Anti-VEGF protein composition and method for producing the same

Abstract

This invention provides a composition containing an anti-VEGF protein and a method for producing the composition. [Solution] A method for producing aflibercept in which the amount of aflibercept variant expressed in host cells cultured in a synthetic medium (CDM) is reduced, wherein the target value of the aflibercept variant is obtained when the cumulative concentration of iron in the CDM is about 55.0 μM or less, the cumulative concentration of copper in the CDM is about 0.8 μM or less, the cumulative concentration of nickel in the CDM is about 0.4 μM or less, the cumulative concentration of zinc in the CDM is about 56.0 μM or less, the cumulative concentration of cysteine ​​in the CDM is about 10.0 mM or less, and / or, vi. the cumulative concentration of antioxidants in the CDM is about 0.001 mM to about 10 mM for any single antioxidant, and when multiple antioxidants are added, the total antioxidant concentration is about 30 mM or less.

Description

[Technical Field]

[0001] Sequence List This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy was created on 13 August 2020, is denoted as 070816-02251_SL.txt, and has a size of 134,385 bytes.

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

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

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

[0005] The class of cell-derived dimeric mitogens that exhibit selectivity for vascular endothelial cells has been identified as vascular endothelial growth factor (VEGF) and is referred to by this name.

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

[0007] Several eye 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 the accumulation of fluid in the submacula, which can lead to vision loss. Diabetic macular edema (DME) is another eye disorder that involves neovascularization. DME is the most widely recognized cause of moderate vision loss in diabetic patients and is a common complication of diabetic retinopathy, a disease that negatively affects the blood vessels of the retina. Clinically significant DME occurs when fluid leaks into the center of the macula, the photosensitive part of the retina responsible for clear, direct vision. The presence of fluid in the macula can lead to severe vision loss or blindness.

[0008] For example, various VEGF inhibitors, such as the VEGF trap Eylea (aflibercept), are approved to treat these eye disorders. [Overview of the project]

[0009] overview This invention relates to anti-VEGF proteins, including aflibercept, a VEGF trap protein, which is a fusion protein. The invention also relates to novel anti-VEGF proteins, aflibercept MiniTrap, or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise specified). This specification discloses methods for producing these anti-VEGF proteins, including manufacturing modes that provide efficient and effective means for producing the proteins of interest. In one embodiment, the invention is made for the use of a synthetic medium (CDM) for producing anti-VEGF proteins. In a particular embodiment, the CDM of interest is a CDM that, upon use, produces a protein sample, which is yellowish-brown and may contain oxidized species. Furthermore, protein variants of aflibercept and VEGF MiniTrap are disclosed in this application along with their associated manufacturing methods.

[0010] Manufacturing of Aflibercept This disclosure describes the production of aflibercept using a cell culture medium. In one embodiment, the cell culture medium is a synthetic medium ("CDM"). CDM is often used because it is a synthetic preparation that does not contain proteins or animal-derived components and provides certainty regarding the composition of the medium. In another embodiment, the cell culture medium is a soy hydrolyzed medium.

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

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

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

[0014] In one embodiment, a clarified recovered sample from a CDM culture containing aflibercept is subjected to a capture chromatography procedure. In one embodiment, the capture step is an affinity chromatography procedure using, for example, protein A. In a further embodiment, the eluate from the affinity procedure exhibits a specific color. For example, this eluate may be yellowish-brown. As will be described in more detail below, the color can be (i) determined by the European color standard "BY" for qualitative visual inspection, or (ii) determined by a colorimetric assay (CIE L) which is more quantitative than the BY system. * a * , b * (or CIELAB) can be used for evaluation. However, in either case, color evaluations between multiple samples must be normalized to protein concentration to ensure that the investigation is meaningful. For example, referring to Example 9 below, the eluate of protein A was approximately 2.52 "b * It has a value of "", which corresponds to approximately the BY value of BY5 (when measured at a protein concentration of 5 g / L in the protein A eluate). When comparing the color of the protein A eluate with another sample, the comparison must then be performed at the same protein concentration. b in the CIELAB color space * The value is used to represent the color of the sample, covering a range from blue (-) to yellow (+). A higher b* value for a sample compared to another sample indicates a deeper yellowish-brown coloration in that sample compared to the other samples.

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

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

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

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

[0019] Ion exchange can be used to reduce or minimize color. In one aspect of this embodiment, the clarified recovered sample is subjected to capture chromatography using, for example, protein A affinity chromatography. The affinity column is eluted and has a first color assigned a specific BY and / or b * value. Subsequently, this protein A eluate is subjected to ion exchange chromatography such as anion exchange chromatography (AEX). The ion exchange column is washed, the flow-through is collected, and has a second color assigned a specific BY and / or b * value. In certain aspects, the color value of the first color (either "BY" or "b * ") is different from the second color. In a further aspect, the first color of the protein A eluate has a more yellowish-brown color when compared to the second color of the AEX flow-through as reflected by the respective BY and / or b * values. Typically, the yellowish-brown color of the second color after AEX is reduced when compared to the first color of the protein A eluate. For example, by using anion exchange, after AEX, the b * value went from approximately 3.06 (first color) to approximately 0.96 (second color), and the yellowish-brown color observed in the protein A eluate sample decreased. See Examples 2, Tables 2 - 3 below.

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

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

[0022] This embodiment may include the addition of one or more steps, in any particular order, such as hydrophobic interaction chromatography (HIC), affinity chromatography, multimodal chromatography, virus inactivation (e.g., using a low pH), virus filtration, and / or ultrafiltration / dialysis filtration, and other well-known chromatographic steps.

[0023] In one embodiment, the anti-VEGF protein is glycosylated with one or more asparagines as follows: G0-GlcNAc glycosylation, G1-GlcNAc glycosylation, G1S-GlcNAc glycosylation, G0 glycosylation, G1 glycosylation, G1S glycosylation, G2 glycosylation, G2S glycosylation, G2S2 glycosylation, G0F glycosylation, G2F2S glycosylation, G2F2S2 glycosylation, G1F glycosylation, G1FS glycosylation, G2F Lycosylation, G2FS glycosylation, G2FS2 glycosylation, G3FS glycosylation, G3FS3 glycosylation, G0-2GlcNAc glycosylation, Man4 glycosylation, Man4_A1G1 glycosylation, Man4_A1G1S1 glycosylation, Man5 glycosylation, Man5_A1G1 glycosylation, Man5_A1G1S1 glycosylation, Man6 glycosylation, Man6_G0+ phosphate glycosylation, Man6+ phosphate glycosylation, and / or Man7 glycosylation. In one embodiment, the anti-VEGF protein may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0024] In one embodiment, the glycosylation profile of the anti-VEGF protein composition is as follows: approximately 40% to approximately 50% total fucosylated glycans, approximately 30% to approximately 55% total sialylated glycans, approximately 6% to approximately 15% mannose-5, and approximately 60% to approximately 79% galactosylated glycans (see Example 6). In one embodiment, the anti-VEGF protein has Man5 glycosylation at approximately 32.4% asparagine 123 residues and / or approximately 27.1% asparagine 196 residues.

[0025] In one embodiment, this process may further include formulating the active pharmaceutical ingredient using pharmaceutically acceptable excipients. In one embodiment of this embodiment, pharmaceutically acceptable excipients may be selected from: water, buffers, sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. Other excipients well known to those skilled in the art are within the scope of this embodiment.

[0026] In one embodiment of this design, the formulation may be suitable for administration to human subjects. In particular, administration may be affected by intravitreal injection. In one embodiment, the formulation may contain approximately 40 to approximately 200 mg / mL of the 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 of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, It may be used as a method to treat or prevent neovascular ophthalmic disorders, which may include, for example, non-proliferative diabetic retinopathy (e.g., characterized by a diabetic retinopathy severity score (DRSS) level of approximately 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects without DME).

[0028] Manufacturing of VEGF MiniTrap This disclosure describes the manufacture of a modified version of aflibercept in which the Fc portion is removed or absent, and which is referred to as aflibercept MiniTrap or VEGF MiniTrap. This MiniTrap can be produced in cell culture media containing synthetic medium (CDM) or soy hydrolyzed 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 further processed so that the Fc portion is enzymatically removed, resulting in MiniTrap. Alternatively, the gene encoding MiniTrap (e.g., a nucleotide sequence encoding aflibercept without its Fc portion) can be produced using a suitable host cell and under suitable conditions.

[0030] In one embodiment, a method for producing MiniTrap involves the 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 a so-called full-length aflibercept fusion protein (see U.S. Patent No. 7,279,159, the full teaching of which is incorporated herein by reference), which comprises (a) providing genetically modified host cells to express full-length aflibercept; (b) culturing the host cells in CDM under favorable conditions for the cells to express full-length aflibercept; (c) recovering a preparation of full-length aflibercept produced by these cells; and (d) subjecting the full-length aflibercept to enzymatic cleavage specific to the removal of the Fc portion of the fusion protein. In another embodiment, the nucleotide sequence encoding aflibercept minus its Fc portion is expressed from a suitable host cell under favorable conditions well known to those skilled in the art (see U.S. Patent No. 7,279,159).

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

[0032] Suitable CDMs include Dulbecco's Modified Eagle (DME) medium, Ham Nutritional Mixture, EX-CELL medium (SAFC), and IS CHO-CD medium (Irvine). Other CDMs known to those skilled in the art are also intended within the scope of this invention. In certain embodiments, preferred CDMs are CDM1B (Regeneron) or Excell medium (SAFC).

[0033] In one embodiment, during the production of MiniTrap, a sample containing the protein of interest (i.e., aflibercept fusion protein and / or MiniTrap) and its variants (including oxovariants) may exhibit a specific color characteristic, namely yellowish-brown. For example, the eluate sample from the affinity chromatography step may be BY and / or b * A specific yellowish-brown color can be measured using the system (see Examples 2 and 9 below). Exemplary sources for the "sample" include affinity chromatography, such as protein A; this sample can be obtained from the flow-through fraction of an ion-exchange chromatography procedure; or it can be obtained from a strip of an ion-exchange column. Other sources exist during manufacturing processes, as is well known to those skilled in the art, from which the sample can be analyzed. As described above and in further detail below, the color can be evaluated using (i) the European color standard "BY" for qualitative visual inspection, or (ii) a colorimetric assay (CIELAB) which is more quantitative than the BY system. In either case, however, color evaluations between multiple samples must be normalized, for example, using protein concentration, to ensure that the investigation between samples is meaningful.

[0034] In one embodiment of this embodiment, the full-length aflibercept fusion protein may be subjected to enzymatic processing ("cleavage operation") to produce a VEGF MiniTrap using proteolytic digestion, for example, with a protease or an enzymatically active variant thereof. In one embodiment of this embodiment, the protease may be an immunoglobulinase from Streptococcus pyogenes (IdeS). In another embodiment, the protease may be thrombin trypsin, endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C, outer membrane protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain, pronase, or a protease derived from Aspergillus Saitoi. In one embodiment, the protease may be a cysteine ​​protease. In a particular embodiment of this embodiment, the protease may be IdeS. In another embodiment, the protease may be a variant of IdeS. Non-limiting examples of IdeS variants are described below. These variants include polypeptides having amino acid sequences such as those described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In one embodiment, the protease may be immobilized on agarose or another suitable matrix.

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

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

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

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

[0039] In a further embodiment, the second capture flow-through may be subjected to ion exchange chromatography such as AEX. The AEX column may be washed with a suitable buffer to collect an AEX flow-through fraction that essentially contains MiniTrap. This AEX flow-through fraction is then used for specific BY and / or b * It may have a second color which is yellowish-brown with a value. In a further embodiment, the first color (flow-through from the second capture step) and the second color (flow-through from the ion exchange procedure) are BY and / or b * It has different colors as measured by one of the systems. In one embodiment, the second color is BY and / or b after AEX. * When comparing either of the values ​​to the first color, a decrease in yellowish-brown is demonstrated.

[0040] In another embodiment, the cleavage operation in step (b) may be carried out using a chromatographic column, where the cleavage operation is, for example, enzymatic activity, attached to or immobilized on the column matrix. The column used in step (b) may contain one or more proteases that have already been mentioned and are more fully described below.

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

[0042] In one example, samples from different stages of the manufacturing process were analyzed for color and the presence of oxovaleans. Referring to Example 9, the affinity flow-through pool (flow-through from the second protein A affinity step) was approximately 1.58 of the first b * It had a value (see Table 9-3). This second affinity flowthrough was applied to AEX. The AEX flowthrough was approximately 0.50 for the second b * The value is present, indicating a significant reduction in yellowish-brown color after the use of AEX. Strip samples were obtained by stripping the AEX column, and the 3b was approximately 6.10. * A value was observed, indicating that this strip sample had a more yellowish-brown color compared to either the loaded or flow-through sample.

[0043] Referring again to Example 9, oxovalean analysis was also performed. The samples analyzed were the affinity flow-through pool (protein A affinity eluate), AEX flow-through, and AEX strip. Referring to Tables 9-5 and 9-6, changes in oxovalean 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 oxovalean (specifically oxo-tryptophan) decreased from approximately 0.055% in the loaded sample to approximately 0.038% in the flow-through sample after AEX chromatography, indicating a decrease in oxovalean after AEX. Analysis of the AEX strip revealed that the proportion of oxotryptophan species was approximately 0.089%. Comparing this strip value to the loaded amount (and flow-through), it was clear that the majority of this oxovalean was retained on the AEX column.

[0044] This embodiment may include the addition of one or more steps, in any particular order, such as hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, virus inactivation (e.g., using a low pH), virus filtration, and / or ultrafiltration / dialysis filtration.

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

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

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

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

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

[0050] The formulation of the present invention is suitable for administration to human subjects. In one embodiment of this invention, administration can be performed by intravitreal injection. In one embodiment, the formulation may contain about 40 to about 200 mg / mL of the protein of interest. In a particular embodiment, 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 of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, and vitreous neovascularization. It may be used in methods to treat or prevent neovascular ophthalmic disorders that may include, for example, non-proliferative diabetic retinopathy (e.g., characterized by a diabetic retinopathy severity score (DRSS) level of approximately 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects without DME).

[0052] Variants 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) for producing VEGF MiniTrap. IdeS(SEQ ID NO: 1) contains asparagine residues at positions 87, 130, 182, and / or 274 (in SEQ ID NO: 1 below, the "N" is shown in bold and italics). * (This is indicated as "). Asparagine at these positions can be mutated with amino acids other than asparagine to form IdeS variants (one or more mutated amino acids are indicated as one or more amino acids highlighted in italics and underline): TIFF2026094272000002.tif115165TIFF2026094272000003.tif209165TIFF20260942720 00004.tif216165TIFF2026094272000005.tif201165TIFF2026094272000006.tif165165

[0053] In one embodiment, the polypeptide has an isolated amino acid sequence having at least 70% sequence identity over the entire length of the isolated amino acid sequence, as described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In one embodiment, 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 yet another embodiment, the isolated amino acid sequence has about 100% sequence identity over the entire length of the isolated amino acid sequence. In one embodiment, the polypeptide may be capable of cleaving a target protein into fragments. In a particular embodiment, the target protein is IgG. In another embodiment, the target protein is a fusion protein. In yet another embodiment, the fragments may include Fab fragments and / or Fc fragments.

[0054] Sequence ID 1, Sequence ID 2, Sequence ID 3, Sequence ID 4, Sequence ID 5, Sequence ID 6, Sequence ID 7, Sequence ID 8, Sequence ID 9, Sequence ID 10, Sequence ID 11, Sequence ID 12, Sequence ID 13, Sequence ID 14, Sequence ID 15, and Sequence ID 16.

[0055] The disclosure also includes isolated nucleic acid molecules encoding an isolated amino acid sequence having at least 70% sequence identity over the entire length of the isolated amino acid sequence, as described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In one embodiment, 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 yet another embodiment, the isolated amino acid sequence has about 100% sequence identity over the entire length of the isolated amino acid sequence. In one embodiment, the polypeptide may be capable of cleaving a target protein into fragments. In a particular embodiment, the target protein is IgG. In another particular embodiment, the target protein is a fusion protein. In yet another particular embodiment, the fragments may include Fab fragments and / or Fc fragments.

[0056] The disclosure also includes a vector comprising a nucleic acid encoding a polypeptide having an isolated amino acid sequence having at least 70% sequence identity over the entire length of the isolated amino acid sequence, as described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In one embodiment, the nucleic acid molecule is operably ligated to an expression control sequence capable of directing its expression in a host cell. In one embodiment, the vector is a plasmid. In one embodiment, the isolated amino acid sequence 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 embodiment, the polypeptide may be capable of cleaving a target protein into fragments. In a particular embodiment, the target protein is IgG. In another embodiment, the target protein is a fusion protein. In yet another embodiment, the fragment may include a Fab fragment and / or an Fc fragment.

[0057] In one embodiment, the isolated amino acid may include a parent amino acid sequence defined by Sequence ID No. 1, having asparagine residues at positions 87, 130, 182, and / or 274 mutated to amino acids other than asparagine. In one embodiment, the mutation may result in increased chemical stability at alkaline pH values ​​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 ​​compared to the parent amino acid sequence. In one embodiment, the amino acids may be selected from aspartic acid, leucine, and arginine. In a particular embodiment, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another embodiment, the asparagine residue at position 130 is mutated to an arginine residue. In yet another embodiment, the asparagine residue at position 182 is mutated to a leucine residue. In yet another embodiment, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another embodiment, the asparagine residues at positions 87, 130, and 182 are mutated. In yet another embodiment, the asparagine residues at positions 87, 182, and 274 are mutated. In yet another embodiment, the asparagine residues at positions 130, 182, and 274 are mutated. In yet another embodiment, the asparagine residues at positions 87, 130, 182, and 274 are mutated.

[0058] In related embodiments, the disclosure includes isolated nucleic acid molecules encoding isolated amino acid sequences having a parent amino acid sequence defined by Sequence ID No. 1, which has asparagine residues at positions 87, 130, 182, and / or 274 mutated to amino acids other than asparagine. See above. The mutations may result in increased chemical stability at alkaline pH values ​​compared to the parent amino acid sequence.

[0059] In more relevant embodiments, the disclosure includes a vector. This vector comprises a nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence including a parent amino acid sequence defined by SEQ ID NO: 1, which has asparagine residues at positions 87, 130, 182, and / or 274 mutated to amino acids other than asparagine. See above. The mutation may 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 ligated to an expression regulatory sequence capable of directing its expression within a host cell. In one embodiment, the vector may be a plasmid.

[0060] Affinity-based production This disclosure also provides a method for reducing host cell proteins, as well as other undesirable proteins and nucleic acids, while producing anti-VEGF proteins using affinity chromatography.

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

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

[0063] In one embodiment of this invention, the recombinant protein of interest is cultured in a CDM. Preferred CDMs include Dulbecco's Modified Eagle (DME) medium, Ham Nutritional Mixture, Excell medium, IS CHO-CD medium, and CDM1B. Other CDMs known to those skilled in the art are also intended to be within the scope of this invention.

[0064] The product may contain at least one contaminant, which includes one or more host cell proteins in addition to the recombinant protein of interest. The at least one contaminant may originate from the cell substrate, cell culture, or downstream processes.

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

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

[0067] In one embodiment of this embodiment, the method may include the step of subjecting a biological sample containing an anti-VEGF protein and one or more host cell proteins / contaminants to affinity chromatography under suitable conditions, wherein the stationary phase of the affinity chromatography contains a protein capable of selectively or specifically binding to the anti-VEGF protein. In a particular embodiment, the protein may be an antibody, a fusion protein, an scFv, or an antibody fragment. In a particular embodiment, the protein may be VEGF 165 VEGF 121, for example, VEGF forms derived from other species, such as rabbits. For example, as illustrated in Tables 7-1 and 7-10, VEGF may be a protein that can selectively or specifically bind to or interact with anti-VEGF proteins. 165 The use of these proteins led to the successful production of MT5 (anti-VEGF protein), aflibercept, and anti-VEGF scFv fragments. In another specific embodiment, the protein may be one or more proteins having the amino acid sequences shown in SEQ ID NOs. 73-80. Table 7-1 also discloses the successful production of MT5 using proteins having the amino acid sequences shown in SEQ ID NOs. 73-80 as proteins that can selectively or specifically bind to the anti-VEGF protein (MT5).

[0068] In one embodiment of this invention, the method may include the step of subjecting a biological sample containing an anti-VEGF protein and one or more host cell proteins / contaminants to affinity chromatography under suitable conditions, wherein the stationary phase of the affinity chromatography contains a protein that is selectively or specifically able to bind to or interact with the anti-VEGF protein, and this anti-VEGF protein may be selected from aflibercept, VEGF MiniTrap, or anti-VEGF antibody. In a particular embodiment, the VEGF MiniTrap may be further obtained from a VEGF receptor component, which may be formed by recombinant expression of the VEGF MiniTrap in host cells. By carrying out 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 a sample containing MT5 (anti-VEGF protein) when five different affinity chromatography columns are used. These five different affinity chromatography columns use different proteins that are selectively or specifically able to bind to MT5, such as (i) VEGF 165The mice anti-VEGFR1 mAb human IgG1 (SEQ ID NO: 72), (ii)mAb1 (SEQ ID NO: 73 is the heavy chain and SEQ ID NO: 74 is the light chain), (iii)mAb2 (SEQ ID NO: 75 is the heavy chain and SEQ ID NO: 76 is the light chain), (iv)mAb3 (SEQ ID NO: 77 is the heavy chain and SEQ ID NO: 78 is the light chain), and (v)mAb4 (SEQ ID NO: 79 is the heavy chain and SEQ ID NO: 80 (v)mAb4 is the mouse anti-VEGFR1 mAb human IgG1). As seen in Figures 35A and 35B, the elutes from each affinity-based manufacturing process reduced host cell proteins from over 7000 ppm to about 25 ppm and about 55 ppm, respectively.

[0069] Preferred conditions for using affinity chromatography include, but are not limited to, equilibration of the affinity chromatography column using an equilibration buffer. For example, after equilibration using 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, it can be washed once or multiple times using an equilibration buffer, such as Dulbecco's phosphate-buffered saline (DPBS). Other washes, including washes using different buffers, may be used before eluting the column. Column elution may be affected by the type, pH, and conductivity of the buffer, and other elution conditions well known to those skilled in the art may be applied. For example, after elution using one or more elution buffers, such as glycine at a pH of about 2.0 to about 3.0, the eluted fraction can be neutralized by adding a neutralizing buffer, such as 1M Tris at pH 7.5.

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

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

[0072] This embodiment may include the addition of one or more steps, not necessarily in a specific order, and may include, for example, hydrophobic interaction chromatography, affinity-based chromatography, multimodal chromatography, virus inactivation (e.g., using low pH), virus filtration, and / or ultrafiltration / dialysis filtration.

[0073] In one embodiment, the glycosylation properties of the anti-VEGF protein composition are as follows: approximately 40% to approximately 50% total fucosylated glycans, approximately 30% to approximately 55% total sialylated glycans, approximately 6% to approximately 15% mannose-5, and approximately 60% to approximately 79% galactosylated glycans.

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

[0075] In one embodiment, the method may further include a step of formulating the active pharmaceutical ingredient using pharmaceutically acceptable excipients. In one embodiment, pharmaceutically acceptable excipients may be selected from: water, buffers, sugars, salts, surfactants, amino acids, polyols, chelating agents, emulsifiers, and preservatives. Other excipients well known to those skilled in the art are within the scope of this embodiment.

[0076] In one embodiment of this embodiment, the formulation may be suitable for administration to human subjects. In one embodiment of this embodiment, administration can be carried out by intravitreal injection. In one embodiment, the formulation may contain about 40 to about 200 mg / mL of the protein of interest. In a particular embodiment, the protein of interest may be aflibercept, an 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 of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, and vitreous neovascularization. It may be used in methods to treat or prevent neovascular ophthalmic disorders that may include, for example, non-proliferative diabetic retinopathy (e.g., characterized by a diabetic retinopathy severity score (DRSS) level of approximately 47 or 53) or proliferative diabetic retinopathy (e.g., in subjects without DME).

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

[0079] In one embodiment of this model, the sample comprises a protein of interest, such as an aflibercept fusion protein having a minimal oxovalean or no oxovalean. The sample is subjected to photostress to synthesize an oxidized species of aflibercept. In a particular embodiment, the sample is subjected to photostress by using cool white light. In another particular embodiment, the sample is subjected to photostress by using ultraviolet light.

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

[0081] In a specific embodiment of this model, a sample containing aflibercept or another anti-VEGF protein is exposed to ultraviolet light for approximately 4 to 40 hours, resulting in an increase of approximately 1.5 to 25 times in the modified oligopeptide product (obtained upon digestion). In this case, the sample is DKTH * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), TNYLTH * R (Sequence ID 21), SDTGRPFVEMYSEIPEIIH* MTEGR (SEQ ID NO: 22), VH * EKDK (SEQ ID NO: 23), SDTGRPFVEM * YSEIPEIIHMTEGR (Sequence No. 64), SDTGRPFVEMYSEIPEIIHM * TEGR (SEQ ID NO: 65), TQSGSEM * K (Sequence ID 66), SDQGLYTC * A ASSGLM * TK (Sequence ID 67), IIW * DSR / RIIW*DSR / IIW * DSRK (Sequence ID 28), TELNVGIDFNW * EYPSSK (Sequence ID 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK(SEQ ID NO: 70)F * LSTLTIDGVTR (Sequence ID 32) It comprises one or more modified oligopeptides selected from the group consisting of H * It is histidine and is oxidized to 2-oxo-histidine, C * It is cysteine ​​and is carboxymethylated, M * It is oxidized methionine, W * It is oxidized tryptophan, Y * is tyrosine oxide, F * It is phenylalanine oxide. Digestion can be carried out by the proteases mentioned so far, for example, trypsin. The oligopeptides can be analyzed using mass spectrometry.

[0082] Method to minimize yellowish-brown color This disclosure provides a method for reducing the yellowish-brown coloration during the production of aflibercept, MiniTrap, or equivalents produced in CDM.

[0083] In one embodiment, the method comprises the steps of culturing host cells expressing a recombinant protein of interest in CDM under preferred conditions, and subsequently recovering a preparation containing the recombinant protein of interest. In one embodiment, the recombinant protein of interest is an anti-VEGF protein. In a particular embodiment, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (an example thereof is disclosed in U.S. Patent No. 7,279,159, which is incorporated herein by reference in whole), scFv, and other anti-VEGF proteins. In one embodiment, the method produces a preparation of the recombinant protein of interest, the color of which is determined by the European BY method or the CIELAB method (b * It is characterized using ). In addition, the presence of oxovaleans can be analyzed using, for example, LC-MS.

[0084] In one embodiment of this model, the mitigation conditions include increasing or decreasing the cumulative concentration of one or more culture medium components, such as amino acids, metals, or antioxidants, including, for example, salts and precursors. This corresponds to reductions in color and protein variants of aflibercept and VEGF MiniTrap. Non-limiting examples of amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In certain embodiments, a decrease in cysteine ​​may be effective in reducing the yellowish-brown color of the preparation. Cysteine ​​concentration may also affect oxovariants.

[0085] In one embodiment, the method comprises the steps of culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under preferred conditions, and recovering a preparation of the protein of interest produced by the cells, the preferred conditions being partially achieved by reducing the cumulative concentration of cysteine ​​in the CDM to about 10 mM or less. Examples of preferred 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 the bioreactor throughout the process of cell culture forming the CDM, including the amount added at the start of the culture (CDM on day 0) and the amounts of components added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or starter culture before production in the bioreactor (i.e., before CDM on day 0) is also included. The cumulative amount is not affected by the loss of components over time during culture (e.g., by metabolism or chemical degradation). Therefore, for example, if a component is added to two cultures at different times (e.g., all components are added at the beginning in one culture, and components are added over time in another), even if the two cultures have the same cumulative amount of the component, they may have different absolute levels. The cumulative amount is also not affected by the situ synthesis of the component over time during cultivation (e.g., by metabolism or chemical transformation). Therefore, for example, if a component is synthesized in situ in one of the two cultures during a biotransformation process, even if the two cultures have the same cumulative amount of a given component, they may have different absolute levels. The cumulative amount can be expressed in units such as grams or moles of the component.

[0086] As used herein, the term “cumulative concentration” refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the start of a production batch, including additions to the initial volume from any seed culture used in the culture. For example, if the bioreactor contains 2 liters of cell culture medium at the start of a production batch, and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onward is 2 g / L (i.e., 4 grams divided by 2 liters). Even if an additional 1 liter of liquid without component X is added to the bioreactor on day 4, the cumulative concentration remains 2 g / L. Even if some liquid is lost from the bioreactor on day 5 (e.g., by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentration may be expressed in units such as grams / liter or moles / liter.

[0087] In one embodiment of this invention, the method comprises the steps of culturing host cells expressing a recombinant protein of interest in CDM under preferred conditions, and recovering a protein preparation produced by the cells, wherein preferred conditions are obtained by reducing the ratio of the cumulative cysteine ​​concentration to the cumulative total amino acid concentration of about 1:50 to about 1:30, which is about 1:10 to 1:29.

[0088] In one embodiment, this method comprises (i) culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under preferred conditions, and (ii) recovering a preparation of the recombinant protein of interest produced by the cells, wherein preferred conditions are obtained by reducing the cumulative iron concentration in the CDM to less than about 55.0 μM. In one embodiment of this present invention, the preparation obtained by this method shows a reduced yellowish-brown color compared to the preparation obtained by a method where the cumulative iron concentration in the CDM is greater than about 55.0 μM.

[0089] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under preferred conditions. The method further includes recovering a preparation of the recombinant protein of interest produced by the cells, preferred conditions which are obtained by reducing the cumulative concentration of copper in the CDM to about 0.8 μM or less. In one embodiment of this present invention, the preparation obtained by this method shows a reduced yellowish-brown color compared to the preparation obtained by a method where the cumulative concentration of copper in the CDM is greater than about 0.8 μM.

[0090] In one embodiment, this method includes the steps of culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under preferred conditions, and recovering a preparation of the recombinant protein of interest produced by the cells, wherein preferred conditions are obtained by reducing the cumulative concentration of nickel in the CDM to about 0.40 μM or less. In one embodiment of this present invention, the preparation obtained by this method shows a reduced yellowish-brown color compared to the preparation obtained by a method where the cumulative concentration of nickel in the CDM is greater than about 0.40 μM.

[0091] In one embodiment, the method includes culturing host cells expressing a recombinant protein of interest, such as aflibercept, in CDM under preferred conditions. The method further includes recovering a preparation of the recombinant protein of interest produced by the cells, preferred conditions which are obtained by reducing the cumulative concentration of zinc in the CDM to about 56 μM or less. In one embodiment of this present invention, the preparation obtained by this method shows a reduced yellowish-brown color compared to the preparation obtained by a method where the cumulative concentration of zinc in the CDM is greater than about 56 μM.

[0092] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under preferred conditions. The method further comprises recovering a preparation of the recombinant protein of interest produced by the cells, preferred conditions being the presence of an antioxidant in the CDM at a cumulative concentration of about 0.001 mM to about 10 mM for a single antioxidant, and at a cumulative concentration of about 30 mM or less when multiple antioxidants are added to the CDM. In one embodiment of this example, the preparation obtained by this method shows reduced yellowish-brown color compared to a preparation obtained by a method in which the antioxidant is not present in the CDM at a cumulative concentration of about about 0.01 mM or more than about 100 mM. Non-limiting examples of antioxidants may include taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, vitamin C, vitamin E, chelating agents, catalase, S-carboxymethyl-L-cysteine, and combinations thereof. Non-exclusive examples of chelating agents include auryntricarboxylic acid (ATA), deferoxamine (DFO), EDTA, and citrate.

[0093] In one embodiment, the method comprises culturing host cells expressing a recombinant protein of interest, such as aflibercept, in a CDM under preferred conditions. The method further comprises recovering a preparation of the recombinant protein of interest produced by the cells, wherein preferred conditions include a CDM having a cumulative concentration of iron less than about 55 μM, a cumulative concentration of copper less than or equal to about 0.8 μM, a cumulative concentration of nickel less than or equal to about 0.40 μM, a cumulative concentration of zinc less than or equal to about 56 μM, a cumulative concentration of cysteine ​​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, if multiple antioxidants are added to the CDM, a CDM having a cumulative concentration of about 30 mM or less.

[0094] In one embodiment of this embodiment, the preparation obtained using preferred conditions results in a reduction of the aflibercept and VEGF MiniTrap protein variants to a desired amount (referred to as the "target value" of the aflibercept and VEGF MiniTrap protein variants). In a further embodiment of this embodiment, the preparation obtained using preferred conditions results in a desired b when normalizing the protein preparation containing the aflibercept and VEGF MiniTrap variants to a concentration of 5 g / L or 10 g / L. * Value or BY value (each "Target b") * This brings about a reduction in the color of the preparation to a value (referred to as the "target BY value"). In a further embodiment of this embodiment, target b * The value (or target BY value) and / or the target value of the variant can be obtained in the preparation if the titer does not increase or decrease significantly.

[0095] These and other aspects of the present invention will be better recognized and understood in conjunction with the following description and accompanying drawings. The following description illustrates various embodiments and numerous specific details thereof, but these are given as examples only and are not limiting. Many substitutions, modifications, additions, or rearrangements can be made within the scope of the present invention. [Brief explanation of the drawing]

[0096] [Figure 1] A VEGF MiniTrap generated using an exemplary embodiment is shown. This VEGF MiniTrap contains Fc fragments cleaved from VEGFR1 (SEQ ID NO: 34), VEGFR2 (SEQ ID NO: 36, hinge domain fragment (SEQ ID NO: 60)), and aflibercept (SEQ ID NO: 55). [Figure 2] The proposed mechanism for histidine oxidation to 2-oxo-histidine (14Da) is shown. [Figure 3] The proposed mechanism for histidine oxidation to 2-oxo-histidine (16Da) is shown. [Figure 4] The proposed mechanism for the oxidation of tryptophan to N-formylkynurenine and kynurenine is shown. [Figure 5] An exemplary embodiment for the production of aflibercept is shown. [Figure 6] An exemplary embodiment for the production of VEGF MiniTrap is shown. [Figure 7] An exemplary embodiment for the production of aflibercept is shown. [Figure 8] An exemplary embodiment for the production of VEGF MiniTrap is shown. [Figure 9] As an exemplary embodiment, a chart of the calculated BY standard for the calculated b* value is shown. [Figure 10] The results of experiments conducted to evaluate the proportion of 2-oxo-histidine and tryptophan oxidation (underlined portions indicate residue oxidation) in oligopeptides from protease-digested AEX loading and flow-through, including a reduced and alkylated aflibercept (SEQ ID NO: 55) fragment, are shown. [Figure 11] This shows the relative abundance of peptides identified from peptide mapping analysis performed using oligopeptides from protease-digested AEX loading and flow-through (underlined portions represent oxidation of residues in the peptide sequence), including a fragment of aflibercept (SEQ ID NO: 55). [Figure 12A] The overall chromatogram chart of absorbance as time (minutes) for MT4 and MT1 at 350 nm is shown. [Figure 12B] This shows an enlarged view of the chromatogram chart of absorbance over time (16-30 minutes) for MT4 and MT1 at 350 nm, including SEQ ID NOs. 21 and 28. [Figure 12C] This shows an enlarged view of the chromatogram chart of absorbance over time (30-75 minutes) for MT4 and MT1 at 350 nm, including sequence numbers 17, 18, 19, and 20. [Figure 13]The results of experiments conducted to evaluate the proportion of 2-oxo-histidine (and tryptophan dioxide) in oligopeptides from protease-digested MT1 treated by AEX chromatography and in oligopeptides from protease-digested MT1 stripped from AEX chromatography, including SEQ ID NOs. 17, 18, 19, 20, 21, and 28, are shown. [Figure 14] This paper presents the results of experiments conducted to compare the acidic species present in different production lots of MT1, including SEQ ID NOs. 17, 18, 19, 20, 21, and 28, with the acidic fractions obtained when performing strong cation exchange (CEX) chromatography. [Figure 15] This document describes an exemplary method for concentrating acidic species and other variants present in a cell culture recovery sample using strong cation exchange chromatography. [Figure 16] The fractions from a strong cation exchange chromatography operation according to an exemplary embodiment are shown. [Figure 17] The strong cation exchange chromatograms performed according to exemplary embodiments are shown for MT1 production subjected to CEX (before any production procedure, below BY3) and for concentrated variants of desialylated MiniTrap (dsMT1) using a salt-pH dual gradient. [Figure 18A] A 3D chromatogram of an unfractionated parent control, performed by strong cation exchange chromatography according to an exemplary embodiment, is shown. [Figure 18B] A 3D chromatogram for MT1 and fraction 1, representing some of the tailing features, is shown for an experiment performed by strong cation exchange chromatography according to an exemplary embodiment. [Figure 18C] A 3D chromatogram of the features of MT1 and fraction 2, performed by strong cation exchange chromatography according to an exemplary embodiment, is shown. [Figure 18D] A 3D chromatogram of the features of MT1, fraction 3, performed by strong cation exchange chromatography according to an exemplary embodiment is shown. [Figure 18E] A 3D chromatogram of the features of MT1, fraction 4, performed by strong cation exchange chromatography according to an exemplary embodiment is shown. [Figure 18F] A 3D chromatogram of the features of MT1, fraction 5, performed by strong cation exchange chromatography according to an exemplary embodiment is shown. [Figure 18G] A 3D chromatogram of the features of MT1, fraction 6, performed by strong cation exchange chromatography according to an exemplary embodiment is shown. [Figure 18H] A 3D chromatogram of the features of MT1, fraction 7, performed by strong cation exchange chromatography according to an exemplary embodiment is shown. [Figure 19] The electrophoretic diagram of image capillary isoelectric focusing (icIEF) performed according to an exemplary embodiment for MT1 production is shown. [Figure 20] The results of a study correlating the appearance of oxidized amino acid residues, including sequence numbers 17, 18, 19, 20, 21, 28, 29, and 83, with exposure of MT1 to cold white light or UVA light are shown. [Figure 21] A 3D SEC-PDA (size exclusion chromatography coupled with photodiode array detection) chromatogram of CWL-stressed MT1 having an absorbance at approximately 350 nm (see, for example, the circle highlighting approximately 350 nm) is shown according to an exemplary embodiment. In this case, A shows the chromatogram at T=0, B shows the chromatogram at 0.5 × ICH, C shows the chromatogram at 2.0 × ICH, and D depicts MT1 in a vial (normalized to 80 mg / mL) stressed by CWL at different time intervals. [Figure 22]A 3D SEC-PDA chromatogram in UVA-stressed MT1 having an absorbance at approximately 350 nm (see, for example, the circle highlighting approximately 350 nm) is shown according to an exemplary embodiment. In this case, A shows the chromatogram at T=0, B shows the chromatogram at 0.5 × ICH, C shows the chromatogram at 2.0 × ICH, and D depicts MT1 in vials (normalized to 80 mg / mL) stressed with UVA at different time intervals. [Figure 23A] The A320 / 280 absorbance ratio quantified from SEC-PDA chromatograms of samples stressed using CWL (upper panel) is shown. [Figure 23B] This chart shows the A320 / 280 absorbance ratio for size variants in a sample stressed using CWL (lower panel), where the sample was stressed according to an exemplary embodiment. [Figure 24A] The A320 / 280 absorbance ratio quantified from SEC-PDA chromatograms of samples stressed using UVA (upper panel) is shown. [Figure 24B] This chart shows the A320 / 280 absorbance ratio for size variants in a sample stressed using UVA (lower panel), where the sample was stressed according to an exemplary embodiment. [Figure 25A] This shows estimated values ​​of the effects that incubation of various components containing aflibercept has on the generation of color (predicted by CIE L*, a*, and b* values). [Figure 25B] The plot shows the actual values ​​against the predicted b values. [Figure 26A] This study shows the effects of CDM containing low cysteine ​​and low metal content on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmotic pressure (E). [Figure 26B] This study shows the effects of CDM containing low cysteine ​​and low metal content on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmotic pressure (E). [Figure 26C] This study shows the effects of CDM containing low cysteine ​​and low metal content on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmotic pressure (E). [Figure 26D] This study shows the effects of CDM containing low cysteine ​​and low metal content on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmotic pressure (E). [Figure 26E] This study shows the effects of CDM containing low cysteine ​​and low metal content on aflibercept potency (A), viable cell concentration (B), viability (C), ammonia (D), and osmotic pressure (E). [Figure 27] This chart shows a predictive profile of the color of the recovered material (confirmed as the b* value on day 13) in relation to the increase / decrease in metal and cysteine ​​concentrations, according to an exemplary embodiment. [Figure 28A] This shows the effect of incubation of various components containing aflibercept in CDM consumed during the generation of color (predicted by CIE L*, a*, and b* values). [Figure 28B] The plot shows the estimated predicted impact at the b-value. [Figure 28C] This study demonstrates the estimated incubation effect of various components containing aflibercept in CDM during the generation of color (predicted by CIE L*, a*, and b* values) in shaking flask culture. [Figure 28D] This shows the effect of incubation of hypotaurine containing aflibercept and deferoxamine mesylate (DFO) in CDM consumed during the generation of color (CIE L*, a*, and b* predicted by the "b" value). [Figure 28E] This shows the effect of incubation on various components containing aflibercept from shaking flask cultures during color generation (predicted by CIE L*, a*, and b* values). [Figure 29] This chart shows the effect of adding uridine, manganese, galactose, and dexamethasone to CDM on the potency of aflibercept production. [Figure 30] This chart shows the effects of adding uridine, manganese, galactose, and dexamethasone to CDM on the viability of cells expressing aflibercept. [Figure 31] This chart shows the effect of adding uridine, manganese, galactose, and dexamethasone to CDM on the number of viable cells expressing aflibercept. [Figure 32] This chart shows a standard curve of absorbance against host cell protein concentration (ng / mL) prepared using a standard host cell protein solution from Cygnus 3G (F550). [Figure 33] This is an image of an SDS-PAGE analysis performed using a sample buffer for non-reducing SDS-PAGE. [Figure 34] This is an image of an SDS-PAGE analysis performed using a reduced SDS-PAGE sample buffer. [Figure 35A] This chart shows the total host cell proteins detected in the elution fractions from the loading solution, VEGF165, mAb1, and mAb2, respectively, from affinity chromatography columns 1-3. [Figure 35B] This chart shows the total host cell proteins detected in the elution fractions from affinity chromatography columns 1, 2, 4, and 5, respectively, containing the loading solution, VEGF165, mAb1, mAb3, and mAb4. [Figure 36A] This shows the SEC profile of the VEGF MiniTrap before affinity chromatography generation. [Figure 36B] The SEC profile of the VEGF MiniTrap after affinity chromatography generation is shown. [Figure 37] This diagram illustrates the dynamics analysis of VEGF MiniTrap against VEGF165. In this case, the VEGF MiniTrap constructs being tested were derived from before and after affinity chromatography generation according to several exemplary embodiments. [Figure 38] Figure showing the SPR sensorgram from the kinetic assay of VEGF MiniTrap against VEGF165. In this case, the VEGF MiniTrap constructs being tested were derived before and after performing affinity chromatography generation according to some exemplary embodiments. [Figure 39] Chart of total host cell proteins detected in elution fractions from an affinity chromatography column repeatedly used for columns containing a loading solution, VEGF165, mAb1 and mAb2. [Figure 40] Structure of VEGF MiniTrap MT1 (SEQ ID NO: 46) according to an exemplary embodiment. [Figure 41] Structure of VEGF MiniTrap MT6 (SEQ ID NO: 51) according to an exemplary embodiment. [Figure 42] Total ion chromatogram (TIC) of relative absorbance versus time (minutes) for native SEC-MS analysis of MT1, MT5 and MT6, and an enlarged view of the low molecular weight region from the TIC. [Figure 43] Deconvoluted mass spectra of the main peaks for MT1 and MT5 to confirm the identity of the MiniTrap dimer along with the elucidation of some PTMs. [Figure 44] Deconvoluted mass spectra of the main peaks for MT6 to confirm the identity of the single-chain MiniTrap along with the elucidation of some PTMs. [Figure 45A] Chart of relative absorbance versus time (minutes) for MT1 for low molecular weight impurities in MT1. [Figure 45B] Mass spectrum for low molecular weight impurities in MT1. [Figure 46] Relative absorbance versus time (minutes) for MT1 showing the absence of the FabRICATOR enzyme used to cleave aflibercept to MT1. [Figure 47]Relative absorbance against time (minutes) for low molecular weight impurities in MT5 is shown. [Figure 48] Relative absorbance against time (minutes) for low molecular weight impurities in MT6 is shown. [Figure 49A] A chart of absorbance against time (minutes) obtained by performing HILIC-UV / MS on VEGF MiniTrap MT6 is shown. This chart shows the elution of the main peak at 21 minutes and the O-glycan at approximately 21.5 minutes. [Figure 49B] A mass spectrum obtained by performing HILIC-UV / MS on VEGF MiniTrap MT6 showing the main peak at 47985.8 Da is shown. [Figure 49C] A mass spectrum of the O-glycan of VEGF MiniTrap MT6 obtained by performing HILIC-UV / MS is shown. [Figure 50] An image of the VEGF MiniTrap dimer, where the disulfide bridges in the hinge region (SEQ ID NO: 83) of VEGF MiniTrap can be parallel or cross. [Figure 51] The relative abundance of the distribution of the glycan observed at Asn36, including SEQ ID NO: 100, in MT1, MT5 and MT6 is shown. [Figure 52] The relative abundance of the distribution of the glycan observed at Asn68, including SEQ ID NO: 101, in MT1, MT5 and MT6 is shown. [Figure 53] The relative abundance of the distribution of the glycan observed at Asn123, including SEQ ID NO: 102, in MT1, MT5 and MT6 is shown. [Figure 54] The relative abundance of the distribution of the glycan observed at Asn196, including SEQ ID NO: 103, in MT1, MT5 and MT6 is shown. [Figure 55] Analysis of released N-linked glycans by hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection and mass spectrometry analysis (maximum measurement limit and stacking) is shown. [Figure 56]The HILIC-FLR chromatograms for MT1, MT5, and MT6 are shown. [Figure 57] This shows the analysis of released N-linked glycans by HILIC linked to fluorescence detection and mass spectrometry (maximum detection limit, stacking, and normalization). [Figure 58A] This table shows detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5, and MT6. [Figure 58B] This table shows detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5, and MT6. [Figure 58C] This table shows detailed glycan identification and quantification from VEGF MiniTrap samples MT1, MT5, and MT6. [Figure 59] An exemplary production procedure for manufacturing MiniTrap is shown according to an exemplary embodiment. [Modes for carrying out the invention]

[0097] Detailed explanation Angiogenesis, the proliferation of new blood vessels from the existing vascular system, is a highly organized process crucial for proper fetal and postnatal angiogenesis. Abnormal or pathological angiogenesis is characteristic of cancer and several retinal diseases. In these cases, 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 fluid and retinal vessels of patients with various eye diseases. Blocking VEGF activity is also an optimal treatment for DME, wet AMD, CNV, retinal vein occlusion, and other eye diseases in which abnormal angiogenesis underlies the pathogenesis.

[0098] As used herein, aflibercept is one of those anti-VEGF proteins that contain the entire human amino acid sequence, including the second Ig domain of human VEGFR1 and the third Ig domain of human VEGFR2, expressed as an inline fusion with human IgG1 (Fc). Aflibercept binds to all forms of VEGF-A (VEGF), as well as PlGF and VEGF-B. Several other homodimers, VEGF MiniTraps, are produced as products of enzymatic cleavage from aflibercept or as recombinant expression directly from host cell lines. An example of such a VEGF MiniTrap is shown in Figure 1. In this figure, terminal lysine is shown (k), and some culture processes remove this terminal lysine, while others do not. Figure 1 illustrates a process in which terminal lysine remains. In general, aflibercept encompasses both cases: with and without terminal lysine.

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

[0100] In injectable therapeutic solutions, a yellowish-brown color may be an undesirable characteristic. This can be an important parameter used to determine whether a drug product meets the required levels of purification and quality for a particular treatment. Yellowish-brown colors observed during the manufacturing process of biopharmaceuticals may result from chemical modifications of the biopharmaceutical, degradation products of formulation excipients, or degradation products formed by the reaction of the biopharmaceutical with the formulation excipients. However, this information can be helpful in understanding the cause of the color change. It can also assist in designing short-term and long-term storage conditions to prevent modifications that promote such color changes.

[0101] The inventors observed that using AEX during the preparation of the anti-VEGF protein solution minimized the yellowish-brown discoloration. In addition, the inventors discovered that the yellowish-brown discoloration could be reduced by modifying the cell culture used to produce recombinant proteins such as aflibercept or modified aflibercept such as MiniTrap.

[0102] This invention encompasses the production of anti-VEGF proteins and CDMs. In addition, this invention is based on identifying and optimizing upstream and downstream process technologies for protein production.

[0103] Where applicable, some of the examples described below illustrate the production of anti-VEGF proteins (Example 1), the production of oxidized species of anti-VEGF proteins (Example 4), a method for reducing oxidized species of anti-VEGF proteins by optimizing the culture medium (Example 5), and a method for reducing oxidized species of anti-VEGF proteins by optimizing the manufacturing method (Example 2).

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

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

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

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

[0108] As used herein, the terms “chemically defined medium” or “chemically defined media” (both abbreviated as “CDM”) refer to a synthetic growth medium in which all components are identified and their concentrations defined. A synthetic medium does not contain microorganisms, yeasts, animal or plant extracts, animal serum or plasma, but individual plant or animal-derived components (e.g., proteins, polypeptides) may be added. A synthetic medium may contain inorganic salts such as phosphates, sulfates, and their equivalents as needed to support growth. The carbon source is defined and is typically a sugar such as glucose, lactose, galactose, and their equivalents, or other compounds such as glycerol, lactates, acetates, and their equivalents. Certain synthetic media may also use phosphates as buffers, while other buffers such as sodium bicarbonate, HEPES, citrate, triethanolamine, and their equivalents may be used. Examples of commercially available synthetic media include, but are not limited to, various Dulbecco's Modified Eagle (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Ham Nutritional 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, in U.S. Patent Nos. 6,171,825 and 6,936,441, International Publication No. 2007 / 077217, and U.S. Patent Application Publication Nos. 2008 / 0009040 and 2007 / 0212770, the entire teachings thereof 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 throughout the process of cell culture forming a CDM, including the amount added at the start of the culture (CDM on day 0) and the amounts of components added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or starter culture before production in the bioreactor (i.e., before CDM on day 0) is also included. The cumulative amount is not affected by the loss of the component over time during culture (e.g., by metabolism or chemical degradation). Therefore, for example, if a component is added to two cultures at different times (e.g., all of the component is added at the beginning in one culture, and the component is added over time in another culture), the two cultures may have different absolute levels even if they have the same cumulative amount of the component. The cumulative amount is also not affected by the situ synthesis of the component over time during culture (e.g., by metabolism or chemical transformation). Therefore, for example, if a component is synthesized in situ in one of two cultures during a bioconversion process, two cultures having the same cumulative amount of a given component may have different absolute levels. The cumulative amount can be expressed in units such as grams or moles of the component.

[0110] As used herein, the term “cumulative concentration” refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the start of a production batch, including additions to the initial volume derived from any seed culture used in the culture. For example, if the bioreactor contains 2 liters of cell culture medium at the start of a production batch, and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onward is 2 g / L (i.e., 4 grams divided by 2 liters). Even if an additional 1 liter of liquid without component X is added to the bioreactor on day 4, the cumulative concentration remains 2 g / L. Even if some liquid is lost from the bioreactor on day 5 (e.g., by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentration may be expressed in units such as grams / liter or moles / liter.

[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 may 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 may be 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 mL, approximately 0.6 mg / mL, approximately 0.7 mg / mL, approximately 0.8 mg / mL, approximately 0.9 mg / mL, approximately 1 mg / mL, approximately 2 mg / mL, approximately 3 mg / mL, approximately 4 mg / mL, approximately 5 mg / mL, approximately 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, approximately 50 mg / mL, approximately 55 mg / mL, approximately 60 mg / mL, approximately 65 mg / mL, approximately 70 mg / mL, approximately 5 mg / mL, approximately 80 mg / mL, approximately 85 mg / mL, approximately 90 mg / mL, approximately 100 mg / mL, approximately 110 mg / mL, approximately 120 mg / mL, approximately 130 mg / mL, approximately 140 mg / mL, approximately 150 mg / mL, approximately 160 mg / mL, approximately 170 mg / mL, approximately 180 mg / mL, approximately 190 m The pH may be g / 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 may be greater than about 5.0. In one exemplary embodiment, the pH may be greater than about 5.0, greater than about 5.5, greater than about 6, greater than about 6.5, greater than about 7, greater than about 7.5, greater than about 8, or greater than about 8.5.

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

[0113] As used herein, the term “ultrafiltration” or “UF” may include membrane filtration processes similar to reverse osmosis that use hydrostatic pressure to force water through a semipermeable membrane. Ultrafiltration is described in detail in Leos J. Zeman & Andrew L. Zydney, Microfiltration and ultrafiltration: principles and applications (1996), the entire teaching of which is incorporated herein by reference. Filters with pore sizes smaller than 0.1 μm can be used for ultrafiltration. By using filters with such small pore sizes, the volume of the sample can be reduced by permeating the sample buffer through the filter while retaining the protein behind the filter.

[0114] As used herein, “diafiltration” or “DF” may include methods using an ultrafilter to remove and replace salts, sugars, and non-aqueous solvents, separate them from bound species, remove low molecular weight materials, and / or cause a rapid change in the ionic and / or pH environment. Microsolutes are most efficiently removed by adding a solvent to the ultrafiltered solution at a rate approximately equal to the ultrafiltration rate. In this way, microspecies are washed out of the solution in a constant amount. In certain exemplary embodiments of the present invention, a diafiltration step can be used to replace various buffers used in connection with the present invention, for example, before chromatography or other manufacturing steps, to remove impurities from the protein preparation. As used herein, the term “downstream process technique” refers to one or more techniques used after the upstream process technique that produces the protein. Downstream process technologies include, for example, affinity chromatography using a solid phase having clearly defined molecules such as protein A affinity chromatography and VEGF receptors (VEGF®) that can interact with its congeners; ion exchange chromatography such as anion exchange chromatography or cation chromatography; hydrophobic interaction chromatography; or substitution chromatography, for example, the production of protein products.

[0115] The term “recombinant host cell” (or simply “host cell”) includes cells into which a recombinant expression vector encoding the protein of interest is introduced. It should be understood that these terms are intended to refer not only to the specific cells of interest but also to the offspring of such cells. Since certain modifications can occur over generations due to either mutation or environmental influences, these offspring may not actually be identical to the parent cells, but this remains within the scope of the term “host cell” as used herein. In one embodiment, host cells include prokaryotic and eukaryotic cells selected from any of the kingdoms of life. In one embodiment, eukaryotic cells include protist cells, fungal cells, plant cells, and animal cells. In a further embodiment, host cells include eukaryotic cells such as plant cells and / or animal cells. Cells may be mammalian cells, fish cells, insect cells, amphibian cells, or avian cells. In a particular embodiment, the host cell is a mammalian cell. A variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and other depositary institutions, as well as commercial suppliers. Cells that can be used in the process of the present invention include MK2.7 cells; PER-C6 cells; Chinese hamster ovary cells (CHO) such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. CellMolec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and International Publication No. 01 / 92337A2), dihydrofolate reductase-negative CHO cells (CHO / -DHFR, Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (US Patent No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); SV40 (COS cells, COS-7, ATCC) Monkey kidney CV1 cells transformed with 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, renal epithelial cells and retinal epithelial cells), as well as established cell lines and their cell lines (e.g., human fetal 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); Canine kidney cells (MDCK, ATCC CCL-34); Human lung cells (W138, ATCC CCL-75); Human liver cancer cells (HEP-G2, HB8065); Mouse mammary 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;PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH3 cells, L2 cells, LLC-RC256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or their derivatives), fibroblasts from any tissue or organ (heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lungs, vascular tissue (arteries, veins, capillaries), Lymphoid tissue (lymph glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen, and fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit551 cells, Detroit510 cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC)). CRL-1587;VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB / 3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB / 3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H / IOTI / 2 cells, HSDM1C3 cells, KLN 205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L) cells, LM strain (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss / 3T3 cells, Indian muntjac muntac) cells, SIRC cells, C;II This includes, but is not limited to, cells and Jensen cells or their derivatives (cell lines), or any other cell types known to those skilled in the art.

[0116] As used herein, the term “host cell protein” (HCP) includes proteins derived from host cells and may be unrelated to the desired protein of interest. Host cell proteins may be process-related impurities that may originate from the manufacturing process, and these may include three main categories: cell substrate-derived, cell culture-derived, and downstream-derived. Cell substrate-derived impurities include, but are not limited to, proteins derived from host organs and nucleic acids (host cell genome, vector, or total DNA). Cell culture-derived impurities include, but are not limited to, inducers, antimicrobial agents, serum, and other culture medium components. Downstream-derived impurities include, but are not limited to, enzymes, chemical and biological reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, nonmetallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leached substances.

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

[0118] As used herein, the term “hydrolyzed agent” refers to one or a combination of a number of different agents capable of performing protein digestion. Non-limiting examples of hydrolyzed agents capable of performing enzymatic digestion include proteases, elastases, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergylopepsin 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), immunoglobulinase (IdeS) of Streptococcus pyogenes, thermolysin, papain, pronase, V8 protease, or biologically active fragments, homologs thereof, or combinations thereof. Non-enzymatic digestion can be performed using hydrolyzing agents, but are not limited to those used in this context. Examples include high temperature, microwave, ultrasound, high pressure, infrared radiation, solvents (non-limited examples include ethanol and acetonitrile), immobilized enzyme digestion (IMER), magnetic particle immobilized enzymes, and on-chip immobilized enzymes. For a recent review describing available techniques for protein digestion, see Switzar et al., “Protein Digestion: An Overview of the Available Techniques and Recent Developments” (Linda Switzar, Martin Giera & Wilfried MANiessen, *Protein Digestion: An Overview of the Available Techniques and Recent Developments*, 12 *Journal of Proteome Research* 1067-1077 (2013), the entire teaching is incorporated herein). One or more hydrolyzing agents can cleave peptide bonds in a sequence-specific manner, producing predictable collections of shorter peptides. The ratio of hydrolyzing agent to protein and the time required for digestion can be appropriately selected to obtain optimal protein digestion.If the enzyme-to-substrate ratio is inappropriately high, the digestion rate will be correspondingly fast, potentially impairing sequence coverage by not providing sufficient time for peptide analysis with a mass spectrometer. On the other hand, a low E / S ratio requires a long digestion time, which increases data acquisition time. The enzyme-to-substrate ratio can range from approximately 1:0.5 to approximately 1:200. As used herein, the term “digestion” refers to the hydrolysis of one or more peptide bonds of a protein. Several approaches exist for performing the digestion of proteins in biological samples using appropriate hydrolysants, which are, for example, 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 possessing unique characteristics in terms of specificity, efficiency, and optimal digestion conditions. Proteases refer to both endopeptidases and exopeptidases and are classified based on their ability to cleave peptides at non-terminal or terminal amino acids. Alternatively, proteases also refer to six different classes, as they are classified based on their catalytic mechanism, including aspartate proteases, glutamine proteases, and metalloproteases, cysteine ​​proteases, serine proteases, and threonine proteases. The terms "protease" and "peptidase" are used interchangeably to refer to enzymes that hydrolyze peptide bonds.

[0119] The term "in conjunction with" indicates that components such as the anti-VEGF composition of the present invention can be formulated as a single composition for simultaneous delivery, or separately as two or more compositions (e.g., kits containing each component), together with another agent such as an anti-ANG2 agent. Components administered in conjunction with each other can be administered to the subject at different times, apart from when the other components are administered. For example, each administration may be given non-simultaneously (e.g., separately or sequentially) with intervals over a given period. Separate components administered in conjunction with each other may also be administered essentially simultaneously (e.g., at exactly the same time or divided by nonclinically significant periods) during the same administration session. Furthermore, separate components administered in conjunction with each other may be administered to the subject via the same or different routes. For example, a composition of aflibercept may be administered together with another agent such as an anti-ANG2 agent, and this aflibercept composition may contain less than approximately 15% of its variant.

[0120] As used herein, the term “liquid chromatography” refers to a process that enables the separation of a fluid-transported biological / chemical mixture into its components as a result of differential distribution while the mixture flows through (or into) a stationary fluid phase or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion exchange chromatography, size exclusion chromatography, affinity chromatography, mixed-mode chromatography, hydrophobic chromatography, or mixed-mode chromatography.

[0121] As used herein, “affinity chromatography” may include separation of two substances based on their affinity for a chromatographic material. This may include feeding substances onto a column containing a suitable affinity chromatography medium. Non-limiting examples of such chromatographic media include, but are not limited to, a protein A resin, a protein G resin, an affinity support containing an antigen that has produced a binding molecule (e.g., an antibody) to the same, a protein capable of binding to a protein of interest, and an affinity support containing an Fc-binding protein. In one embodiment, the affinity column may be equilibrated with a suitable buffer before loading the sample. An example of a suitable buffer may be Tris / NaCl buffer (pH approximately 7.0–8.0). Those skilled in the art can develop a suitable buffer without excessive effort. After this equilibration, the sample can be loaded onto the column. After loading the column, the column may be washed once or multiple times, for example, using the equilibration buffer. Other washings, including washing with different buffers, may be used before eluting the column. The affinity column can then be eluted using a suitable elution buffer. A suitable elution buffer may be an acetate / NaCl buffer (pH approximately 2.0–3.5). Furthermore, those 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, and the absorbance at 280 nm, for example, is particularly useful when the sample of interest contains aromatic rings (e.g., proteins with aromatic amino acids such as tryptophan).

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

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

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

[0125] As used herein, the term “mass spectrometer” includes an instrument capable of identifying specific molecular species and measuring their precise masses. This term implies the inclusion of any molecular detector capable of characterizing polypeptides or peptides. A mass spectrometer may include three main components: an ion source, a mass spectrometer, and a detector. The role of the ion source is to produce ions in the gas phase. Atoms, molecules, or clusters of the analyte can be transferred to the gas phase and ionized simultaneously (by electrospray ionization) or through a separation process. The choice of ion source varies depending on the application. In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. As used herein, the term “tandem mass spectrometry” includes a technique for obtaining structural information on sample molecules by using multiple steps, such as mass selection and mass separation. A requirement is that the sample molecules are transferred to the gas phase and ionized to form fragments in a predictable and controllable manner after the initial mass selection step. Multi-stage MS / MS or MS n First, Precursorion (MS 2 ) are selected and separated, and then fragmented to obtain the first fragment ion (MS 3) is separated and fragmented into a second fragment (MS 4 Tandem MS is performed as long as important information can be obtained by separating fragments or other components, or as long as the fragment ion signal is detectable. Tandem MS is successfully performed by combining many types of analyzers. Which analyzers to combine for a particular application is determined by many different factors, such as size, cost, and effectiveness, as well as sensitivity, selectivity, and speed. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but hybrids also exist in which temporal tandem analyzers are spatially linked, or linked with spatial tandem analyzers. A spatial tandem mass spectrometer comprises an ion source, a precursor ion activator, and at least two non-trapped mass spectrometers. Separation by a specific m / z can be designed to select ions in one section of the instrument, dissociate them in an intermediate region, and then send the resulting ions to another analyzer for m / z separation and data acquisition. In a temporal tandem, mass spectrometer ions generated by an ion source can be confined, isolated, fragmented, and separated at m / z within the same physical apparatus. Peptides identified by mass spectrometry can be used as representative substitutes for untreated 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, but is not limited to, amino acid sequencing of protein fragments, determination of protein sequencing, determination of de novo sequencing of proteins, locating or identifying post-translational modifications, or comparative analysis, or a combination thereof.

[0126] As used herein, "Mini-Trap" or "MiniTrap-binding molecule" is capable of binding to a VEGF molecule. Examples of such MiniTraps include (i) a chimeric polypeptide and (ii) a multimer (e.g., dimer) molecule containing two or more polypeptides linked non-covalently, for example, by one or more disulfide crosslinks. MiniTraps can be produced by chemical modification, enzymatic activity, or recombinant manufacturing.

[0127] As used herein, “VEGF MiniTrap” or “VEGF MiniTrap-binding molecule” may be a molecule or complex of molecules that binds to VEGF, and which has one or more sets of VEGF receptor Ig-like domains (or their variants) (e.g., VEGFR1 Ig domain 2 and / or VEGFR2 Ig domains 3 and / 4) and modified multimerized components (MCs), or does not have these modified multimerized components, where the MCs are modified immunoglobulin Fc. This modification may result from proteolytic digestion of a VEGF trap (e.g., aflibercept or convercept) or from the direct expression of the resulting polypeptide chain having a shortened MC sequence. (See the molecular structure shown in Figure 1.) Figure 1 shows the VEGF MiniTrap molecule, which is the product of proteolytic digestion of aflibercept in Streptococcus pyogenes IdeS. The homodimeric molecule is shown having Ig hinge domain fragments linked by two parallel disulfide bonds. The VEGFR1 domain, VEGFR2 domain, and hinge domain fragment (MC) are shown. Points in aflibercept where IdeS cleavage occurs are indicated by " / / ". The Fc fragment cleaved from aflibercept is also shown. Such single chimeric polypeptides that are not dimerized may also be VEGF MiniTraps if they have VEGF-binding activity. The term "VEGF MiniTrap" comprises a single polypeptide containing a first set of one or more VEGF receptor Ig domains (or variants thereof), lacking an MC, but fused with a linker (e.g., a peptide linker) to one or more further sets of one or more VEGF receptor Ig domains (or variants thereof). The VEGF-binding domains in the VEGF MiniTrap of the present invention may be identical or different from others (see International Publication 2005 / 00895, the entire teaching thereof is incorporated herein).

[0128] For example, in one embodiment of the present invention, the unmodified immunoglobulin Fc domain includes an amino acid sequence or amino acids 1 to 226 thereof: TIFF2026094272000007.tif26165 (Sequence ID 33, where X1 is L or P, and X2 is A or T).

[0129] Examples of VEGF inhibition include VEGF (for example, VEGF 110 VEGF 121 , and / or VEGF 165 One example is the antagonistic effect of VEGF binding to the VEGF receptor, due to competition with the VEGF receptor for binding. Such inhibition can inhibit the activation of VEGFR by VEGF, for example, in cell lines (e.g., HEK293) that express a chimeric VEGF receptor (e.g., a homodimer thereof) having an extracellular domain of VEGFR that fuses to the intracellular domains of IL18Rα and / or IL18Rβ on the cell surface and an NFκB-luciferase-IRES-eGFP reporter gene, such as the cell line HEK293 / D9 / Flt-IL18Rα / Flt-IL18Rβ as described herein, thereby inhibiting luciferase expression.

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

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

[0132] The present invention provides a VEGF MiniTrap polypeptide 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 ; wherein, - R1D2 is VEGF receptor 1 (VEGFR1) Ig domain 2 (D2); - R2D3 is VEGFR2 Ig domain 3; - R2D4 is VEGFR2 Ig domain 4; - MC is a multimerization component (e.g., derived from IgG1, e.g., an IgG hinge domain or a fragment thereof); - The linker is a peptide containing approximately 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acids, for example (GGGS) g and; and, Independent, 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 present invention, R1D2 has the amino acid sequence: Includes TIFF2026094272000008.tif11165 (SEQ ID NO: 34). In one embodiment, R1D2 lacks the N-terminal SDT.

[0134] In one embodiment of the present invention, R1D2 has the amino acid sequence: Includes TIFF2026094272000009.tif11165 (sequence number 35).

[0135] In one embodiment of the present invention, R2D3 has the amino acid sequence: Includes TIFF2026094272000010.tif11164 (sequence number 36).

[0136] In one embodiment of the present invention, R2D4 has the amino acid sequence: Includes TIFF2026094272000011.tif12164 (sequence number 37).

[0137] In one embodiment of the present invention, R2D4 has the amino acid sequence: Includes TIFF2026094272000012.tif11164 (sequence number 38).

[0138] In one embodiment, the polymerizing component (MC) for use in VEGF MiniTrap is a peptide, for example, a modified Fc immunoglobulin (e.g., derived from IgG1) that can bind to another polymerizing component. In one embodiment, the MC is a modified Fc immunoglobulin comprising an immunoglobulin hinge region. For example, in one embodiment, the MC is a peptide comprising one or more cysteines (e.g., 1, 2, 3, 4, 5, or 6) that can form one or more cysteine ​​crosslinks having cysteine ​​in another MC, for example, DKTHTCPPC (SEQ ID NO: 39), DKTHTCPPCPPC (SEQ ID NO: 40), DKTHTCPPCPPCPPC (SEQ ID NO: 41), DKTHTC(PPC) h (wherein h is 1, 2, 3, 4, or 5), DKTHTCPPCPAPELLG (sequence number 60), DKTHTCPLCPAPELLG (sequence number 43), DKTHTC (sequence number 44), or DKTHTCPLCPAP (sequence number 45).

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

[0140] In one embodiment of the present invention, the VEGF MiniTrap polypeptide has the amino acid sequence: This includes TIFF2026094272000013.tif165166 and TIFF2026094272000014.tif55165. As stated, such polypeptides can be multimerized (e.g., dimerization (e.g., homodimerization)), in which case the binding between polypeptides is mediated via the multimerizing component.

[0141] In one embodiment of the present invention, the VEGFR1 Ig-like domain 2 of the monomer VEGF MiniTrap of the present invention has N-linked glycosylation at N36 and / or N68; and / or an intrachain disulfide bridge between C30 and C79; and / or the VEGFR2 Ig-like domain 3 of the monomer VEGF MiniTrap of the present invention has N-linked glycosylation at N123 and / or N196; and / or an intrachain disulfide bridge between C124 and C185.

[0142] In one embodiment of the present invention, the VEGF MiniTrap has the following structure: ·(R1D2)1-(R2D3)1-(G4S)3-(R1D2)1-(R2D3)1; ·(R1D2)1-(R2D3)1-(G4S)6-(R1D2)1-(R2D3)1; ·(R1D2)1-(R2D3)1-(G4S)9-(R1D2)1-(R2D3)1; or ·(R1D2)1-(R2D3)1-(G4S) 12 -(R1D2)1-(R2D3)1 It includes. G4S is -Gly-Gly-Gly-Gly-Ser-.

[0143] In one embodiment of the present invention, VEGF MiniTrap has the amino acid sequence: The following are included: TIFF2026094272000015.tif25165TIFF2026094272000016.tif213166TIFF2026094272000017.tif52165 (in this case, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). As described herein, these polypeptides may include a secondary structure in which similar VEGFR Ig domains bind to form an intrachain VEGF-binding domain (e.g., Figure 2). In one embodiment of the present invention, two or more such polypeptides are polymerized (e.g., dimerized (e.g., homodimerized)), in which the VEGFR Ig domain of each chain binds to a similar Ig domain of another chain to form an intrachain VEGF-binding domain.

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

[0145] In one embodiment of the present invention, the polypeptide comprises a secondary structure, in this case a single chimeric polypeptide (e.g., (R1D2) a -(R2D3) b -Linker-(R1D2) c -(R2D3) d ; or (R1D2) a -(R2D3) b -(R2D4) c -Linker-(R1D2) d -(R2D3) e -(R2D4) f ) or similar VEGFR Ig domains in a separate chimeric polypeptide (e.g., a homodimer) align to form a VEGF-binding domain. For example in this case, (i) The R1D2 domains in question are aligned, (ii) The R2D3 domains are aligned and / or (iii) The R2D4 domains are aligned, It forms a VEGF-binding domain. Figure 2 shows a single-stranded VEGF MiniTrap exhibiting such a domain configuration. VEGFR1, VEGFR2, and a linker domain are shown. The linker shown is (G4S)6. The present invention relates to (G4S)3; (G4S)9; or (G4S) 12 Contains a single-stranded VEGF MiniTrap with a linker.

[0146] In addition, the present invention also provides a complex comprising a VEGF MiniTrap as described herein, which forms a complex with a VEGF polypeptide or a fragment or fusion thereof. In one embodiment of the present 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. Examples of complexes include homodimerized VEGF molecules bound to the homodimerized VEGF MiniTrap polypeptide. In one embodiment of the present invention, the complex is in vitro (e.g., immobilized on a solid substrate) or present in the body of the subject. The present invention also relates to VEGF dimers (e.g., VEGF) complexed with VEGF MiniTrap. 165 This also includes a composite composition of ).

[0147] As used herein, the terms “protein” or “protein of interest” may include any amino acid polymer having covalently linked amide bonds. Examples of proteins of interest include, but are not limited to, aflibercept and MiniTrap. A protein comprises one or more amino acid polymer chains, which are generally known in the art as “polypeptides.” “Polypeptides” refers to polymers composed of amino acid residues, related naturally occurring structural variants, and their synthetic analogs that do not exist in nature and are linked via peptide bonds, related naturally occurring structural variants, and their synthetic analogs that do not exist in nature. “Synthetic peptides or polypeptides” refers to peptides or polypeptides that do not exist in nature. Synthetic peptides or polypeptides may be synthesized, for example, using automated polypeptide synthesizers. Various 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 embodiment, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and their equivalents. Proteins of interest may include any of the following: biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In certain embodiments, the protein of interest is an anti-VEGF fusion protein (e.g., aflibercept or MiniTrap). Proteins may be produced using recombinant cell-based production systems such as insect baculovirus lines, yeast lines (e.g., Pichia sp.), or mammalian lines (e.g., CHO cells and CHO-K1 cells and other CHO derivatives).For recent reviews on biotherapeutic proteins and their production, see Ghaderi et al., “Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation” (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012), the entire teaching is incorporated herein). In some exemplary embodiments, the protein includes modifiers, adducts, and other covalently bound moieties. Examples of such modifications, adducts, and parts include avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose-binding proteins (MBPs), chitin-binding proteins (CBPs), glutathione-S-transferase (GST) myc-epitopes, fluorescent labels, and other dyes, and their equivalents. Proteins can be classified based on their composition and solubility, and therefore, proteins include simple proteins such as globular and fibrous proteins, complex proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins, and inducer proteins such as primary and secondary inducer proteins.

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

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

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

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

[0152] The term "bispecific antibody" refers to an antibody capable of selectively binding to two or more epitopes. A bispecific antibody generally comprises two distinct heavy chains, each having a heavy chain that specifically binds to a different epitope on either two different molecules (e.g., multiple antigens) or the same molecule (e.g., the same antigen). If 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 of the first epitope is generally at least one to two orders of magnitude, one to three orders of magnitude, or one to four orders of magnitude lower than the affinity of the first heavy chain of the second epitope, and vice versa. The epitopes recognized by a bispecific antibody may be on the same target or different targets (e.g., the same protein or different proteins). A bispecific antibody can be constructed, for example, by combining heavy chains that recognize different epitopes on the same antigen. For example, nucleic acid sequences encoding heavy chain variable regions that recognize different epitopes of the same antigen can be fused with nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in cells that express immunoglobulin light chains.

[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, and an immunoglobulin light chain that can bind to each heavy chain but none of these domains provide antigen-binding specificity; or an immunoglobulin light chain that can bind to each heavy chain and to one or more epitopes linked by the heavy chain antigen-binding domains; or an immunoglobulin light chain that can bind to each heavy chain and to one or both epitopes to which one or both heavy chains can bind. BsAbs can be divided into two main classes: those with an Fc region (IgG-like) and those without an Fc region, the latter of which are usually smaller than IgG-like bispecific molecules containing IgG and Fc. IgG-like bsAbs may have different formats, such as triomab, knob-into-hole IgG (kih IgG), crossumab, orth-Fab IgG, dual variable-domain Ig (DVD-Ig), two-in-one or dual-action Fab (DAF), IgG single-strand Fv (IgG-scFv), or κλ-body. Non-IgG-like alternative formats include tandem scFv, diabody format, single-stranded diabody, tandem diabody (TandAb), dual-affinity retargeting molecule (DART), DART-Fc, nanobody, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Muller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the full instructions are incorporated herein).Methods for producing bsAb are not limited to quadroma technology based on somatic cell fusions of two different hybridoma cell lines, chemical conjugation including chemical crosslinking linkers, or genetic approaches utilizing recombinant DNA technology. Examples of bsAb include: U.S. Patent No. 12 / 823838 filed June 25, 2010; U.S. Patent No. 13 / 488628 filed June 5, 2012; U.S. Patent No. 14 / 031075 filed September 19, 2013; U.S. Patent No. 14 / 808171 filed July 24, 2015; U.S. Patent No. 15 / 713574 filed September 22, 2017; Examples of disclosures are made in patent applications such as U.S. Patent No. 15 / 713569, U.S. Patent No. 15 / 386453 filed on December 21, 2016, U.S. Patent No. 15 / 386443 filed on December 21, 2016, U.S. Patent No. 15 / 22343 filed on July 29, 2016, and U.S. Patent No. 15814095 filed on November 15, 2017, which are incorporated herein by reference. Low levels of homodimeric impurities may be present in multiple steps during the production of bispecific antibodies. Because these impurities co-elute with the major species when present in low amounts and performed using conventional liquid chromatography, detection of such homodimeric impurities can be difficult when performed using intact mass spectrometry.

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

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

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

[0157] In some exemplary embodiments, the protein of interest may be produced from mammalian cells. Mammalian cells may be of human origin, or non-human origin, such as primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), established cell lines and their strains (e.g., 293 fetal kidney cells, BHK cells, HeLa cervical epithelial cells, and PER-C6 retinal cells, MDBK(NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit562 cells, HeLa229 cells, HeLaS3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells, NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 cells).VA13, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Yl cells, LLC-PKi cells, PK(15) cells, GHi cells, G H3 cells, L2 cells, LLC-RC256 cells, MHiCi cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells, RK-cells, PK-15 cells or derivatives thereof), any Fibroblasts from the tissues or organs of the heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lungs, vascular tissue (arteries, veins, capillaries), lymphoid tissue (lymph glands, pharyngeal tonsils, tonsils, bone marrow, and blood), spleen, and fibroblasts and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinocytes, Dempsey cells, Detroit551 cells, Detroit510 cells). cells, Detroit525 cells, Detroit529 cells, Detroit532 cells, Detroit539 cells, Detroit548 cells, Detroit573 cells, HEL299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB / 3T3 cells, F This may include, but is not limited to, 9 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, mouse L cell line 2071, mouse L cell line LM, 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 a protein. Non-limiting examples of protein alkylating agents include iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methylmethanethiosulfonate (MMTS), and 4-vinylpyridine or combinations thereof.

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

[0160] As used herein, the term “protein reducing agent” refers to a drug used to reduce disulfide crosslinks in proteins. Non-exclusive examples of protein reducing agents used to reduce proteins include dithiothreitol (DTT), β-mercaptoethanol, Elman’s reagent, hydroxylamine hydrochloride, sodium borohydride cyanohydride, tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl), or combinations thereof.

[0161] As used herein, the term "variant" of a polypeptide (e.g., a variant of the VEGFR Ig domain) refers to a polypeptide containing an amino acid sequence that is identical or similar to at least approximately 70–99.9% (e.g., 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) to the reference amino acid sequence or native amino acid sequence of the protein of interest. Sequence comparison may be performed, for example, by the BLAST algorithm, in which case the parameters of this algorithm are selected to yield the greatest match between each sequence across the full length of each reference sequence (e.g., assuming maximum match with threshold: 10, word size: 3, query width: 0, BLOSUM62 matrix, gap cost: present 11, extension 1, and adjustment of the conditional composition score matrix). A polypeptide variant (e.g., a variant of the VEGFR Ig domain) also refers to a polypeptide containing a reference amino acid sequence with one or more mutations removed, such as missense substitutions (e.g., conservative substitutions), nonsense mutations, deletions, or insertions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). The following references pertain to the BLAST algorithm, which is frequently used in sequence analysis: BLAST ALGORITHMS: Altschul et al. (2005) FEBS J. 272 ​​(20): 5101-5109; Altschul, SF, et al., (1990) J. Mol. Biol. 215: 403-410; Gish, W., et al., (1993) Nature Genet. 3: 266-272; Madden, TL, et al., (1996) Meth. EnzyMol. 266: 131-141; Altschul, SF, et al., (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J., et al., (1997) Genome Res. 7: 649-656; Wootton, JC, et al. al.,(1993)Comput.Chem.17:149-163;Hancock,JMet al.,(1994)Comput.Appl.Biosci.10:67-70; Alignment Scoring Systems: Dayhoff, M.O., et al., 「A model of evolutionary change in proteins.」 In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, D.C.; Schwartz, R.M., et al., 「Matrices for detecting distant relationships」 In Atlas of Protein Sequence and Structure, (1978) vol. 5, suppl. 3. M.O. Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, D.C.; Altschul, S.F., (1991) J. Mol. Biol. 219:555-565; States, D.J., et al., (1991) Methods 3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Altschul, S.F., et al., (1993) J. Mol. Evol. 36:290-300; Alignment Statistics: Karlin, S., et al., (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob. 22:2022-2039; and Altschul, S.F. 「Evaluating the statistical significance of multiple distinct local alignments.」 In Theoretical and Computational Methods in Genome Research (S. Suhai, ed.), (1997) pp. 1-14, Plenum, N.Y.These entire instructions are incorporated herein.

[0162] Some variants may be covalent modifications that polypeptides undergo either during their ribosome synthesis (simultaneous translation modification) or after their ribosome synthesis (post-translational modification "PTM"). PTMs are generally introduced by specific enzymes or enzymatic pathways. Many occur at specific characteristic protein sequences (e.g., signature sequences) within the protein backbone. Hundreds of PTMs have been recorded, and these modifications consistently affect several aspects of the protein's structure or function (Walsh, G. "Proteins" (2014), 2nd edition, published by Wiley and Sons, Ltd., ISBN: 9780470669853; the entire teaching is incorporated herein). In certain exemplary embodiments, a protein composition may contain two or more protein variants of the protein of interest.

[0163] In the case of aflibercept, protein variants (and structural characteristics of aflibercept, e.g., proteins sharing one or more heavy or light chain regions of aflibercept) may include, but are not limited to, oxidative variants resulting from the oxidation of one or more amino acid residues at histidine, cysteine, methionine, tryptophan, phenylalanine, and / or tyrosine residues, and deamidation variants resulting from deamidation at asparagine residues and / or deoxyglucosonated arginine residues.

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

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

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

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

[0168] As used herein, the terms “acidic species,” “AS,” “acidic region,” and “AR” refer to protein variants characterized by an overall acidic charge. For example, in the preparation of recombinant proteins, such acidic species can be detected by various methods, such as ion exchange, including 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 include, but are not limited to, deamidated variants, defucosylated variants, oxidized variants, methylglyoxal (MGO) variants, glycated variants, and citrate variants. Exemplary structural variants include, but are not limited to, glycosylated variants and acetonelated variants. Exemplary fragmentation variants include, but are not limited to, Fc and Fab fragments, Fab-deficient fragments, and fragments deficient in the heavy chain variable domain; any modified protein species from the target molecule by peptide chain dissociation, enzymatic and / or chemical modification; C-terminal cleavage variants; variants with excision of the N-terminal Asp in the light chain; and variants with N-terminal cleavage of the light chain. Other acidic species variants include unpaired disulfides, host cell proteins, and host nucleic acids, chromatography materials, and culture medium components. Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17).

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

[0170] The terms “acidic species” or “AS” do not refer to process-related impurities. As used herein, “process-related impurities” refer to impurities present in a protein-containing composition that are not derived from proteins themselves. Examples of process-related impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatography materials, and culture medium components.

[0171] In one exemplary embodiment, the amount of acidic species in the anti-VEGF composition compared to the protein of interest may be up to about 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and is within one or more of the above ranges. Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition may include an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept.

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

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

[0174] In one exemplary embodiment, the amount of oxidized species in the anti-VEGF composition compared to the protein of interest may be up to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, within one or more of the above ranges. Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition may include an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept or MiniTrap.

[0175] Cysteine ​​residues undergo spontaneous oxidation, which can lead to the formation of either intramolecular or intermolecular disulfide bonds, or single-molecule byproducts such as sulfenic acid.

[0176] Histidine residues are also highly susceptible to oxidation via reactions involving their imidazole rings, which can sequentially 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 teaching is incorporated herein). The proposed mechanism for histidine oxidation is highlighted in Figures 2 and 3. Detailed mechanistic studies are available in Anal. Chem. 2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. 21(2000) 1093-1097, the entire teaching is incorporated herein.

[0177] Oxidation of methionine can lead to the formation of methionine sulfoxides (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 are 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 teaching is incorporated herein).

[0178] Oxidation of tryptophan can result in a complex mixture of products. Primary products may be N-formylkynurenine and kynurenine with monoxide, dioxide, and / or trioxide products (Figure 4). Peptides with oxidative Trp modification are generally kynurenine (KYN) and hydroxytryptophan (W). OX1 ), and N-formyl kynurenine / dihydroxytryptophan (NFK / W OX2 (Also known as "double oxidized Trp"), trihydroxytryptophan (W OX3 (also known as "triply oxidized TRP"), and, for example, hydroxykynurenine (KYN OX1 Corresponding to the formation of these combinations, such as +20Da, it exhibits mass increases of 4Da, 16Da, 32Da, and 48Da. Hydroxytryptophan (W OX1Oxidation of tryptophan (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 is incorporated herein). It was found that tryptophan oxidation, rather than methionine and histidine oxidation, produces color changes in protein products (Characterization of the Degradation Products of a Color-Changed Monoclonal Antibody: Tryptophan-Derived Chromophores.dx.doi.org / 10.1021 / ac404218t|Anal.Chem.2014,86,6850-6857). Similar to tryptophan, tyrosine oxidation initially produces 3,4-dihydroxyphenylalanine (DOPA) and dityrosine (Li,S,C Schoneich, and RT. Borchardt. 1995. Chemical Instability of Protein Pharmaceuticals: Mechanisms of Oxidation and Strategies for Stabilization. Biotechnol. Bioeng. 48:490-500).

[0179] As used herein, the terms “basic species,” “basic region,” and “BR” refer to protein variants, such as their antibody or antigen-binding moieties, characterized by their overall basic charge compared to primary charge variant species present in the protein. For example, in the preparation of recombinant proteins, such basic species can be detected by various methods, such as ion exchange, including WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Exemplary variants include, but are not limited to, lysine variants, aspartic acid isomerization, succinimide formation at asparagine, methionine oxidation, amidation, incomplete disulfide bond formation, serine-to-arginine mutation, glycosylation, fragmentation, and aggregation. Generally, basic species elute later than the main peak during CEX or earlier than the main peak during AEX analysis. (Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, MAbs. 2012 Sep 1;4(5):578-585. doi:10.4161 / mabs.21328; the entire instruction is incorporated herein.)

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

[0181] As described with respect to acidic species, the term “basic species” does not include process-related impurities, and basic species may be 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 may be up to about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, within one or more of the above ranges. Examples of anti-VEGF compositions are described in Section III below. In one embodiment, the anti-VEGF composition may include an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (an example of which is disclosed in U.S. Patent No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred embodiment, the recombinant protein of interest is aflibercept.

[0183] Where used herein, “sample matrix” or “biological sample” can be obtained from any step in a bioprocess, such as cell culture fluid (CCF) or recovered cell culture fluid (HCCF), or any step in a downstream process (including the final formulated product, such as the active pharmaceutical ingredient (DS) or drug product (DP)). In some other specific exemplary embodiments, the biological sample may be selected from any step in a downstream process, such as clarification, chromatographic generation, virus inactivation, or filtration. In some specific exemplary embodiments, the drug product may be selected from a drug product manufactured in a clinic, during transport, storage, or handling.

[0184] As used herein, the term “Subject” means a mammal (e.g., rat, mouse, cat, dog, cattle, sheep, horse, goat, rabbit), preferably a human being in need of prevention and / or treatment of cancer or neovascular eye disease. The Subject may have cancer or neovascular eye disease, or may be predisposed to developing cancer or neovascular eye disease.

[0185] With respect to the formulation of proteins, as used herein, the term “stable” means that the protein of interest in a formulation can retain an acceptable degree of its chemical structure or biological function after being stored under the exemplary conditions defined herein. A formulation may be stable even if, after being stored for a specified period, the protein of interest contained therein does not maintain 100% of its chemical structure or biological function. Under certain circumstances, the retention of about 90%, 95%, 96%, 97%, 98%, or 99% of the protein structure or function after being stored for a specified period may be considered “stable.”

[0186] The term “to treat” or “treatment” refers to a therapeutic action that, for example, improves, stabilizes, or eliminates an undesirable disease or disorder (e.g., neovascular ophthalmopathy or cancer) to any clinically measurable degree by causing regression, stabilization, or elimination of one or more symptoms or signs of such disease or disorder (for example, in the case of neovascular ophthalmopathy, by causing a reduction or maintenance of the Diabetic Retinopathy Severity Score (DRSS), by improving or maintaining vision (e.g., with respect to best-corrected visual acuity, such as as measured by an increase in letters on the ETDRS visual acuity test), by increasing or maintaining the visual field, and / or decreasing or maintaining the thickness of the central retina, and in the case of cancer, by stopping or regressing the growth, survival time, and / or metastasis of cancer cells in the subject). Typically, therapeutic measurement is the administration of a therapeutically effective dose of VEGF MiniTrap to a subject with the disease or disorder in one or more doses.

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

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

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

[0190] Examples of VEGF receptor-based chimeric molecules include chimeric polypeptides containing two or more immunoglobulin (Ig)-like domains of VEGF receptors, such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), which may also contain a multimerizing domain (e.g., an Fc domain that promotes the multimerization [e.g., dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is a molecule called VEGFR1R2-FcΔC1(a) (also known as aflibercept and marketed under the trade name EYLEA®). In certain exemplary embodiments, aflibercept is It contains the amino acid sequence described as TIFF2026094272000018.tif43166 (SEQ ID NO: 55).

[0191] As used herein, "viral filtration" may include, but is not limited to, filtration using suitable filters such as Planova 20N®, Planova 50N®, or BioEx filters from Asahi Kasei Pharma, Viresolve® filters from EMD Millipore, ViroSart CPV filters from Sartorius, or Ultipor DV20 or Ultipor DV50® filters from Pall Corporation. Selecting a suitable filter to obtain the desired filtration performance will be apparent to those skilled in the art.

[0192] II. Color determination As used herein, the color observed during the production of recombinant proteins, particularly anti-VEGF proteins, can be measured by a variety of methods. Non-limiting examples include the iodine color number, Hazen color number, Gardner color number, Robibond color number, Saybolt color number, mineral oil color number, European Pharmacopoeia color number, United States Pharmacopoeia color number, and CIE L. * a * , b *Examples of scales used include (or CIELAB), Crett colorimetric, Hess-Ives colorimetric, yellowness, ADMI colorimetric, and ASBC and EBC brewery colorimetric. Details on these scales can be found in Lange's Application Report No. 3.9e, the entire teaching of which is incorporated herein.

[0193] Visual color matching based on the standards of the European Pharmacopoeia (Ph Eur) (European color standards, European Pharmacopoeia. Chapter 2.2.2. Degree of coloration of liquids. 8) th See ed.EP. This entire instruction is incorporated herein by reference.) The instruction may include the preparation of color reference solutions as described in the European Pharmacopoeia (EP 2.2.2. Degree of Coloration of Liquids 2), namely, the preparation of five reference solutions: three parent solutions of red (cobalt(II) chloride), yellow (iron(III) chloride), and blue (copper(II) sulfate), and 1% hydrochloric acid, with hues of yellow (Y), greenish-yellow (GY), yellowish-brown (BY), brown (B), and red (R). Using these five reference solutions in sequence, a total of 37 reference solutions are prepared (Y1-Y7, GY1-GY7, BY1-BY7, B1-B9, and R1-R7). Each reference solution is clearly defined in the CIE-Lab color space, for example, by lightness, hue, and chroma. Of the seven tan standards (BY standards), BY1 is the darkest standard and BY7 is the lightest dark. Matching of BY color standard samples with a given sample is typically performed under diffused sunlight. The compositions of the European tan standards are described in Table 1 below.

[0194] (Table 1) Composition of the European Yellow-Brown Standard TIFF2026094272000019.tif51166 Yellow-brown standard solution (BY): 10.8 g / L FeCl3.6H2O, 6.0 g / L CoCl2.6H2O, and 2.5 g / L CuSO4.5H2O

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

[0196] In contrast to EP color measurement, United States Pharmacopeia Article 1061, Color-Instrumental Measurement, is defined as CIE L * a * , b * The color is accurately and objectively quantified by referring to the use of (or CIELAB) color measurement. A total of 20 reference solutions (identified sequentially by the letters A-T) are defined by the United States Pharmacopeia. The color of the measured sample is automatically correlated with the color reference solution. This is the color reference solution closest to the sample (i.e., the one with the smallest color difference ΔE relative to the sample's color). * This means that a reference solution containing ΔL is displayed. * Value, Δa * Value and Δb * The value is L of the sample. * value, a * value, b * The value and the displayed L of the United States Pharmacopeia solution * value, a * value, b * Shows the quantitative difference from the value. CIE L * a * , b * In the coordinate system, L * The brightness of a color is represented on a scale from 0 to 100 (where 0 is the darkest and 100 is the brightest), a * (a) represents the colors red and green. * A positive value of represents red, but on the other hand, a *(Negative values ​​of represent green), b * (b) represents the yellow or blue color of the sample. * A positive value of b represents yellow, but b * (Negative values ​​represent blue.) The color difference from the standard or the first sample used for evaluation is ΔL for each color component. * Δa * and Δb * It can be represented by a change in . A compound change, or color difference, is expressed by the formula: It can be calculated as a simple Euclidean distance in space using TIFF2026094272000020.tif5128. CIE L * a * , b * Color coordinates can be generated, for example, using a Hunter Labs UltraScanPro (Hunter Associates Laboratory, Reston, Virginia) or on a BYK-Gardner LCS IV (BYK-Gardner, Columbia, Maryland). For the Hunter Labs UltraScanPro, a Didymium Filter Test can be performed for wavelength calibration. This instrument can be standardized before use with a TTRAN equipped with a 0.780-inch port insert and DIW. This establishes the upper (L=100) and lower (L=0) points of the photometric scale using a light trap and blackboard. 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 teaching is incorporated herein. BY standard colors are also CIE L * a * , b * This can occur under a specific color space ("CIELAB" or "CIELab" color space). See Table 2.

[0197] (Table 2) CIE L * a * , b * Characterization of the European standard for yellowish-brown in color space TIFF2026094272000021.tif63170 ^ Reported by Pack et al. ~ Regarding each BY color standard, this specification refers to L * Value and b * The value was measured experimentally.

[0198] To enable high-throughput screening of color assays, spectrophotometric assays (CIELAB) are preferable to BY color standards for quantitative measurement. Alternative assays have been further optimized as described in the Examples section.

[0199] For any sample evaluated for color, the protein concentration of the test sample must be standardized for comparison with the protein concentration in the sample, for example, 5 g / L, 10 g / L, and equivalents.

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

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

[0202] The inventors discovered that producing an anti-VEGF protein (e.g., aflibercept) in a specific CDM resulted in the creation of a biological sample exhibiting a distinct color. This distinct color characteristic was observed during different manufacturing steps and even in the final formulation containing the anti-VEGF protein. As observed in Example 9, for the production of VEGF MiniTrap, culturing cells in CDM produced an anti-VEGF protein (e.g., aflibercept) with a strong yellowish-brown color. The affinity capture step after recovery also produced an eluate exhibiting a specific color, such as yellowish-brown. Further manufacturing steps using AEX also exhibited a yellowish-brown color, although the intensity was reduced.

[0203] As described in detail below, color can be evaluated using (i) the European color standard "BY" for qualitative visual inspection, or (ii) CIELAB, a colorimetric assay that is more quantitative than the BY system. However, in either case, color evaluations between multiple samples were normalized to protein concentration to ensure that the investigation / comparison was meaningful. For example, referring to Example 9, particularly Table 9-2, the eluate of protein A was approximately 2.52 "b *It has a value of ', which corresponds to the approximate BY value of BY5 (when measured at a protein concentration of 5 g / L in the protein A eluate). When comparing the color of the protein A eluate with another sample, the comparison must then be performed using the same protein concentration. Therefore, the protein A eluate and approximately 0.74 b * When compared to an AEX pool with a value (measured at a protein concentration of 5 g / L in the protein A eluate), this production method shows a substantial reduction in the yellowish-brown color of the sample from the protein A eluate compared to the AEX pool after AEX chromatography.

[0204] The compositions of the present invention may be characterized by the yellowish-brown color described herein, for example, having a darkness / intensity equivalent to the European yellowish-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), with a b of 17-23, 10-17, 5-10, 3-5, or 1-3. * The composition has a value and contains approximately 5 g / L of anti-VEGF protein or approximately 10 g / L of anti-VEGF protein, and this composition can be obtained as a sample from the clarified recovered material or as a protein A eluate from the clarified recovered material.

[0205] In one embodiment, the composition of the present invention produced using CDM produces a biological sample having a distinct yellowish-brown color, and this sample is (i) Not yellowish-brown according to the European color standard BY2; (ii) Not yellowish-brown according to the European color standard BY3; (iii) Not yellowish-brown according to the European color standard BY4; (iv) Not yellowish-brown according 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) Between BY4 and BY5 in European color standards It can be characterized by standard color characteristic evaluation recognized by [method name]. This composition contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and this composition is obtained as a sample from the clarified recovered protein A eluate.

[0206] In another embodiment, the composition of the present invention produced using CDM produces a biological sample having different yellowish-brown properties, and this composition is CIELAB scale: (i) Approximately 22-23 b * Not as yellowish-brown as the value suggests; (ii) about 16-17 b * Not as yellowish-brown as the value suggests; (iii) b of 9-10 * Not as yellowish-brown as the value suggests; (iv) 4-5 b * Not as yellowish-brown as the value suggests; (v) b of 2-3 * Not as yellowish-brown as the value suggests; (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 It can be characterized by standard color characteristic evaluation recognized by [method name]. This composition contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and this composition is obtained as a sample from the clarified recovered protein A eluate.

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

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

[0209] Before digestion of the anti-VEGF protein, the sulfhydryl group of cysteine ​​is blocked by a reaction with iodoacetamide (IAM), resulting in the following chemical structure: This produces residues represented by TIFF2026094272000022.tif17128. Such modifications prevent free thiols from reforming disulfide crosslinks and prevent scrambling of disulfide bonds. The present invention comprises compositions (e.g., aqueous compositions) containing anti-VEGF proteins and their variants. When modified with IAM, digested with proteases (e.g., Lys-C and trypsin), and analyzed by mass spectrometry, this is the following peptide: EIGLLTC contains approximately 0.004-0.013% 2-oxo-histidine. * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH contains approximately 0.006-0.028% 2-oxo-histidine. * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH contains approximately 0.049-0.085% 2-oxo-histidine. * QHK (SEQ ID NO: 20), DKTH contains approximately 0.057-0.092% 2-oxohistidine. * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH contains approximately 0.008-0.022% 2-oxo-histidine. * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56), containing approximately 0.185-0.298% tryptophan dioxide. or EIGLLTC contains approximately 0.008% 2-oxo-histidine. * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH contains approximately 0.02% 2-oxo-histidine. * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH contains approximately 0.06% 2-oxo-histidine. * QHK (SEQ ID NO: 20), DKTH contains approximately 0.07% 2-oxohistidine. * TC * PPC * PAPELLG (SEQ ID NO: 17), TNYLTH contains approximately 0.01% 2-oxo-histidine. * R (SEQ ID NO: 21), and / or IIWDSR (SEQ ID NO: 56) contains approximately 0.23% dioxotryptophan. This includes H * It is 2-oxo-histidine, C * This is carboxymethylated cysteine. In one embodiment of the present invention, the peptide is deglycosylated with PNGase F.

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

[0211] The present invention comprises a composition containing an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in this anti-VEGF protein is modified to kynurenine. Furthermore, the color of the composition is equivalent in darkness / intensity to the European yellow-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b * The composition has a value and contains approximately 5 g / L of anti-VEGF protein or approximately 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from the clarified recovery or the protein A eluate of the clarified recovery. Such a composition can be obtained from the clarified recovery when subjected to a capture chromatography procedure. This capture procedure is, for example, an affinity chromatography procedure using a protein A affinity column. When the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.

[0212] The present invention comprises a composition containing an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in this anti-VEGF protein is modified to mono-hydroxytryptophan. Furthermore, the color of the composition is equivalent in darkness / intensity to the European yellow-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b is characterized by *The composition contains approximately 5 g / L of anti-VEGF protein or approximately 10 g / L of anti-VEGF protein. This composition is obtained as a sample derived from the clarified recovery or the protein A eluate of the clarified recovery. Such a composition can be obtained from the clarified recovery when subjected to a capture chromatography procedure. This capture procedure is, for example, an affinity chromatography procedure using a protein A affinity column. When the sample extracted from the affinity procedure is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.

[0213] The present invention comprises a composition containing an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in this anti-VEGF protein is modified to dihydroxytryptophan. Furthermore, this color is equivalent in darkness / intensity to the European yellowish-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b, characterized by using * The composition has a value and contains approximately 5 g / L of anti-VEGF protein or approximately 10 g / L of anti-VEGF protein. The composition is obtained as a sample derived from a clarified recovery or the protein A eluate of a clarified recovery. Such a composition can be obtained from a clarified recovery prepared using a CDM containing anti-VEGF protein and its oxo variants, which is subjected to a capture chromatography procedure. This capture procedure is, for example, an affinity chromatography procedure using a protein A affinity column. When the sample extracted from the affinity procedure is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.

[0214] The present invention comprises a composition containing an anti-VEGF protein, wherein about 0.1% to 10% of all tryptophan in this anti-VEGF protein is modified to tri-hydroxytryptophan. Furthermore, the color of the composition is equivalent in darkness / intensity to the European yellow-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6), and / or CIE L of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b is characterized by * The composition contains approximately 5 g / L of anti-VEGF protein or approximately 10 g / L of anti-VEGF protein. This composition is obtained as a sample derived from a clarified recovery or the protein A eluate of a clarified recovery. Such a composition can be obtained using capture chromatography, for example, an affinity chromatography procedure using a protein A affinity column. When the sample extracted from affinity is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.

[0215] In one embodiment, the composition of the present invention may contain an anti-VEGF protein, in which case the anti-VEGF protein may include modifications of one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acid is 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 arginine is converted to Arg3-deoxyglucosone; a C-terminal glycine is absent; and / or one or more non-glycosylated glycosites are present.

[0216] Such compositions can be obtained, for example, from a clarified recovery prepared using a CDM containing anti-VEGF proteins and their variants subjected to a capture chromatography procedure. This capture procedure is, for example, an affinity chromatography procedure using a protein A column. When the sample extracted from the affinity procedure is analyzed, for example, using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected.

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

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

[0219] In some exemplary embodiments, the compositions of the present invention may contain anti-VEGF proteins and their variants (including oxovariants), the amount of protein variants in the composition may be up to about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, within one or more of the above ranges. Such compositions can be obtained from clarified recoveries prepared using a CDM containing anti-VEGF proteins and their variants subjected to a capture chromatography procedure. The capture procedure is, for example, an affinity chromatography procedure using a Protein A column, and one or more of these variants may be detected when analyzed using liquid chromatography-mass spectrometry (LC-MS). In one embodiment, the color of such compositions is, for example, equivalent in darkness / intensity to the European yellow-brown standards (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or L of the CIE of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b is characterized by * The composition has a value of approximately 5 g / L or approximately 10 g / L of anti-VEGF protein.

[0220] In other exemplary embodiments, the composition of the present invention comprises an anti-VEGF protein and its variants, the amount of protein variants in the composition may be about 0% to about 20%, for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2 %~approx. 20%, approx. 3%~approx. 20%, approx. 4%~approx. 20%, approx. 5%~approx. 20%, approx. 6%~approx. 20%, approx. 7%~approx. 20%, approx. 8%~approx. 20%, approx. 9%~approx. 20%, approx. 10%~approx. 20%, approx. 0%~approx. 10%, approx. 0.05%~approx. 10%, approx. 0.1%~approx. 10%, approx. 0.2%~approx. 10%, approx. 0.3%~approx. 10%, approx. 0.4%~approx. 10%, approx. 0.5%~approx. 10%, approx. 0.6%~approx. 10%, approx. 0.7%~approx. 10%, approx. 0.8%~approx. 10%, approx. 0.9%~approx. 10%, approx. 1%~approx. 10%, approx. 1.5%~approx. 10%, approx. 2%~approx. 1 0%, approximately 3% to 10%, approximately 4% to 10%, approximately 5% to 10%, approximately 6% to 10%, approximately 7% to 10%, approximately 8% to 10%, approximately 9% to 10%, approximately 0% to 7.5%, approximately 0.05% to 7.5%, approximately 0.1% to 7.5%, approximately 0.2% to 7.5%, approximately 0.3% to 7.5%, approximately 0.4% to 7.5%, approximately 0.5% to 7.5%, approximately 0.6% to 7.5%, approximately 0.7% to 7.5%, approximately 0.8% to 7.5%, approximately 0.9% to 7.5%, approximately 1% to 7.5%, approximately 1.5% to 7.5%, approximately 2% to 7 The concentration can be 0.5%, approximately 3% to 7.5%, approximately 4% to 7.5%, approximately 5% to 7.5%, approximately 6% to 7.5%, approximately 7% to 7.5%, approximately 0% to 5%, approximately 0.05% to 5%, approximately 0.1% to 5%, approximately 0.2% to 5%, approximately 0.3% to 5%, approximately 0.4% to 5%, approximately 0.5% to 5%, approximately 0.6% to 5%, approximately 0.7% to 5%, approximately 0.8% to 5%, approximately 0.9% to 5%, approximately 1% to 5%, approximately 1.5% to 5%, approximately 2% to 5%, approximately 3% to 5%, and approximately 4% to 5%, and is within one or more of the above ranges. Such compositions can be obtained by performing capture chromatography on the recovered sample. This capture step is, for example, an affinity chromatography procedure using a protein A column.When a sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants may be detected. In one embodiment, the color of such compositions is, for example, equivalent in darkness / intensity to the European yellow-brown standard (BY2-BY3, BY3-BY4, BY4-BY5, or BY5-BY6) and / or L of the CIE of about 17-23, 10-17, 5-10, 3-5, or 1-3. * a * , b * b is characterized by * The composition has a value of approximately 5 g / L or approximately 10 g / L of anti-VEGF protein.

[0221] In one embodiment, the composition of the present invention may contain an anti-VEGF protein containing the acidic species, the amount of the acidic species in the composition may be about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and is within one or more of the above ranges. As described above, these acidic species can be detected by various methods, such as ion exchange (WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing). Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17). Compositions containing acidic species can be obtained from biological materials, such as recovered material or affinity-generated material using ion exchange chromatography.

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

[0223] In another embodiment, the composition of the present invention comprises an anti-VEGF protein containing its acidic species, the amount of the acidic species in the composition can be about 0% to about 20%, for example, about 0% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% %~approx. 20%, approx. 4%~approx. 20%, approx. 5%~approx. 20%, approx. 6%~approx. 20%, approx. 7%~approx. 20%, approx. 8%~approx. 20%, approx. 9%~approx. 20%, approx. 10%~approx. 20%, approx. 0%~approx. 10%, approx. 0.05%~approx. 10%, approx. 0.1%~approx. 10%, approx. 0.2%~approx. 10%, approx. 0.3%~approx. 10%, approx. 0.4%~approx. 10%, approx. 0.5%~approx. 10%, approx. 0.6%~approx. 10%, approx. 0.7%~approx. 10%, approx. 0.8%~approx. 10%, approx. 0.9%~approx. 10%, approx. 1%~approx. 10%, approx. 1.5%~approx. 10%, approx. 2%~approx. 10%, approx. 3% ~10%, 4%~10%, 5%~10%, 6%~10%, 7%~10%, 8%~10%, 9%~10%, 0%~7.5%, 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%~7.5%, 0.7%~7.5%, 0.8%~7.5%, 0.9%~7.5%, 1%~7.5%, 1.5%~7.5%, 2%~7.5% It is possible that the percentages fall within one or more of the following ranges: approximately 3% to approximately 7.5%, approximately 4% to approximately 7.5%, approximately 5% to approximately 7.5%, approximately 6% to approximately 7.5%, approximately 7% to approximately 7.5%, approximately 0% to approximately 5%, approximately 0.05% to approximately 5%, approximately 0.1% to approximately 5%, approximately 0.2% to approximately 5%, approximately 0.3% to approximately 5%, approximately 0.4% to approximately 5%, approximately 0.5% to approximately 5%, approximately 0.6% to approximately 5%, approximately 0.7% to approximately 5%, approximately 0.8% to approximately 5%, approximately 0.9% to approximately 5%, approximately 1% to approximately 5%, approximately 1.5% to approximately 5%, approximately 2% to approximately 5%, approximately 3% to approximately 5%, and approximately 4% to approximately 5%. As mentioned above, these acidic species can be detected by various methods, such as ion exchange (e.g., WCX - WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing).Typically, acidic species elute earlier than the main peak during CEX analysis, 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 an acidic region, and all peaks eluting after the protein of interest were summed as a basic region. In exemplary embodiments, acidic species may elute as two or more acidic regions, which may be numbered AR1, AR2, AR3, etc., based on the specific retention time of the peaks and the ion exchange column used.

[0225] In one embodiment, the composition may contain an anti-VEGF protein including an acidic species, and AR1 is present in amounts of 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 ranges. In one embodiment, the composition may contain an anti-VEGF protein including its acidic species, where AR1 is present in amounts of approximately 0.0% to 10%, 0.0% to 5%, 0.0% to 4%, 0.0% to 3%, 0.0% to 2%, 3% to 5%, 5% to 8%, or 8% to 10%, or 10% to 15%, within one or more of the above ranges. As described above, these acidic regions can be detected by various methods, such as ion exchange (e.g., WCX - WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing). Generally, acidic species elute earlier than the main peak during CEX or later than the main peak during AEX analysis (see Figures 16 and 17).

[0226] In another embodiment, the composition may contain an anti-VEGF protein including an acidic species, where AR2 is present in amounts of 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 ranges. In one embodiment, the composition may contain an anti-VEGF protein including an acidic species, wherein AR2 is present in an amount of 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 is within one or more of the above ranges.

[0227] In one embodiment, the composition may contain an anti-VEGF protein containing its basic species, the amount of the basic species in the composition may be up to about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and is within one or more of the above ranges.In one aspect, the composition may contain anti-VEGF protein and its basic species, and the amount of basic species in this composition compared to anti-VEGF protein may be 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% to about 20%, about 2%~approx. 20%, approx. 3%~approx. 20%, approx. 4%~approx. 20%, approx. 5%~approx. 20%, approx. 6%~approx. 20%, approx. 7%~approx. 20%, approx. 8%~approx. 20%, approx. 9%~approx. 20%, approx. 10%~approx. 20%, approx. 0%~approx. 10%, approx. 0.05%~approx. 10%, approx. 0.1%~approx. 10%, approx. 0.2%~approx. 10%, approx. 0.3%~approx. 10%, approx. 0.4%~approx. 10%, approx. 0.5%~approx. 10%, approx. 0.6%~approx. 10%, approx. 0.7%~approx. 10%, approx. 0.8%~approx. 10%, approx. 0.9%~approx. 10%, approx. 1%~approx. 10%, approx. 1.5%~approx. 10%, approx. 2%~approx. 10%, approximately 3% to 10%, approximately 4% to 10%, approximately 5% to 10%, approximately 6% to 10%, approximately 7% to 10%, approximately 8% to 10%, approximately 9% to 10%, approximately 0% to 7.5%, approximately 0.05% to 7.5%, approximately 0.1% to 7.5%, approximately 0.2% to 7.5%, approximately 0.3% to 7.5%, approximately 0.4% to 7.5%, approximately 0.5% to 7.5%, approximately 0.6% to 7.5%, approximately 0.7% to 7.5%, approximately 0.8% to 7.5%, approximately 0.9% to 7.5%, approximately 1% to 7.5%, approximately 1.5% to 7.5%, approximately 2% to 7 Possible ranges include 0.5%, approximately 3% to 7.5%, approximately 4% to 7.5%, approximately 5% to 7.5%, approximately 6% to 7.5%, approximately 7% to 7.5%, approximately 0% to 5%, approximately 0.05% to 5%, approximately 0.1% to 5%, approximately 0.2% to 5%, approximately 0.3% to 5%, approximately 0.4% to 5%, approximately 0.5% to 5%, approximately 0.6% to 5%, approximately 0.7% to 5%, approximately 0.8% to 5%, approximately 0.9% to 5%, approximately 1% to 5%, approximately 1.5% to 5%, approximately 2% to 5%, approximately 3% to 5%, and approximately 4% to 5%, and one or more of the above ranges are included.

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

[0229] In one embodiment, the composition may contain an anti-VEGF protein including a basic species, and BR1 is present in amounts of 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. In one embodiment, the composition may contain anti-VEGF protein and its basic species, wherein BR1 is present in an amount of 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 is within one or more of the above ranges.

[0230] In another embodiment, the composition may contain anti-VEGF proteins and their basic species, wherein BR2 is present in amounts of 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. In one embodiment, the composition may contain an anti-VEGF protein and its basic species, wherein BR2 is present in an amount of 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 is within one or more of the above ranges.

[0231] In another embodiment, the composition may contain anti-VEGF proteins and their basic species, wherein BR3 is present in amounts of 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. In one embodiment, the composition may contain an anti-VEGF protein and its basic species, wherein BR3 is present in an amount of 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 is within one or more of the above ranges.

[0232] Photo-induced oxidation of Aflibercept In addition to discovering different color properties or variants of anti-VEGF protein compositions produced using CDM, the inventors also discovered that these compositions can be artificially generated in the laboratory by exposure to light.

[0233] Variants of modified anti-VEGF compositions, including oxidation, can be generated by exposing anti-VEGF proteins to cool white light or ultraviolet light. In one embodiment, the anti-VEGF composition contains an increase of approximately 1.5 to 50 times in one or more modified oligopeptides compared to the sample, and these oligopeptides are DKTH * TC * PPC * PAPELLG (SEQ ID NO: 17), EIGLLTC * EATVNGH * LYK (SEQ ID NO: 18), QTNTIIDVVLSPSH * GIELSVGEK (SEQ ID NO: 19), TELNVGIDFNWEYPSSKH * QHK (SEQ ID NO: 20), TNYLTH *R (Sequence ID 21), SDTGRPFVEMYSEIPEIIH * MTEGR (SEQ ID NO: 22), VH * EKDK (SEQ ID NO: 23), SDTGRPFVEM * YSEIPEIIHMTEGR (Sequence No. 64), SDTGRPFVEMYSEIPEIIHM * TEGR (SEQ ID NO: 65), TQSGSEM * K (Sequence ID 66), SDQGLYTC * A ASSGLM * TK (Sequence ID 67), IIW * DSR / RIIW * DSR / IIW * DSRK (Sequence ID 28), TELNVGIDFNW * EYPSSK (Sequence ID 29), GFIISNATY * K (SEQ ID NO: 69), KF * PLDTLIPDGK(SEQ ID NO: 70)F * LSTLTIDGVTR (Sequence ID 32) A group consisting of H is selected, where H * It is histidine and is oxidized to 2-oxo-histidine, C * It is cysteine ​​and is carboxymethylated, M * It is oxidized methionine, W * It is oxidized tryptophan, Y * is tyrosine oxide, F *This is phenylalanine oxide. In a further embodiment, the anti-VEGF composition may include an increase of about 1.5 to about 10 times in one or more modified oligopeptides by exposing the anti-VEGF composition to cool white light for a certain period of time, for example, about 30 hours. In another embodiment, the anti-VEGF composition may include an increase of about 1.5 to about 10 times in one or more modified oligopeptides by exposing the sample to cool white light for about 75 hours. In yet another embodiment, the anti-VEGF composition may include an increase of about 1.5 to about 20 times in one or more of the previously described oligopeptides by exposing the sample to cool white light for about 100 hours. In yet another embodiment, the anti-VEGF composition may include an increase of about 1.5 to about 20 times in one or more of the previously described oligopeptides by exposing the sample to cool white light for about 150 hours. In yet another embodiment, the anti-VEGF composition may increase by approximately 1.5 to 50 times in one or more oligopeptides by exposing the sample to cool white light for approximately 300 hours (see Example 4 below).

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

[0235] Anti-VEGF protein generated using carbohydrate diversity-CDM The composition of the present invention contains an anti-VEGF protein, and the anti-VEGF protein produced in CDM has a variety of carbohydrate diversity. Different glycosylation profiles of the anti-VEGF protein are within the scope of the present invention.

[0236] In some exemplary embodiments of the present invention, this composition may contain one or more asparagine-glycosylated anti-VEGF proteins, such as: G0-GlcNAc glycosylation, G1-GlcNAc glycosylation, G1S-GlcNAc glycosylation, G0 glycosylation, G1 glycosylation, G1S glycosylation, G2 glycosylation, G2S glycosylation, G2S2 glycosylation, G0F glycosylation, G2F2S glycosylation, G2F2S2 glycosylation, G1F glycosylation, G1 FS glycosylation, G2F glycosylation, G2FS glycosylation, G2FS2 glycosylation, G3FS glycosylation, G3FS3 glycosylation, G0-2GlcNAc glycosylation, Man4 glycosylation, Man4_A1G1 glycosylation, Man4_A1G1S1 glycosylation, Man5 glycosylation, Man5_A1G1 glycosylation, Man5_A1G1S1 glycosylation, Man6 glycosylation, Man6_G0+ phosphate glycosylation, Man6+ phosphate glycosylation, and / or Man7 glycosylation. In one embodiment, the protein of interest may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

[0237] In one embodiment, the composition may have the following glycosylation properties: 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 may contain an anti-VEGF protein, the protein of interest having Man5 glycosylation at approximately 32.4% of asparagine 123 residues and / or approximately 27.1% of asparagine 196 residues. In one embodiment, the protein of interest may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap.

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

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

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

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

[0243] In one embodiment, the anti-VEGF protein may have fucosylated glycans at levels reduced by approximately 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Compared to the levels of fucosylated glycans in anti-VEGF proteins produced using soy hydrolysates, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2 The percentage falls within one or more of the above ranges, such as ~49%, 2~50%, 3~10%, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44%, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25%, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47%, 4~48%, 4~49%, 4~50%, or 1~99%.

[0244] In one embodiment, the anti-VEGF protein may have sialylated glycans at levels reduced by approximately 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Compared to the levels of sialylated glycans in anti-VEGF proteins produced using soy hydrolysates, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2 The percentage falls within one or more of the above ranges, such as ~49%, 2~50%, 3~10%, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44%, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25%, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47%, 4~48%, 4~49%, 4~50%, or 1~99%.

[0245] In another embodiment, the anti-VEGF protein may have galactosyl glycans at levels reduced by approximately 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Compared to the levels of galactosyl glycans in anti-VEGF proteins produced using soy hydrolysates, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, The value falls within one or more of the following ranges: 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, or 1-99%.

[0246] In one embodiment, the anti-VEGF protein may have mannosylated glycans increased by approximately 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. Compared to the levels of mannosylated glycans in anti-VEGF proteins produced using soy hydrolysates, for example, 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2 The percentage falls within one or more of the above ranges, such as ~49%, 2~50%, 3~10%, 3~15%, 3~20%, 3~25%, 3~30%, 3~35%, 3~40%, 3~41%, 3~42%, 3~43%, 3~44%, 3~45%, 3~46%, 3~47%, 3~48%, 3~49%, 3~50%, 4~10%, 4~15%, 4~20%, 4~25%, 4~30%, 4~35%, 4~40%, 4~41%, 4~42%, 4~43%, 4~44%, 4~45%, 4~46%, 4~47%, 4~48%, 4~49%, 4~50%, or 1~99%.

[0247] The compositions described in this section may be produced by a number of upstream and downstream parameters, as described in sections IV and V below.

[0248] IV. Preparation of compositions using upstream process technologies In the case of biopharmaceuticals, the implementation of robust and flexible upstream processes is desirable. Efficient upstream processes can lead to the desired production and scale-up of the protein of interest. The inventors of the present invention, which includes an anti-VEGF protein, can be produced by adjusting the conditions during protein production upstream, such as by changing the components of the culture medium of CDM. Each step of the upstream process can affect the quality, purity, and quantity of the protein produced.

[0249] This disclosure demonstrates the existence of specific variants of aflibercept and / or MiniTrap produced using CDM. These variants include isoforms comprising 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 CDM can produce preparations of anti-VEGF proteins with desired target values ​​of the aflibercept and / or MiniTrap protein variants. As mentioned above, a yellowish-brown color associated with fragments produced using CDM may also exist. (As mentioned above, not all CDMs examined by the inventors showed clear discoloration.)

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

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

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

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

[0254] In some embodiments, compositions produced by using modified CDM can generate preparations of anti-VEGF proteins with desired target BY values. The color of these preparations is as follows: (i) Not yellowish-brown according to the European color standard BY2; (ii) Not yellowish-brown according to the European color standard BY3; (iii) Not yellowish-brown according to the European color standard BY4; (iv) Not yellowish-brown according 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) Between BY4 and BY5 in European color standards Characterized as such, this composition contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and a sample of this composition can be obtained as a sample from the clarified recovered protein A eluate. As can be seen in Example 9 below, Table 9-3, the protein A eluate containing 5 g / L of aflibercept exhibits a yellowish-brown color, which is 1.77 b * It is measured to have a value of 0.50 b. When generated downstream after AEX, such samples have a b value of 0.50. * The value was obtained. This demonstrates the usefulness of AEX in reducing the yellowish-brown color of the sample (Table 9-3).

[0255] Compositions produced using modified CDM can yield an anti-VEGF protein preparation, the color of which is measured on the CIELAB scale: (i) Approximately 22-23 b * Not as yellowish-brown as the value suggests; (ii) about 16-17 b * Not as yellowish-brown as the value suggests; (iii) b of 9-10 * Not as yellowish-brown as the value suggests; (iv) 4-5 b * Not as yellowish-brown as the value suggests; (v) b of 2-3 * Not as yellowish-brown as the value suggests; (vi) b of 17-23 * value; (vii) b of 10-17 * value; (viii) b of 5-10 * value; (ix) 3-5 b* value; or (x) b of 1-3 * value Characterized by recognized standard color characteristics, in this case, the composition contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and this composition is obtained as a sample from the clarified recovered protein A eluate. See Example 9, Table 9-3.

[0256] With respect to components added to cell cultures to form a modified CDM, the term "cumulative amount" refers to the total amount of a particular component added to the bioreactor throughout the cell culture process that forms the CDM, including the amount added at the start of the culture (CDM on day 0) and the amounts of components added sequentially. When calculating the cumulative amount of a component, the amount of the component added to the seed train culture or starter culture before production in the bioreactor (i.e., before CDM on day 0) is also included. The cumulative amount is not affected by the loss of components over time during culture (e.g., by metabolism or chemical degradation). Therefore, for example, if a component is added to two cultures at different times (e.g., all components are added at the beginning in one culture, and components are added over time in another culture), the two cultures may have different absolute levels even if they have the same cumulative amount of the component. The cumulative amount is also not affected by the situ synthesis of components over time during culture (e.g., by metabolism or chemical transformation). Therefore, if, for example, a component is synthesized in situ in one of two cultures during the bioconversion process, two cultures having the same cumulative amount of a given component may have different absolute levels. The cumulative amount can be expressed in units such as grams or moles of the component. The term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the start of a production batch, including additions to the initial volume from any seed culture used in the culture. For example, if a bioreactor contains 2 liters of cell culture medium at the start of a production batch, and 1 gram of component X is added on days 0, 1, 2, and 3, the cumulative concentration from day 3 onwards is 2 g / L (i.e., 4 grams divided by 2 liters). Even if an additional 1 liter of liquid without component X is added to the bioreactor on day 4, the cumulative concentration remains 2 g / L. Even if some liquid is lost from the bioreactor on day 5 (e.g., by evaporation), the cumulative concentration remains 2 g / L. Cumulative concentration can be expressed in units such as grams / liter or moles / liter.

[0257] A. Amino acids: In some embodiments, modified CDM can be obtained by decreasing or increasing the cumulative concentration of amino acids in the CDM. Non-limiting examples of such amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine (or salts thereof). The increase or decrease in the cumulative amount of these amino acids in the modified CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 100% compared to the starting CDM, and is within one or more of the above ranges. Alternatively, the increase or decrease in the cumulative amount of one or more amino acids in the modified CDM can be approximately 5-20%, 10-30%, 30-40%, 30-50%, 40-60%, 60-70%, 70-80%, 80-90%, or 90-100% compared to the unmodified CDM, and is within one or more of the above ranges (see Figures 25-27 and Example 5).

[0258] In some embodiments, modified CDM can be obtained by reducing the cumulative concentration of cysteine ​​in the CDM. To form modified CDM, the reduction in the amount of cysteine ​​in the CDM can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to unmodified CDM, and is within one or more of the above ranges. Alternatively, the reduction in the cumulative amount of cysteine ​​in the modified CDM can be approximately 5-20%, 10-30%, 30-40%, 30-50%, 40-60%, 60-70%, 70-80%, 80-90%, or 90-100% compared to CDM, and is within one or more of the above ranges. In one embodiment, the amount of cumulative cysteine ​​in the modified CDM is less than approximately 1 mM, less than approximately 2 mM, less than approximately 3 mM, less than approximately 4 mM, less than approximately 5 mM, less than approximately 6 mM, less than approximately 7 mM, less than approximately 8 mM, less than approximately 9 mM, or less than approximately 10 mM (see Figures 25-27 and Example 5).

[0259] In some embodiments, modified CDM can be obtained by substituting at least a certain percentage of the cumulative cysteine ​​in the CDM with cystine. The substitution can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to unmodified CDM, and is within one or more of the above ranges. Alternatively, the substitution can be approximately 5% to 20%, 10% to 30%, 30% to 40%, 30% to 50%, 40% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 100% compared to the unmodified CDM, and is within one or more of the above ranges (see Figures 25-27 and Example 5).

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

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

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

[0263] In some embodiments, the modified CDM contains about 0.01 mM to about 20 mM of taurine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and one or more of the above ranges.

[0264] In some embodiments, the modified CDM contains about 0.01 mM to about 20 mM of hypotaurine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and one or more of the above ranges.

[0265] In some embodiments, the modified CDM contains about 0.01 mM to about 20 mM glycine, i.e., 0.01 mM to about 1 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 10 mM, 0.1 mM to about 1 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 10 mM, and one or more of the above ranges.

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

[0267] In some embodiments, the modified CDM contains 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 one or more of the above ranges.

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

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

[0270] D. Modifications to the culture medium to adjust glycosylation: This disclosure also includes a method for modulating the glycosylation of anti-VEGF proteins by changing the cumulative concentration of certain components in the CDM. Based on the cumulative amount of components added to the CDM, the total percentage of fucosylation, total percentage of galactosylation, total percentage of sialylation, and mannose-5 can be changed.

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

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

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

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

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

[0276] V. Preparation of compositions using downstream process technology The compositions containing the anti-VEGF protein of the present invention can be produced by adjusting the conditions during downstream protein production. The inventors have found that optimizing the downstream procedure minimizes specific variants of the anti-VEGF protein, and these variants cause discoloration. Optimization of the downstream process can produce compositions with reduced oxovariants and optimized color properties.

[0277] Downstream process technologies may be used alone or in combination with upstream process technologies as described in Section IV above.

[0278] A. Anion exchange chromatography: In some embodiments, the compositions of the present invention may be involved in a process comprising expressing an anti-VEGF protein in host cells in CDM, in which case the anti-VEGF protein is secreted from the host cells into the culture medium to obtain a clarified recovery. This recovery is then subjected to the following steps: (a) loading the biological sample obtained from the recovery onto anion exchange chromatography (AEX); (b) washing the AEX column with a suitable washing buffer; (c) collecting (one or more) flow-through fractions; optionally (d) washing the column with a suitable strip buffer; and (e) collecting the stripped fractions.

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

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

[0281] Protein variants may include modifications of one or more residues, such as: one or more asparagines being deamidated; one or more aspartic acid being converted to isoaspartate and / or Asn; one or more methionines being oxidized; one or more tryptophans being converted to N-formylkynurenine; one or more tryptophans being mono-hydroxytryptophan; one or more tryptophans being di-hydroxytryptophan; one or more tryptophans being tri-hydroxytryptophan; one or more arginine being converted to Arg3-deoxyglucosone; the absence of a C-terminal glycine; and / or the presence of one or more non-glycosylated glycosites.

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

[0283] The flow-through fraction may contain one or more of the following: (a) The proportion of histidine residues oxidized to 2-oxo-histidine, whose color properties are as follows: (i) Not yellowish-brown according to the European color standard BY2; (ii) Not yellowish-brown according to the European color standard BY3; (iii) Not yellowish-brown according to the European color standard BY4; (iv) Not yellowish-brown according 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) Between BY4 and BY5 in European color standards This composition contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and this composition is obtained as a sample from the flow-through fraction. (b) The percentage of histidine residues oxidized to 2-oxo-histidine. Furthermore, these colors are characterized by having a yellowish-brown color that is close to the colors of BY2, BY3, BY4, BY5, BY6, and BY7, or not darker / stronger than BY2, not darker than BY3, not darker than BY4, not darker than BY5, not darker than BY6, not darker than BY7, or between BY2 and BY3, between BY2 and BY4, between BY3 and BY4, or between BY3 and BY5. (c) The percentage of histidine residues oxidized to 2-oxo-histidine, and their colors are as follows: (i) Approximately 22-23 b * Not as yellowish-brown as the value suggests; (ii) about 16-17 b * Not as yellowish-brown as the value suggests; (iii) b of 9-10 * Not as yellowish-brown as the value suggests; (iv) 4-5 b * Not as yellowish-brown as the value suggests; (v) b of 2-3 * Not as yellowish-brown as the value suggests; (vi) b of 17-23 * value; (vii) b of 10-17 * value; (viii) b of 5-10 * value; (ix) 3-5 b * value; or (x) b of 1-3 * value Like CIE's L * a * , b * Characterized by its color in color space, the composition in this case contains approximately 5 g / L or approximately 10 g / L of anti-VEGF protein, and the composition is obtained as a sample from the flow-through fraction. (d) In the composition, histidine residues in amounts of approximately 1% or less, approximately 0.1% or less, or approximately 0.1-1%, approximately 0.2-1%, approximately 0.3-1%, approximately 0.4-1%, approximately 0.5-1%, approximately 0.6-1%, approximately 0.7-1%, approximately 0.8-1%, or approximately 0.9-1% are oxidized to 2-oxo-histidine. The calculation of the percentages is described in Section II.

[0284] B. Affinity Chromatography: In some embodiments, the compositions of the present invention may be produced using a process comprising expressing an anti-VEGF protein in host cells, in which case the anti-VEGF protein is secreted from the host cells into a culture medium to obtain a clarified recovery. This recovery is subjected to the following steps: (a) loading a biological sample obtained from the clarified recovery onto an affinity chromatography column, wherein the affinity chromatography comprises a protein capable of selectively or specifically binding to the anti-VEGF protein; (b) washing the affinity chromatography column with a suitable elution buffer; and (c) collecting (one or more) elution fractions. For example, as illustrated in Tables 7-1 and 7-7 to 7-10, VEGF may be used as a protein capable of selectively or specifically binding to the anti-VEGF protein. 165By using and collecting the eluted fraction according to the above method, successful generation of MT5 (anti-VEGF protein), aflibercept, and anti-VEGF scFv fragments was achieved. Table 7-1 also discloses successful generation of MT5 using different proteins that can selectively or specifically bind to MT5: (i) mAb1 (sequence number 73 is the heavy chain and sequence number 74 is the light chain, mouse anti-VEGFR1 mAb human IgG1), (ii) mAb2 (sequence number 75 is the heavy chain and sequence number 76 is the light chain, mouse anti-VEGFR1 mAb human IgG1), (iii) mAb3 (sequence number 77 is the heavy chain and sequence number 78 is the light chain, mouse anti-VEGFR1 mAb mouse IgG1), and (iv) mAb4 (sequence number 79 is the heavy chain and sequence number 80 is the light chain, mouse anti-VEGFR1 mAb mouse IgG1).

[0285] With respect to step (a) above, the biological sample loaded onto the affinity column may be a sample from which a clarified recovery can be produced before affinity chromatography, including but not limited to ion exchange chromatography (either anionic or cation). Other chromatographic procedures known to those skilled in the art can also be used before the affinity step. Importantly, biological samples containing anti-VEGF proteins can be subjected to affinity chromatography.

[0286] In some embodiments, the compositions of the present invention may be produced using a process comprising expressing the VEGF MiniTrap protein in host cells, in which case the VEGF MiniTrap is secreted from the host cells into a culture medium, which can be further processed to form a clarified recovery. This recovery can be further processed by known chromatographic procedures for producing a biological sample containing VEGF MiniTrap. This biological sample can be further processed using a step comprising: (a) loading the biological sample onto an affinity chromatography column, wherein the affinity chromatography includes a protein that can selectively or specifically bind to or interact with the VEGF MiniTrap protein; (b) washing the affinity chromatography column with a suitable elution buffer; and (c) collecting one or more elution fractions. Referring again to Table 7-1, different proteins that can selectively or specifically bind to or interact with MT5 include (i) VEGF 165 The successful production of MT5 (VEGF MiniTrap) using (ii) mAb1 (sequence number 73 is the heavy chain and sequence number 74 is the light chain, mouse anti-VEGFR1 mAb human IgG1), (iii) mAb2 (sequence number 75 is the heavy chain and sequence number 76 is the light chain, mouse anti-VEGFR1 mAb human IgG1), (iv) mAb3 (sequence number 77 is the heavy chain and sequence number 78 is the light chain, mouse anti-VEGFR1 mAb mouse IgG1), and (v) mAb4 (sequence number 79 is the heavy chain and sequence number 80 is the light chain, mouse anti-VEGFR1 mAb mouse IgG1) is disclosed in this table.

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

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

[0289] In some exemplary embodiments, proteins that can selectively or specifically bind to ("binding agents") or interact with anti-VEGF proteins, such as aflibercept or MiniTrap, may originate from humans or mice.

[0290] The affinity generation process may further include equilibrating the affinity column using an equilibration buffer before loading the biological sample. Exemplary equilibration buffers may include 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), and DPBS (pH 7.4).

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

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

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

[0294] In some embodiments, the eluted fraction can be neutralized using a neutralizing buffer. An example of such a neutralizing buffer is tris-tris-HCl (pH approximately 7.0 to 9.0).

[0295] C.IdeS mutant IdeS proteases, used to cleave Fc fusion proteins such as aflibercept, rapidly lose enzymatic activity under basic pH conditions, which may limit their use during the production of VEGF MiniTrap. Therefore, variants have been developed to be more stable at basic pH in the presence of a strong base, such as NaOH. Such basic conditions may be 1 hour in 0.05 N NaOH or 0.5 hours in 0.1 N NaOH.

[0296] In some embodiments, the IdeS variant may have an amino acid sequence that has at least about 70% sequence identity over its entire length to the amino acid sequences described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%, or about 100% sequence identity over its entire length to the amino acid sequences directly mentioned above.

[0297] In some embodiments, the IdeS variant may have an isolated nucleic acid molecule encoding a polypeptide having an amino acid sequence that has at least 70% sequence identity over its entire length to an amino acid sequence such as those described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%, or about 100% sequence identity over its entire length to the amino acid sequences directly mentioned above.

[0298] In some embodiments, the polypeptide can be expressed by a host cell having a suitable vector containing a nucleic acid encoding the identified peptide, having an amino acid sequence that has at least 70% sequence identity over its entire length to the amino acid sequences described in the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, and 16. In one embodiment, the nucleic acid molecule is operably ligated to an expression control sequence that can direct its expression within the host cell. In one embodiment, the vector is a plasmid. In some embodiments, the amino acid sequence has about 75%, 80%, 85%, 90%, 95%, or about 100% sequence identity over its entire length to the amino acid sequences directly mentioned above. In some embodiments, the isolated nucleic acid molecule can be used to encode the polypeptide.

[0299] In some embodiments, the IdeS mutant may have an amino acid sequence comprising the parent amino acid sequence defined by Sequence ID No. 1 (IdeS), having asparagine residues at positions 87, 130, 182, and / or 274 mutated to amino acids other than asparagine. In one embodiment, the mutation may result in increased chemical stability at alkaline pH values ​​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 ​​compared to the parent amino acid sequence. In one embodiment, the amino acids may be selected from aspartic acid, leucine, and arginine. In a particular embodiment, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another particular embodiment, the asparagine residue at position 130 is mutated to an arginine residue. In yet another particular embodiment, the asparagine residue at position 182 is mutated to a leucine residue. In yet another particular embodiment, the asparagine residue at position 274 is mutated to an aspartic acid residue. In yet another particular embodiment, the asparagine residues at positions 87 and 130 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 130 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 182 and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 130, and 182 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 182, and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 130, 182, and 274 are mutated. In yet another specific embodiment, the asparagine residues at positions 87, 130, 182, and 274 are mutated. In some embodiments, the amino acid sequence has approximately 75%, 80%, 85%, 90%, 95%, or approximately 100% sequence identity over its entire length with respect to the amino acid sequence described above. In some embodiments, the isolated nucleic acid molecule can be used to encode a polypeptide.

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

[0301] VI. General Protein Production A variety of different production techniques, including but not limited to affinity chromatography, ion exchange chromatography, mixed-mode chromatography, size exclusion chromatography, and hydrophobic interaction chromatography, used alone or in combination, are assumed to be within the scope of the present invention. These chromatographic steps separate a mixture of proteins, which are a biological sample, based on their charge, hydrophobicity, size, or a combination thereof, depending on the particular form of separation. Multiple different chromatographic resins are available for each of the techniques suggested above, thereby allowing for precise production schemes for the specific proteins involved. Each separation method achieves physical separation or selective adhesion to the separation medium, increasing with each further passage through the column, as the proteins pass through the column at different rates. The proteins are then collected from a flow-through fraction obtained by (i) differentially eluting using a suitable elution buffer and / or (ii) washing the column with a suitable equilibration buffer, optionally from the column used. In some cases, impurities preferentially adhere to the column, while the protein of interest does not adsorb as much; that is, the protein of interest does not adsorb to the solid phase of a particular column and therefore passes through the column. In such cases, the protein of interest is separated from the impurities (such as HCPs and protein variants). In other cases, if impurities cannot adsorb to the column and therefore pass through the column, these impurities are separated from the protein of interest.

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

[0303] A. Initial recovery and virus inactivation In certain embodiments, the initial steps of the production methods disclosed herein involve the clarification and primary recovery of the protein of interest from a biological sample. Primary recovery includes one or more centrifugation steps to separate the protein of interest from host cells and associated cell fragments. Centrifugation of the sample may be carried out at, for example, 7,000 xg to approximately 12,750 xg. In terms of large-scale production, such centrifugation may be carried out in a production line at a flow rate set to achieve, for example, a turbidity level of 150 NTU in the resulting supernatant. Such supernatant may then be collected for further processing or filtered into the line through one or more depth filters for further clarification of the sample.

[0304] In certain embodiments, primary recovery may involve the use of one or more deep filtration steps to clarify the sample, thereby assisting in the processing of the protein of interest. In other embodiments, primary recovery may include the use of one or more deep filtration steps after centrifugation. Non-limiting examples of depth filters that may be used in view of the present invention include Millistak+X0HC, F0HC, D0HC, A1HC, B1HC depth filters (EMD Millipore), 3M® Model 30 / 60ZA, 60 / 90 ZA, VR05, VR07, and Delipid depth filters (3M Corp.). 0.2 μm filters such as Sartorius' 0.45 / 0.2 μm Sartopore® bilayer or Millipore's Express SHR or SHC filter cartridges typically follow the depth filter. Other filters known to those skilled in the art may also be used.

[0305] In certain embodiments, the primary recovery process may also be a point for reducing or inactivating viruses that may be present in the biological sample. One or more of the various methods of virus reduction / inactivation may be used during the primary recovery step of the production, including thermal inactivation (pasteurization), pH inactivation, buffer / surfactant treatment, ultraviolet and gamma irradiation, and the addition of certain chemical inactivators such as β-propiolactone or copper phenanthroline, as described in U.S. Patent No. 4,534,972 (the entire teaching of which is incorporated herein by reference). In certain exemplary embodiments of the present invention, the sample is exposed to virus inactivation with a surfactant during the primary recovery step. In other embodiments, the sample may be exposed to inactivation by low pH during the primary recovery step.

[0306] In embodiments where viral reduction / inactivation is used, the biological sample may be adjusted for further production steps as needed. For example, after low-pH viral inactivation, the pH of the sample is typically adjusted to a more neutral pH, such as about 4.5 to about 8.5, before continuing the production process. In addition, 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 the generation of a protein of interest. The chromatographic material is selectively or specifically capable of binding to or interacting with the protein of interest. Non-limiting examples of such chromatographic materials include protein A and protein G. Also, chromatographic materials may include proteins or moieties thereof that are capable of binding to or interacting with the protein of interest, for example. In one embodiment, the protein of interest is an anti-VEGF protein such as aflibercept, MiniTrap, or a protein related thereto.

[0308] Affinity chromatography involves subjecting a biological sample to a column containing a suitable protein A resin. As used herein, "protein A" means protein A recovered from its natural source, protein A produced by synthesis (e.g., by peptide synthesis or recombinant technology), and C H 2 / C H It encompasses variants that retain the ability to bind to proteins having three regions. In certain embodiments, the protein A resin is useful for affinity base production and isolation of various antibody isotypes by specifically interacting with the Fc portion of molecules that would have the regions.

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

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

[0311] In one embodiment, the eluate may be subjected to virus inactivation with, for example, a surfactant or a low pH. A suitable surfactant concentration or a suitable pH (and time) may be selected to obtain the desired virus inactivation result. After virus inactivation, the eluate is usually adjusted in pH and / or conductivity 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 before an additional chromatographic polishing step. Suitable depth filters include, but are not limited to, Millistak+XOHC, FOHC, DOHC, AIHC, X0SP, and BIHC pod filters (EMD Millipore) or Zeta Plus 30ZA / 60ZA, 60ZA / 90ZA, Delipid, VR07, and VR05 filters (3M). Emphaze AEX Hybrid Purifier multi-mechanism filters may also be used to clarify the eluate. An eluate pool may be required to adjust to a specific pH and conductivity to obtain the desired impurity removal and product recovery from the deep filtration step.

[0313] C. Anion exchange chromatography In a particular embodiment, the protein of interest is generated by subjecting a biological sample to at least one anion exchange separation step. In one scenario, the anion exchange step may occur after an affinity chromatography (e.g., protein A affinity) procedure. In another scenario, the anion exchange step may occur before the affinity chromatography step. In yet another protocol, anion exchange may occur both before and after the affinity chromatography step. In one embodiment, the protein of interest is either aflibercept or MiniTrap.

[0314] The use of anion exchange materials, in contrast to cation exchange materials, is partly based 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] When performing separation, the initial protein composition (biological sample) can be placed in contact with anion exchange material using one of various techniques, such as batch production techniques or chromatography techniques.

[0316] From a batch production perspective, the anion exchange material is prepared in or equilibrated with the desired starting buffer. Preparation yields a slurry of the anion exchange material. The biological sample is brought into contact with the slurry to allow protein adsorption onto 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 stand and removing the supernatant. The slurry can then be subjected to one or more washing and / or elution steps.

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

[0318] The washing step is typically carried out by AEX chromatography, using conditions similar to the loading conditions, or alternatively, by decreasing the pH of the washing solution and / or increasing its ionic strength / conductivity in a stepwise or linear gradient manner. In one embodiment, the salt aqueous solutions used for both the loading buffer and the washing buffer have a pH at or near the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0 to 2 units higher or lower than the pI of the protein of interest, but this may be within the range of 0 to 0.5 units higher or lower. This may also be present at the pI of the protein of interest.

[0319] The anionic agent may be selected from the group consisting of acetates, chlorides, formates, and combinations thereof. The cationic agent may be selected from the group consisting of Tris, arginine, sodium, and combinations thereof. In certain examples, the buffer solution is a Tris / formate buffer. The buffer may be selected from the group consisting of pyridine, piperazine, L-histidine, bis-Tris, bis-trispropane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol), piperazine, 1,3-diaminopropane, and piperidine.

[0320] A packed anion exchange chromatography column, anion exchange membrane device, anion exchange monolithic device, or depth filter medium may be operated in binding-elution mode, flow-through mode, or hybrid mode, in which case the protein will exhibit binding to the chromatography material and may be washed from such material using a buffer similar to or substantially similar to the loading buffer.

[0321] In the binding-elution mode, under conditions where a specific protein adsorbs to a resin-based matrix, the column or membrane device is initially conditioned with a buffer having appropriate ionic strength and pH. For example, during feed loading, the protein of interest may adsorb to the resin by electrostatic attraction. After washing the column or membrane device with an equilibration buffer or another buffer having a different pH and / or conductivity, product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion exchange matrix. Changing the pH, thereby altering the charge of the solute, is another way to achieve solute elution. The change in conductivity or pH may be stepwise (gradient elution) or stepwise (step elution).

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

[0323] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amine (Q) groups. Additional non-limiting examples include Poros 50PI and Poros 50HQ, which are rigid polymer beads with a cross-linked poly[styrene-divinylbenzene] backbone; Capto Q Impres and Capto DEAE, which are highly fluid agarose beads; Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are polymer-based beads; Fractogel® EMD TMAE Hicap, a synthetic polymer resin with tentacle-like ion exchangers; Sartobind STIC® PA nano, a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q nano, a strong anion exchange chromatography membrane; CUNO BioCap, a zeta-plus depth filter constructed from inorganic filter aids, purified cellulose, and ion exchange resins; and XOHC, a deep filtration medium constructed from inorganic filter aids, cellulose, and mixed cellulose esters.

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

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

[0326] In certain embodiments, including those relating to aflibercept and / or VEGF MiniTrap, the method of the present invention can be used to selectively remove, significantly reduce, or essentially remove at least 10% of protein variants, 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 being deamidated; one or more aspartic acid being converted to aspartate-glycine and / or Asn-Gly; one or more methionines being oxidized; one or more tryptophans being converted to N-formylkynurenine; one or more tryptophans being mono-hydroxytryptophan; one or more tryptophans being di-hydroxytryptophan; one or more tryptophans being tri-hydroxytryptophan; one or more arginine being converted to Arg3-deoxyglucosone; the absence of a C-terminal glycine; and / or the presence of one or more non-glycosylated glycosites. The use of AEX was also observed to reduce the oxidized and acidic species of anti-VEGF variants in the affinity eluate. Compared to affinity elution, after using AEX, the flow-through fraction may show a reduction of at least about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% in the oxidized and / or acidic species of anti-VEGF variants.

[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 Trp58 and / or Trp138; (iii) Oxidized tyrosine residue of Tyr64; (iv) Oxidized phenylalanine residues selected from Phe44 and / or Phe166; and / or (v) Oxidized methionine residues selected from Met10, Met20, Met163, and / or Met192. One or more of these may be cited.

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

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

[0331] When performing cation exchange, the sample containing the protein of interest can be brought into contact with the cation exchange material using one of various techniques, such as batch production techniques or chromatography techniques, as described above for AEX.

[0332] A saline solution may be used as both a loading buffer and a washing buffer, having a pH lower than the isoelectric point (pI) of the protein of interest. In one embodiment, the pH is about 0 to 5 units lower than the pI of the protein. In another embodiment, the pH is in the range of 1 to 2 units lower than the pI of the protein. In yet another embodiment, the pH is in the range of 1 to 1.5 units lower than the pI of the protein.

[0333] In certain embodiments, the concentration of the anionic agent in the aqueous salt solution is increased or decreased to achieve a pH of about 3.5 to about 10.5, or about 4 to about 10, or about 4.5 to about 9.5, or about 5 to about 9, or about 5.5 to about 8.5, or about 6 to about 8, or about 6.5 to about 7.5. In one embodiment, the concentration of the anionic agent is increased or decreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Suitable buffer systems for use in the CEX method include, but are not limited to, tris formate, tris acetate, ammonium sulfate, sodium chloride, or sodium sulfate.

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

[0335] Packed cation exchange chromatography columns or anion exchange membrane devices can be operated in either a binding-elution mode, a flow-through mode, or a hybrid mode, in which case the product will bind to or interact with the chromatography material and can be washed away from such material using a buffer similar to or substantially similar to the loading buffer (details of these modes are outlined above).

[0336] The cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include: Capto SP ImpRes, a highly fluid agarose bead; CM Hyper D grade F, a ceramic bead coated and impregnated with a functionalized hydrogel, with 250-400 μeq / mL of ionic groups; Eshmuno S, a hydrophilic polyvinyl ether base matrix with an ion exchange capacity of 50-100 μeq / mL; Nuvia C Prime, a hydrophobic cation exchange medium composed of a highly crosslinked macroporous hydrophilic polymer matrix with 55-75 με / mL of ionic groups; Nuvia S, which has an UNOsphere base matrix with 90-150 με / mL of ionic groups; Poros HS, a rigid polymer bead with a crosslinked poly[styrene-divinylbenzene] backbone; Poros XS, a rigid polymer bead with a crosslinked poly[styrene-divinylbenzene] backbone; Toyo Pearl Giga Cap CM 650M, a polymer-based bead with an ion exchange capacity of 0.225 meq / mL; and Toyo Pearl Giga Cap S, a polymer-based bead. Examples include, but are not limited to, 650M polymer-based beads such as Toyo Pearl MX TRP. Note that CEX chromatography may be used in conjunction with MM resins as described herein.

[0337] The protein load of a sample containing the protein of interest is adjusted to a total protein load on the column of approximately 5 g / L to 150 g / L, or approximately 10 g / L to 100 g / L, approximately 20 g / L to 80 g / L, approximately 30 g / L to 50 g / L, or approximately 40 g / L to 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 of approximately 0.5 g / L to 50 g / L, or approximately 1 g / L to 20 g / L.

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

[0339] In certain embodiments, methods of the present invention, including those related to aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap, may be used to selectively remove, significantly reduce, or essentially remove all oxovaleans in a sample, in which case the protein of interest is essentially present during the flow-through of the CEX procedure, while the oxovaleans are substantially captured by the column medium.

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

[0341] The column resin used for mixed-mode separation may be Capto Adhere. Capto Adhere is a strong anion exchanger with multimodal functionality. The base matrix of this strong anion exchanger is a highly crosslinked agarose having ligands (N-benzyl-N-methylethanolamine) that exhibit different functionalities for interactions such as ionic interactions, hydrogen bonding, and hydrophobic interactions. In certain embodiments, the resin used for mixed-mode separation is selected from PPA-HyperCel and HEA-HyperCel. The base matrices of PPA-HyperCel and HEA-HyperCel are highly porous crosslinked cellulose. Their ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine offer different selectivity and hydrophobicity options for protein separation. Additional mixed-mode chromatography supports include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain embodiments, a mixed-mode chromatographic resin may consist of ligands, sometimes referred to as a base matrix, that are bound directly or via spacers to an organic or inorganic support. The support may take the form of particles, such as essentially spherical particles, monoliths, filters, membranes, surfaces, capillaries, and their equivalents. In certain embodiments, the support may be prepared from crosslinked carbohydrate materials, such as natural polymers including agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, guerane, arginate, and their equivalents. To obtain high adsorption capacity, the support may be porous, and the ligands are then bound to the outer surface and pore surfaces. Such natural polymer supports may be prepared according to standard methods, e.g., reverse suspension gelation (S. Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire teaching of which is incorporated herein by reference).Alternatively, supports may be prepared from crosslinked synthetic polymers, such as styrene or styrene derivatives, divinylbenzene, acrylamide, acrylic acid esters, methacrylic acid esters, vinyl esters, vinylamides, and their equivalents. Such synthetic polymers may 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 teaching is incorporated herein by reference). Porous natural or synthetic polymer supports are also available from manufacturers such as GE Healthcare (Uppsala, Sweden).

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

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

[0344] In certain embodiments, including those relating to aflibercept and / or MiniTrap, the methods of the present invention may be used to selectively remove, significantly reduce, or essentially remove all PTMs, including oxovaleans.

[0345] The method for producing the compositions of the present invention can also be carried out in continuous chromatography mode. In this mode, at least two columns are used (referred to as the "first" column and the "second" column). In certain embodiments, this continuous chromatography mode can be carried out so that elution fractions and / or strip fractions containing PTMs, such as oxovarians, can be loaded sequentially or simultaneously onto the second column (with or without dilution).

[0346] In one embodiment, the medium for continuous mode may be one of many chromatography resins having hydrophobic pendant functional groups and anion exchange functional groups, monolithic media, membrane adsorption media, or deep filtration media.

[0347] F. Hydrophobic Interaction Chromatography The compositions of the present invention may also be prepared using hydrophobic interaction chromatography (HIC).

[0348] During separation, biological samples are brought into contact with the HIC material, for example, using batch production techniques or column or membrane chromatography. Before HIC treatment, it may be desirable to adjust the concentration of the salt buffer to obtain the desired protein binding / interaction to the resin or membrane.

[0349] While ion-exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography utilizes the hydrophobic properties of the protein to achieve selective separation. Hydrophobic groups on or within the protein interact with the hydrophobic groups of the chromatography resin or membrane. Typically, under favorable conditions, the more hydrophobic the protein (or part of the protein), the stronger the interaction with the column or membrane. Therefore, under favorable conditions, HIC can be used to facilitate the separation of process-related impurities (e.g., HCP) and product-related substances (e.g., aggregates or fragments) from the protein of interest in a sample.

[0350] Similar to ion exchange chromatography, HIC columns or HIC membrane devices can also be operated in elution mode, flow-through mode, or hybrid mode, in which case the product exhibits binding or interaction with the chromatographic material and can even be washed from these materials using buffers similar to or substantially similar to the loading buffer. (Details of these modes are outlined above in relation to AEX processing.) Since hydrophobic interactions are strongest at high ionic strengths, this form of separation is conveniently used after a salt elution step, such as the steps typically used in relation to ion exchange chromatography. Alternatively, the salt can be added to the sample before using HIC. While protein adsorption to HIC columns works favorably with higher salt concentrations, the actual concentration can vary widely depending on the properties of the protein of interest, the type of salt, and the specific HIC ligand selected. Various ions can be arranged in a so-called soluphobic series, depending on whether they promote hydrophobic interactions (salting out) or weaken hydrophobic interactions by disrupting the structure of water (chaotropic effect). Cations include Ba 2+ Ca 2+ Mg 2+ ;Li + ;Cs + ;Na + ;K + ;Rb + ;NH4 + They are ranked in terms of increasing salting-out activity, etc. On the other hand, anions are PO4 3- ;SO4 2- ;CH3CO3 - ;CI - ;Br - NO3 - ClO4 - I - ;SCN - They are ranked based on factors such as increasing the chaotropic effect.

[0351] Generally, Na + , K + or NH4 +The sulfates effectively promote ligand-protein interactions using HIC. Salts that influence the strength of the interaction may be formulated, as given by the following relationship: (NH4)2SO4 > Na2SO4 > NaCl > NH4C1 > NaBr > NaSCN. Generally, salt concentrations of approximately 0.75 M to 2 M ammonium sulfate or approximately 1 M to 4 M NaCl are useful.

[0352] HIC media typically consist of a base matrix (e.g., crosslinked agarose or synthetic copolymer material) to which hydrophobic ligands (e.g., alkyl or aryl groups) are bonded. Preferred HIC media include phenyl-functionalized agarose resins or membranes (e.g., Phenyl Sepharose® from GE Healthcare or Phenyl Membrane from Sartorius). Many HIC resins are commercially available. Examples include, but are not limited to, Capto Phenyl, 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 Column or Macro-Prep® t-Butyl Column (Bio-Rad, California); WP HI-Propyl(C3)® (JTBaker, New Jersey); and Toyopearl® Ether, Phenyl or Butyl (TosoHaas, PA); ToyoScreen PPG; ToyoScreen Phenyl; ToyoScreen Butyl; ToyoScreen Hexyl; GE HiScreen and Butyl FF HiScreen Octyl FF.

[0353] The protein loading of a sample containing the protein of interest is adjusted to a total protein loading on the column of approximately 50 g / L to 1000 g / L, 5 g / L to 150 g / L, 10 g / L to 100 g / L, 20 g / L to 80 g / L, 30 g / L to 50 g / L, or 40 g / L to 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, of approximately 0.5 g / L to 50 g / L, or 1 g / L to 20 g / L.

[0354] The pH selected for any particular generation process can be specific to its application, as it must be compatible with the stability and activity of the protein. However, between pH 5.0 and 8.5, certain pH values ​​may be advantageous because they have little significance for the final selectivity and resolution of HIC separation. Increasing pH weakens hydrophobic interactions, and pH levels above 8.5 or below 5.0 more significantly alter protein retention. Furthermore, changes in ionic strength, the presence of organic solvents, temperature, and pH (especially at the isoelectric point pI when no net surface charge is present) can affect protein structure and solubility, and consequently affect interactions with other hydrophobic surfaces, such as those in HIC media. Therefore, in certain embodiments, the present invention incorporates a generation strategy that adjusts one or more of the above to achieve a desired reduction in process-related impurities and / or product-related substances.

[0355] In certain embodiments, spectroscopy such as UV, NIR, FTIR, fluorescence, and Raman can be used to monitor proteins and impurities of interest in online, at-line, or in-line modes, which can then be used to control the level of aggregates in the pooled material collected from the HIC-adsorbent eluate. In certain embodiments, the online, at-line, or in-line monitoring method can be used either on the eluate line of the chromatography process or in the collection container, thereby enabling the achievement of the desired product quality / recovery. In certain embodiments, a UV signal can be used as a ...

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