatography.

[0222] In one embodiment, the color of such compositions is, for example, of a darkness / intensity equivalent to a European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or a CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3.* , a * , b * characterized by b * The composition has a pH of about 5 g / L or about 10 g / L. As an example, referring to Figures 16 and 17, fractions F1 and F2 represent acidic fractions containing the majority of acidic species. Peaks 1 and 2 of MT1 in Figure 17 contain acidic species, and fractions F1 and F2 contain the majority of the acidic fraction. The fractions containing such acidic species (F1 and F2) also exhibited a yellow-brown color compared to the other fractions (Figures 18B and 18C).

[0223] In another embodiment, the composition of the present invention comprises an anti-VEGF protein including its acidic species, and the amount of acidic species in the composition can be from about 0% to about 20%, for example, from about 0% to about 20%, from about 0.05% to about 20%, from about 0.1% to about 20%, from about 0.2% to about 20%, from about 0.3% to about 20%, from about 0.4% to about 20%, from about 0.5% to about 20%, from about 0.6% to about 20%, from about 0.7% to about 20%, from about 0.8% to about 20%, from about 0.9% to about 20%, from about 1% to about 20%, from about 1.5% to about 20%, from about 2% to about 20%, from about 3% to about 20%, or from about 3% to about 20%. % to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, 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 may be within one or more of the above ranges. As mentioned above, these acidic species can be detected by various methods, such as ion exchange, for example WCX (WCX-10 HPLC, weak cation exchange chromatography), or IEF (isoelectric focusing).Typically, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17).

[0224] Using a cation exchange column, all peaks eluting before the main peak of interest were summed as the acidic region, and all peaks eluting after the protein of interest were summed as the basic region. In exemplary embodiments, acidic species can elute as two or more acidic regions, which can be numbered AR1, AR2, AR3, etc., based on the specific retention time of the peaks and the ion exchange column used.

[0225] In one embodiment, a composition can include an anti-VEGF protein that includes an acidic species, and AR1 is 20%, 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%, or within one or more of the above ranges. In one embodiment, a composition can include an anti-VEGF protein containing its acidic species, wherein AR1 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%, about 8% to about 10%, or about 10% to about 15%, including any one or more of the above ranges. As noted above, such acidic regions can be detected by various methods, such as ion exchange, e.g., WCX (WCX-10 HPLC, weak cation exchange chromatography), or IEF (isoelectric focusing). Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17).

[0226] In another embodiment, a composition can include an anti-VEGF protein that includes an acidic species, and AR2 is 20%, 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%, or within one or more of the above ranges. In one embodiment, a composition can include an anti-VEGF protein that includes an acidic species, and AR2 is between about 0.0% and about 10%, between about 0.0% and about 5%, between about 0.0% and about 4%, between about 0.0% and about 3%, between about 0.0% and about 2%, between about 3% and about 5%, between about 5% and about 8%, or between about 8% and about 10%, or between about 10% and about 15%, including any one or more of the above ranges.

[0227] In one embodiment, a composition can include an anti-VEGF protein including a basic species thereof, wherein the amount of basic species in the composition can be up to about 20%, 19%, 18%, 17%, 16%, 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%, or within one or more ranges thereof.In one embodiment, a composition can include an anti-VEGF protein and a basic species thereof, and the amount of the basic species in the composition relative to the anti-VEGF protein can be from about 0% to about 20%, e.g., 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, 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% 0.5%, 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 may be within one or more of the above ranges.

[0228] Basic species can elute as two or more basic regions, which can be numbered BR1, BR2, BR3, etc., based on the particular retention time of the peak and the ion exchange used.

[0229] In one embodiment, a composition can include an anti-VEGF protein that includes a basic species, 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%, or within one or more of the above ranges. In one embodiment, a composition can include an anti-VEGF protein and a basic species thereof, wherein BR1 is between about 0.0% and about 10%, between about 0.0% and about 5%, between about 0.0% and about 4%, between about 0.0% and about 3%, between about 0.0% and about 2%, between about 3% and about 5%, between about 5% and about 8%, or between about 8% and about 10%, or between about 10% and about 15%, including any one or more of the above ranges.

[0230] In another embodiment, a composition can include an anti-VEGF protein and a basic species thereof, wherein 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%, or within one or more of the above ranges. In one embodiment, a composition can include an anti-VEGF protein and a basic species of the anti-VEGF protein, wherein BR2 is between about 0.0% and about 10%, between about 0.0% and about 5%, between about 0.0% and about 4%, between about 0.0% and about 3%, between about 0.0% and about 2%, between about 3% and about 5%, between about 5% and about 8%, or between about 8% and about 10%, or between about 10% and about 15%, including any one or more of the above ranges.

[0231] In another embodiment, a composition can include an anti-VEGF protein and a basic species thereof, wherein BR3 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%, or within one or more of the above ranges. In one embodiment, a composition can include an anti-VEGF protein and a basic species of the anti-VEGF protein, wherein BR3 is between about 0.0% and about 10%, between about 0.0% and about 5%, between about 0.0% and about 4%, between about 0.0% and about 3%, between about 0.0% and about 2%, between about 3% and about 5%, between about 5% and about 8%, or between about 8% and about 10%, or between about 10% and about 15%, including any one or more of the above ranges.

[0232] Light-induced oxidation of aflibercept In addition to discovering different color characteristics of anti-VEGF protein compositions or variants thereof produced using CDM, the inventors have also discovered that such compositions can be artificially produced in the laboratory by exposure to light.

[0233] Modified variants of the anti-VEGF composition, including oxidation, can be generated by exposing the anti-VEGF protein to cool white light or ultraviolet light. In one embodiment, the anti-VEGF composition comprises about a 1.5 to about a 50-fold increase in one or more modified oligopeptides compared to the sample, wherein the oligopeptides are: DKTH * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), TNYLTH *R (SEQ ID NO: 21), SDTGRPFVEMYSEIPEIIH * MTEGR (SEQ ID NO: 22), VH * EKDK (SEQ ID NO: 23), SDTGRPFVEM * YSEIPEIIHMTEGR (SEQ ID NO: 64), SDTGRPFVEMYSEIPEIIHM * TEGR (SEQ ID NO: 65), TQSGSEM * K (SEQ ID NO: 66), SDQGLYTC * AASSGLM * TK (SEQ ID NO: 67), IIW * DSR (SEQ ID NO: 28), RIIW * DSR (SEQ ID NO: 115), IIW * DSRK (SEQ ID NO: 114), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO: 70) F * LSTLTIDGVTR (SEQ ID NO: 32) wherein H * is histidine and is oxidized to 2-oxo-histidine, and C * is a cysteine ​​and is carboxymethylated, and M * is oxidized methionine, and W * is oxidized tryptophan, and Y * is oxidized tyrosine, and F *is oxidized phenylalanine. In a further embodiment, the anti-VEGF composition can comprise about a 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing the anti-VEGF composition to cool white light for a period of time, e.g., about 30 hours. In another embodiment, the anti-VEGF composition can comprise about a 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing the sample to cool white light for about 75 hours. In yet another embodiment, the anti-VEGF composition can comprise about a 1.5 to about 20-fold increase in one or more oligopeptides described above by exposing the sample to cool white light for about 100 hours. In yet another embodiment, the anti-VEGF composition can comprise about a 1.5 to about 20-fold increase in one or more oligopeptides described above by exposing the sample to cool white light for about 150 hours. In yet another embodiment, the anti-VEGF composition can comprise about a 1.5 to about a 50-fold increase in one or more oligopeptides upon exposing the sample to cool white light for about 300 hours (see Example 4 below).

[0234] The anti-VEGF composition can include about a 1.5 to about a 3-fold increase in one or more oligopeptides as described above by exposing a sample of the anti-VEGF composition to UV light for about 4 hours. In another embodiment, the anti-VEGF composition can include about a 1.5 to about a 10-fold increase in one or more oligopeptides by exposing the sample to UV light for about 10 hours. In yet another embodiment, the anti-VEGF composition can include about a 1.5 to about a 10-fold increase in one or more described oligopeptides by exposing the sample to UV light for about 16 hours. In yet another embodiment, the anti-VEGF composition can include about a 1.5 to about a 25-fold increase in one or more oligopeptides by exposing the sample to UV light for about 20 hours. In yet another embodiment, the anti-VEGF composition can include about a 1.5 to about a 25-fold increase in one or more oligopeptides by exposing the sample matrix to UV light for about 40 hours. See Example 4.

[0235] Anti-VEGF proteins generated using glycodiversity-CDM The compositions of the invention comprise anti-VEGF proteins, and the anti-VEGF proteins produced in CDM have various glycodiversities. Different glycosylation profiles of the anti-VEGF proteins are within the scope of the invention.

[0236] In some exemplary embodiments of the invention, the composition can include an anti-VEGF protein glycosylated at one or more asparagines, such as 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, G1 FS 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. In one embodiment, the protein of interest can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0237] In one embodiment, the composition can have the following glycosylation profile: about 40% to about 50% total fucosylated glycans, about 30% to about 50% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (Example 6).

[0238] In one embodiment, the composition can include an anti-VEGF protein, wherein the protein of interest has Man5 glycosylation at about 32.4% of the asparagine 123 residue and / or about 27.1% of the asparagine 196 residue. In one aspect, the protein of interest can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0239] In another embodiment, the composition may have about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% total fucosylated glycans.

[0240] In yet another embodiment, the composition may have about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, or about 50% total sialylated glycans.

[0241] In one embodiment, the composition may comprise about 6%, about 7%, about 8%, about 8%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15% mannose-5.

[0242] In another embodiment, the composition may have about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, or about 79% total galactosylated glycans.

[0243] In one embodiment, the anti-VEGF protein may have levels of fucosylated 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%. Compared to the levels of fucosylated glycans in anti-VEGF proteins produced using soy hydrolysate, 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%, 2-50%, 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- and one or more of the above values, such as 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 one embodiment, the anti-VEGF protein 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%. Compared to the levels of sialylated glycans in anti-VEGF proteins produced using soy hydrolysates, 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%, 2-50%, 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%, 2-9 and one or more of the above values, such as 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] In another embodiment, the anti-VEGF protein may have levels 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%. Compared to the levels of galactosylated glycans in anti-VEGF proteins produced using soy hydrolysates, 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%, and one or more of the above values, such as 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%.

[0246] In one embodiment, the anti-VEGF protein 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%. Compared to the levels of mannosylated glycans in anti-VEGF proteins produced using soy hydrolysates, 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%, 2–50%, 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%, 2–9 and one or more of the above values, such as 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%.

[0247] The compositions described in this section can be produced by multiple upstream and downstream parameters, which are described in Sections IV and V, respectively, below.

[0248] IV. Preparation of Compositions Using Upstream Process Technology For biopharmaceuticals, implementing a robust and flexible upstream process is desirable. An efficient upstream process can lead to desirable production and scale-up of the protein of interest. The inventors have demonstrated that compositions of the present invention containing anti-VEGF proteins can be produced by adjusting conditions during upstream protein production, such as changing the media components of the CDM. Each step in the upstream process can affect the quality, purity, and quantity of the protein produced.

[0249] The present disclosure demonstrates the existence of specific variants of aflibercept and / or MiniTrap produced using CDM. These variants include isoforms containing one or more oxidized amino acid residues. Examples of oxidized residues include, but are not limited to, one or more histidine, tryptophan, methionine, phenylalanine, or tyrosine residues. Compositions produced using modified CDM can produce anti-VEGF protein preparations with desired target levels of aflibercept and / or MiniTrap protein variants. As noted above, a yellow-brown color associated with fragments produced using CDM may also be present. (As noted above, not all CDMs tested by the inventors showed significant discoloration.)

[0250] The present invention involves culturing host cells in the modified CDM under suitable conditions such that the cells express a recombinant protein of interest, followed by recovering a preparation of the recombinant protein of interest produced by the cells. Such modified CDM can be used to produce the compositions described in Section III above. (Note that the CDM is a medium that is yellow-brown when aflibercept is expressed.)

[0251] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under suitable conditions. The method further includes recovering a preparation of the recombinant protein of interest produced by the cells, the suitable conditions including a CDM having a cumulative iron concentration in the CDM of less than about 55 μM, a cumulative copper concentration in the CDM of less than about 0.8 μM, a cumulative nickel concentration in the CDM of less than about 0.40 μM, a cumulative zinc concentration in the CDM of less than about 56 μM, a cumulative cysteine ​​concentration in the CDM of less than about 10 mM, and / or an antioxidant in the CDM at a concentration of about 0.001 mM to about 10 mM for a single antioxidant, and a cumulative concentration of less than about 30 mM if multiple antioxidants are added to the CDM.

[0252] In one aspect of this embodiment, the preparation obtained using suitable conditions results in a reduction of aflibercept and VEGF MiniTrap protein variants to a desired amount of aflibercept and VEGF MiniTrap protein variants (referred to as the "target value" of aflibercept and VEGF MiniTrap protein variants). In a further aspect of this embodiment, the preparation obtained using suitable conditions results in a reduction in the color of the preparation to a desired BY value (referred to as the "target BY value") when the preparation of proteins containing aflibercept and VEGF MiniTrap variants is normalized to a concentration of 5 g / L, 10 g / L or more.

[0253] In a further aspect of this embodiment, the target BY value and / or target value of the variant can be achieved in a preparation if the titer increases or does not decrease significantly (see Example 5).

[0254] In some embodiments, the compositions produced by using the modified CDM can produce preparations of anti-VEGF proteins with desired target BY values, the color of which is as follows: (i) a yellow-brown color similar to the European color standard BY2; (ii) a tan color similar to the European color standard BY3; (iii) a tan color similar to the European color standard BY4; (iv) a tan color similar to the European color standard BY5; (v) European color standard between BY2 and BY3; (vi) European color standard between BY3 and BY4; (vii) European color standard between BY4 and BY5 The composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, and a sample of the composition can be obtained from the clarified Protein A eluate. As shown in Example 9, Table 9-3 below, the Protein A eluate containing 5 g / L aflibercept exhibited a yellow-brown color, which exhibited a b of 1.77. * When produced downstream after AEX, these samples are measured to have a b value of 0.50. * This demonstrates the usefulness of AEX in reducing the tan color of the samples (Table 9-3).

[0255] The composition produced by using the modified CDM can produce a preparation of anti-VEGF protein, the color of which is measured on the CIELAB scale: (i) About 22-23 b * Yellow-brown color comparable to the value; (ii) about 16-17 b * Yellow-brown color comparable to the value; (iii) 9-10 b * Yellow-brown color comparable to the value; (iv) 4-5 b * Yellow-brown color comparable to the value; (v) 2-3 b * Yellow-brown color comparable to the value; (vi) 17-23 b * value; (vii) 10-17 b * value; (viii) 5 to 10 b * value; (ix) 3 to 5 b * value; or (x) 1 to 3 b * value The compositions are characterized by accepted standard color characterization at 100°C / 200°F, where the compositions contain about 5 g / L or about 10 g / L of anti-VEGF protein, and are obtained as samples from clarified harvest Protein A eluate. See Example 9, Table 9-3.

[0256] With respect to components added to a cell culture to form a modified CDM, the term "cumulative amount" refers to the total amount of a particular component added to a bioreactor throughout the course of cell culture to form a CDM, including the amount added at the beginning of the culture (CDM on Day 0) and the amount of the component added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or inoculum prior to production in the bioreactor (i.e., prior to CDM on Day 0) is also included. The cumulative amount is not affected by loss of the component (e.g., due to metabolic or chemical degradation) over time during culture. Thus, for example, if a component is added to two cultures at different times (e.g., in one culture, all of the components are added initially, and in another culture, the components are added sequentially), even if the two cultures have the same cumulative amount of the component, they may have different absolute levels. The cumulative amount is also not affected by in situ synthesis of the component (e.g., due to metabolic or chemical conversion) over time during culture. Thus, for example, if a component is synthesized in situ in one of two cultures during the bioconversion process, two cultures with the same cumulative amount of a given component may have different absolute levels. The cumulative amount can be expressed in units such as grams or moles of the component. The term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the start of a production batch, including additions to the starting volume from any inoculum used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the start of a production batch and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onwards is 2 g / L (i.e., 4 grams divided by 2 liters). If an additional 1 liter of liquid not containing component X is added to the bioreactor on day 4, the cumulative concentration will remain at 2 g / L. If some amount of liquid is lost from the bioreactor (e.g., due to evaporation) on day 5, the cumulative concentration will remain at 2 g / L. The cumulative concentration may be expressed in units such as grams / liter or moles / liter, for example.

[0257] A. Amino acids: In some embodiments, the modified CDM can be obtained by decreasing 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 increase or decrease in the cumulative amount of these amino acids in the modified CDM compared to the starting 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%, or within one or more of the above ranges. Alternatively, the increase or decrease in the cumulative amount of one or more amino acids in the modified CDM compared 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%, within one or more of the above ranges (see Figures 25 to 27 and Example 5).

[0258] In some embodiments, the modified CDM can be obtained by reducing the cumulative concentration of cysteine ​​in the CDM. To form the modified CDM, the reduction in the amount of cysteine ​​in the CDM compared to the unmodified 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%, or within one or more of the above ranges. Alternatively, the cumulative amount of cysteine ​​in the modified CDM can 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%, relative to the CDM, and can range anywhere from 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 (see Figures 25-27 and Example 5).

[0259] In some embodiments, the modified CDM can be obtained by substituting at least a certain percentage of the cumulative cysteines in the CDM with cystine, 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%, or within one or more of the above ranges, relative to the unmodified CDM. Alternatively, the substitution 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%, relative to unmodified CDM, within one or more of the above ranges (see Figures 25-27 and Example 5).

[0260] In some embodiments, the modified CDM can be obtained by substituting 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%, or within one or more of the above ranges, relative to the unmodified CDM. Alternatively, the substitution 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%, relative to unmodified CDM, and can be within one or more of the above ranges.

[0261] B. Metal: 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 unmodified 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%, or within one or more of the above ranges. Alternatively, the increase or decrease in the cumulative amount of one or more metals in the modified CDM compared 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%, within one or more of the above ranges (see Figures 25-27 and Example 5).

[0262] C. Antioxidants: In some embodiments, the modified CDM comprises one or more antioxidants, non-limiting examples of which can be taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, vitamin C, vitamin E, and combinations thereof (see Figures 28A-E and Example 5).

[0263] In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM taurine, 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 within one or more of the above ranges.

[0264] In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM hypotaurine, 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 within one or more of the above ranges.

[0265] In some embodiments, the modified CDM comprises about 0.01 mM to about 20 mM glycine, 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 within one or more of the above ranges.

[0266] In some embodiments, the modified CDM comprises about 0.01 μM to about 5 μM thioctic acid, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM, about 1 μM to about 5 μM, and within one or more of the above ranges.

[0267] In some embodiments, the modified CDM comprises about 0.01 M to about 5 mM glutathione, 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 within one or more of the above ranges.

[0268] In some embodiments, the modified CDM comprises about 0.01 μM to about 5 μM hydrocortisone, i.e., about 0.01 μM to about 0.1 μM, about 0.1 μM to about 1 μM, about 1 μM to about 2.5 μM, about 1 μM to about 3 μM, about 1 μM to about 5 μM, and within one or more of the above ranges.

[0269] In some embodiments, the modified CDM comprises about 1 μM to about 50 μM vitamin C, i.e., about 1 μM to about 5 μM, about 5 μM to about 20 μM, about 10 μM to about 30 μM, about 5 μM to about 30 μM, about 20 μM to about 50 μM, about 25 μM to about 50 μM, and within one or more of the above ranges.

[0270] D. Modifications to Media to Modulate Glycosylation: The present disclosure also includes methods for modulating the glycosylation of anti-VEGF proteins by varying the cumulative concentration of certain components in the CDM. Based on the cumulative amount of components added to the CDM, the total % fucosylation, total % galactosylation, total % sialylation, and mannose-5 can be varied.

[0271] In an exemplary embodiment, a method for modulating glycosylation of an anti-VEGF protein can include adding a CDM containing uridine. The anti-VEGF protein can have about 40% to about 55% total fucosylated glycans, about 30% to about 50% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (see Example 6, below).

[0272] In some embodiments, the method for modulating glycosylation of an anti-VEGF protein can include adding manganese to the CDM. In one aspect, the CDM lacks manganese prior to addition. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (see Example 6, below).

[0273] In some embodiments, the method for modulating glycosylation of an anti-VEGF protein can include adding galactose to the CDM. In one aspect, the CDM lacks galactose prior to addition. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (Example 6).

[0274] In some embodiments, the method for modulating glycosylation of an anti-VEGF protein can include adding dexamethasone to the CDM. In one aspect, the CDM lacks dexamethasone prior to addition. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (see Example 6, below).

[0275] In some embodiments, the method for modulating glycosylation of an anti-VEGF protein can include adding one or more of uridine, manganese, galactose, and dexamethasone to the CDM. In one aspect, the CDM lacks one or more of uridine, manganese, galactose, and dexamethasone prior to addition. The anti-VEGF protein can have about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 2% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans (Example 6).

[0276] V. Preparation of Compositions Using Downstream Process Technologies Compositions containing the anti-VEGF proteins of the present invention can be produced by adjusting conditions during downstream protein production. The inventors have discovered that optimizing downstream procedures minimizes certain variants of anti-VEGF proteins that cause discoloration. Optimizing downstream processes can produce compositions with reduced oxo variants and optimized color properties.

[0277] The downstream process techniques may be used alone or in combination with the upstream process techniques described in Section IV above.

[0278] A. Anion Exchange Chromatography: In some embodiments, the compositions of the invention may be involved in a process comprising expressing an anti-VEGF protein in host cells in a CDM, where the anti-VEGF protein is secreted from the host cells into the medium, and obtaining a clarified harvest, which is subjected to the following steps: (a) loading a biological sample obtained from the harvest onto an anion exchange chromatography (AEX) column, (b) washing the AEX column with a suitable wash buffer, (c) collecting the flow-through fraction(s), optionally (d) washing the column with a suitable strip buffer, and (e) collecting the stripped fraction(s).

[0279] The flow-through fraction may contain about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the oxo variants of the anti-VEGF protein sample compared to the oxo variants in the strip fractions of the anion exchange chromatography column. For example, referring to Tables 9-5 and 9-6, the flow-through fraction contains oxidized variants of the anti-VEGF protein. In this case, some histidine and tryptophan residues are oxidized by approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and within one or more of the above ranges) compared to the oxidized variants in the stripped fraction.

[0280] The pH of both the equilibration buffer and the wash buffer for the AEX column can be about 8.20 to about 8.60. In another embodiment, the conductivity of both the equilibration buffer and the wash buffer for the AEX column can be about 1.50 to about 3.0 mS / cm. In one embodiment, the equilibration buffer and the wash buffer can be about 50 mM Tris-HCl. In one embodiment, the strip buffer contains 2 M sodium chloride or 1 N sodium hydroxide, or both (see Table 2-2). Example 2 further illustrates optimization of the concentration and conductivity of the equilibration buffer and the wash buffer.

[0281] Protein variants may include modifications of one or more residues, such as: one or more asparagines are deamidated; one or more aspartates are converted to isoaspartate and / or Asn; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyltryptophan; one or more tryptophans are di-hydroxyltryptophan; one or more tryptophans are tri-hydroxyltryptophan; one or more arginines are converted to Arg3-deoxyglucosone; the C-terminal glycine is absent; and / or one or more non-glycosylated glycosites are present.

[0282] The protein of interest may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap. Protein variants may be formed by one or more of the following: (i) oxidation of histidine from a histidine residue selected from His86, His110, His145, His209, His95, His19, and / or His203 (or equivalent residue positions in proteins that share certain structural characteristics of aflibercept); (ii) oxidation of a tryptophan residue selected from tryptophan residues at Trp58 and / or Trp138 (or equivalent residue positions in proteins that share certain structural characteristics of aflibercept). (iii) oxidation of a tyrosine residue at Tyr64 (or an equivalent position on proteins sharing certain structural characteristics of aflibercept); (iv) oxidation of a phenylalanine residue selected from Phe44 and / or Phe166 (or an equivalent residue position on proteins sharing certain structural characteristics of aflibercept); and / or (v) oxidation of a methionine residue selected from Met10, Met20, Met163, and / or Met192 (or an equivalent residue position on proteins sharing certain structural characteristics of aflibercept).

[0283] The flow-through fraction may contain one or more of the following: (a) The percentage of histidine residues that are oxidized to 2-oxo-histidine, the color properties of which are as follows: (i) a yellow-brown color similar to the European color standard BY2; (ii) a tan color similar to the European color standard BY3; (iii) a tan color similar to the European color standard BY4; (iv) a tan color similar to the European color standard BY5; (v) European color standard between BY2 and BY3; (vi) European color standard between BY3 and BY4; (vii) European color standard between BY4 and BY5 and the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the flow-through fraction. (b) the percentage of histidine residues that are oxidized to 2-oxo-histidine.Furthermore, these colors are characterized by having a tan color that is close to the color of BY2, BY3, BY4, BY5, BY6, BY7, or is not darker / stronger 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, or between BY3 and BY5. (c) The percentage of histidine residues that are oxidized to 2-oxo-histidine, and their colors are as follows: (i) About 22-23 b * Yellow-brown color comparable to the value; (ii) about 16-17 b * Yellow-brown color comparable to the value; (iii) 9-10 b * Yellow-brown color comparable to the value; (iv) 4-5 b * Yellow-brown color comparable to the value; (v) 2-3 b * Yellow-brown color comparable to the value; (vi) 17-23 b * value; (vii) 10-17 b * value; (viii) 5 to 10 b* value; (ix) 3 to 5 b * value; or (x) 1 to 3 b * value CIE L like * , a * , b * The composition is characterized by a color in a color space, where the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the flow-through fraction. (d) about 1% or less, about 0.1% or less, or about 0.1-1%, about 0.2-1%, about 0.3-1%, about 0.4-1%, about 0.5-1%, about 0.6-1%, about 0.7-1%, about 0.8-1%, or about 0.9-1% of the histidine residues in the composition are oxidized to 2-oxo-histidine. Calculation of percentages is described in Section II.

[0284] B. Affinity Chromatography: In some embodiments, the compositions of the present invention can be produced using a process comprising expressing an anti-VEGF protein in a host cell, where the anti-VEGF protein is secreted from the host cell into the culture medium, and obtaining a clarified harvest. This harvest is then subjected to the following steps: (a) loading a biological sample obtained from the clarified harvest onto an affinity chromatography column, where the affinity chromatography contains proteins capable of selectively or specifically binding to the anti-VEGF protein; (b) washing the affinity chromatography column with a suitable elution buffer; and (c) collecting the elution fraction(s). For example, as exemplified in Table 7-1 and Tables 7-7 to 7-10, VEGF is used as a protein capable of selectively or specifically binding to the anti-VEGF protein. 165Using mAb1 (murine anti-VEGF mAb human IgG1, SEQ ID NO:73 is the heavy chain and SEQ ID NO:74 is the light chain), (ii) mAb2 (murine anti-VEGFR1 mAb human IgG1, SEQ ID NO:75 is the heavy chain and SEQ ID NO:76 is the light chain), (iii) mAb3 (murine anti-VEGFR1 mAb mouse IgG1, SEQ ID NO:77 is the heavy chain and SEQ ID NO:78 is the light chain), and (iv) mAb4 (murine anti-VEGFR1 mAb mouse IgG1, SEQ ID NO:79 is the heavy chain and SEQ ID NO:80 is the light chain) as different proteins capable of selectively or specifically binding to MT5.

[0285] With respect to step (a) above, the biological sample loaded onto the affinity column can be derived from a sample that can be subjected to clarification prior to affinity chromatography, including, but not limited to, ion exchange chromatography (either anionic or cationic). Other chromatographic procedures well known to those skilled in the art can also be utilized prior to use in the affinity step. Importantly, the biological sample containing the anti-VEGF protein can be subjected to affinity chromatography.

[0286] In some embodiments, compositions of the present invention can be produced using a process comprising expressing a VEGF MiniTrap protein in a host cell, where the VEGF MiniTrap protein is secreted from the host cell into the medium, and the medium can be further processed to form a clarified harvest. This harvest can be further processed by known chromatography procedures to produce a biological sample containing the VEGF MiniTrap protein. The biological sample can be further processed using steps including: (a) loading the biological sample onto an affinity chromatography column, where the affinity chromatography contains a protein capable of selectively or specifically binding to or interacting with the VEGF MiniTrap protein; (b) washing the affinity chromatography column with a suitable elution buffer; and (c) collecting one or more elution fractions. Referring again to Table 7-1, different proteins capable of selectively or specifically binding to or interacting with MT5 include: (i) VEGF 165 Disclosed in this table is the successful generation of MT5 (VEGF MiniTrap) using (ii) mAb1 (murine anti-VEGFR1 mAb human IgG1, where SEQ ID NO:73 is the heavy chain and SEQ ID NO:74 is the light chain), (iii) mAb2 (murine anti-VEGFR1 mAb human IgG1, where SEQ ID NO:75 is the heavy chain and SEQ ID NO:76 is the light chain), (iv) mAb3 (murine anti-VEGFR1 mAb mouse IgG1, where SEQ ID NO:77 is the heavy chain and SEQ ID NO:78 is the light chain), and (v) mAb4 (murine anti-VEGFR1 mAb mouse IgG1, where SEQ ID NO:79 is the heavy chain and SEQ ID NO:80 is the light chain).

[0287] In one embodiment, affinity chromatography can also be used to isolate other MiniTrap proteins. After cleavage of aflibercept, the sample containing the cleaved aflibercept can be subjected to affinity chromatography using a binding agent specific for the cleaved aflibercept. In one embodiment, the binding agent can be an antibody or a portion thereof.

[0288] Cleavage of aflibercept can be facilitated using proteolytic digestion of aflibercept, for example, with IdeS protease (FabRICATOR) or a variant thereof, to generate VEGF MiniTrap. Cleavage of aflibercept with IdeS protease or a variant thereof can generate a mixture of products containing an Fc fragment and VEGF MiniTrap. The VEGF MiniTrap can be further processed using one or more of the purification strategies described herein.

[0289] In some exemplary embodiments, proteins capable of selectively or specifically binding ("binding agents") or interacting with anti-VEGF proteins, such as aflibercept or MiniTrap, may be of human or murine origin.

[0290] The affinity production process may further include equilibrating the affinity column with an equilibration buffer prior to loading with the biological sample. Exemplary equilibration buffers may be 20 mM sodium phosphate (pH 6-8, particularly pH 7.2), 10 mM sodium phosphate, 500 mM NaCl (pH 6-8, particularly pH 7.2), 50 mM Tris (pH 7-8), DPBS (pH 7.4).

[0291] The biological sample may be loaded using a suitable buffer such as DPBS.

[0292] The affinity generation process may further include washing the affinity column with one or more wash buffers. The column may be washed one or more times. Furthermore, the wash may also be collected as a wash fraction. The pH of both wash buffers may be about 7.0 to about 8.60. In one embodiment, the wash buffer may be DPBS. In another embodiment, the wash buffer may be 20 mM sodium phosphate (pH 6-8, particularly pH 7.2), 10 mM sodium phosphate, 500 mM NaCl (pH 6-8, particularly pH 7.2), 50 mM Tris (pH 7-8), or DPBS (pH 7.4).

[0293] The affinity process may further include washing the affinity column with one or more suitable elution buffers and collecting the eluted fractions. The column may be washed one or more times. Non-limiting examples of suitable elution buffers include ammonium acetate (pH of about 2.0 to about 3.0), acetic acid (pH of about 2.0 to about 3.2), glycine-HCl (pH of about 2.0 to about 3.0), sodium citrate (pH of about 2.0 to about 3.0), citric acid (pH of about 2.0 to about 3.0), potassium isothiocyanate (pH of about 2.0 to about 3.0), or combinations thereof.

[0294] In some embodiments, the eluted fractions may be neutralized using a neutralization buffer, such as Tris-Tris-HCl (pH of about 7.0 to about 9.0).

[0295] C. IdeS mutant The IdeS protease used to cleave Fc-fusion proteins such as aflibercept rapidly loses enzymatic activity under basic pH conditions, which may limit its use during the manufacture of VEGF MiniTrap. Therefore, variants have been developed that are more stable at basic pH in the presence of a strong base, e.g., NaOH. Such basic conditions can be 0.05 N NaOH for 1 hour or 0.1 N NaOH for 0.5 hours.

[0296] In some embodiments, an IdeS variant may have an amino acid sequence that comprises at least about 70% sequence identity over its entire length to an amino acid sequence set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO: 16. In some aspects, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its entire length to the amino acid sequence directly referenced above.

[0297] In some embodiments, an IdeS variant may comprise an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence comprising at least 70% sequence identity over its entire length to an amino acid sequence as set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO: 16. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95% or about 100% sequence identity over its entire length to the amino acid sequence directly referenced above.

[0298] In some embodiments, the polypeptide has an amino acid sequence that comprises at least 70% sequence identity over its entire length to the amino acid sequence set forth in the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, and can be expressed by a host cell containing a suitable vector containing a nucleic acid encoding for the identified peptide. In one embodiment, the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in the host cell. In one embodiment, the vector is a plasmid. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%, or about 100% sequence identity over its entire length to the amino acid sequence directly referenced above. In some embodiments, an isolated nucleic acid molecule can be used to encode the polypeptide.

[0299] In some embodiments, the IdeS variant may have an amino acid sequence that includes the parent amino acid sequence defined by SEQ ID NO: 1 (IdeS), with the asparagine residues at positions 87, 130, 182, and / or 274 mutated to amino acids other than asparagine. In one embodiment, the mutations may result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In another embodiment, the mutations may result in a 50% increase in chemical stability at alkaline pH values ​​compared to the parent amino acid sequence. In one embodiment, the amino acids may be selected from aspartic acid, leucine, and arginine. In a particular embodiment, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another particular embodiment, the asparagine residue at position 130 is mutated to an arginine residue. In yet another particular embodiment, the asparagine residue at position 182 is mutated to a leucine residue. In yet another particular embodiment, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another particular embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another particular embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another particular embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another particular embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another particular embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another particular embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another particular embodiment, the asparagine residues at positions 87, 130 and 182 are mutated. In yet another particular embodiment, the asparagine residues at positions 87, 182 and 274 are mutated. In yet another particular embodiment, the asparagine residues at positions 130, 182 and 274 are mutated. In yet another particular embodiment, the asparagine residues at positions 87, 130, 182 and 274 are mutated. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%, or about 100% sequence identity over its entire length to the amino acid sequence set forth above. In some embodiments, the isolated nucleic acid molecule can be used to encode a polypeptide.

[0300] Those skilled in the art, familiar with standard molecular biology techniques, can prepare and use the IdeS variants of the present invention without undue burden. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, tissue culture, and transformation (e.g., electroporation, lipofection). See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, supra, which is incorporated herein by reference for any purpose. Enzymatic reactions and purification techniques can be performed according to manufacturer's specifications or as described herein.

[0301] VI. General Protein Production A variety of different purification techniques, alone or in combination, are contemplated within the scope of the present invention, including, but not limited to, affinity chromatography, ion exchange chromatography, mixed-mode chromatography, size exclusion chromatography, and hydrophobic interaction chromatography. These chromatography steps separate a mixture of proteins representing a biological sample based on their charge, hydrophobicity, or size, or a combination thereof, depending on the specific type of separation. Different chromatography resins are available for each of the techniques suggested above, allowing purification schemes to be tailored precisely for the specific proteins involved. Each separation method allows proteins to pass through the column at different rates, resulting in physical separation or selective adhesion to the separation medium, which increases with each additional pass through the column. Proteins are then (i) differentially eluted using an appropriate elution buffer and / or (ii) collected from the flow-through fractions obtained from the column used, optionally by washing the column with an appropriate equilibration buffer. In some cases, impurities preferentially adhere to the column and the protein of interest does not adsorb to a great extent, i.e., if the protein of interest does not adsorb to the solid phase of a particular column and thus passes through the column, the protein of interest is separated from the impurities (such as HCPs, protein variants, etc.). In some cases, if the impurity cannot adsorb to the column and thus passes through the column, the impurity is separated from the protein of interest.

[0302] After a recombinant protein has been produced using the upstream production methods described above and / or alternative production methods common in the art, the production process may begin with a separation step. Once a clarified solution or mixture containing the protein of interest, e.g., a fusion protein, is obtained, separation of the protein of interest from process-related impurities (e.g., other proteins produced by the cell (e.g., HCPs) and product-related substances, such as acidic or basic variants) is performed. A combination of one or more different purification techniques may be used, including affinity chromatography, ion exchange chromatography (e.g., CEX, AEX), mixed-mode (MM) chromatography, and / or hydrophobic interaction chromatography. These purification steps separate mixtures of components within a biological sample based on, for example, charge, hydrophobicity, and / or apparent size. Numerous chromatography resins are commercially available for each of the chromatography techniques mentioned herein, allowing for precise tailoring of purification schemes for the specific proteins involved. Each separation method allows proteins to either pass through the column at different rates, achieving increasing physical separation with each additional pass through the column, or to selectively adsorb to the separation resin (or medium). The protein can then be differentially collected, and in some cases, the protein of interest is separated from components of the biological sample when other components specifically adsorb to the column resin while the protein of interest does not.

[0303] A. Primary Recovery and Viral Inactivation In certain embodiments, the initial steps of the production methods disclosed herein involve the clarification and primary recovery of the protein of interest from a biological sample. Primary recovery involves one or more centrifugation steps to separate the protein of interest 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 the context of large-scale production, such centrifugation is performed in-line, 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 processing or filtered in-line through one or more depth filters for further sample clarification.

[0304] In certain embodiments, primary recovery can involve the use of one or more depth filtration steps to clarify the sample, thereby aiding in the processing of the protein of interest. In other embodiments, primary recovery can involve the use of one or more depth filtration steps after centrifugation. 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.). 0.2 μm filters, such as Sartorius' 0.45 / 0.2 μm Sartopore™ bilayer or Millipore's Express SHR or SHC filter cartridges, typically follow the depth filter. Other filters known to those skilled in the art can also be used.

[0305] In certain embodiments, the primary recovery process may also be a point for reducing or inactivating viruses that may be present in the biological sample. Any one or more of a variety of viral reduction / inactivation methods may be used during the primary recovery stage of production, including heat inactivation (pasteurization), pH inactivation, buffer / detergent treatment, ultraviolet and gamma irradiation, and the addition of certain chemical inactivating agents, such as β-propiolactone or copper phenanthroline, as described in U.S. Pat. No. 4,534,972, the entire teachings of which are incorporated herein by reference. In certain exemplary embodiments of the present invention, the sample is subjected to detergent viral inactivation during the primary recovery stage. In other embodiments, the sample may be subjected to low pH inactivation during the primary recovery stage.

[0306] In those embodiments in which viral reduction / inactivation is used, the biological sample may be adjusted, if necessary, for further purification steps. For example, after low pH viral inactivation, the pH of the sample is typically adjusted to a more neutral pH, such as about 4.5 to about 8.5, before continuing the purification process. Additionally, the mixture may be diluted with water for injection (WFI) to obtain the desired conductivity.

[0307] B. Affinity Chromatography In certain exemplary embodiments, it may be advantageous to subject a biological sample to affinity chromatography to produce a protein of interest. The chromatography material can selectively or specifically bind or interact with the protein of interest. Non-limiting examples of such chromatography materials include Protein A and Protein G. Also included are chromatography materials containing proteins or portions thereof that can bind or interact with the protein of interest. In one embodiment, the protein of interest is an anti-VEGF protein, such as aflibercept, MiniTrap, or a protein related thereto.

[0308] Affinity chromatography involves applying a biological sample to a column containing a suitable Protein A resin. As used herein, "Protein A" refers to Protein A recovered from its natural source, synthetically produced Protein A (e.g., by peptide synthesis or by recombinant technology), C H 2 / C H The term "protein A resin" encompasses variants thereof that retain the ability to bind to proteins having three domains. In certain embodiments, Protein A resins are useful for affinity-based generation and isolation of various antibody isotypes by specifically interacting with the Fc portion of molecules that may have the three domains.

[0309] There are several manufacturers of Protein A resins. One suitable resin is MabSelect™ from GE Healthcare. Suitable resins include, but are not limited to, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, and rProtein A Sepharose from GE Healthcare; ProSep HC, ProSep Ultra, and ProSep Ultra Plus from EMD Millipore; and MapCapture from Life Technologies. A non-limiting example of a suitable column packed with MabSelect™ is a column approximately 1.0 cm in diameter by approximately 21.6 cm in length (17 mL bed volume). Suitable columns may contain resins such as MabSelect™ SuRe or similar resins. Protein A is commercially available from Repligen, Pharmacia, and Fermatech.

[0310] The affinity column can be equilibrated with a suitable buffer before loading the sample. After loading the column, the column can be washed one or more times using a suitable wash buffer. The column can then be eluted using an appropriate elution buffer, such as glycine-HCl, acetic acid, or citric acid. The eluate can be monitored using techniques well known to those skilled in the art, such as a UV detector. The eluted fraction of interest can be collected and then prepared for further processing.

[0311] In one embodiment, the eluate may be subjected to viral inactivation, for example, by either a detergent or low pH. A suitable detergent concentration or a suitable pH (and time) may be selected to achieve the desired viral inactivation results. After viral inactivation, the eluate is typically adjusted for pH and / or conductivity for subsequent purification steps.

[0312] To remove turbidity and / or various impurities from the protein of interest, the eluate may be subjected to filtration through a depth filter prior to an additional chromatographic polishing step. Examples of suitable 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, Delipid, VR07, and VR05 filters (3M). Emphaze AEX Hybrid Purifier multi-mechanism filters may also be used to clarify the eluate. To achieve the desired impurity removal and product recovery from the depth filtration step, the eluate pool may need to be adjusted to a specific pH and conductivity.

[0313] C. Anion Exchange Chromatography In certain embodiments, the protein of interest is produced by subjecting biological samples to at least one anion exchange separation step.In some scenarios, the anion exchange step can occur after affinity chromatography (e.g., Protein A affinity) procedure.In other scenarios, the anion exchange step can occur before affinity chromatography step.In still other protocols, the anion exchange can occur both before and after affinity chromatography step.In one aspect, the protein of interest is either aflibercept or MiniTrap.

[0314] The use of anion exchange materials, as opposed to cation exchange materials, is based in part on the local charge of the protein of interest. Anion exchange chromatography can be used in combination with other chromatographic procedures, such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and other modes of chromatography known to those skilled in the art.

[0315] In carrying out the separation, the initial protein composition (biological sample) can be placed in contact with the anion exchange material using any of a variety of techniques, for example, using batch production techniques or chromatographic techniques.

[0316] In terms of batch production, the anion exchange material is prepared in or equilibrated with the desired starting buffer. Once prepared, a slurry of the anion exchange material is obtained. The biological sample is contacted with the slurry to allow protein adsorption to the anion exchange material. The solution containing acidic species that do not bind to the AEX material is separated from the slurry by allowing the slurry to settle and removing the supernatant. The slurry may be subjected to one or more washing and / or elution steps.

[0317] In the context of chromatographic separation, a chromatographic column is used to contain a chromatographic support material (resin or solid phase). A sample containing the protein of interest is loaded onto a particular chromatographic column. The column may then be subjected to one or more washing steps using a suitable wash buffer. Components of the sample that are not adsorbed onto the resin may flow through the column. Components that are adsorbed to the resin may be differentially eluted using an appropriate elution buffer.

[0318] Wash steps are typically performed in AEX chromatography using conditions similar to the loading conditions, or alternatively, by decreasing the pH and / or increasing the ionic strength / conductivity of the wash solution in a stepwise or linear gradient fashion. In one embodiment, the aqueous salt solutions used for both the loading and wash buffers have a pH that is at or near the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0-2 units higher or lower than the pI of the protein of interest, although it can be within a range of 0-0.5 units higher or lower. It can also be at the pI of the protein of interest.

[0319] The anionic agent may be selected from the group consisting of acetate, chloride, formate, and combinations thereof. The cationic agent may be selected from the group consisting of Tris, arginine, sodium, and combinations thereof. In a specific example, the buffer solution is a Tris / formate buffer. The buffer may be 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-propaol), piperazine, 1,3-diaminopropane, and piperidine.

[0320] 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, where proteins exhibit binding to the chromatographic material and can be washed from such material using a buffer that is the same as or substantially similar to the loading buffer.

[0321] In bind-elute mode, a column or membrane device is first conditioned with a buffer of appropriate ionic strength and pH under conditions that allow a specific protein to adsorb to the resin-based matrix. For example, during feed loading, the protein of interest may be adsorbed 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, which competes with the solute for the charged sites of the anion exchange matrix. Changing the pH, thereby altering the charge of the solute, is another way to achieve solute elution. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).

[0322] In flow-through mode, the column or membrane device is operated at a selected pH and conductivity such that the protein of interest does not bind to the resin or membrane, while the acidic species are either retained on the column or have a different elution profile compared to the protein of interest. In this regard, the acidic species interact or bind to the chromatographic material under suitable conditions, while the protein of interest and certain aggregates and / or fragments of the protein of interest pass through the column.

[0323] 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, which are rigid polymer beads with a backbone made of cross-linked poly[styrene-divinylbenzene]; Capto Q Impres and Capto DEAE, which are high-flow 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-like 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 constructed from inorganic filter aids, purified cellulose, and ion exchange resins; and XOHC, a depth filtration medium constructed from inorganic filter aids, cellulose, and mixed cellulose esters.

[0324] In certain embodiments, the protein load of the sample may be adjusted to a total protein load on the column of about 50 g / L to about 500 g / L, or about 75 g / L to about 350 g / L, or about 200 g / L to about 300 g / L. In other embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded on the column of about 0.5 g / L to about 50 g / L, about 1 g / L to about 20 g / L, or about 3 g / L to about 10 g / L. In yet other embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material on the column of about 37 g / L.

[0325] Additives such as polyethylene glycol (PEG), surfactants, amino acids, sugars, chaotropic agents, etc. may be added to improve the performance of the separation to achieve better separation, recovery, and / or product quality.

[0326] In certain embodiments, including those related to aflibercept and / or VEGF MiniTrap, the methods of the invention can be used to selectively remove, significantly reduce, or essentially remove at least 10% of protein variants, thereby producing a protein composition having reduced protein variants.

[0327] Protein variants may contain modifications of one or more residues, such as: one or more asparagines are deamidated; one or more aspartates are converted to aspartate-glycines and / or Asn-Gly; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenines; one or more tryptophans are mono-hydroxyltryptophans; one or more tryptophans are di-hydroxyltryptophans; one or more tryptophans are tri-hydroxyltryptophans; one or more arginines are converted to Arg3-deoxyglucosone; the C-terminal glycine is absent; and / or one or more non-glycosylated glycosites are present. The use of AEX has also been observed to reduce oxidized and acidic species of anti-VEGF variants in the affinity eluate. Compared to the affinity eluate, after use of AEX, the flow-through fraction may exhibit at least about a 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% reduction in oxidized and / or acidic species of the anti-VEGF variant.

[0328] Protein variants of aflibercept and / or VEGF MiniTrap include: (i) an oxidized histidine from a histidine residue selected from His86, His110, His145, His209, His95, His19, and / or His203; (ii) an oxidized tryptophan residue selected from a tryptophan residue at Trp58 and / or Trp138; (iii) an oxidized tyrosine residue at Tyr64; (iv) an oxidized phenylalanine residue selected from Phe44 and / or Phe166; and / or (v) an oxidized methionine residue selected from Met10, Met20, Met163, and / or Met192. One or more of the following may be mentioned.

[0329] D. Cation Exchange Chromatography The composition of the present invention can be produced by subjecting the biological sample containing the protein of interest to at least one cation exchange (CEX) step.In certain exemplary embodiments, the CEX step is added to the AEX step, and is carried out either before or after the AEX step.In one aspect, the protein of interest is aflibercept, MiniTrap, or any of the molecules related thereto.

[0330] The use of a cation exchange material versus an anion exchange material, such as those described above, is based in part on the local charge of the protein of interest in a given solution and desired separation conditions. The use of a cation exchange step before an anion exchange step, or the use of an anion exchange step before an anion exchange step, is within the scope of the present invention. Furthermore, the use of a cation exchange step alone in combination with other chromatographic procedures is also within the scope of the present invention.

[0331] In performing cation exchange, a sample containing the protein of interest can be contacted with the cation exchange material using any of a variety of techniques, such as batch production techniques or chromatographic techniques, as described above for AEX.

[0332] Aqueous salt solutions may be used as both loading and washing buffers with a pH lower than the isoelectric point (pI) of the protein of interest. In one embodiment, the pH is about 0-5 units lower than the pI of the protein. In another embodiment, the pH is within the range of 1-2 units lower than the pI of the protein. In yet another embodiment, the pH is within the range of 1-1.5 units lower than the pI of the protein.

[0333] In certain embodiments, the concentration of the anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of about 3.5 to about 10.5, or about 4 to about 10, or about 4.5 to about 9.5, or about 5 to about 9, or about 5.5 to about 8.5, or about 6 to about 8, or about 6.5 to about 7.5. In one aspect, the concentration of the anionic agent is increased or decreased in the aqueous salt solution 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, or sodium sulfate.

[0334] In certain embodiments, the conductivity and pH of the aqueous salt solution are adjusted by increasing or decreasing the concentration of a cationic agent. In one aspect, the cationic agent is maintained at a concentration ranging from about 20 mM to about 500 mM, from about 50 mM to about 350 mM, from about 100 mM to about 300 mM, or from 100 mM to about 200 mM. Non-limiting examples of cationic agents are selected from the group consisting of sodium, Tris, triethylamine, ammonium, arginine, and combinations thereof.

[0335] Packed cation exchange chromatography columns or anion exchange membrane devices may be operated in either bind-elute mode, flow-through mode, or hybrid mode, in which the product binds to or interacts with the chromatographic material and may be washed from such material using a buffer the same as or substantially similar to the loading buffer (details of these modes are outlined above).

[0336] Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include Capto SP ImpRes, high-fluidity agarose beads; CM Hyper D grade F, ceramic beads coated and impregnated with a functionalized hydrogel, with 250-400 μeq / mL of ionic groups; Eshmuno S, a hydrophilic polyvinyl ether-based matrix with an ion exchange capacity of 50-100 μeq / mL; Nuvia C Prime, a hydrophobic cation exchange medium composed of a highly crosslinked macroporous hydrophilic polymer matrix with an ion exchange capacity of 55-75 μeq / mL; Nuvia S, a UNOsphere-based matrix with ionic groups of 90-150 μeq / mL; Poros HS, rigid polymer beads with a crosslinked poly[styrene-divinylbenzene] backbone; Poros XS, rigid polymer beads with a crosslinked poly[styrene-divinylbenzene] backbone; Toyo Pearl Giga Cap CM 650M, a polymer-based bead with an ion exchange capacity of 0.225 meq / mL; and Toyo Pearl Giga Cap S, a polymer-based bead. 650M; Toyo Pearl MX TRP, a polymer-based bead. Note that CEX chromatography can be used with MM resins, as described herein.

[0337] The protein load of the sample containing the protein of interest is adjusted to a total protein load on the column of about 5 g / L to about 150 g / L, or about 10 g / L to about 100 g / L, about 20 g / L to about 80 g / L, about 30 g / L to about 50 g / L, or about 40 g / L to about 50 g / L. In certain embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded onto the column of about 0.5 g / L to about 50 g / L, or about 1 g / L to about 20 g / L.

[0338] Additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents, etc. may be added to improve the performance of the separation to achieve better separation, recovery, and / or product quality.

[0339] In certain embodiments, methods of the invention, including those involving aflibercept or anti-VEGF antibodies or VEGF MiniTrap, can be used to selectively remove, significantly reduce, or essentially remove all of the oxo variants in a sample, where the protein of interest is essentially present in the flow-through of the CEX procedure, while the oxo variants are substantially captured by the column media.

[0340] E. Mixed-mode chromatography Mixed-mode ("MM") chromatography may also be used to prepare the compositions of the invention. MM chromatography, also referred to herein as "multimodal chromatography," is a chromatographic strategy that utilizes a support containing a ligand capable of providing at least two different interactions with an analyte or protein of interest from a sample. One of these sites provides an attractive charge-charge interaction between the ligand and the protein of interest, while the other provides an electron acceptor-donor interaction and / or a hydrophobic and / or hydrophilic interaction. Electron donor-acceptor interactions include hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole, and other interactions.

[0341] The column resin used for mixed-mode separations can be Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. The base matrix of this strong anion exchanger is highly cross-linked agarose with a ligand (N-benzyl-N-methylethanolamine) that exhibits different functionalities for interactions, such as ionic interactions, hydrogen bonding, and hydrophobic interactions. In certain embodiments, the resin used for mixed-mode separations 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 selectivity and hydrophobicity options for protein separations. Additional mixed-mode chromatography supports include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain embodiments, mixed-mode chromatography resins, sometimes referred to as base matrices, can be composed of ligands bound directly or via spacers to organic or inorganic supports. The supports can be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, and the like. In certain embodiments, the supports can be prepared from cross-linked carbohydrate materials, e.g., natural polymers such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, and the like. To achieve high adsorption capacity, the support can be porous, and the ligands are then bound 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), the entire teachings of which are incorporated herein by reference).Alternatively, the support can be prepared from a crosslinked synthetic polymer, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, and the like. Such synthetic polymers can be prepared according to standard methods. See, "Styrene-based polymer supports developed by suspension polymerization" (R. Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire teachings of which are incorporated herein by reference). Porous natural or synthetic polymer supports are also available from manufacturers such as GE Healthcare (Uppsala, Sweden).

[0342] The protein load of the biological sample mixture containing the protein of interest can be adjusted to a total protein load on the column of about 25 g / L to about 750 g / L, or about 75 g / L to about 500 g / L, or about 100 g / L to about 300 g / L. In certain exemplary embodiments, the protein concentration of the load protein mixture is adjusted to a protein concentration of the material loaded onto the column that is about 1 g / L to about 50 g / L, or about 9 g / L to about 25 g / L.

[0343] Additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents, etc. may be added to improve the performance of the separation to achieve better separation, recovery, and / or product quality.

[0344] In certain embodiments, including those related to aflibercept and / or MiniTrap, the methods of the invention can be used to selectively remove, significantly reduce, or essentially remove all PTMs, including oxo variants.

[0345] The methods for preparing the compositions of the present i...

Claims

1. 1. A method for producing aflibercept, comprising: (a) producing a clarified harvest of cells cultured in a cell culture medium comprising a defined medium (CDM); (b) binding aflibercept from the clarified harvest using an affinity chromatography column comprising a polypeptide capable of binding to or interacting with aflibercept, wherein the polypeptide is an antibody comprising a heavy chain of SEQ ID NO: 73 and a light chain of SEQ ID NO: 74; an antibody comprising a heavy chain of SEQ ID NO: 75 and a light chain of SEQ ID NO: 76; An antibody comprising a heavy chain of SEQ ID NO: 77 and a light chain of SEQ ID NO: 78, and An antibody comprising a heavy chain of SEQ ID NO: 79 and a light chain of SEQ ID NO: 80 a bonding step selected from the group consisting of: (c) eluting the aflibercept of step (b) to form an affinity eluate; Optionally, (d) subjecting the eluted aflibercept of (c) to a second chromatographic capture step; (e) collecting the flow-through fraction, wherein the flow-through fraction comprises aflibercept; A method comprising:

2. 10. The method of claim 1, further comprising the step of equilibrating the affinity chromatography column of (b) using an equilibration buffer.

3. 3. The method of claim 2, wherein the equilibration buffer is Dulbecco's phosphate buffered saline or Tris-HCl.

4. 4. The method of claim 3, wherein the equilibration buffer has a pH of 8.3 to 8.

6.

5. 10. The method of claim 1, further comprising washing the column (d) with an equilibration buffer to obtain one or more flow-through fractions.

6. 6. The method of claim 5, wherein the equilibration buffer is Dulbecco's phosphate buffered saline and has a pH of 7.0 to 8.

6.

7. 10. The method of claim 1, further comprising subjecting the column of (b) to an elution buffer to obtain one or more elution fractions.

8. 8. The method of claim 7, wherein the elution buffer comprises a 100 mM glycine buffer having a pH of 2.

5.

9. 8. The method of claim 7, wherein the pH of the elution buffer is between 2.0 and 3.

5.

10. 8. The method of claim 7, further comprising neutralizing the elution fraction by adding a neutralization buffer.

11. 11. The method of claim 10, wherein the neutralization buffer is Tris-HCl.

12. 10. The method of claim 1, wherein the amount of host cell protein in (c) is significantly reduced by at least 90%, at least 95%, at least 98%, or at least 99% compared to the amount of host cell protein in the clarified harvest.

13. 10. The method of claim 1, wherein the second capture chromatography in (d) comprises anion exchange chromatography (AEX).

14. The method of claim 13, further comprising the steps of equilibrating an AEX column with an equilibration buffer and washing the AEX column with a wash buffer, wherein the conductivity of both the equilibration buffer and the wash buffer for the AEX column can be 1.50 to 3.0 mS / cm.

15. 14. The method of claim 13, wherein the aflibercept from the flow-through fraction of (e) comprises less than 20% total acidic species of aflibercept, the acidic species corresponding to a peak eluting earlier than the main peak in a cation exchange chromatography (CEX) chromatogram of aflibercept, the chromatogram being generated using a first mobile phase of 20 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7, and a second mobile phase of 40 mM sodium phosphate, 100 mM sodium chloride, pH 9.0, and the chromatogram being generated using detection at 280 nm.

16. 1. A method for producing aflibercept, comprising: (a) providing a host cell genetically modified to express aflibercept; (b) culturing the host cells under conditions suitable for expression of the aflibercept; (c) recovering a preparation comprising aflibercept and at least one impurity produced by the host cell; (d) subjecting the preparation to affinity chromatography under suitable conditions, wherein the affinity chromatography uses a column containing a polypeptide capable of binding to or interacting with the aflibercept, the polypeptide comprising: an antibody comprising a heavy chain of SEQ ID NO: 73 and a light chain of SEQ ID NO: 74; an antibody comprising a heavy chain of SEQ ID NO: 75 and a light chain of SEQ ID NO: 76; An antibody comprising a heavy chain of SEQ ID NO: 77 and a light chain of SEQ ID NO: 78, and An antibody comprising a heavy chain of SEQ ID NO: 79 and a light chain of SEQ ID NO: 80 and a step of providing the A method comprising:

17. 17. The method of claim 16, wherein the antibody comprises a heavy chain of SEQ ID NO: 73 and a light chain of SEQ ID NO:

74.

18. 17. The method of claim 16, wherein the antibody comprises a heavy chain of SEQ ID NO: 75 and a light chain of SEQ ID NO:

76.

19. 17. The method of claim 16, wherein the antibody comprises a heavy chain of SEQ ID NO: 77 and a light chain of SEQ ID NO:

78.

20. 17. The method of claim 16, wherein the antibody comprises a heavy chain of SEQ ID NO: 79 and a light chain of SEQ ID NO:

80.

21. The method of claim 16, further comprising subjecting the column of (d) to an elution buffer to obtain one or more elution fractions, wherein the amount of host cell protein in the elution fractions is significantly reduced by at least 90%, at least 95%, at least 98%, or at least 99% compared to the amount of host cell protein in (c).

Images