RU2795973C1 - Protein compositions against vegf and methods for their production - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to polypeptides with protease activity, and can be used to obtain recombinant immunoglobulins. Proposed polypeptides with protease activity of the enzyme that destroys immunoglobulin, Streptococcus pyogenes (IdeS).

EFFECT: production of polypeptides capable of cleaving a target protein with an Fc domain, such as aflibercept, into fragments.

18 cl, 59 dwg, 9 tbl, 9 ex

Description

SEQUENCE LIST

This application contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The specified ASCII copy, created on August 13, 2020, is named 070816-02351_SL.txt and is 134385 bytes in size.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority and benefit of U.S. Provisional Application No. 63/065012, filed August 13, 2020, the contents of which are incorporated herein by reference in its entirety.

FIELD OF TECHNOLOGY

The present invention generally relates to compositions against VEGF and methods for their preparation.

BACKGROUND OF THE INVENTION

Protein-based biopharmaceutical compositions have become important products for research, treatment of ophthalmic diseases, cancer, autoimmune disease, infection, and other diseases and disorders. Biopharmaceuticals represent one of the fastest growing segments of the pharmaceutical industry.

A class of cell-derived dimeric mitogens with selectivity for vascular endothelial cells has been identified and designated as vascular endothelial cell growth factor (VEGF).

Sustained angiogenesis can cause or exacerbate certain diseases such as psoriasis, rheumatoid arthritis, hemangiomas, angiofibromas, diabetic retinopathy, and neovascular glaucoma. An inhibitor of VEGF activity may be useful as a treatment for such diseases and other VEGF-induced pathological angiogenesis and vascular permeability conditions such as tumor vascularization. Angiopoietins and members of the vascular endothelial growth factor (VEGF) family are the only growth factors thought to be highly specific for vascular endothelial cells.

Some eye diseases are associated with pathological angiogenesis. For example, the development of age-related macular degeneration (AMD) is associated with a process called choroidal neovascularization (CNV). Leakage from the CNV causes macular edema and fluid accumulation under the macula, resulting in loss of vision. Diabetic macular edema (DME) is another eye disease with an angiogenic component. DME is the most common cause of moderate vision loss in patients with diabetes and a common complication of diabetic retinopathy, a disease that affects the blood vessels in the retina. Clinically significant DME occurs if fluid seeps into the center of the macula, the light-sensitive part of the retina responsible for sharp, direct vision. Fluid in the macula can cause severe vision loss or blindness.

Various VEGF inhibitors, such as the Eylea VEGF Trap (aflibercept), have been approved for the treatment of such eye conditions.

SUMMARY OF THE INVENTION

The present invention relates to anti-VEGF proteins, including the VEGF decoy protein aflibercept, which is a fusion protein. The present invention also relates to a novel anti-VEGF protein, aflibercept MiniTrap or VEGF MiniTrap (collectively referred to as MiniTrap unless otherwise indicated). Disclosed herein are methods for the production of these anti-VEGF proteins, including production conditions that provide efficient and effective methods for the production of proteins of interest. In one aspect, the present invention is directed to the use of a chemically defined environment (COS) for the production of anti-VEGF proteins. In a specific aspect, the HOS of interest are those HOS that, when used, provide a protein sample in which the sample is tan in color and may contain oxidized forms. In addition, this application describes the protein variants of aflibercept and VEGF MiniTrap, along with related methods of obtaining.

Getting aflibercept

The present invention describes the production of aflibercept using a cell culture medium. In one embodiment, the cell culture medium is a chemically defined medium ("COS"). HOS is often used due to the fact that it is a protein-free, chemically defined formula containing no animal components, and there is certainty regarding the composition of the medium. In another embodiment, the cell culture medium is soy hydrolysate medium.

In one embodiment, a method for producing a recombinant protein comprises (a) providing a host cell genetically engineered to express the recombinant protein of interest; (b) culturing the host cell in COS under suitable conditions in which the cell expresses the recombinant protein of interest; and (c) collecting a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a specific aspect, the anti-VEGF protein is selected from the group consisting of aflibercept and recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159), aflibercept scFv, and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.

In one aspect of the present embodiment, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells, and BHK.

Suitable HOSs include Dulbecco's Modified Eagle's Medium (DME), Hem's Formula, Excell's medium, and IS CHO-CD medium. Other HOS known to those skilled in the art are also considered to be within the scope of the present invention. In a particular aspect, a suitable HOS is HOS1B (Regeneron) or Excell Enhanced Environment (SAFC).

In one embodiment, a decontaminated COS culture sample containing aflibercept is subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, Protein A. In another aspect, the affinity procedure eluate has a specific color, for example, the eluate may be tan. As described in more detail below , color can be assessed using (i) European color standard "BY" in which a qualitative visual inspection is made, or (ii) colorimetric analysis, CIE L*, a*, b* (or CIELAB) , which is more quantitative than the BY system. However, in any case, the color score between multiple samples must be normalized to protein concentration in order to provide an informative estimate. For example, with respect to Example 9 below, the Protein A eluate has a "b*" value of about 2.52, which corresponds to a BY value of about BY5 (when measured at a protein concentration of 5 g/L in the Protein A eluate). If the color of the Protein A eluate is to be compared with another sample, then the comparison should be made with respect to the same protein concentration. The b* value in the CIELAB color space is used to express the color of samples and ranges from blue (-) to yellow (+). A higher b* value of one sample than another indicates a more intense tan coloration in the sample than the other.

In one embodiment, aflibercept is derived from a host cell genetically engineered to express aflibercept using COS. In one aspect, other types or variants of aflibercept are also prepared. These variants include isoforms of aflibercept that contain one or more oxidized amino acid residues, collectively referred to as oxo variants. A decontaminated COS harvest containing aflibercept as well as its oxo variants can be subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, a protein A column. -spectrophotometry (LC-MS), one or more oxidized variants of aflibercept can be detected. Several amino acid residues of modified aflibercept have been shown to be oxidized, including, without limitation, histidine and/or tryptophan residues. In one aspect, the variants may include the oxidation of one or more methionine residues, as well as other residues, see below .

In another aspect, the variants may include the oxidation of one or more tryptophan residues to form N-formylkynurenine. In a further aspect, the variants may include the oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In a specific aspect, the protein variants may include the oxidation of one or more tryptophan residues to form di-hydroxyl tryptophan. In a specific aspect, protein variants may include the oxidation of one or more tryptophan residues to form tri-hydroxyl tryptophan.

In another aspect, the variants may include one or more modifications selected from the group consisting of deamidation of, for example, one or more asparagines; one or more aspartic acids converted to iso-aspartate and/or Asn; oxidation of one or more methionines; oxidation of one or more tryptophans to N-formylkynurenine; oxidation of one or more tryptophans in mono-hydroxyltryptophan; oxidation of one or more tryptophans in di-hydroxyltryptophan; oxidation of one or more tryptophans in tri-hydroxyltryptophan; 3-deoxyglucosonation Arg of one or more arginines; removal of the C-terminal glycine; and the presence of one or more non-glycosylated glycosides.

In another embodiment, the present invention is directed to methods for preparing aflibercept. In one aspect, the decontaminated collection sample containing aflibercept and variants thereof is subjected to a capture step, such as protein A affinity chromatography. After the affinity step, the affinity eluate may be subjected to ion exchange chromatography. Ion exchange chromatography can be either cation or anion exchange chromatography. It is also contemplated that the scope of the present embodiment includes mixed or multimodal chromatography, as well as other chromatographic procedures, which are discussed further below. In a specific aspect, the ion exchange chromatography is anion exchange chromatography (AEX). Suitable conditions for using AEX include, but are not limited to, tris hydrochloride at a pH of about 8.3 to about 8.6. After equilibration using, for example, tris hydrochloride at a pH of about 8.3 to about 8.6, a sample is loaded onto the AEX column. After loading the column, it can be washed one or more times, using, for example, an equilibrating buffer. In a particular aspect, the conditions employed may provide for differential chromatographic behavior of aflibercept and its oxidized variants such that a fraction containing aflibercept in the absence of significant amounts of oxo variants can be collected in the flow-through fraction, while a significant proportion of oxo variants are retained on the solid phase of the AEX column and can be obtained by cleaning the column - see example 2 below, fig. . 11 . Referring to FIG. 11 and Example 2, changes in oxo variants can be observed between different stages of production. For example, this change can be illustrated by the data in the section "Tryptophan oxidation level (%)", in particular, in the column "W138(+16)". It can be observed that oxo variants (particularly oxo-tryptophan) go from about 0.131% in the loading sample to about 0.070% in the running sample after AEX chromatography (AEX split 2), indicating that there is a reduction in oxo variants. aflibercept using AEX.

The use of ion exchange can be used to reduce or minimize color. In one aspect of the present embodiment, the decontaminated collection sample is subjected to capture chromatography, such as using protein A affinity chromatography. The affinity column is eluted and has a first color assigned to it with a specific BY and/or b* value. This protein A eluate is then subjected to ion exchange chromatography, eg anion exchange chromatography (AEX). The ion exchange column is washed and the wet end is collected and has a second color assigned to it with a specific BY and/or b* value. In a specific aspect, the color value (either "BY" or "b*") of the first color is different from the second color. In a further aspect, the first color of the protein A eluate is more tan than the second color of the AEX flow path, as reflected by the corresponding BY and/or b* value. Typically, there is a decrease in the yellow-brown color of the second color after AEX, compared to the first color of the protein A eluate. For example, the use of anion exchange reduced the yellow-brown color observed in the protein A eluate sample from a b* value of about 3.06 (first color) to about 0.96 (second color) after AEX - see example 2, table 2-3 below.

In one aspect of the embodiment, the pH of both buffers for equilibrating and washing the AEX column can be from about 8.30 to about 8.60. In another aspect, the conductivity of both of the equilibration and wash buffers for the AEX column may be from about 1.50 to about 3.00 mS/cm.

In one aspect of the embodiment, the equilibration and wash buffers may be 50 mM tris hydrochloride. In one aspect, the purification buffer contains 2 mol sodium chloride or 1 N sodium chloride. sodium hydroxide or both ( see Table 2-2 ).

The present embodiment may include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography (HIC), affinity chromatography, multimodal chromatography, viral inactivation ( e.g. using low pH), virus filtration and/or ultra-/ diafiltration, as well as other well-known chromatographic steps.

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

In one aspect, the glycosylation profile of the anti-VEGF protein composition is about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and from about 60% to about 79% galactosylated glycans (see example 6). In one aspect, the anti-VEGF protein has Man5 glycosylation at about 32.4% asparagine 123 residues and/or about 27.1% asparagine 196 residues.

In one embodiment, the method may further include formulating the drug substance using a pharmaceutically acceptable excipient. In one aspect of the embodiment, the pharmaceutically acceptable excipient may be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier, and preservative. Other excipients well known to the person skilled in the art are within the scope of this embodiment.

In one aspect of the embodiment, the formulation may be suitable for administration to a human subject. In particular, administration may be by intravitreal injection. In one aspect, the composition may have from about 40 to about 200 mg/ml of the protein of interest.

The composition can be used as a method of treating or preventing angiogenic eye diseases, which may include: age-related macular degeneration ( for example , 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 glaucoma fibrosis, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygoid hymen, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathy ( eg , non-proliferative diabetic retinopathy ( eg , characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53, or proliferative diabetic retinopathy; eg , in a subject not suffering from DME).

Obtaining MiniTrap VEGF

The present invention describes the preparation of a modified version of aflibercept with the Fc portion removed or missing and is referred to as MiniTrap or MiniTrap VEGF aflibercept. This MiniTrap can be prepared in a cell culture medium containing Chemically Defined Composition (COS) medium or soy hydrolysate medium.

In one embodiment, MiniTrap is produced using HOS. In one aspect of making MiniTrap, a full-length aflibercept is prepared using a suitable host and under suitable conditions, and further processed whereby the Fc portion is enzymatically removed, thereby obtaining a MiniTrap. Alternatively, the gene encoding MiniTrap ( eg , the nucleotide sequence encoding aflibercept in the absence of its Fc portion) can be generated under suitable conditions using a suitable host cell.

In one embodiment, the method for manufacturing MiniTrap comprises making a full-length aflibercept fusion protein, followed by cleavage of the Fc region. In one aspect, the method includes generating a recombinant protein, namely a full-length aflibercept fusion protein ( see U.S. Patent No. 7,279,159, the entire contents of which are incorporated herein by reference), comprising (a) providing a host cell genetically engineered to express the full-length aflibercept; (b) culturing the host cell in HOS under suitable conditions, in which the cell expresses full length aflibercept; (c) harvesting a cell-produced full length aflibercept preparation; and (d) subjecting the full length aflibercept to an enzymatic cleavage specific to remove the Fc portion of the fusion protein. In another aspect, the nucleotide sequence encoding aflibercept in the absence of its Fc portion is expressed from an appropriate host cell under appropriate conditions well known to one of skill in the art ( see US Pat. No. 7,279,159).

In one aspect of the present embodiment, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells, and BHK.

Suitable HOS include Dulbecco's Modified Eagle's Medium (DME), Hem's Nutritional Formula, EX-CELL Medium (SAFC), and IS CHO-CD Medium (Irvine). Other HOS known to those skilled in the art are also considered to be within the scope of the present invention. In a specific aspect, a suitable HOS is HOS1B (Regeneron) or Excell Environment (SAFC).

In one aspect, during the production of MiniTrap, a sample containing a protein of interest ( i.e. , an aflibercept and/or MiniTrap fusion protein), along with its variants (including oxo variants), may exhibit certain color properties - tan . For example, an eluate sample from the affinity chromatography step may show a distinct tan color as measured using the BY and/or b* system ( see examples 2 and 9 below). Illustrative sources for "sample" may include affinity chromatography, eg protein A, eluate; the sample may be obtained from the flow fraction of an ion exchange chromatography procedure; it can also be obtained from cleaning the ion exchange column - there are other sources during the manufacturing process, well known to those skilled in the art, from which a sample can be analyzed. As noted above and further described below, color can be assessed using (i) the European color standard "BY", which performs a qualitative visual inspection, or (ii) a colorimetric analysis, CIELAB, which is more quantitative than the BY system. However, in any case, the color score between multiple samples should be normalized, eg using protein concentration, in order to provide an informative score between samples.

In one aspect of the present embodiment, the full-length aflibercept fusion protein can be subjected to an enzymatic treatment (“cleavage activity”) to form MiniTrap VEGF, for example, using a proteolytic cleavage using a protease or an enzymatically active variant thereof. In one aspect of this embodiment, the protease may be a pyogenic streptococcus immunoglobulin-degrading enzyme (IdeS). In another aspect, the protease may be thrombin trypsin, Arg-C endoprotease, Asp-N endoprotease, Glu-C endoprotease, outer membrane protease T (OmpT), IdeS, chymotrypsin, pepsin, thermolysin, papain, pronase, or a protease from Aspergillus Saitoi. In one aspect, the protease may be a cysteine protease. In a specific aspect of the embodiment, the protease may be IdeS. In another aspect, the protease may be a variant of IdeS. Non-limiting examples of IdeS variants are described below and include a polypeptide, having the amino acid sequence set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14 , SEQ ID NO.: 15 and SEQ ID NO.: 16. In one aspect, the protease can be immobilized on agarose or other suitable matrix.

In one aspect, the protein of interest (together with its variants) is produced using HOS. In a specific aspect, the protein of interest includes aflibercept or MiniTrap. Variants include one or more oxidized amino acid residues, collectively referred to as oxo variants. Examples of oxidized residues include, without limitation, one or more histidine and/or tryptophan residues, other oxidized residues were detected by LC-MS and are described below, for example, oxidized methionine. Subsequent chromatography, eg AEX, can be used to isolate these oxo variants from a protein of interest in a given sample and are described herein.

In one aspect, the variants may include the oxidation of one or more tryptophan residues to form N-formylkynurenines. In a further aspect, the variants may include the oxidation of one or more tryptophan residues to form mono-hydroxyl tryptophan. In a specific aspect, the protein variants may include the oxidation of one or more tryptophan residues to form di-hydroxyl tryptophan. In a specific aspect, protein variants may include the oxidation of one or more tryptophan residues to form tri-hydroxyl tryptophan.

In another aspect, oxo variants may include one or more modifications selected from the group consisting of deamidation of, for example, one or more asparagine residues; one or more aspartic acids converted to iso-aspartate and/or Asn; oxidation of one or more methionine residues; oxidation of one or more tryptophan residues to N-formylkynurenine; oxidation of one or more tryptophan residues in mono-hydroxyltryptophan; oxidation of one or more tryptophan residues in di-hydroxyltryptophan; oxidizing one or more tryptophan residues from tri-hydroxyltryptophan; 3-deoxyglucosonation Arg of one or more arginine residues; removal of the C-terminal glycine; and the presence of one or more non-glycosylated glycosides.

In one embodiment, the method for producing the MiniTrap protein comprises (a) capturing the full-length aflibercept fusion protein on a first chromatography platform, and (b) cleaving the aflibercept to thereby form the MiniTrap protein, i.e. , aflibercept in the absence of an Fc domain. In one aspect, the first chromatography support comprises an affinity chromatography medium, an ion exchange chromatography medium, or a hydrophobic interaction chromatography medium. In a specific aspect, the first chromatography platform comprises an affinity chromatography platform, eg, protein A. In a further aspect, the protein of the capture step (a) is eluted from the first chromatography platform prior to the cleavage step (b). In a further aspect, the second capture step follows the cleavage step (b). In a specific aspect, this second capture step can be performed using affinity chromatography, such as protein A affinity chromatography. The flow path of this second capture step (containing MiniTrap) has a first color, e.g. or b* - see example 9 below. In addition, LC-MS analysis of this second capture flow path may demonstrate the presence of oxo variants, with one or more MiniTrap residues oxidized ( see Example 9 below).

In a further aspect, the second capture flow path may be subjected to ion exchange chromatography, such as AEX. This AEX column can be washed with an appropriate buffer and an AEX run-through fraction containing mostly MiniTrap can be collected. This AEX flow cut may have a second color that is yellow-brown with a specific BY and/or b* value. In a further aspect, the first color (the flow-through fraction from the second capture step) and the second color (the flow-through fraction from the ion exchange procedure) have different colors measured either by the BY or b* system. In one aspect, the second color has a reduced yellow-brown color compared to the first color using either a BY or b* value and then AEX.

In another embodiment, the cleavage activity in step (b) can be carried out using a chromatographic column, wherein the cleavage activity, eg, enzymatic activity, is attached or immobilized in the matrix of the column. The column used in step (b) may contain one or more of the proteases mentioned and described in more detail below.

In one embodiment, the ion exchange chromatography procedure may include an anion exchange (AEX) chromatography medium. In another aspect, the ion exchange chromatography medium includes a cation exchange (CEX) chromatography medium. Suitable conditions for using AEX include, but are not limited to, tris hydrochloride at a pH of about 8.3 to about 8.6. After equilibration using, for example, tris hydrochloride at a pH of about 8.3 to about 8.6, a sample is loaded onto the AEX column. After loading the column, it can be washed one or more times, using, for example, an equilibrating buffer. In a particular aspect, the applied conditions may allow differential chromatographic behavior of MiniTrap and its oxo variants using AEX such that MiniTrap is essentially in the flow-through fraction while oxo variants are essentially retained on the AEX column and can be controlled by column cleanup ( see below). example 9 below).

In one example, samples from various preparation steps were analyzed for color and the presence of oxo variants. Relative to Example 9, the affinity flow-through pool (the flow-through fraction from the second protein A affinity step) had a first b* value of about 1.58 ( see Table 9-3 ). This second affinity flow fraction was subjected to AEX. The run-through fraction of AEX had a second b* value of about 0.50, indicating a significant reduction in tan after application of AEX. Purification of the AEX column provided a purification sample and a third b* value of about 6.10 was observed, indicating that this purification sample was more tan than either the batch or the run-through.

With regard to example 9, also carried out the analysis of the oxo-variant. The samples analyzed were the affinity flow-through pool (second protein A affinity eluate), the AEX flow-through, and the AEX purification. With respect to Table 9-5 and Table 9-6 , changes in oxo variants can be observed between different preparation steps. For example, this change can be illustrated by the data in the section "Tryptophan oxidation level (%)", in particular, in the column "W58(+16)". It can be observed that oxo variants (particularly oxo-tryptophan) go from about 0.055% in the loading sample to about 0.038% in the flow sample after AEX chromatography, indicating that there is a decrease in oxo variants after AEX. The purification of AEX was analyzed and the percentage of oxo-tryptophan was found to be about 0.089%. If the value of this band was compared with the load (as well as the flow-through fraction), it turned out that a significant part of this oxo-variant remained on the AEX column.

The present invention may include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, viral inactivation ( e.g. using low pH), virus filtration, and/or ultra/diafiltration.

One embodiment of the present invention is directed to a method for regenerating a chromatographic column containing resin. In one aspect of the embodiment, the resin has an immobilized hydrolyzing agent. In another aspect of the embodiment, the resin contains an immobilized protease enzyme. In another aspect of the embodiment, the resin is a FabRICATOR® resin or a resin mutant. In one aspect of the embodiment, the method for regenerating a column containing resin includes the reactivity of the resin.

In one aspect of an embodiment, a method for regenerating a resin containing column comprises incubating the resin in an acetic acid column. In one aspect, the concentration of acetic acid used is from about 0.1 mol to about 2 mol. In one aspect, the concentration of acetic acid is about 0.5 mol. In one aspect, the resin is incubated for at least about 10 minutes. In another aspect, the resin is incubated for at least about 30 minutes. In another aspect of the present embodiment, the resin is incubated for at least about 50 minutes. In another aspect of the present embodiment, the resin is incubated for at least about 100 minutes. In another aspect of the present embodiment, the resin is incubated for at least about 200 minutes. In another aspect of the present embodiment, the resin is incubated for at least about 300 minutes.

Optionally, the resin on the column is further incubated with guanidine hydrochloride (Gu-HCl). In one aspect, Gu-HCl without acetic acid is used to regenerate the resin in the column. The concentration of Gu-HCl used is from about 1 N. up to about 10 n. In another aspect, the concentration of Gu-HCl is about 6N. In a further aspect, the resin in the column may be incubated for at least about 10 minutes with regenerating agents (acetic acid, Gu-HCl). In another aspect, the resin is incubated for at least about 30 minutes. In another aspect, the resin is incubated for at least about 50 minutes. In another aspect, said resin is incubated for at least about 100 minutes.

In one embodiment, the column containing the resin is stored in ethanol. In one aspect, the column is stored in ethanol, and the percentage of ethanol is from about 5% vol./about. up to about 20% vol./about. In a specific aspect, the column is stored using 20% v/v. ethanol.

In one embodiment, the method may further comprise formulating a MiniTrap VEGF using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient may be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier, and preservative. Other excipients well known to the person skilled in the art are within the scope of this embodiment.

The composition of the present invention is suitable for administration to a human subject. In one aspect of the present embodiment, administration may be by intravitreal injection. In one aspect, the composition may have from about 40 to about 200 mg/ml of the protein of interest. In a specific aspect, the protein of interest is either aflibercept or MiniTrap aflibercept.

The composition can be used in a method of treating or preventing angiogenic eye diseases, which may include: 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 ), retinal branch vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), papilledema neovascularization, corneal neovascularization, neovascularization retina, vitreous neovascularization, pannus, pterygoid hymen, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathy ( eg , non-proliferative diabetic retinopathy ( eg , characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53, or proliferative diabetic retinopathy; eg , in a subject not suffering from DME).

Options

This disclosure describes the use of IdeS (FabRICATOR) (SEQ ID NO.: 1) or other polypeptides that are variants of IdeS (SEQ ID NO.: 2-16) to obtain MiniTrap VEGF. IdeS (SEQ ID NO.: 1) contains asparagine residues at position 87, 130, 182 and/or 274 (shown as "N*" in bold and italics in SEQ ID NO.: 1 below). Asparagine at these positions can be mutated to an amino acid other than asparagine to form IdeS variants (and the mutated amino acid(s) are shown in italics and underlined):

SEQ ID NO.: 1

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRL SLTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 2

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 3

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 4

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 5

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFN*GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFN*GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSDGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 6

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTFDGKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINFRGEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFIN*GYRLSLTNH GPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDSN*GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 7

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLT NHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 8

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 9

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N * GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLT NHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 10

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 11

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLS LTNHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 12

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLTNH GPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS N* GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 13

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI N* GYRLSLT NHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 14

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF N* GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLT NHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 15

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF N* GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLT NHGPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

SEQ ID NO.: 16

MRKRCYSTSAAVLAAVTLFVLSVDRGVIADSFSANQEIRYSEVTPYHVTSVWTKGVTPPANFTQGEDVFHAPYVANQGWYDITKTF D GKDDLLCGAATAGNMLHWWFDQNKDQIKRYLEEHPEKQKINF R GEQMFDVKEAIDTKNHQLDSKLFEYFKEKAFPYLSTKHLGVFPDHVIDMFI L GYRLSLTNH GPTPVKEGSKDPRGGIFDAVFTRGDQSKLLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLWGADFDS D GNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKEDNIGAQVLGLFTLSTGQDSWNQTN

In one embodiment, the polypeptide has an isolated amino acid sequence that is at least 70% identical throughout the entire length of the isolated amino acid sequence shown in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11 , SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16. In one aspect, the isolated amino acid sequence is at least about 80% identical along the entire length of the selected amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 90% identical over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 100% identical over the entire length of the isolated amino acid sequence. In one aspect, the polypeptide may be capable of cleaving the target protein into fragments. In a specific aspect, the target protein is an IgG. In another aspect, the target protein is a fusion protein. In another aspect, the fragments may contain a Fab fragment and/or an Fc fragment.

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

The present invention also includes an isolated nucleic acid molecule encoding a polypeptide having an isolated amino acid sequence at least 70% identical throughout the entire length of the selected amino acid sequence shown in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16. In one aspect, an isolated amino acid sequence of at least about 80% identical over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 90% identical over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 100% identical over the entire length of the isolated amino acid sequence. In one aspect, the polypeptide may be capable of cleaving the target protein into fragments. In a specific aspect, the target protein is an IgG. In another specific aspect, the target protein is a fusion protein. In another specific aspect, the fragments may contain a Fab fragment and/or an Fc fragment.

The present disclosure also includes a vector that contains a nucleic acid encoding a polypeptide having an isolated amino acid sequence at least 70% identical throughout the entire length of the selected amino acid sequence shown in the group consisting of SEQ ID NO.: 2, SEQ ID NO. : 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16. In one aspect, the nucleic acid molecule operably linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector may be a plasmid. In one aspect, the isolated amino acid sequence is at least about 80% identical over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 90% identical over the entire length of the isolated amino acid sequence. In another aspect, the isolated amino acid sequence is at least about 100% identical over the entire length of the isolated amino acid sequence. In one aspect, the polypeptide may be capable of cleaving the target protein into fragments. In a specific aspect, the target protein is an IgG. In another aspect, the target protein is a fusion protein. In another aspect, the fragments may contain a Fab fragment and/or an Fc fragment.

In one embodiment, the isolated amino acid may contain the original amino acid sequence as defined in SEQ ID NO.: 1 with the asparagine residue at position 87, 130, 182 and/or 274 mutated to an amino acid other than asparagine. In one aspect, the mutation may provide increased chemical stability at alkaline pH values compared to the original amino acid sequence. In another aspect, the mutation may provide a 50% increase in chemical stability at alkaline pH values compared to the original amino acid sequence. In one aspect, the amino acid may be selected from aspartic acid, leucine, and arginine. In a specific aspect, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another aspect, the asparagine residue at position 130 is mutated to an arginine residue. In another aspect, the asparagine residue at position 182 is mutated to a leucine residue. In another aspect, the asparagine residue at position 274 is mutated to an aspartic acid residue. In another aspect, the asparagine residues at positions 87 and 130 are mutated. In another aspect, the asparagine residues at positions 87 and 182 are mutated. In another aspect, the asparagine residues at positions 87 and 274 are mutated. In another aspect, the asparagine residues at positions 130 and 182 are mutated. In another aspect, the asparagine residues at positions 130 and 274 are mutated. In another aspect, the asparagine residues at positions 182 and 274 are mutated. In another aspect, the asparagine residues at positions 87, 130 and 182 are mutated. In another aspect, the asparagine residues at positions 87, 182 and 274 are mutated. In another aspect, the asparagine residues at positions 130, 182 and 274 are mutated. In another aspect, the asparagine residues at positions 87, 130, 182 and 274 are mutated.

In a related embodiment, the present invention includes an isolated nucleic acid molecule encoding a polypeptide with an isolated amino acid sequence comprising the parent amino acid sequence as defined in SEQ ID NO.: 1 with an asparagine residue at position 87, 130, 182 and/or 274 mutated to the amino acid , other than asparagine - see above. The mutation may provide increased chemical stability at alkaline pH values compared to the original amino acid sequence.

In a further related embodiment, the present invention includes a vector that contains a nucleic acid molecule encoding a polypeptide with an isolated amino acid sequence comprising the parent amino acid sequence as defined in SEQ ID NO.: 1 with an asparagine residue at position 87, 130, 182 and/or 274 , mutated to an amino acid other than asparagine - see above. The mutation may provide increased chemical stability at alkaline pH values compared to the original amino acid sequence. In one aspect, the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector may be a plasmid.

Affinity based acquisition

The present invention also provides methods for reducing host cell proteins as well as other undesirable proteins and nucleic acids during the production of an anti-VEGF protein using affinity chromatography.

In one embodiment, a method for producing a recombinant protein comprises (a) providing a host cell genetically engineered to express the recombinant protein of interest; (b) culturing the host cell under suitable conditions in which the cell expresses the recombinant protein of interest; and (c) collecting a preparation of the recombinant protein of interest produced by the cell. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a specific aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (an example of which is disclosed in US Pat. No. 7,279,159), scFv, and other anti-VEGF proteins.

In one aspect of the present embodiment, the recombinant protein of interest is expressed in a suitable host cell. Non-limiting examples of suitable host cells include, but are not limited to, CHO, CHO K1, EESYR®, NICE®, NS0, Sp2/0, embryonic kidney cells, and BHK.

In one aspect of the present embodiment, the recombinant protein of interest is cultured in HOS. Suitable HOS includes Dulbecco's Modified Eagle's Medium (DME), Hem's formula, Excell medium, IS CHO-CD medium, and HOS1B. Other HOS known to those skilled in the art are also considered to be within the scope of the present invention.

The formulation may contain at least one contaminant, including one or more host cell proteins in addition to the recombinant protein of interest. At least one impurity may be formed from a cell substrate, cell culture, or further processes.

In one embodiment, the present invention is directed to methods for obtaining an anti-VEGF protein from a biological sample using affinity chromatography. In a specific aspect, the methods disclosed herein can be used to separate, at least in part, an anti-VEGF protein from one or more host cell proteins and nucleic acids ( e.g. , DNA) generated during a method for culture of an anti-VEGF protein.

In one aspect, the method may include subjecting the biological sample containing the anti-VEGF protein, together with concomitant impurities, to affinity chromatography under suitable conditions. In a particular aspect, affinity chromatography may include material capable of selectively or specifically binding to an anti-VEGF protein ("capture"). Non-limiting examples of such a chromatographic material include protein A, protein G, a chromatographic material containing, for example, a protein capable of binding to an anti-VEGF protein, and a chromatographic material containing an Fc-binding protein. In a specific aspect, a protein capable of binding to or interacting with an anti-VEGF protein may be an antibody, a fusion protein, or a fragment thereof. Non-limiting examples of such a material capable of selectively or specifically binding to an anti-VEGF protein are described in Example 7.

In one aspect of the present embodiment, the method may include subjecting a biological sample containing an anti-VEGF protein and one or more host cell proteins/contaminants to affinity chromatography under suitable conditions, wherein the stationary phase of the affinity chromatography contains a protein capable of selectively or specifically binding to the anti-VEGF protein. VEGF. In a specific aspect, the protein may be an antibody, fusion protein, scFv, or antibody fragment. In a specific aspect, the protein may be forms of VEGF ₁₆₅ , VEGF ₁₂₁ , VEGF from other species such as the rabbit. For example, as illustrated in Table 7-1 and Table 7-10 , the use of VEGF ₁₆₅ as a protein capable of selectively or specifically binding to or interacting with an anti-VEGF protein results in the successful production of MT5 (anti-VEGF protein), aflibercept, and an anti-VEGF scFv fragment. . In another specific aspect, the protein may be one or more proteins with the amino acid sequence shown in SEQ ID NO.: 73-80. Table 7-1 also discloses the successful production of MT5 using proteins with the amino acid sequence shown in SEQ ID NO.: 73-80 as a protein capable of selectively or specifically binding to an anti-VEGF protein (MT5).

In one aspect of the present embodiment, the method may include subjecting a biological sample comprising an anti-VEGF protein and one or more host cell proteins/contaminants to affinity chromatography under suitable conditions, wherein the stationary phase of the affinity chromatography comprises a protein capable of selectively or specifically binding to or interacting with an anti-VEGF protein, wherein the anti-VEGF protein can be selected from aflibercept, MiniTrap VEGF, or an anti-VEGF antibody. In a specific aspect, MiniTrap VEGF can be obtained, in addition, from components of the VEGF receptor; it can be generated by recombinant expression of MiniTrap VEGF in a host cell. The implementation of the method may reduce the amount of one or more proteins of the host cell in the sample. For example, in FIG . 35A and FIG. 35B shows a significant reduction in total host cell proteins in a sample containing MT5 (anti-VEGF protein) using five different affinity chromatography columns containing (i) VEGF ₁₆₅ (SEQ ID NO.: 72); (ii) mAb1 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 73 is the heavy chain and SEQ ID NO.: 74 is the light chain); (iii) mAb2 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 75 is the heavy chain and SEQ ID NO.: 76 is the light chain); (iv) mAb3 (mouse IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 77 is the heavy chain and SEQ ID NO.: 78 is the light chain) and (v) mAb4 (mouse IgG1 against mouse anti-VEGFR1 mAb, where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) because various proteins are able to selectively or specifically bind to MT5. As seen in FIG. 35A and FIG. 35B , eluates from each of the affinity-based preparation methods reduced host cell backs from over 7000 ppm to about 25 ppm and to about 55 ppm, respectively.

Suitable conditions for using affinity chromatography may include, without limitation, equilibration of the affinity chromatography column using an equilibration buffer. After equilibration using, for example, tris hydrochloride at a pH of about 8.3 to about 8.6, the biological sample is loaded onto the affinity chromatography column. After loading the column, the column can be washed one or more times using, for example, an equilibration buffer such as Dulbecco's phosphate-buffered saline (DPBS). Other washes, including those with various buffers, may be used prior to column elution. The elution of the column may depend on the type of buffer, pH and conductivity, other elution conditions may be used, well known to the person skilled in the art. After elution using one or more types of elution buffers, eg glycine at a pH of about 2.0 to about 3.0, the eluted fractions can be neutralized by adding a neutralizing buffer, eg 1 mol Tris at pH 7.5.

In one aspect of the embodiment, the pH of both the wash buffer and the equilibration buffer may be from about 7.0 to about 8.6. In one aspect of the embodiment, the wash buffer may be DPBS. In one aspect, the elution buffer may contain 100 mmol glycine buffer with a pH of about 2.5. In another aspect, the elution buffer may be a buffer with a pH of about 2.0 to about 3.0. In one aspect, the neutralizing buffer may contain 1 mol of Tris with a pH of about 7.5.

In one aspect of the present embodiment, the method may further comprise washing the column with wash buffer. In one aspect of the present embodiment, the method may further include eluting the column with an elution buffer to obtain eluted fractions. In a specific aspect, the amount of host cell proteins in the eluted fractions is significantly reduced compared to the amount of host cell proteins in the biological sample, for example, by about 70%, about 80%, 90%, about 95%, about 98%, or about 99% .

The present invention may include the addition of one or more steps, in no particular order, such as hydrophobic interaction chromatography, affinity chromatography, multimodal chromatography, viral inactivation ( e.g. using low pH), virus filtration, and/or ultra/diafiltration.

In one aspect, the glycosylation profile of the anti-VEGF protein composition is about 40% to about 50% total fucosylated glycans, about 30% to about 55% total sialylated glycans, about 6% to about 15% mannose-5, and about 60% to about 79% galactosylated glycans.

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

In one embodiment, the method may further include formulating the drug substance using a pharmaceutically acceptable excipient. In one aspect, the pharmaceutically acceptable excipient may be selected from the following: water, buffering agents, sugar, salt, surfactant, amino acid, polyol, chelating agent, emulsifier, and preservative. Other excipients well known to the person skilled in the art are within the scope of this embodiment.

In one aspect of the embodiment, the formulation may be suitable for administration to a human subject. In one aspect of the present embodiment, administration may be by intravitreal injection. In one aspect, the composition may have from about 40 to about 200 mg/ml of the protein of interest. In a specific aspect, the protein of interest may be aflibercept, an anti-VEGF antibody, or MiniTrap VEGF.

The composition can be used in a method of treating or preventing angiogenic eye diseases, which may include: 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 ), retinal branch vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, postoperative glaucoma fibrosis, proliferative vitreoretinopathy (PVR), papilledema neovascularization, corneal neovascularization, neovascularization retina, vitreous neovascularization, pannus, pterygoid hymen, vascular retinopathy, diabetic retinopathy in a subject with diabetic macular edema; or diabetic retinopathy ( eg , non-proliferative diabetic retinopathy ( eg , characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53), or proliferative diabetic retinopathy; eg , in a subject not suffering from DME).

Synthesis of oxo-forms

One embodiment of the present invention is directed to one or more methods for synthesizing oxidized forms of a protein using light. In one aspect of the present embodiment, the protein of interest is an anti-VEGF protein. In a specific aspect, the anti-VEGF protein is aflibercept. In another aspect, the anti-VEGF protein is MiniTrap VEGF, including recombinant MiniTrap VEGF. In another aspect of the present embodiment, the anti-VEGF protein is a single chain variable fragment (scFv).

In one aspect of the present embodiment, the sample contains a protein of interest, such as an aflibercept fusion protein with minimal or no oxo variants. The sample is subjected to photo-stress for the synthesis of oxidized forms of aflibercept. In a specific aspect, the sample is subjected to photo-stress through the application of cold white light. In another specific aspect, the sample is subjected to photo-stress through the use of ultraviolet light.

In a specific aspect of the embodiment, a sample containing aflibercept or other anti-VEGF protein is exposed to cold white light for about 30 hours to about 300 hours to produce about 1.5 to about 50-fold enhancement of the modified oligopeptide. These peptides are enzymatically cleaved and analyzed and contain one or more of the group consisting of

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO. : 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIHMTEGR (SEQ ID NO.: 64 ), SDTGRPFVEMYSEIPEIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR/RIIW*DSR/IIW* DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), where H* is histidine oxidized to 2-oxo-histidine, where C* is carboxymethylated cysteine, where M* is oxidized methionine, where W* is oxidized tryptophan, where Y* is is an oxidized tyrosine and where F* is an oxidized phenylalanine. Cleavage can be carried out by the proteases mentioned earlier, for example, trypsin. Oligopeptides can be analyzed using mass spectrometry.

In a certain aspect of an embodiment, a sample containing aflibercept or other anti-VEGF protein is exposed to ultraviolet light for about 4 hours to about 40 hours to produce about 1.5-fold to about 25-fold increase in modified oligopeptide products (obtained from cleavage), wherein the sample contains one or more modified oligopeptides selected from the group consisting of

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO. : 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIHMTEGR (SEQ ID NO.: 64 ), SDTGRPFVEMYSEIPEIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR/RIIW*DSR/IIW* DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), where H* is histidine oxidized to 2-oxo-histidine, where C* is carboxymethylated cysteine, where M* is oxidized methionine, where W* is oxidized tryptophan, where Y* is is an oxidized tyrosine and where F* is an oxidized phenylalanine. Cleavage can be carried out by the proteases mentioned earlier, for example, trypsin. Oligopeptides can be analyzed using mass spectrometry.

Ways to Minimize Yellow-Brown

The present invention provides methods for reducing tan coloration during the production of aflibercept, MiniTrap or the like produced in HOS.

In one embodiment, the method includes culturing a host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, and then harvesting a preparation containing the recombinant protein of interest. In one aspect, the recombinant protein of interest is an anti-VEGF protein. In a specific aspect, the anti-VEGF protein is selected from the group consisting of aflibercept, MiniTrap, recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159, which is incorporated herein by reference in its entirety), scFv, and other anti-VEGF proteins. In one aspect, the method may provide for the production of a recombinant protein of interest, wherein the color of the preparation is characterized using the European BY method or the CIELAB method (b*). In addition, the presence of oxo variants can be analyzed using, for example, LC-MS.

In one aspect of the present embodiment, the mitigation conditions include an increase or decrease in cumulative concentrations of one or more environmental components, e.g., amino acids, metals, or antioxidants, including salts and precursors, corresponding to a decrease in color and protein variants of aflibercept and MiniTrap VEGF. 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 a particular aspect, the reduction of cysteine may be effective in reducing the yellow-brown color of the preparation. Cysteine concentration may also influence oxo variants.

In one embodiment, the method comprises culturing a host cell in COS under suitable conditions, wherein the host cell expresses a recombinant protein of interest, e.g. concentration of cysteine in HOS to no more than about 10 mmol. Examples of a suitable medium include, without limitation, XOC1B, Excell, or the like. As used herein, the term “cumulative amount” refers to the total amount of a particular component added to the bioreactor during cell culture to form COS, including amounts added at the start of culture (COS on day 0) and amounts added subsequently of the component. The amounts of component added to the culture in the seed fermenter system or inoculum prior to production in the bioreactor ( ie , before COS on day 0) are also included when calculating the cumulative amount of the component. The cumulative amount does not depend on the loss of the component over time during cultivation (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may still have different absolute levels, for example, if the component is added to two cultures at different points in time ( for example , if in one culture the entire component is added from the beginning, and in the other culture component is added over time). The cumulative amount is also independent of the in situ synthesis of the component over time during cultivation (eg, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a listed component may still have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by a biological transformation process. The cumulative amount may be expressed in units such as grams or moles of a component, for example.

As used herein, the term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of fluid in the bioreactor at the start of the production batch, including consumption of the initial volume from any inoculum used in culture. For example, if the bioreactor contains 2 liters of cell culture medium at the start of the production batch, and one gram of component X is added on days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L ( i.e. , 4 grams divided into 2 liters). If on day 4 another liter of liquid containing no component X is added to the bioreactor, the cumulative concentration will still be 2 g/l. If on day 5 some liquid has been lost from the bioreactor (eg due to evaporation), the cumulative concentration will still be 2 g/l. The cumulative concentration may be expressed in units such as grams per liter or moles per liter, for example.

In one aspect of this embodiment, the method comprises culturing a host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, harvesting a preparation of the protein produced by the cell, wherein suitable conditions are obtained by reducing the cumulative cysteine concentration ratio from about 1: 10 to 1:29 to a cumulative total amino acid concentration of about 1:50 to about 1:30.

In one embodiment, the method comprises (i) culturing a host cell in COS under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept, and (ii) harvesting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions are obtained by reducing the cumulative concentration of iron in HOS to less than about 55.0 μmol. In one aspect of this embodiment, the preparation obtained by this method has a less yellow-brown color than the preparation obtained by a method in which the cumulative concentration of zinc in COS is more than about 55.0 μmol.

In one embodiment, the method comprises culturing the host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, such as aflibercept. The method further includes harvesting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions are obtained by reducing the cumulative concentration of zinc in the COS to no more than about 0.8 µmol. In one aspect of this embodiment, the preparation obtained by this method has a less yellow-brown color than the preparation obtained by a method in which the cumulative concentration of zinc in COS is more than about 0.8 μmol.

In one embodiment, the method comprises culturing a host cell in COS under suitable conditions, wherein the host cell expresses a recombinant protein of interest, such as aflibercept, and harvesting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions are obtained by reducing the cumulative nickel concentration in HOS to no more than about 0.40 µmol. In one aspect of this embodiment, the preparation obtained by this method has a less yellow-brown color than the preparation obtained by a method in which the cumulative concentration of zinc in COS is more than about 0.40 μmol.

In one embodiment, the method comprises culturing the host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, such as aflibercept. The method further includes harvesting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions are obtained by reducing the cumulative concentration of zinc in the COS to no more than about 56 µmol. In one aspect of this embodiment, the preparation obtained by this method has a less yellow-brown color than the preparation obtained by a method in which the cumulative concentration of zinc in COS is more than about 56 μmol.

In one embodiment, the method comprises culturing the host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, such as aflibercept. The method further includes collecting a preparation of the recombinant protein of interest produced by the cell, wherein the appropriate conditions are obtained by having antioxidants in the COS at a cumulative concentration of from about 0.001 mmol to about 10 mmol for one antioxidant and a cumulative concentration of not more than about 30 mmol if in said COS add some antioxidants. In one aspect of this embodiment, the preparation obtained by this method is less yellow-brown in color than the preparation obtained by the method, and the method does not use antioxidants in COS at a cumulative concentration of less than about 0.01 mmol or more than about 100 mmol. Non-limiting examples of an antioxidant 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-limiting examples of chelating agents include auryl tricarboxylic acid (ATA), deferoxamine (DFO), EDTA, and citrate.

In one embodiment, the method comprises culturing the host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, such as aflibercept. The method further comprises collecting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions include a COS with a cumulative concentration of iron in said COS of less than about 55 μmol, a cumulative concentration of copper in said COS of no more than about 0.8 μmol, a cumulative a nickel concentration in said HOS of not more than about 0.40 µmol, a cumulative zinc concentration in said HOS of no more than about 56 µmol, a cumulative cysteine concentration in said HOS of less than 10 mmol; and/or an antioxidant in said HOS at a concentration of from about 0.001 mmol to about 10 mmol for one antioxidant, and a cumulative concentration of not more than about 30 mmol if several antioxidants are added to said HOS.

In one aspect of the present embodiment, the formulation, made under suitable conditions, reduces the aflibercept and MiniTrap VEGF protein variants to the desired amount of aflibercept and MiniTrap VEGF protein variants (referred to as the "target value" of the aflibercept and MiniTrap VEGF protein variants). In a further aspect of this embodiment, the formulation obtained by applying the appropriate conditions provides a color reduction of the formulations to the desired b* value or BY value (referred to as "target b* value" "target BY value", respectively) if the protein formulation, including including aflibercept and MiniTrap VEGF variants normalized to 5 g/L or 10 g/L. In a further aspect of the present embodiment, a b* target (or BY target) and/or a variant target can be obtained in a formulation in which the titer increases or does not decrease significantly.

These and other aspects of the invention will be better appreciated and understood when viewed in conjunction with the following description and accompanying drawings. The following description, while indicating various embodiments and their many specific details, is provided by way of illustration and not limitation. Many substitutions, modifications, additions or permutations can be made within the scope of the present invention.

BRIEF DESCRIPTION OF GRAPHICS

In FIG. 1 depicts a VEGF MiniTrap generated using an exemplary embodiment containing VEGFR1 (SEQ ID NO.: 34), VEGFR2 (SEQ ID NO.: 36, hinge domain fragment (SEQ ID NO.: 60)) and an Fc fragment cleaved from aflibercept (SEQ ID NO.: 55).

In FIG. 2 shows the proposed mechanism for the oxidation of histidine to 2-oxo-histidine (14 Da).

In FIG. 3 depicts the proposed mechanism for the oxidation of histidine to 2-oxo-histidine (16 Da).

In FIG. 4 shows the proposed mechanism for the oxidation of tryptophan to N-formylkynurenine and kynurenine.

In FIG. 5 depicts an exemplary embodiment for the preparation of aflibercept.

In FIG. 6 depicts an exemplary embodiment of obtaining MiniTrap VEGF.

In FIG. 7 depicts an exemplary embodiment for preparing aflibercept.

In FIG. 8 depicts an exemplary embodiment of making MiniTrap VEGF.

In FIG. 9 is a block diagram of the calculated BY standards compared to the b* value calculated as an exemplary embodiment.

In FIG. 10 depicts the results of an experiment performed to evaluate the percentage of 2-oxo-histidines and tryptophan oxidation (where the underline denotes oxidation of the residue) in oligopeptides from the protease-cleaved AEX loading and flow-through fraction, including reduced and alkylated aflibercept fragments (SEQ ID NO.: 55) .

In FIG. 11 depicts the relative abundance of peptides identified from a peptide mapping assay performed using oligopeptides from protease-cleaved AEX loading and a flow-through fraction (where the underline denotes oxidation of a residue in the peptide sequence), including fragments of aflibercept (SEQ ID NO.: 55).

In FIG. 12A shows a complete overview of the absorbance versus time (minutes) chromatogram for MT4 and MT1 at 350 nm.

In FIG. 12B is an expanded view of the absorbance versus time chromatogram (16-30 minutes) for MT4 and MT1 at 350 nm, including SEQ ID NOs: 21 and 28.

In FIG. 12C shows an expanded view of the absorbance versus time chromatogram (30-75 minutes) for MT4 and MT1 at 350 nm, including SEQ ID NO.: 17, 18, 19, 20.

In FIG. 13 shows the results of an experiment performed to evaluate the percentage of 2-oxo-histidines (and tryptophan double oxidation) in oligopeptides from protease digested MT1 that was processed by AEX chromatography and oligopeptides from protease digested MT1 that was purified from AEX chromatography, including SEQ ID NO.: 17, 18, 19, 20, 21, 28.

In FIG. 14 depicts the results of an experiment carried out to compare the acid forms present in various production batches of MT1 and the acid fractions of the acid obtained by performing strong cation exchange (CEX) chromatography, including SEQ ID NO.: 17, 18, 19, 20, 21, 28 .

In FIG. 15 depicts an exemplary method for enriching acid forms and other variants present in cell culture harvest samples using strong cation exchange chromatography.

In FIG. 16 depicts the fractions formed when performing strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 17 depicts strong cation exchange chromatograms obtained in accordance with an exemplary embodiment for CEX-treated MT1 (before any manufacturing procedure, ≤BY3) and enriched versions of desiallylated MiniTrap (dsMT1) using a dual salt and pH gradient.

In FIG. 18A depicts a 3D chromatogram for an unfractionated baseline control performed by strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 18B depicts a 3D chromatogram for MT1, with fraction 1 showing some of the tail features of the experiment carried out by strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 18C depicts a 3D chromatogram for MT1, fraction 2 function, performed by strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 18D depicts a 3D chromatogram for MT1, fraction 3 function, performed by strong cation exchange chromatography in accordance with an exemplary embodiment.

In FIG. 18E shows a 3D chromatogram for MT1, fraction 4 function, performed by strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 18F depicts a 3D chromatogram for MT1, fraction 5 function, performed by strong cation exchange chromatography in accordance with an exemplary embodiment.

In FIG. 18G shows a 3D chromatogram for MT1, fraction 6 function, performed by strong cation exchange chromatography in accordance with an illustrative embodiment.

In FIG. 18H depicts a 3D chromatogram for MT1, a function of fraction 7, performed by strong cation exchange chromatography in accordance with an exemplary embodiment.

In FIG. 19 shows electropherograms of capillary isoelectric focusing with detection (icIEF) directly in the capillary, made in accordance with an exemplary embodiment to obtain MT1.

In FIG. 20 shows the results of a study on the correlation of exposure to cold white light on MT1 or UVA light with the appearance of oxidized amino acid residues, including SEQ ID NOs: 17, 18, 19, 20, 21, 28, 29, and 83.

In FIG. 21 shows 3D chromatograms of SEC-PDA (size exclusion chromatography coupled with a photodiode array detector) MT1 exposed to CBS with absorption at ~350 nm ( see e.g. ~350 nm circular highlight) according to an exemplary embodiment, where A is chromatogram at T=0, B denotes chromatogram at 0.5x ICH, C denotes chromatogram at 2.0x ICH and D denotes MT1 vials (normalized to 80 mg/ml) exposed to CBS for various time intervals.

In FIG. 22 shows 3D SEC-PDA chromatograms of UVA-exposed MT1 with absorption at ~350 nm ( see e.g. ~350 nm circular highlight) according to an illustrative embodiment, where A denotes the chromatogram at T=0, B denotes the chromatogram at 0 .5x ICH, C denotes the chromatogram at 2.0x ICH and D denotes MT1 in vials (normalized to 80 mg/ml) exposed to UVA for various time intervals.

In FIG. 23 A shows the A320/280 absorbances calculated from SEC-PDA chromatograms for CWL stressed samples (top panel) and B is a plot of the A320/280 absorbances of size variants in CWL-stressed samples (bottom panel). ), and the samples are exposed in accordance with an illustrative embodiment.

In FIG. 24 A shows A320/280 absorbances calculated from SEC-PDA chromatograms for UVA stressed samples (upper panel) and B is a plot of A320/280 absorbances for size variants in UVA exposed samples (lower panel). ), and the samples are exposed in accordance with an illustrative embodiment.

In FIG. 25A depicts scale assessment of the effect of incubation of various components with aflibercept on color formation (CIE L*, a*, b*, predicted b value); and B is a graph of the actual value versus the predicted value b.

In FIG. 26 shows the effect of low cysteine and low metal COS on aflibercept titer ( A ), viable cell concentration ( B ), viability ( C ), ammonia content ( D ) and osmolality ( E ).

In FIG. 27 is a graph showing predicted crop color profile (visible as day 13 b* values) with increase/decrease in metal and cysteine content in accordance with an exemplary embodiment.

In FIG. 28 ( AB ) depicts the effect of incubation of various components with aflibercept in spent COS on color formation (CIE L*, a*, b*, predicted value b) ( A ); and plotted with scaled predicted impacts on the value of b( B ).

In FIG. 28C depicts scaled estimated effects of incubation of various components with aflibercept in COS on color development (CIE L*, a*, b*, predicted value b) in shake flask culture.

In FIG. 28D depicts the effect of incubation of mesylate salt of hypotaurine and deferoxamine (DFO) with aflibercept in spent COS on color generation (CIE L*, a*, b*, predicted value "b").

In FIG. 28E shows the effect of incubating the various components alone with aflibercept from a shake flask culture on color formation (CIE L*, a*, b*, predicted value "b").

In FIG. 29 is a graph showing the effect of adding uridine, manganese, galactose and dexamethasone to COS on the titer of aflibercept produced.

In FIG. 30 is a graph showing the effect of adding uridine, manganese, galactose, and dexamethasone to COS on the viability of aflibercept-expressing cells that form aflibercept.

In FIG. 31 is a graph showing the effect of adding uridine, manganese, galactose, and dexamethasone to COS on the number of viable aflibercept-expressing cells in which aflibercept is formed.

In FIG. 32 is a graph showing standard host cell protein uptake versus protein (ng/mL) curve prepared using host cell protein standard solutions from Cygnus 3G (F550).

In FIG. 33 is an image of an SDS-PAGE analysis performed using a non-reducing SDS-PAGE sample buffer.

In FIG. 34 is an image of an SDS-PAGE analysis performed using SDS-PAGE Sample Recovery Buffer.

In FIG. 35A is a graph of total host cell protein found in loading solution, eluted fractions from affinity column 1-3 containing VEGF ₁₆₅ , mAb1 and mAb2, respectively.

In FIG. 35B is a graph of total host cell proteins found in loading solution, eluted fractions from affinity column 1, 2, 4 and 5 containing VEGF ₁₆₅ , mAb1, mAb3 and mAb4, respectively.

In FIG. 36 shows SEC profiles of VEGF MiniTrap A before and B after affinity chromatography.

In FIG. 37 is a cartoon depiction of a VEGF MiniTrap kinetic study on VEGF ₁₆₅ where the studied VEGF MiniTrap constructs were generated before and after affinity chromatography was obtained according to some exemplary embodiments.

In FIG. 38 shows SPR sensorgrams from a VEGF MiniTrap kinetic study to VEGF ₁₆₅ , where the studied VEGF MiniTrap constructs were generated before and after affinity chromatography was obtained according to some exemplary embodiments.

In FIG. 39 is a graph of the total host cell protein found in the loading solution, eluted fractions from the affinity chromatography column, reapplied to columns containing VEGF ₁₆₅ , mAb1 and mAb2.

In FIG. 40 depicts the structure of VEGF MiniTrap MT1 (SEQ ID NO: 46) according to an exemplary embodiment.

In FIG. 41 depicts the structure of a VEGF MiniTrap MT6 (SEQ ID NO: 51) according to an exemplary embodiment.

In FIG. 42 shows relative absorbance total ion chromatograms (TIC) versus time (minutes) for native SEC-MS analysis of MT1, MT5 and MT6 and an enlarged view of the low molecular weight region from TIC.

In FIG. 43 shows the main peak deconvolution mass spectra for MT1 and MT5 to confirm the identity of the MiniTrap dimer with clarification for some PTMs.

In FIG. 44 shows deconvoluted mass spectra of the main peak for MT6, confirming the identity of single stranded MiniTrap with elucidation for some PTMs.

In FIG. 45A is a plot of relative absorbance versus time (minutes) for low molecular weight impurities in MT1.

In FIG. 45B shows the mass spectra of low molecular weight impurities in MT1.

In FIG. 46 shows relative absorbance versus time (minutes) for MT1, indicating the absence of the FabRICATOR enzyme used to cleave aflibercept on MT1.

In FIG. 47 shows the relative absorbance versus time (minutes) for low molecular weight impurities in MT5.

In FIG. 48 shows relative absorbance versus time (minutes) for low molecular weight impurities in MT6.

In FIG. 49A is a graph of absorbance versus time (minutes) obtained from a HILIC-UV/MS run on a VEGF MiniTrap MT6, where the graph shows the elution of the main peak at 21 minutes and O-glycans at about 21.5 minutes.

In FIG. 49B depicts a mass spectrum obtained from HILIC-UV/MS for VEGF MiniTrap MT6 showing the main peak at 47985.8 Da.

In FIG. 49C depicts VEGF MiniTrap MT6 O-glycan mass spectra obtained by HILIC-UV/MS.

In FIG. 50 is a depiction of a VEGF MiniTrap dimer where the disulfide bridge at the hinge region (SEQ ID NO.: 83) can be parallel or crossed.

In FIG. 51 shows the relative frequency distribution of glycans observed in Asn36, including SEQ ID NO: 100, among MT1, MT5 and MT6.

In FIG. 52 shows the relative frequency distribution of glycans observed in Asn68, including SEQ ID NO: 101, among MT1, MT5 and MT6.

In FIG. 53 shows the relative distribution frequency of glycans observed in Asn123, including SEQ ID NO: 102, among MT1, MT5 and MT6.

In FIG. 54 shows the relative distribution frequency of glycans observed in Asn196, including SEQ ID NO: 103, among MT1, MT5 and MT6.

In FIG. 55 shows the analysis of the released N-linked glycan by hydrophilic interaction chromatography (HILIC) combined with fluorescence detection and mass spectrometric analysis (full scale and reduced).

In FIG. 56 shows HILIC-FLR chromatograms for MT1, MT5 and MT6.

In FIG. 57 shows the analysis of the released N-linked glycan by HILIC in combination with detection by fluorescence and mass spectrometric analysis (full-scale, reduced and normalized).

In FIG. 58A is a table detailing the identification and quantification of glycans from VEGF MiniTrap MT1, MT5, and MT6 samples.

In FIG. 58B is a table detailing the identification and quantification of glycans from VEGF MiniTrap MT1, MT5, and MT6 samples.

In FIG. 58C is a table detailing the identification and quantification of glycans from VEGF MiniTrap MT1, MT5 and MT6 samples.

In FIG. 59 depicts an exemplary manufacturing procedure for making a MiniTrap in accordance with an exemplary embodiment.

DETAILED DESCRIPTION OF THE INVENTION

Angiogenesis, the growth of new blood vessels from a pre-existing vasculature, is a highly organized process that is critical for proper embryonic and postnatal vascular development. Abnormal or pathological angiogenesis is a hallmark of cancer and certain retinal diseases where upregulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) results in increased endothelial proliferation, changes in vascular morphology, and increased vascular permeability. Elevated levels of VEGF have been found in the vitreous and retinal vasculature in patients with various ocular diseases. Blocking VEGF activity has also become the therapy of choice for the treatment of DME, wet AMD, CNV, retinal vein occlusion, and other angiogenesis-related ocular diseases.

As used herein, aflibercept is one such anti-VEGF protein comprising a fully human amino acid sequence comprising a second human VEGFR1 Ig domain and a third human VEGFR2 Ig domain expressed as a linear fusion to (Fc) human IgG1. Aflibercept binds all forms of VEGF-A (VEGF) but also binds PlGF and VEGF-B. Several other homodimeric MiniTrap VEGFs have been generated as enzymatically digested products from aflibercept or recombinantly expressed directly from host cell lines. One such example of MiniTrap VEGF is shown in FIG. . 1 . This figure shows terminal lysine (k), some culture processes remove this terminal lysine and others do not. Fig. 1 illustrates a process in which terminal lysine remains. In general, aflibercept covers both the presence and absence of terminal lysine.

As shown in this document, the present invention partially discloses the production of proteins against VEGF (example 1) using HOS. Analysis of aflibercept-containing solutions prepared using certain COSs showed a certain color property, eg an intense yellow-brown color. The color intensity of the solution depends on the applied HOS. Not all COSs tested provided a distinct yellow-brown color after the solutions were normalized to 5 g/L.

A color, eg yellow-brown, in an injectable therapeutic drug solution may be an undesirable characteristic. This can be an important parameter used to determine whether a drug product satisfies a given level of purity and quality for a particular therapeutic agent. A color such as yellow-brown observed during the preparation of a biological product may be caused by chemical modifications of this biological product, degradation products of formulation excipients, or degradation products formed as a result of the reaction of biological and formulation excipients. However, such information may be useful in understanding the cause of the color change. It can also help in developing both short and long term storage conditions to prevent modifications that contribute to such color change.

The present inventors observed that the use of AEX during preparation of the anti-VEGF protein solution minimized tan coloration. In addition, the present inventors have found that tan coloration can be reduced by modifying the cell culture used to produce a recombinant protein such as aflibercept or a modified aflibercept such as MiniTrap.

The present invention encompasses anti-VEGF proteins and their production using COS. Furthermore, the present invention is based on the identification and optimization of upstream and downstream processes for protein production.

As shown in this document, some of the examples below describe the production of anti-VEGF protein (Example 1), the production of oxidized anti-VEGF proteins (Example 4), methods for reducing oxidized anti-VEGF proteins by optimizing the culture medium (Example 5), and for by optimizing methods of obtaining (example 2).

A number of recent patent applications and granted patents are directed to the description of various forms of aflibercept and methods for its preparation, but none of them describe or suggest the anti-VEGF compositions and methods for their preparation described in this document. See, for example , U.S. Patent Application No. 16/566847 from Coherus Biosciences Inc., U.S. Patent No. 10646546 from Sam Chun Dang Pharm. Co., Ltd., U.S. Patent No. 10576128 from Formycon AG, International Application No. PCT/US2020/015659 from Amgen Inc. and US Pat. Nos. 8,956,830; 9217168; 9487810; 9663810; 9926583 and 10144944 from Momenta Pharmaceuticals, Inc.

I. Explanation of selected terms

Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning, which is usually understood by a person skilled in the field to which the present invention belongs. Methods and materials similar or equivalent to those described in this document, known to a person skilled in the art, can be used in the practice of specific embodiments described in this document. All referenced publications are incorporated herein by reference in their entirety.

The singular forms are to be understood as meaning "at least one" and the terms "about" and "approximately" are to be understood as subject to standard variations, as will be understood by those skilled in the art, and where ranges are indicated, the final terms are included. points.

Used in this document, the term "angiogenic disease of the eye" means any disease of the eye caused by or associated with the growth or proliferation of blood vessels or the permeability of blood vessels.

As used herein, "chemically defined media" or "chemically defined media" (abbreviated "XOS" in both cases) refers to a synthetic culture medium in which the identity and concentration of all ingredients are determined. The chemically defined medium does not contain bacterial, yeast, animal or plant extracts, animal serum or plasma, although individual components of plant or animal origin ( eg proteins, polypeptides , etc. ) may be added. Chemically defined media may contain inorganic salts such as phosphates, sulfates, and the like, necessary to support growth. The carbon source is specified and is typically a sugar such as glucose, lactose, galactose, and the like, or other compounds such as glycerol, lactate, acetate, and the like. While some chemically defined culture media also use phosphate salts as a buffer, other buffers such as sodium bicarbonate, HEPES, citrate, triethanolamine, and the like can be used. Examples of commercially available c media include, but are not limited to, various Dulbecco's Modified Eagle's (DME) media (Sigma-Aldrich Co; SAFC Biosciences, Inc.), Hem's formula (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various EX- CELL (Sigma-Aldrich Co; SAFC Biosciences, Inc.), various IS CHO-CD media (FUJIFILM Irvine Scientific), combinations thereof, and the like. Methods for preparing chemically defined culture media are known in the art, for example, in US Pat. Nos. 6,171,825 and 6,936,441; through a link.

As used herein, the term “cumulative amount” refers to the total amount of a particular component added to the bioreactor during cell culture to form COS, including amounts added at the start of culture (COS on day 0) and amounts added subsequently of the component. The amounts of component added to the culture in the seed fermenter system or inoculum prior to production in the bioreactor ( ie , before COS on day 0) are also included when calculating the cumulative amount of the component. The cumulative amount does not depend on the loss of the component over time during cultivation (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may still have different absolute levels, for example, if the component is added to two cultures at different points in time ( for example , if in one culture the entire component is added from the beginning, and in the other culture component is added over time). The cumulative amount is also independent of the in situ synthesis of the component over time during cultivation (eg, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a listed component may still have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by a biological transformation process. The cumulative amount may be expressed in units such as grams or moles of a component, for example.

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 the production batch, including consumption of the initial volume from any inoculum used in culture. For example, if the bioreactor contains 2 liters of cell culture medium at the start of the production batch, and one gram of component X is added on days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L ( i.e. , 4 grams divided into 2 liters). If on day 4 another liter of liquid containing no component X is added to the bioreactor, the cumulative concentration will still be 2 g/l. If on day 5 some liquid has been lost from the bioreactor (eg due to evaporation), the cumulative concentration will still be 2 g/l. The cumulative concentration may be expressed in units such as grams per liter or moles per liter, for example.

As used herein, the term “composition” refers to a protein of interest that is formulated together with one or more pharmaceutically acceptable media. In one aspect, the protein of interest is aflibercept and/or MiniTrap. In some illustrative embodiments, the amount of protein of interest in the formulation may vary 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, about 0.6 mg/ml, about 0.7 mg/ml, about 0.8 mg/ml , about 0.9 mg/ml, about 1 mg/ml, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml , about 8 mg/ml, about 9 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 5 mg/ml, about 80 mg /ml, about 85 mg/ml, about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml , about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, about 300 mg/ml, about 325 mg/ml, about 350 mg/ml, about 375 mg/ml, about 400 mg/ml, about 425 mg/ml, about 450 mg/ml, about 475 mg/ml, about 500 mg /ml, about 525 mg/ml, about 550 mg/ml, about 575 mg/ml or about 600 mg/ml. In some exemplary embodiments, the pH of the composition 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.

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

As used herein, the term "ultrafiltration" or "UF" may include a membrane filtration process similar to reverse osmosis, using hydrostatic pressure to force water through a semi-permeable membrane. Ultrafiltration is described in detail in Leos J. Zeman & Andrew L. Zydney, Microfiltration and ultrafiltration: principles and applications (1996), the full contents of which are incorporated herein. For ultrafiltration, filters with a pore size of less than 0.1 µm can be used. When using filters having such a small pore size, sample volume can be reduced by allowing sample buffer to pass through the filter while proteins remain behind the filter.

As used herein, "diafiltration" or "DF" may include the method of using ultrafilters to remove and replace salts, sugars, and non-aqueous solvents, to separate free forms from bound forms, to remove low molecular weight material, and/or to provide a rapid change in ionic and/or pH avg. Dissolved microparticles are most effectively removed by adding a solvent to the ultrafiltration solution at a rate approximately equal to the ultrafiltration rate. This ensures that the microparticles are washed out of the solution in a constant volume. In certain illustrative embodiments of the present invention, the diafiltration step can be used to replace various buffers used in conjunction with the present invention, for example, prior to chromatography or other manufacturing steps, as well as to remove impurities from a protein preparation. Used in this document, the term "technology further processes" refers to one or more techniques used after technologies of previous processes to obtain protein. Further process technology includes, for example, obtaining a protein product, using, for example, affinity chromatography, including protein A affinity chromatography, as well as affinity chromatography, which uses a solid phase with a well-defined molecule, such as VEGF, which can interact with its related form such as VEGF receptor (VEGF R), ion exchange chromatography such as anion or cation exchange chromatography, hydrophobic interaction chromatography or displacement chromatography.

The expression "recombinant host cell" (or simply "host cell") includes a cell into which a recombinant expression vector encoding a protein of interest has been introduced. It should be understood that such a term is intended to refer not only to the particular cell in question, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may not actually be identical to the original cell, but may still be included within the scope of the term "host cell" as used herein. In one embodiment, the host cells include prokaryotic and eukaryotic cells selected from any biological kingdom. In one aspect, eukaryotic cells include protozoan, fungal, plant, and animal cells. In a further aspect, host cells include eukaryotic cells, such as plant and/or animal cells. The cells may be a mammalian cell, a fish cell, an insect cell, an amphibian cell, or a feathered cell. In a specific aspect, the host cell is a mammalian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Virginia) and other repositories, as well as from commercial suppliers. Cells that can be used in the processes of the present invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese Hamster Ovary (CHO) cells such as CHO-K1 (ATCC CCL-61), DG44 (Chasin et al . , 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al ., 1997, Biochemistry, 36: 10901-10909; and WO 01/92337 A2), dihydrofolate reductase-negative CHO cells (CHO/-DHFR , Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci. USA , 77:4216), and dp12.CHO cells (US Pat. No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney CV1 cells transformed with SV40 (COS cells, COS-7, ATCC CRL-1651); HEK 293 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) and resistant cell lines and strains thereof ( e.g. human embryonic kidney cells ( e.g. 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al ., 1977, J. Gen. Virol. , 36:59), newborn hamster kidney cells (BHK, ATCC CCL-10), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23 :243-251) ; human cervical carcinoma cells (HELA, ATCC CCL-2); dog kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP- G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC CCL-51), Buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci ., 383:44-68); MCR 5 cells; FS4 cells; retinal PER-C6 cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, cells LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13 cells, 2RA cells, WISH cells, BS-CI cells, LLC-MK ₂ cells, Clone cells M-3, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK ₁ cells, PK(15) cells, GH ₁ cells, GH ₃ cells, L2 cells, LLC-RC 256 cells , MH ₁ C ₁ cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof), fibroblast cells from any tissue or organ (including, without limitation, heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblastic and fibroblast-like cell lines ( eg . , TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, Citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, cells Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl ₁ cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C ₃ H/IOTI cells /2, HSDM ₁ C ₃ cells, KLN205 cells, McCoy cells, mouse L cells, 2071 strain cells (mouse L cells), LM strain cells (mouse L cells), L-MTK cells (mouse L cells) , clones NCTC 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, C _II cells, and Jensen cells, or derivatives thereof), or any other cell type known to the person skilled in the art.

The term "host cell proteins" (HCP) as used herein includes a protein derived from a host cell and may not be related to the required protein of interest. Host cell proteins can be a manufacturing contaminant that can be generated during a manufacturing process and can include three main categories: cell substrate derivatives, cell culture derivatives, and downstream derivatives. Impurities derived from the cell substrate include, but are not limited to, host-derived proteins and nucleic acid (genomic, vector, or total DNA of the host cell). Impurities derived from cell culture include, without limitation, inducers, antibiotics, serum, and other media components. Impurities formed during subsequent steps include, but are not limited to, enzymes, chemical and biochemical processing agents ( e.g. , cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts ( e.g. , heavy metals, arsenic, non-metal ion), solvents, carriers, ligands ( e.g. monoclonal antibodies) and other leachable products.

In some exemplary embodiments, the host cell protein may have a pI in the range of about 4.5 to about 9.0. In an exemplary embodiment, 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.

As used herein, the term "hydrolysing agent" refers to any one or combination of a large number of different agents that can effect protein cleavage. Non-limiting examples of hydrolyzing agents that can effect enzymatic cleavage include the protease fromAspergillus saitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N, chymotrypsin, aspergillopepsin I, protease LysN (Lys-N), endoprotease LysC (Lys-C), endoprotease Asp-N (Asp-N), endoprotease Arg- C (Arg-C), Glu-C (Glu-C) endoprotease or outer membrane protein T (OmpT), Streptococcus pyogenes immunoglobulin-cleaving enzyme (IdeS), thermolysin, papain, pronase, V8 protease or biologically active fragments or homologues thereof , or combinations thereof. Non-limiting examples of hydrolyzing agents that can effect non-enzymatic digestion include the use of high temperature, microwave radiation, ultrasound, high pressure, infrared radiation, solvents (ethanol and acetonitrile are non-limiting examples), immobilized enzyme digestion (IMER), enzymes with immobilized magnetic particles, and embedded immobilized enzymes. For a recent review discussing available methods for protein digestion see Switzaret al., "Protein Digestion: An Overview of the Available Techniques and Recent Developments" (Linda Switzar, Martin Giera & Wilfried M. A. Niessen,Protein Digestion:An Overview of the Available Techniques and Recent Developments, 12 Journal of Proteome Research 1067-1077 (2013), the full contents of which are incorporated herein). One or a combination of hydrolyzing agents can cleave peptide bonds in a protein or polypeptide in a sequence-specific manner, creating a predictable collection of shorter peptides. The ratio of hydrolysing agent to protein and the time required for cleavage can be appropriately selected to obtain optimal protein cleavage. If the ratio of enzyme to substrate is unacceptably high, the corresponding high cleavage rate does not allow sufficient time for analysis of the peptides by the mass spectrometer and the coating sequence will be compromised. On the other hand, a low E/S ratio would require a long split and hence a long acquisition time. The ratio of enzyme to substrate may be from about 1:0.5 to about 1:200. As used herein, the term "cleavage" refers to the hydrolysis of one or more peptide bonds of a protein. There are several approaches to carrying out protein digestion in a biological sample using a suitable hydrolysing agent, for example, enzymatic digestion or non-enzymatic digestion. One of the widely used methods for digesting proteins in a sample involves the use of a protease. There are many proteases, and each of them has its own characteristics in terms of specificity, efficiency and optimal cleavage conditions. Proteases refer to both endopeptidases and exopeptidases, which are classified based on the ability of the protease to cleave non-terminal or terminal amino acids in a peptide. Alternatively, proteases also belong to six different classes - aspartic, glutamic and metalloprotease, cysteine, serine and threonine proteases, which are classified based on the mechanism of catalysis. The terms "protease" and "peptidase" are used interchangeably to refer to enzymes that hydrolyze peptide bonds.

The term “co-with” indicates that the components, an anti-VEGF composition of the present invention together with another agent, such as an anti-ANG2 antibody, may be combined in a single composition for simultaneous delivery, or may be combined separately in two or more compositions ( e.g. , a set including each component). Components administered together may be administered to a subject at a different time than when the other component is administered; for example, each administration may be administered non-simultaneously ( eg , separately or sequentially) at intervals over a specified period. Individual components administered together may also be administered substantially simultaneously ( eg , at exactly the same time or after a clinically non-significant period) during the same administration session. In addition, the individual components co-administered can be administered to the subject by the same or different route, for example, an aflibercept formulation together with another agent, such as an anti-ANG2 antibody, where the aflibercept formulation contains about 15% or less of its variants.

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

As used herein, "affinity chromatography" may include separations, including any method by which two substances are separated based on their affinity for a chromatographic material. This may involve exposing the substances to a column containing a suitable chromatographic affinity medium. Non-limiting examples of such a chromatographic medium include, but are not limited to, protein A resin, protein G resin, affinity supports containing an antigen against which the binding molecule ( e.g. , an antibody) has been raised, a protein capable of binding to a protein of interest, and affinity supports, containing an Fc-binding protein. In one aspect, the affinity column may be equilibrated with an appropriate buffer prior to sample loading. An example of a suitable buffer would be Tris/NaCl buffer pH 7.0 to 8.0. A skilled person can design a suitable buffer without undue burden. After this equilibration, the sample can be loaded onto the column. After loading the column, it can be washed one or more times, using, for example, an equilibrating buffer. Other washes, including those with various buffers, may be used prior to column elution. The affinity column can then be eluted using an appropriate elution buffer. An example of a suitable elution buffer would be acetic acid/NaCl buffer, pH about 2.0 to 3.5. Again, the skilled artisan can design an appropriate elution buffer without undue burden. The eluate can be controlled using techniques well known to those skilled in the art, including UV, for example absorbance at 280 nm can be used, especially if the sample of interest contains aromatic rings ( e.g. proteins with aromatic amino acids such as tryptophan).

In the context of this document, "ion exchange chromatography" may refer to separations, including any method by which two substances are separated based on the difference in their respective ionic charges, either on the molecule of interest and/or the chromatographic material as a whole, or locally in specific regions. molecule of interest and/or chromatographic material, and thus either a cation exchange material or an anion exchange material can be used. Ion exchange chromatography separates molecules based on differences between the local charges of the molecules of interest and the local charges of the chromatographic material. A packaged ion exchange chromatography column or ion exchange membrane device can be operated in bind-elution mode, flow fraction mode, or hybrid mode. After washing the column or membrane device with equilibration buffer or other buffer, product recovery can be achieved by increasing the ionic strength ( i.e., conductivity) of the elution buffer so that it can compete with the solute for charged sites on the ion exchange matrix. Changing the pH and therefore changing the charge of the solute may be another way to achieve elution of the solute. The change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution). Anionic or cationic substituents may be attached to matrices in order to obtain anionic or cationic chromatography supports. Non-limiting examples of anion exchange substituents include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE), and quaternary amino (Q) groups. Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Cellulosic ion exchange media or support may include DE23™, DE32™, DE52™, CM-23™, CM-32™, and CM-52™ available from Whatman Ltd. Maidstone, Kent, UK Crosslinked and SEPHADEX® based ion exchange materials are also known. For example, DEAE-, QAE-, CM- and SP-SEPHADEX® and DEAE-, Q-, CM- and S-SEPHAROSE® and SEPHAROSE® Fast Flow, and Capto™ S, all available from GE Healthcare. In addition, both DEAE and CM derived ethylene glycol methacrylate copolymer such as TOYOPEARL™ DEAE-650S or M and TOYOPEARL™ CM-650S or M are available from Toso Haas Co., Philadelphia, Pa., or Nuvia S and UNOSphere ™ S from BioRad, Hercules, Calif., Eshmuno® S from EMD Millipore, MA.

As used herein, the term "hydrophobic interaction chromatography resin" may include a solid phase that may be covalently modified with phenyl, octyl, butyl, and the like. It can use the properties of hydrophobicity to separate molecules from each other. In this type of chromatography, hydrophobic groups such as phenyl, octyl, hexyl, or butyl can form the stationary phase of the column. Molecules such as proteins, peptides, and the like pass through a HIC column (Hydrophobic Interaction Chromatography) which has one or more hydrophobic regions on its surface or has hydrophobic pockets and can 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 phenyl Sartobind (Sartorius Corporation, New York, USA).

As used herein, the term "mixed mode chromatography" or "multimodal chromatography" (abbreviation for both "MMC") includes a chromatography method in which solutes interact with a stationary phase through more than one interaction mode or mechanism. MMC can be used as an alternative or complementary tool to conventional reverse phase (RP), ion exchange (IEX) and normal phase (NP) chromatography. Unlike RP, NP, and IEX chromatography, in which hydrophobic interaction, hydrophilic interaction, and ionic interaction, respectively, are the dominant interaction modes, a combination of two or more of these interaction modes can be used in mixed mode chromatography. The mixed mode chromatography medium can provide unique selectivity that cannot be reproduced by single mode chromatography. Mixed mode chromatography can also offer potential cost savings, longer column life and operational flexibility compared to affinity based methods. In some exemplary embodiments, the mixed mode chromatography medium may be composed of mixed type ligands bound to an organic or inorganic support, sometimes referred to as a host matrix, either directly or through a spacer. The support may be in the form of particles such as substantially spherical particles, a monolith, a filter, a membrane, a surface, capillaries, etc. In some exemplary embodiments, the support may be made from a natural polymer such as a cross-linked carbohydrate material such as agarose , agPV, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc. To obtain high adsorption capacity, the substrate can be porous and the ligands are then attached to the outer surfaces as well as the pore surfaces. Such base polymer supports can be prepared according to standard methods such as reverse slurry gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire contents of which are incorporated herein). Alternatively, the support may be made from a synthetic polymer such as cross-linked synthetic polymers such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, and the like. Such synthetic polymers can be obtained by standard methods, for example, "Styrene based polymer supports developed by suspension polymerization" (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire content of which is incorporated herein) . Porous natural or synthetic polymer substrates are also available from commercial sources such as GE Healthcare, Uppsala, Sweden.

As used herein, the term "mass spectrometer" includes a device capable of identifying specific types of molecules and accurately measuring their masses. The term is intended to include any molecular detector by which a polypeptide or peptide can be characterized. A mass spectrometer can include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to form gas phase ions. Atoms, molecules or clusters of the analyte can be transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or by separate methods. The choice of ion source depends on the application. In some exemplary embodiments, the mass spectrometer may be a tandem mass spectrometer. Used in this document, the term "tandem mass spectrometry" includes a technique in which structural information about the molecules of the sample is obtained through the application of several stages of mass selection and isotope separation. A prerequisite is the transformation of the sample molecules into the gas phase and ionization in such a way that the fragments are formed in a predictable and controlled way after the first stage of mass selection. Multi-step MS/MS, or MS ⁿ , can be performed by first selecting and isolating the precursor ion (MS ² ), fragmenting it, isolating the primary fragment ion (MS ³ ), fragmenting it, isolating the secondary fragment (MS ⁴ ), and so on. until meaningful information can be obtained or the fragment ion signal is detectable. Tandem MS has been successfully performed with a wide variety of analyzer combinations. The choice of analyzer for a particular application can be determined by many different factors, such as sensitivity, selectivity, and speed, as well as size, cost, and availability. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but there are also hybrids where temporal tandem parsers are coupled spatially or to spatial tandem parsers. A spatially tandem mass spectrometer includes an ion source, a precursor ion activation device, and at least two non-capturing mass analyzers. Specific mass/charge separation functions can be designed such that ions are sampled in one section of the instrument, dissociated in the intermediate region, and product ions are then transferred to another analyzer for mass/charge separation and data acquisition. In a time-tandem mass spectrometer, ions produced in an ion source can be captured, separated, fragmented, and separated by mass/charge in the same physical device. The peptides detected by the mass spectrometer can be used as surrogate representatives of the intact protein and their post-translational modifications. They can be used to characterize proteins by comparing experimental and theoretical MS/MS data, the latter generated from candidate peptides in a protein sequence database. Characterization includes, without limitation, amino acid sequencing of protein fragments, protein sequencing determination, de novo protein sequencing determination, post-translational modification location or post-translational modification determination, or comparability analysis, or combinations thereof.

As used herein, a "Mini-Trap" or "MiniTrap" or "MiniTrap binding molecule" is capable of binding to a VEGF molecule. Such MiniTraps may include (i) chimeric polypeptides as well as (ii) multimeric ( eg dimeric) molecules containing two or more polypeptides that are non-covalently linked, eg via one or more disulfide bridges. MiniTrap can be produced through chemical modification, enzymatic activity, or produced recombinantly.

As used herein, a "VEGF MiniTrap" or "VEGF MiniTrap binding molecule" can be a molecule or complex of molecules that binds to VEGF and has one or more sets of VEGF receptor Ig-like domains (or variants thereof) ( e.g. , Ig domain 2 VEGFR1 and/or Ig domain 3 and/or 4 VEGFR2) and a modified or absent multimerizing component (MC), for example, in which MC is a modified immunoglobulin Fc. The modification may result from proteolytic cleavage of a VEGF decoy ( e.g. aflibercept or conbercept) or direct expression of the resulting polypeptide chains with a truncated MC sequence. (See the molecular structure depicted in Fig . 1. ) Fig. 1 is a molecular image of VEGF MiniTrap, which is a proteolysis product of aflibercept with IdeS Streptococcus pyogenes . Depicted is a homodimeric molecule having an Ig hinge domain fragment linked by two parallel disulfide bonds. The VEGFR1 domain, the VEGFR2 domain, and the hinge domain fragment (MC) are indicated. The point in the aflibercept where IdeS cleavage occurs is indicated by the "//" sign. The Fc fragment cleaved from aflibercept is also labeled. One such chimeric polypeptide that does not dimerize can also be a VEGF MiniTrap if it has VEGF binding activity. The term "VEGF MiniTrap" includes a single polypeptide containing a first set of one or more Ig domains of the VEGF receptor (or variants thereof), without an MC, but fused to a linker ( e.g. , a peptide linker) with one or more additional sets of one or more Ig -domains of the VEGF receptor (or variants thereof). The VEGF binding domains in the VEGF MiniTrap of the present invention may be identical or different (see WO2005/00895, which is incorporated herein in its entirety).

For example, in an embodiment of the present invention, the unmodified immunoglobulin Fc domain contains the amino acid sequence, or amino acids 1-226 thereof:

TPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO.: 33; where X ₁ is L _or P and _{X 2} is A or T)

Inhibition of VEGF includes, for example, antagonizing VEGF that binds to the VEGF receptor, for example, by competing with the VEGF receptor for binding to VEGF ( eg , VEGF ₁₁₀ , VEGF ₁₂₁ and/or VEGF ₁₆₅ ). Such inhibition may result in inhibition of VEGF-mediated VEGFR activation, such as inhibition of luciferase expression in a cell line ( eg , HEK293) expressing a chimeric VEGF receptor ( eg , its homodimer) having extracellular VEGFR domains fused to intracellular domains of IL18Rα and/or IL18Rβ on the cell surface, as well as having an NFkB-luciferase-IRES-eGFP reporter gene, such as the HEK293/D9/Flt-IL18Rα/Flt-IL18Rβ cell line, as set forth herein.

VEGF receptor Ig domain components from the VEGF MiniTrap of the present invention may include

(i) one or more immunoglobulin-like (Ig) domains 2 of VEGFR1 (Flt1) (R1D2),

(ii) one or more VEGFR2 Ig domains 3 (Flk1 or KDR) (Flk1D3) (R2D3),

(iii) one or more VEGFR2 Ig domains 4 (Flk1 or KDR) (Flk1D4) (R2D4) and/or

(iv) one or more VEGFR3 Ig domains 3 (Flt4) (Flt1D3 or R3D3).

Immunoglobulin-like domains of VEGF receptors may be referred to herein as "Ig" VEGFR domains. VEGFR Ig domains referred to herein, e.g., R1D2 (which may be referred to herein as VEGFR1(d2)), R2D3 (which may be referred to herein as VEGFR2(d3)), R2D4 (which may be referred to herein as VEGFR2( d4)) and R3D3 (which may be referred to herein as VEGFR3(d3)) are intended to encompass not only the entire wild-type Ig domain, but also variants thereof that substantially retain the functional characteristics of the wild-type domain, e.g., retain the ability to form a functional domain VEGF binding when incorporated into VEGF MiniTrap. One skilled in the art will appreciate that numerous variants of the above Ig domains can be generated that retain substantially the same functional characteristics as the wild-type domain.

The present invention provides a VEGF MiniTrap polypeptide containing the following domain structure:

((R1D2)-(R2D3)) _a -linker-((R1D2)-(R2D3)) _b ;

((R1D2)-(R2D3)-(R2D4)) _c -linker-((R1D2)-(R2D3)-(R2D4)) _d ;

((R1D2)-(R2D3)) _e -(MC) _g ;

((R1D2)-(R2D3)-(R2D4)) _f -(MC) _g ;

Where

R1D2 is the Ig domain 2 (D2) of the VEGF 1 receptor (VEGFR1);

R2D3 is the Ig domain 3 of VEGFR2;

R2D4 is the Ig domain 4 of VEGFR2;

MC is a multimerizing component ( eg IgG hinge domain or fragment thereof, eg from IgG1);

the linker is a peptide containing about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, for example, (GGGS) _g ;

And,

regardless,

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.

In one embodiment of the present invention, R1D2 contains the amino acid sequence: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIID (SEQ ID NO.: 34). In one aspect, R1D2 does not contain an N-terminal SDT.

In one embodiment of the present invention, R1D2 contains the amino acid sequence: PFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQT (SEQ ID NO.: 35).

In one embodiment of the present invention, R2D3 contains the amino acid sequence: VVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 36).

In one embodiment of the present invention, R2D4 contains the amino acid sequence: PFVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG (SEQ ID NO.: 37).

In one embodiment of the present invention, R2D4 contains the amino acid sequence: FVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPIKSEKQSHVVSLVVYVP (SEQ ID NO.: 38).

In one embodiment of the present invention, the multimerizing component (MC) for use in VEGF MiniTrap is a peptide, eg, a Fc-modified immunoglobulin ( eg , from IgG1), that is capable of binding to another multimerizing component. In one aspect, the MC is an Fc-modified immunoglobulin that includes an immunoglobulin hinge region. For example, in one embodiment of the present invention, the MC is a peptide containing one or more ( for example , 1, 2, 3, 4, 5, or 6) cysteines that are capable of forming one or more cysteine bridges with cysteines in another MC, for example, DKTHTCPPC (SEQ ID NO.: 39), DKTHTCPPCPPC (SEQ ID NO.: 40), DKTHTCPPCPPCPPC (SEQ ID NO.: 41), DKTHTC(PPC) _h where h is 1, 2, 3, 4 or 5, DKTHTCPPCPAPELLG (SEQ ID NO.: 60), DKTHTCPLCPAPELLG (SEQ ID NO.: 43), DKTHTC (SEQ ID NO.: 44) or DKTHTCPLCPAP (SEQ ID NO.: 45).

The present invention also provides a VEGF MiniTrap polypeptide having the following domain structure:

(i) (R1D2) _a -(R2D3) _b -(MC) _c ; or

(ii) (R1D2) _a -(R2D3) _b -(R2D4) _c -(MC) _d ;

which can be homodimerized with a second of said polypeptides, for example, by linking between the MCs of each polypeptide,

Where

(i) said R1D2 domains match;

(ii) said R2D3 domains match; and/or

(iii) said R2D4 domains are consistent,

with the formation of a dimeric VEGF-binding domain.

In one embodiment of the present invention, the VEGF MiniTrap polypeptide contains the following amino acid sequence: SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP N ₃₆ ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS N ₆₈ ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL N ₁₂₃ CTARTELNVGIDFNWEYPSSKHQ HKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKK N ₁₉₆ STFVRVHEK DKTHTCPPC PAPELLG (SEQ ID NO.: 46; MC underlined); GRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRV HENLSVAFGSGMESLVEATVGERVRIPAKYLGYPPPEIKWYKNGIPLESNHTIKAGHVLTIMEVSERDTGNYTVILTNPISKEKQSHVVSLVVYVPPGPG DKTHTCPLCPAPELLG (SEQ ID NO.: 47; MC underlined); SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP N ₃₆ ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS N ₆₈ ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL N ₁₂₃ CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKK N ₁₉₆ STFVRVHEK DKTHTCPPC (SEQ ID NO.: 48; MS underlined); SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP N ₃₆ ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS N ₆₈ ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL N ₁₂₃ CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKK N ₁₉₆ STFVRVHEK DKTHTCPPC PPC (SEQ ID NO.: 49; MC underlined); SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSP N ₃₆ ITVTLKKFPLDTLIPDGKRIIWDSRKGFIIS N ₆₈ ATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVL N ₁₂₃ CTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSD QGLYTCAASSGLMTKK N ₁₉₆ STFVRVHEK DKTHTCPPC PPCPPC (SEQ ID NO.: 50; MC underlined); or SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTF VRVHEK DKTHTC-(PPC)x (MC underlined; where x is 1, 2, 3, 4, or 5). As already discussed, such polypeptides can be multimerized ( eg , dimerized ( eg , homodimerized)), wherein the binding between polypeptides is mediated by multimerizing moieties.

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

In one embodiment of the present invention, the VEGF MiniTrap contains the following structure:

(R1D2) ₁ -(R2D3) ₁ -(G ₄ S) ₃ -(R1D2) ₁ -(R2D3) ₁ ;

(R1D2) ₁ -(R2D3) ₁ -(G ₄ S) ₆ -(R1D2) ₁ -(R2D3) ₁ ;

(R1D2) ₁ -(R2D3) ₁ -(G ₄ S) ₉ -(R1D2) ₁ -(R2D3) ₁ ; or

(R1D2) ₁ -(R2D3) ₁ -(G ₄ S) ₁₂ -(R1D2) ₁ -(R2D3) ₁ .

G ₄ S is -Gly-Gly-Gly-Gly-Ser-

In one embodiment of the present invention, the VEGF MiniTrap contains the following amino acid sequence:

(i)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVR VHEK GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKT QSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 51; linker underlined);

(iii)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVR VHEK GGGGSGGGGSGGGGS SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTR SDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 52; linker underlined);

(iv)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVR VHEK GGGGSGGGGSGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEY PSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 53; linker underlined)

(v)

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVR VHEK GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLV LNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK (SEQ ID NO.: 54; linker underlined);

or

(vi)

(где x составляет 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 или 15). Как обсуждается в данном документе, данные полипептиды могут содержать вторичную структуру, в которой подобные домены Ig VEGFR связаны с образованием внутрицепочечного VEGF-связывающего домена (например, фиг. 2). В одном варианте осуществления настоящего изобретения два или более таких полипептидов мультимеризуются (например, димеризуются (например, гомодимеризуются)), причем Ig-домены VEGFR каждой цепочки, связываются с подобными Ig-доменами другой цепи с образованием межцепочечного связывающего-VEGF домена.

(where x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15). As discussed herein, these polypeptides may contain a secondary structure in which similar VEGFR Ig domains are linked to form an intrachain VEGF binding domain ( eg , Figure 2 ). In one embodiment of the present invention, two or more such polypeptides are multimerized ( e.g. , dimerized ( e.g. , homodimerized)), wherein the VEGFR Ig domains of each chain bind to similar Ig domains of the other chain to form an interchain-VEGF-binding domain.

In certain embodiments of the present invention, the VEGF MiniTrap of the present invention does not contain any significant modification of the amino acid residues of the VEGF MiniTrap polypeptide ( e.g. , targeted chemical modification such as pegylation or iodoacetamidation, e.g., at the N- and/or C-terminus).

In one embodiment _of the present invention _{, the} polypeptide comprises a secondary structure in which VEGFR _Ig domains in one chimeric polypeptide ( e.g. _a -(R2D3) _b -(R2D4) _c -linker-(R1D2) _d -(R2D3) _e -(R2D4) _f ) or in individual chimeric polypeptides ( eg homodimers) coordinate to form the VEGF binding domain. For example, in which

(i) said R1D2 domains match;

(ii) said R2D3 domains match; and/or

(iii) said R2D4 domains are consistent,

with the formation of a VEGF-binding domain. In FIG. 2 is a description of a single strand VEGF MiniTrap depicting such domain matching. VEGFR1, VEGFR2 and linker domains are indicated. The linker shown is (G ₄ S) ₆ . The present invention includes single chain VEGF MiniTrap with a (G ₄ S) ₃ linker; (G ₄ S) ₉ or (G ₄ S) ₁₂ .

In addition, the present invention also provides a complex containing a VEGF MiniTrap, as discussed herein, complexed with a VEGF polypeptide or fragment or fusion. In one embodiment of the present invention, VEGF ( eg VEGF ₁₆₅ ) is homodimerized and/or VEGF MiniTrap is homodimerized to a 2:2 complex (2 VEGF:2 MiniTrap) and/or VEGF MiniTrap is homodimerized to a 1:1 complex. The complexes may include homodimerized VEGF molecules associated with homodimerized VEGF MiniTrap polypeptides. In one embodiment of the present invention, the complex is present in vitro ( eg , immobilized on a solid support) or is in the subject's body. The present invention also includes the composition of complexes of a VEGF dimer ( eg VEGF ₁₆₅ ) complexed with a VEGF MiniTrap.

As used herein, the term "protein" or "protein of interest" may include any amino acid polymer with covalently linked amide bonds. Examples of a protein of interest include, without limitation, aflibercept and MiniTrap. Proteins contain one or more amino acid polymeric chains commonly known in the art as "polypeptides". "Polypeptide" refers to a polymer composed of amino acid residues, corresponding naturally occurring structural variants and their synthetic non-natural counterparts linked through peptide bonds, the corresponding naturally occurring structural variants, and their synthetic non-naturally occurring counterparts. "Synthetic peptides or polypeptides" refers to a non-naturally occurring peptide or polypeptide. Synthetic peptides or polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. A person skilled in the art is aware of various methods of solid phase peptide synthesis. A protein may contain one or more polypeptides to form a single functioning biomolecule. In another exemplary aspect, a protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. The protein of interest may include any of biotherapeutic proteins, recombinant proteins used in research or therapy, decoys and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, and bispecific antibodies. In a specific aspect, the protein of interest is an anti-VEGF fusion protein ( eg , aflibercept or MiniTrap). Proteins can be produced using recombinant cell based production systems such as the insect baculovirus system, yeast systems ( e.g. Pichia sp.), mammalian systems ( e.g. CHO cells and CHO cell derivatives such as CHO-K1 cells ). For a recent review of 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 full content of which is included in this document). In some exemplary embodiments, the proteins contain modifications, adducts, and other covalently linked moieties—these modifications, adducts, and moieties include, for example, avidin, streptavidin, biotin, glycans ( e.g. , N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose , mannose and other monosaccharides), PEG, polyhistidine, FLAGtag, maltose binding protein (MBP), chitin binding protein (CBP), glutathione S-transferase (GST) myc epitope, fluorescent labels and other dyes, etc. Proteins can be classified based on composition and solubility and thus can include simple proteins such as globular proteins and fibrillar proteins; conjugated proteins such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins and lipoproteins; and derived proteins such as primary derived proteins and secondary derived proteins.

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, scFv, and combinations thereof.

As used herein, the term "recombinant protein" refers to a protein resulting from the transcription and translation of a gene contained in a recombinant expression vector that has been introduced into a suitable host cell. In certain exemplary embodiments, the recombinant protein may be a fusion protein. In a specific aspect, the recombinant protein is an anti-VEGF fusion protein ( eg aflibercept or MiniTrap). In certain exemplary embodiments, the recombinant protein may be an antibody, such as a chimeric, humanized, or fully human antibody. In certain exemplary embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain exemplary embodiments, the antibody molecule is a full-length antibody ( eg , IgG1) or, alternatively, the antibody may be a fragment ( eg , an Fc fragment or a Fab fragment).

As used herein, the term "antibody" includes immunoglobulin molecules containing four polypeptide chains, two heavy chains (H) and two light chains (L), linked by disulfide bonds, as well as their multimers (For example, IgM). Each heavy chain contains a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In various embodiments, the anti-big-ET-1 FR (or antigen-binding portion thereof) may be identical to human germline sequences or may be naturally or artificially modified. The amino acid consensus sequence can be determined based on parallel analysis of two or more CDRs. As used herein, the term "antibody" also includes antigen-binding fragments of complete antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and the like, as used herein, include any naturally occurring, enzymatically produced, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody can be generated, for example, from whole antibody molecules using any suitable standard techniques such as proteolytic cleavage or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding the variable and optionally constant domains of the antibody. Such DNA is known and/or readily available, e.g. from commercial sources, DNA libraries (including,For example, antibody phage libraries), or can be synthesized. The DNA can be sequenced and processed chemically or using molecular biology techniques, for example, to place one or more variable and/or constant domains in an appropriate configuration, or to introduce codons, create cysteine residues, modify, add or remove amino acids, etc. d.

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

The term "bispecific antibody" includes an antibody that can selectively bind two or more epitopes. Bispecific antibodies usually include two different heavy chains, each of which binds specifically to different epitopes—either to two different molecules ( eg , antigens) or to the same molecule ( eg , on the same antigen). If the bispecific antibody is capable of selectively binding two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will typically be one to two, three, or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, and vice versa . The epitopes recognized by a bispecific antibody may be on one or another target ( for example , on the same or a different protein). Bispecific antibodies can be obtained, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding variable heavy chain sequences that recognize different epitopes on the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain.

A typical bispecific antibody has two heavy chains, each with three heavy chain CDRs, followed by a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, and an immunoglobulin light chain that either does not confer antigen-binding specificity but can bind to each heavy chain or which can bind to each heavy chain and which can bind one or more epitopes linked by antigen-binding regions of the heavy chain, or which can bind to each heavy chain and bind one or both heavy chains to one or both epitopes. BsAbs can be divided into two main classes: those that contain an Fc region (IgG-like) and those that do not have an Fc region, the latter usually being smaller than IgG and IgG-like Fc-containing bispecific molecules. IgG-like bsAbs can come in a variety of formats such as, but not limited to, triomab, IgG hole-protrusions (kih IgG), crossMab, IgG orth-Fab, Ig dual variable domains (DVD-Ig), Fab two-in-one, or dual action (DAF), IgG single chain Fv (IgG scFv) or κλ bodies. Non-IgG-like various formats include tandem scFv, diabody format, single chain diabody, tandem diabodies (TandAb), dual affinity retargeting molecule (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock method ( DNL) (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Müller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), full content included in this document). Methods for producing bsAbs are not limited to quadroma technology based on somatic fusion of two different hybridoma cell lines, chemical conjugation that includes chemical crosslinkers, and genetic approaches that use recombinant DNA technology. Examples of bsAbs include those disclosed in the following patent applications, which are incorporated herein by reference: US No. 12/823838, filed June 25, 2010; U.S. No. 13/488628, filed June 5, 2012; U.S. No. 14/031075, filed September 19, 2013; U.S. No. 14/808171, filed July 24, 2015; U.S. No. 15/713574, filed September 22, 2017; U.S. No. 15/713569, filed September 22, 2017; U.S. No. 15/386453, filed December 21, 2016; U.S. No. 15/386443, filed December 21, 2016; U.S. No. 15/22343 filed July 29, 2016; US No. 15814095, filed November 15, 2017. Low levels of homodimer impurities may be present at several steps in the production of bispecific antibodies. Detection of such homodimer impurities can be problematic when performed using intact mass analysis due to the low content of homodimer impurities and the co-elution of these impurities with major species when carried out using the conventional liquid chromatographic method.

As used herein, "multispecific antibody" refers to an antibody with binding specificity for at least two different antigens. Although such molecules typically bind only two antigens ( i.e., bispecific antibodies, bsAbs), antibodies with additional specificity, such as trispecific antibodies and trispecific KIHs, can also be considered using the system and method described herein. .

Used in this document, the term "monoclonal antibody" is not limited to antibodies obtained using hybridoma technology. A monoclonal antibody can be obtained from a single clone, including any eukaryotic, prokaryotic or phage clone, by any means available or known in the art. Monoclonal antibodies suitable for use in the present invention can be obtained using a wide range of methods known in the art, including the use of hybridoma, recombinant and phage display technologies, or combinations thereof.

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 particular exemplary embodiment, 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 more than one type of protein of interest in the compositions.

In some exemplary embodiments, the protein of interest may be produced by a mammalian cell. Mammalian cells may be of human or non-human origin, may include primary epithelial cells ( e.g. , keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells), resistant cell lines, and strains thereof ( e.g. , 293 embryonic kidney cells, BHK cells, HeLa cervical epithelial cells and retinal PER-C6 cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, CHO cells, BeWo cells, Chang cells, Detroit cells 562, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 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-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells and TH-I cells, B1 cells, BSC-1 cells, RAf cells, RK cells, PK cells -15 or derivatives thereof), fibroblast cells from any tissue or organ (including, without limitation, heart, liver, kidney, colon, intestine, esophagus, stomach, nervous tissue (brain, spinal cord), lung, vascular tissue (artery, vein , capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, and fibroblast and fibroblast-like cell lines ( e.g. , CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells , citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC cells -5, 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, 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, KLN205 cells, cells McCoy, Mouse L cells, 2071 strain cells (mouse L cells), LM strain cells (mouse L cells), L-MTK' cells (mouse L cells), NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells and Jensen cells, Sp2/0, NS0, NS1 cells or derivatives thereof).

As used herein, the term "protein alkylating agent" refers to an agent used to alkylate certain free amino acid residues in a protein. Non-limiting examples of protein alkylating agents are iodoacetamide (IOA), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM), methylmethanethiosulfonate (MMTS), and 4-vinylpyridine, or combinations thereof.

In the context of this document, "protein denaturation" may refer to a process in which the three-dimensional shape of a molecule changes from its original state. Protein denaturation can be carried out using a protein denaturing agent. Non-limiting examples of a protein denaturing agent include heat, high or low pH, reducing agents such as DTT (see below), or exposure to chaotropic agents. Several chaotropic agents can be used as protein denaturants. Chaotropic solutes increase the entropy of a system by inhibiting intramolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic effects. Non-limiting examples of chaotropic agents include butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, N-lauryl sarcosinate, urea, and salts thereof.

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

As used herein, the term "variant" polypeptide ( e.g. , VEGFR Ig domain) refers to a polypeptide containing an amino acid sequence that is at least about 70-99.9% ( e.g. , 70, 71, 72, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 5, 99.9%) is identical or similar to the specified or parental amino acid sequence of the protein of interest. Sequence comparison can be performed, for example, using the BLAST algorithm, in which the algorithm parameters are chosen to provide the highest match between matching sequences over the entire length of the matching check sequences ( e.g. , expected threshold: 10; word length: 3; maximum number of matches in the query range : 0; BLOSUM matrix 62; gap cost: presence 11, extension 1, conditional compositional adjustment of the substitution matrix). Variants of a polypeptide ( e.g. VEGFR Ig domain) can also refer to a polypeptide containing the specified amino acid sequence, excluding one or more ( e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) mutations such as, for example, sense-changing mutations ( eg , conservative substitutions), non-sense mutations, deletions, or insertions. The following references refer to BLAST algorithms often used for sequential 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. , (1993) Comput. Chem. 17:149-163; Hancock, JM et al. , (1994) Comput. Appl. biosci. 10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, MO, et al. , “A model of evolutionary change in proteins.” in Atlas of Protein Sequence and Structure, (1978) vol. 5 suppl. 3. MO Dayhoff (ed.), pp. 345-352, Natl. Biomed. Res. Found., Washington, DC; Schwartz, R.M., et al. , “Matrices for detecting distant relationships.” in Atlas of Protein Sequence and Structure, (1978) vol. 5 suppl. 3.'' MO Dayhoff (ed.), pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, SF, (1991) J. Mol. Biol. 219:555-565; States, DJ, et al. , (1991) Methods 3:66-70; Henikoff, S., et al. , (1992) Proc. Natl. Acad. sci. USA 89:10915-10919; Altschul, SF, 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, SF “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, NY; the contents of which are included in this document.

Some variants may be covalent modifications that polypeptides undergo, either during (co-translational modification) or after (post-translational modification “PTM”) their ribosome synthesis. PTMs are usually administered by specific enzymes or enzyme pathways. Many of these occur at the site of a specific characteristic protein sequence ( eg signature sequence) within a protein scaffold. Several hundred PTMs have been reported and these modifications invariably affect some aspect of protein structure or function (Walsh, G. "Proteins" (2014) second edition, published in Wiley and Sons, Ltd., ISBN: 9780470669853, the contents of which are incorporated herein ). In certain exemplary embodiments, the protein composition may comprise more than one type of protein variant of the protein of interest.

Protein variants in the case of aflibercept (and proteins with the same structural characteristics of aflibercept, e.g., one or more aflibercept heavy or light chain regions) may include, without limitation, oxidation variants that may result from the oxidation of one or more amino acid residues arising, for example, on residues histidine, cysteine, methionine, tryptophan, phenylalanine and/or tyrosine; deamidation variants, which may result from deamidation at asparagine residues and/or deoxyglucosonation of an arginine residue.

Regarding aflibercept (and proteins with the same structural characteristics of aflibercept, e.g., one or more aflibercept heavy or light chain regions), oxidation variants may include the oxidation of histidine residues at His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent positions residue on proteins with the same specific structural characteristics of aflibercept); oxidation of tryptophan residues at Trp58 and/or Trp138 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); oxidation of tyrosine residues at Tyr64 (or equivalent positions on proteins with the same specific structural characteristics of aflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); and/or oxidation of methionine residues to Met10, Met20, Met163 and/or Met192 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept).

With respect to aflibercept (and proteins with the same structural characteristics of aflibercept, e.g., one or more regions of the heavy or light chain of aflibercept), deamidation options may include deamidation of the asparagine residue at Asn84 and/or Asn99 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept) .

With respect to aflibercept (and proteins with the same structural characteristics of aflibercept, e.g., one or more regions of the heavy or light chain of aflibercept), a deoxyglucosonation variant may include 3-deoxyglucosonation of an arginine residue in Arg5 (or the equivalent position of the residue on proteins with the same specific structural characteristics of aflibercept).

Protein variants can include both acidic forms and basic forms. The acidic forms are typically variants that elute earlier than the main peak from CEX or later than the main peak from AEX, while the basic varieties are variants that elute later than the main peak from CEX or earlier than main pick from AEX.

As used herein, the terms "acid forms", "AS", "acidic region", and "AR" refer to protein variants that are characterized by an overall acidic charge. For example, in recombinant protein preparations, such acid forms can be detected by various methods such as ion exchange, for example WCX-10 HPLC (weak cation exchange chromatography) or IEF (isoelectric focusing). Acid forms of an antibody may include variants, structural variants, and/or fragmentation variants. Illustrative variants may include, without limitation, deamidation variants, afucosylation variants, oxidation variants, methylglyoxal (MGO) variants, glycation variants, and citric acid variants. Exemplary structural variants include, without limitation, glycosylation variants and acetonation variants. Exemplary fragmentation variants include any modified forms of the protein from the target molecule due to dissociation of the peptide chain, enzymatic and/or chemical modifications, including, but not limited to, Fc and Fab fragments, Fab-less fragments, fragments without heavy chain variable domain, C-terminal truncation variants, c-terminal truncation variants, excision of the N-terminal Asp in the light chain, and variants with N-terminal truncation of the light chain. Other acid form variants include those containing unpaired disulfides, host cell proteins and host cell nucleic acids, chromatographic materials, and media components. Typically, acid forms elute before the main peak during CEX or after the main peak during AEX analysis ( see FIGS. 16 and 17 ).

In certain embodiments, the implementation of the protein composition may contain more than one type of variant acid forms. For example, and not by way of limitation, the total number of acid forms can be categorized based on the chromatographic retention time of the peaks that appear. Another example in which general acid forms can be classified may be based on the type of variant - variants, structure variants, or fragmentation variant.

The term "acid forms" or "AS" does not refer to process impurities. The term "manufacturing impurity" in the context of this document refers to impurities that are present in the composition containing the protein, but do not come from the protein itself. Process impurities include, but are not limited to, host cell proteins (HCPs), host cell nucleic acids, chromatographic materials, and media components.

In one illustrative embodiment, the amount of acid forms in the anti-VEGF composition compared to the protein of interest may be no more than 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 varies within one or more of the previous ones. Examples of the composition against VEGF are discussed below in section III. In one aspect, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.

Among the chemical degradation pathways responsible for acidic or basic forms, the two most commonly observed covalent modifications occurring in proteins and peptides are deamination and oxidation. Methionine, cysteine, histidine, tryptophan and tyrosine are among the amino acids that are most sensitive to oxidation: Met and Cys due to their sulfur atoms and His, Trp and Tyr due to their aromatic rings.

Used in this document, the terms "oxidative form", "OS" or "oxidation variant" refers to protein variants formed by oxidation. Such oxidizing species can also be detected by various methods such as ion exchange, for example WCX-10 HPLC (weak cation exchange chromatography), or IEF (isoelectric focusing). Oxidation variants may be formed due to oxidation occurring at histidine, cysteine, methionine, tryptophan, phenylalanine and/or tyrosine residues. In particular, with respect to aflibercept (and proteins with the same structural characteristics of aflibercept, e.g. one or more aflibercept heavy or light chain regions), oxidation variants may include the oxidation of histidine residues at His86, His110, His145, His209, His95, His19, and/or His203 ( or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); oxidation of tryptophan residues at Trp58 and/or Trp138 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); oxidation of tyrosine residues at Tyr64 (or equivalent positions on proteins with the same specific structural characteristics of aflibercept); oxidation of phenylalanine residues at Phe44 and/or Phe166 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); and/or oxidation of methionine residues to Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept).

In one exemplary embodiment, the amount of oxidative species in the anti-VEGF composition relative to the protein of interest may be no more than 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% and varies within one or more of the previous ones. Examples of the composition against VEGF are discussed below in section III. In one aspect, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept or MiniTrap.

Cysteine residues can undergo spontaneous oxidation to form either intra- or intermolecular disulfide bonds or monomolecular by-products such as sulfonic acid.

Histidine residues are also very susceptible to oxidation by reaction with their imidazole rings, which can subsequently generate additional hydroxyls (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 contents of which are incorporated herein in their entirety). Proposed mechanisms for histidine oxidation are highlighted in FIG. 2 and FIG. 3 . Detailed mechanism studies are available at Anal. Chem. 2014, 86, 4940-4948 and J. Pharm. Biomed. Anal. 21 (2000) 1093-1097, the contents of which are incorporated herein in their entirety.

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

Tryptophan oxidation can provide a complex mixture of products. The primary products may be N-formylkynurenine and kynurenine along with mono-oxidation, di-oxidation and/or tri-oxidation products ( FIG. 4 ). Peptides carrying oxidized Trp modifications typically show mass gains of 4, 16, 32, and 48 Da, corresponding to the formation of kynurenine (KYN), hydroxytryptophan (W _ox1 ) and N-formylkynurenine/dihydroxytryptophan (NFK/W _ox2 , also referred to as "doubly oxidized Trp”), trihydroxytryptophan (W _ox3 , also called “tri-oxidized Trp”) and combinations thereof such as hydroxykynurenine (KYN _ox1 , +20 Da). Oxidation to hydroxytryptophan (W _ox1 ) (Mass spectrometric identification of oxidative modifications of tryptophan residues in proteins: chemical artifact or post-translational modification? J. Am. Soc. Mass Spectrom. 2010 Jul; 21(7): 1114-1117, full the content of which is included in this document). Tryptophan oxidation, but not methionine and histidine oxidation, has been found to cause 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, 685-6857). Like tryptophan, tyrosine oxidation is primarily mediated by 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).

As used herein, the terms “basic forms”, “basic region”, and “BR” refer to variants of a protein, e.g., an antibody or antigen-binding region thereof, which are characterized by an overall basic charge relative to the primary charge variant present in the protein. For example, in recombinant protein preparations, such basic forms can be detected by various methods such as ion exchange, for example, WCX-10 HPLC (weak cation exchange chromatography) or IEF (isoelectric focusing). Illustrative variants may include, without limitation, lysine variants, aspartic acid isomerization, succinimide formation on asparagine, methionine oxidation, amidation, incomplete disulfide bond formation, serine to arginine mutation, aglycosylation, fragmentation, and aggregation. Typically, major species elute later than the major peak during CEX or earlier than the major 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 full contents of which are incorporated herein.)

In certain embodiments, the implementation of the protein composition may contain more than one type of variant of the main form. For example, and not by way of limitation, the common base forms can be categorized based on the chromatographic retention time of the peaks that appear. Another example in which common basic forms can be separated may be based on the type of variant - variants, structure variants, or fragmentation variant.

As discussed for acid forms, the term "base forms" does not include process impurities, and base forms may be the result of product preparation (referred to in this document as "preparation derived base forms"), or the result of storage (referred to in this document as " the basic forms formed as a result of storage”).

In one exemplary embodiment, the amount of base forms in the anti-VEGF composition compared to the protein of interest may be no more than 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% and varies within one or more of the previous ones. Examples of the composition against VEGF are discussed below in section III. In one aspect, the anti-VEGF composition may comprise an anti-VEGF protein selected from the group consisting of aflibercept, recombinant MiniTrap (examples of which are disclosed in US Pat. No. 7,279,159), scFv, and other anti-VEGF proteins. In a preferred aspect, the recombinant protein of interest is aflibercept.

In the context of this document, "sample matrix" or "biological sample" can be obtained at any stage of the bioprocess, e.g., cell culture fluid (CCF), harvested cell culture fluid (HCCF), at any stage of the subsequent processing process, drug substance ( DS) or drug product (DP) containing the final formulated product. In some other specific exemplary embodiments, the biological sample may be selected from any step in the subsequent clarification, chromatographic preparation, viral inactivation, or filtration process. In some specific exemplary embodiments, the drug product may be selected from a manufactured drug product during clinical trials, transport, storage, or circulation.

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

In terms of protein composition, the term "stable" as used herein refers to a protein of interest in a composition capable of maintaining an acceptable level of chemical structure or biological function after storage under the exemplary conditions defined herein. The composition may be stable even if the protein of interest contained therein does not retain 100% of its chemical structure or biological function after storage for a certain time. Under certain circumstances, maintaining about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of protein structure or function after storage for a certain time may be considered "stable".

The term "treat" or "treatment" refers to therapeutic measures that reverse, stabilize, or eliminate an unwanted disease or disorder ( e.g. , angiogenic eye disease or cancer), for example, causing regression, stabilization, or elimination of one or more of the symptoms or signs of such disease or disorders to any clinically measurable degree, such as in relation to angiogenic eye disease, causing a reduction or maintenance of a diabetic retinopathy severity score (DRSS), by improving or maintaining vision ( for example , in the best correction of visual acuity, for example, by measuring the increase in letters ETDRS) , increasing or maintaining the field of view and/or reducing or maintaining central retinal thickness and, in relation to cancer, arresting or reversing the growth, survival and/or metastasis of cancer cells in a subject. Typically, the therapeutic measure is the administration of one or more doses of a therapeutically effective amount of VEGF MiniTrap to a subject with a disease or disorder.

As used herein, the term "upstream process technology" in the context of protein production refers to activities involving the production and collection of proteins from cells during or after cell culture of the protein of interest. As used herein, the term "cell culture" refers to a method of generating and maintaining a population of host cells capable of producing a recombinant protein of interest, as well as methods and techniques for optimizing the production and collection of a protein of interest. For example, once the expression vector has been introduced into an appropriate host cell, the host cell may be maintained under conditions suitable for expressing the appropriate nucleotide-encoding sequences and collecting and producing the desired recombinant protein.

Using the cell culture techniques of the present invention, the protein of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In embodiments in which the protein of interest is produced intracellularly, solid residues, either host cells or lysed cells ( eg , as a result of homogenization), can be removed by a variety of methods including, but not limited to, centrifugation or ultrafiltration. If a protein of interest is secreted into the medium, supernatants from such expression systems can first be concentrated using a commercially available protein filter, such as an Amicon™ or Millipore Pellicon™ ultrafiltration unit. In one aspect, a protein of interest can be collected by centrifugation followed by depth filtration followed by capture affinity chromatography.

As used herein, a "VEGF antagonist" is any protein or peptide that binds or interacts with VEGF. Typically, this binding or interaction inhibits the binding of VEGF to its receptors (VEGFR1 and VEGFR2), and/or inhibits the biological signaling pathways and activity of VEGF. VEGF antagonists include molecules that interfere with the interaction between VEGF and the natural VEGF receptor, for example, molecules that bind to VEGF or the VEGF receptor and prevent or otherwise interfere with the interaction between VEGF and the VEGF receptor. Specific exemplary VEGF antagonists include anti-VEGF antibodies ( e.g. , ranibizumab [LUCENTIS®]), antibodies to the VEGF receptor ( e.g. , anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.), and chimeric VEGF receptor-based or VEGF-inhibiting fusion molecules. proteins (also referred to herein as "VEGF-Trap" or "VEGF MiniTrap") such as aflibercept, Ziv-aflibercept, and protein having the amino acid of SEQ ID NO.: 60. Other examples of VEGF-Trap are ALT-L9, M710 , FYB203 and CHS-2020. Additional examples of VEGF-Trap can be found in US Pat. Nos. 7,070,959; 7306799; 7374757; 7374758; 7531173; 7608261; 5952199; 6100071; 6383486; 6897294 and 7771721, which are specifically incorporated herein by reference in their entirety.

VEGF receptor-based chimeric molecules include chimeric polypeptides that contain two or more immunoglobulin (Ig)-like domains of a VEGF receptor, such as VEGFR1 (also referred to as Flt1) and/or VEGFR2 (also referred to as Flk1 or KDR), and may also contain a multimerizing domain ( eg , an Fc domain that allows multimerization [ eg , dimerization] of two or more chimeric polypeptides). An exemplary VEGF receptor-based chimeric molecule is referred to as VEGFR1R2-FcΔC1 (a) (also known as aflibercept; sold under the trade name EYLEA®). In certain exemplary embodiments, aflibercept comprises the following amino acid sequence:

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVR VHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFMWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. (SEQ ID NO.: 55)

As used herein, “viral filtration” may include filtration using suitable filters including, but not limited to Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50 filter ™ from Pall Corporation. One of ordinary skill in the art will recognize the selection of a suitable filter to obtain the desired filtration performance.

II. Color definition

In the context of this document, the color observed during the production of a recombinant protein, more specifically an anti-VEGF protein, can be measured by various methods. Non-limiting examples include the use of iodine color index, Hazen color index, Gardner color index, Lovibond color index, Saybolt color index, mineral oil color index, European Pharmacopeia color index, USP color index, CIE L*, a*, b* (or CIELAB), Klett Color Index, Hess-Yves Color Index, Yellowness Index, ADMI and ASBC Color Index and EBC Brewing Color Index. A detailed description of these scales can be found in Application Report No. 3.9 e from Lange, the full contents of which are included in this document.

Visual color matching based on the European Pharmacopoeia (Ph Eur) (European color standard, see European Pharmacopoeia. Chapter 2.2.2. Degree of coloration of liquids. 8 ^th ed. EP, the contents of which are incorporated herein) may include solution preparation with a reference color as described in Ph. Eur. (EP 2.2.2. Degree of Coloration of Liquids 2) - prepare three stock solutions for red (cobalt (II) chloride), yellow (iron (III) chloride), and blue (copper (II) sulfate) and 1% hydrochloric acid , five color reference solutions for yellow (Y), greenish yellow (GY), brownish yellow (BY), brown (B) and red (R) shades. Using these five reference solutions, a total of thirty-seven color reference solutions (Y1-Y7, GY1-GY7, BY1-BY7, B1-B9 and R1-R7) are prepared in turn. Each reference solution is clearly defined in the CIE-Lab color space, such as luminance, hue, and chromaticity. Of the seven tan standards (BY standards), BY1 is the darkest standard and BY7 is the least dark. Compliance of a given sample with a sample of the BY color standard is usually done in diffuse daylight. The compositions of the European standards of yellow-brown color are described below in table 1 .

Table 1. Composition of European standards in yellow-brown color Reference solution Volumes in ml BY standard solution Hydrochloric acid (10 g/1 HCl) BY1 100.0 0.0 BY2 75.0 25.0 BY3 50.0 50.0 BY4 25.0 75.0 BY5 12.5 87.5 BY6 5.0 95.0 BY7 2.5 97.5

Brown-yellow standard solution 10.8 g/l FeCl ₃ .6H ₂ O, 6.0 g/l CoCl ₂ .6H ₂ O and 2.5 g/l CuSO ₄ .5H ₂ O

Testing the color of liquids is carried out by comparing the test solution with a standard color solution. The composition of the standard color solution is selected depending on the shade and intensity of the color of the test solution. As a rule, the comparison is carried out in flat-bottomed, colorless, transparent, neutral glass tubes that are as close as possible to the internal diameter and in all other respects ( for example , tubes with a diameter of about 12, 15, 16, or 25 mm). For example, the comparison could be between 2 or 10 ml of test solution and standard color solution. The depth of liquids, for example, can be 15, 25, 40 or 50 mm. The color assigned to the test solution should not be more intense than the standard color. Color comparisons are usually made in diffused light ( e.g. daylight) against a white background. Colors can be compared along the vertical or horizontal axis of the tubes.

. Цветовые координаты CIE L*, a*, b* могут быть сгенерированы, например, с использованием Hunter Labs UltrascanPro (Hunter Associates Laboratory, Рестон, Вирджиния) или на BYK Gardner LCS IV (BYK-Gardner, Колумбия, Мэриленд). Для Hunter Labs UltraScan Pro можно выполнить испытание дидимиевого фильтра для калибровки длины волны. Прибор может быть стандартизирован в TTRAN с использованием вставки порта 0,780 дюйма и DIW перед использованием; таким образом устанавливая верхнее (L=100) и нижнее (L=0) значения фотометрической шкалы с использованием световой ловушки и черной карты. См. 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), полное содержание которой включено в настоящий документ. Цвет стандартов BY также может быть выражен в цветовом пространстве CIE L*, a*, b* (цветовое пространство «CIELAB» или «CIELab»). См. таблицу 2. Unlike EP color measurement, the monograph USP 1061 Color - Instrumental Measurement refers to the use of CIE L*, a*, b* (or CIELAB) color measurement to accurately and objectively quantify colors. The USP defines a total of twenty color reference solutions (named consecutively A through T). The color of the measured sample is automatically correlated with the color reference solutions. This means that the reference color solution closest to the sample is displayed ( i.e. , the standard solution with the smallest color difference ΔE* from the color of the sample). The ΔL*, Δa* and Δb* values give quantitative differences between the L*, a* and b* values of the sample and displayed solutions according to USP. CIE coordinate system L* a* b*, L* represents the degree of lightness of a color on a scale of 0 to 100, where 0 is the darkest and 100 is the lightest, a* represents the redness or greenishness of the color (positive a* values represent red color, while negative a* values represent green) and b* represents the yellowness or blueness of the sample, positive b* values represent yellow, and negative b* values represent blue. The difference in color from the standard or original sample during evaluation can be represented by the change in the individual color components ΔL*, Δa* and Δb*. A complex change or difference in color can be calculated as a simple Euclidean distance in space according to the formula:

. CIE color coordinates L*, a*, b* can be generated using, for example, Hunter Labs UltrascanPro (Hunter Associates Laboratory, Reston, VA) or BYK Gardner LCS IV (BYK-Gardner, Columbia, MD). For the Hunter Labs UltraScan Pro, you can test the didymium filter for wavelength calibration. The instrument can be standardized to TTRAN using a 0.780 in. port insert and DIW before use; thus setting the upper (L=100) and lower (L=0) values of the photometric scale using a light trap and a black card. 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 content of which is incorporated herein. BY standards color can also be expressed in the CIE L*, a*, b* color space ("CIELAB" or "CIELab" color space). See table 2 .

Table 2. Characteristics of European standards for brown-yellow color in the CIE L*, a*, b* color space Standard L* ^{^} a* ^{^} b* ^{^} L* ^~ a* ^~ b* ^~ BY1 93.95 -2.76 28.55 92.84 -3.16 31.15 BY2 94.76 -2.96 22.69 94.25 -3.77 26.28 BY3 96.47 -2.84 16.41 95.92 -3.44 18.52 BY4 97.17 -1.94 9.07 97.67 -2.63 10.70 BY5 98.91 -1.19 4.73 98.75 -1.61 5.77 BY6 99.47 -0.59 2.09 99.47 -0.71 2.38 BY7 99.37 -0.31 1.13 99.71 -0.37 1.17 ^{^} According to Packet al.
^~ Measured experimentally in this document are the L* and b* values for each BY color standard.

To provide high throughput screening for color analysis, the spectrophotometric analysis method (CIELAB) is more appropriate and quantitative than BY color standards. The surrogate analysis was further optimized as described in the Examples section.

For any of the samples judged by color, the protein in the samples must be standardized to the protein in the sample, eg 5 g/l, 10 g/l, etc. for comparison.

III. Compositions against VEGF

There are at least five members of the VEGF protein family that regulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor (PlGF). Anti-VEGF compositions may contain a VEGF antagonist that specifically interacts with one or more members of the VEGF protein family and inhibits one or more of its biological activities, for example, its mitogenic, angiogenic and/or vascular permeability activities.

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

The inventors of the present invention found that the production of anti-VEGF proteins ( eg aflibercept) in certain COSs provided a biological sample having a distinct color. Different color properties were observed at different stages of production and even in the final formulation containing the anti-VEGF protein. As observed in Example 9, for VEGF MiniTrap production, cell culture in COS provided an anti-VEGF protein ( eg , aflibercept) with an intense tan color. The affinity of the post-harvest capture step was also provided by the eluate, which had a specific color, tan. Further production steps using AEX also showed a yellow-brown color, but with less intensity.

As described in more detail below, color can be assessed using (i) the European BY standard, which is a qualitative visual inspection, or (ii) a colorimetric analysis, CIELAB, which is more quantitative than the BY system. However, in any case, the color score between multiple samples was normalized to protein concentration in order to provide meaningful evaluation/comparison. For example, with respect to Example 9 below, specifically Table 9-2 , the Protein A eluate has a "b*" value of about 2.52, which corresponds to a BY value of about BY5 (when measured at a protein concentration of 5 g/L in the Protein A eluate) . If the color of the Protein A eluate is to be compared with another sample, then the comparison should be made using the same protein concentration. Thus, when comparing the protein A eluate with an AEX pool that has an ab* value of about 0.74 (when measured with 5 g/l protein in the protein A eluate), the preparation method shows a significant reduction in sample tan color from the protein eluate A to AEX pool after AEX chromatography.

The compositions of the present invention may be characterized by a yellow-brown color as discussed herein, for example, no darker/intense than the European standard brown-yellow color BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value of 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/l of anti-VEGF protein or about 10 g/l of anti-VEGF protein, and wherein the composition obtained as a sample from a clarified harvest or protein A eluate from a clarified harvest.

In one embodiment, the compositions of the present invention, obtained using COS provides a biological sample with a distinct yellow-brown color, and the sample can be characterized by a recognized standard color characteristic:

(i) no more yellow-brown than the European color standard BY2;

(ii) no more yellow-brown than the European color standard BY3;

(iii) no more yellow-brown than the European color standard BY4;

(iv) no more yellow-brown than the European color standard BY5;

(v) between the values of the European color standard BY2 and BY3;

(vi) between the values of the European color standard BY3 and BY4;

(vii) between the values of the European color standard BY4 and BY5, and the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein, and the composition is obtained as a sample from the clarified collection protein A eluate.

In another embodiment, the compositions of the present invention, obtained using A HOS provides a biological sample with a distinct yellow-brown color, and the composition is characterized by a recognized standard color characteristic on the CIELAB scale:

(i) no more yellow-brown than a b* value of about 22-23;

(ii) no more yellow-brown than a b* value of about 16-17;

(iii) no more yellow-brown than a b* value of 9-10;

(iv) no more yellow-brown than a b* value of 4-5;

(v) no more yellow-brown than a b* value of 2-3;

(vi) a b* value from 17 to 23;

(vii) b* value from 10 to 17;

(viii) b* value from 5 to 10;

(ix) b* value from 3 to 5; or

(x) a b* value from 1 to 3, wherein the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein, and the composition is obtained as a sample from a protein A eluate of a clarified harvest.

In one embodiment, the compositions of the present invention, obtained using XOS, may contain other forms or variants of the protein against VEGF. These variants include isoforms of the anti-VEGF protein that contain one or more oxidized amino acid residues, collectively referred to as oxo variants. Enzymatic cleavage of such compositions containing the anti-VEGF protein and its oxo variants may include one or more of the following:

EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), which contains about 0.004-0.013% 2-oxo-histidines,

QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), which contains about 0.006-0.028% 2-oxo-histidines,

TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), which contains about 0.049-0.085% 2-oxo-histidines,

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), which contains about 0.057-0.092% 2-oxo-histidines,

TNYLTH*R (SEQ ID NO.: 21), which contains about 0.008-0.022% 2-oxo-histidines, and/or

IIWDSR (SEQ ID NO.: 56), which contains about 0.185-0.298% dioxidized tryptophan;

or

EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), which contains about 0.008% 2-oxo-histidines,

QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), which contains about 0.02% 2-oxo-histidines,

TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), which contains about 0.06% 2-oxo-histidines,

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), which contains about 0.07% 2-oxo-histidines,

TNYLTH*R (SEQ ID NO.: 21), which contains about 0.01% 2-oxo-histidines, and/or

IIWDSR (SEQ ID NO.: 56), which contains about 0.23% di-oxo-tryptophans, where H* is histidine, which can be oxidized to 2-oxo-histidine, and where C* is cysteine, which may be carboxymethylated. In specific embodiments, the anti-VEGF protein is aflibercept. In another embodiment, the anti-VEGF protein is VEGF MiniTrap.

In one illustrative embodiment of the present invention, the compositions of the present invention may contain an anti-VEGF protein, and no more than about 1%, no more than about 0.1%, or about 0.1-1%, 0.2-1%, 0.3 -1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1%, or 0.9-1% anti-VEGF protein histidine residues are 2-oxo-histidine. In such compositions, there may be a heterogeneous population of anti-VEGF protein variants, each having a variable number of 2-oxo-histidine residues and non-oxidized histidine residues. Thus, the percentage of anti-VEGF 2-oxo-histidine protein in the composition refers to the site-specific 2-oxo-histidines among the anti-VEGF molecules divided by the total number of site-specific histidines in the anti-VEGF protein molecules (oxidized plus non-oxidized) multiplied by 100. One way to quantify the level of 2-oxo-histidines in the composition is to cleave the polypeptide with a protease ( for example , Lys-C and/or trypsin) and analyze the amount of 2-oxo-histidines in the resulting peptides, for example, by mass spectrometry (MS).

Before protein cleavage against VEGF, the sulfhydryl groups of cysteines are blocked by reaction with iodoacetamide (IAM), resulting in a residue represented by the following chemical structure:

.

.

This modification protects the free thiols from the re-formation of disulfide bridges and prevents the cleavage of disulfide bonds. The present invention includes compositions ( e.g. , aqueous compositions) containing an anti-VEGF protein and variants thereof which, when modified with IAM and digested with a protease ( e.g. , Lys-C and trypsin) and analyzed by mass spectrometry, contain the following peptides:

EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), which contains about 0.004-0.013% 2-oxo-histidines,

QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), which contains about 0.006-0.028% 2-oxo-histidines,

TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), which contains about 0.049-0.085% 2-oxo-histidines,

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), which contains about 0.057-0.092% 2-oxo-histidines,

TNYLTH*R (SEQ ID NO.: 21), which contains about 0.008-0.022% 2-oxo-histidines, and/or

IIWDSR (SEQ ID NO.: 56), which contains about 0.185-0.298% dioxidized tryptophan;

or

EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), which contains about 0.008% 2-oxo-histidines,

QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), which contains about 0.02% 2-oxo-histidines,

TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), which contains about 0.06% 2-oxo-histidines,

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), which contains about 0.07% 2-oxo-histidines,

TNYLTH*R (SEQ ID NO.: 21), which contains about 0.01% 2-oxo-histidines, and/or

IIWDSR (SEQ ID NO.: 56), which contains about 0.23% di-oxo-tryptophans,

where H* is 2-oxo-histidine, and where C* is carboxymethylated cysteine. In one embodiment of the present invention, the peptides are deglycosylated by PNGase F.

The present invention includes compositions comprising an anti-VEGF protein wherein about 0.1%-10% of all histidines of the anti-VEGF protein are modified to 2-oxo-histidine. In addition, the color of the composition is not darker/intense than, for example, the European Brown-Yellow Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6, alternatively having a b* value as characterized by CIE L*, a*, b* from about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein. The composition is obtained either as a sample from a clarified harvest or as a protein A eluate from a clarified harvest. Such compositions can be obtained from a clarified harvest if the harvested material is subjected to a capture chromatography procedure. In one aspect, the capture step is an affinity chromatography procedure using, for example, a protein A affinity column. If the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more variants can be detected.

The present invention includes compositions comprising an anti-VEGF protein wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to kynurenine. In addition, the color of the composition is not darker/intense than the European Brown-Yellow Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as characterized by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/L of anti-VEGF protein or about 10 g/L of anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or protein A eluate from a clarified harvest. Such compositions may be obtained from a clarified harvest upon subjecting to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A affinity column. If the affinity sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

The present invention includes compositions comprising an anti-VEGF protein wherein about 0.1%-10% of the total tryptophans of the anti-VEGF protein are modified to mono-hydroxyl tryptophan. In addition, the color of the composition is not darker/intense than the European Brown-Yellow Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as characterized by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/L of anti-VEGF protein or about 10 g/L of anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or protein A eluate from a clarified harvest. Such compositions may be obtained from a clarified harvest upon subjecting to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A affinity column. If the sample recovered from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

The present invention includes compositions comprising an anti-VEGF protein wherein about 0.1%-10% of the total tryptophans of the anti-VEGF protein are modified to di-hydroxyl tryptophan. In addition, the color is not darker/intense than the European Brown-Yellow Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as characterized by CIE L*, a* , b* from about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/L of anti-VEGF protein or about 10 g/L of anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or protein A eluate from a clarified harvest. Such compositions can be obtained from a clarified harvest obtained using COS containing the anti-VEGF protein, as well as its oxo variants, subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A affinity column. If the sample recovered from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

The present invention includes compositions containing an anti-VEGF protein, wherein about 0.1%-10% of all tryptophans of the anti-VEGF protein are modified to tri-hydroxyl tryptophan. In addition, the color of the composition is not darker/intense than the European Brown-Yellow Standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as characterized by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5, or 1-3, wherein the composition contains about 5 g/L of anti-VEGF protein or about 10 g/L of anti-VEGF protein. The composition is obtained as a sample from a clarified harvest or protein A eluate from a clarified harvest. Such compositions can be obtained using capture chromatography. The capture step is an affinity chromatography procedure using, for example, a protein A affinity column. If the sample recovered from the affinity step is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

In one embodiment, the compositions of the present invention may comprise an anti-VEGF protein, wherein the anti-VEGF protein may have one or more residue modifications as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to iso-aspartate and/or Asn; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; C-terminal glycine is absent; and/or there is one or more non-glycosylated glycosites.

Such compositions can be obtained from a clarified harvest obtained using COS containing an anti-VEGF protein, as well as its variants, subjected, for example, to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A column. If the sample recovered in the affinity step is analyzed using, for example, liquid chromatography-mass spectrometry (LC-MS), one or more options.

In one illustrative embodiment, the compositions of the present invention may contain an anti-VEGF protein having the same structural characteristics of aflibercept, which may be oxidized at one or more of the following: His86, His110, His145, His209, His95, His19, and/or His203 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); Trp58 and/or Trp138 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); Tyr64 (or equivalent positions on proteins with the same specific structural characteristics of aflibercept); Phe44 and/or Phe166 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); and/or Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept. Such compositions can be prepared from a clarified harvest using COS containing aflibercept, as well as its oxo variants subjected to The capture step may be an affinity chromatography procedure using, for example, a protein A column. one or more of these variants are found.

In one embodiment, the compositions of the present invention may contain VEGF MiniTrap with the amino acid sequence of SEQ ID NO.: 46, which can be oxidized to His86, His110, His145, His209, His95, His19 and/or His203; Trp58 and/or Trp138; Tyr64; Phe44 and/or Phe166; and/or Met10, Met 20, Met163 and/or Met192. Such compositions can be obtained from a clarified harvest obtained using HOS containing VEGF MiniTrap, as well as variants subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A column - when analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected.

In some illustrative embodiments, the compositions of the present invention may contain an anti-VEGF protein and variants thereof (including oxo variants), wherein the amount of protein variants in the composition may be no more than 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 varies within one or more of the previous ones. Such compositions can be obtained from a clarified harvest obtained using COS containing an anti-VEGF protein, as well as variants thereof, subjected to a capture chromatography procedure. The capture step is an affinity chromatography procedure using, for example, a protein A column - when analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected. In one aspect, the color of such a composition is not darker/more intense than, for example, the European brown-yellow color standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as defined by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5 or 1-3, and the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein.

In other illustrative embodiments, the compositions of the present invention may contain an anti-VEGF protein and variants thereof, wherein the amount of protein variants in the composition may be from about 0% to about 20%, for example, from about 0% to about 20%, from 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%, from about 0.9 % to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, from about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, from about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, from about 2% up to about 10%, from about 3% to about 10%, from about 4% to about 10%, from about 5% to about 10%, from about 6% to about 10%, from about 7% to about 10%, about 8% to about 10%, about 9% to about 10%, about 0% to about 7.5%, about 0.05% to about 7.5%, about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5% up to about 7.5%, from about 0.6% to about 7.5%, from about 0.7% to about 7.5%, from about 0.8% to about 7.5%, from about 0, 9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, from about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5% , from about 0.4% to about 5%, from about 0.5% to about 5%, from about 0.6% to about 5%, from about 0.7% to about 5%, from about 0.8 % to about 5%, from about 0.9% to about 5%, from about 1% to about 5%, from about 1.5% to about 5%, from about 2% to about 5%, from about 3% up to about 5%, from about 4% to about 5% and varies within one or more of the previous ones. Such compositions can be obtained by capture chromatography on the collected sample. The capture step is an affinity chromatography procedure using, for example, a protein A column. If the sample is analyzed using liquid chromatography-mass spectrometry (LC-MS), one or more of these variants can be detected. In one aspect, the color of such a composition is not darker/more intense than, for example, the European brown-yellow color standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as defined by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5 or 1-3, and the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein.

In one embodiment, the compositions of the present invention may contain an anti-VEGF protein, including its acidic forms, and the amount of acidic forms 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 varies within one or more of the previous ones. As discussed above , such acid forms can be detected by various methods such as ion exchange, for example WCX (WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing). Typically, the acid forms elute before the main peak during CEX or after the main peak during AEX analysis ( see Fig. 16 and Fig. 17 ). Compositions containing acid forms can be obtained from biological material, such as harvest or affinity-derived material using ion exchange chromatography.

In one aspect, the color of such a composition is not darker/more intense than, for example, the European brown-yellow color standard BY2-BY3, BY3-BY4, BY4-BY5 or BY5-BY6 and/or having a b* value as defined by CIE L*, a *, b* from about 17-23, 10-17, 5-10, 3-5 or 1-3, and the composition contains about 5 g/l or about 10 g/l. As an example, referring to FIG. 16 and FIG. 17 , fractions F1 and F2 denote acid fractions which contain most of the acid forms. Peaks 1 and 2 of MT1 in FIG. 17 contain the acidic forms, while fractions F1 and F2 contain the majority of the acidic fractions. Fractions containing these acid forms (F1 and F2) also showed a tan color compared to other fractions ( FIGS. 18B and FIG. 18C ).

In other illustrative embodiments, the compositions of the present invention may contain an anti-VEGF protein and its acid forms, and the amount of acid forms in the composition may be from about 0% to about 20%, for example, from about 0% to about 20%, from 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 %, from about 0.5% to about 20%, from about 0.6% to about 20%, from about 0.7% to about 20%, from about 0.8% to about 20%, from about 0, 9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, from about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, from about 0.8% to about 10%, from about 0.9% to about 10%, from about 1% to about 10%, from about 1.5% to about 10%, from about 2 % to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10% , from about 8% to about 10%, from about 9% to about 10%, from about 0% to about 7.5%, from about 0.05% to about 7.5%, from about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5 % to about 7.5%, from about 0.6% to about 7.5%, from about 0.7% to about 7.5%, from about 0.8% to about 7.5%, from about 0 .9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, from about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5 %, from about 0.4% to about 5%, from about 0.5% to about 5%, from about 0.6% to about 5%, from about 0.7% to about 5%, from about 0, 8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, about 3 % to about 5%, from about 4% to about 5% and varies within one or more of the previous ones. As discussed above, such acid forms can be detected by various methods such as ion exchange, for example WCX (WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing). Typically, the acid forms elute before the main peak during CEX or after the main peak during AEX analysis ( see Fig. 16 and Fig. 17 ).

When using a cation exchange column, all peaks eluting before the main peak of interest were summed as the acid region, and all peaks eluting after the protein of interest were summed as the main region. In exemplary embodiments, acidic forms may be eluted as two or more acidic regions and may be numbered AR1, AR2, AR3, etc. based on the specific retention time of the peaks and the ion exchange column used.

In one embodiment, the compositions may contain an anti-VEGF protein, including acid forms, wherein AR1 is 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0, 5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.0%, and varies within one or more of the previous ones. In one aspect, the compositions may contain an anti-VEGF protein, including acidic forms thereof, wherein AR1 is from about 0.0% to about 10%, from about 0.0% to about 5%, from 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 from about 10% to about 15%, and varies within one or more of the previous ones. As discussed above, such acidic regions can be detected by various methods such as ion exchange, for example WCX (WCX-10 HPLC, weak cation exchange chromatography) or IEF (isoelectric focusing). Typically, the acid forms elute before the main peak during CEX or after the main peak during AEX analysis ( see Fig. 16 and Fig. 17 ).

In another embodiment, the compositions may contain an anti-VEGF protein, including acidic forms, with AR2 being 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 varies within one or more of the previous ones. In one aspect, the compositions may contain an anti-VEGF protein, including acid forms, wherein AR2 is from about 0.0% to about 10%, from about 0.0% to about 5%, from 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 from about 10% to about 15%, and varies within one or more of the previous ones.

In one embodiment, the compositions may contain an anti-VEGF protein, including its main forms, and the amount of the main forms in the composition may be no more than 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 varies within one or more of the previous ones. In one aspect, the compositions may comprise an anti-VEGF protein and base forms thereof, wherein the amount of base forms in the composition compared to the anti-VEGF protein may be from 0% to about 20%, e.g. , from about 0% to about 20%, from 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 %, from about 0.5% to about 20%, from about 0.6% to about 20%, from about 0.7% to about 20%, from about 0.8% to about 20%, from about 0, 9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 3% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, from about 10% to about 20%, about 0% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, from about 0.8% to about 10%, from about 0.9% to about 10%, from about 1% to about 10%, from about 1.5% to about 10%, from about 2 % to about 10%, about 3% to about 10%, about 4% to about 10%, about 5% to about 10%, about 6% to about 10%, about 7% to about 10% , from about 8% to about 10%, from about 9% to about 10%, from about 0% to about 7.5%, from about 0.05% to about 7.5%, from about 0.1% to about 7.5%, about 0.2% to about 7.5%, about 0.3% to about 7.5%, about 0.4% to about 7.5%, about 0.5 % to about 7.5%, from about 0.6% to about 7.5%, from about 0.7% to about 7.5%, from about 0.8% to about 7.5%, from about 0 .9% to about 7.5%, about 1% to about 7.5%, about 1.5% to about 7.5%, about 2% to about 7.5%, about 3% to about 7.5%, about 4% to about 7.5%, about 5% to about 7.5%, about 6% to about 7.5%, about 7% to about 7.5%, about 0% to about 5%, about 0.05% to about 5%, about 0.1% to about 5%, about 0.2% to about 5%, about 0.3% to about 5%, about 0.4% to about 5%, about 0.5% to about 5%, about 0.6% to about 5%, about 0.7% to about 5%, about 0 .8% to about 5%, about 0.9% to about 5%, about 1% to about 5%, about 1.5% to about 5%, about 2% to about 5%, from about 3% to about 5%, from about 4% to about 5% and varies within one or more of the previous ones.

The main forms may be eluted as two or more main regions and may be numbered as BR1, BR2, BR3, etc. based on the determined peak retention time and the ion exchange applied.

In one embodiment, the compositions may contain an anti-VEGF protein, including basic forms, wherein BR1 is 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5 %, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or 0.0%, and varies within one or more of the previous ones. In one aspect, the compositions may contain an anti-VEGF protein and its main forms, and BR1 is from about 0.0% to about 10%, from about 0.0% to about 5%, from 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 from about 10% to about 15%, and varies within one or more of the previous ones.

In one embodiment, the composition may contain an anti-VEGF protein and its main forms, with BR2 being 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 varies within one or more of the previous ones. In one aspect, the compositions may comprise an anti-VEGF protein and its major forms of an anti-VEGF protein, wherein the BR2 is from about 0.0% to about 10%, from about 0.0% to about 5%, from 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 from about 10% to about 15%, and varies within one or more of the previous ones.

In one embodiment, the composition may contain an anti-VEGF protein and its main forms, with BR3 being 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 varies within one or more of the previous ones. In one aspect, the compositions may comprise an anti-VEGF protein and its major anti-VEGF protein forms, wherein BR3 is from about 0.0% to about 10%, from about 0.0% to about 5%, from 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 from about 10% to about 15%, and varies within one or more of the previous ones.

Photoinduced oxidation of aflibercept

In addition to discovering different color characteristics or variants of anti-VEGF protein compositions produced using COS, the present inventors have also discovered that such compositions can be artificially produced in the laboratory by exposure to light.

Modified, including oxidized, anti-VEGF formulations can be obtained by exposing anti-VEGF to cold white light, white light, or ultraviolet light. In one aspect, the anti-VEGF composition may comprise from about 1.5 to about 50-fold increase in the content of one or more modified oligopeptides compared to the sample, and the oligopeptides are selected from the group consisting of

DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), EIGLLTC*EATVNGH*LYK (SEQ ID NO.: 18), QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO. : 20), TNYLTH*R (SEQ ID NO.: 21), SDTGRPFVEMYSEIPEIH*MTEGR (SEQ ID NO.: 22), VH*EKDK (SEQ ID NO.: 23), SDTGRPFVEM*YSEIPEIHMTEGR (SEQ ID NO.: 64 ), SDTGRPFVEMYSEIPEIHM*TEGR (SEQ ID NO.: 65), TQSGSEM*K (SEQ ID NO.: 66), SDQGLYTC*AASSGLM*TK (SEQ ID NO.: 67), IIW*DSR/RIIW*DSR/IIW* DSRK (SEQ ID NO.: 28), TELNVGIDFNW*EYPSSK (SEQ ID NO.: 29), GFIISNATY*K (SEQ ID NO.: 69), KF*PLDTLIPDGK (SEQ ID NO.: 70) F*LSTLTIDGVTR (SEQ ID NO.: 32), where H* is histidine oxidized to 2-oxo-histidine, where C* is carboxymethylated cysteine, where M* is oxidized methionine, where W* is oxidized tryptophan, where Y* is is an oxidized tyrosine and where F* is an oxidized phenylalanine. In a further aspect, the anti-VEGF composition may comprise from about 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing the anti-VEGF composition to cold white light for a period of, for example, about 30 hours. In another aspect, the anti-VEGF composition may comprise about 1.5 to about 10-fold increase in one or more modified oligopeptides by exposing the sample to cold white light for about 75 hours. In another aspect, the anti-VEGF composition may include about 1.5 to about 20-fold increase in one or more of the described oligopeptides before exposing the sample to cold white light for about 100 hours. In another aspect, the anti-VEGF composition may include about 1.5 to about 20-fold increase in one or more of the described oligopeptides before exposing the sample to cold white light for about 150 hours. In a further aspect, the anti-VEGF composition may comprise from about 1.5 to about 50-fold increase in one or more oligopeptides by exposing the sample to cold white light for about 300 hours - see Example 4 below.

The anti-VEGF composition may comprise from about 1.5 to about 3-fold increase in one or more of the oligopeptides described above by exposing a sample of the anti-VEGF composition to ultraviolet light for about 4 hours. In another aspect, the anti-VEGF composition may comprise from about 1.5 to about 10-fold increase in one or more oligopeptides by exposing the sample to ultraviolet light for about 10 hours. In another aspect, the anti-VEGF composition may comprise about 1.5 to about 10-fold increase in one or more of the described oligopeptides by exposing the sample to ultraviolet light for about 16 hours. In another aspect, the anti-VEGF composition may comprise about 1.5 to about 25-fold increase in one or more of the described oligopeptides by exposing the sample to ultraviolet light for about 20 hours. In another aspect, the anti-VEGF composition may comprise about 1.5 to about 25-fold increase in one or more of the described oligopeptides by exposing the sample to ultraviolet light for about 40 hours. See example 4.

Glycodiversity - anti-VEGF protein produced using COS

The compositions of the present invention comprise an anti-VEGF protein, wherein the anti-VEGF protein produced in HOS has different glycodiversity. Various anti-VEGF protein glycosylation profiles are within the scope of the present invention.

In some exemplary embodiments of the present invention, the composition may comprise an anti-VEGF protein glycosylated on one or more asparagines as follows: G0-GlcNAc glycosylation; glycosylation of G1-GlcNAc; glycosylation of G1S-GlcNAc; G0 glycosylation; G1 glycosylation; G1S glycosylation; G2 glycosylation; G2S glycosylation; G2S2 glycosylation; G0F glycosylation; G2F2S glycosylation; glycosylation of G2F2S2; G1F glycosylation; G1FS glycosylation; G2F glycosylation; G2FS glycosylation; glycosylation of G2FS2; G3FS glycosylation; G3FS3 glycosylation; G0-2GlcNAc glycosylation; glycosylation of Man4; glycosylation of Man4_A1G1; glycosylation of Man4_A1G1S1; glycosylation of Man5; glycosylation of Man5_A1G1; glycosylation of Man5_A1G1S1; glycosylation of Man6; glycosylation of Man6_G0+phosphate; glycosylation of Man6 + phosphate; and/or Man7 glycosylation. In one aspect, the protein of interest may be aflibercept, an anti-VEGF antibody, or MiniTrap VEGF.

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

In one embodiment, the composition may comprise an anti-VEGF protein, wherein the protein of interest has Man5 glycosylation at about 32.4% asparagine 123 residues and/or about 27.1% asparagine 196 residues. In one aspect, the protein of interest may be aflibercept, anti-VEGF antibody or MiniTrap VEGF.

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.

In 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% of total sialylated glycans.

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% mannose-5 .

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% of total galactosylated glycans.

In one embodiment, the anti-VEGF protein may have fucosylated glycans reduced by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of the previous values, e.g. 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41 %, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10 %, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44 %, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25 %, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47 %, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40 %, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50 % or 1-99% compared to the level of fucosylated glycans in the anti-VEGF protein obtained using soy hydrolysate.

In one embodiment, the anti-VEGF protein may have sialylated glycans reduced by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of the previous values, e.g. 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41 %, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10 %, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44 %, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25 %, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47 %, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40 %, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50 % or 1-99% compared to the level of sialylated glycans in the anti-VEGF protein obtained using soy hydrolyzate.

In one embodiment, the anti-VEGF protein may have a level of galactosylated glycans reduced by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of the previous values, e.g. 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41 %, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10 %, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44 %, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25 %, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47 %, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40 %, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50 % or 1-99% compared to the level of galactosylated glycans in the anti-VEGF protein obtained using soy hydrolyzate.

In one embodiment, the anti-VEGF protein may have a level of mannosylated glycans increased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of the previous values, e.g. 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43 %, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20 %, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46 %, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35 %, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49 %, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42 %, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99% compared to the level mannosylated glycans in an anti-VEGF protein prepared using soy hydrolyzate.

The compositions described in this section can be produced through several preceding and subsequent parameters, as described below in sections IV and V, respectively.

IV. Obtaining compositions using technologies of the previous process

For biologics, a reliable and flexible bottom-up process must be implemented. An efficient upstream process can provide the desired production and scale-up of the protein of interest. The present inventors have found that compositions of the present invention containing an anti-VEGF protein can be obtained by modulating conditions during upstream protein production, such as changes in the components of the COS environment. Each step of the upstream process can affect the quality, purity, and quantity of protein produced.

The present invention provides evidence for the existence of certain variants of aflibercept and/or MiniTrap obtained using HOS. These variants include isoforms that contain one or more oxidized amino acid residues. Examples of oxidized residues include, without limitation, one or more histidine, tryptophan, methionine, phenylalanine, or tyrosine residues. Compositions obtained by using modified HOS can provide an anti-VEGF protein preparation with the desired target value of aflibercept and/or MiniTrap protein variants. As mentioned above, a yellow-brown color can also be observed associated with fractions obtained using COS. (As mentioned above, not all COS tested by the present inventors showed a distinct color change.)

The present invention includes culturing a host cell in modified HOS under suitable conditions in which the cell expresses the recombinant protein of interest, followed by harvesting a preparation of the recombinant protein of interest produced by the cell. This modified HOS can be used to prepare compositions as described above in section III. (It should be noted that COS is the medium in which a yellow-brown color appears when aflibercept is expressed.)

In one embodiment, the method comprises culturing the host cell in COS under suitable conditions, wherein the host cell expresses the recombinant protein of interest, such as aflibercept. The method further comprises collecting a preparation of the recombinant protein of interest produced by the cell, wherein suitable conditions include a COS with a cumulative concentration of iron in said COS of less than about 55 μmol, a cumulative concentration of copper in said COS of no more than about 0.8 μmol, a cumulative a nickel concentration in said HOS of not more than about 0.40 µmol, a cumulative zinc concentration in said HOS of no more than about 56 µmol, a cumulative cysteine concentration in said HOS of less than about 10 mmol; and/or an antioxidant in said HOS at a concentration of from about 0.001 mmol to about 10 mmol for one antioxidant, and a cumulative concentration of not more than about 30 mmol if several antioxidants are added to said HOS.

In one aspect of the present embodiment, the formulation, made under suitable conditions, reduces the aflibercept and MiniTrap VEGF protein variants to the desired amount of aflibercept and MiniTrap VEGF protein variants (referred to as the "target value" of the aflibercept and MiniTrap VEGF protein variants). In a further aspect of this embodiment, the formulation resulting from the application of suitable conditions provides a color reduction of the formulations to the desired BY value (referred to as the "target BY value") if the protein formulation, including aflibercept and VEGF MiniTrap variants, is normalized to a concentration of 5 g/ l, 10 g/l or even higher.

In a further aspect of the present embodiment, the target BY value and/or the target value of the variants can be obtained in a preparation in which the titer increases or does not decrease significantly ( see Example 5).

In some embodiments, the compositions obtained by using the modified HOS can provide an anti-VEGF protein preparation with the desired target BY value, the color of the preparation being characterized as follows:

(i) no more yellow-brown than the European color standard BY2;

(ii) no more yellow-brown than the European color standard BY3;

(iii) no more yellow-brown than the European color standard BY4;

(iv) no more yellow-brown than the European color standard BY5;

(v) between the values of the European color standard BY2 and BY3;

(vi) between the values of the European color standard BY3 and BY4;

(vii) between European Color Standard values BY4 and BY5, wherein the composition contains about 5 g/L or about 10 g/L of anti-VEGF protein, and wherein a sample of the composition can be obtained as a sample from a protein A eluate of a clarified harvest. As can be seen in Example 9, Table 9-3 below, the protein A eluate containing 5 g/l aflibercept was yellow-brown in color with a measured b* value of 1.77. Such a sample, if obtained after AEX, had a b* value of 0.50, demonstrating the utility of AEX in reducing sample tan color ( Table 9-3 ).

Compositions made using the modified HOS can provide an anti-VEGF protein preparation, the color of the preparation having a recognized standard CIELAB color score:

(i) no more yellow-brown than a b* value of about 22-23;

(ii) no more yellow-brown than a b* value of about 16-17;

(iii) no more yellow-brown than a b* value of 9-10;

(iv) no more yellow-brown than a b* value of 4-5;

(v) no more yellow-brown than a b* value of 2-3;

(vi) a b* value from 17 to 23;

(vii) b* value from 10 to 17;

(viii) b* value from 5 to 10;

(ix) b* value from 3 to 5; or

(x) a b* value from 1 to 3, wherein the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein, and the composition is obtained as a sample from a protein A eluate of a clarified harvest. See example 9, table 9-3 .

For components added to cell culture to form modified COS, as used herein, the term "cumulative amount" refers to the total amount of a particular component added to the bioreactor during cell culture to form COS, including amounts added at the start of culture (COS per day 0) and subsequently added quantities of the component. The amounts of component added to the culture in the seed fermenter system or inoculum prior to production in the bioreactor ( ie , before COS on day 0) are also included when calculating the cumulative amount of the component. The cumulative amount does not depend on the loss of the component over time during cultivation (for example, through metabolism or chemical degradation). Thus, two cultures with the same cumulative amounts of a component may still have different absolute levels, for example, if the component is added to two cultures at different points in time ( for example , if in one culture the entire component is added from the beginning, and in the other culture component is added over time). The cumulative amount is also independent of the in situ synthesis of the component over time during cultivation (eg, via metabolism or chemical conversion). Thus, two cultures with the same cumulative amounts of a listed component may still have different absolute levels, for example, if the component is synthesized in situ in one of the two cultures by a biological transformation process. The cumulative amount may be expressed in units such as grams or moles of a component, for example. The term "cumulative concentration" refers to the cumulative amount of a component divided by the volume of liquid in the bioreactor at the beginning of the production batch, including the consumption of the initial volume from any inoculum used in culture. For example, if the bioreactor contains 2 liters of cell culture medium at the start of the production batch, and one gram of component X is added on days 0, 1, 2, and 3, then the cumulative concentration after day 3 is 2 g/L ( i.e. , 4 grams divided into 2 liters). If on day 4 another liter of liquid containing no component X is added to the bioreactor, the cumulative concentration will still be 2 g/l. If on day 5 some liquid has been lost from the bioreactor (eg due to evaporation), the cumulative concentration will still be 2 g/l. The cumulative concentration may be expressed in units such as grams per liter or moles per liter, for example.

Amino acids:

In some embodiments, the implementation of the modified HOS can be obtained by reducing or increasing the cumulative concentrations of amino acids in HOS. 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 HOS can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 %, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to the original HOS, and varies within one or more of the previous ones. Alternatively, the increase or decrease in the cumulative amount of one or more amino acids in the modified HOS can be from about 5% to about 20%, from about 10% to about 30%, from about 30% to about 40%, from about 30% to about 50%. %, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to unmodified HOS, and varies within one or more of the previous ones ( see Fig. 25-27 and example 5).

In some embodiments, the implementation of the modified HOS can be obtained by reducing the cumulative concentration of cysteine in the HOS. The decrease in the amount of cysteine in COS with the formation of modified COS can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to unmodified HOS, and varies within one or more of the previous ones. Alternatively, the reduction in the cumulative amount of cysteine in the modified HOS can be from about 5% to about 20%, from about 10% to about 30%, from about 30% to about 40%, from about 30% to about 50%, from about 40 % to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to COS, and varies within one or more of the previous ones. In one aspect, the amount of cumulative cysteine in the modified HOS is less than about 1 mmol, about 2 mmol, about 3 mmol, about 4 mmol, about 5 mmol, about 6 mmol, about 7 mmol, about 8 mmol, about 9 mmol, or about 10 mmol ( see Fig. 25-27 and example 5).

In some embodiments, a modified HOS can be obtained by replacing at least a certain percentage of the cumulative cysteine in the HOS with cysteine. Substitution can be around 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 100% compared to unmodified HOS, and varies within one or more of the previous ones. Alternatively, the substitution may be from about 5% to about 20%, from about 10% to about 30%, from about 30% to about 40%, from about 30% to about 50%, from about 40% to about 60% , from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 100% compared to unmodified HOS, and varies within one or more of previous ( see Fig. 25-27 and example 5).

In some embodiments, a modified HOS can be obtained by replacing at least a certain percentage of the cumulative cysteine in the HOS with cysteine sulfate. Substitution can be around 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 100% compared to unmodified HOS, and varies within one or more of the previous ones. Alternatively, the substitution may be about 5% to about 20%, about 10% to about 30%, about 30% to about 40%, about 30% to about 50%, about 40% to about 60%, from about 60% to about 70%, from about 70% to about 80%, from about 80% to about 90%, or from about 90% to about 100% compared to unmodified HOS, and varies within one or more of the previous .

Metals:

In some embodiments, the implementation of the modified HOS can be obtained by reducing or increasing the cumulative concentration of metals in the HOS. Non-limiting examples of metals include iron, copper, manganese, molybdenum, zinc, nickel, calcium, potassium, and sodium. The increase or decrease in the amount of one or more metals in the modified HOS can be about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% compared to unmodified HOS, and varies within one or more of the previous ones. Alternatively, the increase or decrease in the cumulative amount of one or more metals in the modified HOS can be from about 5% to about 20%, from about 10% to about 30%, from about 30% to about 40%, from about 30% to about 50%. %, about 40% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% compared to unmodified HOS, and varies within one or more of the previous ones ( see Fig. 25-27 and example 5).

Antioxidants:

In some embodiments, the modified HOS contains one or more antioxidants. Non-limiting examples of antioxidants can include taurine, hypotaurine, glycine, thioctic acid, glutathione, choline chloride, hydrocortisone, vitamin C, vitamin E, and combinations thereof ( see FIGS. 28A-E and Example 5).

In some embodiments, the modified HOS contains from about 0.01 mmol to about 20 mmol taurine, i. e. 0.01 mmol to about 1 mmol, about 0.01 mmol to about 5 mmol, about 0.01 mmol to about 10 mmol, 0.1 mmol to about 1 mmol, about 0.1 mmol to about 5 mmol, from about 0.1 mmol to about 10 mmol, from about 1 mmol to about 5 mmol, from about 1 mmol to about 10 mmol and varies within one or more of the previous ones.

In some embodiments, the modified HOS contains from about 0.01 mmol to about 20 mmol hypotaurine, i. 0.01 mmol to about 1 mmol, about 0.01 mmol to about 5 mmol, about 0.01 mmol to about 10 mmol, 0.1 mmol to about 1 mmol, about 0.1 mmol to about 5 mmol, from about 0.1 mmol to about 10 mmol, from about 1 mmol to about 5 mmol, from about 1 mmol to about 10 mmol and varies within one or more of the previous ones.

In some embodiments, the implementation of the modified HOS contains from about 0.01 mmol to about 20 mmol of glycine, i. 0.01 mmol to about 1 mmol, about 0.01 mmol to about 5 mmol, about 0.01 mmol to about 10 mmol, 0.1 mmol to about 1 mmol, about 0.1 mmol to about 5 mmol, from about 0.1 mmol to about 10 mmol, from about 1 mmol to about 5 mmol, from about 1 mmol to about 10 mmol and varies within one or more of the previous ones.

In some embodiments, the modified HOS contains from about 0.01 µmol to about 5 µmol of thioctic acid, i.e. , from about 0.01 µmol to about 0.1 µmol, from about 0.1 µmol to about 1 µmol, from about 1 µmol to about 2.5 µmol, from about 1 µmol to about 3 µmol, from about 1 µmol to about 5 µmol and varies within one or more of the previous ones.

In some embodiments, the implementation of the modified HOS contains about 0.01 M to about 5 mmol of glutathione, i.e. , from 0.01 mmol to about 1 mmol, from 0.1 mmol to about 1 mmol, from about 0.1 mmol to about 5 mmol, from about 1 mmol to about 5 mmol and varies within one or more of the previous ones.

In some embodiments, the modified HOS contains from about 0.01 µmol to about 5 µmol hydrocortisine, i.e. , from about 0.01 µmol to about 0.1 µmol, from about 0.1 µmol to about 1 µmol, from about 1 µmol to about 2.5 µmol, from about 1 µmol to about 3 µmol, from about 1 µmol to about 5 µmol and varies within one or more of the previous ones.

In some embodiments, the modified HOS contains from about 1 µmol to about 50 µmol of vitamin C, i.e. , from about 1 µmol to about 5 µmol, from about 5 µmol to about 20 µmol, from about 10 µmol to about 30 µmol, from about 5 µmol to about 30 µmol, from about 20 µmol to about 50 µmol, from about 25 µmol up to about 50 µmol and varies within one or more of the previous ones.

Environmental changes to modulate glycosylation:

The present disclosure also includes methods for modulating anti-VEGF protein glycosylation by altering the cumulative concentrations of certain components in the COS. Based on the cumulative amounts of components added to COS, total % fucosylation, total % galactosylation, total % sialylation, and mannose-5 may vary.

In exemplary embodiments, a method for modulating anti-VEGF protein glycosylation may include adding uridine to the COS. An 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. ( See example 6 below).

In some embodiments, a method for modulating anti-VEGF protein glycosylation may include adding manganese to the COS. In one aspect, the HOS does not contain manganese prior to filing. An 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. ( See example 6 below).

In some embodiments, a method for modulating anti-VEGF protein glycosylation may include adding galactose to the COS. In one aspect, HOS does not contain galactose prior to filing. An 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).

In some embodiments, a method for modulating anti-VEGF protein glycosylation may include adding dexamethasone to the COS. In one aspect, HOS does not contain dexamethasone prior to filing. An 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. ( See example 6 below).

In some embodiments, a method for modulating glycosylation of an anti-VEGF protein may include feeding one or more of uridine, manganese, galactose, and dexamethasone to the COS. In one aspect, HOS does not contain one or more of uridine, manganese, galactose, and dexamethasone prior to filing. An 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).

V. Preparation of compositions using downstream technologies

Compositions containing the anti-VEGF protein of the present invention can be obtained by modulating conditions during subsequent production of the protein. The present inventors have found that optimizing downstream procedures can minimize certain anti-VEGF protein variants as well as discoloration. Optimization of the subsequent process can provide a composition with reduced oxo variants as well as optimized color characteristics.

The downstream technologies may be used alone or in combination with the downstream technologies described in Section IV above .

Anion exchange chromatography:

In some embodiments, a composition of the present invention may include a process comprising expressing an anti-VEGF protein in a host cell in COS, wherein the anti-VEGF protein is secreted from the host cell into the medium to obtain a clarified harvest. The harvest is subjected to the following (a) loading the biological sample obtained from the harvest onto an anion exchange chromatography (AEX) column; (b) washing the AEX column with an appropriate wash buffer, (c) collecting the run-through fraction(s), optionally, (d) washing the column with an appropriate purification buffer, and (e) collecting the purified fractions.

The flow fractions may contain oxo variants of the anti-VEGF protein, which are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% anti-VEGF protein sample versus oxo variants in the purified fraction of an anion exchange chromatography column. For example, with respect to Table 9-5 and Table 9-6 , the flow fractions contain oxidized variants of the anti-VEGF protein, where some histidine and tryptophan residues are about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (and varies within one or more of the previous ones), compared to the oxidized variants in the purified fractions.

The pH of both the equilibration buffer and the wash for the AEX column may be from about 8.20 to about 8.60. In another aspect, the conductivity of both of the equilibrate and wash buffers for the AEX column may be from about 1.50 to about 3.0 mS/cm. In one aspect, the equilibration and wash buffers may be 50 mmol tris hydrochloride. In one aspect, the purification buffer contains 2 mol sodium chloride or 1 N sodium chloride. sodium hydroxide or both ( see Table 2-2 ). Example 2 further illustrates the optimization of the conductivity of the equilibration and wash buffer.

Protein variants may include modifications to one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to iso-aspartate and/or Asn; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; C-terminal glycine is absent; and/or there is one or more non-glycosylated glycosites.

The protein of interest may be aflibercept, an anti-VEGF antibody, or a VEGF MiniTrap. Protein variants can be formed by one or more of the following: (i) oxidation of histidines from histidine residues selected from His86, His110, His145, His209, His95, His19 and/or His203 (or equivalent residue positions on proteins with the same defined structural characteristics aflibercept); (ii) oxidizing tryptophan residues selected from tryptophan residues in Trp58 and/or Trp138 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); (iii) oxidation of a tyrosine residue at Tyr64 (or equivalent positions on proteins with the same specific structural characteristics of aflibercept); (iv) oxidation of phenylalanine residues selected from Phe44 and/or Phe166 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept); and/or (v) oxidation of methionine residues selected from Met10, Met 20, Met163 and/or Met192 (or equivalent residue positions on proteins with the same specific structural characteristics of aflibercept).

The flow fractions may contain one or more of the following:

percentage of histidine residues that have been oxidized to 2-oxo-histidine, and their color characteristic is as follows:

(i) no more yellow-brown than the European color standard BY2;

(ii) no more yellow-brown than the European color standard BY3;

(iii) no more yellow-brown than the European color standard BY4;

(iv) no more yellow-brown than the European color standard BY5;

(v) between the values of the European color standard BY2 and BY3;

(vi) between the values of the European color standard BY3 and BY4;

(vii) between the values of the European color standard BY4 and BY5, and the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein, and the composition is obtained as a sample from flow-through fractions.

Percentage of histidine residues that have been oxidized to 2-oxo-histidine. In addition, their color is characterized by the presence of a yellow-brown color, which approximately corresponds to BY2, BY3, BY4, BY5, BY6, BY7; or it is not darker/more intense than BY2, not darker than BY3, not darker than BY4, not darker than BY5, not darker than BY6, not darker than BY7; or is between BY2 and BY3, between BY2 and BY4, between BY3 and BY4, or between BY3 and BY5.

The percentage of histidine residues that have been oxidized to 2-oxo-histidine, and their color is characterized by the color in the CIE L*, a*, b* color space as follows:

(i) no more yellow-brown than a b* value of about 22-23;

(ii) no more yellow-brown than a b* value of about 16-17;

(iii) no more yellow-brown than a b* value of 9-10;

(iv) no more yellow-brown than a b* value of 4-5;

(v) no more yellow-brown than a b* value of 2-3;

(vi) a b* value from 17 to 23;

(vii) b* value from 10 to 17;

(viii) b* value from 5 to 10;

(ix) b* value from 3 to 5; or

(x) between b* values of 1-3, wherein the composition contains about 5 g/l or about 10 g/l of anti-VEGF protein, and the composition is obtained as a sample from flow-through fractions.

Not more than about 1%, not more than about 0.1%, or about 0.1-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1% or 0.9-1% of histidine residues in the composition are oxidized to 2-oxo-histidine. The percentage calculation is described in Section II.

Affinity Chromatography:

In some embodiments, the compositions of the present invention may be prepared by a process comprising expressing an anti-VEGF protein in a host cell, wherein the anti-VEGF protein is secreted from the host cell into the medium to form a clarified harvest. Harvesting is subjected to the following steps comprising (a) loading a biological sample obtained from the cleaned crop onto an affinity chromatography column, the affinity chromatography comprising a protein capable of selectively or specifically binding to an anti-VEGF protein; (b) washing the affinity column with a suitable elution buffer, and (c) collecting the eluted fraction(s). For example, as illustrated in Table 7-1 and Table 7-7 to 7-10 , using VEGF ₁₆₅ as a protein capable of selectively or specifically binding to an anti-VEGF protein and collecting the eluted fractions as in the method described above resulted in successful obtaining MT5 (anti-VEGF protein), aflibercept and an anti-VEGF scFv fragment. Table 7-1 also discloses the successful production of MT5 using (i) mAb1 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 73 is the heavy chain and SEQ ID NO.: 74 is the light chain); (ii) mAb2 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 75 is the heavy chain and SEQ ID NO.: 76 is the light chain); (iii) mAb3 (mouse IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 77 is the heavy chain and SEQ ID NO.: 78 is the light chain) and (iv) mAb4 (mouse IgG1 against mouse anti-VEGFR1 mAb, where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain) because various proteins are able to selectively or specifically bind to MT5.

With respect to step (a) above, a biological sample for loading into an affinity column can be obtained from a sample in which a clarified harvest can be subjected to affinity preparation, including, without limitation, ion exchange chromatography (anionic or cationic). Other chromatographic procedures well known to the person skilled in the art may also be used prior to the application of the affinity step. The important point is that a biological sample containing an anti-VEGF protein can be subjected to affinity chromatography.

In some embodiments, the compositions of the present invention may be prepared using a process comprising the following: expression of the VEGF MiniTrap protein in a host cell, wherein the VEGF MiniTrap is secreted from the host cell into the medium, and the medium can be further processed to form a clarified harvest . This collection can be further processed through known chromatographic procedures to obtain a biological sample containing VEGF MiniTrap. This biological sample can be further processed by applying the following steps, including (a) loading the biological sample into an affinity chromatography column, wherein the affinity chromatography contains a protein capable of selectively or specifically binding to or interacting with the VEGF MiniTrap protein; (b) washing the affinity column with a suitable elution buffer; and (c) collecting the eluted fraction(s). With respect to Table 7-1 , this table discloses the successful production of MT5 (VEGF MiniTrap) using (i) VEGF ₁₆₅ ; (ii) mAb1 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 73 is the heavy chain and SEQ ID NO.: 74 is the light chain); (iii) mAb2 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 75 is the heavy chain and SEQ ID NO.: 76 is the light chain); (iv) mAb3 (mouse IgG1 anti-mouse anti-VEGF-R1 mAb, wherein SEQ ID NO.: 77 is the heavy chain and SEQ ID NO.: 78 is the light chain) and (v) mAb4 (mouse IgG1 anti-mouse anti-VEGF-R1 mAb). VEGFR1, where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain), since various proteins are able to selectively or specifically bind or interact with MT5.

In one embodiment, affinity chromatography can also be used to isolate other MiniTrap proteins. After digestion of aflibercept, a sample containing digested aflibercept can be subjected to affinity chromatography using a binder specific for digested aflibercept. In one aspect, the binder may be an antibody or a portion thereof.

Cleavage of aflibercept can be facilitated using proteolytic cleavage of aflibercept, for example, IdeS protease (FabRICATOR) or a variant thereof to generate a VEGF MiniTrap. Cleavage of aflibercept by the IdeS protease or variant may provide a mixture of products including an Fc fragment and VEGF MiniTrap. VEGF MiniTrap can be further processed using one or more of the production strategies described herein.

In some exemplary embodiments, a protein capable of selectively or specifically binding ("binder") or interacting with an anti-VEGF protein, such as aflibercept or MiniTrap, may be from a human or a mouse.

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

The biological sample can be loaded using a suitable buffer such as DPBS.

This affinity preparation process may further include washing the affinity column with one or more wash buffers. The column can be washed one or more times. In addition, washings can also be collected as washing fractions. The pH of both wash buffers may be from about 7.0 to about 8.60. In one aspect, the wash buffer may be DPBS. In another aspect, the wash buffer may be 20 mM sodium phosphate, pH 6-8 (especially 7.2), 10 mM sodium phosphate, 500 mM NaCl, pH 6-8 (especially 7.2), 50 mM Tris pH 7- 8 or DPBS pH 7.4.

This affinity process may further include washing the affinity column with one or more suitable elution buffers and collecting the eluted fractions. The column can be washed one or more times. Non-limiting examples of such a suitable elution buffer 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 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 their combinations.

In some aspects, the eluted fractions may be neutralized using a neutralization buffer. An example of such a neutralizing buffer is Tris-Tris-HCl (pH about 7.0 to about 9.0).

IdeS mutants:

The IdeS protease used to cleave an Fc fusion protein such as aflibercept will rapidly lose enzymatic activity at basic pH conditions, which may limit its use in the manufacture of VEGF MiniTrap. Thus, variants have been developed that are more stable at basic pH, for example in the presence of a strong base such as NaOH. Such basic conditions may be 0.05N. NaOH for 1 hour or 0.1 N. NaOH for 0.5 h.

In some embodiments, IdeS mutants may have an amino acid sequence that is at least about 70% identical throughout its length to the amino acid sequences set forth in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO. : 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16. In some aspects, the amino acid sequence of about 75%, 80%, 85%, 90%, 95%, or about 100% identical throughout its length to the amino acid sequences directly listed above.

In some embodiments, IdeS mutants may have an isolated nucleic acid molecule encoding a polypeptide with an amino acid sequence at least 70% identical throughout its length to the amino acid sequences shown in the group consisting of SEQ ID NO.: 2, SEQ ID NO. : 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16. In some aspects, the amino acid sequence on about 75%, 80%, 85%, 90%, 95%, or about 100% identical throughout its length to the amino acid sequences directly listed above.

In some embodiments, the polypeptide has an amino acid sequence at least 70% identical throughout its length to the amino acid sequences listed in the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16 and is expressed by the host cell with a suitable vector containing a nucleic acid encoding the identified peptides. In one aspect, the nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. In one aspect, the vector may be a plasmid. In some aspects, the amino acid sequence is about 75%, 80%, 85%, 90%, 95%, or about 100% identical throughout its length to the amino acid sequences directly listed above. In some aspects, an isolated nucleic acid molecule can be used to encode a polypeptide.

In some embodiments, IdeS mutants may have an amino acid sequence containing the original amino acid sequence identified in SEQ ID NO.: 1 (IdeS) with an asparagine residue at position 87, 130, 182, and/or 274 mutated to an amino acid other than asparagine. In one aspect, the mutation may provide increased chemical stability at alkaline pH values compared to the original amino acid sequence. In another aspect, the mutation may provide a 50% increase in chemical stability at alkaline pH values compared to the original amino acid sequence. In one aspect, the amino acid may be selected from aspartic acid, leucine, and arginine. In a specific aspect, the asparagine residue at position 87 is mutated to an aspartic acid residue. In another specific aspect, the asparagine residue at position 130 is mutated to an arginine residue. In another specific aspect, the asparagine residue at position 182 is mutated to a leucine residue. In another specific aspect, the asparagine residue at position 274 is mutated to aspartic acid residues. In another specific aspect, the asparagine residues at positions 87 and 130 are mutated. In another specific aspect, the asparagine residues at position 87 and 182 are mutated. In another specific aspect, the asparagine residues at positions 87 and 274 are mutated. In another specific aspect, the asparagine residues at positions 130 and 182 are mutated. In another specific aspect, the asparagine residues at positions 130 and 274 are mutated. In another specific aspect, the asparagine residues at positions 182 and 274 are mutated. In another specific aspect, the asparagine residues at positions 87, 130 and 182 are mutated. In another specific aspect, the asparagine residues at position 87, 182 and 274 are mutated. In another specific aspect, the asparagine residues at position 130, 182 and 274 are mutated. In another specific aspect, the asparagine residues at position 87, 130, 182 and 274 are mutated. In some aspects, the amino acid sequence is about 75%, 80%, 85%, 90%, 95%, or about 100% identical throughout its length to the amino acid sequences described above. In some aspects, an isolated nucleic acid molecule can be used to encode a polypeptide.

Those skilled in the art familiar with standard molecular biology techniques can make and use the IdeS mutants of the present invention without undue burden. For recombinant DNA, oligonucleotide synthesis, tissue culture and transformation, standard methods ( eg , electroporation, lipofection) can be used. see , for example, Sambrook et al. , Molecular Cloning : A Laboratory Manual, supra , incorporated herein by reference for all purposes. Enzymatic reactions and methods of obtaining can be carried out in accordance with the manufacturer's description or as described in this document.

VI. Total protein intake

A variety of different preparation techniques, including, without limitation, affinity, ion exchange, mixed mode chromatography, size exclusion chromatography, and hydrophobic interaction chromatography, alone or in combination, are considered to be within the scope of the present invention. These chromatographic steps make it possible to separate mixtures of biological sample proteins based on their charge, degree of hydrophobicity, size, or combinations thereof, depending on the specific form of separation. For each of the methods mentioned above , several different chromatographic resins are available, allowing the manufacturing scheme to be precisely tailored to the particular protein involved. Each separation method causes the protein to pass at different rates through the column to achieve a physical separation that increases as they pass further through the column or selectively adhere to the separation medium. The proteins are then either (i) differentially eluted using the appropriate elution buffers and/or (ii) collected from the flow-through fractions obtained from the column used, optionally by washing the column with the appropriate equilibration buffer. In some cases, the protein of interest is separated from impurities (HCP, protein variants , etc. ) if the impurities preferentially adhere to the column and the protein of interest to a lesser extent, i.e. , the protein of interest is not adsorbed to the solid phase of a particular column and thus flows through the column. In some cases, impurities are separated from the protein of interest when they are not adsorbed to the column and thus flow through the column.

The manufacturing process may begin at the separation step after the recombinant protein has been produced using the prior manufacturing methods described above and/or alternative manufacturing methods conventional in the art. Once a clarified solution or mixture containing a protein of interest, such as a fusion protein, is obtained, the protein of interest is separated from process contaminants (for example, other proteins produced by the cell (for example, HCP) as well as related compounds such as acidic or main options). A combination of one or more different preparation methods may be used, including affinity, ion exchange chromatography ( eg , CEX, AEX), mixed mode (MM) chromatography, and/or hydrophobic interaction chromatography. Such preparation steps allow mixtures of components within a biological sample to be separated based on, for example, charge, degree of hydrophobicity and/or apparent size. For each of the chromatography techniques mentioned herein, numerous chromatographic resins are commercially available to allow precise tailoring of the manufacturing scheme to the particular protein involved. Each of the separation methods allows proteins to either pass at different rates through the column, achieving a physical separation that increases as they pass further through the column, or to be adsorbed selectively by the separation resin (or media). The proteins can then be harvested in different ways. In some cases, the protein of interest separates from components of the biological sample when other components are specifically adsorbed to the resin in the column and the protein of interest is not adsorbed.

Primary recovery and inactivation of viruses

In some embodiments, the initial steps of the production methods disclosed herein include clarification and initial recovery of a protein of interest from a biological sample. Primary recovery will include one or more centrifugation steps to separate the protein of interest from the host cell and associated cellular debris. Sample centrifugation can be performed at a speed of, for example, but not limited to, from 7000 x g to about 12750 x g. In the context of large scale production, such centrifugation can occur on-line at a set flow rate to achieve, for example, a turbidity level of 150 NTU in the resulting supernatant. This supernatant can then be collected for further processing or in-line filtration through one or more depth filters to further clarify the sample.

In some embodiments, primary recovery may include the use of one or more depth filtration steps to clarify the sample and thus facilitate processing of the protein of interest. In another embodiment, the primary recovery may include the use of one or more stages of depth filtration after centrifugation. Non-limiting examples of depth filters that can be used in the context of the present invention include Millistak+ X0HC, F0HC, D0HC, A1HC, B1HC (EMD Millipore), 3M™ model 30/60ZA, 60/90 ZA, VR05, VR07 depth filters, Delipid depth filters (3M Corp.). Depth filters are typically followed by a 0.2 µm filter such as a 0.45/0.2 µm Sartopore™ Sartorius dual layer or Millipore Express SHR or SHC filter cartridges. Other filters well known to those skilled in the art may also be used.

In certain embodiments, the primary recovery process may also be a point to reduce or inactivate viruses that may be present in a biological sample. Any one or more of a variety of virus reduction/inactivation techniques may be used in the primary recovery phase of production, including heat inactivation (pasteurization), pH inactivation, buffer/detergent treatment, UV and γ-irradiation, and the addition of certain chemical inactivators, such as β-propiolactone or, for example, copper phenanthroline as described in US Pat. No. 4,534,972, the entire contents of which are incorporated herein by reference. In some exemplary embodiments of the present invention, the sample is subjected to viral inactivation by a detergent during the primary recovery phase. In another embodiment, the sample may be subjected to low pH inactivation during the primary recovery phase.

In those embodiments that use virus reduction/inactivation, the biological sample can be adjusted, if necessary, for further production steps. For example, following 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 proceeding with the preparation process. Additionally, the mixture can be diluted with water for injection (WFI) to obtain the desired conductivity.

Affinity chromatography

In certain exemplary embodiments, it may be preferable to subject a biological sample to affinity chromatography to obtain a protein of interest. The chromatographic material is capable of selectively or specifically binding or interacting with a protein of interest. Non-limiting examples of such a chromatographic material include protein A and protein G. In addition, the chromatographic material, including, for example, a protein or a portion thereof, is capable of binding to or interacting with a protein of interest. In one aspect, the protein of interest is an anti-VEGF protein such as aflibercept, MiniTrap, or a related protein.

Affinity chromatography may involve exposing a biological sample to a column containing a suitable protein A resin. As used herein, the term "protein A" includes protein A isolated from its natural source, protein A obtained synthetically ( for example , through peptide synthesis or through recombinant techniques), and variants thereof that retain the ability to bind proteins that have a C _H 2/ _CH 3 region. In certain aspects, Protein A resin is suitable for affinity-based generation and isolation of various isotypes of antibodies through specific interaction with the Fc portion of the molecule. if it contains such a region.

There are several commercial sources of protein A resin. One suitable resin is MabSelect™ from GE Healthcare. Suitable resins include, without limitation, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose from GE Healthcare, ProSep HC, ProSep Ultra and ProSep Ultra Plus from EMD Millipore, MapCapture from Life Technologies. A non-limiting example of a suitable column packaged with MabSelect™ is a column about 1.0 cm diameter x about 21.6 cm long (bed volume 17 ml). A suitable column may contain a resin such as MabSelect™ SuRe or a similar resin. Protein A can also be purchased commercially from Repligen, Pharmacia and Fermatech.

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

In one aspect, the eluate can be subjected to viral inactivation, for example, either with a detergent or low pH. A suitable detergent concentration or pH (and time) can be chosen to obtain the desired result regarding viral inactivation. After viral inactivation, the eluate is usually subjected to pH and/or conductivity adjustment for subsequent production steps.

The eluate may be filtered through a depth filter to remove turbidity and/or various impurities from the protein of interest prior to additional chromatographic further purification steps. Examples of suitable depth filters include, without limitation, Millistak+ XOHC, FOHC, DOHC, AIHC, X0SP and BIHC Pod (EMD Millipore) filters, or Zeta Plus 30ZA/60ZA, 60ZA/90ZA, delipid, VR07 and VR05 (3M) filters. The Emphaze AEX Hybrid Purifier multi-stage filter can also be used to purify the eluate. The eluate pool may need to be adjusted to a specific pH and conductivity to ensure proper impurity removal and product recovery in the depth filtration step.

Anion exchange chromatography

In certain embodiments, a protein of interest is obtained by subjecting a biological sample to at least one anion exchange separation step. In one case, the anion exchange step may occur after an affinity chromatography procedure ( e.g. affinity with protein A). In other cases, the anion exchange step may occur before the affinity chromatography step. In other protocols, anion exchange can occur either before or after the affinity chromatography step. In one aspect, the protein of interest is either aflibercept or MiniTrap.

The use of an anion exchange material versus a cation exchange material is based in part on the local charges 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, as well as other chromatography modes known to the person skilled in the art.

When performing separation, the original protein composition (biological sample) can be brought into contact with an anion exchange material using any of a variety of techniques, for example, using a batch production technique or a chromatographic technique.

In the context of batch production, the anion exchange material is prepared in or equilibrated with the required starting buffer. Upon receipt receive a suspension of anion-exchange material. The biological sample is brought into contact with the suspension to ensure that the protein is adsorbed by the anion exchange material. A solution containing acid forms that do not bind to the AEX material is obtained from the slurry by allowing the slurry to settle and removing the supernatant. The suspension may be subjected to one or more washing steps and/or elution steps.

In the context of chromatographic separation, a chromatographic column is used to contain a chromatographic support material (resin or solid phase). A sample containing a protein of interest is loaded onto a specific chromatographic column. The column can then be set up for one or more wash steps using the appropriate wash buffer. Sample components not adsorbed by the resin are more likely to flow through the column. Components adsorbed on the resin can be differentially eluted using a suitable elution buffer.

The wash step is typically carried out by AEX chromatography using conditions similar to those of the batch, or alternatively by lowering the pH and/or increasing the ionic strength/conductivity of a step or linear gradient wash. In one aspect, the aqueous salt solution used in both the loading and wash buffer has a pH equal to or close to the isoelectric point (pI) of the protein of interest. Typically, the pH is about 0-2 units above or below the pI of the protein of interest, however, it may be 0-0.5 units above or below. It may also be equal to the pI of the protein of interest.

The anionic agent may be selected from the group consisting of acetate, chloride, formate, and combinations thereof. The cationic agent may be selected from the group consisting of tris, arginine, sodium, and combinations thereof. In a specific example, the buffer solution is tris/formate buffer. The buffer may be selected from the group consisting of pyridine, piperazine, L-histidine, bis-tris, bis-tris-propane, imidazole, N-ethylmorpholine, TEA (triethanolamine), tris, morpholine, N-methyldiethanolamine, AMPD (2- amino-2-methyl-l,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-l-propanol), piperazine, 1,3-diaminopropane and piperidine.

A packaged anion exchange chromatography column, an anion exchange membrane device, an anion exchange monolithic device, or depth filter media can operate in either eluted binding mode, flow through fraction mode, or a hybrid mode in which proteins interact with the chromatographic material and can be purified from such material using a buffer that is the same or substantially the same as the boot buffer.

In the binding-elution mode, the column or membrane device is first conditioned with a buffer of the appropriate ionic strength and pH under conditions where certain proteins will be adsorbed onto the resin matrix. For example, during loading, a protein of interest may be adsorbed onto the resin due to electrostatic attraction. After washing the column or membrane device with an equilibration buffer or another buffer with a different pH and/or conductivity, recovery of the product is achieved by increasing the ionic strength ( i.e. , conductivity) of the elution buffer to compete with the solute for charged sites on the anion exchange matrix. Changing the pH and thus changing the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH can be gradual (gradient elution) or stepwise (step elution).

In flow cut mode, the column or membrane device is operated at a selected pH and conductivity such that the protein of interest does not bind to the resin or membrane, while the acidic forms will either remain on the column or have a different elution profile compared to the protein. of interest. In the context of this strategy, the acidic forms will interact with or bind to the chromatographic material under appropriate conditions, while the protein of interest and certain aggregates and/or fragments of the protein of interest will flow through the column.

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 crosslinked poly[styrene-divinylbenzene] backbone; Capto Q Impres and Capto DEAE, which are high flow agarose beads; Toyopearl QAE-550, Toyopearl DEAE-650 and Toyopearl GigaCap Q-650, which are polymer based pellets; Fractogel® EMD TMAE Hicap, which is a synthetic polymer resin with a tentacle type ion exchange material; Sartobind STIC® PA nano, which is a salt-resistant chromatographic membrane with a primary amine ligand; Sartobind Q nano, which is a strong anion exchange chromatographic membrane; CUNO BioCap, which is a Zeta-Plus depth filter media made from inorganic filter aids, purified cellulose and ion exchange resin; and XOHC, which is a depth filter media made from an inorganic filter aid, cellulose, and mixed cellulose esters.

In certain embodiments, sample protein loading can be adjusted to a total protein loading on the column of about 50 g/L to about 500 g/L, or about 75 g/L to about 350 g/L, or about 200 g/L to about 300 g/l. In other exemplary embodiments, the protein concentration of the protein loading mixture is adjusted to a protein concentration of the material loaded into the column from about 0.5 g/L to about 50 g/L, from about 1 g/L to about 20 g/L, or from about 3 g/l to about 10 g/l. In other embodiments, the protein concentration of the protein loading mixture is adjusted to a protein concentration in the column material of about 37 g/L.

Additives such as polyethylene glycol (PEG), detergents, amino acids, sugars, chaotropic agents may be added to increase separation efficiency to achieve better separation, recovery and/or product quality.

In some embodiments, including those related to aflibercept and/or VEGF MiniTrap, the methods of the present invention can be used to selectively remove, significantly reduce, or substantially remove at least 10% of protein variants, thereby obtaining protein compositions that have reduced content of protein variants.

Protein variants may include modifications to one or more residues as follows: one or more asparagines are deamidated; one or more aspartic acids are converted to aspartate-glycine and/or Asn-Gly; one or more methionines are oxidized; one or more tryptophans are converted to N-formylkynurenine; one or more tryptophans are mono-hydroxyl tryptophan; one or more tryptophans are di-hydroxyl tryptophan; one or more tryptophans are tri-hydroxyl tryptophan; one or more arginines are converted to Arg 3-deoxyglucosone; C-terminal glycine is absent; and/or there is one or more non-glycosylated glycosites. It was also noted that the use of AEX reduces the oxidized and acidic forms of anti-VEGF variants in said affinity eluate. Compared to the affinity eluate, after application of 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% oxidized and/or acidic forms of anti-VEGF variants.

Protein variants of aflibercept and/or VEGF MiniTrap may include one or more of (i) oxidized histidines from histidine residues selected from His86, His110, His145, His209, His95, His19 and/or His203; (ii) oxidized tryptophan residues selected from tryptophan residues in Trp58 and/or Trp138; (iii) an oxidized tyrosine residue in Tyr64; (iv) oxidized phenylalanine residues selected from Phe44 and/or Phe166; and/or (v) oxidized methionine residues selected from Met10, Met 20, Met163 and/or Met192.

Cation exchange chromatography

The compositions of the present invention can be prepared by subjecting a biological sample containing a protein of interest to at least one cation exchange (CEX) step. In certain exemplary embodiments, the CEX step will complement the AEX step and is performed either before or after the AEX step. In one aspect, the protein of interest is either aflibercept or MiniTrap or a related molecule.

The use of a cation exchange material versus an anion exchange material, such as the anion exchange materials discussed above , is based in part on the local charges of the protein of interest in a given solution and the required separation conditions. It is within the scope of the present invention to use a cation exchange step prior to the use of the anion exchange step or an anion exchange step prior to the use of the cation exchange step. Furthermore, it is within the scope of the present invention to use only the cation exchange step in combination with other chromatographic procedures.

When performing a cation exchange, a sample containing a protein of interest may be contacted with a cation exchange material using any of a variety of techniques, such as using a batch production technique or a chromatographic technique as described above for AEX.

An aqueous saline solution can be used as both loading and wash buffer with a pH below the isoelectric point (pI) of the protein of interest. In one aspect, the pH is about 0-5 units below the pI of the protein. In another aspect, it is 1-2 units below the pI of the protein. In another aspect, it is 1-1.5 units below the pI of the protein.

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 from about 5 to about 9, or about 5.5 to about 8.5, or about 6 to about 8, or about 6.5 to about 7.5. In one aspect, the concentration of the anionic agent is increased or decreased in the aqueous salt solution to achieve a pH of 5, or 5.5, or 6, or 6.5, or 6.8, or 7.5. Buffer systems suitable for use in CEX processes include, but are not limited to, tris-formate, tris-acetate, ammonium sulfate, sodium chloride, and sodium sulfate.

In certain embodiments, the conductivity and pH of the aqueous salt solution is adjusted by increasing or decreasing the concentration of the cationic agent. In one aspect, the concentration of the cationic agent is maintained in the range of about 20 mmol to about 500 mmol, about 50 mmol to about 350 mmol, about 100 mmol to about 300 mmol, or about 100 mmol to about 200 mmol. Non-limiting examples of the cationic agent are selected from the group consisting of sodium, tris, triethylamine, ammonia, arginine, and combinations thereof.

A packaged cation exchange chromatography column or cation exchange membrane device can operate in both coupling-elution mode, flow through mode, or hybrid mode, with the product exhibiting binding or interaction with the chromatographic material, but can be purified from such material using a buffer that is the same or substantially similar to the boot buffer (the details of these modes are set out above).

Cationic substituents include carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P), and sulfonate (S). Additional cationic materials include, without limitation, Capto SP ImpRes, which is a high flow agarose beads; CM Hyper D Grade F, which is a functionalized hydrogel coated and impregnated ceramic beads, 250-400 ion groups mEq/mL; Eshmuno S, which is a hydrophilic polyvinyl ether base matrix with 50-100 µmeq/ml ionic capacity; Nuvia C Prime, which is a hydrophobic cation exchange medium consisting of a macroporous highly cross-linked hydrophilic polymer matrix 55-75 με/ml; Nuvia S, which has a UNOsphere base matrix with 90 -150 με/ml of ionic groups; Poros HS, which is a rigid polymer beads with a crosslinked poly[styrene-livinylbenzene] backbone; Poros XS, which is a rigid polymer beads with a crosslinked poly[styrene-divinylbenzene] backbone; Toyo Pearl Giga Cap CM 650M, which is a 0.225 meq/ml ionic capacity polymer-based bead; Toyo Pearl Giga Cap S 650M, which is a resin based pellet; Toyo Pearl MX TRP, which is a resin based pellet. It is noted that CEX chromatography can be used with the MM resins described herein.

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

Additives such as polyethylene glycol, detergents, amino acids, sugars, chaotropic agents may be added to increase separation efficiency, thus achieving better separation, recovery and/or product quality.

In certain embodiments, including those relating to aflibercept or an anti-VEGF or VEGF MiniTrap antibody, the methods of the present invention can be used to selectively remove, significantly reduce, or substantially remove all oxo variants in a sample where the protein of interest will be essentially represent a flow-through fraction of the CEX procedure, while the oxo variants will essentially be captured by the column medium.

Mixed Mode Chromatography

Mixed mode chromatography (“MM”) can be used to prepare the compositions of the present invention. MM chromatography, also referred to herein as “multimodal chromatography”, is a chromatographic strategy that uses a support containing a ligand that is capable of providing at least two different interactions with an analyte or protein of interest from a sample. One of these sites provides an attractive type of charge interaction between the ligand and the protein of interest, and the other site provides electron donor-acceptor interaction and/or hydrophobic and/or hydrophilic interaction. Donor-acceptor interactions of electrons include interactions such as hydrogen bonding, π-π, cation-π, charge transfer, dipole-dipole, induced dipole , etc.

The resin in the column used for the mixed mode separation may be Capto Adhere. Capto Adhere is a strong anion exchange material with multimodal performance. Its main matrix is a highly cross-linked agarose with a ligand (N-benzyl-N-methylethanolamine) that exhibits various interaction functionalities such as ionic interaction, hydrogen bonding, and hydrophobic interaction. In certain aspects, the resin used for the mixed mode separation is selected from PPA-HyperCel and HEA-HyperCel. The PPA-HyperCel and HEA-HyperCel base matrices are cross-linked cellulose with high porosity. Their ligands are phenylpropylamine and hexylamine, respectively. Phenylpropylamine and hexylamine provide different selectivity and hydrophobicity options for protein separation. Optional mixed mode chromatography supports include, but are not limited to, Nuvia C Prime, Toyo Pearl MX Trp 650M, and Eshmuno® HCX. In certain aspects, a mixed mode chromatography resin consists of ligands bound to an organic or inorganic support, sometimes referred to as a base matrix, either directly or through a spacer. The support may be in the form of particles such as substantially spherical particles, a monolith, a filter, a membrane, a surface, capillaries, and the like. In certain aspects, the support is derived from a parent polymer such as a crosslinked hydrocarbon material, eg agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, and the like. To obtain a high adsorption capacity, the substrate may be porous and the ligands are then attached to the outer surfaces as well as to the surfaces of the pores. Such base polymer supports can be prepared according to standard methods such as reverse slurry gelation (S Hjerten: Biochim Biophys Acta 79(2), 393-398 (1964), the entire contents of which are incorporated herein by reference). Alternatively, the support may be made from a synthetic polymer such as cross-linked synthetic polymers such as styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, and the like. Such synthetic polymers can be prepared by standard methods, see “Styrene based polymer supports developed by suspension polymerization” (R Arshady: Chimica e L'Industria 70(9), 70-75 (1988), the entire contents of which are incorporated herein. via link). Porous virgin or synthetic polymer substrates are also available from commercial sources such as GE Healthcare, Uppsala, Sweden.

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 about 25 g/L to about 750 g/L, or about 75 g/L to about 500 g/L, or about 100 g/l to about 300 g/l. In certain illustrative embodiments, the protein concentration of the protein loading mixture is adjusted to a protein concentration of the material loaded into the column from about 1 g/L to about 50 g/L, or from about 9 g/L to about 25 g/L.

Additives such as polyethylene glycol, detergents, amino acids, sugars, chaotropic agents may be added to increase separation efficiency, thus achieving better separation, recovery and/or product quality.

In certain embodiments, including those relating to aflibercept and/or MiniTrap, the methods of the present invention can be used to selectively remove, significantly reduce, or substantially remove all PTMs, including oxo variants.

The methods for preparing the compositions of the present invention can also be applied to continuous chromatography. This mode uses at least two columns (referred to as the "first" column and the "second" column). In certain embodiments, this continuous chromatography mode can be performed such that eluted fractions and/or purified fractions containing PTMs, e.g., oxo variants, can then be loaded sequentially or in parallel to a second column (with or without dilution).

In one embodiment, the selected continuous mode media can be one of many hydrophobic and anion exchange pendant chromatographic resins, a monolithic media, a membrane adsorbing media, or a depth filtration media.

Hydrophobic Interaction Chromatography

The compositions of the present invention can also be obtained using hydrophobic interaction chromatography (HIC).

When performing separation, the biological sample is brought into contact with the HIC material, for example, using a batch production technique or using column or membrane chromatography. Before processing with HIC, it may be necessary to adjust the concentration of saline buffer to achieve the necessary binding/interaction of proteins with the resin or membrane.

Because ion exchange chromatography relies on the local charge of the protein of interest for selective separation, hydrophobic interaction chromatography exploits the hydrophobic properties of proteins to achieve selective separation. The hydrophobic groups on or within the protein interact with the hydrophobic groups of the chromatographic resin or membrane. Generally, under the right conditions, the more hydrophobic a protein (or parts of a protein) is, the more strongly it interacts with the column or membrane. Thus, under suitable conditions, HIC can be used to facilitate the separation of process impurities ( eg HCP) as well as related substances ( eg aggregates and fragments) from the protein of interest in the sample.

Similar to ion exchange chromatography, a HIC column or HIC membrane device can also operate in elution, flow or hybrid mode, in which the product binds or reacts with chromatographic material, but can be purified from such material using a buffer that is the same or essentially similar to the boot buffer. (Details of these modes are described above in connection with the AEX treatment.) Since hydrophobic interactions are strongest at high ionic strength, this form of separation is conveniently carried out after a salt elution step, such as those commonly used in connection with ion exchange chromatography. Alternatively, salts can be added to the sample before the HIC step is applied. Protein adsorption to the HIC column is favored by a high salt concentration, but the actual content can vary widely depending on the nature of the protein of interest, the type of salt, and the particular HIC ligand chosen. Various ions can be organized into the so-called solophobic series, depending on whether they promote hydrophobic interactions (salting out effects) or disrupt the structure of water (chaotropic effect) and lead to a weakening of the hydrophobic interaction. Cations are classified from the point of view of increasing the effect of salting out in the form of Ba ²⁺ ; Ca2 ⁺ ; Mg2 ⁺ ; Li ⁺ ; Cs ⁺ ; Na ⁺ ; K ⁺ ; Rb ⁺ ; NH4 ⁺ , while anions can be categorized as chaotropic enhancement as PO ₄ ^3- ; SO ₄ ^2- ; CH ₃ CO ₃ ^- ; CI ^- ; ^Br- ; NO ₃ ^- ; ClO ₄ ^- ; I ^- ; ^SCN- .

In general, Na ⁺ , K ⁺ or NH4 ⁺ sulfates efficiently provide the interaction of the ligand with the protein using HIC. Salts can be formulated to influence the strength of interaction defined by the following relationship: (NH ₄ ) ₂ SO ₄ > Na ₂ SO ₄ > NaCl > NH ₄ C1 > NaBr > NaSCN. Salt concentrations of 0.75M to about 2M ammonium sulfate or 1M to about 4M NaCl are generally useful.

HIC media usually contain a base matrix ( eg cross-linked agarose or a synthetic copolymer material) to which hydrophobic ligands ( eg alkyl or aryl groups) are attached. Suitable media for HIC include agarose resin or a membrane functionalized with phenyl groups ( eg 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, Low or High Substitution Phenyl Sepharose™ 6 Fast Flow, Phenyl Sepharose™ High Performance, Octyl Sepharose™ High Performance (GE Healthcare); propyl Fractogel™ EMD or phenyl Fractogel™ EMD (E. Merck, Germany); methyl Macro-Prep™ or Macro-Prep™ tert-butyl columns (Bio-Rad, CA); WP HI-Propyl (C3)™ (JT Baker, New Jersey); and a simple ether, phenyl or butyl Toyopearl™ (TosoHaas, PA); Toyo Screen PPG; phenyl ToyoScreen; butyl ToyoScreen; hexyl ToyoScreen; GE HiScreen and Butyl FF HiScreen Octyl FF.

The protein load of the sample containing the protein of interest is adjusted to a total protein load on the column from about 50 g/L to about 1000 g/L, from about 5 g/L to about 150 g/L, from about 10 g/L to about 100 g/l, about 20 g/l to about 80 g/l, about 30 g/l to about 50 g/l, or about 40 g/l to about 50 g/l. In certain embodiments, the protein concentration of the protein loading mixture is adjusted to a protein concentration of the material to be loaded into the column from about 0.5 g/L to about 50 g/L, or from about 1 g/L to about 20 g/L.

Since the pH chosen for any particular manufacturing process must be compatible with protein stability and activity, specific pH conditions may be specific to each application. However, since at pH 5.0-8.5, specific pH values have very little bearing on the ultimate selectivity and resolution of HIC separation, such conditions may be preferred. Raising the pH attenuates hydrophobic interactions and protein retention changes more dramatically at pH values above 8.5 or below 5.0. In addition, changes in ionic strength, the presence of organic solvents, temperature and pH (especially at the isoelectric point pI when there is no net surface charge) can affect the structure and solubility of the protein, and therefore the interaction with other hydrophobic surfaces, such as those in the environment. HIC, and therefore, in some embodiments, the present invention includes production strategies in which one or more of the above are adjusted to achieve the desired reduction in process impurities and/or related substances.

In certain embodiments, spectroscopy techniques such as UV, NIR, FTIR, fluorescence, Raman can be used to monitor the protein of interest and impurities in a system, on-line or in-loop mode, which can then be used to monitor the level of aggregates in the pooled material collected from the HIC adsorbent effluent. In certain embodiments, in-system, at-stream or in-loop monitoring methods may be applied either in the effluent line of the chromatography stage or in the collection vessel to ensure that the desired quality/recovery is achieved. In certain embodiments, the UV signal can be used as a surrogate to achieve the appropriate product quality/recovery, and the UV signal can be processed in an appropriate manner, including, without limitation, processing methods such as integration, differentiation, moving average, so that the normal variability of the process can be eliminated and the target product quality can be achieved. In certain embodiments, such measurements can be combined with in-line dilution methods so that the ion concentration/feed/wash conductivity can be controlled using feedback and therefore facilitate product quality control.

size exclusion chromatography

Size exclusion chromatography or gel filtration is based on the separation of components depending on their molecular size. The separation depends on the amount of time the substances spend in the porous stationary phase compared to the time spent in the liquid. The probability that a molecule will be in a pore depends on the size of the molecule and the pore. In addition, the ability of a substance to penetrate into pores is determined by the diffusion mobility of macromolecules, which is higher for small macromolecules. Very large macromolecules may not penetrate the pores of the stationary phase at all; and for very small macromolecules, the penetration probability is close to unity. While the larger molecular weight components move faster through the stationary phase, the smaller molecular size components have a longer path length through the pores of the stationary phase and thus stay longer in the stationary phase.

The chromatographic material may comprise a sizing material, where the sizing material is a resin or a membrane. The matrix used for size exclusion is preferably an inert gel medium, which may be a composite of cross-linked polysaccharides, such as cross-linked agarose and/or dextran, in the form of spherical beads. The degree of crosslinking determines the size of the pores that are present in the swollen gel granules. Molecules larger than a certain size do not enter the gel beads and thus pass through the chromatographic layer most rapidly. Smaller molecules such as detergent, protein, DNA, etc., which penetrate the gel beads to varying degrees depending on their size and shape, are retained as they pass through the layer. Thus, the molecules are usually eluted in order of decreasing molecular size.

Porous chromatographic resins suitable for gel chromatography of viruses may be composed of dextrose, agarose, polyacrylamide, or silica, which have different physical characteristics. Combinations of polymers can also be used. The most commonly used are those sold under the trade name "SEPHADEX" from Amersham Biosciences. Other substrate sizes of other construction materials are also suitable, such as Toyopearl 55F (polymethacrylate from Tosoh Bioscience, Montgomery Pa.) and Bio-Gel P-30 Fine (BioRad Laboratories, Hercules, CA).

The protein load of the sample containing the protein of interest can be adjusted to a total protein load on the column from about 50 g/L to about 1000 g/L, from about 5 g/L to about 150 g/mL, from about 10 g/L up to about 100 g/l, from about 20 g/l to about 80 g/l, from about 30 g/l to about 50 g/l, or from about 40 g/l to about 50 g/l. In certain embodiments, the protein concentration of the protein loading mixture is adjusted to a protein concentration of the material to be loaded into the column from about 0.5 g/L to about 50 g/L, or from about 1 g/L to about 20 g/L.

Virus filtration

Viral filtration is a special step in the manufacturing process that reduces the viral load. This step is usually performed after chromatographic polishing. Virus reduction can be achieved using suitable filters including, but not limited to Planova 20N™, 50 N or BioEx from Asahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPV from Sartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation. One of ordinary skill in the art will recognize the selection of a suitable filter to obtain the desired filtration performance.

Ultrafiltration / Diafiltration

In certain embodiments of the present invention, ultrafiltration and diafiltration are used to further concentrate and assemble the protein of interest. Ultrafiltration is described in detail in Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9); the entire contents of which are incorporated herein by reference. One filtration process is tangential flow filtration as described in the Millipore Pharmaceutical Process Filtration Catalogue, pages 177-202 (Bedford, Mass., 1995/96), the entire contents of which are hereby incorporated by reference. Ultrafiltration is generally understood to mean filtration using filters with a pore size of less than 0.1 µm. When using filters having such a small pore size, the sample volume can be reduced by permeating the sample buffer through the pores of the filter membrane while the proteins remain above the membrane surface.

A person skilled in the art can select a suitable membrane filter device for the UV/DF operation. Examples of membrane cassettes suitable for the present invention include, without limitation, Pellicon 2 or Pellicon 3 cassettes with 10 kDa, 30 kDa or 50 kDa membranes from EMD Millipore, Kvick 10 kDa, 30 kDa or 50 kDa membrane cassettes from GE Healthcare, and Centramate cassettes or Centrasette 10 kDa, 30 kDa, or 50 kDa from Pall Corporation.

Illustrative Acquisition Strategies

Primary recovery can occur by sequentially applying pH reduction, centrifugation, and filtration to remove cells and cellular debris (including HCP) from the production bioreactor harvest. The present invention is directed to subjecting a biological sample containing a protein of interest from primary recovery to one or more production steps, including (in no particular order) AEX, CEX, SEC, HIC, and/or MM. Certain aspects of the present invention include additional processing steps. Examples of additional workup procedures include ethanol precipitation, isoelectric focusing, reverse phase HPLC, silica gel chromatography, Sepharose™ heparin chromatography, post anion exchange chromatography and/or post cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxyapatite chromatography, gel- electrophoresis, dialysis, and affinity chromatography ( eg using protein A or G, antibody, specific substrate, ligand, or antigen as the capture reagent). In certain aspects, the column temperature (as well as other parameters) can be independently varied to improve the separation efficiency and/or yield of any particular production step.

In certain embodiments, the unbound run-through fraction and wash fractions may be further fractionated and a combination of fractions that provide the desired product purity may be combined.

The column loading and washing steps can be controlled by measuring in-loop, down-stream, or off-loop levels of product-related impurities/substances, either in the column effluent, or in the collected pool, or both, to achieve a particular goal of product quality and/or exit. In some embodiments, the loading concentration can be dynamically controlled by loop dilution, serial or continuous dilution with buffers or other solutions to achieve the separation required to improve separation efficiency and/or yield.

Examples of such manufacturing processes are shown in Fig. 5-8 .

In FIG. 5 shows one exemplary embodiment used to prepare aflibercept. Referring to FIG. 5 , the method comprises (a) expressing aflibercept in a host cell cultured in COS; (b) capturing aflibercept using a first chromatography support, which may include a capture affinity resin; and (c) contacting at least a portion of the aflibercept with a second chromatography support, which may include anion exchange chromatography. Step (c) may further include washing the AEX column and collecting the running fraction(s) of the sample containing aflibercept. Optionally, step (c) may include cleaning the second chromatographic support and collecting the purified fractions. These steps can be performed using the standard procedure in combination with the methodology mentioned above . It should be understood that a person skilled in the art may prefer the use of CEX instead of or in addition to AEX. Optionally, additional chromatographic steps may be used, including, without limitation, HIC and SEC.

In addition to the illustrative embodiment in FIG. 5 , another further exemplary embodiment (d) contacting at least a portion of said aflibercept obtained in step (c) with a third chromatographic support. In one aspect, the protocol may include (e) contacting at least a portion of the aflibercept from step (d) with a fourth chromatographic support. In one aspect of this embodiment, the protocol may optionally include exposing the sample containing aflibercept from step (c) to a pH of less than 5.5. In one aspect, the present method includes a clarification step prior to step (a).

In FIG. 6 shows one exemplary embodiment used to make a VEGF MiniTrap. The method comprises (a) expressing aflibercept in a host cell cultured in COS; (b) capturing aflibercept using a first chromatographic support, which may include an affinity chromatography resin; (c) cleavage of aflibercept, thereby removing the Fc domain and forming a sample containing VEGF MiniTrap; (d) contacting the sample from step (c) with a second chromatographic support, which may be affinity chromatography; and (e) contacting the flow fraction from step (d) with a third chromatographic support, which may include anion exchange chromatography. Step (d) involves collecting the flow-through fraction(s) where, due to the lack of an Fc domain, MiniTrap must reside while aflibercept or any other protein having an Fc domain must substantially interact with the affinity column in step (d). Optionally, step (d) may include cleaning the third chromatographic substrate and collecting the purified fractions. These steps can be performed according to the standard methodology in combination with the methodology set forth above. Optionally, additional chromatographic steps such as HIC and SEC may be included without limitation.

In FIG. 7 shows one exemplary embodiment for making aflibercept. The method comprises (a) expressing aflibercept in a host cell cultured in COS; (b) capturing aflibercept using a first chromatographic support, which may include cation exchange chromatography; and (c) contacting the flow fraction from step (b) with a second chromatographic support, which may include anion exchange chromatography. Optionally, step (c) may include cleaning the second chromatographic support and collecting the purified fractions. These steps can be performed according to standard procedures in combination with the protocols mentioned above. Optionally, additional chromatographic procedures may be applied, including, without limitation, HIC and SEC.

In FIG. 8 shows one exemplary embodiment for making a VEGF MiniTrap. The method comprises (a) expressing aflibercept in a host cell cultured in COS; (b) capturing aflibercept using a first chromatographic support, which may include ion exchange chromatography; (c) subjecting the flow fraction from (b) containing aflibercept to affinity chromatography; elution, and the elution includes aflibercept; (d) subjecting aflibercept to (c) cleavage activity, whereby the Fc domain is cleaved to form a VEGF MiniTrap. In one aspect, the ion exchange from step (b) includes AEX. Alternatively, step (b) may include CEX. Optionally, additional chromatographic steps may be included, such as additional ion exchange chromatography steps after step (d), addition of HIC and/or SEC.

VII. Pharmaceutical formulations containing compositions

The present invention also discloses compositions containing compositions against VEGF (as described above). Suitable anti-VEGF protein formulations include, but are not limited to, those described in US Pat. No. 7,608,261, US Pat. No. 7,807,164, US Pat. 9580489, U.S. Patent No. 10400025, U.S. Patent No. 8110546, U.S. Patent No. 8404638, U.S. Patent No. 8710004, U.S. Patent No. 8921316, U.S. Patent No. 9416167, U.S. Patent No. 9511140, U.S. Patent No. 9636400 and U.S. Patent No. 104 06226, all of which incorporated herein by reference in their entirety.

The upstream process technologies (described in section IV above ), the downstream process technologies (described in section V above ) can be used alone or in combination with each other to effect formulation preparation.

The present invention discloses compositions containing compositions against VEGF, together with one or more ingredients/auxiliaries, as well as methods of use and methods for obtaining such compositions. In one embodiment of the present invention, the pharmaceutical composition of the present invention has a pH of about 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, or 6.2.

To prepare pharmaceutical formulations of anti-VEGF compositions, the anti-VEGF composition is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, N.Y.; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.; the entire contents of which are incorporated herein by reference. In one embodiment of the present invention, the pharmaceutical formulation is sterile.

Pharmaceutical formulations of the present invention include an anti-VEGF composition and a pharmaceutically acceptable carrier including, for example, water, buffers, preservatives and/or detergents.

The present invention provides a pharmaceutical composition comprising any of the anti-VEGF compositions set forth herein and a pharmaceutically acceptable carrier, for example, wherein the concentration of the polypeptide is about 40 mg/ml, about 60 mg/ml, about 80 mg/ml, 90 mg/ml. ml, about 100 mg/ml, about 110 mg/ml, about 120 mg/ml, about 130 mg/ml, about 140 mg/ml, about 150 mg/ml, about 200 mg/ml, or about 250 mg/ml.

The scope of the present invention includes dehydrated, eg, lyophilized, compositions comprising an anti-VEGF protein and a pharmaceutically acceptable carrier substantially (from about 85% to about 99% or more) free of water.

In one embodiment, an additional therapeutic agent that is administered to a subject in conjunction with an anti-VEGF composition disclosed herein is administered to the subject in accordance with Physicians' Desk Reference 2003 (Thomson Healthcare; 57th edition (November 1, 2002), the contents of which incorporated herein by reference).

The present invention provides a vessel ( e.g. , a plastic or glass vial with a cap or chromatographic column, a cannula or syringe barrel) containing any of the anti-VEGF compositions or a pharmaceutical formulation containing a pharmaceutically acceptable carrier described herein. The present invention also provides an injectable device containing an anti-VEGF composition or formulation as described herein, such as a syringe, pre-filled syringe, or pen. In one aspect, the vessel is colored ( eg brown) to block light, natural or otherwise.

The present invention includes combinations comprising anti-VEGF compositions together with one or more additional therapeutic agents. The anti-VEGF composition and the additional therapeutic agent may be in the same composition or in separate compositions. For example, the therapeutic agent is an Ang-2 inhibitor ( eg , nesvacumab), a Tie-2 receptor activator, an anti-PDGF antibody or antigen-binding fragment thereof, an anti-PDGF receptor or an anti-PDGF receptor beta antibody or antigen-binding fragment thereof, and/or an additional VEGF antagonist, such as aflibercept, conbercept, bevacizumab, ranibizumab, anti-VEGF aptamer such as pegaptanib ( eg pegaptanib sodium), single chain ( eg VL-VH) anti-VEGF antibody such as brolucizumab, DARPin to VEGF such as DARPin abicipar pegol, a bispecific anti-VEGF antibody, for example, which also binds to ANG2, such as RG7716, or a soluble form of human vascular endothelial growth factor receptor 3 (VEGFR-3) containing extracellular domains 1-3 expressed as an Fc fusion protein.

VIII. Methods of treatment

The present invention provides methods for treating or preventing cancer ( e.g. , growth and/or metastasis of which is mediated at least in part by VEGF, e.g., VEGF-mediated angiogenesis) or angiogenic eye disease in a subject, comprising administering a therapeutically effective amount of the compositions described in this document (section III above ).

The upstream process technologies (Section IV above ), downstream process technologies (Sections V and VI above ) can be used alone or in combination with each other to produce the compositions described in Section III and/or the compositions described in Section VII that can be used. for the treatment or prevention of various disorders, including ophthalmic and oncological disease.

The present invention also provides a method of administering the compositions described herein (section III and section VII) to a subject ( e.g. , a human) comprising administering compositions of about 0.5 mg, 2 mg, 4 mg, 6 mg, 8 mg, 10 mg, 12 mg, 14 mg, 16 mg, 18 mg, or 20 mg of the protein of interest ( e.g. aflibercept or MiniTrap) in a maximum of about 100 µl, e.g., about 50 µl, about 70 µl, or about 100 µl, and optionally an additional therapeutic agent, into the body of the subject via, for example, intraocular injection, such as via intravitreal injection.

The present invention provides a method for treating cancer whose mouth and/or metastasis is mediated at least in part by VEGF, for example, VEGF-mediated angiogenesis or angiogenic eye disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the compositions given in this document (Section III and Section VII above), for example, 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg of the protein of interest, in no more than about 100 μl, and optionally additional therapeutic agent, to the subject. In one embodiment of the present invention, administration is by intravitreal injection. Non-limiting examples of angiogenic eye diseases that can be treated or prevented using those described herein include

age-related macular degeneration ( for example , wet or dry),

macular edema,

macular edema after retinal vein occlusion,

retinal vein occlusion (RVO),

central retinal vein occlusion (CRVO),

retinal vein occlusion (BRVO),

diabetic macular edema (DME),

choroidal neovascularization (CNV),

iris neovascularization,

neovascular glaucoma,

postoperative fibrosis in glaucoma,

proliferative vitreoretinopathy (PVR),

neovascularization of the optic disc,

corneal neovascularization,

retinal neovascularization,

vitreous neovascularization,

pannus,

pterygoid hymen,

vascular retinopathy,

diabetic retinopathy in a subject with diabetic macular edema; And

diabetic retinopathy ( eg , non-proliferative diabetic retinopathy ( eg , characterized by a Diabetic Retinopathy Severity Scale (DRSS) level of about 47 or 53) or proliferative diabetic retinopathy; eg , in a subject who does not suffer from DME).

The route of administration of such compositions or formulations (Section III and Section VII) may vary and is determined by the skilled artisan. Routes of administration include parenteral, non-parenteral, oral, rectal, transmucosal, enteric, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, external, dermal, intraocular, intravitreal, percutaneous, or intra-arterial.

In one embodiment of the present invention, intravitreal injection of a pharmaceutical composition of the present invention (which includes the compositions or formulations of the present invention) comprises the step of puncturing the eye with a syringe and needle ( e.g. , 30 gauge injection needle) containing the composition, and injecting the composition ( e.g. , no more than about 100 microliters; about 40, 50, 55, 56, 57, 57.1, 58, 60, or 70 microliters) into the vitreous of the eye in sufficient volume to deliver the therapeutically effective amount set forth herein, e.g., about 2, 4, 6, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8 or 8.9, 10 or 20 mg of protein of interest . Optionally, the methods include the steps of administering a local anesthetic ( eg , proximethacaine, lidocaine, or tetracaine), an antibiotic ( eg , a fluoroquinolone), an antiseptic ( eg , povidone-iodine), and/or a pupil dilator into the eye to be injected. In one aspect, prior to injection, a sterile field is created around the eye to be injected. Following intravitreal injection, the subject is monitored for increased intraocular pressure, inflammation, and/or blood pressure.

An effective or therapeutically effective amount of a protein of interest for an angiogenic eye disease refers to an amount of a protein of interest sufficient to cause regression, stabilization, or elimination of a cancer or angiogenic eye disease, for example, by regression, stabilization, or elimination of one or more symptoms or signs of cancer or angiogenic eye disease to any clinically measurable degree, e.g. in relation to angiogenic eye disease, causing reduction or maintenance of the Diabetic Retinopathy Severity Scale (DRSS), by improving or maintaining vision ( e.g. , in best corrected visual acuity, measured increasing the letters ETDRS), increasing or maintaining the field of view and/or decreasing or maintaining central retinal thickness and, in relation to cancer, arresting or reversing the growth, survival and/or metastasis of cancer cells in a subject.

In one embodiment of the present invention, an effective or therapeutically effective amount of a protein of interest, such as aflibercept, for treating or preventing angiogenic eye disease is about 0.5 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 7.25 mg, 7.7 mg, 7.9 mg, 8.0 mg, 8.1 mg, 8.2 mg, 8.3 mg, 8.4 mg, 8.5 mg, 8.6 mg, 8.7 mg, 8.8 mg, 8.9 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, or 19 mg 20 mgFor example, in no more than about 100 μl. The amount may vary depending on the age and size of the administered subject, the target disease, conditions, route of administration, and the like. In certain exemplary embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the protein of interest in an amount that may be approximately the same as, or less than, or greater than the initial dose, with successive doses separated by at least 1- 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated. It should also be understood that for any given subject, specific dosage regimens may be adjusted over time according to individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges given herein are illustrative only and are not intended to be limiting. or practice of the claimed composition.

IX. Method for analysis of protein variants

The levels of protein variants in a chromatographic sample obtained using the techniques described herein can be analyzed as described in the examples below. In some embodiments, the cIEF method is used using an iCE3 analyzer (ProteinSimple) with a fluorocarbon coated capillary cartridge (100 µm x 5 cm). The ampholyte solution consists of a mixture of 0.35% methylcellulose (MC), 4% Pharmalyte 3-10 carrier ampholytes, 4% Pharmalyte 5-8 carrier ampholytes, 10 mM L-arginine HCl, 24% formamide, and pI 5.12 markers and 9.77 in purified water. The anolyte is 80 mmol phosphoric acid and the catholyte is 100 mmol sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted with purified water to 10 mg/ml. The samples were mixed with the ampholyte solution and then focused by applying a potential of 1500 V for one minute and then a potential of 3000 V for 7 minutes. The in-focus variants were imaged by passing 280 nm ultraviolet light through the capillary and into the digital camera lens of the CCD. This image was then analyzed to determine the distribution of different charge options. Specialists in this field can change the exact parameters, while achieving the desired result.

Various publications are cited in the description text, including patents, patent applications, published patent applications, access numbers, technical articles, and scientific articles. Each of these cited references is incorporated by reference in its entirety.

The present invention will become clearer from the following examples. However, they should not be construed as limiting the scope of the invention.

EXAMPLES

MiniTrap (MT) 1-6 discussed in the examples are as follows:

MT1: VEGF MiniTrap obtained by cleavage of aflibercept obtained using XOC1.

MT2: VEGF MiniTrap obtained by cleavage of aflibercept obtained using XOS2.

MT3: VEGF MiniTrap obtained by cleavage of aflibercept obtained using XOS3.

MT4: VEGF MiniTrap obtained by cleavage of aflibercept obtained using soy hydrolysate.

MT5: Recombinant VEGF MiniTrap (dimer).

MT6: Recombinant VEGF MiniTrap (scFv).

The characteristics of MT1, MT5 and MT6 are described below in Example 8.

Sample Color Evaluation

The spectrophotometric analysis method for measuring the b* value (CIELAB) proved to be suitable for performing color evaluation.

The absorbance of a 1 ml protein sample was quantified from the visible light spectrum (380 to 780 nm) and the absorbance curve was converted to the CIELAB color space using a set of matrix operations. The instrument can process approximately 6 samples per hour. The high throughput assay format used a CLARIOstar plate reader (BMG Labtech). Up to 96 samples can be analyzed using a 96-well plate that requires 0.3 ml of sample.

To convert BY standards to b* values, BY reference standards (BY1 to BY7) were quantified using a high throughput analysis format.

Solutions were prepared according to the BY standards described above. The b* value for each of the standards is shown in FIG. 9 . This method provided faster analysis with less sample requirements and shorter analysis times, as shown in Table 3 below. For all samples evaluated using this method, the protein concentration in the test samples was standardized to 5 g/l or 10 g/l.

Table 3 original High throughput Quantity/sample 1 ml 0.3 ml Measurement format cuvette (separate) 96-well plate (main part) lead time 6 samples per hour 96 samples in 5 minutes Data input manual automated Data storage excel LIMS

Example 1 Protein Preparation Using a Chemically Defined Medium

1.1 Source and collection of cells

In the current study, an aflibercept-producing cell line was used. Aflibercept-producing cell lines were cultured and harvested using Chemically Defined Medium (COS).

1.2 Proteolytic cleavage of aflibercept

An immobilized IdeS enzyme column (FabRICATOR® obtained from Genovis (Cambridge, MA)) was used to generate MT1. Aflibercept derived from a cell culture harvest (20 mg in 1.0 ml digestion buffer) was added to the column and incubated for 30 min at 18°C. After 30 min. the column was washed with cleavage buffer (1.0 ml). The digestion mixture and washing solutions were combined. The mixture was loaded onto a Protein A analytical column and eluted (Applied Biosystems™, POROS™ 20 µmol protein A, 2.1×30 mmol, 0.1 ml cartridge (catalog # 2-1001-00). Applied Biosystems™ Protocol for POROS™ Cartridge 20 µmol Protein A 2.1 x 30 mmol, 0.1 ml (P/N 2-1001-00) Column height was 20 ± 1.0 cm, residence time was 15 minutes, and equilibration/wash was performed using 40 mmol Tris buffer, 54 mmol acetate pH 7.0 ± 0.1.

Example 2 Anion Exchange Chromatography (AEX) to Minimize Color

(A) AEX was applied to reduce color formation

AEX chromatography was used to remove the color obtained during the preparation of aflibercept expressed using XOC1.

2.1 Design

For this study, five AEX splits were performed as detailed in Table 2-1 with an AEX protocol as described in Table 2-2 . 15.7 ml Sepharose Fast Flow Q column (19.5 cm bed height, 1.0 cm id) and 14.1 ml POROS 50 HQ column (18.0 cm bed height, 1.0 cm id) integrated into the desktop liquid chromatography controller AKTA Avant.

The pH of the AEX load was adjusted to the target value of ± 0.05 pH units using 2 mol Tris base or 2 mol acetic acid. The electrical conductivity of the AEX load was adjusted to the target value of ± 0.1 mS/cm using 5 mol sodium chloride or deionized water. All pool samples were analyzed for high molecular weight (HMW), color and yield.

Table 2-1. Summary of AEX Color Reduction Study Design Separation via AEX Estimated condition Resin 1 pH 8.30-8.50, 1.90-2.10 mS/cm POROS 50 HQ 2 pH 7.90-8.10, 2.40-2.60 mS/cm Q Sepharose FF 3 pH 7.90-8.10, 2.40-2.60 mS/cm POROS 50 HQ 4 pH 7.70-7.90, 3.90-4.10 mS/cm Q Sepharose FF 5 pH 7.70-7.90, 3.90-4.10 mS/cm POROS 50 HQ

Table 2-2. AEX protocol for color reduction Stage Description mobile phase Column volumes (CV) Line speed
(cm/h) 1 Prebalancing 2 mol sodium chloride (NaCl) 2 200 2 Balancing 50 mmol Tris buffer, variable mmol NaCl
Variable pH and Conductivity 2 200 3 Loading Download AEX
Variable pH and Conductivity 40 g/l-resin 200 4 FT/flush 50 mmol Tris buffer, variable mmol NaCl
Variable pH and Conductivity 2 200 5 Cleaning 1 2 mol sodium chloride (NaCl) 2 200 6 Cleaning 2 1 n. sodium hydroxide (NaOH) 2 200

2.2 Results

Application of the AEX separation for production showed significant color reduction. (Table 2-3). For example, as seen in Table 2-3 , the color observed in the flow through fraction (FT) and wash in the AEX 1 separation (pH 8.30-8.50, 1.90-2.10 mS/cm) was ab * 1.05 compared to AEX Load Color ("AEX Load") with ab* value of 3.06. An increase in the b* value reflects the intensity of the yellow-brown color of the sample.

Five AEX separations were performed to evaluate resin effect (Q Sepharose FF or POROS 50 HQ) and pH and conductivity setpoint (pH 8.40 and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm or pH 7 .80 and 4.00 mS/cm) for color reduction. For POROS 50 HQ, yields (64.4, 81.9 and 91.4%) and HMW pool levels (1.02, 1.29 and 1.83%) increased when the setpoint was changed to lower pH and higher conductivity . The color (b* values) also increased (1.05, 1.33 and 1.55) as the set point was changed to lower pH and higher conductivity. Higher pH levels and lower conductivity produced the most color reduction compared to the AEX separation for POROS 50 HQ.

For Q Sepharose Fast Flow, yields (49.5 and 77.7%) and HMW pool levels (0.59 and 1.25%) also increased as the set point was changed to lower pH and higher conductivity. The color (b* values) also increased (0.96 and 1.35) as the set point was changed to lower pH and higher conductivity.

The use of AEX reduces yellow-brown chromaticity - see table 2-3 . In addition, Q Sepharose Fast Flow was determined to reduce color more than POROS 50 HQ for two target values evaluated in both resins. At a set pH of 8.00 and 2.50 mS/cm, the POROS 50 HQ pool had a b* value of 1.33 while the Sepharose Fast Flow Q pool had a b* value of 0.96. Similarly, at a set pH of 7.80 and 4.00 mS/cm, the POROS 50 HQ pool had a b* value of 1.55 while the Sepharose Fast Flow Q pool had a b* value of 1.35 ( Table 2-3 ) .

Table 2-3. Summary of AEX color reduction experiment results Separation via AEX Fraction Exit (%) VM
(%) Color (L*) Color (a*) Color (b*) 1 FT/flush 64.4 1.02 98.89 0.01 1.05 2 FT/flush 49.5 0.59 98.30 -0.03 0.96 3 FT/flush 81.9 1.29 99.07 -0.07 1.33 4 FT/flush 77.7 1.25 99.42 -0.04 1.35 5 FT/flush 91.4 1.83 99.19 -0.09 1.55 - Filtered Pool (AEX Boot) - 3.66-3.98 98.73 -0.21 3.06

AEX, anion exchange chromatography; HMW, macromolecular compounds; N/A, not specified

To measure the color, the fractions were corrected to a protein concentration of 10 g/L.

2.3 Conclusion

The use of AEX has been found to reduce tan coloration, see Table 2-3 . Referring to Table 2-3 , the AEX loading has a b* value of 3.06, but upon exposure to AEX chromatography (AEX separation 1-5), the b* value decreases, indicating a decrease in tan color. Again, as the value of b* decreases, so does the coloring; an increase in the b* value reflects an increase in yellow-brown color in this sample.

Color reduction was assessed using two AEX resins (POROS 50 HQ and Q Sepharose Fast Flow) and three set points (pH 8.40 and 2.00 mS/cm, pH 8.00 and 2.50 mS/cm, and pH 7 .80 and 4.00 mS/cm). For both resins, the color reduction was higher for higher pH values and lower conductivity values. In addition, Q Sepharose Fast Flow provided greater color reduction than POROS 50 HQ at two test points evaluated on both resins (pH 8.00 and 2.50 mS/cm and pH 7.80 and 4.00 mS/cm ). However, all five AEX separation methods provided a significant reduction in color compared to the AEX loading solution ("AEX Loading"), demonstrating the importance of obtaining AEX in the process of obtaining aflibercept expressed using COS. The initial value b* of the load AEX (when weighing 10 g/l) may vary from about 0.5 to about 30, more specifically from about 1.0 to about 25.0, and even more specifically from about 2.0 to about 20 ,0. After application of AEX, the b* value for the flow fraction (at a concentration of 10 g/l) may vary from 0.5 to about 10.0, more specifically from about 0.5 to about 7.0, and even more specifically from about 0.5 up to about 5.0.

2.4 Peptide mapping

Getting a sample . Tryptic mapping of reduced and alkylated aflibercept samples derived from the AEX load and the run-through fraction of the above experiment ( Table 2-3 ) was performed to detect and quantify post-translational modification (PTM). An aliquot of each sample (load and running fraction) was denatured with 8.0 M urea, 0.1 M Tris-HCl, pH 7.5, reduced with DTT, and then alkylated with iodoacetamide. The denatured, reduced and alkylated sample was first digested with recombinant Lys-C (rLys-C) at an enzyme to substrate ratio of 1:100 (w/w) at 37°C for 30 min, diluted with 0.1 mol Tris-HCl , pH 7.5, so that the final concentration of urea was 1.8 mol, then digested with trypsin at a ratio of enzyme to substance of 1:20 (w/w) at 37°C for 2 hours, and then deglycosylated using PNG -ase F at a ratio of enzyme substrates of 1:5 (wt./wt.) at 37°C for 1 hour. Cleavage was stopped by bringing the pH below 2.0 using formic acid (FA).

Analysis by LC-MS . A 20 μg aliquot of the resulting rLys-C/tryptic peptides from each sample was separated and analyzed by reverse phase ultra-performance liquid chromatography (UPLC) using a Waters ACQUITY UPLC CSH C18 column (130 Å, 1.7 μmol, 2.1 × 150 mm) followed by detection of CPC in the system (at wavelengths of 280 nm, 320 nm and 350 nm) and analysis by mass spectrometry. Mobile phase A contained 0.1% FA in water and mobile phase B contained 0.1% FA in acetonitrile. After the introduction of the sample, the gradient was initiated with exposure for 5 min. at 0.1% B followed by a linear increase to 35% B over 75 minutes for optimal peptide separation. MS and MS/MS experiments were performed using a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with higher energy impact dissociation (HCD) used for peptide fragmentation for MS/MS experiments. Peptide identity assignment was based on the experimentally determined exact mass of a given peptide in the full MS spectrum as well as b and y ion fragments in the corresponding MS/MS HCD spectrum. Extracted ion chromatograms of the peptides from the feed and flow fraction were obtained ( see Fig. 10 ). As seen in the extracted ion chromatogram in FIG. 10 shows peptide fragments identified in "AEX Load" and "FT/AEX Wash" from AEX partitions 1-5 (as defined in Table 2-3 ). The relative amount of some of these peptides identified in FIG. 10 from the peptide mapping analysis are shown in FIG. 11 .

Referring to FIG. 11 , this number identifies the various peptide fragments analyzed and their relative levels of oxidation. In particular, the third column identifies the amino acid residues ("peptide sequence") of the peptide fragments that have been isolated and analyzed. Each peptide sequence has an amino acid residue which is underlined. An underlined amino acid residue indicates an amino acid in the peptide sequence that is oxidized. Oxidized amino acids correspond to either histidine (H) oxidation or tryptophan (W) oxidation. This figure also shows the lines to the right of each of the peptide sequences, showing the number of oxidized forms. This shading in the rows indicates differences in the relative amount of oxidized residues in a particular sample using different AEX divisions indicated in the respective column headings. For example, referring to the second peptide (EIGLLTCEATVNGHLYK) in FIG. 11 , when read horizontally, the relative total population of a given peptide in a particular sample ("aflibercept AEX loading") that is oxidized is approximately 0.013% oxidized. As you move along the same line, a shift in hatching is observed, indicating a change in the relative content of oxidized forms. For example, when using the same peptide sequence, the relative amount of oxidized forms for AEX separation is from 0.006% to 0.010%, following different AEX separation protocols. Thus, it can be understood that AEX chromatography reduces the content of oxidized species.

(B) AEX was used to reduce color formation when making MiniTrap

AEX chromatography was performed to remove the color obtained during the preparation of MT1, which was obtained by performing cleavage of the full-length aflibercept expressed using XOC1.

2.5 Design

For this study, four AEX splits were performed as described in Table 2-4 . The AEX load was obtained from the MT1 sample filtration ("MT1 filtered pool"). 15.7 ml Capto Q column (20.0 cm bed height, 1.0 cm id), 14.1 ml POROS 50 HQ column (18.0 cm bed height, 1.0 cm id) and column with 16.5 ml Q Sepharose FF (layer height 21.0 cm, inner diameter 1.0 cm) were integrated into an AKTA Avant tabletop liquid chromatography controller for this experiment.

The pH of the AEX load was adjusted to the target value of ± 0.05 pH units using 2 mol Tris base or 2 mol acetic acid. The conductivity of the AEX load was adjusted to the target value of ± 0.1 mS/cm using 5 mol sodium chloride or deionized water. All pool samples were analyzed for HMW, color and yield.

Table 2-4. Study design summary for the AEX color reduction study Separation via AEX Resin AEX Protocol 1 Capto Q Table 2-6 2 POROS 50 HQ Table 2-6 3 Q Sepharose FF Table 2-6 4 POROS 50 HQ Table 2-5

Table 2-5. AEX Flow Fraction Protocol Used for Color Reduction Study Stage Description mobile phase Column volumes (CV) Line speed
(cm/h) 1 Prebalancing 2 mol sodium chloride (NaCl) 2 200 2 Balancing 50 mM Tris buffer, 40 mM NaCl
pH 7.90-8.10, 6.50-7.50 mS/cm 2 200 3 Loading Download AEX
pH 7.90-8.10, 6.50-7.50 mS/cm 30 g/l-resin 200 4 Washing 50 mM Tris buffer, 40 mM NaCl
pH 7.90-8.10, 6.50-7.50 mS/cm 2 200 5 Cleaning 1 2 mol sodium chloride (NaCl) 2 200 6 Cleaning 2 1 n. sodium hydroxide (NaOH) 2 200

AEX, anion exchange chromatography; CV, column volume

Table 2-6. AEX Binding and Elution Protocol Used for Color Reduction Study Stage Description mobile phase Column volumes (CV) Line speed
(cm/h) 1 Prebalancing 2 mol sodium chloride (NaCl) 2 200 2 Balancing 50 mmol tris buffer
pH 8.30-8.50, 1.90-2.10 mS/cm 2 200 3 Loading Download AEX
pH 8.30-8.50, 1.90-2.10 mS/cm 30 g/l-resin 200 4 Washing 50 mmol tris buffer
pH 8.30-8.50, 1.90-2.10 mS/cm 2 200 5 Elution 50 mM Tris buffer, 70 mM NaCl
pH 8.30-8.50, 8.50-9.50 mS/cm 2 200 6 Cleaning 1 2 mol sodium chloride (NaCl) 2 200 7 Cleaning 2 1 n. sodium hydroxide (NaOH) 2 200

AEX, anion exchange chromatography; CV, column volume

2.6 Results

All four AEX separations provided color reduction as seen by staining the running fraction and washing for AEX separations 1-4 ( Table 2-7 ). Although the first three AEX separations were evaluated in the binding and elution mode ( Table 2-6 ), it was observed that most of the product was present in the loading and washing blocks (62%-94%).

The first three separations were used to evaluate pH targets of 8.4 and 2.0 mS/cm for Capto Q, POROS 50 HQ and Q Sepharose FF resins. All three separations provided a good yield (more than 80%). The POROS 50 HQ AEX pool showed the lowest yellow in the AEX pool (b* value of 2.09), followed by the Q Sepharose FF AEX pool (b* value of 2.22) and the Capto Q AEX pool (b* value of 2.55 ).

Table 2-7. Summary of AEX color reduction experiment results Separation via AEX Fraction Exit (%) VM (%) Color (L*) Color (a*) Color (b*) 1 FT/flush 90.7 0.49 99.11 -0.27 2.55 2 FT/flush 93.8 0.33 99.20 -0.28 2.09 3 FT/flush 86.7 0.23 98.88 -0.23 2.22 4 FT/flush 99.5 1.13 98.90 -0.39 3.40 - Filtered MT1 pool (AEX download) - 0.65 98.18 -0.37 4.17

AEX, anion exchange chromatography; HMW, macromolecular compounds.

To measure the color, the fractions were corrected to a protein concentration of 5 g/l.

2.7 Conclusion

As seen for aflibercept (see section 2.3 above), the use of AEX was found to reduce tan coloration ( Table 2-7 ) in MiniTrap preparation. Referring to Table 2-7 , the AEX loading has a b* value of 4.17, but upon exposure to AEX chromatography (AEX separation 1-4), the b* value decreases, indicating a decrease in tan color. Again, as the value of b* decreases, so does the coloring. The initial value b* of the load AEX (at 5 g/l weight) may vary from about 0.5 to about 25, more specifically from about 1.0 to about 20.0, and even more specifically from about 1.5 to about 15 ,0. After application of AEX, the b* value for the flow fraction (at a concentration of 5 g/l) can vary from 0.5 to about 10.0, more specifically from about 0.5 to about 7.0, and even more specifically from about 0.5 up to about 5.0.

Example 3 Oxidized Peptide Assay

3.1 Peptide mapping

Getting a sample . Tryptic mapping of samples of reduced and alkylated MiniTrap (MT1) and MT4 (MiniTrap, similar to MT1 using another full length soy hydrolysate cell culture aflibercept) was performed to identify and quantify post-translational modification. An aliquot of the sample was denatured with 8.0 M urea in 0.1 M Tris-HCl, pH 7.5, reduced with DTT, and then alkylated with iodoacetamide. The denatured, reduced and alkylated drug substance was first cleaved with recombinant Lys-C (rLys-C) at an enzyme to substrate ratio of 1:100 (w/w) at 37°C for 30 min., diluted with 0.1 mol Tris- HCl, pH 7.5, so that the final concentration of urea was 1.8 mol, then digested with trypsin at a ratio of enzyme to substance of 1:20 (w/w) at 37°C for 2 hours, and then deglycosylated using PNGase F at a ratio of enzyme substrates 1:5 (wt./wt.) at 37°C for 1 hour. Cleavage was stopped by bringing the pH below 2.0 using formic acid (FA).

Analysis by LC-MS . A 20 μg aliquot of the resulting rLys-C/tryptic peptides from each sample was separated and analyzed by reverse phase ultra-performance liquid chromatography (UPLC) using a Waters ACQUITY UPLC CSH C18 column (130 Å, 1.7 μmol, 2.1 × 150 mm) followed by detection of CPC in the system (at wavelengths of 280 nm, 320 nm and 350 nm) and analysis by mass spectrometry. Mobile phase A contained 0.1% FA in water and mobile phase B contained 0.1% FA in acetonitrile. After sample injection, a gradient was started with a 5 min exposure at 0.1% B followed by a linear increase to 35% B over 75 minutes for optimal separation of the peptides. MS and MS/MS experiments were performed on a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer with higher energy impact dissociation (HCD) used for peptide fragmentation for MS/MS experiments. Peptide identity assignment was based on the experimentally determined exact mass of a given peptide in the full MS spectrum as well as b and y ion fragments in the corresponding MS/MS HCD spectrum. Received extracted ion chromatograms of oxidized peptides and the corresponding parent peptide with integrated areas of the peaks to calculate the site-specific percentage of oxidized(s) amino acid(s) residue(s) in the sample MT1.

Peptide fragments linked to increase absorbance at 350 nm

PTM on MT1 was observed by comparing tryptic peptide maps for MT1 and MT4 ( FIG . 12A shows the absorbance of peptides eluted from 20.0 to 75 minutes). Peptides with different UV peaks have been isolated. An enlarged view of the chromatogram is shown in Fig. 12B showing the uptake of peptides eluted from 16 to 30 minutes. Peptides with a sharp contrast in UV absorption between MT1 and MT4 were TNYLTH*R, IIW (+4) DSR, and IIIW (+132) DSR (* or underline indicates oxidation of residues). Further, an enlarged view of the chromatogram is shown in Fig. 12C showing the absorbance of peptides eluted from 30 to 75 minutes. Peptides with a sharp contrast in UV absorption between MT1 and MT4 were DKTH*TC*PPC*PAPELLG (SEQ ID NO.: 17), TELNVGIDFNWEYPSSKH*QHK (SEQ ID NO.: 20), EIGLLTCEATVNGH*LYK (SEQ ID NO.: 18) and QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19) (* means residue oxidation). Peptide mapping revealed the identity of the peptides, the number of which varies significantly in the VEGF MiniTrap. The relative amount of peptides identified by peptide mapping analysis is shown in Table 3-1 . The amount of 2-oxo-histidines in MT1 (produced in COS) was higher than in MT4 (produced in soy hydrolyzate), suggesting that the medium used to express aflibercept may have a significant effect on the relative content of peptides with oxidized histidines or oxidized tryptophans. For example, for the peptide QTNTIIDVVLSPSH*GIELSVGEK (SEQ ID NO.: 19), the percentage of peptide in MT1 (produced in COS) was 0.015% compared to the percentage of peptide in MT4 (produced in soy hydrolysate; which is about 15 times less compared to MT1).

Table 3-1. Peptide Modified peptide sequence MT1 MT4 Multiplicity of change
MT1/MT4 EIGLLTCEATVNGHLYK (SEQ ID NO.: 57) EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18) 0.011% 0.004% 2.75 QTNTIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 58) QTNTIIDVVLSPSH[+14]GIELSVGEK (SEQ ID NO.: 19) 0.015% 0.001% 15.00 TELNVGIDFNWEYPSSKHQHK (SEQ ID NO.: 59) TELNVGIDFNWEYPSSKH[+14]QHK (SEQ ID NO.: 20) 0.204% 0.026% 7.85 DKTHTCPPCPAPELLG (SEQ ID NO.: 60) DKTH[+14]TC[+57]PPC[+57]PAPELLG (SEQ ID NO.: 17) 0.115% 0.018% 6.39 TNYLTHR (SEQ ID NO.: 61) TNYLTH[+14]R (SEQ ID NO.: 21) 0.130% 0.020% 6.50

Quantification of color and 2-oxo-histidine . Also shown is the percentage of 2-oxo-histidines in the oligopeptides formed as a result of protease cleavage, measured by mass spectrometry ( Table 3-2 ). (Values have been normalized to unmodified peptides.) Table 3-2(I) shows the percentage of oxidized histidines/tryptophans observed for the AEX:MT1 batch 1 run-through, MT1 AEX run-through batch 2, and MT1 AEX run-through MT1 batch 3. Table 3-2(II) shows the percentage of oxidized histidines/tryptophans observed for Acid Cut 1, Acid Cut 2, and Base Cut obtained by performing a CEX separation for batch MT1 3. It is clear from this table that the acid variants are composed of oxidized species. . It is clear from Table 3-2(I) that the % 2-oxo-histidines and tryptophan dioxidation containing peptides/protein is reduced in the AEX flow-through fraction compared to the AEX purification. Obviously, purification of the AEX column increases the percentage of such modified peptides. For example, the % modified peptide "EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18)" in the AEX run-through fraction (MT1 batch 1) was 0.013% and in the "AEX purification" was 0.080%. This also confirms that the AEX column captures modified peptides, thus reducing the percentage of modified peptides in the AEX run-through fraction.

Table 3-2 (I). Percentage of 2-oxo-histidines/tryptophans Modified peptides Cleaning AEX Flow fraction AEX intense yellow MT1 batch 1
BY1, 110 mg/ml MT1 batch 2 ≤BY3, 110mg/mL MT1 batch 3 ≤BY3, 110mg/mL EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18) 0.080% 0.013% 0.008% 0.006% QTNTIIDVVLSPSH[+14]GIELSVGEK (SEQ ID NO.: 19) 0.054% 0.028% 0.023% 0.019% TELNVGIDFNWEYPSSKH[+14]QHK (SEQ ID NO.: 20) 0.235% 0.085% 0.049% 0.049% DKTH[+14]TC[+57]PPC[+57]PAPELLG (SEQ ID NO.: 17) 0.544% 0.092% 0.077% 0.057% TNYLTH[+14]R (SEQ ID NO.: 21) 0.089% 0.022% 0.011% 0.010% IIW[+32]DSR (SEQ ID NO.: 28) 0.738% 0.252% 0.198% 0.298%

Table 3-2 (II). Percentage of 2-oxo-histidines/tryptophans Modified peptides Flow fraction CEX Acid cut 1 from MT1 batch 3
Yellow Acid cut 2 from MT1 lot 3
Yellow Main faction from MT1 batch 3
No color EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18) 0.009% 0.008% 0.004% QTNTIIDVVLSPSH[+14]GIELSVGEK (SEQ ID NO.: 19) 0.013% 0.015% 0.006% TELNVGIDFNWEYPSSKH[+14]QHK (SEQ ID NO.: 20) 0.131% 0.151% 0.049% DKTH[+14]TC[+57]PPC[+57]PAPELLG (SEQ ID NO.: 17) 0.117% 0.132% 0.068% TNYLTH[+14]R (SEQ ID NO.: 21) 0.014% 0.008% 0.008% IIW[+32]DSR (SEQ ID NO.: 28) 0.458% 0.269% 0.185%

In Table 3-2(II), [+57] is the alkylation of cysteine with iodoactamide with the addition of a carboxymethylmine moiety on cysteine, representing an increase in net weight of about +57 over unmodified cysteine:

Table 3-2 (II) , [+14] shows the conversion from His to 2-oxo-His, one oxygen is added to carbon 2 but two hydrogens are lost (one from carbon 2, the other from nitrogen 3), which represents an increase in net weight of about +14 compared to unmodified histidine.

In Table 3-2 (II) , [+32] represents the dioxidation of tryptophan resulting in the formation of N-formylkynurenine, which represents an increase in net weight of about +32 compared to unmodified tryptophan ( Fig. 4 ).

A second set of experiments was performed to evaluate the percentage of 2-oxo-histidines (and tryptophan dioxidation) in oligopeptides from protease-cleaved FabRICATOR-cleaved aflibercept (MT4), which was processed by AEX chromatography ( Figure 13 and Table 3-3 below). The percentage of 2-oxo-histidines and tryptophan dioxidation in AEX purification for oligopeptides from protease-cleaved FabRICATOR-cleaved aflibercept (MT4) was significantly greater than the percentage of 2-oxo-histidines and tryptophan dioxidation in the AEX flow-through fraction (referring to "MT1" in table 3-3 below).

Table 3-3. Percentage of 2-oxo-histidines Modified peptides Full size aflibercept MT1 Clearing AEX from MT1 AEX/MT1 clearing multiple change IIW[+32]DSR (SEQ ID NO.: 28) 0.22% 0.34% 0.81% 2.4 EIGLLTC[+57]EATVNGH[+14]LYK (SEQ ID NO.: 18) 0.00% 0.02% 0.08% 4.0 QTNTIIDVVLSPSH[+14]GIELSVGEK (SEQ ID NO.: 19) 0.01% 0.04% 0.07% 1.8 TELNVGIDFNWEYPSSKH[+14]QHK (SEQ ID NO.: 20) 0.01% 0.19% 0.42% 2.2 DKTH[+14]TC[+57]PPC[+57]PAPELLG (SEQ ID NO.: 17) 0.01% ^a 0.11% 0.63% 5.7 TNYLTH[+14]R (SEQ ID NO.: 21) 0.00% 0.03% 0.10% 3.3

^a value calculated using a different peptide for full-length aflibercept, since the C-terminal peptide is different from MiniTrap.

The percentage of 2-oxo-histidines and tryptophan dioxidation in the AEX band was significantly greater than the percentage of 2-oxo-histidines and tryptophan dioxidation in the AEX flow-through fraction during MT1 production (referring to “MT1” in Table 3-3 above). Compared to Table 3-2 , Table 3-3 shows similar results that AEX column purification provided a sample with a significantly higher percentage of 2-oxo-histidines and tryptophan dioxidation compared to the percentage of 2-oxo-histidines and tryptophan dioxidation. in the AEX run-through fraction, suggesting that the 2-oxo-histidine and tryptophan dioxidation forms are bound to the AEX column during separation and removed when the AEX column is cleaned. This is also evident in the extracted ion chromatogram as seen in FIG . 14 .

Strong cation exchange chromatogram (CEX)

Carried out a series of experiments to identify acid forms and other options present in samples containing proteins against VEGF.

Strong cation exchange chromatography was performed using a MonoS (10/100) GL column (GE Life Sciences, Marlborough, MA). Mobile phases were used to separate the samples: 20 mmol 2-(N-morpholino)ethanesulfonic acid (MES), pH 5.7 (mobile phase A) and 40 mmol sodium phosphate, 100 mmol sodium chloride, pH 9.0 (mobile phase B ). A non-linear pH gradient was used to elute charged MT1 variants with detection at 280 nm. Peaks that elute at a relative residence time before the main peak are referred to herein as acid forms.

A sample from MT1 batch 2 (≤BY3) was subjected to CEX prior to any enrichment using the method described in FIG. 15 . The sample was desiallylated to simplify the MT1 variants. This was followed by preparative treatment with SEC (Superdex 200, prep grade XK26/100) using 1X DPBS, pH 7.2 ± 0.2, as the mobile phase. Fractions from a preparative SEC column containing desialylated MiniTrap (dsMT1) were pooled and further subjected to strong cation exchange chromatography (SCX) for enrichment with charged MT1 variants using a double salt and pH gradient. As a result, a total of 7 fractions (F1-F7; MC is the method control, Fig. 16 and Fig. 17 ) were obtained.

When performing CEX, the acid forms elute before the main peaks, and the main forms elute after the main peaks. As seen in FIG. 17 , peaks 3-5 are the main peaks. Peaks 1 and 2 elute before eluting the basic forms of MT1 (peaks 3-5) and thus contain the acid forms. Peak 6 elutes after eluting the main MT1 species (peaks 3-5) and thus includes the main forms. Table 3-4 shows the relative content of the peaks in MC (as shown in Fig. 16 ). For example, row two in Table 3-4 (labeled MC) shows that the total relative amount of acid forms in MC is about 19.8% ( ie , peak 1+peak 2). Table-3-4 also shows the relative content of the peaks for each individual fraction. Although there are overlapping species in different fractions (as shown in Fig . 16 and Fig. 17 ), most fractions F1 and F2 are acid forms (ie, peak 1 and peak 2). For example, the F1 fraction consists of 63.7% peak 1 and 19.2% peak 2 (total 82.9% acid forms). Fraction F2 consists of 9.6% of peak 1 and 75.9%. peak 2 (total 85.5% acid forms). Most of the F3-F5 fractions are the main forms of MT1 (peaks 3-5). Finally, most fractions F6-F7 are the main forms (peak 6), but include some parts of the main species ( eg peaks 4 and 5).

It was also observed that fractions F1 and F2 (which constitute the acid forms) had an intense yellow-brown color compared to fractions F3-F5 (which comprise the main forms or "MT1"). All fractions were checked for color at a concentration of at least 13 mg/ml. As can be seen from this example, the presence of acid forms in the sample is monitored with the appearance of a yellow-brown color, the removal (or minimization) of which can be done by removing (or minimizing) the acid forms from MT1.

Table 3-4. Relative number of peaks based on analytical CEX Sample Peak area (%) Peak 1 Peak 2 Peak 3 Peak 4 Peak 5 Peak 6 MC 5.9 13.9 15.0 25.4 20.9 19.0 MT1 F1 63.7 19.2 17.1 n/a n/a n/a MT1 F2 9.6 75.9 10.6 2.2 1.6 n/a MT1 F3 n/a 5.0 57.2 37.8 n/a n/a MT1 F4 n/a n/a 16.3 56.3 27.4 n/a MT1 F5 n/a n/a n/a 33.1 50.4 16.5 MT1 F6 n/a n/a n/a 16.0 27.7 56.3 MT1 F7 2.8 7.7 8.0 16.1 16.5 48.9

n/a: not revealed

3D chromatograms for MT1 batch 2 and fractions F1 - F7 are shown in FIG . 18A-H . MT1 batch 2 did not show any significant spectral features ( FIG. 18A ). Fractions 1 and 2 (containing the acid forms) showed a spectral response from 320-360 nm (see circle in Fig. 18B ). This feature was more prominent in Fraction 1 compared to Fraction 2 ( Figs . 18B and 18C ) and absent in Fractions 3 and Fractions 4-7 (major species, MT1) ( Figs . 18D and 18H ), which did not show tan coloring.

Thus, as noted above, CEX led to the identification of acid forms/acid fractions (fractions 1 and 2) which show an intense yellow-brown color compared to the basic forms/fractions (fractions 3-6). This result was also observed as a distinct spectral characteristic present in the 3D chromatograms of the F1-F2 fractions and absent in the F3-F7 fractions.

Visualized electropherograms of capillary isoelectric focusing (icIEF)

The distribution of variants across F1-7 and MC fractions (from MT1 - Batch 2 after CEX) was further assessed using icIEF ( FIG. 19 ).

The distribution of variants in fractions F1-7 and MC (from MT1 - batch 2 after CEX) was further assessed by icIEF using an iCE3 analyzer (ProteinSimple) with a fluorocarbon coated capillary cartridge (100 μm x 5 cm). The ampholyte solution consisted of a mixture of 0.35% methylcellulose (MC), 0.75% Pharmalyte 3-10 carrier ampholytes, 4.2% Pharmalyte 8-10.5 carrier ampholytes and 0.2% pI marker 7.40 and 0.15% pI 9.77 marker in purified water. The anolyte is 80 mmol phosphoric acid and the catholyte is 100 mmol sodium hydroxide, both in 0.10% methylcellulose. Samples were diluted with purified water and sialidase A was added to each diluted sample at a ratio of enzyme to substrate of 1:200 (sialidase A units per milligram MT1), followed by incubation at ambient temperature for approximately 16 hours. Sialidase A treated samples were mixed with the ampholyte solution and then focused to a potential of 1500 V for one minute followed by a potential of 3000 V for 7 minutes. Focused MT1 variants were imaged by passing 280 nm ultraviolet light through a capillary and into a CCD digital camera lens. This image was then analyzed to determine the distribution of different charge options. ( FIG. 19 ). Referring to FIG. 19 , fractions F1 and F2 (or acid fractions) showed no peak for MT1, which is clearly observed for MC and fractions F3-F7 (basic forms, MT1). Thus, it was believed that icIEF electrophoregrams were able to detect and determine the distribution of different charge variants of the protein in question, in this case MT1. Thus, it was evident that the acid fraction during CEX analysis showed (a) an increased relative content of 2-oxo-histidine or dioxo-tryptophan ( Table 3-2(II) ); (b) increased yellow-brown coloration (data not shown); and (c) the presence of a spectral signature on the 3D chromatograms for fractions 1 and 2 ( Fig. 18B and Fig. 18C ).

Example 4 Photoinduction Study

In this example, photoinduction of a VEGF MiniTrap (MT), such as MT1, was performed by exposing the protein sample to varying amounts of cold white (CW) fluorescent light or ultraviolet A (UVA) light. The color content and oxidized amino acid content of the samples exposed to light were determined. LCMS analysis was performed after exposure as described above. Exposure of the MT to cold white light or UVA light results in an increase in oxidized amino acid residues, such as histidine ( Table 4-1, Table 4-2 and Table 4-3 ).

Table 4-1. Photoinduction study design Cumulative Impact 0.2(xICH) 0.5(xICH) 0.8(xICH) 1.0(xICH)H 2.0(xICH) Exposure to cool white fluorescent light (lux*h) 0.24 million lux*h. 0.6 million lux*h. 0.96 million lux*h. 1.2 million lux*h. 2.4 million lux*h. Incubation time with cold white fluorescent light (at 8 klux) 30 hours 75 hours 100 hours 150 hours 300 hours Exposure to UVA (Wh/m ² ) 40 100 160 200 400 Incubation time with UVA (at 10 W/m ² ) 4 hours 10 hours 16 hours 20 hours 40 hours

The ICH refers to the ICH Harmonized Tripartite Guidelines: Stability Testing: Photostability Testing for New Medicinal Substances and Products Q1B, which specifies photostability studies to be carried out with cold white fluorescent light of at least 1.2 million lux*h and near ultraviolet energy of at least 200 W*h/ ^m2 .

Table 4-2 shows the color enhancement of the MT sample under cold white light and ultraviolet light. For example, the b value for the sample (t=0) was 9.58. When this sample is exposed to cold white light with an intensity of 2.4 million lux*h. the value of b increases to 22.14. This increase in the value of b indicates that exposure to cold white light on the MT at 2.4 million lux*h. increases the yellow-brown color of the sample compared to the sample (t=0). Similarly, exposing the MT sample (t=0) to ultraviolet light at 400 Wh/m ² increases the b value to 10.72 from 9.58. This increase in the value of b indicates that exposure to ultraviolet light on the MT sample at 400 W*h/m ² leads to an increase in the yellow-brown color of the sample compared to the sample (t=0).

Table 4-2. Color of samples exposed to cold white light and ultraviolet light Photo-exposure xICH
(lux*h) L* a* b* BY value cold white light T=0 97.37 -1.12 9.58 4.0 0.2x (0.24 million lux*h) 96.46 -0.72 11.75 3.7 0.5x (0.6 million lux*h) 95.47 -0.4 11.3 3.7 0.8x (0.96 million lux*h) 95.33 -0.38 11.96 3.6 1.0x (1.2 million lux*h) 94.42 -0.2 13.72 3.3 2.0x (2.4 million lux*h) 92.70 0.41 22.14 2.0 UFA 0.2x (40 Wh/m ² ) 97.26 -0.92 12.66 3.5 0.5x (100 Wh/m ² ) 100.39 -1.01 11.83 3.7 0.8x (160 Wh/m ² ) 79.69 -0.18 10.1 3.6 1.0x (200 Wh/m ² ) 97.48 -0.95 11.36 3.7 2.0x (400 Wh/m ² ) 97.76 -0.98 10.72 3.8

The color samples are specified using the CIELAB color space (variables L*, a* and b*) and relative to the EP BY color standard; L*=white to black (L* - lightness); a*=purple to aqua; b*=yellow to blue, the higher the b value, the more yellow.

Table 4-3 (I). 2-oxo-His levels in peptides from UV-exposed MiniTrap Peptides Website t0 UV_4 h UV_10 h UV_16 h UVF_20 h UV_40 h DKTHTCPPCPAPELLG (SEQ ID NO.: 17) H209 0.056% 0.067% 0.081% 0.088% 0.077% 0.091% EIGLLTCEATVNGHLYK (SEQ ID NO.: 18) H86 0.010% 0.020% 0.034% 0.037% 0.033% 0.035% QTNTIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 19) H110 0.024% 0.031% 0.028% 0.028% 0.027% 0.027% TELNVGIDFNWEYPSSKHQHK (SEQ ID NO.: 20) H145 0.096% 0.147% 0.163% 0.173% 0.147% 0.125% TNYLTHR (SEQ ID NO.: 21) H95 0.014% 0.032% 0.044% 0.056% 0.058% 0.078% SDTGRPFVEMYSEIPEIHMTEGR (SEQ ID NO.: 22) H19 0.007% 0.013% 0.021% 0.025% 0.024% 0.034% VHEKDK (SEQ ID NO.: 23) H203 0.040% 0.105% 0.238% 0.255% 0.269% 0.324%

Table 4-3 (II). 2-oxo-His levels in peptides from cold white light exposed MiniTrap Peptides Website t0 HB_30 h HB_75 h HB_100 h HB_150 h HB_300 h DKTHTCPPCPAPELLG (SEQ ID NO.: 17) H209 0.056% 0.152% 0.220% 0.243% 0.258% 0.399% EIGLLTCEATVNGHLYK (SEQ ID NO.: 18) H86 0.010% 0.063% 0.110% 0.132% 0.170% 0.308% QTNTIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 19) H110 0.024% 0.085% 0.120% 0.128% 0.148% 0.180% TELNVGIDFNWEYPSSKHQHK (SEQ ID NO.: 20) H145 0.096% 0.423% 0.585% 0.634% 0.697% 0.748% TNYLTHR (SEQ ID NO.: 21) H95 0.014% 0.103% 0.175% 0.198% 0.267% 0.437% SDTGRPFVEMYSEIPEIHMTEGR (SEQ ID NO.: 22) H19 0.007% 0.025% 0.043% 0.049% 0.058% 0.115% VHEKDK (SEQ ID NO.: 23) H203 0.040% 0.426% 0.542% 0.622% 0.702% 1.309%

Exposure of aflibercept MT to cold white light or UVA light was monitored by the appearance of oxidized histidines (2-oxo-His). ( Table 4-3 ). Referring to Table 4-3 , the "SDTGRPFVEMYSEIPEIIHMTEGR (SEQ ID NO.: 22)" peptide with oxo-histidine was 0.007% in the MT sample (t=0), while its content increased to 0.324% when exposed to ultraviolet light for 40 hours ( Table 4-3 (I) ) and up to 1.309% when exposed to cold white light for 300 hours ( Table 4-3 (II) ).

Two forms of 2-oxo-histidine were observed, the 13.98 Da forms (as shown in Fig. 2 ) and the 15.99 Da forms (as shown in Fig. 3 ), with the 13.98 Da forms being predominant in light-exposed samples. minitrap. The 15.99 Da forms are known to be the product of a copper catalyzed process. Schöneich, J. Pharm. Biomed Anal. 21:1093-1097 (2000) . In addition, the 13.98 Da shapes are the product of a light-driven process. Liu et al., Anal. Chem. 86(10: 4940-4948 (2014)).

Similar to the increased content of oxidized histidines in samples exposed to cold white light and UVA light, exposure to cold white light or UVA light on MT also caused the formation of other PTMs ( Table 4-4 and Table 4-5 ).

Table 4-4 (I). Other PTMs in peptides from UV-exposed MiniTrap Peptides Website t0 UV_4 h UV_10 h UV_16 h UVF_20 h UV_40 h Deamidation EIGLLT C EATV N GHLYK (SEQ ID NO.: 62) N84 20.8% 21.7% 21.5% 21.5% 22.7% 22.4% QT N TIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 63) N99 5.3% 5.4% 5.5% 5.4% 5.5% 5.6% Oxidation SDTGRPFVE M YSEIPEIHMTEGR (SEQ ID NO.: 64) M10 4.5% 8.2% 11.1% 13.3% 13.8% 19.3% SDTGRPFVEMYSEIPEIH M TEGR (SEQ ID NO.: 65) M20 1.1% 2.0% 2.8% 3.4% 3.4% 4.6% TQSGSE M K (SEQ ID NO.: 66) M163 2.0% 2.7% 4.1% 4.6% 7.9% 8.7% SDQGLYT C AASSGL M TK (SEQ ID NO.: 67) M192 5.4% 8.1% 10.9% 12.1% 12.5% 18.3% 3-deoxygluconazone SDTG R PFVEMYSEIPEIHMTEGR (SEQ ID NO.: 68) R5 9.9% 10.0% 9.9% 9.7% 9.8% 9.3%

Table 4-4 (II). Other PTMs in peptides from cold white exposed MiniTrap Peptides Website t0 HB_30 h HB_75 h HB_100 h HB_150 h HB_300 h Deamidation EIGLLT C EATV N GHLYK (SEQ ID NO.: 62) N84 20.8% 22.0% 22.9% 20.3% 21.8% 21.3% QT N TIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 63) N99 5.3% 5.6% 5.2% 5.6% 5.5% 5.8% Oxidation SDTGRPFVE M YSEIPEIHMTEGR (SEQ ID NO.: 64) M10 4.5% 11.7% 17.3% 19.9% 25.1% 39.7% SDTGRPFVEMYSEIPEIH M TEGR (SEQ ID NO.: 65) M20 1.1% 3.1% 4.3% 5.1% 6.1% 8.2% TQSGSE M K (SEQ ID NO.: 66) M163 2.0% 3.3% 15.7% 11.7% 26.4% 20.5% SDQGLYT C AASSGL M TK (SEQ ID NO.: 67) M192 5.4% 10.7% 15.3% 18.7% 22.8% 37.6% 3-deoxygluconazone SDTG R PFVEMYSEIPEIHMTEGR (SEQ ID NO.: 68) R5 9.9% 9.9% 9.6% 9.3% 9.3% 9.0%

Table 4-5 (I). Tryptophan/Tyrosine/Phenylalanine oxidation levels in peptides from UV-exposed MiniTrap Peptides Modification Website t0 UV_4 h UV_10 h UV_16 h UVF_20 h UV_40 h SDQGLYTCAASSGLMTK (SEQ ID NO.: 67) +4 W58 0.016% 0.049% 0.089% 0.119% 0.132% 0.221% +16 0.047% 0.109% 0.177% 0.225% 0.242% 0.514% +32 0.200% 0.487% 0.415% 0.481% 0.423% 0.498% +48 0.000% 0.000% 0.000% 0.001% 0.001% 0.001% TELNVGIDFNWEYPSSK (SEQ ID NO.: 29) +4 W138 0.435% 0.462% 0.550% 0.557% 0.502% 0.512% +16 0.083% 0.100% 0.161% 0.206% 0.239% 0.448% +32 0.009% 0.018% 0.027% 0.039% 0.044% 0.115% +48 0.284% 0.278% 0.270% 0.302% 0.343% 0.275% GFIISNATYK (SEQ ID NO.: 69) +16 Y64 0.032% 0.041% 0.046% 0.053% 0.054% 0.073% KFPLDTLIPDGK (SEQ ID NO.: 70) +16 F44 0.068% 0.077% 0.087% 0.084% 0.070% 0.096% FLSTLTIDGVTR (SEQ ID NO.: 71) +16 F166 0.066% 0.075% 0.085% 0.089% 0.089% 0.124%

Table 4-5 (II). Tryptophan/Tyrosine/Phenylalanine Oxidation Levels in Peptides from MiniTrap Cold White Light Exposure Peptides Modification Website t0 HB_30 h HB_75 h HB_100 h HB_150 h HB_300 h SDQGLYT C AASSGL M TK (SEQ ID NO.: 67) +4 W58 0.016% 0.063% 0.124% 0.161% 0.228% 0.526% +16 0.047% 0.129% 0.227% 0.283% 0.377% 0.795% +32 0.200% 1.601% 2.706% 3.139% 3.925% 6.974% +48 0.000% 0.001% 0.002% 0.002% 0.003% 0.005% TELNVGIDFN W EYPSSK (SEQ ID NO.: 29) +4 W138 0.435% 0.555% 0.481% 0.490% 0.429% 0.522% +16 0.083% 0.109% 0.364% 0.251% 0.399% 0.753% +32 0.009% 0.019% 0.027% 0.033% 0.048% 0.135% +48 0.284% 0.284% 0.330% 0.308% 0.347% 0.316% GFIISNAT Y K (SEQ ID NO.: 69) +16 Y64 0.032% 0.043% 0.057% 0.063% 0.078% 0.127% K F PLDTLIPDGK (SEQ ID NO.: 70) +16 F44 0.068% 0.087% 0.072% 0.088% 0.079% 0.144% F LSTLTIDGVTR (SEQ ID NO.: 71) +16 F166 0.066% 0.091% 0.088% 0.101% 0.112% 0.168%

Thus, MT exposure to cold white light or UVA light was monitored by the appearance of oxidized residues (such as histidines/tryptophans (oxo-Trp)). Four types of oxo-trp were observed: +4 Da, +16 Da, +32 Da, and +48 Da. The +4 Da forms are explained by the formation of kynurenine ( Fig. 4 ), while 16 Da, +32 Da, and +48 Da represent mono-oxidation, di-oxidation, and tri-oxidation of tryptophan residues. Peptide mapping of tryptic digests of MT samples observed at 320 nm as shown in FIG . 20 . The relative presence of oxidized residues containing peptides can be compared in FIG . 20 . For example, for the peptide, IIW(+4) DSRK, a significant difference in its presence can be seen for the MT sample at t=0, and the MT1 sample exposed to UVA for 40 hours, and the MT sample exposed to cold white light for 300 hours.

MT exposure to cold white light or UVA light was also assessed for the presence of HMW/low molecular weight (LMW) particles ( Table 4-6 ).

Table 4-6. HMW/LMW shapes were formed after extended exposure to UVA and cold white light Sample: MT1, 80 mg/mL, pH 5.8 Samples Exposed to Light Dark controls Samples Exposed to Light Dark controls Samples Exposed to Light Dark controls Cumulative exposure to UVA (x ICH) % HMW % native % NMK t=0 2.1 ND 96.7 ND 1.2 ND 0.2x ICH (40Wh/m2) 2.1 2.1 96.7 96.7 1.2 1.3 0.5x ICH (100Wh/m2) 11.6 2.2 86.5 96.6 1.9 1.2 0.8x ICH (160Wh/m2) 14.5 2.2 83.4 96.6 2.1 1.2 1.0x ICH (200Wh/m2) 15.8 2.2 81.9 96.6 2.3 1.3 2.0x ICH (400Wh/m2) 22.7 2.3 74.5 96.7 2.8 1.0 Cumulative exposure to cold white light (x ICH) % HMW % native % NMK 0.2x ICH (0.24 million lux*h) 12.1 2.2 86.6 96.6 1.4 1.2 0.5x ICH (0.6 million lux*h) 20.4 2.3 77.9 96.4 1.6 1.3 0.8x ICH (0.96 million lux*h) 23.2 2.4 75.1 96.2 1.7 1.4 1.0x ICH (1.2 million lux*h) 30.1 2.6 68.1 96.2 1.9 1.3 2.0x ICH (2.4 million lux*h) 45.0 2.9 52.6 95.8 2.4 1.4

To monitor color against HMW/LMW forms for each sample, analytical size exclusion chromatography with full spectrum PDA detection (SEC-PDA) as shown above was performed on all stressed samples (cold white light and UVA). SEC-PDA analysis of cold white light-exposed MTs shows a significant increase in absorbance at ~350 nm for all size variants except LMW forms ( Fig. 21 ), while SEC-PDA on UVA-exposed MTs shows no increase in absorbance at ~350 nm ( Fig. 22 ). In contrast to the samples exposed to cold white light, the samples exposed to UVA did not provide any significantly quantifiable yellow-brown color.

A similar result was obtained after studying the absorption coefficients at 320 nm and 280 nm for samples exposed to UVA and cold white light. The A320/A280 ratios, analyzed by either raw intensity or total peak area, were tracked with increasing yellow intensity in samples exposed to cold white light ( Fig. 23 ), whereas A320/A280 ratios were not tracked with increasing yellow intensity in samples exposed to UVA radiation ( Fig. 24 ). This confirms the previous observation that MT1 samples exposed to UVA do not provide the same yellow-brown color that is observed after exposure to cold white light.

Example 5 Previous Color Reduction Methods

5.1 Study of the incubation of a medium with a chemically defined composition

The effect of various components added to fresh chemically defined medium (COS) containing aflibercept on color was observed.

One or more 50 ml shake tubes with vent caps with 10 ml working volume (fresh XOC1) were incubated for 7 days, sampling on day 0 and day 7. Samples of aflibercept (aflibercept recombinant protein in aqueous buffer, pH 6.2 , containing 5 mmol sodium phosphate, 5 mmol sodium citrate and 100 mmol sodium chloride) was added to shaking tubes at a concentration of 6 g/l.

Components added to achieve cumulative concentration:

Cysteine: 16.6 mmol

Iron: 0.23 mmol

Copper: 0.0071 mmol

Zinc: 0.54 mmol

The scale effect of each component added to the b* value (CIE L*, a*, b* color space) is shown in FIG. 25A and a plot of actual b* versus predicted b* is shown in FIG. 25B . The addition of cysteine resulted in the largest increase in yellow-brown color. Iron and zinc also form the color. Folic acid and B vitamins (including thiamine, niacinamide, D-pantothenic acid, D-biotin, and pyridoxine) increased tan color. Riboflavin and vitamin B12 did not significantly affect color.

5.2 Effect of cysteine and metal reduction on b* value

Bioreactors ( eg 2L) were inoculated from a seed culture of an aflibercept-producing cell line. The inoculated cultures were grown at 35.5°C, pH 7.1 ± 0.25, and a bubbling set point of 22 cc. see If necessary, glucose, defoamer and basic nutrition were added to the bioreactors. The effect of the decrease in the concentration of cysteine and metals on the color during the expression of aflibercept was evaluated in XOC1.

Wednesday on day 0=XOS1 including 1.48 mmol cysteine

Nutrient media:

Day 2=CDF + 1.3-2.1 mmol cysteine

Day 4=CDF+1.6-1.7 mmol cysteine

Day 6=CDF+1.6-1.7 mmol cysteine

Day 8=CDF+1.6-1.7 mmol cysteine

The bioreactor conditions were as follows:

cysteine was added at a cumulative concentration of about 6-7 millimoles per liter of culture, 8-9 millimoles per liter of culture, or 10-11 millimoles per liter of culture.

Metals in XOS1 (levels 0.5x, 1x, or 1.5x XOS1) at 1x levels are listed below (where the values precede the addition of the inoculum):

Fe=68-83 micromoles per liter of culture

Zn=6-7 micromoles per liter of culture

Cu=0.1-0.2 micromoles per liter of culture

Ni=0.5-1 micromoles per liter of culture

Reducing the cumulative levels of cysteine to 6-7 mmol/l reduces the yellow-brown color without significantly affecting the titer. Reducing the metal content up to 0.5 times in the medium reduced the color with a significant increase in titer. The effect on titer, VCC (viable cell concentration), viability, ammonia, or osmolality was minimal ( see Fig . 26A-E ). The predicted scale effect of metal and cysteine content on b* value and titer is shown in FIG . 27 .

5.3 Evaluation of the effect of antioxidants on the b* value

The effect of antioxidants, taurine, hypotaurine, thioctic acid, glutathione, glycine, and vitamin C on the color added to spent COS containing aflibercept was evaluated. One or more 50 ml vent cap shake tubes with 10 ml working volume (XOC1) were incubated for 7 days, sampling on day 0 and day 7.

Conditions for adding components to lean XOS1 were as follows:

aflibercept sample (aflibercept recombinant protein in aqueous buffer, pH 6.2, containing 5 mM sodium phosphate, 5 mM sodium citrate, and 100 mM sodium chloride) added to shake tubes at a concentration of 6 g/L

antioxidants added to lean XOS1 at the following concentrations:

taurine=10 mmol culture

hypotaurine=10 mmol culture

glycine=10 mmol culture

thioctic acid=0.0024 mmol culture

glutathione, reduced=2 mmol culture

hydrocortisone=0.0014 mmol culture

vitamin C (ascorbic acid)=0.028 mmol culture

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

Table 5-1. Summary of Antioxidant Effect on Color Formation in MiniTrap Condition b* value Lean Wednesday to day 0 0.37 Lean environment control on day 7 1.47 Lean Wednesday on Day 7 + Antioxidants ^* 1.02

^* antioxidants that significantly reduced the b * value: hypotaurine / taurine / glycine, thioctic acid, vitamin C.

A summary of the predicted effect of various antioxidants on the b* value (CIE L*, a*, b* color space) is shown in FIG . 28(AC) .

The effect of further addition of antioxidants on the color of depleted COS containing aflibercept was evaluated. One or more 50 ml vent cap shake tubes with 10 ml working volume (XOC1) were incubated for 7 days, sampling on day 0 and day 7.

Conditions for adding components to lean XOS1 were as follows:

aflibercept sample (aflibercept recombinant protein in aqueous buffer, pH 6.2, containing 5 mM sodium phosphate, 5 mM sodium citrate and 100 mM sodium chloride) added to shake tubes at a concentration of 6 g/L

Two DOE experiments were carried out:

(i) antioxidants added to lean XOS1 at the following concentrations:

taurine=10 mmol culture

hypotaurine=10 mmol culture

glycine=10 mmol culture

thioctic acid=0.0024 mmol culture

vitamin C (ascorbic acid)=0.028 mmol culture

(ii) antioxidants added to achieve the following cumulative concentrations:

ATA=2.5 µmol - 5 µmol

deferoxamine mesylate (DFO)=5 µmol - 10 µmol

catalase=101.5 mg/l

S-carboxymethyl-L-cysteine \u003d 10 mmol

hypotaurine reduces lean medium color formation ( Figure 28D ). DFO also significantly reduced lean color formation ( FIG. 28D ). The rest of the antioxidants had no statistical effect on color formation.

Table 5-2. Summary of Antioxidant Effect on Color Formation in MiniTrap Condition b* value Lean Wednesday to day 0 0.44 Lean environment control on day 7 1.73 Lean Wednesday on Day 7 + hypotaurine 1.32 Lean Wednesday on Day 7+DFO 0.92

Antioxidant Shake Bottle Study:

taurine, hypotaurine, glycine, thioctic acid, and vitamin C were evaluated alone and in combination for their ability to reduce color formation in cell culture ( Table 5-3 ).

250 ml shake flasks were inoculated from a seed culture of an aflibercept-producing cell line. The inoculated cells were grown at 35.5° C. in 5% CO ₂ controlled incubators. If necessary, glucose and basic nutrition were added to shake flasks. The process described above was used in which the metals were present at a concentration of 0.5x in XOC1 and cysteine was added at a cumulative concentration of 6-7 mmol.

Table 5-3. Antioxidant Level 1
0x Level 1
0.5x Level 1
1x Taurine 0 3.75 mmol 7.5 mmol hypotaurine 0 3.75 mmol 7.5 mmol Glycine 2.0 mmol 5.75 mmol 9.5 mmol Thioctic acid 1.0 µmol 1.9 µmol 2.8 µmol Vitamin C 0 11.0 µmol 21.0 µmol

In FIG. 28E shows the predicted effect of the antioxidants in Table 5-3 on the b* value (CIE L* color space, a*, b*) and final titer. Taurine, hypotaurine, glycine significantly reduced the b* value without a negative effect on the titer.

Example 6 Glycosylation and Viability Studies for Aflibercept Using COS

Bioreactors ( eg 2L) were inoculated from a seed culture of an aflibercept-producing cell line. The inoculated cultures were grown at 35.5°C, pH 7.1 ± 0.25, and a bubbling set point of 22 cc. see If necessary, glucose, defoamer and basic nutrition were added to the bioreactors. The preparation of the aflibercept protein was carried out using XOS1 (proprietary). The production of a host cell line expressing the aflibercept fusion protein was performed using XOS1 (proprietary), XOS2 (commercially available) and XOS3 (commercially available). A number of experiments were carried out using HOS 1, 2, and 3 without additional medium components. Another series of experiments was carried out using HOS 1-3, to which were added manganese (manganese chloride tetrahydrate, Sigma, 3.2 mg/l), galactose (Sigma, 8 g/l) and uridine (Sigma, 6 g/l) in nutrient media to change the profile of galactosylation. Finally, a series of experiments were carried out using HOS 1-3 to which manganese (manganese chloride tetrahydrate, Sigma, 3.2 mg/l), galactose (Sigma, 8 g/l) and uridine (Sigma, 6 g/l) were added. in nutrient media to change the profile of galactosylation and added dexamethasone (Sigma, 12 mg/l) to nutrient media to change the sialylation profile of the composition. A clarified harvest using each HOS was obtained by centrifugation followed by 0.45 µm filtration.

Samples were treated with protein A before analysis with N-glycan.

Titer measurements

In these examples, unless otherwise noted, aflibercept titers were measured daily using an Agilent 1200 Series HPLC (Santa Clara, CA) or equivalent low pH step gradient elution with detection at 280 nm. Concentrations were assigned relative to a reference calibration curve.

Viable cell density (VCD) and cell viability values

In these examples, unless otherwise indicated, viable cell density (VCD) and cell viability values were measured by trypan blue exclusion using Nova BioProfile Flex automated cell counters (Nova Biomedical, Waltham, MA). Glucose, lactate, off-loop pH, dissolved oxygen (DO), pCO2 measurements and osmolality were measured using Nova BioProfile Flex (Nova Biomedical, Waltham, MA).

Profiling of N-glycan-oligosaccharides

[1] Approximately 15 μg of processed protein A samples from XOS collections 1-3 were prepared for N-glycan analysis according to the Waters GlycoWorks protocol using the GlycoWorks Rapid Deglycosylation and GlycoWorks RapiFluor-MS Label kits (Waters part numbers 186008939 and 186008091, respectively) . N-glycans were removed from the aflibercept protein by treating the samples with PNGase-F at 50.5°C for 5 minutes followed by cooling at 25°C for 5 minutes. The released glycans were labeled with RapiFluor-MS fluorescent dye by reaction at room temperature for 5 minutes. The protein was precipitated by adding acetonitrile to the reaction mixture and precipitated to the bottom of the well by centrifugation at 2204 x g for 10 minutes. The supernatant containing labeled glycans was collected and analyzed by UPLC using hydrophilic interaction liquid chromatography (Waters BEH Amide column) with post-column fluorescence detection. After binding to the column, the labeled glycans were separated and eluted using a binary mobile phase gradient consisting of acetonitrile and 50 mM aqueous ammonium formate (pH 4.4). Labeled glycans were detected using a fluorescent detector with an excitation wavelength of 265 nm and an emission wavelength of 425 nm. Using the relative percentages of N-glycan peak areas in the obtained chromatograms, the distribution of N-glycans is presented as the total percentage of N-glycans (1) containing fucose core residues (total fucosylation, Table 6-1 ), (2) containing at least one residue salicylic acid (total sialylation, table 6-2 ), (3) identified as mannose-5 (mannose-5, table 6-3 ), (4) containing at least one galactose residue (total galactosylation, table 6-4 ) , and (5) with known identity (total peaks identified, Table 6-5 ).

results

Viable cell count (VCC), viability and collection titers are shown in FIG. 29-31 for HOS 1-3 with and without additional components.

Among the nine cultures, the XOS1 culture containing uridine, manganese and galactose showed the highest titer after 12 days (5.5 g/l). XOS1 without additional components also showed a high titer after 12 days (about 4.25 g/l) compared to other seven cultures ( Fig. 29 ).

Cell viability results were similar under different conditions up to day 6 of the process. After day 7 of the process, cultures of XOS2 and XOS3 with or without additional media components showed a viability of over 90% ( Fig. 30 ).

The XOC1 culture with uridine, manganese and galactose showed the highest VCC around day 6 ( Fig. 31 ).

The influence of cultures and supplements had a significant effect on the overall distribution of N-glycans ( Tables 6-1 to 6-5 ). Glycan levels were compared using protein A treated aflibercept (two samples evaluated) made using soy hydrolysate. The common identified peaks are listed in Table 6-5 .

Table 6-1. Total fucosylation (%) Condition Day 6 Day 10 Day 12 XOS1 48.75 - 46.26 XOS1 +UMG 49.21 - 44.38 XOS1+UMG+Dex 48.88 - 46.23 HOS2 - 45.68 45.14 XOS2 +UMG - 46.36 45.27 XOS2+UMG+Dex - 46.92 - HOS3 49.24 - 45.59 XOS3 +UMG 48.71 - 42.61 XOS3+UMG+Dex 49.36 - 44.56 Soy hydrolyzate 1 51.37 Soy hydrolyzate 2 52.43

U is uridine, M is manganese, G is galactose, Dex is dexamethasone

Table 6-2. Total sialylation (%) Condition Day 6 Day 10 Day 12 XOS1 44.06 - 39.14 XOS1 +UMG 43.72 - 35.8 XOS1+UMG+Dex 43.2 - 36.72 HOS2 - 37.62 36.67 XOS2 +UMG - 37.57 36.29 XOS2+UMG+Dex - 38.06 - HOS3 44 - 31.21 XOS3 +UMG 42.48 - 30.84 XOS3+UMG+Dex 43.82 - 32.74 Soy hydrolyzate 1 58.24 Soy hydrolyzate 2 59.23

U is uridine, M is manganese, G is galactose, Dex is dexamethasone

Table 6-3. Mannose-5 (%) Condition Day 6 Day 10 Day 12 XOS1 6.76 - 10.1 XOS1 +UMG 6.9 - 13.17 XOS1+UMG+Dex 6.23 - 8.86 HOS2 - 9.71 11.96 XOS2 +UMG - 9.44 10.93 XOS2+UMG+Dex - 8.21 - HOS3 2.31 - 12.63 XOS3 +UMG 2.71 - 13.38 XOS3+UMG+Dex 2.05 - 11.98 Soy hydrolyzate 1 5.19 Soy hydrolyzate 2 5.24

U is uridine, M is manganese, G is galactose, Dex is dexamethasone

Table 6-4. Total galactosylation (%) Condition Day 6 Day 10 Day 12 XOS1 68.44 - 62.9 XOS1 +UMG 69.25 - 59.02 XOS1+UMG+Dex 69.05 - 63.26 HOS2 - 65.33 63.68 XOS2 +UMG - 68.13 66 XOS2+UMG+Dex - 69.35 - HOS3 74.57 - 62.28 XOS3 +UMG 74.82 - 62.2 XOS3+UMG+Dex 76.48 - 65.18 Soy hydrolyzate 1 79.64 Soy hydrolyzate 2 80.55

U is uridine, M is manganese, G is galactose, Dex is dexamethasone

Table 6-5. Total Peaks Identified (%) Condition Day 6 Day 10 Day 12 XOS1 87.28 - 84.67 XOS1 +UMG 88.43 - 83.82 XOS1+UMG+Dex 87.36 - 83.44 HOS2 - 86.23 86.67 XOS2 +UMG - 87.81 86.87 XOS2+UMG+Dex - 87.53 - HOS3 86.38 - 86.31 XOS3 +UMG 87.07 - 86.13 XOS3+UMG+Dex 87.18 - 87.43 Soy hydrolyzate 1 93.93 Soy hydrolyzate 2 94.74

U is uridine, M is manganese, G is galactose, Dex is dexamethasone

Total fucosylation, total sialylation, total galactosylation and mannose-5 observed on day 12 of different COS cultures ranged from 42.61% to 46.26%, from 30.84% to 39.14%, from 59.02 to 66 % and from 8.86% to 13.38%, respectively. These glycosylation values differ from the glycosylation values obtained using soy hydrolyzate.

Finally, color measurements were made for clarified harvests derived from cells expressing aflibercept in XOS1, XOS2 and XOS3 supplemented with uridine, manganese and galactose. The operating parameters for the bioreactor research steps will be known to one of ordinary skill in the art.

Example 7 Affinity Production of Proteins Against VEGF

7.1 Expression of VEGF MiniTrap

Recombinant VEGF MiniTrap coding regions (eg, MT5, SEQ ID NO.: 46) were operably linked to a signal sequence cloned into a mammalian expression vector and transfected into Chinese Hamster Ovary (CHO-K1) cells; stably transfected pools were isolated after selection using hygromycin 400 μg/ml for 12 days. Stable pools of CHO cells grown in protein-free media of defined chemistry were used to obtain proteins for testing. Recombinant polypeptides were secreted from the cells into the nutrient medium.

VEGF MiniTrap Constituent Domain Sequences

Human Flt1 (access number NP_001153392.1)

Human Flk1 (accession number NP_002244.1)

Human Fc (IGHG1, accession number P01857-1)

Recombinant VEGF MiniTrap with (MT5) was obtained from this process and further processed.

7.2 Preparation of columns for affinity chromatography

Five distinct proteins capable of binding to VEGF MiniTrap (MT5) were evaluated. The proteins used include VEGF ₁₆₅ (SEQ ID NO.: 72), mAb1 (human IgG1 against mouse anti-VEGFR1 mAb, where SEQ ID NO.: 73 is the heavy chain and SEQ ID NO.: 74 is the light chain); mAb2 (human IgG1 against mouse anti-VEGFR1 mAb, wherein SEQ ID NO.: 75 is the heavy chain and SEQ ID NO.: 76 is the light chain); mAb3 (mouse IgG1 against mouse anti-VEGFR1 mAb, where SEQ ID NO.: 77 is the heavy chain and SEQ ID NO.: 78 is the light chain) and mAb4 (mouse IgG1 against mouse anti-VEGFR1 mAb, where SEQ ID NO.: 79 is a heavy chain and SEQ ID NO.: 80 is a light chain).

The column was activated by washing the columns with 6 column volumes (CV) of 1 mmol glacial hydrochloric acid at a flow rate not exceeding 1 ml/min. Ten mg of each protein was loaded into three HiTrap NHS-Activated HP affinity columns (1 ml, GE Healthcare, Cat # 17-0716-01) and the columns were capped to allow binding for 30 minutes at room temperature. The columns were washed with 18 column volumes of 0.5 M sodium acetate, 0.5 M NaCl, pH 4.0 and the open areas were blocked with 18 column volumes of 0.5 M Tris-HCl, 0.5 M NaCl pH 8.3 (washing was carried out in in the following order: 6 column volumes 0.5M Tris-HCl, 0.5M NaCl, pH 8.3, 6 column volumes 0.5M sodium acetate (sodium acetate: JT Baker, Cat # 3470-01) 0 .5 M NaCl, pH 4.0; 6 column volumes 0.5 M Tris buffer, pH 8.3; column incubated for 30 minutes at room temperature; 6 column volumes 0.5 M sodium acetate buffer, 0.5 M NaCl, pH 4.0; 6 column volumes 0.5 M Tris-HCl, 0.5 NaCl, pH 8.3 and 6 column volumes 0.5 M sodium acetate buffer, 0.5 M NaCl, pH 4.0) . The columns were stored in DPBS, pH 7.5. The five evaluated columns were designated as column 1 (containing VEGF ₁₆₅ ), column 2 (containing mAb1), column 3 (containing mAb2), column 4 (containing mAb3) and column 5 (containing mAb4).

7.3 Preparation of MiniTrap using affinity chromatography

Getting a sample. Two different acquisition processes were performed for MiniTrap. In one case, material containing a MiniTrap sample was prepared using each affinity column where the starting material (MiniTrap) was diluted 1X with DPBS buffer to 20 mg/mL and loaded onto the column and run at room temperature for 30 minutes. Using an affinity column, MiniTrap was isolated from ~7000 ppm HCP.

Alternatively, harvested culture supernatant that contained 0.4 mg/ml protein in the supernatant was used and loaded onto different affinity columns (1-5) separately. No further dilution was performed. The affinity columns were then washed with 9 CV 1x DPBS buffer followed by dilution of the proteins using IgG elution buffer, pH 2.8 (Thermo, Cat # 21009).

The MiniTrap material prepared as described above was then filtered through a 0.45 µM filter or centrifuged before loading onto the columns prepared as described in section 7.2 above. Twenty-five ml loading solution containing approximately 0.4 mg/ml was loaded into each of the columns and incubated for 20 minutes. Each column was washed with 9 CV DPBS (Invitrogen, Cat # 14190-144) before elution to equilibrate. The amount of MT5 in the wash fractions is shown in Table 7-1 . The washes were followed by elution using 6 CV at pH 2.8 (commercial elution buffer, (Thermo, cat. no. 21009)) and 100 mmol glycine buffer pH 2.5 and the fractions were quickly neutralized by adding 1 M Tris buffer, pH 7.5 (Invitrogen, cat. no. 15567-027). The amount of MiniTrap in the eluted fractions is also shown in Table 7-1 .

MiniTrap (MT5) was successfully generated from all five affinity columns. The yield from the VEGF ₁₆₅ column was large compared to the mAb1 and mAb2 columns. MAb3 and mAb4 containing humanized anti-VEGFR1 mAbs also showed successful production of MT5 in similar yields to mAb1 and mAb2. In Table 7-1, the expected yield was calculated based on 100% conjugation efficiency and a 1:1 molar ratio captured based on protein affinity for MT5.

Table 7-1. affine column Column 1
VEGF ₁₆₅ Column 2
mAb1 Column 3
mAb2 Column 4
mAb3 Column 5
mAb4 Amount of conjugation (mg) 10 10 10 10 10 Loading (mg) 21.2 21.2 21.2 20.1 20.1 Expected amount (mg) ~12 ~3.2 ~3.2 ~3.2 ~3.2 Wash (mg) 14.9 13.2 12.6 15.2 14.7 Eluate (mg) 4.8 1.6 1.8 1.5 1.6

7.4 Column stability study

Several cycles were performed using columns 1 and 2 following the method described in section 7.3 ( table 7-2 for column 1 and table 7-3 for column 2).

Table 7-2 Product yield cycle number 1 2 3 4 5 6 7 Loading (mg) 21.2 19.7 19.7 19.7 19.6 19.6 19.6 Wash (mg) 14.9 13.5 15.0 15.0 14.6 14.6 14.3 Eluate (mg) 4.8 5.4 5.2 4.8 5.2 4.6 4.8

Table 7-3 Product yield cycle number 1 2 3 4 5 6 7 Loading (mg) 21.2 19.7 19.7 19.7 19.6 19.6 19.6 Wash (mg) 13.2 16.0 16.4 16.4 17.1 17.2 17.2 Eluate (mg) 1.6 1.8 1.8 1.8 2.0 2.0 2.0

The columns were stored at 4° for about 5 weeks. A similar amount of MT5 was eluted from each product, demonstrating good column stability.

7.5 Stability study of the generated VEGF MiniTrap

SDS-PAGE analysis of eluted fractions from three columns (column 1, column 2 and column 3) was performed. Samples were prepared in non-reducing and reducing exemplary SDS-PAGE buffer and run on a 4-12% NuPage Bis-Tris gel gradient using 1X MES (Cat. No. NP0322, Invitrogen, Carlsbad, CA).

The wells were loaded with (1) molecular weight standard, (2) loading solution, (3) column wash from column 1, (4) eluted fraction from column 2, (5) eluted fraction from column 1, (6) eluted fraction from column 3, (7) MT5 stored at pH 2.8 for 1 min, (8) MT5 stored at pH 2.8 for 30 min, and (9) molecular weight standard ( Fig . 33 and Fig. 34 ). The analysis showed that the fractions obtained from the eluted fractions from all three affinity columns (columns 1-3) showed the same size profiles, and the use of affinity columns did not destabilize the MiniTrap.

7.6 Calculation of host cell protein level

A standard host cell protein concentration curve was generated using the CHO HCP ELISA kit, 3G (F550) (Cygus Technologies) ( Figure 32 and Table 7-4 ). The amount of HCP in the loading solutions and eluted fractions was calculated using the standard curve as shown in FIG. 32 and the curve formulas shown in Table 7-4 .

Table 7-4. Curve formula Low asymptote Incline EU ₅₀ (ng/ml) High asymptote R ² Y=(A-D)/(1+(X/C)^B) + D 0.2 1.9 32.9 2.3 1

The total amount of HCP was calculated using the standard curve, and the graph with the total number of proteins of the host cell is presented in Fig . 35A . In FIG. 35B also shows the total host cell proteins in the load compared to washes and eluted fractions from columns 1, 2, 4, and 5. Several runs were performed using columns and (#) in FIG. 35B represents the run from which a fraction was eluted.

The use of affinity capture using proteins capable of binding to MiniTrap showed an effective reduction in HCP from about 7000 ppm to about 25-50 ppm. As observed for yield, the VEGF ₁₆₅ column showed a higher purity of MiniTrap from HCP than shown for the mAb1 and mAb2 columns.

7.7 VEGF MiniTrap SEC profiles before and after affinity preparation

The SEC profiles of the eluted fractions from three columns (columns 1-3) were compared to the SEC profile of MiniTrap in the loading solution. As seen in FIG. 36 and in Table 7-5 , the MT5 SEC profiles before or after affinity generation were largely similar.

Table 7-5. The peak number according to FIG. 36 retention time % peak area retention time % peak area retention time % peak area retention time % peak area Boot Solution Column 1 eluate Column 2 eluate Column 3 eluate 1 6.8 1.8 6.8 1.2 6.9 1.1 7.0 1.2 2 7.8 94.6 7.9 97.2 7.9 97.3 7.9 98.3 3 9.4 3.6 10.2 1.7 11.4 1.6 11.3 0.5

7.8 Kinetic characteristics of VEGF MiniTrap before and after binding of mAb1, mAb2 and VEGF column samples ₁₆₅

Kinetic studies were performed using the Biacore T200 instrument.

Equilibrium dissociation constants (K _D values) for VEGF165 binding to MiniTrap in the eluates from columns 1 and 2 and the loading solution were determined using a real-time surface plasmon resonance biosensor using a Biacore T200 instrument. All binding studies were performed in 10 mmol HEPES, 150 mmol NaCl, 3 mmol EDTA and 0.05% v/v. Tween-20 surfactant, pH 7.4 (HBS-ET), running buffer at 25°C. The Biacore sensor surface was first prepared by coupling an amine compound to mAb1 to capture MT5. A simplified depiction of the binding study is shown in FIG . 37 .

Briefly, eluates from columns and loading solution were diluted in HBS-EP buffer (Biacore) and injected through immobilized protein matrices at ~70 RU capture level. Then VEGF ₁₆₅ was injected with a flow rate of 50 μl/min. An equivalent volume of the analyte was simultaneously injected onto the untreated control surface to serve as blank sensograms to subtract the volume refractive index background. The sensor chip surface was regenerated between cycles with two 5-minute injections of 10 mmol glycine at a rate of 25 μl/min. The resulting experimental binding sensograms were then evaluated using the BIA Assessment 4.0.1 software to determine the kinetic velocity parameters. The datasets for each sample corresponded to the 1:1 Langmuir model. For these studies, binding and dissociation data were analyzed according to the Global Fit analysis protocol, while choosing a local fit for the maximum analyte binding capacity (RU) or Rmax attribute. In this case, the software calculated a single dissociation constant (kd), an association constant (ka), and an affinity constant (Kd). The equilibrium dissociation constant was K _D =kd/ka. The kinetic rate of the forward reaction, the kinetic rate of the reverse reaction and the overall affinity were determined using various concentrations of VEGF ₁₆₅ in the range of 0.03-2 nmol ( table 7-6 ). The dissociation half-lives (t½) were calculated from the kinetic rate constants as t _1/2= ln(2)/60*Kd. Binding kinetic parameters for MT5 to VEGF ₁₆₅ obtained from pre and post affinity chromatography at 25° C. are shown in Table 7-6 .

Affinity (KD), forward reaction rate (ka, M-1c-1) and reverse reaction rate (kd) for MT5 obtained by affinity chromatography compared to the loading solution for evaluating the effect(s) of the affinity chromatography step did not demonstrate changes in the kinetic characteristics of MT5 from other samples. The SPR sensorgrams of the VEGF MiniTrap constructs are shown in FIG . 38 .

Table 7-6. VEGF MiniTrap Samples k _a
(10 ⁶ M ^-1 With ^-1 ) k _d
(10 ^-5 With ^-1 ) K _D
(10 ^-12 M) t½
(min.) Chi ²
(RU ² ) R _max (RU) Boot Solution 9.44 1.74 1.84 664 0.10 20 Column 2 eluate 8.83 1.49 1.69 775 0.17 28 Column 1 eluate 12.18 1.80 1.48 641 0.18 19

7.9 Multiple production runs

Chromatographic preparation of the collection obtained in step 7.1 was carried out using column 1 (hVEGF ₁₆₅ ) and column 2 (mAb1) as shown in 7.3. The columns were used for several chromatographic runs. The output of the columns did not change significantly due to additional runs, indicating that the columns retained their binding capacity ( Table 7-7 ).

Table 7-7. Product yield affine column Column 1 Column 2 cycle number 1 2 3 4 1 2 3 4 Loading (mg) 21.2 19.7 19.7 19.7 21.2 19.7 19.7 19.7 Wash (mg) 14.9 13.5 15.0 15.0 13.2 16.0 16.4 16.4 Eluate (mg) 4.8 5.4 5.2 4.8 1.6 1.8 1.8 1.8

Calculations of HCP in the loading solution, wash fractions and eluted fractions for columns 1 and 2 were performed using the method described in 7.4 ( Fig. 39 ). The calculated total amount of HCP indicated that column reuse did not reduce the column's ability to bind to MiniTrap.

7.10 Optimizing affinity chromatography columns

Chromatographic preparation of the collected material in accordance with obtaining 7.1 was carried out using column 1 (VEGF ₁₆₅ ) and column 2 (mAb1). For optimization studies, 14 mg or 45 mg instead of 10 mg of VEGF165 or anti-VEGF R1 mAb was loaded into two HiTrap NHS-Activated HP affinity columns (1 ml, GE Healthcare) and the columns were capped to allow the compound to be held for 30 minutes at room temperature . Column preparation and harvesting, including MiniTrap, was performed as described in steps 7.2 and 7.3 above. The amount of MT5 in the wash and eluted fractions is shown in Table 7-8 . Comparison of the affinity column with a conjugation amount of 14 mg or 45 mg (VEGF ₁₆₅ or anti-VEGF R1 mAb1 (mAb1)) instead of 10 mg shows increased MiniTrap yield from both columns. Thus, column yield using the described method can be improved by optimizing the ratio of protein to column, or by increasing conjugation efficiency by changing pH, incubation time, incubation temperature , etc.

Table 7-8. affine column (hVEGF ₁₆₅ ) (Mouse anti-VEGF R1 mAb) MW (kDa) ~ 40 (dimer) 145 Amount of conjugation (mg) 10 14 10 45 Loading (mg) 21.2 45.5 21.2 45.5 Wash (mg) 14.9 36.8 13.2 29.2 Eluate (mg) ~5.0 7.6 ~1.8 5.5

7.11 Use of CEX with affinity chromatography

A cell culture sample from MT5 expression was generated using column 1 as described in section 7.3 above. The resulting eluate was subjected to cation exchange chromatography (CEX) column (HiTrap Capto S, 1 ml). Column operating conditions are shown in Table 7-9 .

Table 7-9. Stages affinity Cation exchange (CEX) Column Affinity column, 1 ml HiTrap Capto S, 1 ml Loading MT5 CM2926 20 mmol acetate, pH 5.0 (load/wash 1) Washing 1X DPBS pH 7.2 10 mmol phosphate, pH 7.0 Elution Pierce™ IgG Elution Buffer 50 mmol Tris buffer, 62.5 mmol (NH ₄ ) ₂ SO ₄ , pH 8.5 Regeneration/purification 10 mmol glycine pH 2.5 50 mmol Tris buffer, 1 mol (NH ₄ ) ₂ SO ₄ , pH 8.5

The total amount of HCP in the initial/starter cell culture sample, affinity column 1 eluate, and CEX eluate was about 230,000 ng/mL, from about 9,000 ng/mL to about 850 ng/mL, respectively. The amounts of HCP were quantified using the Cygnus CHO HCP ELISA kit, 3G, as above.

7.12 Use of affinity chromatography to generate another anti-VEGF protein

Column 1 was evaluated to examine its ability to produce other anti-VEGF proteins. Aflibercept and a scFv fragment with VEGF binding potential were used for this study. The manufacturing processes were carried out as described in section 7.3. Table 7-10 shows that column 1 successfully bound and eluted other anti-VEGF proteins.

Table 7-10. Affine column 1 scFv Aflibercept Loading (mg) 10 20 Wash (mg) 4.5 10.6 Eluate (mg) 3.6 10.2

Example 8 Characterization of VEGF MiniTrap Constructs Based on Mass Spectrometry

Materials . VEGF MiniTrap (MT1) was generated from aflibercept as described in Example 1. VEGF MiniTrap 5 (MT5) was generated as described in Example 7. VEGF MiniTrap (MT6) was generated by the following method: Recombinant VEGF MiniTrap (MT5) coding regions were operably linked with a signal sequence and cloned into a mammalian expression vector, transfected into Chinese hamster ovary (CHO-K1) cells and stably transfected pools were isolated after selection using hygromycin 400 μg/ml for 12 days. Stable pools of CHO cells grown in HOS were used to obtain proteins for analysis.

8.1 Deglycosylation of glycoproteins.

Samples from the MT1, MT2 and MT3 clarified harvest were diluted or reconstituted to 0.52 mg/ml in 28.8 µl of 1% (w/v) RG surfactant solution (RapiGest SF, Waters, Milford, MA) and 50 mmol HEPES (pH 7.9). These solutions were heated to approximately 95° C. for 2 minutes, allowed to cool to 50° C. and mixed with 1.2 μl of PNGase F solution (GlycoWorks Rapid PNGase F, Waters, Milford, MA). Deglycosylation was completed by incubating the samples at 50°C for 5 min.

8.2 Analysis by HILIC-fluorescence-ESI-MS (MS/MS).

MT1 was analyzed by HILIC separation coupled with fluorescent and mass spectrometric detection. MT2 and MT3 were analyzed using only HILIC. Chromatography was performed using a Waters 2D Acquity UPLC equipped with a photodiode array and fluorescence detectors (FLR) and coupled to a Waters Synapt G2-S mass spectrometer (MS conditions). Hydrophilic Interaction Chromatography (HILIC) separation mode was used with a Waters UPLC Glycan BEH Amide column, 150×2.1 mmol, 1.7 µm. The column temperature was set to 60°C and the autosampler temperature was set to 5°C. The injection volume was 50 µl. The scanning range of the photodiode array was 190–700 nm. FLR was set to 265 nm excitation, 425 nm emission for RapiFluor labeled glycans, and 274 nm excitation and 303 nm emission for tyrosine present in glycopeptides. The initial flow rate was 0.4 ml/min. with mobile phase A containing 100 mmol ammonium formate (pH 4.4) and mobile phase B being acetonitrile.

8.3 MC conditions

Liquid chromatography/mass spectrometry (LC/MS) experiments were performed using a Waters Synapt G2-S mass spectrometer. The scan range was a mass-to-charge ratio of 100-2400 for positive and negative ion assays. The scan time was 1 s, and glufibrinopeptide B was continuously injected (2 μl/min.) as a calibrating substance ("fixing mass"). The capillary voltage was set to 2.5 kV at a source temperature of 120°C and a desolvation temperature of 500°C. The gas flow rate of the nitrogen atomizer was set to 700 l/h.

8.4 Original SEC-MS

An ACQUITY UPLC class I system (Waters, Milford, MA) was linked to a Q Exactive HF hybrid quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) for all online SEC-MS analyses. An ACQUITY UPLC Protein BEH SEC column (200Å, 1.7 µm, 4.6 x 300 mm) was set at 30°C and used for protein separation. The mobile phase was 100 mmol ammonium acetate at pH 6.8. Each separation took 30 minutes at a flow rate of 0.3 ml/min and the injection volume was set to 40 μg. The following MS parameters were used for online SEC-nano-ESI-MS data collection. Each data acquisition lasted 25 minutes, starting immediately after sample injection. The deglycosylated samples were positively ionized with a spray voltage of 3 kV, a capillary temperature of 200° C., and an RF 70 S-lens level. The internal CID was set to 75 eV. A full MS scan was obtained at a resolution of 15K with a mass mass/charge range of 2000-8000. For a full MS scan, a maximum injection time of 100 ms, an automatic gain control target of 3e6, and 10 microscans were used.

8.5 Peptide mapping

Obtaining a sample for peptide mapping. Reductions were achieved by adding 500 mmol/L dithiothreitol (DTT) to a final concentration of 5 mmol/L followed by incubation at 4° C. for 60 minutes. Alkylation was carried out by adding 500 mmol/l iodoacetamide (IAM) to a final concentration of 10 mmol/l and incubating at 4°C for 60 min. In the dark. Denaturing buffer was exchanged for digestion buffer (1 mol/L urea in 0.1 mol/L Tris buffer, pH 7.8) using Zeba™ Spin 7 K MWCO desalting size-exclusion columns (P/N 89882) (Thermo Scientific, Waltham , Massachusetts) according to the manufacturer's instructions. Recombinant porcine trypsin (purchased from Sigma, Cat # 03708985001) was added at a 1:18 weight ratio (enzyme:sample) (based on VEGF MiniTrap protein measured using UV-Vis spectrophotometry after buffer exchange), the concentration of VEGFTrap proteins was adjusted to 0.5 μg/μl and digestion was allowed to proceed for 4 hours of incubation at room temperature. After the splitting was completed, 0.1% formic acid was added to LC-MS grade water in a ratio by volume of 1:1. The hydrolysates were stored at -80°C until analysis.

Analysis by LC-MS/MS of tryptic hydrolysates . One or more 2.5 μg (10 μl) of the digested peptides was loaded through an autosampler onto a C18 column placed in a thermostatically controlled column oven set at 40°C. In the queue for injection, the samples were kept at 7°C. The chromatography was started with 98% mobile phase A (0.1% v/v formic acid in water) and 2% mobile phase B (0.1% v/v formic acid in acetonitrile) at a constant flow rate of 0.200 ml/min. After washing for 10 minutes, the peptides eluted over a gradient of 110 minutes, at which the content of mobile phase B increased at a rate of 0.39% per minute until a final composition containing 45% of mobile phase B was reached. Before the next injection of the sample, the column was washed for 15 min. 97% of mobile phase B, then equilibrated with 98% of mobile phase A over 25 minutes. The eluate was sent for testing during the first 1.5 minutes and the last 5 minutes of the run. Peptides eluting from the chromatographic column were analyzed by UV absorbance at 214 nm followed by mass spectrometry on an LTQ Orbitrap Elite or Discovery XL. Replication peptide mapping data was collected for samples PS 8670 and RM 8671 to include three tandem MS (MS/MS) analyzes and one MS only analysis. MS/MS analyzes were performed to identify peptides in a data-dependent manner in which one cycle of experiments consisted of one full MS scan from 300 w/charge to 2000 w/charge followed by five consecutive MS/MS events performed for ions from the first to the fifth in intensity, detected at a minimum threshold count of 500 during the MS scan that initiates this cycle. The AGC serial mass spectrometry (MSn) target was set to 1E4 with microscan=3. The ion trap was used in centroid mode at normal scan speed to analyze the MS/MS fragments. Full MS scans were collected in profile mode using a high resolution FTMS analyzer ( R =30,000) for AGC 1E6 full scan and microscans=1. Ions were sampled for MS/MS using an isolation width of 2 Da, then fragmented by collision-induced dissociation (CID) with helium gas using normalized CID energy of 35, an activation Q of 0.25, and an activation time of 10 ms. The default state of charge has been set to z = 2 . Data-dependent masses were placed on the exclusion list for 45 seconds if the precursor ion triggered the phenomenon twice within 30 seconds; the exclusion width was set to ±1 Da. State of Charge Rejection is enabled for unassigned states of charge. The mass reject list included common impurities at 122.08 mass/charge , 185.94 mass/charge , 355.00 mass/charge , 371.00 mass/charge , 391.00 mass/charge , 413.30 mass/charge , 803.10 mass/charge , 1222.10 mass/charge , 1322.10 mass /charge , 1422.10 mass /charge , 1822.10 mass/charge and 1922.10 mass/charge . MS-only analyzes were performed to create a map of unreduced TIC peptides and reduced maps.

8.6 Results

Structure of VEGF MiniTrap constructs. The structure of VEGF MiniTrap MT1, MT5 and MT6 is shown in FIG. 40 , fig. 41 , fig. 43 and FIG. 44 .

Initial mass analysis using SEC-MS confirmed the identity of all three molecules at the intact protein level after deglycosylation ( Fig. 42 ). The total ion count (TIC) chromogram of the native SEC-MS assay demonstrates the detection of intact VEGF MiniTrap molecules at approximately 12-13 minutes. The expansion of the low molecular weight (LMW) region of the TIC showed the presence of LMW impurities in all three protein samples.

Deconvoluted VEGF MiniTrap mass spectra further confirmed their identity and provided data to elucidate the major PTMs present in the sample, containing MT1 and MT5 ( Figure 43 ), which are dimers, and MT6 ( Figure 44 ), which is a single-stranded protein.

Analysis of the MT1 sample. LMW forms identified by TIC analysis of SEC-MS samples containing MT1 were extracted to determine three different LMW impurities - LMW1, LMW2 and LMW3 ( FIG. 45A and FIG. 45B ). Forms of LMW1 included truncated forms of aflibercept. The LMW2 forms included the Fc contaminant present in the sample from the aflibercept digestion that was performed to produce MiniTrap. The LMW3 form includes a monomer possibly cleaved from an MT1 molecule (dimer).

The MT1 sample did not show the presence of the FabRICATOR enzyme, which was used to cleave aflibercept to form the MiniTrap protein. The enzyme, if present, is detectable at about 11.5 and 12.5 minutes. Such a peak was not detected during SEC-MS analysis of the MT1 sample ( Fig. 46 ).

MT5 sample analysis. Forms of LMW identified by TIC analysis of SEC-MS samples containing MT5 were extracted for the presence of two different LMW contaminants, LMW1 and LMW2 ( Fig. 47 ).

MT6 sample analysis. Forms of LMW identified by TIC analysis of SEC-MS samples containing MT1 were extracted for the presence of three different LMW impurities - LMW1, LMW2 and LMW3 ( Fig. 48 ). Forms of LMW2 include an MT6 fragment, and cleavage results in a VEGF MiniTrap fragment with a G4S linker. The LMW5 forms are an MT6 fragment in which the cleavage occurred immediately before or after the G4S linker.

The glycans in the MT6 sample were identified by their mass and elution order in the HILIC method using the glucose unit value pioneered by Waters and the National Institute for Research and Training in Biotechnology (Dublin, Ireland) ( FIG. 49A and FIG. 49B ).

Quantitative determination of free thiol. The cysteine residues of the VEGF MiniTrap constructs may be involved in the formation of intra- and intermolecular disulfide bonds, or they may exist as free thiols. The presence of sulfide bonds in peptides and proteins has been shown to impose conformational rigidity on the protein. Thiols can be detected by various reagents and separation methods. Analysis of three VEGF MiniTrap constructs for relatively very low free thiol levels is shown in Table 8-1 .

Table 8-1. Location Peptide (free cysteine site) MT1 MT5 MT6 VEGF R1 ELVIP C R (SEQ ID NO.: 81) < 0.1% < 0.1% < 0.1% VEGF R2 LVLN C TAR (SEQ ID NO.: 82) 0.3% 0.3% 0.3% Hinge Fc THT C PP C PAPELLG (SEQ ID NO.: 83) 0.0% 0.0% N/A

Quantitative determination of trisulfide. Like free thiols in Cys residues of VEGF MiniTrap constructs, trisulfide bonds can influence protein structure. Analysis of the three VEGF MiniTrap constructs under very low free thiol conditions is shown in Table 8-2.

Table 8-2. Location Peptide MT1 MT5 MT6 VEGF R1 ELVIP C R - EIGLLT C EATVNGHLYK (SEQ ID NO.: 84) 0.1% < 0.1% 0.1% VEGF R2 LVLN C TAR - SDQGLYT C AASSGLMTK(K) (SEQ ID NO.: 85) < 0.1% < 0.1% < 0.1% Hinge Fc THT C PP C PAPELLG - THT C PP C PAPELL(G) (SEQ ID NO.: 86) 1.5% 3.7% N/A

Intrachain disulfide in the hinge region. Unpaired disulfide bonds in the hinge region can affect the structure, function, and stability of VEGF MiniTrap constructs. Analysis of the three VEGF MiniTrap constructs for very low or no intrachain disulfide bonds in the hinge region of the VEGF MiniTrap constructs [THTC*PPC*PAPELLG, C* shows where an intrachain sulfide bond can form] is shown in Table 8-3.

Table 8-3. Peptide MT1 MT5 MT6 disulfide < 0.1% < 0.1% N/A Trisulfide < 0.1% < 0.1% N/A

Quantitative determination of isomers of transverse and parallel disulfide bonds. For MT1 and MT5, which are dimers connected by parallel disulfide bonds at the hinge regions, there is the possibility of isomers where the disulfide bonds at the hinge regions can cross ( Fig. 50 ).

Quantification of disulfide bond types, parallel and crossed, showed that the recombinantly expressed MT2 protein had a slightly higher level of crossed disulfide bridge in the Fc hinge region compared to MT1, which is a FabRICATOR cleaved molecule ( Table 8-4 ).

Table 8-4. disulfide MT1 MT5 MT6 transverse 0.2% 3.9% N/A Parallel 99.8% 96.1% N/A

Post-translational modifications (PTM).

Table 8-5. PTM Website Modified peptide MT1 MT5 MT6 Deamidation Asn84
(Asn319) EIGLLTCEATV N GHLYK (SEQ ID NO.: 87) Succinimide 3.1% 3.2% 3.2% Asp/iso Asp 21.9% 18.9% 20.9% Asn99
(Asn334) QT N TIIDVVLSPSHGIELSVGEK (SEQ ID NO.: 88) Succinimide 4.6% 4.6% 4.0% Asp/iso Asp 0.7% 0.5% 0.6% Oxidation Met10 SDTGRPFVE M YSEIPEIHMTEGR (SEQ ID NO.: 64) 1.8% 2.1% 2.1% Met20 SDTGRPFVEMYSEIPEIH M TEGR (SEQ ID NO.: 65) 2.9% 3.0% 2.7% Met245 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSSDTGRPFVE M YSEIPEIHMTEGR (SEQ ID NO.: 91) - - 1.4% Met255 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSSDTGRPFVEMYSEIPEIIH M TEGR (SEQ ID NO.: 92) - - 2.7% Met163 (Met398) TQSGSE M K (SEQ ID NO.: 66) 4.3% 4.3% 3.8% Met192
(Met427) SDQGLYTCAASSGL M TK (SEQ ID NO.: 94) 5.0% 5.0% 4.2% Loss of C-terminal glycine Gly211 THTCPPCPAPELL G (SEQ ID NO.: 95) 0.1% 2.0% -

PTM evaluation in all three VEGF MiniTrap designs showed comparable PTM levels ( Table 8-5 ). The deamidation observed at Asn84 to form succinimide ranged from 3.1 to 3.2% and to form aspartic acid/isoaspartic acid was 18.9-21.9%. Oxidation of several methionine residues ( eg Met10, Met 20m Met163 and Met192) was observed in the range of about 0.7-6.8% for all three VEGF MiniTrap constructs. MT6, which, unlike MT1 and MT5, contains a linker, has shown additional oxidation of methionine residues on the linker ( eg Met245 and Met255). About 0.1% and 2.0% of the C-terminal glycine (Gly211) in MT1 and MT5 showed a loss of glycine. This was not observed for MT6, which lacks the C-terminal glycine.

Modifications of the advanced glycation end product associated with the glycation of lysine and arginine. Glycation of VEGF MiniTrap constructs can alter their structure and function, resulting in impaired anti-VEGF activity.

Table 8-6. Website PTM MT1 MT5 MT6 Arg5 3-deoxyglucoson 8.0% 8.1% 9.2% glycation 0.1% 0.1% 0.1% Carboxymethylation 1.5% 1.4% 1.4% Arg153 3-deoxyglucoson < 0.1% < 0.1% < 0.1% Arg96 3-deoxyglucoson < 0.1% < 0.1% < 0.1% Lys62 glycation 1.1% 1.1% 1.3% Carboxymethylation < 0.1% < 0.1% < 0.1% Lys68 glycation 0.4% 0.3% 0.5% Lys149 glycation 0.6% 0.5% 0.6% Carboxymethylation < 0.1% < 0.1% < 0.1% Lys185 glycation < 0.1% < 0.1% < 0.1%

Evaluation of modifications in all three VEGF MiniTrap designs showed comparable levels ( Table 8-6 ).

modified sites. Altered sites on the VEGF MiniTrap constructs, as revealed by intact mass analysis according to Section 8.4, were confirmed and quantified using abbreviated peptide mapping as shown in Section 8.5 ( Table 8-7 ). The T90N91 site for the TNYLTHR** peptide sequence indicates that the asparagine has been converted to aspartic acid after truncation, while the N99T100 site for the QTNTIIDVVLSPSHGIELSVGEK* peptide sequence indicates a high level of non-specific trypsin cleavage. These two truncation sites were found in the formation of impurities of low molecular weight forms in the evaluation of MT1 and MT5. Truncation at M245Y246 was only found on MT3, which had a unique linker and was responsible for LMW2 impurity during MT3 production.

Table 8-7. Website Peptide sequence MT1 MT5 MT6 N99T100 QTNTIIDVVLSPSHGIELSVGEK* (SEQ ID NO.: 96) 12.6% 13.2% 13.6% T90N91 TNYLTHR** (SEQ ID NO.: 97) 0.5% 0.1% 0.3% M245Y246 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSSSDTGRPFVEMYSEIPEIHMTEGR (SEQ ID NO.: 98) - - 1.8% M10Y11 SDTGRPFVEMYSEIPEIHMTEGR (SEQ ID NO.: 99) 0.2% 1.5% 1.7%

[2] Quantification of glycoside occupancy . N-glycosylation is a common PTM. The characterization of site-specific N-glycosylation, including N-glycan macroheterogeneity (glycosylation site occupancy) and microheterogeneity (site-specific glycan structure), is important for understanding glycoprotein biosynthesis and function. The degree of glycosylation can vary depending on how the protein is expressed. Glycosylation levels at N36 were similar for all three VEGF MiniTraps ( Table 8-8 and FIG. 51 ). Similarly, levels of glycosylation at N68 were also similar for all three VEGF MiniTraps ( Table 8-8 and FIG. 52 ). Glycosylation levels at N123 were also similar for all three VEGF MiniTraps ( Table 8-8 and FIG. 53 ), but mannose-5 was found to be elevated in the MT1 preparation. For VEGF MiniTrap constructs, Asn196 glycosylation was lower for MT5 and MT6 compared to MT1 ( Table 8-8 and Fig. 54 ). In addition, mannose-5 levels were also increased for MT1 than for MT4 and MT6.

Table 8-8. Website Peptide MT1 MT5 MT6 N36 (R)VTSP N ITVTLK (SEQ ID NO.: 100) 98.3% 98.1% 99.4% N68 (K)GFIIS N ATYK (SEQ ID NO.: 101) 51.9% 55.4% 64.9% N123 (K)LVL N CTAR (SEQ ID NO.: 102) 99.9% 99.4% 98.4% N196 (K) N STFVR (SEQ ID NO.: 103) 98.6% 44.5% 55.1%

[3] N-glycan analysis . Glycosylation at N36 is shown in Table 8-9 . G2F, G2FS, G2FS2 were the main N-glycans found in all three VEGF MiniTraps. For glycosylation at N68 shown in Table 8-10 , G2F and G2FS were the main N-glycans found in all three VEGF MiniTraps. For glycosylation at N123 shown in Table 8-11 , G2F and G2S were the major N-glycans found in all three VEGF MiniTraps, and mannose-5 was found at high levels of MT1 compared to MT5 and MT6. For glycosylation at N196 shown in Table 8-12 , G2, G2S, G2S2 were the major N-glycans found in all three VEGF MiniTraps, and mannose-5 was found at high levels of MT1 compared to MT5 and MT6.

Table 8-9. Glycans on N36 MT1 MT5 MT6 G0F-GlcNAc 2.0% 1.8% 1.8% G1F 3.2% 1.0% 1.4% G1F-GlcNAc 4.8% 4.6% 4.9% G1FS-GlcNAc 3.1% 3.8% 3.1% G2F 17.4% 15.1% 19.8% G2F2S 1.7% 2.0% 2.2% G2FS 34.2% 31.5% 31.9% G2FS2 20.4% 25.8% 19.0% G3FS 2.3% 4.0% 5.5% G3FS2 2.6% 4.7% 5.0% G3FS3 1.1% 2.4% 1.9% G1_Man5+Phos 1.2% 0.3% 0.2% Man6+Phos 5.7% 2.5% 2.8%

Table 8-10. Glycans on N68 MT1 MT5 MT6 G0F-GlcNAc 1.2% 1.1% 1.1% G1F 5.1% 1.4% 1.7% G1F-GlcNAc 3.9% 3.9% 4.0% G1FS 1.2% 0.4% 0.4% G1FS1-GlcNAc 1.2% 1.6% 1.4% G2F 27.4% 23.6% 28.6% G2F2S 2.2% 3.0% 3.4% G2FS 52.4% 55.2% 50.2% G2FS2 3.9% 6.9% 5.8% G3FS 0.5% 1.2% 1.6% G3FS2 0.4% 1.1% 1.2%

Table 8-11. Glycans on N123 MT1 MT5 MT6 G0-GlcNAc 3.5% 3.7% 3.5% G1-GlcNAc 6.2% 6.8% 6.4% G1S-GlcNAc 4.1% 3.5% 2.8% G2 10.6% 16.7% 17.1% G2F 1.5% 7.2% 7.0% G2FS 2.1% 13.6% 14.2% G2S 12.7% 26.1% 25.5% G2S2 1.3% 5.0% 6.6% G1_Man4 3.8% 1.3% 1.4% G1S_Man4 3.9% 2.1% 1.8% G1_Man5 4.0% 1.2% 1.1% G1S_Man5 3.2% 1.4% 1.4% Man4 2.6% 1.9% 1.8% Man5 35.5% 4.3% 3.1% Man6 1.1% 0.1% 0.1% Man7 2.8% 0.1% 0.1%

Table 8-12. Glycans on N196 MT1 MT5 MT6 G0-GlcNAc 1.9% 1.8% 1.9% G1 4.1% 3.6% 4.2% G1-GlcNAc 1.9% 2.5% 2.4% G1S-GlcNAc 2.9% 2.6% 1.8% G2 20.7% 28.2% 32.1% G2F 2.0% 5.1% 6.0% G2FS 2.0% 6.1% 6.2% G2FS2 0.5% 1.6% 1.3% G2S 17.7% 31.2% 29.9% G2S2 4.4% 9.7% 6.7% G3S 0.1% 0.7% 1.0% G1S_Man4 1.0% 0.3% 0.3% G1_Man5 2.3% 0.5% 0.5% Man3 3.1% 0.7% 0.6% Man4 2.7% 0.8% 0.6% Man5 30.4% 3.6% 3.4%

O-glycans on a linker for MT3 . Linker GS for MT3 was evaluated for the study of O-glycans on MT3. O-xylosylation was detected at serine residues located on the MT3 GS linker (GGGG S GGGG S GGGG S GGGG S GGGG S GGGG S SDTGRPFVEMYSEIPEIIHMTEGR, the underlined serine residues were glycosylated). The composition of the O-glycan is shown in Table 8-13 .

Table 8-13. Composition Mass Note Number Level Xylosylation +132.0 Three- < 0.1% Di- 1.5% Mono- 15% xylose + galactose +294.1 Mono- 0.9% Xylose + galactose + sialic acid +585.2 Mono- 0.7%

HILIC-FLR-MS analysis . HILIC-FLR-MS analysis was performed for all VEGF MiniTrap proteins as described in section 8.2. Analysis showed that the N-linked glycans for MT5 and MT6 were similar but different from those obtained for MT1 ( Figure 55 shows full and reduced chromatograms, Figure 56 shows full and overlapped chromatograms, and Figure 57 shows full , reduced and normalized chromatograms).

Finally, percent glycosylation and detailed glycan identification and quantification for all three VEGF MiniTrap proteins are listed in Table 8-14 and FIG. 58A-C , respectively. As observed in all glycan assays, the glycosylation profile and mannose levels for MT5 and MT6 are similar but different from MT1.

Table 8-14. MT1 MT2 MT3 % fucosylation 42.9% 57.8% 57.2% % galactosylation 71.6% 92.9% 93.7% % sialylation 33.1% 47.6% 44.8% % high-mannose forms 17.6% 2.6% 2.3% % separation 1.9% 0.4% 0.4%

Example 9 Color acquisition and quantification using ascending media and optimization of the delivery process.

(A) Non-optimized HOS (control bioreactor)

The MiniTrap fabrication described in Example 5 was used.

The operating parameters for the study steps are known to the average person skilled in the art.

Wednesday on day 0=XOS1 and included the following nutrients, antioxidants, and metals:

Cysteine was added at a cumulative concentration of 8-9 mmol

The metals in the original medium are listed below at 1x concentration (if the elements are listed before the addition of the inoculum):

Fe=68-83 micromoles per liter of culture

Zn=6-7 micromoles per liter of culture

Cu=0.1-0.2 micromoles per liter of culture

Ni=0.5-1 micromoles per liter of culture

The collection of MT1 followed the acquisition procedure shown in FIG . 59 . The operating parameters of the chromatography are known to those of ordinary skill in the art. Operating parameters for affinity capture (stage 3 in Fig. 59 ), affinity flow fraction (stage 5 in Fig. 59 ), AEX (stage 8 in Fig. 59 ) and HIC (stage 9 in Fig. 59 ) are set out in Table 9- 1. Proteolytic cleavage of aflibercept after the affinity capture and filtration step was performed using the procedure described in Example 1.2.

Table 9-1. Stages Affine capture Affinity flow fraction AEX HIC Resin MabSelect SuRe MabSelect SuRe POROS 50 HQ Capto Phenyl HS Loading 30 g/l resin 30 g/l resin pH 6.80-7.20 40 g/l resin
pH 8.30-8.50, 1.90-2.10 mS/cm 100 g/l resin pH 4.40-4.60 7.50-10.50 mS/cm Balancing 20 mmol sodium phosphate pH 7.10-7.30, 2.60-3.20 mS/cm 26 mmol Tris buffer, 16 mmol sodium phosphate, 18 mmol acetate pH 6.90-7.10, 2.00-4.00 mS/cm 50 mmol tris buffer
pH 8.30-8.50, 1.90-2.10 mS/cm 40 mM Tris buffer, 30 mM sodium citrate, 74 mM acetate pH 4.40-4.60, 7.50-10.50 mS/cm Wash 1 10 mmol sodium phosphate, 500 mmol NaCl pH 7.10-7.30, 40-50 mS/cm 26 mmol Tris buffer, 16 mmol sodium phosphate, 18 mmol acetate pH 6.90-7.10, 2.00-4.00 mS/cm 50 mmol tris buffer
pH 8.30-8.50, 1.90-2.10 mS/cm 40 mM Tris buffer, 30 mM sodium citrate, 74 mM acetate pH 4.40-4.60, 7.50-10.50 mS/cm Flushing 2 20 mmol sodium phosphate pH 7.10-7.30, 2.60-3.20 mS/cm N/A N/A N/A Elution 40 mmol acetic acid pH 2.80-3.20, 0.28-0.36 mS/cm 40 mmol acetic acid pH 2.80-3.20, 0.28-0.36 mS/cm N/A N/A Regeneration/purification 1 500 mmol acetic acid, pH 2.25-2.65, 0.90-1.25 mS/cm 500 mmol acetic acid, pH 2.25-2.65, 0.90-1.25 mS/cm 2 mol sodium chloride (NaCl) proprietary buffer Regeneration/purification 2 N/A N/A 1 n. sodium hydroxide (NaOH) N/A

Table 9-2 shows the color quantification of the pools obtained from the various chromatographic steps. Color quantification was performed using pooled samples having a protein spike of 5 g/L.

The affinity capture pool refers to the eluate collected from the affinity capture step (step 3 in FIG. 59 ). Enzymatic pool refers to the flow-through fraction collected during the enzymatic cleavage step (step 4 in FIG. 59 ). The affinity flow-through pool refers to the flow-through fraction collected in the affinity flow-cut step (step 5 in FIG. 59 ) and the affinity flow-through fraction eluate refers to the eluate collected in the affinity flow-cut step (step 5 in FIG. 59 ). The AEX pool and AEX purification refer to the flow through fraction and the purified fraction obtained from the anion exchange chromatography step (step 8 in FIG. 59 ). The HIC pool refers to the flow fraction collected during the hydrophobic interaction chromatography step (step 9 in FIG. 59 ).

As seen in Table 9-2 , each stage shows a decrease in color (as seen from the decrease in pool b* values). For example, when performing flow fraction affinity chromatography, the flow fraction has a b* value of 2.16 (reduced from the b* value of 2.52 for the flow fraction resulting from the affinity capture step). The run-through and washing after AEX separation further reduced the color, as evidenced by the decrease in b* value from 2.16 to 0.74. As expected, AEX column cleanup resulted in a tan sample that was significantly more intense than the run-through and wash after AEX separation as seen from the b* values (8.10 vs. 0.74). Finally, the HIC step provided further color reduction (the b* value can be normalized to 5 g/l protein safe from the b* value obtained for the HIC pool at a protein concentration of 28.5 g/l).

Table 9-2. Quantification of the color of samples at various stages of production Sample Conc. (g/l) L* a* b* Affine Capture Pool 5.0±0.1 98.75 -0.12 2.52 Pool of enzymatic cleavage 5.0±0.1 99.03 -0.07 1.61 Affinity flow fraction pool 5.0±0.1 98.95 -0.08 2.16 Eluate of the affinity flow fraction 5.0±0.1 98.92 -0.01 0.83 AEX pool 5.0±0.1 99.72 -0.03 0.74 Purification AEX 2 mol NaCl 5.0±0.1 96.25 -0.42 8.10 Pool HIC 28.5 98.78 -0.28 3.11

(B) Optimized HOS (low cysteine, low metals and high antioxidants, bioreactor)

The effect of cysteine reduction, metal reduction, and increase in antioxidants on color was assessed using the following protocols:

Average per day 0=XOS1

Cysteine was added at a cumulative concentration of 5-6 mmol

Antioxidants were added to XOC1 to achieve the following cumulative concentrations (where data is present prior to inoculum addition):

taurine=10 mmol culture

glycine=10 mmol culture

thioctic acid=0.0024 mmol culture

vitamin C (ascorbic acid)=0.028 mmol culture

The metals in the starting environment are listed below for level 1x.

Fe=68-83 micromoles per liter of culture

Zn=6-7 micromoles per liter of culture

Cu=0.1-0.2 micromoles per liter of culture

Ni=0.5-1 micromoles per liter of culture

All metal reductions are included using the 0.25x concentrations listed above for the medium.

After collecting the MT1 sample, the preparation procedure shown in FIG. 59 . The operating parameters of the chromatography are known to those of ordinary skill in the art. The operating parameters for affinity capture, affinity flow fraction and HIC are set out in Table 9-1. Proteolytic cleavage of aflibercept after the affinity capture and filtration step was performed using the procedure described in Example 1.2.

Table 9-3 shows the color quantification of the pools obtained from the various chromatographic steps. Color quantification was performed using pooled samples having a protein spike of 5 g/L. As can be seen in Table 9-3 , the steps provided a similar preparation as for the steps in Table 9-2 .

Table 9-3. Quantification of the color of samples at various stages of obtaining MiniTrap Sample Conc. (g/l) L* a* b* Affine Capture Pool 5.0±0.1 99.18 -0.09 1.77 Pool of enzymatic cleavage 5.0±0.1 99.44 -0.06 1.17 Affinity flow fraction pool 5.0±0.1 99.32 -0.10 1.58 Eluate of the affinity flow fraction 5.0±0.1 99.74 -0.05 0.60 AEX pool 5.0±0.1 99.63 -0.07 0.50 Purification AEX 2 mol NaCl 5.0±0.1 97.63 -0.49 6.10 Pool HIC 27.6 99.07 -0.29 2.32

Comparing Table 9-2 and Table 9-3 , it is clear that the “low cysteine, low metal, high antioxidant bioreactor condition” has a lower color in the affinity capture pool (b* value of 1.77) compared to “ control bioreactor condition” (b* value 2.52).

For a 160 g/L MT sample where the MT is formed using the steps listed in Table 9-2 and Table 9-3 , an ab* value of 13.45 is predicted for the “low cysteine, low metal, and high antioxidant condition.” in the bioreactor" and an ab* value of 17.45 for the "control bioreactor condition". A 23% color reduction is achieved by optimizing stock media and culture media. For a 110 g/L MT sample where the MT is formed using the steps listed in Table 9-2 and Table 9-3 , an ab* value of 9.25 is predicted for the "low cysteine, low metal, and high antioxidant" condition. in bioreactor" and an ab* value of 12 for "control bioreactor condition".

To understand how each operation of the production setup contributes to color reduction, the b* value for each intermediate in the production process was calculated as a percentage of the color of the affine capture pool ( Table 9-4 ).

Table 9-4. Sample Conc. (g/l) b* ∆b * b* as % of affine capture pool Control bioreactor Affine Capture Pool 5.0±0.1 2.52 N/A 100.0 Pool of enzymatic cleavage 5.0±0.1 1.61 -0.91 63.8 Affinity flow fraction pool 5.0±0.1 2.16 0.55 85.7 AEX pool 5.0±0.1 0.74 -1.42 29.4 Pool HIC 5.0±0.1 0.55 -0.19 21.8 Low cysteine, low metals and high antioxidants in the bioreactor Affine Capture Pool 5.0±0.1 1.77 N/A 100.0 Pool of enzymatic cleavage 5.0±0.1 1.17 -0.60 66.1 Affinity flow fraction pool 5.0±0.1 1.58 0.41 89.2 AEX pool 5.0±0.1 0.50 -1.08 28.2 Pool HIC 5.0±0.1 0.42 -0.08 23.7

The AEX module operation provides the maximum color reduction (b* change from 1.08 to 1.42), while the HIC module operation provides some additional color reduction (b* change from 0.08 to 0.19). The setup operations estimated an overall removal of 76.3% - 78.2% of the color present in the affinity capture pool.

The color of various manufacturing process intermediates for "bioreactor control conditions" and "low cysteine, low metals and high antioxidants in the bioreactor" also studied the percentage of 2-oxo-histidines and the percentage of oxo-tryptophans in the oligopeptides that were formed by cleavage. protease as measured by mass spectrometry as shown in Table 9-5 and Table 9-6 respectively. Peptide mapping was performed as described in Example 3.

Referring to Table 9-5 , when comparing histidine oxidation levels in pools at different stages of production, it is apparent that the percentage of histidine oxidation levels formed for MT decreases in the pool as the production progresses. For example, for H209 in "Bioreactor Control Condition", the percentage histidine oxidation was 0.062 for the enzymatic cleavage pool, which was reduced to 0.029 for the AEX flow-through fraction and further reduced to 0.020 for the HIC pool. Similarly, for H209 under "low cysteine, low metal and high antioxidant bioreactor" conditions, the percent histidine oxidation was 0.039 for the enzymatic cleavage pool, and it was reduced to 0.023 for the AEX flow-through fraction and further reduced to 0.016 for the HIC pool. . Thus, the manufacturing strategy resulted in a reduction in the percentage of histidine oxidation in MT. As color decreases, so does the presence of some oxidized residues in the sample. Similar to histidine oxidation, tryptophan oxidation levels were also monitored for pools at different stages of production for both "control bioreactor conditions" and "low cysteine, low metal and high antioxidant bioreactor" (Table 9-6).

Table 9-5 Fraction Color (b*) Histidine oxidation levels (%) H19 (+14) H86 (+14) H95 (+14) H110 (+14) H145 (+14) H209 (+14) Control Bioreactor Conditions Pool of enzymatic cleavage 1.61 0.023 0.018 0.011 0.014 0.007 0.062 Affinity flow fraction pool (AEX loading) 2.16 0.030 0.027 0.018 0.015 0.011 0.067 Eluate of the affinity flow fraction 0.83 0.030 0.022 0.000 0.018 0.004 0.046 Flow fraction AEX 0.74 0.026 0.025 0.013 0.016 0.010 0.029 Purification AEX 2 mol NaCl 8.10 0.024 0.063 0.033 0.019 0.012 0.063 Pool HIC 0.55 0.018 0.009 0.002 0.021 0.005 0.020 Condition of low cysteine, low metals and high antioxidants in the bioreactor Pool of enzymatic cleavage 1.17 0.019 0.017 0.009 0.014 0.008 0.039 Affinity flow fraction pool (AEX loading) 1.58 0.026 0.025 0.013 0.014 0.010 0.043 Eluate of the affinity flow fraction 0.60 0.031 0.017 0.007 0.020 0.003 0.016 Flow fraction AEX 0.50 0.020 0.022 0.009 0.014 0.010 0.023 Purification AEX 2 mol NaCl 6.10 0.020 0.055 0.025 0.016 0.011 0.042 Pool HIC 0.42 0.013 0.009 0.002 0.017 0.003 0.016

Table 9-6. Fraction Color (b*) Tryptophan oxidation levels (%) W58 (+4) W58 (+16) W58 (+32) W58 (+48) W138 (+4) W138 (+16) W138 (+32) Control Bioreactor Conditions Pool of enzymatic cleavage 1.61 0.006 0.032 0.289 0.000 0.020 1.093 0.106 Affinity flow fraction pool (AEX loading) 2.16 0.016 0.055 0.327 0.000 0.017 0.771 0.111 Eluate of the affinity flow fraction 0.83 0.009 0.031 0.453 0.000 0.025 1.039 0.132 Flow fraction AEX 0.74 0.014 0.038 0.283 0.000 0.023 0.720 0.120 Purification AEX 2 mol NaCl 8.10 0.043 0.089 0.462 0.000 0.031 0.620 0.175 Pool HIC 0.55 0.037 0.126 0.413 0.000 0.020 0.656 0.274 Condition of low cysteine, low metals and high antioxidants in the bioreactor Pool of enzymatic cleavage 1.17 0.009 0.027 0.239 0.001 0.027 1.026 0.136 Affinity flow fraction pool (AEX loading) 1.58 0.013 0.045 0.284 0.000 0.021 0.628 0.107 Eluate of the affinity flow fraction 0.60 0.003 0.026 0.421 0.021 0.025 1.032 0.132 Flow fraction AEX 0.50 0.011 0.031 0.235 0.000 0.022 0.676 0.102 Purification AEX 2 mol NaCl 6.10 0.034 0.073 0.478 0.000 0.032 0.635 0.169 Pool HIC 0.42 0.029 0.122 0.355 0.000 0.022 0.800 0.236

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SEQUENCE LIST

<110> REGENERON PHARMACEUTICALS, INC.

<120> ANTI-VEGF PROTEIN COMPOSITIONS AND METHODS FOR THEIR PRODUCTION

<130> 070816-02359

<140>

<150>

<160> 103

<170> PatentIn version 3.5

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Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

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

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Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

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Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

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Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

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Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

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Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

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Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

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Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

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Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

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Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

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Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

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Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 3

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 3

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 4

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 4

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 5

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 5

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 6

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 6

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 7

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 7

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 8

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 8

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 9

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 9

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 10

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 10

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 11

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 11

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 12

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 12

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asn Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 13

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 13

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Asn Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 14

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 14

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Asn Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 15

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 15

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 16

<211> 339

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 16

Met Arg Lys Arg Cys Tyr Ser Thr Ser Ala Ala Val Leu Ala Ala Val

1 5 10 15

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

20 25 30

Ser Ala Asn Gln Glu Ile Arg Tyr Ser Glu Val Thr Pro Tyr His Val

35 40 45

Thr Ser Val Trp Thr Lys Gly Val Thr Pro Pro Ala Asn Phe Thr Gln

50 55 60

Gly Glu Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln Gly Trp Tyr

65 70 75 80

Asp Ile Thr Lys Thr Phe Asp Gly Lys Asp Asp Leu Leu Cys Gly Ala

85 90 95

Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln Asn Lys Asp

100 105 110

Gln Ile Lys Arg Tyr Leu Glu Glu His Pro Glu Lys Gln Lys Ile Asn

115 120 125

Phe Arg Gly Glu Gln Met Phe Asp Val Lys Glu Ala Ile Asp Thr Lys

130 135 140

Asn His Gln Leu Asp Ser Lys Leu Phe Glu Tyr Phe Lys Glu Lys Ala

145 150 155 160

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

165 170 175

Ile Asp Met Phe Ile Leu Gly Tyr Arg Leu Ser Leu Thr Asn His Gly

180 185 190

Pro Thr Pro Val Lys Glu Gly Ser Lys Asp Pro Arg Gly Gly Ile Phe

195 200 205

Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys Leu Leu Thr Ser Arg

210 215 220

His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile Ser Asp Leu Ile Lys

225 230 235 240

Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu Ser His Thr Tyr Ala

245 250 255

Asn Val Arg Ile Asn His Val Ile Asn Leu Trp Gly Ala Asp Phe Asp

260 265 270

Ser Asp Gly Asn Leu Lys Ala Ile Tyr Val Thr Asp Ser Asp Ser Asn

275 280 285

Ala Ser Ile Gly Met Lys Lys Tyr Phe Val Gly Val Asn Ser Ala Gly

290 295 300

Lys Val Ala Ile Ser Ala Lys Glu Ile Lys Glu Asp Asn Ile Gly Ala

305 310 315 320

Gln Val Leu Gly Leu Phe Thr Leu Ser Thr Gly Gln Asp Ser Trp Asn

325 330 335

Gln Thr Asn

<210> 17

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (4)..(4)

<223> May be oxidized

<220>

<221> OTHER_INDICATIONS

<222>(6)..(6)

<223> May be carboxymethylated

<220>

<221> OTHER_INDICATIONS

<222>(9)..(9)

<223> May be carboxymethylated

<400> 17

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

<210> 18

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be carboxymethylated

<220>

<221> OTHER_INDICATIONS

<222> (14)..(14)

<223> May be oxidized

<400> 18

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

1 5 10 15

Lys

<210> 19

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (14)..(14)

<223> May be oxidized

<400> 19

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

1 5 10 15

Glu Leu Ser Val Gly Glu Lys

20

<210> 20

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (18)..(18)

<223> May be oxidized

<400> 20

Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser

1 5 10 15

Lys His Gln His Lys

20

<210> 21

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(6)..(6)

<223> May be oxidized

<400> 21

Thr Asn Tyr Leu Thr His Arg

15

<210> 22

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (19)..(19)

<223> May be oxidized

<400> 22

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 23

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (2)..(2)

<223> May be oxidized

<400> 23

Val His Glu Lys Asp Lys

15

<210> 24

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (10)..(10)

<223> May be oxidized

<400> 24

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 25

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (20)..(20)

<223> May be oxidized

<400> 25

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 26

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be oxidized

<400> 26

Thr Gln Ser Gly Ser Glu Met Lys

15

<210> 27

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(8)..(8)

<223> May be carboxymethylated

<400> 27

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr

1 5 10 15

Lys

<210> 28

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (3)..(3)

<223> May be oxidized

<400> 28

Ile Ile Trp Asp Ser Arg

15

<210> 29

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (11)..(11)

<223> May be oxidized

<400> 29

Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser

1 5 10 15

Lys

<210> 30

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(9)..(9)

<223> May be oxidized

<400> 30

Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

1 5 10

<210> 31

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (2)..(2)

<223> May be oxidized

<400> 31

Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys

1 5 10

<210> 32

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May be oxidized

<400> 32

Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg

1 5 10

<210> 33

<211> 227

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> MOD_RES

<222>(8)..(8)

<223> Leu or Pro

<220>

<221> MOD_RES

<222> (173)..(173)

<223> Ala or Thr

<400> 33

Asp Lys Thr His Thr Cys Pro Xaa Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Xaa Thr Pro Pro

165 170 175

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

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 34

<211> 103

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 34

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp

100

<210> 35

<211> 93

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 35

Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr

1 5 10 15

Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile

20 25 30

Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly

35 40 45

Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala

50 55 60

Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly

65 70 75 80

His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr

85 90

<210> 36

<211> 102

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 36

Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys

1 5 10 15

Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp

20 25 30

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

35 40 45

Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu

50 55 60

Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr

65 70 75 80

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

85 90 95

Val Arg Val His Glu Lys

100

<210> 37

<211> 97

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 37

Pro Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala Thr

1 5 10 15

Val Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro

20 25 30

Pro Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu Ser Asn His

35 40 45

Thr Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu Arg

50 55 60

Asp Thr Gly Asn Tyr Thr Val Ile Leu Thr Asn Pro Ile Ser Lys Glu

65 70 75 80

Lys Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro Pro Gly Pro

85 90 95

gly

<210> 38

<211> 92

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 38

Phe Val Ala Phe Gly Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val

1 5 10 15

Gly Glu Arg Val Arg Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro

20 25 30

Glu Ile Lys Trp Tyr Lys Asn Gly Ile Pro Leu Glu Ser Asn His Thr

35 40 45

Ile Lys Ala Gly His Val Leu Thr Ile Met Glu Val Ser Glu Arg Asp

50 55 60

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

65 70 75 80

Gln Ser His Val Val Ser Leu Val Val Tyr Val Pro

85 90

<210> 39

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 39

Asp Lys Thr His Thr Cys Pro Pro Cys

15

<210> 40

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 40

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys

1 5 10

<210> 41

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 41

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys

1 5 10 15

<210> 42

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 42

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

<210> 43

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 43

Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

<210> 44

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 44

Asp Lys Thr His Thr Cys

15

<210> 45

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 45

Asp Lys Thr His Thr Cys Pro Leu Cys Pro Ala Pro

1 5 10

<210> 46

<211> 221

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 46

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

210 215 220

<210> 47

<211> 315

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 47

Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His

1 5 10 15

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

20 25 30

Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro

35 40 45

Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser

50 55 60

Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val

65 70 75 80

Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn

85 90 95

Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser

100 105 110

Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn

115 120 125

Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His

130 135 140

Lys Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met

145 150 155 160

Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp

165 170 175

Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys

180 185 190

Asn Ser Thr Phe Val Arg Val His Glu Asn Leu Ser Val Ala Phe Gly

195 200 205

Ser Gly Met Glu Ser Leu Val Glu Ala Thr Val Gly Glu Arg Val Arg

210 215 220

Ile Pro Ala Lys Tyr Leu Gly Tyr Pro Pro Pro Glu Ile Lys Trp Tyr

225 230 235 240

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

245 250 255

Val Leu Thr Ile Met Glu Val Ser Glu Arg Asp Thr Gly Asn Tyr Thr

260 265 270

Val Ile Leu Thr Asn Pro Ile Ser Lys Glu Lys Gln Ser His Val Val

275 280 285

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

290 295 300

Cys Pro Leu Cys Pro Ala Pro Glu Leu Leu Gly

305 310 315

<210> 48

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 48

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys

210

<210> 49

<211> 217

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 49

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Pro Cys

210 215

<210> 50

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 50

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys

210 215 220

<210> 51

<211> 440

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 51

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg

225 230 235 240

Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr

245 250 255

Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile

260 265 270

Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly

275 280 285

Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn Ala

290 295 300

Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly

305 310 315 320

His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile

325 330 335

Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly

340 345 350

Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly

355 360 365

Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys

370 375 380

Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys

385 390 395 400

Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly

405 410 415

Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser

420 425 430

Thr Phe Val Arg Val His Glu Lys

435 440

<210> 52

<211> 425

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 52

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly

210 215 220

Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met

225 230 235 240

Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn

245 250 255

Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp

260 265 270

Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile Ile Ser Asn

275 280 285

Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn

290 295 300

Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr

305 310 315 320

Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val

325 330 335

Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val

340 345 350

Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys

355 360 365

Lys Leu Val Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys

370 375 380

Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln

385 390 395 400

Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn

405 410 415

Ser Thr Phe Val Arg Val His Glu Lys

420 425

<210> 53

<211> 455

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 53

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro

245 250 255

Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu

260 265 270

Gly Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr

275 280 285

Val Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys

290 295 300

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

305 310 315 320

Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His

325 330 335

Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile

340 345 350

Asp Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu

355 360 365

Lys Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile

370 375 380

Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu

385 390 395 400

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

405 410 415

Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu

420 425 430

Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr

435 440 445

Phe Val Arg Val His Glu Lys

450 455

<210> 54

<211> 470

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 54

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

245 250 255

Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp Thr Gly Arg Pro Phe

260 265 270

Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly

275 280 285

Arg Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val

290 295 300

Thr Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg

305 310 315 320

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

325 330 335

Lys Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu

340 345 350

Tyr Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp

355 360 365

Val Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys

370 375 380

Leu Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp

385 390 395 400

Phe Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val

405 410 415

Asn Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu

420 425 430

Ser Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr

435 440 445

Thr Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe

450 455 460

Val Arg Val His Glu Lys

465 470

<210> 55

<211> 432

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 55

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

260 265 270

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

275 280 285

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

290 295 300

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

305 310 315 320

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

325 330 335

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

340 345 350

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

355 360 365

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

370 375 380

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

385 390 395 400

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

405 410 415

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

420 425 430

<210> 56

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 56

Ile Ile Trp Asp Ser Arg

15

<210> 57

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 57

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

1 5 10 15

Lys

<210> 58

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 58

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

1 5 10 15

Glu Leu Ser Val Gly Glu Lys

20

<210> 59

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 59

Thr Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser

1 5 10 15

Lys His Gln His Lys

20

<210> 60

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 60

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

<210> 61

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 61

Thr Asn Tyr Leu Thr His Arg

15

<210> 62

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be carboxymethylated

<400> 62

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

1 5 10 15

Lys

<210> 63

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 63

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

1 5 10 15

Glu Leu Ser Val Gly Glu Lys

20

<210> 64

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (10)..(10)

<223> May be oxidized

<400> 64

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 65

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (20)..(20)

<223> May be oxidized

<400> 65

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 66

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be oxidized

<400> 66

Thr Gln Ser Gly Ser Glu Met Lys

15

<210> 67

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(8)..(8)

<223> May be carboxymethylated

<220>

<221> OTHER_INDICATIONS

<222> (15)..(15)

<223> May be oxidized

<400> 67

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr

1 5 10 15

Lys

<210> 68

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 68

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 69

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(9)..(9)

<223> May be oxidized

<400> 69

Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

1 5 10

<210> 70

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (2)..(2)

<223> May be oxidized

<400> 70

Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys

1 5 10

<210> 71

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May be oxidized

<400> 71

Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr Arg

1 5 10

<210> 72

<211> 165

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 72

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys

1 5 10 15

Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu

20 25 30

Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys

35 40 45

Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu

50 55 60

Gly Leu Glu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile

65 70 75 80

Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu Met Ser Phe

85 90 95

Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg

100 105 110

Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe

115 120 125

Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser

130 135 140

Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys

145 150 155 160

Asp Lys Pro Arg Arg

165

<210> 73

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys

85 90 95

Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

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

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

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

435 440 445

Pro Gly Lys

450

<210> 74

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 74

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Thr Thr Ser Gln Asp Ser Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Ala Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Leu Gln Tyr Asp Tyr Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

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

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 75

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys

85 90 95

Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Gly Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

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

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

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

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

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

435 440 445

Pro Gly Lys

450

<210> 76

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 76

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Thr Ser Gln Asp Ser Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

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

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 77

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 77

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Thr Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys

85 90 95

Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

115 120 125

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

130 135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

195 200 205

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

210 215 220

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

245 250 255

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

260 265 270

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

275 280 285

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

290 295 300

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

305 310 315 320

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

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

340 345 350

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

355 360 365

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

370 375 380

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

385 390 395 400

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

405 410 415

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

420 425 430

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210> 78

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 78

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Thr Thr Ser Gln Asp Ser Asn Asn Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Ala Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Leu Gln Tyr Asp Tyr Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe

195 200 205

Asn Arg Gly Glu Cys

210

<210> 79

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 79

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Phe Tyr Cys

85 90 95

Ala Arg Leu Gly Asn Tyr Gly Gly Tyr Tyr Gly Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser

115 120 125

Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val

130 135 140

Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

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

165 170 175

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

180 185 190

Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala His Pro

195 200 205

Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp Cys Gly

210 215 220

Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys

245 250 255

Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln

260 265 270

Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln

275 280 285

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

290 295 300

Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg

305 310 315 320

Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

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

340 345 350

Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr

355 360 365

Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln

370 375 380

Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly

385 390 395 400

Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu

405 410 415

Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn

420 425 430

His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

435 440 445

<210> 80

<211> 200

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 80

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Gly Lys Val Thr Ile Thr Cys Lys Thr Ser Gln Asp Ser Asn Lys Tyr

20 25 30

Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile

35 40 45

His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro

65 70 75 80

Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro

100 105 110

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly

115 120 125

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn

130 135 140

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn

145 150 155 160

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser

165 170 175

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr

180 185 190

Cys Glu Ala Thr His Lys Thr Ser

195 200

<210> 81

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 81

Glu Leu Val Ile Pro Cys Arg

15

<210> 82

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 82

Leu Val Leu Asn Cys Thr Ala Arg

15

<210> 83

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (2)..(2)

<223> May be oxidized

<400> 83

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10

<210> 84

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 84

Glu Leu Val Ile Pro Cys Arg Glu Ile Gly Leu Leu Thr Cys Glu Ala

1 5 10 15

Thr Val Asn Gly His Leu Tyr Lys

20

<210> 85

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (26)..(26)

<223> May or may not be present

<400> 85

Leu Val Leu Asn Cys Thr Ala Arg Ser Asp Gln Gly Leu Tyr Thr Cys

1 5 10 15

Ala Ala Ser Ser Gly Leu Met Thr Lys Lys

20 25

<210> 86

<211> 28

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (28)..(28)

<223> May or may not be present

<400> 86

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Thr His

1 5 10 15

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

20 25

<210> 87

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 87

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

1 5 10 15

Lys

<210> 88

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 88

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

1 5 10 15

Glu Leu Ser Val Gly Glu Lys

20

<210> 89

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (10)..(10)

<223> May be oxidized

<400> 89

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 90

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (20)..(20)

<223> May be oxidized

<400> 90

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 91

<211> 54

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> OTHER_INDICATIONS

<222> (40)..(40)

<223> May be oxidized

<400> 91

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp

20 25 30

Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile

35 40 45

His Met Thr Glu Gly Arg

50

<210> 92

<211> 54

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> OTHER_INDICATIONS

<222> (50)..(50)

<223> May be oxidized

<400> 92

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp

20 25 30

Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile

35 40 45

His Met Thr Glu Gly Arg

50

<210> 93

<211> 8

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be oxidized

<400> 93

Thr Gln Ser Gly Ser Glu Met Lys

15

<210> 94

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (15)..(15)

<223> May be oxidized

<400> 94

Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met Thr

1 5 10 15

Lys

<210> 95

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 95

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10

<210> 96

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 96

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

1 5 10 15

Glu Leu Ser Val Gly Glu Lys

20

<210> 97

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<223> See description as filed for detailed description

substitutions and preferred embodiments

<400> 97

Thr Asn Tyr Leu Thr His Arg

15

<210> 98

<211> 54

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 98

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Asp

20 25 30

Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile

35 40 45

His Met Thr Glu Gly Arg

50

<210> 99

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 99

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg

20

<210> 100

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May or may not be present

<400> 100

Arg Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

1 5 10

<210> 101

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May or may not be present

<400> 101

Lys Gly Phe Ile Ile Ser Asn Ala Thr Tyr Lys

1 5 10

<210> 102

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May or may not be present

<400> 102

Lys Leu Val Leu Asn Cys Thr Ala Arg

15

<210> 103

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(1)

<223> May or may not be present

<400> 103

Lys Asn Ser Thr Phe Val Arg

15

<210> 104

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> OTHER_INDICATIONS

<222> (1)..(60)

<223> This sequence can span 1-15 repeating 1s

Gly Gly Gly Ser.

<400> 104

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

35 40 45

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

50 55 60

<210> 105

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (7)..(21)

<223> This area can cover 1-5 repeated "Pro Pro Cys" units.

<400> 105

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys Pro

1 5 10 15

Pro Cys Pro Pro Cys

20

<210> 106

<211> 226

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> OTHER_INDICATIONS

<222> (212)..(226)

<223> This area can cover 1-5 repeated "Pro Pro Cys" units.

<400> 106

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Asp Lys Thr

195 200 205

His Thr Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys Pro Pro Cys Pro

210 215 220

Pro Cys

225

<210> 107

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 107

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 108

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 108

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 109

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 109

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40 45

<210> 110

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<400> 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

50 55 60

<210> 111

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 111

Gly Gly Gly Gly Ser

15

<210> 112

<211> 485

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> OTHER_INDICATIONS

<222> (206)..(280)

<223> This area can cover 1-15 repeat units

Gly Gly Gly Gly Ser.

<400> 112

Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu

1 5 10 15

Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val

20 25 30

Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr

35 40 45

Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe

50 55 60

Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu

65 70 75 80

Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg

85 90 95

Gln Thr Asn Thr Ile Ile Asp Val Val Leu Ser Pro Ser His Gly Ile

100 105 110

Glu Leu Ser Val Gly Glu Lys Leu Val Leu Asn Cys Thr Ala Arg Thr

115 120 125

Glu Leu Asn Val Gly Ile Asp Phe Asn Trp Glu Tyr Pro Ser Ser Lys

130 135 140

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

145 150 155 160

Ser Glu Met Lys Lys Phe Leu Ser Thr Leu Thr Ile Asp Gly Val Thr

165 170 175

Arg Ser Asp Gln Gly Leu Tyr Thr Cys Ala Ala Ser Ser Gly Leu Met

180 185 190

Thr Lys Lys Asn Ser Thr Phe Val Arg Val His Glu Lys Gly Gly Gly

195 200 205

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

210 215 220

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

245 250 255

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

260 265 270

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

275 280 285

Glu Met Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg

290 295 300

Glu Leu Val Ile Pro Cys Arg Val Thr Ser Pro Asn Ile Thr Val Thr

305 310 315 320

Leu Lys Lys Phe Pro Leu Asp Thr Leu Ile Pro Asp Gly Lys Arg Ile

325 330 335

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

340 345 350

Glu Ile Gly Leu Leu Thr Cys Glu Ala Thr Val Asn Gly His Leu Tyr

355 360 365

Lys Thr Asn Tyr Leu Thr His Arg Gln Thr Asn Thr Ile Ile Asp Val

370 375 380

Val Leu Ser Pro Ser His Gly Ile Glu Leu Ser Val Gly Glu Lys Leu

385 390 395 400

Val Leu Asn Cys Thr Ala Arg Thr Glu Leu Asn Val Gly Ile Asp Phe

405 410 415

Asn Trp Glu Tyr Pro Ser Ser Lys His Gln His Lys Lys Leu Val Asn

420 425 430

Arg Asp Leu Lys Thr Gln Ser Gly Ser Glu Met Lys Lys Phe Leu Ser

435 440 445

Thr Leu Thr Ile Asp Gly Val Thr Arg Ser Asp Gln Gly Leu Tyr Thr

450 455 460

Cys Ala Ala Ser Ser Gly Leu Met Thr Lys Lys Asn Ser Thr Phe Val

465 470 475 480

Arg Val His Glu Lys

485

<210> 113

<211> 211

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

polypeptide

<220>

<221> MOD_RES

<222> (157)..(157)

<223> Any amino acid

<400> 113

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

1 5 10 15

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

20 25 30

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Xaa Thr Pro Pro

145 150 155 160

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

165 170 175

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Pro Ser Cys Ser Val Met

180 185 190

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

195 200 205

Pro Gly Lys

210

<210> 114

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (3)..(3)

<223> May be oxidized

<400> 114

Ile Ile Trp Asp Ser Arg Lys

15

<210> 115

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (4)..(4)

<223> May be oxidized

<400> 115

Arg Ile Ile Trp Asp Ser Arg

15

<210> 116

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 116

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

1 5 10

<210> 117

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(9)..(9)

<223> May be oxidized

<400> 117

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

1 5 10 15

<210> 118

<211> 26

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (2)..(2)

<223> May be oxidized

<220>

<221> OTHER_INDICATIONS

<222> (4)..(4)

<223> May be carboxymethylated

<220>

<221> OTHER_INDICATIONS

<222>(7)..(7)

<223> May be carboxymethylated

<400> 118

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

1 5 10 15

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25

<210> 119

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222>(9)..(9)

<223> May be oxidized

<400> 119

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

1 5 10 15

<210> 120

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 120

Ser Asp Thr Gly Arg Pro Phe Val Glu Met

1 5 10

<210> 121

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 121

Val Thr Ser Pro Asn Ile Thr Val Thr Leu Lys

1 5 10

<210> 122

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<400> 122

Asn Ser Thr Phe Val Arg

15

<210> 123

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (14)..(14)

<223> May or may not be present

<400> 123

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10

<210> 124

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Artificial sequence description: Synthetic

peptide

<220>

<221> OTHER_INDICATIONS

<222> (4)..(4)

<223> May be oxidized

<400> 124

Ile Ile Ile Trp Asp Ser Arg

15

<---

Claims (18)

1. An isolated synthetic or recombinant polypeptide with protease activity of an immunoglobulin-degrading enzyme, Streptococcus pyogenes (IdeS), containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. 2. The polypeptide of claim 1, wherein said polypeptide is capable of cleaving the target Fc domain protein into fragments, wherein at least one fragment contains said Fc domain. 3. The polypeptide of claim 2, wherein the cleavage recognition site comprises glycine-glycine. 4. The polypeptide of claim 2, wherein said target protein is aflibercept. 5. The polypeptide of claim 2, wherein said fragments comprise a Fab fragment and an Fc fragment. 6. An isolated nucleic acid molecule encoding a polypeptide according to claim 1. 7. An expression vector containing said nucleic acid molecule according to claim 6. 8. The vector of claim 7, wherein said nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. 9. The vector of claim 7, wherein said vector is a plasmid. 10. An isolated nucleic acid molecule encoding a polypeptide with protease activity of an immunoglobulin-degrading enzyme, Streptococcus pyogenes (IdeS), containing an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 , SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16. 11. An isolated synthetic or recombinant polypeptide with protease activity of an immunoglobulin-degrading enzyme, Streptococcus pyogenes (IdeS), containing the full-length amino acid sequence shown in SEQ ID NO: 1, with asparagine residues at one or more positions 87, 130, 182, or 274, mutated to an amino acid other than asparagine, wherein said amino acid is aspartic acid, leucine, or arginine. 12. The polypeptide of claim 11, wherein said asparagine residues at two or more positions 87, 130, 182, or 274 are mutated to an amino acid other than asparagine. 13. The polypeptide of claim 12, wherein said asparagine residues at three or more positions 87, 130, 182, or 274 are mutated to an amino acid other than asparagine. 14. The polypeptide of claim 11 wherein said asparagine residues at positions 87, 130, 182 and 274 are mutated to an amino acid other than asparagine. 15. An isolated nucleic acid molecule encoding a polypeptide according to claim 11. 16. An expression vector containing the nucleic acid molecule according to claim 15. 17. The vector of claim 16, wherein said nucleic acid molecule is operably linked to an expression control sequence capable of directing its expression in a host cell. 18. The vector of claim 16, wherein said vector is a plasmid.

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