Anti-VEGF protein composition and method for producing the same

Abstract

The present disclosure relates to compositions comprising anti-VEGF proteins and methods for making the compositions.

Description

Technical field

[0001] sequence listing The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on August 13, 2020, is marked 070816-02451_SL.txt, and is 134,385 bytes in size.

[0002] Cross-reference to related applications This application claims priority to and benefit from U.S. Provisional Patent Application No. 63 / 065,012, filed Aug. 13, 2020, the contents of which are hereby incorporated by reference in their entirety. .

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

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

[0005] A class of cell-derived dimeric mitogens with selectivity for vascular endothelial cells has been identified and referred to by this name as vascular endothelial growth factors (VEGFs).

[0006] Persistent angiogenesis can cause or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibroma, 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. Members of the angiopoietin and vascular endothelial growth factor (VEGF) families are the only growth factors believed to be primarily specific for vascular endothelial cells.

[0007] Several ocular disorders are associated with pathological neovascularization. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from CNV causes macular edema and collection of submacular fluid that causes vision loss. Diabetic macular edema (DME) is another eye disorder with an angiogenic component. DME is the most prevalent cause of moderate vision loss in diabetic patients and 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 portion of the retina that is associated with 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), are approved to treat these eye disorders. [Outline of the invention]

[0009] Overview The present invention relates to anti-VEGF proteins, including the VEGF trap protein aflibercept, which is a fusion protein. The present invention also relates to novel anti-VEGF proteins, aflibercept MiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise stated). Disclosed herein are methods of making these anti-VEGF proteins, including manufacturing modes that provide an efficient and effective means of producing the protein of interest. In one aspect, the present invention is directed to the use of defined media (CDM) to produce anti-VEGF proteins. In certain embodiments, the CDM of interest is a CDM that, when used, produces a protein sample, which is tan in color and may contain oxidized species. Also disclosed in the present application are protein variants of aflibercept and VEGF MiniTrap, along with accompanying methods of manufacture.

[0010] Manufacturing of aflibercept This disclosure describes the production of aflibercept using cell culture media. In one embodiment, the cell culture medium is a defined medium (“CDM”). CDM is often used because it is a synthetic formulation that is protein-free and does not use animal-derived components, and there is certainty regarding the composition of the medium. In another embodiment, the cell culture medium is soy hydrolysis medium.

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

[0012] In one aspect of this embodiment, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells 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 (DME) Medium, Ham 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, the preferred CDM is CDM1B (Regeneron) or Excell Advanced Medium (SAFC).

[0014] In one embodiment, clarified harvested samples from CDM cultures containing aflibercept are subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, protein A. In a further embodiment, the eluate of the affinity procedure exhibits a particular color. For example, the eluate may have a tan color. As described in more detail below, color can be defined as either (i) the European color standard "BY", which provides a qualitative visual inspection, or (ii) a colorimetric assay (CIE L), which is more quantitative than the BY system. * , a * , b * (or CIELAB). In any case, however, color ratings across multiple samples must be normalized to protein concentration to ensure a meaningful study. For example, referring to Example 9 below, the protein A eluate has a "b * , which corresponds to a BY value of approximately BY5 (when measured in protein A eluate at 5 g / L protein concentration). When comparing the color of the Protein A eluate to another sample, then the comparison must be made at the same protein concentration. b in CIELAB color space * The values ​​are used to describe the coloration of the sample, covering blue (-) to yellow (+). A higher b* value for a sample compared to another sample indicates a darker tan coloration in the sample compared to the other sample.

[0015] In one embodiment, aflibercept is produced from host cells that have been 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. A 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 using, for example, a protein A column. When samples extracted from the affinity eluate, which may or may not exhibit a tan color, are analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more Oxidation variants can be detected. Certain amino acid residues of modified aflibercept have been shown to be oxidized, including but not limited to histidine and / or tryptophan residues. In one aspect, variants can include oxidation of one or more methionine residues and other residues. See below.

[0016] In another embodiment, variants can include oxidation of one or more tryptophan residues to form N-formylkynurenine. In further embodiments, variants can include oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In certain embodiments, protein variants may include oxidation of one or more tryptophan residues to form di-hydroxytryptophan. In certain embodiments, protein variants can include oxidation of one or more tryptophan residues to form tri-hydroxytryptophan.

[0017] In another embodiment, the variant can comprise one or more modifications selected from the group consisting of: for example, deamidation of one or more asparagines; isoaspartate of one or more aspartic acids; oxidation of one or more methionines; oxidation of one or more tryptophans to N-formylkynurenine; oxidation of one or more tryptophans to mono-hydroxytryptophan; -oxidation to hydroxyltryptophan; oxidation of one or more tryptophans to tri-hydroxytryptophan; Arg3-deoxyglucosonation of one or more arginines; removal of C-terminal glycines; Presence of glycosites.

[0018] In another embodiment, the present invention relates to a method of making aflibercept. In one aspect, the clarified recovered sample containing aflibercept and variants thereof 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. Ion exchange chromatography can be either cation exchange chromatography or anion exchange chromatography. Mixed-mode or multimodal chromatography, and other chromatographic procedures, discussed further below, are also contemplated within the scope of the present embodiments. In certain embodiments, the ion exchange chromatography is anion exchange chromatography (AEX). Suitable conditions for using AEX include, but are not limited to Tris hydrochloride at a pH of about 8.3 to about 8.6. After equilibration using, for example, Tris hydrochloride 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, eg, using an equilibration buffer. In certain embodiments, the majority of the oxovariants are retained on the stationary phase of the AEX column and can be obtained when the column is stripped, while the fraction containing aflibercept free of significant amounts of oxovariants is flow-through. The conditions used can promote differential chromatographic behavior of aflibercept and its oxidized variants so that they can be collected in fractions. See Example 2 and FIG. 11 below. Referring to FIG. 11 and Example 2, variations regarding oxovariants 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 oxovariant (specifically oxo-tryptophan) went from about 0.131% in the load sample to about 0.070% in the flow-through sample after AEX chromatography (AEX separation 2). Yes, indicating that there was a reduction among the oxovariants of aflibercept using AEX.

[0019] Ion exchange may 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 assigned a specific BY and / or b * It has a primary color with a value. This Protein A eluate is then subjected to ion exchange chromatography, such as anion exchange chromatography (AEX). Wash the ion exchange column, collect the flow-through and assign it a specific BY and / or b * It has a secondary color with a value. In certain embodiments, the color value of the first color (“BY” or “b * ) is different from the secondary color. In a further embodiment, the first color of the protein A eluate is BY and / or b respectively * As reflected by the values, it 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, by using anion exchange, after AEX, b * The tan color observed in the protein A eluate samples decreased from a value of about 3.06 (first color) to about 0.96 (second color). See Example 2, Tables 2-3 below.

[0020] In one aspect of this embodiment, the pH of both the equilibration and wash buffers for the AEX column can be from about 8.30 to about 8.60. In another embodiment, the conductivity of both the equilibration buffer and wash buffer for the AEX column can be from 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 embodiment, the strip buffer contains 2M sodium chloride or 1N sodium hydroxide, or both (see Table 2-2).

[0022] This embodiment includes the addition of one or more steps in no particular order, such as hydrophobic interaction chromatography (HIC), affinity chromatography, multimodal chromatography, virus inactivation (e.g. using low pH), Viral filtration and / or ultrafiltration / diafiltration, as well as other well-known chromatographic steps can be included.

[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. glycosylation, G1S glycosylation, G2 glycosylation, G2S glycosylation, G2S2 glycosylation, G0F glycosylation, G2F2S glycosylation, G2F2S2 glycosylation, G1F glycosylation, G1FS glycosylation, G2F glycosylation, G2FS glycosylation, G2FS2 glycosylation, G3FS glycosylation, G3FS3 glycosylation, G0-2GlcNAc glycosylation, Man4 glycosylation, Man4_A1G1 glycosylation, Man4_A1G1S1 glycosylation, Man5 glycosylation, Man5_A1G1 glycosylation, Man5_A1G1S1 glycosylation, Man6 glycosylation, Man6_G0+ phosphoglycosylation, Man6+ phosphoglycosylation Acid glycosylation and / or Man7 glycosylation. In one aspect, the anti-VEGF protein can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0024] In one aspect, the glycosylation profile of the composition of anti-VEGF proteins is the following: about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 50% total sialylated glycans, 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 residues and / or about 27.1% asparagine-196 residues.

[0025] In one embodiment, the process may further comprise formulating the drug substance using pharmaceutically acceptable excipients. In one aspect of this embodiment, the pharmaceutically acceptable excipients can 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.

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

[0027] The formulation is used for 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 in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, Corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in subjects with diabetic macular edema, or diabetic retinopathy (e.g., nonproliferative diabetes mellitus) vascular 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 suffering from DME) It can be used as a method of treating or preventing neoplastic eye disorders.

[0028] Manufacturing of VEGF MiniTrap This disclosure describes the production of a modified version of aflibercept, in which the Fc portion has been removed or is absent, referred to as aflibercept MiniTrap or VEGF MiniTrap. The MiniTrap can be produced in cell culture media including defined medium (CDM) or soy hydrolysis medium.

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

[0030] In one embodiment, the method for producing MiniTrap comprises production of a full-length aflibercept fusion protein followed by cleavage of the Fc region. In one embodiment, the method involves producing a recombinant protein, such as the so-called full-length aflibercept fusion protein (see US Pat. No. 7,279,159, the teachings of which are incorporated herein by reference in their entirety). , which comprises (a) providing a host cell genetically modified to express full-length aflibercept, (b) subjecting the host cell to CDM under suitable conditions in which the cell expresses full-length aflibercept. (c) recovering a preparation of full-length aflibercept produced by the cells, and (d) subjecting full-length aflibercept to an enzymatic cleavage specific to remove the Fc portion of the fusion protein. A step of subjecting to. In another embodiment, the nucleotide sequence encoding aflibercept minus its Fc portion is expressed from a suitable host cell under suitable conditions well known to those of skill in the art (see U.S. Pat. No. 7,279,159). see).

[0031] In one aspect of this embodiment, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells 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 Medium (DME), Ham 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, the preferred CDM is CDM1B (Regeneron) or Excell medium (SAFC).

[0033] In one aspect, during manufacture of MiniTrap, samples containing the protein of interest (i.e., aflibercept fusion protein and / or MiniTrap) and variants thereof (including oxovariants) develop a specific color characteristic, i.e., a tan color. obtain. For example, an eluate sample from an affinity chromatography step may be BY and / or b * It can exhibit a specific tan color measured using the system (see Examples 2 and 9 below). Exemplary sources for the "sample" include affinity chromatography, such as Protein A; the sample can be obtained from the flow-through fraction of an ion exchange chromatography procedure; It can be obtained from strips. There are other sources from which samples can be analyzed during the manufacturing process that are well known to those skilled in the art. As noted above, and as explained in more detail below, color can be defined as: (i) the European color standard "BY", which provides qualitative visual inspection; or (ii) a ratio that is more quantitative than the BY system. It can be evaluated using a color assay (CIELAB). In any case, however, color assessments between samples must be normalized, eg, using protein concentration, to ensure meaningful inter-sample studies.

[0034] In one aspect of this embodiment, the full-length aflibercept fusion protein is enzymatically treated (" "cutting action"). In one aspect of this embodiment, the protease can be an immunoglobulin degrading enzyme of Streptococcus pyogenes (IdeS). In another embodiment, the protease is thrombin trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain, pronase, or It may be a protease derived from Aspergillus Saitoi. In one aspect, the protease can be a cysteine ​​protease. In certain aspects of this embodiment, the protease can be IdeS. In another embodiment, the protease can be a variant of IdeS. Non-limiting examples of variants of IdeS are described below. This variant is 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 protease can be immobilized on agarose or another suitable matrix.

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

[0036] In one embodiment, variants can include oxidation of one or more tryptophan residues to form N-formylkynurenine. In further embodiments, variants can include oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In certain embodiments, protein variants may include oxidation of one or more tryptophan residues to form di-hydroxytryptophan. In certain embodiments, protein variants can include oxidation of one or more tryptophan residues to form tri-hydroxytryptophan.

[0037] In another embodiment, the oxovariant can comprise one or more modifications selected from the group consisting of: deamidation of one or more asparagine residues; isoaspar of one or more aspartic acids; conversion to tart and / or Asn; oxidation of one or more methionine residues; formation of N-formylkynurenine by oxidizing one or more tryptophan residues; formation of mono-hydroxyl tryptophan; formation of di-hydroxyl tryptophan by oxidation of one or more tryptophan residues; formation of tri-hydroxyl tryptophan by oxidation of one or more tryptophan residues; one or more arginines Arg3-deoxyglucosonation of residues; removal of the C-terminal glycine; and presence of one or more non-glycosylated glycosites.

[0038] In one embodiment, a method for producing a MiniTrap protein comprises the steps of (a) capturing a full-length aflibercept fusion protein on a first chromatography platform, and (b) cleaving aflibercept, thereby producing a MiniTrap protein, That is, forming aflibercept without its Fc domain. In one aspect, the first chromatographic support comprises an affinity chromatographic medium, an ion exchange chromatographic medium, or a hydrophobic interaction chromatographic medium. In certain embodiments, the first chromatography platform comprises an affinity chromatography platform such as Protein A. In a further embodiment, the protein of capturing step (a) is eluted from the first chromatography platform prior to cleaving step (b). Additionally, in a further embodiment, the cleaving step (b) is followed by a second capturing step. In certain embodiments, this second capture step can be facilitated by affinity chromatography, such as protein A affinity chromatography. The flow-through of this second capture step (including the MiniTrap) has a first color, e.g. * It is measured to have value. See, eg, Example 9 below. In addition, LC-MS analysis of this second capture flow-through demonstrates the presence of oxovariants in which one or more residues of MiniTrap are oxidized (see Example 9 below).

[0039] In further embodiments, the second captured flow-through can be subjected to ion exchange chromatography such as AEX. The AEX column can be washed using a suitable buffer and the AEX flow-through fraction, which essentially contains the MiniTrap, collected. This AEX flow-through fraction is a specific BY and / or b * It may have a secondary color that is tan with a value. In a further embodiment, the first color (flowthrough from the second capture step) and the second color (flowthrough of the ion exchange procedure) are BY and / or b * have different colors as measured by any of the systems. In one aspect, the second color is AEX followed by BY and / or b * Demonstrates a reduction in tan when compared to the first color using either of the values.

[0040] In another embodiment, the cleaving action of step (b) can be performed using a chromatographic column, the cleaving action being enzymatically active for example, attached or immobilized to a column matrix. The column used in step (b) may contain one or more of the proteases already mentioned and described more fully below.

[0041] In one embodiment, an ion-exchange chromatography procedure can include anion-exchange (AEX) chromatography media. In another embodiment, the ion exchange chromatography medium can comprise cation exchange (CEX) chromatography medium. Suitable conditions for using AEX include, but are not limited to Tris hydrochloride at a pH of about 8.3 to about 8.6. After equilibration using, for example, Tris hydrochloride 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, eg, using an equilibration buffer. In certain embodiments, the conditions used are such that MiniTrap is substantially present in the flow-through fraction, while oxovariants are substantially retained on the AEX column and can be collected when the column is stripped. AEX can be used to enhance the differential chromatographic behavior of MiniTrap and its oxovariants (see Example 9 below).

[0042] In one example, samples from different stages of manufacture were analyzed for color and the presence of oxovariants. Referring to Example 9, the affinity flow-through pool (flow-through from the second Protein A affinity step) was about 1.58 of 1b * values ​​(see Table 9-3). This second affinity flow-through was subjected to AEX. AEX flow-through is approximately 0.50 secondb * , indicating a significant reduction in tan after the use of AEX. Obtain a strip sample by stripping the AEX column, about 6.10 3b * A value was observed which indicated that this strip sample had more tan color when compared to either load or flow through.

[0043] Referring again to Example 9, oxovariant analysis was also performed. The samples analyzed were the affinity flow-through pool (second Protein A affinity eluate), AEX flow-through, and AEX strips. Referring to Tables 9-5 and 9-6, variations regarding oxovariants 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 "W58(+16)" column). Here, it can be observed that the oxovariant (specifically oxo-tryptophan) went from about 0.055% in the load sample to about 0.038% in the flow-through sample after AEX chromatography, which is Shows that there was a decrease in oxovariants after AEX. AEX strips were analyzed and the percentage of oxotryptophan species was found to be approximately 0.089%. Comparing the strip value to the load (and flow-through), it was evident that the majority of this oxovariant was retained on the AEX column.

[0044] This embodiment includes the addition of one or more steps in no particular order, e.g., hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, virus inactivation (e.g., using low pH), virus filtration, and / or ultrafiltration / diafiltration.

[0045] One embodiment of the present invention relates to a method for regenerating a chromatography column containing resin. In one aspect of this embodiment, the resin has an immobilized hydrolyzing 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 a 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, a method of regenerating a column containing a resin comprises incubating a column resin with acetic acid. In one embodiment, the concentration of acetic acid used is about 0.1M to about 2M. In one aspect, the concentration of acetic acid is about 0.5M. In one embodiment, the resin is incubated for at least about 10 minutes. In another embodiment, 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, Gu-HCl without acetic acid is used to regenerate the column resin. The concentration of Gu-HCl used is about 1N to about 10N. In another embodiment, the concentration of Gu-HCl is about 6N. In a further embodiment, the column resin can be incubated with a 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 resin is stored in ethanol. In one embodiment, the column is stored in ethanol at a percentage of about 5% v / v to about 20% v / v. In certain embodiments, columns are stored using 20% ​​v / v ethanol.

[0049] In one embodiment, the process may further comprise formulating the VEGF MiniTrap using pharmaceutically acceptable excipients. In one aspect, pharmaceutically acceptable excipients 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 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 a protein of interest from about 40 to about 200 mg / mL. In particular embodiments, the protein of interest is either aflibercept or aflibercept MiniTrap.

[0051] The formulation is used for 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 in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, Corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in subjects with diabetic macular edema, or diabetic retinopathy (e.g., nonproliferative diabetes mellitus) vascular 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 suffering from DME) It can be used in methods of treating or preventing neoplastic eye disorders.

[0052] Variant of IdeS This 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 ("N * ”). Asparagine at these positions can be mutated to a non-asparagine amino acid to form an IdeS variant (mutated (one or more) amino acids are italicized and underlined and highlighted (one or more) ): TIFF2022552052000002.tif115165TIFF2022552052000003.tif209165TIFF2022552052000004.tif216165TIFF2022552052000005.tif201165TIFF202255205200000056tif1656

[0053] In one embodiment, the polypeptide is 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, 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 containing at least 70% sequence identity over the entire length of the isolated amino acid sequence It has an amino acid sequence. 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 embodiment, the isolated amino acid sequence has at least about 90% sequence identity over the entire length of the isolated amino acid sequence. In another embodiment, 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 the target protein into fragments. In certain embodiments, the target protein is 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.

[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] 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:12 Having at least 70% sequence identity over the entire length of the isolated amino acid sequence as set forth in the group consisting of: No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16 An isolated nucleic acid molecule that encodes a polypeptide is included. 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 embodiment, the isolated amino acid sequence has at least about 90% sequence identity over the entire length of the isolated amino acid sequence. In another embodiment, 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 the target protein into fragments. In certain embodiments, the target protein is IgG. In another specific embodiment, the target protein is a fusion protein. In yet another specific embodiment, the fragment may comprise a Fab fragment and / or an Fc fragment.

[0056] 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:12 Having at least 70% sequence identity over the entire length of the isolated amino acid sequence as set forth in the group consisting of: No. 13, SEQ ID No. 14, SEQ ID No. 15 and SEQ ID No. 16 A vector containing a nucleic acid that encodes a polypeptide is included. In one embodiment, the nucleic acid molecule is operably linked to expression control sequences capable of directing its expression within a host cell. In one aspect, the vector is a plasmid. 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 embodiment, the isolated amino acid sequence has at least about 90% sequence identity over the entire length of the isolated amino acid sequence. In another embodiment, 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 the target protein into fragments. In certain embodiments, the target protein is 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 is 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. can contain. In one aspect, the mutation may result in increased chemical stability at alkaline pH values ​​as compared to the parent amino acid sequence. In another embodiment, the mutation may result in a 50% increase in chemical stability at alkaline pH values ​​as compared to the parent amino acid sequence. In one aspect, 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 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, asparagine residues at positions 87, 130, 182 and 274 are mutated.

[0058] In a related embodiment, the present disclosure provides 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. An isolated nucleic acid molecule that encodes a polypeptide having an isolated amino acid sequence comprising . See above. Mutations can result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence.

[0059] In further related embodiments, the disclosure includes vectors. The vector comprises an isolated 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. A nucleic acid molecule that encodes a polypeptide having a sequence is included. See above. 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 expression control sequences capable of directing its expression within a host cell. In one aspect, the vector can be a plasmid.

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

[0061] In one embodiment, a method of producing a recombinant protein comprises the steps of: (a) providing a host cell genetically modified to express a recombinant protein of interest; culturing the host cell under suitable conditions to express the protein; 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 certain embodiments, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in US Pat. 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 suitable host cells 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 CDM. Suitable CDMs include Dulbecco's Modified Eagle (DME) Medium, Ham Nutrient Mixture, Excell Medium, and IS CHO-CD Medium, and CDM1B. Other CDMs known to those skilled in the art are also contemplated 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. At least one contaminant may originate from cell substrates, cell culture, or downstream processes.

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

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

[0067] In one aspect of this embodiment, the method can comprise subjecting a biological sample comprising an anti-VEGF protein and one or more host cell proteins / contaminants to affinity chromatography under suitable conditions. The stationary phase of this affinity chromatography comprises a protein capable of selectively or specifically binding to the anti-VEGF protein. In certain embodiments, the protein can be an antibody, fusion protein, scFv or antibody fragment. In certain embodiments, the protein is VEGF 165 , VEGF 121 VEGF forms from other species, such as rabbits. For example, as exemplified in Tables 7-1 and 7-10, VEGF as a protein capable of selectively or specifically binding to or interacting with an anti-VEGF protein 165 led to the successful production of MT5 (anti-VEGF protein), aflibercept and anti-VEGF scFv fragments. In another specific embodiment, the protein can be one or more of the proteins having amino acid sequences shown in SEQ ID NOs:73-80. Table 7-1 also discloses the successful generation of MT5 using proteins having the amino acid sequences shown in SEQ ID NOS: 73-80 as proteins capable of selectively or specifically binding to anti-VEGF proteins (MT5). .

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

[0069] Suitable conditions for using affinity chromatography include, but are not limited to equilibration of the affinity chromatography column using an equilibration buffer. After equilibration using, for example, Tris hydrochloride at a pH of about 8.3 to about 8.6, the biological sample is loaded onto the affinity chromatography column. 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 buffer type and pH and conductivity, and other elution conditions known to those skilled in the art can be applied. After elution using one or more elution buffers, eg, glycine, at a pH of about 2.0 to about 3.0, the eluted fraction can be neutralized by the addition of a neutralizing buffer, eg, 1M 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 from 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 with a pH of about 2.5. In another embodiment, the elution buffer can be a buffer having a pH of about 2.0 to about 3.0. In one embodiment, the neutralization buffer can contain 1M Tris with 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 embodiments, the amount of host cell proteins in the eluted fraction is compared to the amount of host cell proteins in the biological sample, e.g., about 70%, about 80%, about 90%, about 95%, It is significantly reduced by about 98%, or about 99%.

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

[0073] In one embodiment, the glycosylation profile of the composition of anti-VEGF proteins is as follows: about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 50% total sialylated glycans. 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% asparagine-123 residues and / or about 27.1% 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 pharmaceutically acceptable excipients. In one aspect, pharmaceutically acceptable excipients 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 human subjects. In one aspect of this embodiment, administration can be by intravitreal injection. In one aspect, the formulation may have from about 40 to about 200 mg / mL protein of interest. In certain embodiments, the protein of interest can be aflibercept, anti-VEGF antibody, or VEGF MiniTrap.

[0077] The formulation is used for 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 in glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, Corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy in subjects with diabetic macular edema, or diabetic retinopathy (e.g., nonproliferative diabetes mellitus) vascular 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 suffering from DME) It can be used in methods of treating or preventing neoplastic eye disorders.

[0078] Synthesis of oxo species One embodiment of the invention relates to one or more methods of synthesizing oxidized protein species using light. In one aspect of this embodiment, the protein of interest is an anti-VEGF protein. In certain embodiments, the anti-VEGF protein is aflibercept. In another embodiment, 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 comprises a protein of interest, eg, an aflibercept fusion protein with minimal or no oxovariants. A light stress is applied to the sample to synthesize an oxidized species of aflibercept. In certain embodiments, the sample is photostressed by using cool white light. In another particular embodiment, the sample is photostressed by using ultraviolet light.

[0080] In certain aspects of this embodiment, a sample comprising aflibercept or another anti-VEGF protein is exposed to cool white light for about 30 hours to about 300 hours, resulting in about 1.5 to about 50 fold increase in modified oligopeptide. occur. These peptides are enzymatically digested and analyzed, DKTHMore * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EAT VNGH * 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 / RIIW*DSR / IIW * DSRK (SEQ ID NO: 28), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO:70)F * LSTLTIDGVTR (SEQ ID NO: 32) including one or more from the group consisting of H * is histidine and is oxidized to 2-oxo-histidine, C * is a cysteine ​​and is carboxymethylated, M * is methionine oxide and W * is oxidized tryptophan and Y * is tyrosine oxide and F * is oxidized phenylalanine. Digestion can be performed with the proteases mentioned so far, eg with trypsin. Oligopeptides can be analyzed using mass spectrometry.

[0081] In certain aspects of this embodiment, a sample comprising aflibercept or another anti-VEGF protein is exposed to UV light for about 4 hours to about 40 hours to produce a modified oligopeptide product (obtained when performing the digestion) of about resulting in an increase of 1.5 to about 25 fold, in which the sample DKTHMore * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EAT VNGH * 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 / RIIW*DSR / IIW * DSRK (SEQ ID NO: 28), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO:70)F * LSTLTIDGVTR (SEQ ID NO: 32) one or more modified oligopeptides selected from the group consisting of H * is histidine and is oxidized to 2-oxo-histidine, C * is a cysteine ​​and is carboxymethylated, M * is methionine oxide and W * is oxidized tryptophan and Y * is tyrosine oxide and F * is oxidized phenylalanine. Digestion can be performed with the proteases mentioned so far, eg with trypsin. Oligopeptides can be analyzed using mass spectrometry.

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

[0083] In one embodiment, the method comprises culturing host cells expressing the recombinant protein of interest in CDM under suitable conditions, followed by harvesting a preparation containing the recombinant protein of interest. Including process. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In certain embodiments, the anti-VEGF protein is aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159, which is incorporated herein by reference in its entirety), scFv, and selected from the group consisting of other anti-VEGF proteins. In one aspect, the method produces a preparation of the recombinant protein of interest, the color of the preparation being the European BY method or the CIELAB method (b * ). Additionally, the presence of oxovariants can be analyzed using, for example, LC-MS.

[0084] In one aspect of this embodiment, reducing 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, for example. 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. are mentioned. In certain embodiments, reduction of cysteine ​​can be effective in reducing the tan color of preparations. Cysteine ​​concentration can also affect oxovariants.

[0085] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under suitable conditions, a preparation of the protein of interest produced by the cells. Suitable conditions are obtained 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 over the course of cell culture to form CDM, which is the beginning of the culture (day 0 CDM ) and the amounts of ingredients added sequentially. When calculating the cumulative amount of a component, the amount of component added to the seed train culture or inoculum prior to production in the bioreactor (ie, prior to day 0 CDM) is also included. Cumulative amounts are not affected by component loss (eg, by metabolism or chemical degradation) over time during culture. Thus, for example, if components are added to two cultures at different times (e.g., in one culture all the components are added first and in another culture the components are added over time). ), even two cultures with the same cumulative amount for a component may have different absolute levels. Cumulative amounts are also unaffected by in situ synthesis of components (eg, by metabolism or chemical transformations) over time during culture. Thus, for example, if a component is synthesized in situ in one of the two cultures during the biotransformation process, given two cultures with the same cumulative amount of a given component may also have different absolute levels. Cumulative amounts may be expressed in units such as grams or moles of the component.

[0086] As used herein, the term "cumulative concentration" refers to 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. refers to the cumulative amount of an ingredient divided by For example, if a bioreactor contains 2 liters of cell culture medium at the start of a manufacturing 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 (ie 4 grams divided by 2 liters). If, on day 4, 1 liter of additional liquid not containing component X is added to the bioreactor, the cumulative concentration remains 2 g / L. Even if on day 5 some amount of liquid is lost from the bioreactor (eg, by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentrations 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 the recombinant protein of interest in CDM under suitable conditions, recovering a preparation of the protein produced by the cells. Preferred conditions are obtained by reducing the ratio of cumulative cysteine ​​concentration from about 1:10 to 1:29 to cumulative total amino acid concentration from about 1:50 to about 1:30.

[0088] In one embodiment, the method comprises (i) culturing, under suitable conditions, host cells expressing the recombinant protein of interest, e.g., aflibercept, in CDM; Suitable conditions are obtained by reducing the cumulative concentration of iron in the CDM to less than about 55.0 μM. In one aspect of this embodiment, preparations obtained by this method have a reduced tan color than preparations obtained by the method with a cumulative concentration of iron in the CDM up to greater than about 55.0 μM. show.

[0089] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under suitable conditions. The method further comprises recovering the preparation of the recombinant protein of interest produced by the cells, suitable conditions being obtained by reducing the cumulative concentration of copper in the CDM to about 0.8 μM or less. be done. In one aspect of this embodiment, preparations obtained by this method have a reduced tan color than preparations obtained by the method with a cumulative concentration of copper in the CDM up to greater than about 0.8 μM. show.

[0090] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, e.g., aflibercept, in CDM under suitable conditions; Suitable conditions are obtained by reducing the cumulative concentration of nickel in the CDM to about 0.40 μM or less. In one aspect of this embodiment, preparations obtained by this method have a reduced tan color than preparations obtained by the method with a cumulative concentration of nickel in the CDM of up to about 0.40 μM. show.

[0091] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under suitable conditions. The method further comprises recovering the preparation of the recombinant protein of interest produced by the cells, suitable conditions being obtained by reducing the cumulative concentration of zinc in the CDM to about 56 μM or less. . In one aspect of this embodiment, preparations obtained by this method exhibit a reduced tan color than preparations obtained by the method with a cumulative concentration of zinc in the CDM of up to about 56 μM. .

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

[0093] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under suitable conditions. The method further comprises recovering the preparation of the recombinant protein of interest produced by the cells, preferred conditions being a cumulative concentration of iron in the CDM of less than about 55 μM, A cumulative concentration of copper in the CDM that is less than or equal to about 0.40 μM, a cumulative concentration of nickel in the CDM that is less than or equal to about 0.40 μM, a cumulative concentration of zinc in the CDM that is less than or equal to about 56 μM, and a cumulative concentration of cysteine ​​in the CDM that is less than 10 mM and / or antioxidants in the CDM at a concentration of about 0.001 mM to about 10 mM for a single antioxidant, and up to about 30 mM when multiple antioxidants are added to the CDM. Includes CDM with cumulative concentrations.

[0094] In one aspect of this embodiment, the preparation obtained using suitable conditions contains the desired amount of aflibercept and VEGF MiniTrap protein variants (“target value” of aflibercept and VEGF MiniTrap protein variants). ), resulting in a reduction of protein variants of aflibercept and VEGF MiniTrap. In a further aspect of this embodiment, the preparation obtained using suitable conditions normalizes the preparation of protein comprising variants of aflibercept and VEGF MiniTrap to a concentration of 5 g / L or 10 g / L. the desired b * value or BY value (respectively "target b * value”, referred to as the “target BY value”). In a further aspect of this embodiment, goal b * A value (or target BY value) and / or target value for a variant can be obtained in a preparation if the titer does not increase or decrease significantly.

[0095] These and other aspects of the invention will be better appreciated and better understood when considered in conjunction with the following description and accompanying drawings. The following description, while indicating various embodiments and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions or rearrangements may be made within the scope of the invention. [Brief description of the drawing]

[0096]

Figure 1

Figure 2

[0097] detailed description Angiogenesis, the growth of new blood vessels from pre-existing vasculature, is a highly organized process that is critical for proper fetal and postnatal vascular development. Abnormal or pathological neovascularization is a hallmark of cancer and multiple retinal diseases. In this case, upregulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) leads to increased endothelial proliferation, altered 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 the therapy of choice for treating DME, wet AMD, CNV, retinal vein occlusion, and other ocular diseases with an underlying etiology of abnormal angiogenesis.

[0098] As used herein, aflibercept comprises the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2 expressed as an inline fusion with (Fc) of human IgG1, It is one such anti-VEGF protein that contains all human amino acid sequences. Aflibercept binds all forms of VEGF-A (VEGF), but also PlGF and VEGF-B. Several other homodimers, VEGF MiniTrap, are produced either as enzymatically cleaved products from aflibercept or directly recombinantly expressed from host cell lines. An example of such a VEGF MiniTrap is shown in FIG. In this figure the terminal lysine is shown (k), some culture processes remove this terminal lysine while others do not. FIG. 1 illustrates the process by which terminal lysines remain. In general, aflibercept includes both the presence of terminal lysines and the absence of terminal lysines.

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

[0100] Colors such as tan can be an undesirable feature in injectable therapeutic drug solutions. This can be an important parameter used to determine whether a drug product meets a given level of purification and quality for a particular therapy. The yellow-brown color observed along the manufacturing route of biopharmaceuticals may be due to chemical modifications of the biopharmaceutical, degradation products of formulation excipients, or degradation products formed by reactions of biopharmaceuticals and formulation excipients. can be caused by However, such information can be useful in understanding the cause of 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 inventors observed that using AEX during the formation of the anti-VEGF protein solution minimized the tan coloration. In addition, the inventors have found that the tan coloration can be reduced by modifying the cell culture used to produce recombinant proteins such as aflibercept or modified aflibercept such as MiniTrap. I discovered.

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

[0103] As presented herein, some of the examples described below are directed to the production of anti-VEGF proteins (Example 1), the production of oxidized species of anti-VEGF proteins (Example 4), the optimization of culture media. A method for reducing oxidized species of anti-VEGF protein by optimizing (Example 5) and a method for reducing oxidized species of anti-VEGF protein by optimizing the production method (Example 2) are described.

[0104] Although several recent patent applications and issued patents are intended to describe various aflibercept species and methods of making them, the anti-VEGF compositions described herein and methods of making them are There is no description or suggestion of For example, Coherus Biosciences Inc. U.S. Patent Application No. 16 / 566,847; Sam Chun Dang Pharm. Co., Ltd. U.S. Patent No. 10,646,546; / US2020 / 015659, and Momenta Pharmaceuticals, Inc., U.S. Pat.

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

[0106] The term "a (a)" should be understood to mean "at least one," and the terms "about" and "approximately" allow for standard variation as understood by those of ordinary skill in the art. It should then be understood that where a range is provided, the endpoints are included.

[0107] As used herein, the term "angiogenic 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 term "chemically defined medium" or "chemically defined media" (both abbreviated as "CDM") refers to the identification and Refers to a synthetic growth medium with defined concentrations. Defined media do not contain microorganisms, yeast, animal or plant extracts, animal serum or plasma, but individual plant or animal-derived components (eg proteins, polypeptides, etc.) may be added. Synthetic media may contain inorganic salts such as phosphates, sulfates and equivalents required to support growth. Carbon sources are defined and are usually sugars such as glucose, lactose, galactose and their equivalents, or other compounds such as glycerol, lactate, acetate and their equivalents. Certain synthetic media also use phosphate as a buffer, while other buffers such as sodium bicarbonate, HEPES, citric acid, triethanolamine, and their equivalents may be used. Examples of commercially available synthetic media include various Dulbecco's Modified Eagle (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), ham nutrient mixture (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, US Pat. , 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 CDM, which is the beginning of the culture (day 0 CDM ) and the amounts of ingredients added sequentially. When calculating the cumulative amount of a component, the amount of component added to the seed train culture or inoculum prior to production in the bioreactor (ie, prior to day 0 CDM) is also included. Cumulative amounts are not affected by component loss (eg, by metabolism or chemical degradation) over time during culture. Thus, for example, if components are added to two cultures at different times (e.g., in one culture all the components are added first and in another culture the components are added over time). ), even two cultures with the same cumulative amount for a component may have different absolute levels. Cumulative amounts are also unaffected by in situ synthesis of components (eg, by metabolism or chemical transformations) over time during culture. Thus, for example, if a component is synthesized in situ in one of the two cultures during the biotransformation process, given two cultures with the same cumulative amount of a given component may also have different absolute levels. Cumulative amounts can be expressed in units such as grams or moles of an ingredient.

[0110] As used herein, the term "cumulative concentration" refers to the volume of liquid in the bioreactor at the start of the production batch, including additions to the starting volume from any inoculum used in the culture. Refers to the cumulative amount of the divided component. For example, if a bioreactor contains 2 liters of cell culture medium at the start of a manufacturing 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 (ie 4 grams divided by 2 liters). If, on day 4, 1 liter of additional liquid not containing component X is added to the bioreactor, the cumulative concentration remains 2 g / L. Even if on day 5 some amount of liquid is lost from the bioreactor (eg, by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentrations 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 embodiment, the protein of interest is aflibercept and / or MiniTrap. In some exemplary embodiments, the amount of protein of interest in the formulation can range from about 0.01 mg / mL to about 600 mg / mL. In some specific embodiments, the amount of protein of interest in the formulation is 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 0.9 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 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, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 5 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, 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 can be greater than about 5.0. In an exemplary embodiment, the pH can 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, which provides uninterpreted MS-MS spectra for all possible sequences in a database (one or more). provides the possibility to search for 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.scripts.edu / sequest).

[0113] As used herein, the term "ultrafiltration" or "UF" includes membrane filtration processes similar to reverse osmosis that use hydrostatic pressure to force water through a semipermeable membrane. obtain. 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. By using filters with such small pore sizes, the sample volume can be reduced by permeating sample buffer through the filter while retaining proteins behind the filter.

[0114] As used herein, "diafiltration" or "DF" removes and exchanges salts, sugars, and non-aqueous solvents, separates them from bound species, removes low molecular weight materials, and / or Methods may include using ultrafilters to cause rapid changes 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, the microspecies are washed out of the solution in a constant amount. In certain exemplary embodiments of the invention, a diafiltration step is used to exchange various buffers used in connection with the invention, e.g., prior to chromatography or other manufacturing steps, to remove impurities from proteins. It can be removed from the preparation. As used herein, the term "downstream processing technology" refers to one or more techniques used after an upstream processing technology to produce a protein. Downstream processing techniques include, for example, protein A affinity chromatography and affinity chromatography using a solid phase with well-defined molecules such as VEGF that are capable of interacting with their cognate, such as the 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 cells 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's cells, but also to the progeny of such cells. Such progeny may not actually be identical to the parent cell, as certain modifications may occur in successive generations, either due to mutation or environmental influences, but This is still included within 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 embodiments, host cells comprise eukaryotic cells such as plant cells and / or animal cells. Cells can be mammalian, fish, insect, amphibian or avian cells. In certain embodiments, host cells are mammalian cells. Various mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other depositories, as well as commercial suppliers. Cells that can be used in the process of the invention include MK2.7 cells; PER-C6 cells; 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). USA, 77:4216), and Chinese hamster ovary cells (CHO) such as dp12.CHO cells (U.S. Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); Monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCC CRL-1651); 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 cancer cells (HELA, ATCC CCL -2); dog kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human liver cancer cells (HEP-G2, HB8065); mouse breast cancer 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; 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-C-I cells, LLC-MK 2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK 1 cells, PK(15) cells, GH 1 cell, GH 3 cells, L2 cells, LLC-RC256 cells, MH 1 C. 1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblasts from any tissue or organ (heart, liver, kidney, colon, intestine, esophagus, stomach, Nerve tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries), lymphatic tissue (lymph gland, pharyngeal tonsil, tonsil, bone marrow and blood), spleen and fibroblasts and fibroblast-like cell lines (For example, 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, MiCl 1 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, C 3 H / IOTI / 2 cells, HSDM 1 C. 3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, strain L-M (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntac cells, SIRC cells, C II cells, and Jensen cells, or derivatives thereof (including but not limited to cell lines) or any other cell type known to those of skill in the art.

[0116] As used herein, the term "host cell protein" (HCP) includes proteins derived from the host cell and can be unrelated to the desired protein of interest. Host cell proteins can be process-related impurities that can originate from the manufacturing process, which can include three main categories: cell substrate-derived, cell culture-derived, and downstream-derived. Impurities from cell substrates include, but are not limited to, proteins from host organs and nucleic acids (the genome, vector, or total DNA of the host cell). Cell culture derived impurities include, but are not limited to inducers, antimicrobials, serum and other media components. Impurities from downstream sources include 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 (eg, monoclonal antibodies), and other leachable substances.

[0117] In some exemplary embodiments, host cell proteins can have a pI in the range of about 4.5 to about 9.0. In exemplary embodiments, the pI is 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, It can be 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 "hydrolyzing agent" refers to any one or combination of a number of different agents capable of effecting protein digestion. Non-limiting examples of hydrolysing agents capable of enzymatic digestion include protease from Aspergillus niger, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergilopepsin 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), Streptococcus pyogenes immunoglobulin degrading enzyme (IdeS), thermolysin, papain, pronase, V8 protease, or biologically active fragments or homologues thereof, or combinations thereof. . Non-limiting examples of hydrolysing agents capable of performing non-enzymatic digestion include high temperature, microwave, ultrasound, high pressure, infrared, solvents (non-limiting examples are ethanol and acetonitrile), immobilized enzymes. These include the use of digestion (IMER), magnetic particle immobilized enzymes, and on-chip immobilized enzymes. A recent review describing techniques available for protein digestion is Switzar et al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzar, Martin Giera & Wilfried M.A. Niessen, 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 hydrolysing agents can cleave peptide bonds of proteins or polypeptides in a sequence-specific manner to generate a predictable collection of shorter peptides. The ratio of hydrolyzing agent to protein and the time required for digestion can be appropriately selected to obtain optimal digestion of the protein. Inappropriately high enzyme to substrate ratios can result in correspondingly high digestion rates, inadequate time to analyze peptides on the mass spectrometer, and loss of sequence coverage. On the other hand, the low E / S ratio requires long digestion times, which increases the 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 hydrolysis of one or more peptide bonds of a protein. Several approaches exist to carry out the digestion of proteins in biological samples using suitable hydrolysing agents, eg enzymatic 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 properties 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 also refer to six different classes, aspartic proteases, glutamine proteases, and metalloproteases, cysteine ​​proteases, serine proteases, and threonine proteases, as classified based on their mechanism of catalysis. . The terms "protease" and "peptidase" are used interchangeably to refer to enzymes that hydrolyze peptide bonds.

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

[0120] As used herein, the term “liquid chromatography” refers to the flow of a fluid-borne biological / chemical mixture through (or into) a stationary fluid or solid phase, Refers to a process that is separable into components resulting from differential partitioning of the components. Non-limiting examples of liquid chromatography include reverse 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 separations involving any method that separates two substances based on their affinity for a chromatographic material. This may involve applying the substance to a column containing a suitable affinity chromatography medium. Non-limiting examples of such chromatographic media include protein A resins, protein G resins, affinity supports containing antigens against which binding molecules (e.g., antibodies) have been produced, proteins capable of binding to proteins of interest. , and affinity supports comprising Fc binding proteins. In one aspect, the affinity column can be equilibrated with a suitable buffer prior to sample loading. An example of a suitable buffer can be a Tris / NaCl buffer (pH approximately 7.0-8.0). Suitable buffers can be developed by those skilled in the art 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, eg, using an equilibration buffer. Other washes, including washes using different buffers, can be used before eluting the column. The affinity column can then be eluted using a suitable elution buffer. An example of a suitable elution buffer can be an acetic acid / NaCl buffer (pH approximately 2.0-3.5). Moreover, a person skilled in the art can develop a suitable elution buffer 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, to determine if the sample of interest contains aromatic rings (e.g., proteins with aromatic amino acids such as tryptophan). It can be used especially when

[0122] As used herein, "ion exchange chromatography" refers to the separation of two substances either on a molecule of interest and / or on a chromatographic material. may refer to separation including any method that separates, collectively or locally, over specific regions of , based on differences in their respective ionic charges. Therefore, it can use either a cation exchange material or an 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. A packed column or ion exchange membrane device 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, increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the ion-exchange matrix allows the product to can be collected. Altering the pH and thereby altering the charge of the solute can be another method of achieving elution of the solute. The change in conductivity or pH may be stepwise (gradient elution) or stepwise (step elution). Anionic or cationic substituents may be attached to the matrix to form an anionic or cationic support for chromatography. Non-limiting examples of anion 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, U.K. 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 derivitized 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 from BioRad, (Hercules, Calif.), and Eshmuno® S from EMD Millipore (MA).

[0123] As used herein, the term "hydrophobic interaction chromatography resin" can include solid phases that can be covalently modified with phenyl, octyl, butyl, or the like. Hydrophobic interaction chromatography uses properties such as hydrophobicity to separate molecules from each other. 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 their equivalents are passed through a HIC (Hydrophobic Interaction Chromatography) column having one or more hydrophobic regions or hydrophobic pockets on its surface and the stationary phase of the HIC. can interact with the hydrophobic groups that make up the 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). mentioned.

[0124] As used herein, the terms "mixed mode chromatography" or "multimodal chromatography" (both "MMC") refer to the interaction of a solute with a stationary phase through multiple interaction modes or mechanisms. chromatographic methods for MMC can be used as an alternative or complementary tool to conventional 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 each the dominant mode of interaction, mixed-mode chromatography Combinations of two or more of the modes can be used. Mixed-mode chromatographic media can offer unique selectivities that single-mode chromatography cannot reproduce. Mixed-mode chromatography can also offer potential cost savings, extended column lifetime, and operational flexibility when compared to affinity-based methods. In some exemplary embodiments, mixed-mode chromatographic media, sometimes referred to as base matrices, are composed of mixed-mode ligands attached directly or via spacers to an organic or inorganic support. can be configured. The support may be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, and the like. In some exemplary embodiments, supports can be prepared from natural polymers such as cross-linked carbohydrate materials. For example this is agarose, agPV, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate and the like. To obtain high adsorption capacities, the support can be porous and the ligands are then attached to the exterior and pore surfaces. Such natural polymer supports can be prepared by standard methods, such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teaching of which is incorporated herein). ). Alternatively, the support is prepared from synthetic polymers, such as crosslinked synthetic polymers, e.g., styrene or styrene derivatives, divinylbenzene, acrylamides, acrylates, methacrylates, vinyl esters, vinylamides, and the like. can be Such synthetic polymers are prepared by 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 teaching of which is the subject of this publication). incorporated herein). Porous natural or synthetic polymeric supports are also available from manufacturers such as GE Healthcare (Uppsala, Sweden).

[0125] As used herein, the term "mass spectrometer" includes devices capable of identifying specific molecular species and measuring their precise masses. The term is meant to include any molecular detector in which a polypeptide or peptide can be characterized. A mass spectrometer can include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to create ions in the gas phase. Analyte atoms, molecules, or clusters can be transferred to the gas phase and ionized either simultaneously (with electrospray ionization) or through a separation process. The choice of ion source depends on the application. In some exemplary embodiments, the mass spectrometer can 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 stages of mass selection and mass separation. A prerequisite is to transfer the sample molecules into the gas phase and ionize them to form fragments in a predictable and controllable manner after the initial mass selection step. Multi-step MS / MS or MS n is the precursor ion (MS 2 ) is selected and separated, and fragmented to form the first fragment ion (MS 3 ) and fragmented to obtain a second fragment (MS 4), as long as significant information can be obtained or the fragment ion signal is detectable. Tandem MS is successfully performed in combination with many types of analyzers. Which analyzer to combine for a particular application is determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and effectiveness. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but there are also hybrids in which temporal tandem analyzers are coupled spatially or coupled with spatial tandem analyzers. . A spatial tandem mass spectrometer comprises an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. The Separation by Specific m / z function selects ions in one section of the instrument, dissociates them in an intermediate region, and then sends the product ions to another analyzer for separation by m / z and data acquisition. can be designed to In temporal tandem, mass spectrometer ions generated in the ion source can be confined within the same physical device, isolated, fragmented, and separated by m / z. 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 includes amino acid sequencing of protein fragments, determination of protein sequencing, determination of de novo sequencing of proteins, localization of post-translational modifications, or identification of post-translational modifications, or comparative analysis, or combinations thereof. include, but are not limited to.

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

[0127] As used herein, a "VEGF MiniTrap" or "VEGF MiniTrap binding molecule" can be a molecule or complex of molecules that binds to VEGF, which is the Ig-like domain of the VEGF receptor (or variants) (e.g., VEGFR1 Ig domain 2 and / or VEGFR2 Ig domain 3 and / 4) and one or more sets of modified multimerization components (MCs), or modified multimerization thereof Component free, this MC is a modified immunoglobulin Fc. This modification may be the result of proteolytic digestion of the VEGF trap (eg aflibercept or conbercept) or direct expression of the resulting polypeptide chain with a truncated MC sequence. (See the molecular structure shown in Figure 1.) Figure 1 shows the VEGF MiniTrap molecule, the proteolytic product of aflibercept with Streptococcus pyogenes IdeS. A homodimeric molecule is shown having Ig hinge domain fragments connected by two parallel 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 " / / ". Also shown is the Fc fragment cleaved from aflibercept. Such a single chimeric polypeptide that is not dimerized 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 MC but one or more VEGF receptor fused with linkers (eg, peptide linkers) to one or more additional sets of Ig domains (or variants thereof). The VEGF binding domains in the VEGF MiniTrap of the invention may be the same or different from one another (WO 2005 / 00895, the entire teaching of which is incorporated herein). ).

[0128] For example, in one embodiment of the invention, the unmodified immunoglobulin Fc domain comprises the amino acid sequence or amino acids 1-226 thereof: TIFF2022552052000007.tif26165 (SEQ ID NO: 33, in which X 1 is L or P, and X 2 is A or T).

[0129] Inhibition of VEGF includes, for example, VEGF (e.g., VEGF 110 , VEGF 121 , and / or VEGF 165 ) antagonism of VEGF binding to the VEGF receptor, eg, by competing with the VEGF receptor for binding. Such inhibition results, for example, in cells having a VEGFR extracellular domain fused to IL18Rα and / or IL18Rβ intracellular domains on the cell surface and an NFkB-luciferase-IRES-eGFP reporter gene, such as those described herein. VEGFR expression is inhibited in cell lines (e.g. HEK293) expressing chimeric VEGF receptors (e.g. homodimers thereof) with strain HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ. Activation by VEGF can be inhibited.

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

[0131] The immunoglobulin-like domains of VEGF receptors may be referred to herein as VEGFR "Ig" domains. For example, R1D2 (may be referred to herein as VEGFR1(d2))), R2D3 (may be referred to herein as VEGFR2(d3))), R2D4 (may be referred to herein as VEGFR2(d4) )) and R3D3 (which may be referred to herein as VEGFR3(d3))) are referred to herein not only as complete wild-type Ig domains, but also when incorporated into a VEGF MiniTrap, for example Also intended to include those variants that substantially retain the functional properties of the wild-type domain, retaining the ability to form a functional VEGF binding domain. It will be readily apparent to those skilled in the art that numerous variants of the above Ig domains can be obtained which substantially retain the same functional properties as the wild-type domain.

[0132] The present invention provides VEGF MiniTrap polypeptides comprising the following domain structures: ((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. IgG hinge domain or fragments thereof); - the linker is a peptide comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, e.g. (GGGS) g is; 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: Contains TIFF2022552052000008.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 TIFF2022552052000009.tif11165 (SEQ ID NO:35).

[0135] In one embodiment of the invention, R2D3 has the amino acid sequence: Contains TIFF2022552052000010.tif11164 (SEQ ID NO:36).

[0136] In one embodiment of the invention, R2D4 has the amino acid sequence: Includes TIFF2022552052000011.tif11164 (SEQ ID NO:37).

[0137] In one embodiment of the invention, R2D4 has the amino acid sequence: Contains TIFF2022552052000012.tif11164 (SEQ ID NO:38).

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

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

[0140] In one embodiment of the invention, the VEGF MiniTrap polypeptide has the amino acid sequence: Contains TIFF2022552052000013.tif165166TIFF2022552052000014.tif55165. As noted, such polypeptides can be multimerized (e.g., dimerized (e.g., homodimerized)), where binding between the polypeptides is through the multimerization components. mediated by

[0141] In one embodiment of the invention, the VEGFR1 Ig-like domain 2 of the monomeric VEGF MiniTrap of the invention has N-linked glycosylation at N36 and / or N68; and / or VEGFR2 Ig-like domain 3 of the monomeric VEGF MiniTrap of the 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 invention, the VEGF MiniTrap has the structure: (R1D2) 1 -(R2D3) 1 -(G 4 S) 3 -(R1D2) 1 -(R2D3) 1 ; (R1D2) 1 -(R2D3) 1 -(G 4 S) 6 -(R1D2) 1 -(R2D3) 1 ; (R1D2) 1 -(R2D3) 1 -(G 4 S) 9 -(R1D2) 1 -(R2D3) 1 or (R1D2) 1 -(R2D3) 1 -(G 4 S) 12 -(R1D2) 1 -(R2D3) 1 including. G. 4 S is -Gly-Gly-Gly-Gly-Ser-.

[0143] In one embodiment of the invention, the VEGF MiniTrap has the amino acid sequence: TIFF2022552052000015.tif25165TIFF2022552052000016.tif213166TIFF2022552052000017.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 include secondary structures, where similar VEGER Ig domains join to form intrachain VEGF binding domains (e.g., Figure 2 ). In one embodiment of the invention, two or more of such polypeptides are multimerized (e.g., dimerized (e.g., homodimerized)), where the VEGFR Ig domains of each chain are Binds similar Ig domains on different chains to form an internal VEGF binding domain.

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

[0145] In one embodiment of the invention, the polypeptide comprises secondary structure, wherein 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 (eg homodimers) align to form a VEGF binding domain. For example in this case (i) the R1D2 domains are aligned, (ii) the R2D3 domains are aligned and / or (iii) the R2D4 domains are aligned, Forms a VEGF binding domain. Figure 2 shows a single chain VEGF MiniTrap showing such a domain arrangement. VEGFR1, VEGFR2, and linker domains are indicated. The indicated linker is (G 4 S) 6 is. The present invention (G 4 S) 3 ;(G 4 S) 9 ; or (G 4 S) 12 Contains a single chain VEGF MiniTrap with a linker.

[0146] Additionally, the present invention also provides a complex comprising a VEGF MiniTrap as described herein that forms a complex with a VEGF polypeptide or fragment or fusion thereof. In one embodiment of the invention, VEGF (e.g., VEGF 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 be done. 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 (eg, immobilized on a solid substrate) or present within the subject's body. The present invention also provides a VEGF dimer complexed with a VEGF MiniTrap (e.g., VEGF 165 ).

[0147] As used herein, the term "protein" or "protein of interest" can include any amino acid polymer having covalently attached 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" means 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 their synthetic analogues that do not occur in nature. A "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. A variety of solid phase peptide synthesis methods are known to those skilled in the art. A protein may comprise one or more polypeptides to form a single functional biomolecule. In another exemplary aspect, proteins can include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins of interest include biotherapeutic proteins, recombinant proteins for research or therapeutic use, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and Any bispecific antibody may be included. In certain embodiments, the protein of interest is an anti-VEGF fusion protein (eg, aflibercept or MiniTrap). Proteins may be produced in recombinant cell-based systems such as insect bacculovirus systems, yeast systems (e.g. Pichia sp.), mammalian systems (e.g. CHO cells and CHO derivatives such as CHO-K1 cells). It can be produced using a production system. A recent review of biotherapeutic proteins and their production is Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation." 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, proteins include modifications, adducts, and other covalently attached moieties. Such modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans (eg, 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 composition and solubility and thus include simple proteins such as globular and fibrous proteins, nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins. Complex proteins, such as proteins, and induced proteins, such as primary and secondary induced proteins, are included.

[0148] In some exemplary embodiments, the protein of interest can be a recombinant protein, antibody, bispecific antibody, multispecific antibody, antibody fragment, monoclonal antibody, fusion protein, 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 that is incorporated into a suitable host cell. In certain exemplary embodiments, a recombinant protein can be a fusion protein. In certain embodiments, the recombinant protein is an anti-VEGF fusion protein (eg, aflibercept or MiniTrap). In certain exemplary embodiments, the recombinant protein can be an antibody, eg, a chimeric antibody, a humanized antibody, or a fully human antibody. In certain exemplary embodiments, the recombinant protein can 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 (eg IgG1), or alternatively the antibody may be a fragment (eg Fc or Fab fragment).

[0150] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. , as well as multimers thereof (eg, 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 contains 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 contains one domain (CL1). The VH and VL regions are further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of 3 CDRs and 4 FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from amino-terminus to carboxyl-terminus. In different embodiments of the invention, the FRs (or antigen-binding portions thereof) of the anti-big-ET-1 antibody can be identical to human germline sequences, or can be naturally or artificially altered. Amino acid consensus sequences can be defined based on side-by-side analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of full antibody molecules. As used herein, the term "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and equivalents thereof, refers to any antibody that specifically binds to an antigen to form a complex. It includes naturally occurring polypeptides or glycoproteins, enzymatically obtainable polypeptides or glycoproteins, synthetic polypeptides or glycoproteins, or genetically modified polypeptides or glycoproteins. Antigen-binding fragments of antibodies may be prepared using any suitable standard technique, e.g., proteolytic digestion or recombinant genetic modification techniques involving the manipulation and expression of the DNA encoding the variable and, optionally, constant domains, and intact antibodies. It can be derived from molecules. Such DNAs are known and / or readily available, eg, from commercial sources, DNA libraries (including, eg, phage-antibody libraries), or can be synthesized. DNA is used, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, create cysteine ​​residues, modify, add, or delete amino acids. In addition, they can be sequenced and manipulated chemically or using molecular biology techniques.

[0151] As used herein, an "antibody fragment" includes a portion of an intact antibody, eg, the antigen binding region or variable region of an antibody. Examples of antibody fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv bispecific antibodies, dAb fragments, Fd' fragments, Fd fragments, and isolated Multispecific antibodies formed from complementarity determining region (CDR) regions, and triabodies, tetrabodies, linear antibodies, single chain antibody molecules, and antibody fragments, without limitation. An Fv fragment is a combination of immunoglobulin heavy and light chain variable regions, and a scFv protein is a recombinant single-chain polypeptide molecule in which the immunoglobulin light and heavy chain variable regions are connected by a peptide linker. is. In some exemplary embodiments, an antibody fragment comprises the sufficient amino acid sequence of a parent antibody that is a fragment that binds the same antigen as the parent antibody binds; binds the antigen with similar affinity as the parent antibody and competes with the parent antibody for binding to the antigen. Antibody fragments may be produced by any means. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of intact antibodies, and / or can be produced recombinantly from genes encoding partial antibody sequences. Alternatively or additionally, antibody fragments may be wholly or partly synthetically produced. Antibody fragments may optionally include single chain antibody fragments. Alternatively or additionally, an antibody fragment may comprise multiple chains that are linked together, for example, by disulfide bonds. Antibody fragments may optionally include multimolecular complexes. A functional antibody fragment typically contains 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 consist of two different heavy chains that each specifically bind to a different epitope on either two different molecules (e.g. multiple antigens) or the same molecule (e.g. the same antigen). Contains heavy chain. 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 that of the second epitope. It may be at least 1-2 orders of magnitude or 1-3 orders of magnitude or 1-4 orders of magnitude lower than the affinity of a single heavy chain, and vice versa. The epitopes recognized by the bispecific antibody can be on the same target or different targets (eg, 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 such sequences are placed in cells expressing immunoglobulin light chains. can be expressed.

[0153] A typical bispecific antibody has two heavy chains each having three heavy chain CDRs, followed by a CH1 domain, a hinge, a CH2 domain and a CH3 domain, none of which confer antigen-binding specificity but each heavy chain. an immunoglobulin light chain capable of binding the chain, or an immunoglobulin light chain capable of binding each heavy chain and capable of binding one or more epitopes bound by the heavy chain antigen-binding region, or each heavy chain It has an immunoglobulin light chain that is capable of binding and capable of binding one or both heavy chains to one or both epitopes. BsAbs can be divided into two major classes, those with an Fc region (IgG-like) and those lacking an Fc region, the latter usually comprising an IgG and an IgG-like duplex containing an Fc. It is smaller than the specific molecule. IgG-like bsAbs are e.g. triomab, knobs into holes IgG (kih IgG), closumab, 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-bodies, but not limited to these. Different non-IgG-like formats include tandem scFv, diabody format, single-chain diabody, tandem diabody (TandAb), dual affinity retargeting molecule (DART), DART-Fc, nanobody, or 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 teaching of which is in the book. incorporated herein). Methods of producing bsAbs are not limited to quadroma technology, based on the somatic fusion of two different hybridoma cell lines, chemical conjugation involving chemical cross-linkers, genetic approaches utilizing recombinant DNA technology. Examples of bsAbs include: U.S. Patent No. 12 / 823838, filed Jun. 25, 2010; U.S. Patent No. 13 / 488628, filed Jun. 5, 2012; U.S. Patent No. 14 / 031075 filed 19th July, U.S. Patent No. 14 / 808171 filed July 24, 2015, U.S. Patent No. 15 / 713574, U.S. Patent No. 15 / 713569, filed September 22, 2017; U.S. Patent No. 15 / 386453, filed December 21, 2016; US Patent No. 15 / 386443 filed July 29, 2016, US Patent No. 15 / 22343 filed July 29, 2016 and US Patent No. 15814095 filed November 15, 2017. , which are incorporated herein by reference. Low levels of homodimeric impurities can be present at multiple steps during the manufacture of bispecific antibodies. The detection of such homodimer impurities is difficult because of their low abundance and the fact that these impurities co-elute with the major species when performed using conventional liquid chromatography methods. can be difficult when performed using intact mass spectrometry.

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

[0155] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. A monoclonal antibody 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 for this disclosure can 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, a protein of interest can have a pI in the range of about 4.5 to about 9.0. In one particular exemplary embodiment, the pI is 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 6.5, 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, It can be 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, there may be more than one type of protein of interest in the composition.

[0157] In some exemplary embodiments, the protein of interest can be produced from mammalian cells. Mammalian cells can be of human origin, or non-human origin can be primary epithelial cells (e.g. keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, kidney epithelial cells and retinal epithelial cells), established cells Lineages and lines thereof (e.g., 293 embryonic 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 VA13 , 2RA cells, WISH cells, BS-C-I cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-l cells, LLC-PKi cells, PK(15) cells, GHi cells, GH3 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), fibroblasts from any tissue or organ (heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (arteries, veins, capillaries ), lymphoid tissues (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, 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 , F9 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, strain 2071 (mouse L) cells, L-M strain (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), methylmethanethiosulfonate (MMTS), and 4- vinylpyridine or a combination thereof.

[0159] As used herein, "protein denaturation" can refer to a process that alters the three-dimensional shape of a molecule from its native state. Denaturation of proteins can be carried out using protein denaturants. Non-limiting examples of protein denaturants include exposure to heat, high or low pH, reducing agents such as DTT (see below) or chaotropic agents. Some chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of the system by interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen bonding, van der Waals forces, and hydrophobic effects. 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 their salt.

[0160] As used herein, the term "protein reducing agent" refers to agents 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) is at least about 70-99.9% (e.g., a variant of a VEGFR Ig domain) a reference or native amino acid sequence of a protein of interest. 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%) refers to polypeptides containing identical or similar amino acid sequences. Sequence comparisons can be performed, for example, by the BLAST algorithm, wherein the parameters of the algorithm are selected to give the largest match between each sequence over the entire length of each reference sequence (e.g., threshold :10, word size 3, query width: 0 max match, BLOSUM62 matrix, gap cost: presence 11, elongation 1, conditional composition score matrix adjustment). Variants of polypeptides (e.g., variants of the VEGFR Ig domain) may also include one or more (e.g., 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10). The following references relate to BLAST algorithms often used for sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J.272(20):5101-5109; Altschul, S.F., et al. 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., (1993) Comput. Chem. 17:149-163; Hancock, J.M. et al., (1994) Comput. Appl. Biosci. , et al., "A model of evolutionary change in proteins." Atlas of Protein Sequence and Structure, (1978) vol.5, suppl.3. Res.Found., Washington, D.C.; Schwartz, R.M., et al., "Matrices for detecting distant relationships" Atlas of Protein Sequence and Structure, (1978) vol.5, suppl.3. M.O.Dayhoff(ed.) 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. : 10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol. 36: 290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877; :2022-2039; andAltschul, S.F. ``Evaluating the statistical significance of multiple distinct local alignments.'' Theoretical and Computational Methods in Genome Research (S.Suhai, ed.), (1997) pp.1-14, Plenum, N.Y.; The entire teachings of these are incorporated herein.

[0162] Some variants may be covalent modifications that polypeptides undergo either during their ribosomal synthesis (co-translational modifications) or after their ribosomal synthesis (post-translational modifications “PTMs”). PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at sites of particular characteristic protein sequences (eg, 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 ed., Wiley and Sons, Ltd., ISBN: 9780470669853, the entire teachings of which are incorporated herein). In certain exemplary embodiments, a protein composition may contain more than one type of protein variant of the protein of interest.

[0163] Protein variants in the case of aflibercept (and proteins sharing structural features of aflibercept, such as one or more heavy or light chain regions of aflibercept) include, for example, histidine, cysteine, methionine, Oxidative variants that can result from oxidation of one or more amino acid residues occurring at tryptophan, phenylalanine and / or tyrosine residues, deamidation that can result from deamidation at asparagine residues and / or deoxyglucosonated arginine residues. It can include, but is not limited to, variants.

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

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

[0166] With respect to aflibercept (and proteins sharing structural features of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), the deoxyglucosonation variant is Arg5 (or 3-deoxyglucosonation of arginine residues at equivalent residue positions on proteins that share certain structural characteristics).

[0167] Protein variants may contain both acidic and basic species. Acidic species typically elute earlier than the main peak from CEX or later than the main peak from AEX, while basic species elute later than the main peak from CEX or AEX is a variant that elutes earlier than the main peak.

[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, eg, 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 can include, but are not limited to, deamidation variants, defucosylation variants, oxidation variants, methylglyoxal (MGO) variants, glycation variants, and citrate variants. Exemplary structural variants include, but are not limited to glycosylation variants and acetonation variants. Exemplary fragmentation variants include, but are not limited to, Fc and Fab fragments, fragments lacking Fab, fragments lacking the heavy chain variable domain, fragmentation of the peptide chain, enzymatic and / or or any variant protein species from the target molecule by chemical modification; C-terminal truncated variants; variants with truncated N-terminal Asp in the light chain; and variants with N-terminal truncation of the light chain. Other acidic species variants include variants involving unpaired disulfides, host cell proteins, and host nucleic acids, chromatographic materials, and media components. In general, 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 comprise more than one type of acidic species variant. For example, and without limitation, total acidic species can be classified based on the chromatographic retention time at which the peak appears. Another example of how total acidic species can be classified can 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 impurity" refers to an impurity that is present in a composition comprising a protein, but is not itself derived from the protein. Process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatographic materials, and media components.

[0171] In one exemplary embodiment, the amount of acidic species in the anti-VEGF composition compared to the protein of interest is 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% of the above Within one or more ranges. Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition comprises an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), scFv and other anti-VEGF proteins. can include In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0172] Among chemical degradation pathways involving acidic or basic species, the two most commonly observed covalent modifications that occur in proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan, and tyrosine are the most sensitive amino acids 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 a protein formed by oxidation. Such acidic species can also be detected by various methods such as ion exchange, eg WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Oxidative variants can result from oxidation occurring at histidine, cysteine, methionine, tryptophan, phenylalanine and / or tyrosine residues. In particular, with respect to aflibercept (and proteins sharing structural features of aflibercept, e.g., one or more heavy or light chain regions of aflibercept), oxidative variants are His86, His110, His145, His209, Oxidation of histidine residues at His95, His19 and / or His203 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept); oxidation of tryptophan residues at Tyr64 (or equivalent residue positions on proteins sharing certain structural characteristics of aflibercept); oxidation of phenylalanine residues at Phe44 and / or Phe166 (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept); and / or Met10, Met20, Met163 and / or Alternatively, it may involve oxidation at a methionine residue at Met192 (or an equivalent residue position on a protein that shares certain structural features of aflibercept).

[0174] In one exemplary embodiment, the amount of oxidized species in the anti-VEGF composition compared to the protein of interest is 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%, one or more of the above is within the range of Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition comprises an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), scFv and other anti-VEGF proteins. can include In preferred embodiments, the recombinant protein of interest is aflibercept or MiniTrap.

[0175] Cysteine ​​residues can undergo spontaneous oxidation to form either intramolecular or intermolecular disulfide bonds, or unimolecular byproducts such as sulfenic acid.

[0176] Histidine residues are also highly susceptible to oxidation via reactions with their imidazole ring, which in turn can generate additional hydroxyl species (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, the entire teachings of which are incorporated herein). A proposed mechanism for histidine oxidation is highlighted in FIGS. Detailed mechanistic studies are available in Anal. Chem. 2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. be incorporated.

[0177] Oxidation of methionine can lead to the formation of methionine sulfoxide (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 ). 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] Oxidation of tryptophan can lead to a complex mixture of products. Primary products can be N-formylkynurenine and kynurenine with mon-, di- and / or tri-oxidation products (Figure 4). Peptides with oxidative Trp modifications are commonly kynurenine (KYN), hydroxytryptophan (W OX1 ), and N-formylkynurenine / dihydroxytryptophan (NFK / W OX2 , also called “double-oxidized Trp”), trihydroxytryptophan (W OX3 , also called “triply oxidized Trp”) and, for example, hydroxykynurenine (KYN OX1, +20 Da) exhibit mass increases of 4 Da, 16 Da, 32 Da, and 48 Da, corresponding to the formation of these combinations. Hydroxytryptophan (W OX1 ) oxidation (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 teaching of incorporated herein). Tryptophan oxidation, but not methionine and histidine oxidation, was found to produce color changes in protein products (Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores.dx.doi.org / 10.1021 / ac404218t|Anal.Chem.2014,86,6850-6857). Like tryptophan, tyrosine oxidation initially yields 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, which are Characterized by an overall basic charge compared to the primary charge variant species present. For example, in recombinant protein preparations such basic species can be detected by various methods such as ion exchange, eg WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Exemplary variants include lysine variants, aspartic acid isomerization, succinimide formation at asparagine, methionine oxidation, amidation, imperfect disulfide bond formation, serine to arginine mutation, glycosylation, fragmentation and aggregation. but not limited to these. In general, 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 comprise more than one type of basic species variant. For example, without limitation, total basic species can be separated based on the chromatographic retention time at which the peak appears. Another example in which total basic species can be separated can 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 are the result of product preparation (referred to herein as "preparation-derived basic species"), or the result of storage (referred to herein as "storage-derived basic species").

[0182] In one exemplary embodiment, the amount of basic species in the anti-VEGF composition compared to the protein of interest is 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%, one of the above Within the above range. Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition comprises an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in U.S. Pat. No. 7,279,159), scFv and other anti-VEGF proteins. can include In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0183] As used herein, "sample matrix" or "biological sample" refers to any step in a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), downstream processing. It can be obtained from any process, drug substance (DS) or drug product (DP), including the final formulated product. In some other specific exemplary embodiments, the biological sample may be selected from any step of downstream processing such as clarification, chromatographic purification, virus inactivation, or filtration. In some specific exemplary embodiments, drug products may be selected from drug products manufactured in the clinic, transport, storage, or handling.

[0184] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a cancer or an angiogenic eye disorder. Refers to a human in need of prevention and / or treatment of The subject may have cancer or an angiogenic eye disorder, or may be predisposed to developing a cancer or an angiogenic eye disorder.

[0185] As used herein with respect to protein formulation, the term "stable" refers to the ability to retain chemical structure or biological function after storage under exemplary conditions defined herein. Refers to a protein of interest in a formulation that can be retained to an acceptable degree. A formulation may remain stable after storage for a defined period of time even if the protein of interest contained therein does not maintain 100% chemical structure or biological function. . 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 is defined as "stable." can be regarded as

[0186] The terms "treat" or "treatment", for example, to any clinically measurable extent, may be used to treat an undesirable disease or disorder (e.g., angiogenic ocular disorder or cancer), one of such diseases or disorders. By causing regression, stabilization, or elimination of these symptoms or signs (e.g., for neovascular eye disorders, by causing a reduction in or maintenance of the Diabetic Retinopathy Severity Score (DRSS), improve or maintain vision (e.g., in terms of best-corrected visual acuity, e.g., as measured by an increase in character on the ETDRS visual acuity test), increase or maintain visual field, and / or central retina (for cancer, by halting or regressing the proliferation, survival time, and / or metastasis of cancer cells in a subject), ameliorating, stabilizing, or Refers to a therapeutic treatment that removes. Typically, a therapeutic measure is administering a therapeutically effective amount of VEGF MiniTrap in one or more doses to a subject suffering from a disease or disorder.

[0187] As used herein, the term "upstream process technology" in the context of protein preparation refers to the operations involved in producing and collecting proteins from cells during or after cell culture of the protein of interest. Point. As used herein, the term "cell culture" refers to methods for generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as production and collection of the protein of interest. Refers to methods and techniques for optimization. For example, once an expression vector has been incorporated into a suitable host cell, the host cell can be maintained under conditions suitable for expression of the sequences encoding the relevant nucleotides and the harvest and production of the desired recombinant protein.

[0188] When using the cell culture techniques of the invention, the protein of interest can be produced inside the cell, 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., produced by homogenization), either host cells or lysed cells, may be removed by centrifugation or ultrafiltration, including but not limited to. can be removed by a variety of means. If the protein of interest is secreted into the medium, supernatants from such expression systems can be filtered using commercially available protein concentration filters, for example using Amicon™ Millipore Pellicon™ ultrafiltration units. It can be concentrated first. In one aspect, 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 binding to its receptors (VEGFR1 and VEGFR2) and / or inhibits VEGF biological signaling and its activity. VEGF antagonists include molecules that interfere with the interaction of VEGF with native VEGF receptors, e.g., molecules that bind to VEGF or VEGF receptors, and prevent the interaction of VEGF with VEGF receptors or other Molecules that interfere with the process. 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 MiniTrap” herein are referred to herein as, for example, aflibercept, dib-aflibercept, and proteins having amino acids having SEQ ID NO: 60). Other examples of VEGF-Trap are ALT-L9, M710, FYB203, and CHS-2020 Additional examples of VEGF-Trap are described in U.S. Pat. 7,374,757, 7,374,758, 7,531,173, 7,608,261, 5,952,199, 6,100,071, 6,383,486, 6,897,294 & 7,771,721, The entirety of which is specifically incorporated herein by reference.

[0190] VEGF receptor-based chimeric molecules include chimeric polypeptides comprising two or more immunoglobulin (Ig)-like domains of VEGF receptors such as VEGFR1 (also called Flt1) and / or VEGFR2 (also called Flk1 or KDR). , which can also include a multimerization domain (eg, an Fc domain that facilitates multimerization [eg, dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is the molecule designated VEGFR1R2-FcΔC1(a) (also known as aflibercept and sold under the trade name EYLEA®). In certain exemplary embodiments, aflibercept is Contains the amino acid sequence set forth as TIFF2022552052000018.tif43165 (SEQ ID NO: 55).

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

[0192] II. Color judgment As used herein, color observed during production of recombinant proteins, particularly anti-VEGF proteins, can be measured by a variety of methods. Non-limiting examples include iodine color number, Hazen color number, Gardner color number, Lovibond color number, Saybolt color number, mineral oil color number, European Pharmacopoeia color number, US Pharmacopoeia color number, CIE L * , a * , b * (or CIELAB), Klett color number, Hess-Ives color number, yellowness, ADMI color number, and the use of ASBC and EBC brewery color numbers. Details on these 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 Standard, European Pharmacopoeia.Chapter 2.2.2.Degree of coloration of liquids.8 th See ed.EP. The entire teaching of which is incorporated herein) may include the preparation of color reference liquids, as described in the European Pharmacopoeia (EP 2.2.2. Degree of Coloration of Liquids 2), i.e. red ( Cobalt(II) chloride), yellow (iron(III) chloride), and blue (copper(II) sulfate) and 1% hydrochloric acid, yellow (Y), greenish yellow (GY), tan (BY ), brown (B), and red (R) hues are prepared. A total of 37 color reference solutions are prepared by using these 5 kinds of reference solutions in order (Y1 to Y7, GY1 to GY7, BY1 to BY7, B1 to B9, and R1 to R7). Each reference liquid is clearly defined in the CIE-Lab color space, eg 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 BY color standard sample with a given sample is typically performed in diffuse sunlight. The composition of the European tan standard is set forth in Table 1 below.

[0194] (Table 1) Composition of European tan standards TIFF2022552052000019.tif51166 Tan standard solution (BY): 10.8 g / L FeCl 3 .6H 2 O, 6.0g / L CoCl 2 .6H 2 O and 2.5g / L CuSO 4 .5H 2 O

[0195] A liquid color test is performed by comparing a test liquid with a color standard liquid. The composition of the color standard solution is selected according to the hue and intensity of the color of the test solution. Typically, flat-bottom test tubes of colorless, neutral glass (e.g., test tubes of about 12 mm, 15 mm, 16 mm, or 25 mm diameter) with internal diameters and all points that are as closely matched as possible, A comparison is performed. For example, the comparison can be between 2-10 mL of the test solution and a color standard solution. The liquid depth can be, for example, about 15mm, 25mm, 40mm or 50mm. The color assigned to the test liquid should not be darker than the standard color. Color comparisons are typically performed in diffuse light (eg, sunlight) against a white background. Colors can be compared down the vertical or horizontal axis of the test tube.

[0196] In contrast to EP color measurement, USP 1061, Color-Instrumental Measurement is CIE L * , a * , b * (or CIELAB) refers to the use of color measurements 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 Pharmacopoeia. The measured color of the sample is automatically correlated with the color reference liquid. This is the closest color reference solution to the sample (i.e. the smallest color difference ΔE for the color of the sample). * means that a reference solution with ΔL * value, Δa * value and Δb * The value is the L of the sample * value, a * value, b * Values ​​and L for US Pharmacopoeia solutions displayed * value, a * value, b * Quantitative difference between values. 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 * A positive value of represents a red color, while a * negative values ​​indicate green), b * represents the yellow or blue color of the sample (b * Positive values ​​of b represent yellow, while b * negative values ​​represent blue). The color difference from the standard or the first sample at evaluation is the respective color component ΔL * , Δa * and Δb * can be represented by a change in The compound change, or color difference, is the formula: It can be computed as a simple Euclidean distance in space using TIFF2022552052000020.tif5128. CIE L * , a * , b *Color coordinates can be generated, for example, using 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 run for wavelength calibration. This instrument can be standardized on the TTRAN with a 0.780 inch port insert and DIW prior to use. This establishes the top (L=100) and bottom (L=0) of the photometric scale using a light trap and a black plate. 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. BY standard colors are also 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 TIFF2022552052000021.tif63170 ^ Reported by Pack et al. ~ For each BY color standard, L * value and b * Value measured experimentally.

[0198] To enable high-throughput screening of color assays, the Spectrocolorimetric Assay Method (CIELAB) is a better and more quantitative measurement than the BY color standard. A 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 should be normalized to the protein concentration in the sample for comparison, eg 5 g / L, 10 g / L and equivalents.

[0200] III. Anti-VEGF Compositions 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, e.g., mitogenic activity, angiogenic activity, and / or It inhibits one or more of its biological activities, such as vascular permeability activity.

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

[0202] The inventors have discovered that manufacturing anti-VEGF proteins (eg, aflibercept) in certain CDMs produced biological samples with distinct colors. Distinct color characteristics were observed during different manufacturing processes 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 anti-VEGF proteins (eg, aflibercept) with a strong tan color. The post-harvest affinity capture step also produced an eluate with a specific color, such as tan. Further manufacturing steps using AEX also produced a tan color, but the intensity was reduced.

[0203] Color is determined using (i) the European color standard "BY" which provides a qualitative visual inspection, or (ii) CIELAB, a colorimetric assay that is more quantitative than the BY system, as detailed below. can be evaluated as In all cases, however, color ratings across multiple samples were normalized to protein concentration to ensure meaningful investigations / comparisons. For example, referring to Example 9, and particularly Table 9-2, the protein A eluate had a "b * , which corresponds to an approximate BY value of BY5 (measured at 5 g / L protein concentration in protein A eluate). When comparing the color of the Protein A eluate to another sample, then the comparison must be made using the same protein concentration. Therefore, the protein A eluate and a b of approximately 0.74 * (measured at a protein concentration of 5 g / L in the Protein A eluate), this method of production yields a higher AEX pool after AEX chromatography than from the Protein A eluate. A substantial reduction in the tan color of the samples is observed.

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

[0205] In one embodiment, a composition of the invention produced using CDM produces a biological sample having a distinct tan color, the sample comprising: (i) tan equivalent to the European color standard BY2; (ii) tan equivalent to the European color standard BY3; (iii) tan equivalent to the European color standard BY4; (iv) tan equivalent to the European color standard BY5; (v) between BY2 and BY3 in European color standards; (vi) between BY3 and BY4 in European color standards; (vii) European color standards between BY4 and BY5 can be characterized by a standard color characterization recognized by The composition contains about 5 g / L or about 10 g / L of anti-VEGF protein and is obtained as a sample from the clarified harvest Protein A eluate.

[0206] In another embodiment, the composition of the invention produced using CDM produces biological samples having different tan colors, and the composition is on the CIELAB scale: (i) about 22-23b * tan similar to value; (ii) about 16-17b * tan similar to value; (iii) 9~10b * tan similar to value; (iv) 4-5b * tan similar to value; (v) 2~3b * tan similar to 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 can be characterized by a standard color characterization recognized by The composition contains about 5 g / L or about 10 g / L of anti-VEGF protein and is obtained as a sample from the clarified harvest Protein A eluate.

[0207] In one embodiment, compositions of the invention produced using CDM may include other species or variants of anti-VEGF proteins. These variants include anti-VEGF protein isoforms containing one or more oxidized amino acid residues, collectively referred to as oxovariants. Enzymatic digestion of such compositions comprising anti-VEGF proteins and oxovariants thereof is as follows: EIGLLTC containing about 0.004-0.013% 2-oxo-histidine * EAT VNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH containing about 0.006-0.028% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH containing about 0.049-0.085% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing about 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing about 0.008-0.022% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 0.185-0.298% tryptophan dioxide; or EIGLLTC containing about 0.008% 2-oxo-histidine * EAT VNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing about 0.02% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH, containing about 0.06% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing about 0.07% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing about 0.01% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 0.23% di-oxotryptophan can contain one or more of the H * is a 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 invention, a composition of the invention comprises an anti-VEGF protein and comprises about 1% or less, about 0.1% or less, or about 0.1-1%, about 0.2-1%, of the anti-VEGF protein, 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 histidine residues are 2-oxo - is histidine. In such compositions, there may be a heterogeneous population of anti-VEGF protein variants having varying amounts of 2-oxo-histidine and non-oxidized histidine residues, respectively. Therefore, the proportion of 2-oxo-histidine anti-VEGF protein in the composition should be such that the site-specific 2-oxo-histidine in the anti-VEGF molecule is combined with the anti-VEGF protein (oxidized as well as non-oxidized). divided by the total site-specific histidines in the molecule and multiplied by 100. One method of quantifying the level of 2-oxo-histidine in a composition is by digesting the polypeptide with a protease (e.g. Lys-C and / or trypsin), e.g. by mass spectrometry (ms). to analyze the amount of 2-oxo-histidine in the peptides obtained.

[0209] Prior to digestion of the anti-VEGF protein, the cysteine ​​sulfhydryl groups are blocked by reaction with iodoacetamide (IAM) to give the following chemical structure: yields the residue represented by TIFF2022552052000022.tif17128. Such modifications prevent free thiols from reforming disulfide bridges and prevent disulfide bond scrambling. The invention includes compositions (eg, aqueous compositions) comprising anti-VEGF proteins and variants thereof. When modified with IAM, digested with proteases (e.g., Lys-C and trypsin), and analyzed by mass spectrometry, it is the following peptide: EIGLLTC containing about 0.004-0.013% 2-oxo-histidine * EAT VNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH containing about 0.006-0.028% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH containing about 0.049-0.085% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing about 0.057-0.092% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing about 0.008-0.022% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 0.185-0.298% tryptophan dioxide; or EIGLLTC containing about 0.008% 2-oxo-histidine * EAT VNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH, containing about 0.02% 2-oxo-histidine * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH, containing about 0.06% 2-oxo-histidine * QHK (SEQ ID NO: 20), DKTH, containing about 0.07% 2-oxo-histidine * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH, containing about 0.01% 2-oxo-histidine * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing about 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 invention includes compositions comprising anti-VEGF proteins, wherein about 0.1% to 10% of all histidines in the anti-VEGF proteins are modified to 2-oxo-histidines. Further, the color of the composition is, for example, as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), alternatively about 17~23, 10~17, 5~10, 3~5, or 1~3 CIE L * , a * , b * b characterized by using * and the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein. This composition is obtained either as a clarified harvest or as a sample derived from the protein A eluate of the clarified harvest. Such compositions can be obtained from clarified harvests when the harvested material is subjected to a capture chromatography procedure. In one embodiment, the capture step is an affinity chromatography procedure using, for example, a protein A affinity column. One or more variants can be detected when the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS).

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

[0212] The invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in the anti-VEGF protein is modified to mono-hydroxyl tryptophan. Furthermore, the color of the composition is as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or about 17-23 , 10~17, 5~10, 3~5, or 1~3 CIE L * , a * , b * characterized by b *and the composition contains about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. This composition is obtained as a clarified harvest or a sample derived from the protein A eluate of the clarified harvest. Such compositions can be obtained from clarified harvests when subjected to capture chromatography procedures. This capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. One or more of these variants can be detected when samples extracted from the affinity step are analyzed using liquid chromatography-mass spectrometry (LC-MS).

[0213] The invention includes compositions comprising an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in the anti-VEGF protein is modified to di-hydroxyltryptophan. Further, the color is as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or about 17-23,10 ~17, 5~10, 3~5, or 1~3 CIE L * , a * , b * b characterized by using * and the composition contains about 5 g / L of anti-VEGF protein or about 10 g / L of anti-VEGF protein. This composition is obtained as a clarified harvest or a sample derived from the protein A eluate of the clarified harvest. Such compositions can be obtained from clarified harvests made using CDMs containing anti-VEGF proteins and oxovariants thereof that have been subjected to a capture chromatography procedure. This capture step is, for example, an affinity chromatography procedure using a Protein A affinity column. One or more of these variants can be detected when samples extracted from the affinity step are analyzed using liquid chromatography-mass spectrometry (LC-MS).

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

[0215] In one embodiment, a composition of the invention can comprise an anti-VEGF protein, wherein the anti-VEGF protein can comprise one or more residue modifications such as: one or more asparagine is 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-formylkynurenine one or more tryptophans are mono-hydroxytryptophan; one or more tryptophans are di-hydroxytryptophans; one or more tryptophans are tri-hydroxytryptophans; one or more arginines are Arg3-deoxygluco there is no C-terminal glycine; and / or one or more non-glycosylated 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. 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 samples extracted from the affinity step are analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS).

[0217] In one exemplary embodiment, the composition of the invention comprises the following: His86, His110, His145, His209, His95, His19, and / or His203 (or equivalent residue positions), Trp58 and / or Trp138 (or equivalent residue positions on proteins that share certain structural features of aflibercept), Tyr64 (or shares certain structural features of aflibercept) (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept), and / or Met10, Met20, Met163 and / or Met192. (or equivalent residue positions on proteins that share certain structural characteristics of aflibercept) can be oxidized at one or more of the anti-VEGF proteins that share the structural characteristics of aflibercept. Such compositions can be obtained from clarified harvests made using CDM containing aflibercept and its oxovariants that are subjected to a capture chromatography procedure. This capture step can be, for example, an affinity chromatography procedure using a protein A column. One or more of these variants can be detected when samples extracted from the affinity step are analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS).

[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 is His86, His110, His145, His209, His95, His19, and / or His203; Trp58 and Tyr64; Phe44 and / or Phe166; and / or Met10, Met20, Met163, and / or Met192. Such compositions can be obtained from clarified harvests made using CDM containing VEGF MiniTrap and oxovariants thereof subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, 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 can comprise anti-VEGF proteins and variants thereof (including oxovariants), wherein the amount of protein variants in the composition is 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% 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 is an affinity chromatography procedure using, for example, a protein A column, and one or more of these variants can be detected when analyzed using liquid chromatography-mass spectrometry (LC-MS). . In one aspect, the color of such compositions is, for example, as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or Approximately 17-23, 10-17, 5-10, 3-5, or 1-3 CIE L * , a * , b * characterized by b * and the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein.

[0220] In other exemplary embodiments, compositions of the invention comprise anti-VEGF proteins and variants thereof, and the amount of protein variants in the composition can be from about 0% to about 20%, such as about 0%. 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% 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 7.5%, 0.1% to 7.5%, 0.2% to 7.5%, 0.3% to 7.5%, 0.4% to 7.5%, 0.5% to 7.5%, 0.6% 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 5%, 0.7% to 5%, 0.8% to 5%, 0.9% to 5%, 1% to 5%, 1.5% to 5%, 2% can be from about 5%, from about 3% to about 5%, from about 4% to about 5%, and within one or more of the above ranges. Such compositions can be obtained by performing capture chromatography on collected samples. 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 aspect, the color of such compositions is, for example, as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or Approximately 17-23, 10-17, 5-10, 3-5, or 1-3 CIE L * , a * , b * characterized by b * and the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein.

[0221] In one embodiment, a composition of the invention can comprise an anti-VEGF protein comprising acidic species thereof, wherein the amount of acidic species in the composition is 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% and within one or more of the above ranges. As mentioned above, such acidic species can be detected by various methods, eg ion exchange, eg WCX (WCX-10 HPLC, weak cation exchange chromatography), or IEF (isoelectric focusing). In general, 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 harvests or affinity generated materials using ion exchange chromatography.

[0222] In one aspect, the color of such compositions is, for example, as dark / intense as the European tan standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or Approximately 17-23, 10-17, 5-10, 3-5, or 1-3 CIE L * , a * , b * characterized by b * and the composition contains 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 FIG. 17 contain acidic species and fractions F1 and F2 contain the majority of the acidic fraction. Fractions containing such acidic species (F1 and F2) also exhibited a tan color compared to other fractions (FIGS. 18B and 18C).

[0223] In another embodiment, a composition of the invention comprises an anti-VEGF protein comprising an acidic species thereof, and the amount of acidic species in the composition can be from about 0% to about 20%, such as from about 0% to about 20%. 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%~ 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 %~7.5%, 0.1%~7.5%, 0.2%~7.5%, 0.3%~7.5%, 0.4%~7.5%, 0.5%~7.5%, 0.6%~ 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%~ It can be about 5%, about 3% to about 5%, about 4% to about 5%, and within one or more of the above ranges. As mentioned above, such acidic species can be detected by various methods such as ion exchange, eg 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] By using a cation exchange column, all peaks eluting before the main peak of interest were summed as acidic regions and all peaks eluting after the protein of interest were summed as basic regions. . In exemplary embodiments, acidic species can elute as two or more acidic regions, numbered AR1, AR2, AR3, etc., based on the specific retention time of the peak and the ion exchange column used. .

[0225] In one embodiment, the composition can comprise an anti-VEGF protein comprising acidic species, AR1 at 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% within one or more of the above be. In one aspect, the composition can comprise an anti-VEGF protein comprising an acidic species thereof, 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%, or about 8% to about 10%, or about 10% to about 15%, Within one or more of the above. As mentioned above, such acidic regions can be detected by various methods, eg ion exchange, eg WCX (WCX-10 HPLC, weak cation exchange chromatography), or IEF (isoelectric focusing). In general, 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, the composition can comprise an anti-VEGF protein comprising 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% and within one or more of the above is. In one aspect, the composition can comprise an anti-VEGF protein comprising an acidic species, wherein AR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0% ~ about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%; within one or more of

[0227] In one embodiment, a composition can comprise an anti-VEGF protein comprising its basic species, wherein the amount of basic species in the composition is 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%, It may be 0.4%, 0.3%, 0.2%, 0.1% or 0.0% and within one or more of the above ranges. In one aspect, a composition can comprise an anti-VEGF protein and a basic species thereof, and the amount of basic species in the composition relative to the anti-VEGF protein can be from about 0% to about 20%. , for example, 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% ~ about 20%, about 2% ~ about 20%, about 3% ~ about 20%, about 4% ~ about 20%, about 5% ~ about 20%, about 6% ~ about 20%, about 7% ~ 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% 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 7.5%, 3% to 7.5%, 4% to 7.5%, 5% to 7.5%, 6% to 7.5%, 7% to 7.5%, 0% 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 within one or more of the above ranges.

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

[0229] In one embodiment, the composition can comprise an anti-VEGF protein comprising 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% and within one or more of the above. In one aspect, the composition can comprise an anti-VEGF protein and its basic species, wherein BR1 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4%, about 0.0 % to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15%, Within one or more of the above.

[0230] In another embodiment, the composition can comprise an anti-VEGF protein and its basic species, 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% and within one or more of the above . In one aspect, the composition can comprise an anti-VEGF protein and its basic species of an anti-VEGF protein, wherein BR2 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4 %, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15 % and within one or more of the above.

[0231] In another embodiment, the composition can comprise an anti-VEGF protein and its basic species, 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% and within one or more of the above . In one aspect, the composition can comprise an anti-VEGF protein and its basic species of an anti-VEGF protein, wherein BR3 is about 0.0% to about 10%, about 0.0% to about 5%, about 0.0% to about 4 %, about 0.0% to about 3%, about 0.0% to about 2%, about 3% to about 5%, about 5% to about 8%, or about 8% to about 10%, or about 10% to about 15 % and within one or more of the above.

[0232] Photo-induced oxidation of aflibercept In addition to discovering different color characteristics of anti-VEGF protein compositions produced using CDM or variants thereof, the inventors also discovered that such compositions were artificially produced in the laboratory by exposure to light. I also found that it is possible to generate

[0233] Modified anti-VEGF composition variants that include oxidation can be generated by exposing the anti-VEGF protein to cold white light or ultraviolet light. In one embodiment, the anti-VEGF composition comprises from about 1.5 to about 50-fold increase in one or more modified oligopeptides compared to the sample, wherein the oligopeptides are DKTHMore * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EAT VNGH * 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 / RIIW * DSR / IIW * DSRK (SEQ ID NO: 28), TELNVGIDFNW * EYPSSK (SEQ ID NO: 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK (SEQ ID NO:70)F * LSTLTIDGVTR (SEQ ID NO: 32) is selected from the group consisting of H * is histidine and is oxidized to 2-oxo-histidine, C * is a cysteine ​​and is carboxymethylated, M * is methionine oxide and W * is oxidized tryptophan and Y * is tyrosine oxide and F *is oxidized phenylalanine. In further embodiments, the anti-VEGF composition exhibits 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, such as about 30 hours. can contain. In another embodiment, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more modified oligopeptides upon exposing the sample to cool white light for about 75 hours. In yet another embodiment, the anti-VEGF composition comprises about 1.5 to about 20-fold increase in one or more of the previously described oligopeptides by exposing the sample to cool white light for about 100 hours. be able to. In yet another embodiment, the anti-VEGF composition comprises about 1.5 to about 20-fold increase in one or more of the previously described oligopeptides by exposing the sample to cool white light for about 150 hours. be able to. In yet another embodiment, the anti-VEGF composition can comprise from about 1.5 to about 50-fold increase in one or more oligopeptides by exposing the sample to cool white light for about 300 hours (see below See Example 4).

[0234] The anti-VEGF composition comprises 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 ultraviolet light for about 4 hours. be able to. In another embodiment, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more oligopeptides by exposing the sample to ultraviolet light for about 10 hours. In yet another embodiment, the anti-VEGF composition can comprise about 1.5 to about 10-fold increase in one or more of the described oligopeptides by exposing the sample to ultraviolet light for about 16 hours. In yet another embodiment, the anti-VEGF composition can comprise from about 1.5 to about 25 fold increase in one or more oligopeptides upon exposure of the sample to ultraviolet light for about 20 hours. In yet another embodiment, the anti-VEGF composition can comprise from about 1.5 to about 25 fold increase in one or more oligopeptides upon exposure of the sample matrix to ultraviolet light for about 40 hours. See Example 4.

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

[0236] In some exemplary embodiments of the invention, the composition can comprise 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, G1FS glycosylation, G2F glycosylation, G2FS glycosylation, G2FS2 glycosylation, G3FS glycosylation, G3FS3 glycosylation, G0-2GlcNAc glycosylation, Man4 glycosylation, Man4_A1G1 glycosylation, Man4_A1G1S1 glycosylation, Man5 glycosylation, Man5_A1G1 glycosylation, Man5_A1G1S1 glycosylation, Man6 glycosylation, Man6_G0+ phosphate glycosylation, Man6+ phosphate glycosylation, and / or Man7 glycosylation. In one aspect, the protein of interest can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0237] In one embodiment, the composition may have glycosylation properties as follows: 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 comprise an anti-VEGF protein, wherein the protein of interest has Man5 glycosylation at about 32.4% of asparagine 123 residues and / or about 27.1% of asparagine 196 residues. have. In one aspect, the protein of interest can be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0239] In another embodiment, the composition is 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 comprises 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 sialyl can have modified glycans.

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

[0242] In another embodiment, the composition comprises 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% of total galactosylated glycans .

[0243] In one embodiment, the anti-VEGF protein is 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% reduced levels of fucosylated glycans. 1-10%, 1-15%, 1-20%, 1-25%, 1-30% compared to levels of fucosylated glycans in anti-VEGF proteins produced using soy hydrolysates. %, 1~35%, 1~40%, 1~41%, 1~42%, 1~43%, 1~44%, 1~45%, 1~46%, 1~47%, 1~48 %, 1~49%, 1~50%, 2~10%, 2~15%, 2~20%, 2~25%, 2~30%, 2~35%, 2~40%, 2~41 %, 2~42%, 2~43%, 2~44%, 2~45%, 2~46%, 2~47%, 2~48%, 2~49%, 2~50%, 3~10 %, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44 %, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25 %, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47 %, 4-48%, 4-49%, 4-50%, or 1-99% within one or more of the above values.

[0244] In one embodiment, the anti-VEGF protein is 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% reduced levels of sialylated glycans. 1-10%, 1-15%, 1-20%, 1-25%, 1-30% compared to levels of sialylated glycans in anti-VEGF proteins produced using soy hydrolysates. %, 1~35%, 1~40%, 1~41%, 1~42%, 1~43%, 1~44%, 1~45%, 1~46%, 1~47%, 1~48 %, 1~49%, 1~50%, 2~10%, 2~15%, 2~20%, 2~25%, 2~30%, 2~35%, 2~40%, 2~41 %, 2~42%, 2~43%, 2~44%, 2~45%, 2~46%, 2~47%, 2~48%, 2~49%, 2~50%, 3~10 %, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44 %, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25 %, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47 %, 4-48%, 4-49%, 4-50%, or 1-99% within one or more of the above values.

[0245] In another embodiment, the anti-VEGF protein is 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 may have levels of galactosylated glycans reduced by %, 95%, or 99%. 1-10%, 1-15%, 1-20%, 1-25%, 1-30% compared to levels of galactosylated glycans in anti-VEGF proteins produced using soy hydrolysates. %, 1~35%, 1~40%, 1~41%, 1~42%, 1~43%, 1~44%, 1~45%, 1~46%, 1~47%, 1~48 %, 1~49%, 1~50%, 2~10%, 2~15%, 2~20%, 2~25%, 2~30%, 2~35%, 2~40%, 2~41 %, 2~42%, 2~43%, 2~44%, 2~45%, 2~46%, 2~47%, 2~48%, 2~49%, 2~50%, 3~10 %, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44 %, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25 %, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47 %, 4-48%, 4-49%, 4-50%, or 1-99% within one or more of the above values.

[0246] In one embodiment, the anti-VEGF protein is 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% increased levels of mannosylated glycans. 1-10%, 1-15%, 1-20%, 1-25%, 1-30% compared to levels of mannosylated glycans in anti-VEGF proteins produced using soy hydrolysates. %, 1~35%, 1~40%, 1~41%, 1~42%, 1~43%, 1~44%, 1~45%, 1~46%, 1~47%, 1~48 %, 1~49%, 1~50%, 2~10%, 2~15%, 2~20%, 2~25%, 2~30%, 2~35%, 2~40%, 2~41 %, 2~42%, 2~43%, 2~44%, 2~45%, 2~46%, 2~47%, 2~48%, 2~49%, 2~50%, 3~10 %, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44 %, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25 %, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47 %, 4-48%, 4-49%, 4-50%, or 1-99% within one or more of the above values.

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

[0248] IV. Preparation of compositions using upstream process technology For biopharmaceuticals, implementation of robust and flexible upstream processes is desirable. Efficient upstream processes can lead to desirable production and scale-up of proteins of interest. The inventors have found that compositions of the invention comprising anti-VEGF proteins can be produced by adjusting the 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 protein produced.

[0249] The present disclosure demonstrates the existence of certain 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 by using modified CDMs can produce preparations of anti-VEGF proteins with desired target levels of aflibercept and / or MiniTrap protein variants. As mentioned above, there may also be a tan associated with fragments produced using CDM. (As mentioned above, not all of the CDMs examined by the inventors showed distinct discoloration.)

[0250] The present invention involves culturing host cells in modified CDM under suitable conditions in which the cells express the recombinant protein of interest, and subsequently producing a preparation of the recombinant protein of interest produced by the cells. including retrieving. Such modified CDMs can be used to produce the compositions described in Section III above. (Note that CDM is a medium that is tan when aflibercept is expressed.)

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

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

[0253] In a further aspect of this embodiment, target BY values ​​and / or variant target values ​​can be obtained in preparations where titers do not increase or decrease significantly (see Example 5). ).

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

[0255] The composition produced by using the modified CDM can produce a preparation of anti-VEGF protein, the color of this preparation is on the CIELAB scale: (i) about 22-23b * tan similar to value; (ii) about 16-17b * tan similar to value; (iii) 9~10b * tan similar to value; (iv) 4-5b * tan similar to value; (v) 2~3b * tan similar to value; (vi) 17-23b * value; (vii) 10-17b * value; (viii) 5~10b * value; (ix) 3-5b * value; or (x) 1~3b * value wherein the composition comprises about 5 g / L or about 10 g / L of anti-VEGF protein and the composition is a clarified harvest protein Obtained as a sample from the A eluate. See Example 9, Table 9-3.

[0256] With respect to ingredients added to a cell culture to form a modified CDM, the term "cumulative amount" refers to the total amount of a particular ingredient added to the bioreactor over the course of cell culture to form the CDM, which is the beginning of the culture. Include the amount added at (day 0 CDM) and the amount of ingredients added sequentially. When calculating the cumulative amount of a component, the amount of component added to the seed train culture or inoculum prior to production in the bioreactor (ie, prior to day 0 CDM) is also included. Cumulative amounts are not affected by component loss (eg, by metabolism or chemical degradation) over time during culture. Thus, for example, if components are added to two cultures at different times (e.g., in one culture all components are added first and in another culture components are added over time) ), even two cultures with the same cumulative amount for a component may have different absolute levels. Cumulative amounts are also not affected by in situ synthesis of components (eg, by metabolism or chemical transformations) over time during culture. Thus, for example, if a component is synthesized in situ in one of the two cultures during the biotransformation process, given two cultures with the same cumulative amount of a given component may also have different absolute levels. Cumulative amounts can be expressed in units such as grams or moles of an ingredient. 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 the 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 manufacturing 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 (ie 4 grams divided by 2 liters). If, on day 4, 1 liter of additional liquid not containing component X is added to the bioreactor, the cumulative concentration remains 2 g / L. Even if on day 5 some amount of liquid is lost from the bioreactor (eg, by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentrations may be expressed in units such as grams / liter or moles / liter, for example.

[0257] A. Amino acids: In some embodiments, modified CDMs 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) may be mentioned. The increase or decrease in cumulative abundance of these amino acids in the modified CDM is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% compared to the starting CDM. %, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% within one or more of the above be. Alternatively, the increase or decrease in the cumulative amount of one or more amino acids in the modified CDM is about 5 to about 20%, about 10 to about 30%, about 30% to about 40% compared to the unmodified CDM. %, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% and within one or more of the above (see Figures 25-27 and Example 5).

[0258] In some embodiments, modified CDMs can be obtained by decreasing the cumulative concentration of cysteine ​​in the CDM. To form the modified CDM, the reduction in the amount of cysteine ​​in the CDM is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% compared to the unmodified CDM. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and one or more of the above Within range. Alternatively, the reduction in cumulative amount of cysteine ​​in the modified CDM is about 5 to about 20%, about 10 to about 30%, about 30% to about 40%, about 30% to about 50% compared to CDM. %, 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 In one aspect, the amount of cumulative 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, a modified CDM can be obtained by replacing at least a certain percentage of the cumulative cysteines in the CDM with cystine. The substitution was about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and within one or more of the above. Alternatively, the substitution is about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% compared to the unmodified CDM. can be from about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 100%; within the range (see Figures 25-27 and Example 5).

[0260] In some embodiments, a modified CDM can be obtained by replacing at least a certain percentage of the cumulative cysteines in the CDM with cysteine ​​sulfate. The substitution was about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and within one or more of the above. Alternatively, the substitution is 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. is.

[0261] B. Metal: In some embodiments, modified CDMs can be obtained by decreasing 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 modified CDM is about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% compared to unmodified CDM. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% and ranges of one or more of the above is within. Alternatively, the increase or decrease in the cumulative amount of one or more metals in the modified CDM is about 5 to about 20%, about 10 to about 30%, about 30% to about 40% compared to the unmodified CDM. %, about 30% to about 50%, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% and within one or more of the above (see Figures 25-27 and Example 5).

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

[0263] In some embodiments, the modified CDM is about 0.01 mM to about 20 mM taurine, i. 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 is about 0.01 mM to about 20 mM hypotaurine, i. 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 is about 0.01 mM to about 20 mM glycine, i. 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 is about 0.01 μM to about 5 μM thioctic acid, i. , 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.

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

[0269] In some embodiments, the modified CDM is about 1 μM to about 50 μM vitamin C, i. 50 μM, including from about 25 μM to about 50 μM, and within one or more of the above.

[0270] D. Modifications to Media to Adjust Glycosylation: The disclosure also includes methods of modulating glycosylation of anti-VEGF proteins by altering 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 of modulating glycosylation of an anti-VEGF protein can include adding a CDM containing uridine. The anti-VEGF protein contains from about 40% to about 55% total fucosylated glycans, from about 30% to about 50% total sialylated glycans, from about 2% to about 15% mannose-5, and from about 60% to about 79%. % galactosylated glycans (see Example 6 below).

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

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

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

[0275] In some embodiments, methods of 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 contains from about 40% to about 50% total fucosylated glycans, from about 30% to about 55% total sialylated glycans, from about 2% to about 15% mannose-5, and from about 60% to about 79%. % galactosylated glycans (Example 6).

[0276] V. Preparation of Compositions Using Downstream Processing Technologies Compositions comprising anti-VEGF proteins of the invention can be produced by adjusting conditions during downstream protein production. The inventors have found that by optimizing downstream procedures, specific variants of the anti-VEGF protein are minimized and discolored. Downstream process optimization can produce compositions with reduced oxovariants and optimized color properties.

[0277] Downstream processing techniques may be used alone or in combination with the upstream processing techniques described in Section IV above.

[0278] A. Anion Exchange Chromatography: In some embodiments, the compositions of the invention may involve a process involving expressing anti-VEGF proteins in host cells in CDM, where the anti-VEGF proteins are secreted from the host cells into the medium. , a clarified harvest is obtained. This harvest is subjected to: (a) loading the biological sample obtained from the harvest onto an anion exchange chromatography (AEX), (b) washing the AEX column with a suitable wash buffer. (c) collecting the flow-through fraction (one or more), optionally (d) washing the column with a suitable stripping buffer, and (e) collecting the stripped fraction. be.

[0279] The flow-through fraction was approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% compared to the oxovariant in the anion exchange chromatography column strip fraction. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the anti-VEGF protein sample. . 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 approximately 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% compared to the oxidized variant in the stripped fraction. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and within one or more of the above) oxidized .

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

[0281] Protein variants may include 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-formylkynurenine; one or more tryptophans are mono-hydroxytryptophans; one or more tryptophans are di-hydroxytryptophans one or more tryptophans are tri-hydroxytryptophans; one or more arginines are converted to Arg3-deoxyglucosone; no C-terminal glycines are present; and / or one or more non-glycosylated glycosites exists.

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

[0283] The flow-through fraction may contain one or more of: (a) Percentage of histidine residues oxidized to 2-oxo-histidine, whose color properties are: (i) tan equivalent to the European color standard BY2; (ii) tan equivalent to the European color standard BY3; (iii) tan equivalent to the European color standard BY4; (iv) tan equivalent to the European color standard BY5; (v) between BY2 and BY3 in European color standards; (vi) between BY3 and BY4 in European color standards; (vii) European color standards 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) Percentage of histidine residues oxidized to 2-oxo-histidine. Furthermore, these colors are closer to the colors of BY2, BY3, BY4, BY5, BY6, BY7, or not darker / stronger than BY2, darker than BY3, darker than BY4, or darker than BY5. Not dark, not darker than BY6, not darker than BY7, or between BY2 and BY3, between BY2 and BY4, between BY3 and BY4, or between BY3 and BY5, Yellow Characterized by having a brown color. (c) Percentage of histidine residues oxidized to 2-oxo-histidine, whose colors are: (i) about 22-23b * tan similar to value; (ii) about 16-17b * tan similar to value; (iii) 9~10b * tan similar to value; (iv) 4-5b * tan similar to value; (v) 2~3b * tan similar to value; (vi) 17-23b * value; (vii) 10-17b * value; (viii) 5~10b * value; (ix) 3-5b * value; or (x) 1~3b * value CIE L like * , a * , b * Characterized by color in a color space, where the composition contains about 5 g / L or about 10 g / L of anti-VEGF protein, 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 in the composition ~1%, about 0.7-1%, about 0.8-1%, or about 0.9-1% of histidine residues are oxidized to 2-oxo-histidine. Percentage calculations are described in Section II.

[0284] B. Affinity Chromatography: In some embodiments, the compositions of the 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 medium. , a clarified harvest is obtained. This collection is subjected to a step comprising: (a) loading a biological sample obtained from the clarified collection onto an affinity chromatography column, wherein the affinity chromatography (b) washing the affinity chromatography column with a suitable elution buffer; and (c) collecting the (one or more) elution fractions. process to do. For example, as exemplified in Tables 7-1 and Tables 7-7 to 7-10, VEGF as a protein capable of selectively or specifically binding to an anti-VEGF protein 165 and collecting elution fractions according to the above method led to the successful production of MT5 (anti-VEGF protein), aflibercept and anti-VEGF scFv fragments. Table 7-1 also lists (i) mAb1 (SEQ ID NO: 73 is the heavy chain and SEQ ID NO: 74 is the light chain, mouse anti- VEGFR1 mAb human IgG1), (ii) mAb2 (SEQ ID NO:75 is the heavy chain and SEQ ID NO:76 is the light chain, mouse anti-VEGFR1 mAb human IgG1), (iii) mAb3 (SEQ ID NO:77 is the heavy chain SEQ ID NO:78 is the light chain, mouse anti-VEGFR1 mAb mouse IgG1), and (iv) mAb4 (SEQ ID NO:79 is the heavy chain, SEQ ID NO:80 is the light chain, mouse anti-VEGFR1 We disclose the successful generation of MT5 using mAb mouse IgG1).

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

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

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

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

[0289] In some exemplary embodiments, proteins capable of selectively or specifically binding (“binders”) or interacting with anti-VEGF proteins, such as aflibercept or MiniTrap, are of human or murine origin. can have

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

[0291] Biological samples can be loaded using a suitable buffer such as DPBS.

[0292] This affinity generation process may further comprise washing the affinity column with one or more wash buffers. The column can be washed once or multiple times. Additionally, the wash can also be collected as a wash fraction. The pH of both wash buffers can be from about 7.0 to about 8.60. In one aspect, the wash buffer can be DPBS. In another embodiment, the wash buffer is 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 7.2). ~8), or DPBS (pH 7.4).

[0293] The affinity process may further comprise washing the affinity column with one or more suitable elution buffers and collecting elution fractions. The column can be washed once or multiple times. Non-limiting examples of such 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, elution fractions can be neutralized using a neutralizing buffer. An example of such a neutralizing buffer is 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. Variants have therefore been developed to be more stable at basic pH, in the presence of strong bases such as NaOH. Such basic conditions can be 0.05N NaOH for 1 hour or 0.1N NaOH for 0.5 hours.

[0296] In some embodiments, the IdeS variant is 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, an amino acid sequence having at least about 70% sequence identity over its entire length to the amino acid sequence set forth in the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 can have 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 sequences referred to directly above. have.

[0297] In some embodiments, the IdeS variant is 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, an amino acid sequence containing 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: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 can have an isolated nucleic acid molecule that encodes a polypeptide having 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 sequences referred to directly above. have.

[0298] In some embodiments, the polypeptide is 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 with an amino acid sequence that has at least 70% sequence identity over its entire length to the amino acid sequence set forth in the group consisting of can be expressed by a host cell with a suitable vector containing nucleic acid encoding for the identified peptide. In one embodiment, the nucleic acid molecule is operably linked to expression control sequences capable of directing its expression within a host cell. In one aspect, 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 sequences referred to directly above. have. In some embodiments, an isolated nucleic acid molecule can be used to encode a polypeptide.

[0299] In some embodiments, the IdeS variant is the parent as defined by SEQ ID NO: 1 (IdeS) having asparagine residues at positions 87, 130, 182 and / or 274 mutated to an amino acid other than asparagine. It can have an amino acid sequence that contains an amino acid sequence. In one aspect, the mutation may result in increased chemical stability at alkaline pH values ​​as compared to the parent amino acid sequence. In another embodiment, the mutation may result in a 50% increase in chemical stability at alkaline pH values ​​as compared to the parent amino acid sequence. In one aspect, 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 specific embodiment, the asparagine residue at position 182 is mutated to a leucine residue. In yet another specific embodiment, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another specific embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 130 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 182 and 274 are mutated. In yet another specific embodiment, asparagine residues at positions 130, 182 and 274 are mutated. In yet another specific embodiment, 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 sequences set forth above. . In some embodiments, an isolated nucleic acid molecule can be used to encode a polypeptide.

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

[0301] VI. General protein production A variety of different production techniques, alone or in combination, including, but not limited to, affinity chromatography, ion exchange chromatography, mixed mode chromatography, size exclusion chromatography, and hydrophobic interaction chromatography, are contemplated by the present invention. is assumed to be within the range of These chromatographic steps separate mixtures of biological sample proteins based on their charge, hydrophobicity, or size, or a combination thereof, depending on the particular mode of separation. A number of different chromatography resins are available for each of the techniques suggested above, so that a production scheme can be precisely tailored to the particular protein of interest. Each separation method causes the proteins to pass through the column at different velocities, resulting in a physical separation or selective attachment to the separation medium that increases with each further pass through the column. The protein is then (i) differentially eluted using a suitable elution buffer and / or (ii) obtained from the column used, optionally by washing the column with a suitable equilibration buffer. collected from the flow-through fraction. In some cases, impurities preferentially adhere to the column and the protein of interest is less adsorbed, 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 Proteins are separated from impurities (HCPs, protein variants, etc.). In some cases, if an impurity cannot adsorb to the column and thus passes through the column, it will be separated from the protein of interest.

[0302] After the recombinant protein has been produced using the upstream production methods described above, and / or by 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. Separation of the protein of interest from product related substances) is performed. A combination of one or more different purification techniques may be used, including affinity chromatography, ion exchange chromatography (eg, CEX, AEX), mixed mode (MM) chromatography, and / or hydrophobic interaction chromatography. Such a production process separates mixtures of components within a biological sample based on, for example, charge, hydrophobicity, and / or apparent size. Numerous chromatographic resins are commercially available for each of the chromatographic techniques referred to herein, allowing precise tailoring of production schemes to the particular protein of interest. With each separation method, proteins either pass through the column at different velocities, resulting in increasing physical separation with each further pass through the column, or selectively adsorb to the separation resin (or medium). is possible. Proteins can then be collected differentially. Optionally, the protein of interest is separated from the components of the biological sample when the other components specifically adsorb to the column resin while the protein of interest does not.

[0303] A. Primary recovery and virus inactivation In certain embodiments, the initial steps of the production methods disclosed herein involve clarification and primary recovery of the protein of interest from the biological sample. Primary recovery involves one or more centrifugation steps to separate the protein of interest from the host cells and associated cell debris. Centrifugation of the sample can be performed at, for example, without limitation, 7,000xg to approximately 12,750xg. In terms of large-scale production, such centrifugation is performed in production lines, for example, with a flow rate set to achieve a turbidity level of 150 NTU in the resulting supernatant. Such supernatants can then be collected for further processing or filtered into line through one or more depth filters for further clarification of the sample.

[0304] In certain embodiments, primary recovery involves the use of one or more depth filtration steps to clarify the sample, which can aid in processing the protein of interest. In other embodiments, primary recovery can include the use of one or more depth filtration steps after centrifugation. Non-limiting examples of depth filters that may be used in the context of the present invention include Millistak+X0HC, F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), 3M™ models 30 / 60ZA, 60 / 90ZA , VR05, VR07, Delipid depth filters (3M Corp.). A 0.2 μm filter such as a 0.45 / 0.2 μm Sartopore™ bilayer from Sartorius or an Express SHR or SHC filter cartridge from Millipore typically follows a depth filter. Other filters well known to those skilled in the art may also be used.

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

[0306] In those embodiments where viral reduction / inactivation is used, the biological sample can be conditioned for further production steps as necessary. For example, after low pH virus inactivation, the pH of the sample is typically adjusted to a more neutral pH, such as from about 4.5 to about 8.5, before continuing the production 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 for production of a protein of interest. A chromatographic material is capable of selectively or specifically binding or interacting with a protein of interest. Non-limiting examples of such chromatographic materials include protein A, protein G. Also included are chromatographic materials comprising proteins or portions thereof that are capable of binding or interacting with, for example, a protein of interest. In one aspect, the protein of interest is an anti-VEGF protein such as aflibercept, MiniTrap, or their related proteins.

[0308] Affinity chromatography involves subjecting 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 sources, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant technology), C H 2 / C H Those variants that retain the ability to bind proteins with three regions are included. In certain embodiments, Protein A resins are useful for affinity-based production and isolation of various antibody isotypes by specifically interacting with the Fc portion of the molecule that will have the region.

[0309] There are multiple manufacturers of protein A resin. One suitable resin is MabSelect™ from GE Healthcare. Suitable resins include MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose from GE Healthcare; ProSep HC, ProSep Ultra and ProSep Ultra Plus from EMD Millipore; MapCapture of, but not limited to. A non-limiting example of a suitable column packed with MabSelect™ is an approximately 1.0 cm diameter by approximately 21.6 cm long column (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 prior to sample loading. 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 a suitable 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 UV detectors. Elution fractions of interest can be collected and then prepared for further processing.

[0311] In one aspect, the eluate may be subjected to viral inactivation, for example by either detergents or low pH. A suitable detergent concentration or a suitable pH (and time) may be selected to obtain the desired virus inactivation results. After virus inactivation, the eluate is typically pH and / or conductivity adjusted for subsequent production 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 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). but not limited to these. The Emphaze AEX Hybrid Purifier multi-mechanism filter may also be used to clarify the effluent. Eluate pools may be required to be adjusted to specific pH and conductivity to obtain the desired impurity removal and product recovery from the depth filtration step.

[0313] C. Anion exchange chromatography In certain embodiments, the protein of interest is produced by subjecting a biological sample to at least one anion exchange separation step. In some scenarios, an anion exchange step can occur after an affinity chromatography (eg Protein A affinity) procedure. In other scenarios, an anion exchange step can occur before the affinity chromatography step. In still other protocols, anion exchange can occur both before and after the 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 performing the separation, the initial protein composition (biological sample) is contacted with an anion exchange material using any of a variety of techniques, such as using batch production techniques or chromatographic techniques. can be placed in the upright position.

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

[0317] In the context of chromatographic separations, chromatographic columns are used to contain chromatographic support materials (resins or solid phases). A sample containing the protein of interest is loaded onto a specific chromatographic column. The column may then be subjected to one or more washing steps using suitable wash buffers. Components of the sample that are not adsorbed onto the resin can flow through the column. Components adsorbed to the resin can be differentially eluted using a suitable elution buffer.

[0318] By decreasing the pH and / or increasing the ionic strength / conductivity of the wash solution using conditions similar to the loading conditions, or alternatively in a stepwise or linear gradient fashion, the wash step is Typically performed by AEX chromatography. In one aspect, the saline solutions used for both the loading and washing buffers have a pH at or near the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0-2 units above or below the pI of the protein of interest, although it may be in the range of 0-0.5 units above or below. It may also be present at the pI of the protein of interest.

[0319] Anionic agents may be selected from the group consisting of acetates, chlorides, formates and combinations thereof. Cationic agents may be selected from the group consisting of tris, arginine, sodium and combinations thereof. In a particular example, the buffer solution is Tris / formate buffer. Buffers include 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. may be

[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, In some cases, proteins exhibit binding to chromatographic materials and can be washed from such materials using a buffer similar or substantially similar to the loading buffer.

[0321] In the bind-elute mode, a column or membrane device is first conditioned with a buffer having the appropriate ionic strength and pH under conditions in which the specific protein will adsorb to the resin-based matrix. For example, during feed loading, the protein of interest can 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, the ionic strength of the elution buffer competing with the solute for the charged sites of the anion exchange matrix (i.e., conducting The recovery of the product is achieved by increasing the Altering the pH and thereby altering the charge of the solute is another method of achieving elution of the solute. Changes in conductivity or pH may be stepwise (gradient elution) or stepwise (step elution).

[0322] In flow-through mode, the protein of interest is not bound to the resin or membrane, while acidic species are either retained on the column or have different elution profiles compared to the protein of interest. As such, the column or membrane device is operated at a selected pH and conductivity. In terms of this policy, the acidic species interacts or binds with the chromatographic material under suitable conditions, while the protein of interest and specific aggregates and / or fragments of the protein of interest are isolated from the column. pass through.

[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 polymeric beads with a backbone consisting of crosslinked 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 base beads; Fractogel® EMD TMAE Hicap, a synthetic polymer resin with tentacular ion exchangers; ligands of primary amines. Sartobind STIC® PA nano, a salt-tolerant chromatographic membrane having a CUNO BioCap, a filter; and XOHC, a depth filtration media constructed from inorganic filter aids, cellulose, and mixed cellulose esters.

[0324] In certain embodiments, the protein load of the sample is 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. It can be adjusted for protein loading. In other embodiments, the protein concentration of the loaded protein mixture is from about 0.5 g / L to about 50 g / L, from about 1 g / L to about 20 g / L, or from about 3 g / L to about 10 g / L. Adjusted to the protein concentration of the loaded material. In still other embodiments, the protein concentration of the loaded protein mixture is adjusted to a protein centration of material to column of about 37 g / L.

[0325] Additives such as polyethylene glycol (PEG), surfactants, amino acids, sugars, chaotropic agents can be added to improve separation performance 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 are used to selectively remove, substantially reduce, or essentially eliminate at least 10% of protein variants. It can be removed, thereby producing a protein composition with reduced protein variants.

[0327] Protein variants may include modifications of one or more residues such as: one or more asparagines are deamidated; one or more aspartic acids are converted to aspartate-glycine and / or Asn-Gly; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxytryptophans; one or more tryptophans are di- is hydroxyltryptophan; one or more tryptophans are tri-hydroxytryptophan; one or more arginines are converted to Arg3-deoxyglucosone; no C-terminal glycine is present; and / or one or more non-glycosyl polyglycosites are present. The use of AEX was also observed to reduce oxidized and acidic species of anti-VEGF variants in the affinity eluate. After use of AEX, the flow-through fraction contained at least about 20%, 19%, 18%, 17%, 16%, 15% of the oxidized and / or acidic species of the anti-VEGF variant compared to the affinity eluate. , 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or 5%.

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

[0329] D. Cation exchange chromatography A composition of the invention can be produced by subjecting a biological sample containing a protein of interest to at least one cation exchange (CEX) step. In certain exemplary embodiments, the CEX step is in addition to the AEX step and is performed either before or after the AEX step. In one aspect, the protein of interest is aflibercept, MiniTrap, or any of their related molecules.

[0330] The use of cation exchange materials versus anion exchange materials, such as the anion exchange materials mentioned 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 prior to the use of an anion exchange step or the use of an anion exchange step prior to the use of a cation exchange step is within the scope of the present invention. Furthermore, it is within the scope of the invention to use only the cation exchange step in combination with other chromatographic procedures.

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

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

[0333] In certain embodiments, the concentration of the anionic agent in the aqueous salt solution is from about 3.5 to about 10.5, or from about 4 to about 10, or from about 4.5 to about 9.5, or from about 5 to about 9, or from about 5.5 to about increased or decreased to achieve a pH of 8.5, or from about 6 to about 8, or from about 6.5 to about 7.5. In one aspect, the concentration of the anionic agent is increased or decreased in the saline solution to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems suitable 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 saline solution are adjusted by increasing or decreasing the concentration of the cationic agent. In one aspect, the cationic agent is maintained at a concentration that ranges 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] A packed cation-exchange chromatography column or anion-exchange membrane device can be operated in either bind-elute mode, flow-through mode, or hybrid mode, where the product is bound to the chromatographic material or It can interact with this and even be washed from such material using a buffer similar 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 infiltrated with functionalized hydrogel, 250-400 μeq / mL ionic groups; Eshmuno S, a hydrophilic polyvinyl ether base matrix with an ion-exchange capacity of ~100 μeq / mL; Nuvia C Prime, an exchange medium; Nuvia S, which has a UNOsphere base matrix with 90–150 με / mL ionic groups; Poros HS, a rigid polymeric bead with a backbone composed of crosslinked poly[styrene-divinylbenzene]; Poros XS, a rigid polymer bead with a [styrene-divinylbenzene] backbone; Toyo Pearl Giga Cap CM 650M, a polymer-based bead with an ion-exchange capacity of 0.225 meq / mL; Toyo, a polymer-based bead Pearl Giga Cap S 650M; Toyo Pearl MX TRP, a polymer-based bead, but not limited to; Note that CEX chromatography can be used with MM resin as described herein.

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

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

[0339] In certain embodiments, the methods of the invention, including those associated with aflibercept or anti-VEGF antibodies or VEGF MiniTrap, selectively remove or significantly reduce all of the oxovariants in the sample, or It can be used to essentially remove, where the protein of interest is essentially present in the flow-through of the CEX procedure, while the oxovariant is essentially trapped by the column medium.

[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 support comprising a ligand capable of providing at least two different interactions with an analyte from a sample or protein of interest. It is a chromatographic strategy that utilizes the body. One of these sites provides an attractive charge-charge interaction between the ligand and the protein of interest, the other an electron acceptor-donor interaction and / or hydrophobic interaction and / or lead to hydrophilic interactions. Electron donor-acceptor interactions include interactions such as hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, and induced dipole.

[0341] The column resin used for mixed mode separation can be Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal function. The base matrix of this strong anion exchanger consists of ligands (N-benzyl-N-methylethanolamine) that exhibit different functionalities for interactions such as ionic, hydrogen bonding, and hydrophobic interactions. It is a highly crosslinked agarose having In certain embodiments, the resin used for mixed mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrix of PPA-HyperCel and HEA-HyperCel is highly porous crosslinked cellulose. Their ligands are phenylpropylamine and hexylamine respectively. Phenylpropylamine and hexylamine offer different selectivity and hydrophobicity options for protein separations. Additional mixed mode chromatographic supports include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain embodiments, mixed-mode chromatographic resins, sometimes referred to as base matrices, can be composed of ligands attached directly or via spacers to organic or inorganic supports. Supports may be in the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, and the like. In certain embodiments, supports can be prepared from cross-linked carbohydrate materials such as natural polymers such as agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, and the like. To obtain high adsorption capacities, the support can be porous and the ligands are then attached to the exterior and pore surfaces. Such natural polymeric supports can be prepared by standard methods such as inverse suspension gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teaching of which is incorporated herein by reference). incorporated). Alternatively, the support is made from crosslinked synthetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylates, methacrylates, vinyl esters, vinylamides, and the like. can be prepared. 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 polymeric supports are also available from manufacturers such as GE Healthcare (Uppsala, Sweden).

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

[0343] Additives such as polyethylene glycol, surfactants, amino acids, sugars, chaotropic agents may be added to improve separation performance 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 selectively remove, significantly reduce, or essentially eliminate all PTMs, including oxovariants. can be used to remove

[0345] The method of making the compositions of the invention can also be carried out in continuous chromatography mode. In this mode, at least two columns are used (referred to as the 'first' and 'second' columns). In certain embodiments, this continuous chromatography mode is such that elution fractions and / or strip fractions containing PTMs, e.g., oxovariants, can be loaded onto a second column (with or without dilution) sequentially or simultaneously. can be implemented.

[0346] In one embodiment, the choice of media for continuous mode is one of a number of chromatographic resins, monolithic media, membrane adsorption media, or depth filtration media with hydrophobic pendant functional groups and anion exchange functional groups. obtain.

[0347] F. Hydrophobic interaction chromatography Compositions of the invention may also be prepared using hydrophobic interaction chromatography (HIC).

[0348] In performing separations, a biological sample is contacted with a HIC material, for example using batch production techniques or using column or membrane chromatography. Prior to HIC treatment, it may be desirable to adjust the salt buffer concentration to obtain the desired protein binding / interaction to the resin or membrane.

[0349] Hydrophobic interaction chromatography exploits the hydrophobic properties of proteins to achieve selective separation, while ion exchange chromatography relies on the local charge of the protein of interest for selective separation. Hydrophobic groups on or within proteins interact with hydrophobic groups of chromatography resins or membranes. Typically, under suitable conditions, the more hydrophobic a protein (or portion of a protein), the stronger it will interact with a column or membrane. Thus, under suitable conditions, HIC can be used to facilitate the separation of process-related impurities (e.g. HCPs) and product-related substances (e.g. aggregates or fragments) from proteins of interest in a sample. obtain.

[0350] Similar to ion-exchange chromatography, HIC columns or HIC membrane devices can also be operated in elution, flow-through, or hybrid modes, where the product exhibits binding or interaction with the chromatographic material, and A buffer similar or substantially similar to the loading buffer may be used to wash from such materials. (The details of these modes are reviewed above in connection with AEX processing.) Since hydrophobic interactions are strongest at high ionic strength, this form of separation is associated with ion-exchange chromatography. It is conveniently used after a salt elution step, such as the step typically used. Alternatively, salt can be added to the sample prior to using HIC. Adsorption of proteins to HIC columns is favored by high salt concentrations, but the actual concentration may vary over a wide range, depending on the nature of the protein of interest, the type of salt, and the particular HIC ligand chosen. may vary over the Various ions are arranged in so-called soluphobic series, depending on whether they promote hydrophobic interactions (salting out) or weaken hydrophobic interactions by disrupting the water structure (chaotropic effect). can be The cation is Ba 2+ ; Ca 2+ ;Mg 2+ ;Li + ;Cs + ;Na + ;K + ;Rb + ;NH4 + It is ranked in terms of increasing the salting out effect. On the other hand, the anion is PO 4 3- ;SO 4 2- ;CH 3 CO 3 - ;CI - ;Br - ;NO 3 - ;ClO 4 - ;I - ;SCN - etc. are ranked in terms of increasing the chaotropic effect.

[0351] In general, Na + , K + or NH4 + sulfate effectively promotes ligand-protein interactions using HIC. The following relations: (NH 4 ) 2 SO 4 >Na 2 SO 4 >NaCl>NH 4 Salts may be formulated that influence the strength of the interaction, as given by C1>NaBr>NaSCN. Generally, salt concentrations of about 0.75M to about 2M ammonium sulfate, or about 1M to about 4M NaCl are useful.

[0352] HIC media usually comprise a base matrix (eg cross-linked agarose or synthetic copolymer material) to which hydrophobic ligands (eg alkyl or aryl groups) are attached. Suitable HIC media include agarose resins or membranes functionalized with phenyl groups (eg, Phenyl Sepharose™ from GE Healthcare or Phenyl Membrane from Sartorius). Many HIC resins are commercially available. Examples include CaptoPhenyl, Phenyl Sepharose™ 6 Fast Flow with low or high substitution, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance (GE Healthcare); Fractogel™ EMD Propyl or Fractogel™ EMD Phenyl (E. Merck, Germany); Macro-Prep™ methyl columns or Macro-Prep™ t-butyl columns (Bio-Rad, California); WP HI-Propyl (C3)™ (J.T. Baker, New Jersey); and Toyopearl™ ether, phenyl or butyl (TosoHaas, PA); ToyoScreen PPG; ToyoScreen Phenyl; Not limited.

[0353] The protein loading of the sample containing the protein of interest is from about 50 g / L to about 1000 g / L, from about 5 g / L to about 150 g / L, from about 10 g / L to about 100 g / L, from about 20 g / L to about 80 g. / L, from about 30 g / L to about 50 g / L, or from about 40 g / L to about 50 g / L. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to the protein concentration of the material loaded onto the column from about 0.5 g / L to about 50 g / L, or from about 1 g / L to about 20 g / L. be done.

[0354] Particular pH conditions may be specific to each application, as the pH chosen for any particular production process is compatible with protein stability and activity. However, these conditions may work to your advantage because between pH 5.0 and 8.5, certain pH values ​​have little significance to the final selectivity and resolution of the HIC separation. Increasing pH weakens the hydrophobic interactions and changes protein retention more at pH above 8.5 or below 5.0. Furthermore, changes in ionic strength, the presence of organic solvents, temperature and pH (especially at the isoelectric point pI when there is no net surface charge) can affect protein structure and solubility, resulting in that in HIC media. interactions with other hydrophobic surfaces, such as Thus, in certain embodiments, the present invention incorporates production strategies that adjust one or more of the foregoing to achieve desired reductions in process-related impurities and / or product-related substances.

[0355] In certain embodiments, spectroscopic methods such as UV, NIR, FTIR, fluorescence, Raman can be used to monitor proteins and impurities of interest in online, at-line, or in-line modes, which are then can be used to control the level of aggregates in the pooled material collected from the HIC adsorptive eluate. In certain embodiments, on-line, at-line, or in-line monitoring methods can be used either on the eluate line of the chromatography step or in the collection vessel to provide desired product of quality / recovery is achievable. In certain embodiments, a UV signal can be used as a surrogate to achieve adequate product quality / recovery, which UV signal addresses normal process variations and achieves target product quality. In order to make it possible, processing techniques such as, but not limited to, integration method, differentiation method, moving average, etc., can be appropriately processed. In certain embodiments, such measurements can be combined with in-line dilution methods so that the load / wash ionic concentration / conductivity can be controlled by feedback, thus facilitating control of product quality. .

[0356] G. Size exclusion chromatography Size exclusion chromatography or gel filtration relies on the separation of components as a function of molecular size. Separation depends on the amount of time the material spends in the porous stationary phase compared to the time it resides in the fluid. The probability of a molecule being in the pore depends on the size of the molecule and the pore. In addition, the ability of a substance to penetrate into the pores is determined by the diffusional mobility of the macromolecules, which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all. Also, for very small macromolecules, the probability of penetration is close to one. Larger molecular size components migrate past the stationary phase more quickly, while smaller molecular size components are retained longer in the stationary phase due to their longer path through the pores of the stationary phase.

[0357] The chromatographic material can include a size exclusion material, where the size exclusion material is a resin or membrane. The matrix used for size exclusion is preferably an inert gelling medium, which can be, for example, a complex of cross-linked polysaccharides such as cross-linked agarose and / or dextran in the form of particulate beads. The degree of cross-linking determines the size of the pores present in the swollen gel beads. Molecules above a certain size do not enter the gel beads and thus move the fastest across the chromatographic bed. Small molecules, such as detergents, proteins, DNA and the like, which enter gel beads to varying degrees depending on their size and shape, are delayed through the bed. Therefore, the smaller the molecular size, the more generally the molecule is eluted.

[0358] Suitable porous chromatography resins for viral size exclusion chromatography may be made from dextrose, agarose, polyacrylamide, or silica with different physical properties. Combinations of polymers can also be used. The most commonly used is that available from Amersham Biosciences under the trade name "SEPHADEX". Other size exclusion supports derived from materials of different composition are also suitable, such as Toyopearl 55F (polymethacrylate, Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, Calif.). is.

[0359] The protein loading of the sample containing the protein of interest is from about 50 g / L to about 1000 g / L, from about 5 g / L to about 150 g / L, from about 10 g / L to about 100 g / L, from about 20 g / L to about 80 g. / L, from about 30 g / L to about 50 g / L, or from about 40 g / L to about 50 g / L. In certain embodiments, the protein concentration of the loaded protein mixture is adjusted to the protein concentration of the material loaded onto the column from about 0.5 g / L to about 50 g / L, or from about 1 g / L to about 20 g / L. be done.

[0360] H. Virus filtration Viral filtration is a step aimed at virus reduction during the production process. This step is usually performed after chromatographic polishing. Virus reduction was performed using Planova 20N™, Planova 50N, or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius, Ultipor DV20 or Ultipor DV50™ from Pall Corporation. This can be accomplished through the use of suitable filters, including but not limited to. It will be apparent to those skilled in the art to select a suitable filter to obtain the desired filtration performance.

[0361] I. Ultrafiltration / Diafiltration Certain embodiments of the invention utilize ultrafiltration and diafiltration to further concentrate and formulate proteins of interest. Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9), the entire teachings of which are incorporated herein by reference. One filtration process is described in the Millipore Catalog entitled "Pharmaceutical Process Filtration Catalog", pages 177-202 (Bedford, Mass., 1995 / 96), the entire teachings of which are incorporated herein by reference. tangential flow filtration as described in . Ultrafiltration is generally taken to mean filtration using filters with pore sizes less than 0.1 μm. By using filters with such small pore sizes, the sample volume can be reduced by permeating the sample buffer through the membrane pores of the filter while retaining proteins on the membrane surface.

[0362] A person skilled in the art can select the appropriate membrane filter device for UF / DF operation. Examples of membrane cassettes suitable for the present invention include Pellicon2 or Pellicon3 cassettes with 10 kD, 30 kD, or 50 kD membranes from EMD Millipore; Kvick 10 kD, 30 kD, or 50 kD membrane cassettes from GE Healthcare; Centramate or Centrasette 10 kD, 30 kD, or 50 kD cassettes include, but are not limited to.

[0363] J. An exemplary generation policy Primary harvesting proceeds using sequential pH reduction, centrifugation, and filtration to remove cells and cell debris (including HCPs) from the production bioreactor harvest. The present invention subjects the biological sample containing the protein of interest from the primary collection to one or more production steps, including (in no particular order) AEX, CEX, SEC, HIC and / or MM. about things. Certain aspects of the invention include additional processing steps. Examples of additional work-up procedures include ethanol precipitation, isoelectric focusing, reverse phase HPLC, chromatography on silica, chromatography on heparin Sepharose™, further anion exchange chromatography, and / or Further cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography (e.g. protein A or protein G as capture reagents, antibodies, specific substrates , using ligands or antigens). In certain embodiments, column temperature (and other parameters) may be varied independently to improve separation efficiency and / or yield for any particular product step.

[0364] In certain embodiments, the unbound flow-through and wash fractions may be further fractionated and the combination of fractions pooled to provide the target product purity.

[0365] Column loading and washing steps reduce the level of product-related impurities / substances in either or both the column effluent and collection pool to achieve specific target product quality and yield. , in-line, at-line, or off-line measurements. In certain embodiments, the load concentration is dynamically controlled by in-line or batch or serial dilution with buffers or other solutions to achieve the necessary distribution to improve separation efficiency and / or yield. obtain.

[0366] Examples of such generation procedures are shown in FIGS.

[0367] FIG. 5 depicts an exemplary embodiment used to produce aflibercept. Referring to FIG. 5, the method comprises the steps of: (a) expressing aflibercept in host cells cultured in CDM; (b) using a first chromatographic support that may comprise an affinity capture resin; capturing the aflibercept; and (c) contacting at least a portion of the aflibercept with a second chromatographic support, which may include anion exchange chromatography. Step (c) may further comprise washing the AEX column and collecting the flow-through fraction(s) of the sample containing aflibercept. Optionally, step (c) may comprise stripping the second chromatographic support and collecting the stripped fraction. The steps can be carried out by routine methodologies in conjunction with the methodologies mentioned above. Those skilled in the art will appreciate that they may choose to use CEX rather than or in addition to AEX. Additional chromatographic steps, in no particular order, may be utilized, including but not limited to HIC and SEC.

[0368] In addition to the exemplary embodiment of FIG. 5, other additional exemplary embodiments comprise: (d) contacting at least a portion of said aflibercept of step (c) with a third chromatographic support; can include steps. In one embodiment, the protocol may comprise the step of (e) contacting at least a portion of the aflibercept of step (d) with a fourth chromatographic support. In one aspect of this embodiment, the protocol may optionally include subjecting the aflibercept-containing sample of s...

Claims

1. A composition comprising oxoaflibercept, wherein one or more amino acid residues of the aflibercept are oxidized.

2. The composition of claim 1 , wherein the one or more amino acid residues are histidine and / or tryptophan.

3. 2. The composition of claim 1, wherein the oxoaflibercept is enzymatically digested to produce one or more oligopeptides, and the one or more oligopeptides are selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, and combinations thereof.

4. The composition of claim 3 , wherein the enzymatic digestion is carried out using trypsin.

5. A method for producing an oxidized species of aflibercept, comprising: subjecting a sample containing aflibercept to an irradiation of about 240,000 lux; ＊ hour ~ approx. 2.4 million lux ＊ The method includes subjecting the substrate to cool white light for a period of time.

6. 1. A method for producing an oxidized species of aflibercept, comprising: a. A sample containing aflibercept was exposed to approximately 240,000 lux. ＊ hour ~ approx. 2.4 million lux ＊ subjecting the sample to cool white light for a period of time; b. performing the digestion of (a) to form an oligopeptide, wherein the oligopeptide is the following oligopeptide: H ＊ is histidine that has been oxidized to 2-oxo-histidine, and C ＊ is a carboxymethylated cysteine, and M ＊ is oxidized methionine, and W ＊ is oxidized tryptophan, and Y ＊ is oxidized tyrosine, and F ＊ is oxidized phenylalanine, DKTH ＊ T.C. ＊ 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 / RIIW*DSR / IIW ＊ DSRK (SEQ ID NO: 28), TELNVGIDFNW ＊ EYPSSK (SEQ ID NO: 29), GFIISNATY ＊ K (SEQ ID NO: 69), KF ＊ PLDTLIPDGK (SEQ ID NO: 70), F ＊ LSTLTIDGVTR (SEQ ID NO: 32) performing digestion, the digestion comprising one or more of: A method comprising:

7. 7. The method of claim 6, wherein the amount of oxidized species of aflibercept is increased by about 1.5 to about 50 fold after cool white light exposure compared to untreated aflibercept.

8. 7. The method of claim 6, wherein the digestion is carried out using trypsin.

9. 7. The method of claim 6, wherein the oligopeptides are analyzed using mass spectrometry.

10. 7. The method of claim 6, wherein the cool white light has an intensity of about 8 klux.

11. The aflibercept is approximately 240,000 lux. ＊ 7. The method of claim 5 or 6, wherein the amount of oxidized species of aflibercept is increased by about 1.5 to about 10 fold compared to untreated aflibercept, after the treatment with cool white light for a period of time.

12. The aflibercept is approximately 960,000 lux. ＊ 7. The method of claim 5 or 6, wherein the amount of oxidized species of aflibercept is increased by about 1.5 to about 20 fold compared to untreated aflibercept.

13. The aflibercept is about 1.2 million lux ＊ 7. The method of claim 5 or 6, wherein the amount of oxidized species of aflibercept is increased by about 1.5 to about 20 fold compared to untreated aflibercept.

14. The aflibercept is about 2.4 million lux ＊ 7. The method of claim 5 or 6, wherein the amount of oxidized species of aflibercept is increased by about 1.5 to about 50 fold compared to untreated aflibercept.

15. 1. A method for producing an oxidized species of aflibercept, comprising: a. Culturing cells genetically modified to express aflibercept in a synthetic medium (CDM); b. binding aflibercept from the clarified harvest to a Protein A resin; c. eluting the aflibercept of step (b), wherein the eluted aflibercept has a first amount of oxidized oligopeptides; d. subjecting the eluted aflibercept of step (c) to anion exchange chromatography (AEX); e. washing the AEX column of step (d); f. stripping the AEX column of step (e) using a stripping buffer, wherein the aflibercept in the flow-through after the stripping of the AEX column comprises a second amount of oxidized aflibercept; A method comprising:

16. 16. The method of claim 15, wherein the stripping buffer is selected from the group comprising 2 M sodium chloride (NaCl), 1 N sodium hydroxide (NaOH), or a combination thereof.

17. The eluted aflibercept from step (c) is subjected to a fluoride treatment at about 240,000 lux. ＊ hour ~ approx. 2.4 million lux ＊ 16. The method of claim 15, wherein the second amount of oxidized species of aflibercept is increased by about 1.5 to about 50 fold compared to the first amount of oxidized species of aflibercept when normalized for concentration.

18. The eluted aflibercept from step (c) is subjected to a fluoride treatment at about 960,000 lux. ＊ 16. The method of claim 15, wherein the second amount of oxidized species of aflibercept is increased by about 1.5 to about 20 fold compared to the first amount of oxidized species of aflibercept when normalized for concentration.

19. The eluted aflibercept from step (c) is subjected to a fluoride treatment at about 1.2 million lux. ＊ 16. The method of claim 15, wherein the second amount of oxidized species of aflibercept is increased by about 1.5 to about 20 fold compared to the first amount of oxidized species of aflibercept when normalized for concentration.

20. The eluted aflibercept from step (c) is subjected to a fluoride treatment at about 2.4 million lux. ＊ 16. The method of claim 15, wherein the second amount of oxidized species of aflibercept is increased by about 1.5 to about 50 fold compared to the first amount of oxidized species of aflibercept when normalized for concentration.

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