How the product is refined

Abstract

Provided herein are embodiments directed to affinity chromatography purification and separation of contaminant species, including HCPs (host cell proteins), from desired molecular and chemical species.

Description

[Technical field]

[0001] Sequence Listing This application is submitted with an electronic sequence listing, which is provided as a file named KDIAK.141WO.xml, created on February 8, 2023, and having a size of 843,266 bytes. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

[0002] INCORPORATION BY REFERENCE OF PRIORITY APPLICATION This application claims priority to U.S. Provisional Application No. 63 / 267810, filed February 10, 2022.

[0003] All applications for which a foreign or domestic priority claim is identified in the Application Data Sheet filed with this application are hereby incorporated by reference under 37 CFR 1.57.

[0004] The present disclosure relates to methods for purifying antibodies and other cellular products. [Background technology]

[0005] Unit operations involved in processing and purifying molecular species from cell cultures include preferential separation of desired and undesired molecular species. In particular, chromatography and Protein A chromatography allow for preferential separation of certain species based on various molecular binding interactions compared to any wash or flow-through buffer solutions. Optimization of unit operations in this process can increase the overall purity and percent yield of the desired molecular species while reducing impurities. Summary of the Invention [Problem to be solved by the invention]

[0006] Provided herein are methods for purifying a product by administration of a chaotropic agent.

[0007] Some embodiments provided herein provide novel purification methods for use in process steps involving purification of a product, thereby allowing for improved product purity and composition. In some embodiments, the product can be a protein product. In some embodiments, the purification comprises chromatography. In some embodiments, the chromatography can be Protein A or Protein G. In some embodiments, the chaotropic agent can comprise one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0008] Provided herein are methods for purifying protein products using affinity chromatography. Some embodiments provided herein make it possible to overcome certain limitations of the prior art by providing novel methods for purifying protein products after harvesting cell cultures. In some embodiments, the protein product is an antibody. In some embodiments, the protein product is an anti-VEGF antibody. In some embodiments, the anti-VEGF antibody of the present disclosure can be an anti-VEGF antibody conjugate (e.g., KSI-301, KSI-501) or an anti-VEGF protein conjugate that includes a polymer moiety that extends the half-life (e.g., ocular half-life) of the antibody or protein when administered to a subject. In some embodiments, the anti-VEGF antibody can be a VEGF antibody conjugate (e.g., KSI-301, KSI-501) or an anti-VEGF protein conjugate that includes a polymer moiety that extends the half-life (e.g., ocular half-life) of the antibody or protein when administered to a subject. Provided herein are buffer reagent compositions that can be used to purify protein products.

[0009] In some embodiments, provided herein are methods for purifying a product and reducing impurities from a load solution containing a protein and one or more impurities by passing the load solution through an affinity chromatography matrix, followed by at least one wash solution comprising a chaotropic salt, and collecting the protein using an elution solution.

[0010] In some embodiments, provided herein is a method of separating impurities in an eluate comprising a protein of interest, the method comprising loading the eluate comprising the protein of interest onto an affinity chromatography matrix, and washing the affinity chromatography matrix with one or more buffer solutions comprising one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0011] In some embodiments, provided herein is a method of producing a product using affinity chromatography, comprising loading an eluent containing a protein of interest onto an affinity chromatography matrix, then performing a first wash of the affinity chromatography matrix with a first buffer comprising sodium phosphate and a salt, and then performing a second wash of the affinity chromatography matrix with a second buffer comprising a chaotropic agent.

[0012] In some embodiments, provided herein is a method of producing a product using affinity chromatography, the method comprising loading an eluent containing a protein of interest onto an affinity chromatography matrix, performing a first wash with a first buffer comprising Tris and a salt, and performing a second wash with a second buffer comprising Tris and a chaotropic agent, wherein the chaotropic agent of the second buffer is not the same as the salt contained in the first buffer.

[0013] In some embodiments, provided herein is a method of producing a product, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0014] In some embodiments, provided herein is a method of producing a product, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, applying a buffer solution comprising a chaotropic salt to the affinity chromatography matrix, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0015] In some embodiments, a method of producing a product includes harvesting a complex protein, said complex protein comprising an antibody bound to a complex polymer; Provided herein is a method comprising loading the complex protein onto an affinity chromatography matrix, wherein the affinity chromatography matrix binds to the complex protein, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the complex protein, and collecting an eluate, wherein the eluate comprises the complex protein.

[0016] In some embodiments, provided herein is a method of producing a product, the method comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, then eluting and collecting an eluate, the eluate containing the target protein of interest, and removing viral contaminants from the eluate. In some embodiments of the methods provided herein, removing viral contaminants from the eluate comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and / or diafiltration (DF).

[0017] In some embodiments of the methods provided herein, a method of manufacturing a product is provided, comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer containing a chaotropic salt, removing the chaotropic salt, and eluting to collect an eluate, the eluate containing the target protein of interest. In some embodiments of the methods provided herein, the eluate is further combined with an acceptable pharmaceutical excipient to form a pharmaceutical composition. In some embodiments of the methods provided herein, a buffer solution is added to the pharmaceutical composition. In some embodiments of the methods provided herein, a preservative solution is added to the pharmaceutical composition. In some embodiments of the methods provided herein, the pharmaceutical composition is further purified for intravitreal injection.

[0018] In some embodiments, a method for processing a product is provided herein. The method includes loading an eluate onto an affinity chromatography matrix. The method further includes washing with a wash buffer containing a chaotropic salt and collecting an eluate, the concentration of the chaotropic salt is increased from a first concentration to a second concentration, the eluate is collected into at least one fraction, and the at least one fraction includes a product of interest.

[0019] In some embodiments, provided herein is a method for producing a product comprising: collecting a load solution, the load solution comprising a protein of interest; loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest; washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt; eluting and collecting an eluate, the eluate comprising the target protein of interest; and removing viral contaminants from the eluate.

[0020] In some embodiments, provided herein is a method of producing a product comprising loading an eluate onto an affinity chromatography matrix, washing with a first wash buffer, washing with a second wash buffer comprising a chaotropic salt, washing with a third wash buffer, where the third wash buffer removes the chaotropic salt, and eluting with an elution buffer, where the eluate comprises a protein product. In some embodiments, the first wash buffer comprises 50 mM sodium phosphate. In some embodiments, the first wash buffer comprises 50 mM sodium phosphate. The wash buffer further comprises 250 mM sodium chloride. In some embodiments, methods are provided herein, wherein the first wash buffer comprises Tris and salt. In some embodiments, methods are provided herein, further comprising removing viral contaminants from the eluate.

[0021] In some embodiments, methods are provided herein, wherein removing viral contaminants comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and diafiltration (DF). In some embodiments, the eluent comprises a protein of interest. In some embodiments, the protein of interest is an antibody. In some embodiments, the antibody is further conjugated to a polymer to form an antibody conjugate. In some embodiments, the antibody conjugate comprises a bispecific antibody. In some embodiments, the bispecific antibody comprises an anti-VEGF binding moiety and an anti-IL-6 binding moiety.

[0022] In some embodiments, the antibody conjugate has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, and the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains. In the above structure, PC refers to the following structure: [ka] wherein the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0023] In some embodiments, provided herein is a method of producing a product comprising: harvesting a cell culture supernatant, wherein the cell culture supernatant comprises a protein of interest; then treating the cell culture supernatant with an eluent, wherein the eluent comprises the protein of interest; then loading the eluent onto an affinity chromatography matrix and washing with a first wash buffer comprising Tris or sodium phosphate, then washing with a second wash buffer comprising a chaotropic salt; eluting with an elution buffer, wherein the eluate comprises the protein product; then inactivating viral contaminants present in the eluate with a low pH viral buffer to generate a viral inactivated eluate; filtering the viral inactivated eluate; performing at least one round of ion exchange chromatography on the viral inactivated eluate; and filtering the viral inactivated eluate to obtain a retentate, wherein the retentate comprises the protein of interest.

[0024] In some embodiments, the cell culture supernatant is produced in a bioreactor using an animal component-free cell culture. In some embodiments, processing the cell culture supernatant comprises harvesting a cell product from the cell culture. In some embodiments, the cell culture is clarified to remove cells and cell debris. In some embodiments, the eluent comprises the clarified cell culture supernatant.

[0025] In some embodiments, provided herein is a method for purifying a protein using affinity chromatography, comprising contacting a load solution with a medium, said medium being an affinity chromatography matrix that binds a protein of interest, then washing the medium with a buffer solution comprising a chaotropic agent, said chaotropic agent being a salt, and contacting the washed medium with an elution solution under conditions suitable for eluting the protein of interest.

[0026] In some embodiments, provided herein is a method for producing a product comprising applying a solution comprising a protein of interest to an affinity chromatography matrix, washing the affinity chromatography matrix with a first buffer, then washing the affinity chromatography matrix with a second buffer comprising a chaotropic agent, then washing the affinity chromatography matrix with a third buffer to remove the chaotropic agent, then eluting with an elution buffer, wherein the eluate is collected, and wherein the eluate comprises the protein product.

[0027] In some embodiments, provided herein is a system for protein purification comprising a column having a first antigen binding protein bound thereto, a phosphate wash buffer comprising sodium phosphate and a salt, an intermediate wash buffer comprising Tris, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate.

[0028] In some embodiments, provided herein is a system for protein purification, the system comprising a column to which a first antigen binding protein is bound, a first Tris wash buffer comprising Tris and a salt, an intermediate Tris wash buffer, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate. In some embodiments, the column comprises a ligand for affinity chromatography. In some embodiments, the ligand comprises Protein A or Protein G. In some embodiments, the first wash buffer comprising sodium phosphate and a salt has a pH of 5.5 to 9.5. In some embodiments, the phosphate wash buffer comprising sodium phosphate and a salt comprises about 50 mM sodium phosphate. In some embodiments, the phosphate wash buffer comprising sodium phosphate and a salt comprises about 250 mM sodium chloride. In some embodiments, the first Tris wash buffer comprises about 50 mM Tris. In some embodiments, the first Tris wash buffer further comprises about 250 mM sodium chloride. In some embodiments, the intermediate Tris wash buffer comprises about 50 mM Tris. In some embodiments, the pH of the first Tris wash buffer is about 7.2. In some embodiments, the pH of the second wash buffer is about 7.8. In some embodiments, the concentration of magnesium chloride in the second wash buffer is about 2.8 M. In some embodiments, the concentration of sodium formate in the elution buffer is 10 mM.

[0029] In some embodiments, a system for antibody purification is described herein, the system comprising: a column having a Protein A resin bound to an antibody, the antibody comprising a light chain and a heavy chain that are at least one of SEQ ID NOs: 91-93 and 28-30 and at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, respectively; a chaotropic wash buffer comprising a chaotropic salt; and an elution buffer comprising sodium formate.

[0030] In some embodiments, the protein of interest is a bispecific antibody. In some embodiments, the bispecific antibody is specific for VEGF and IL-6. In some embodiments, the protein of interest is an antibody complex. In some embodiments, the affinity chromatography matrix is ​​a Protein A chromatography matrix. In some embodiments, the chaotropic agent in the buffer solution comprises one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts. In some embodiments, the concentration of one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts is 0.05 to 3.5 M. In some embodiments, the buffer solution further comprises Tris. In some embodiments, the concentration of Tris in the buffer solution is at least 5 mM. In some embodiments, the pH of the buffer solution is greater than 5.5. In some embodiments, the eluate further comprises viral impurities.

[0031] In some embodiments of the methods described herein, the methods further comprise removing viral impurities. In some embodiments of the methods described herein, the methods further comprise inactivating the viral impurities. In some embodiments of the methods described herein, the methods further comprise washing the load-loaded affinity chromatography matrix with a pre-wash buffer solution prior to washing with the buffer solution. In some embodiments of the methods described herein, the methods further comprise washing the eluent-loaded affinity chromatography matrix with a post-wash buffer solution after washing with the buffer solution. In some embodiments, the pre-wash buffer solution comprises sodium phosphate. In some embodiments, the pre-wash buffer solution comprises Tris and salt. In some embodiments, the antibody complex has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC has the structure: [ka] where the wavy line indicates the point of attachment to the remainder of the polymer and X is a) -OR (R is -H, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0032] In some embodiments, the antibody complex comprises an anti-VEGF antibody complex comprising an anti-VEGF-A light chain and an anti-VEGF-A heavy chain, wherein the anti-VEGF-A antibody heavy chain comprises CDRH1 which is CDRH1 of SEQ ID NO: 172, CDRH2 which is CDRH2 of SEQ ID NO: 173, and CDRH3 which is CDRH3 of SEQ ID NO: 174, and the anti-VEGF-A antibody light chain comprises CDRL1 which is CDRL1 of SEQ ID NO: 199, CDRL2 which is CDRL2 of SEQ ID NO: 200, and CDRL3 which is CDRL3 of SEQ ID NO: 201.

[0033] In some embodiments, the anti-VEGF antibody conjugate comprises an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30, and the antibody is conjugated to the polymer at C449.

[0034] In some embodiments, the target protein of interest is produced by a cell culture. In some embodiments, the cell culture comprises CHO cells. In some embodiments, the methods described herein further comprise washing the affinity chromatography matrix loaded with the eluent with a post-wash buffer solution after washing with the buffer solution. In some embodiments, washing the affinity chromatography matrix with a buffer solution removes nucleic acids, endotoxins, antifoaming agents, or other small molecules other than the target protein of interest. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes impurities while leaving the target protein of interest bound to the affinity chromatography matrix. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes host cell proteins other than the target protein of interest. In some embodiments, the addition of a chaotropic agent to the buffer solution does not elute the target protein of interest. In some embodiments of the methods described herein, the method further comprises one or more of viral inactivation, tangential flow filtration, diafiltration, ultrafiltration, ion exchange chromatography, or viral reduction filtration. In some embodiments, the eluate is produced in a bioreactor using an animal component-free cell culture. In some embodiments, the product is a protein of interest. In some embodiments, the impurities include host cell protein impurities. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 is a diagram illustrating a general protocol for some embodiments of the collection and purification of a protein of interest. [Diagram 2] FIG. 2 is a diagram illustrating a typical protocol for harvesting and purifying a protein of interest. [Diagram 3] FIG. 3 illustrates a typical protocol for column chromatography according to an embodiment of the present disclosure. [Figure 4] FIG. 4 shows a series of chromatographic profiles performed with different wash buffer solutions. [Diagram 5] FIG. 5 shows a series of chromatographic profiles performed with different wash buffer solutions. [Figure 6] FIG. 6 shows a series of chromatographic profiles run with different wash buffer solutions. [Figure 7] FIG. 7 shows a series of chromatographic profiles run with different wash buffer solutions. [Figure 8] FIG. 8 depicts the amino acid sequences of several embodiments of anti-VEGF antibodies. [Figure 9] 9 depicts several embodiments of an IL-6-VEGF Trap fusion protein, in which the VEGF Trap domain is located at the N-terminus immediately preceding the variable domain (left) or between the Fab and hinge regions of an antibody (right). [Figure 10] FIG. 10 shows the sequence listing of VEGF_trap_variant_1, VEGF_trap_variant_2, and VEGF_trap_variant_3. [Figure 11] Figure 11 depicts an embodiment of an anti-IL-6 heavy chain variable region sequence. The CDRs are underlined. [Figure 12]Figure 12 shows various embodiments of VEGF Trap sequences. Sections that differ between sequences are bold and underlined. Figure 12 also shows some embodiments of linker (GS) sequences. It may be present as a double repeat Gly-Gly-Gly-Gly-Ser linker (GS). [Figure 13] Figure 13 shows several embodiments of heavy chain sequences of anti-IL-6 molecules. The CDRs are underlined. [Figure 14] Figure 14 shows several embodiments of heavy chain sequences of anti-IL-6 molecules. The CDRs are underlined. [Figure 15] Figure 15 shows several embodiments of the light chain sequence of an anti-IL-6 molecule. The CDRs are underlined. [Figure 16-1] Figure 16 shows several embodiments of heavy chain sequences of anti-IL-6 molecules. The CDRs are underlined. [Figure 16-2] Figure 16 shows several embodiments of heavy chain sequences of anti-IL-6 molecules. The CDRs are underlined. [Figure 17A] FIG. 17A shows some embodiments of CDR combinations. [Figure 17B] FIG. 17B shows some embodiments of CDR combinations. [Figure 18] 18 depicts several embodiments of VEGFR-Fc sequence variants, with sections that differ between the sequences in bold and underlined. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] In some embodiments, methods and systems are provided herein for reducing impurities by preferentially separating desired and undesired molecular species using chromatography.

[0037] In some embodiments, a method of purifying a product using affinity chromatography, in some embodiments, the method includes loading an eluent onto an affinity chromatography matrix, the affinity chromatography matrix binding a protein of interest, and washing the affinity chromatography matrix with a buffer solution that includes a chaotropic agent.

[0038] In some embodiments, the affinity chromatography matrix is ​​purified by passing a load solution containing the protein and one or more impurities through the affinity chromatography matrix, followed by at least one wash solution containing a chaotropic salt, and collecting the protein using an elution solution. A method for purifying the product from the load solution to reduce impurities.

[0039] In some embodiments, a method for separating impurities in an eluate containing a protein of interest, in some embodiments, the method comprises loading an eluate containing the protein of interest onto an affinity chromatography matrix and washing the affinity chromatography matrix with one or more buffer solutions comprising magnesium or a magnesium salt.

[0040] In some embodiments, a method of producing a product using affinity chromatography, in some embodiments, the method includes loading an eluent containing a protein of interest onto an affinity chromatography matrix, a first wash of the affinity chromatography matrix with a first buffer comprising sodium phosphate and a salt, and a second wash of the affinity chromatography matrix with a second buffer comprising a chaotropic agent.

[0041] In some embodiments, a method of producing a product using affinity chromatography, in some embodiments, the method includes loading an eluent containing a protein of interest onto an affinity chromatography matrix, performing a first wash with a first buffer containing Tris and a salt, and performing a second wash with a second buffer containing Tris and a chaotropic agent, wherein the chaotropic agent of the second buffer is not the same as the salt contained in the first buffer.

[0042] In some embodiments, a method of producing a product includes (i) collecting a load solution, the load solution comprising a protein of interest, (ii) loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, (iii) washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, (iv) eluting the bound protein of interest, and (v) collecting an eluate, the eluate containing the protein of interest.

[0043] In some embodiments, a method of producing a product includes collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, applying a buffer solution comprising a chaotropic salt to the affinity chromatography matrix, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0044] In some embodiments, provided herein is a method of producing a product, the method comprising: collecting a complex protein, the complex protein comprising an antibody bound to a complex polymer, loading the complex protein onto an affinity chromatography matrix, the affinity chromatography matrix binding to the complex protein, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the complex protein, and collecting the eluate, the eluate comprising the complex protein.

[0045] In some embodiments, the method is a method for producing a product. The method includes washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, eluting and collecting an eluate, the eluate containing the target protein of interest, and removing viral contaminants from the eluate, in some embodiments, removing viral contaminants from the eluate includes one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and / or diafiltration (DF).

[0046] In some embodiments, the method is a method for producing a product. In some embodiments, the method includes washing an affinity chromatography matrix bound to a target protein of interest with a buffer containing a chaotropic salt, removing the chaotropic salt, and eluting and collecting an eluate, the eluate containing the target protein of interest. In some embodiments, the eluate is further combined with an acceptable pharmaceutical excipient to form a pharmaceutical composition. In some embodiments, a buffer solution is added to the pharmaceutical composition. In some embodiments, a preservative solution is added to the pharmaceutical composition. In some embodiments, the pharmaceutical composition is further purified for intravitreal injection.

[0047] In some embodiments, a method of producing a product includes collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding the protein of interest, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting and collecting an eluate, the eluate comprising the target protein of interest, and removing viral contaminants from the eluate.

[0048] In some embodiments, the method comprises loading an eluate onto an affinity chromatography matrix, washing with a first wash buffer, washing with a second wash buffer comprising a chaotropic salt, washing with a third wash buffer, removing the chaotropic salt with the third wash buffer, and eluting with an elution buffer, collecting an eluate, the eluate comprising a protein product. In some embodiments, the first wash buffer comprises 50 mM sodium phosphate. In some embodiments, the first wash buffer further comprises 250 mM sodium chloride. In some embodiments, the first wash buffer comprises Tris and salt. In some embodiments, the method further comprises removing viral contaminants from the eluate. In some embodiments, removing viral contaminants comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and diafiltration (DF). In some embodiments, the eluate comprises a protein of interest. In some embodiments, the protein of interest is an antibody. In some embodiments, the antibody is further conjugated to a polymer to form an antibody conjugate. In some embodiments, the antibody conjugate has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, the antibody conjugate comprises a bispecific antibody. In some embodiments, the bispecific antibody comprises an anti-VEGF binding moiety and an anti-IL-6 binding moiety.

[0049] In some embodiments, the antibody conjugate has the structure of formula (II): [ka] where "n." is an integer from 1 to 50 and "ni" is an integer from 1 to 50; each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody through the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, the antibody conjugate comprises a bispecific antibody. In some embodiments, the bispecific antibody comprises an anti-VEGF binding moiety and an anti-IL-6 binding moiety.

[0050] In some embodiments, a method for producing a product is provided herein, the method comprising: harvesting a cell culture supernatant, the cell culture supernatant comprising a protein of interest; treating the cell culture supernatant with an eluent, the eluent comprising the protein of interest; loading the eluent onto an affinity chromatography matrix; washing with a first wash buffer comprising Tris or sodium phosphate; washing with a second wash buffer comprising a chaotropic salt; eluting with an elution buffer, the eluate comprising the protein product; inactivating viral contaminants present in the eluate with a low pH viral buffer to produce a viral inactivated eluate; filtering the viral inactivated eluate; subjecting the viral inactivated eluate to at least one round of ion exchange chromatography; and filtering the viral inactivated eluate to obtain a retentate, the retentate comprising the protein of interest. In some embodiments, the cell culture supernatant is produced in a bioreactor using an animal component-free cell culture. In some embodiments, processing the cell culture supernatant comprises harvesting a cell product from the cell culture. In some embodiments, the cell culture is clarified to remove cells and cell debris. In some embodiments, the eluent comprises the clarified cell culture supernatant.

[0051] In some embodiments, a method of purifying a protein using affinity chromatography includes contacting a load solution with a medium, the medium being an affinity chromatography matrix that binds a protein of interest, washing the medium with a buffer solution that includes a chaotropic agent, the chaotropic agent being a salt, and contacting the washed medium with an elution solution under conditions suitable for eluting the protein of interest.

[0052] In some embodiments, a method of producing a product, in some embodiments, the method comprises applying a solution comprising a protein of interest to an affinity chromatography matrix, washing the affinity chromatography matrix with a first buffer, washing the affinity chromatography matrix with a second buffer comprising a chaotropic agent, washing the affinity chromatography matrix with a third buffer to remove the chaotropic agent, and eluting with an elution buffer, collecting an eluate, wherein the eluate comprises the protein product.

[0053] In some embodiments is a system for protein purification, hi some embodiments, the system comprises a column having a first antigen binding protein bound thereto, a phosphate wash buffer comprising sodium phosphate and a salt, an intermediate wash buffer comprising Tris, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate.

[0054] In some embodiments, the system is for protein purification. In some embodiments, the system includes a column to which a first antigen binding protein is bound, a first Tris wash buffer comprising Tris and a salt, an intermediate Tris wash buffer, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate. In some embodiments, the column includes a ligand for affinity chromatography. In some embodiments, the ligand includes Protein A or Protein G. In some embodiments, the first wash buffer comprising sodium phosphate and a salt has a pH of 5.5 to 9.5. In some embodiments, the phosphate wash buffer comprising sodium phosphate and a salt has a pH of about 50 mM. In some embodiments, the phosphate wash buffer comprising sodium phosphate and salt comprises about 250 mM sodium chloride. In some embodiments, the first Tris wash buffer comprises about 50 mM Tris. In some embodiments, the first Tris wash buffer further comprises about 250 mM sodium chloride. In some embodiments, the intermediate Tris wash buffer comprises about 50 mM Tris. In some embodiments, the pH of the first Tris wash buffer is about 7.2. In some embodiments, the pH of the second wash buffer is about 7.8. In some embodiments, the concentration of magnesium chloride in the second wash buffer is about 2.8 M. In some embodiments, the concentration of sodium formate in the elution buffer is 10 mM.

[0055] Some embodiments are systems for antibody purification. In some embodiments, the system includes a column having a Protein A resin bound to an antibody, the antibody comprising a light chain and a heavy chain, each of which is at least one of SEQ ID NOs: 91-93 and 28-30 and at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262; a chaotropic wash buffer comprising a chaotropic salt; and an elution buffer comprising sodium formate. In some embodiments, the protein of interest is a bispecific antibody. In some embodiments, the bispecific antibody is specific for VEGF and IL-6. In some embodiments, the bispecific antibody is OG2072. In some embodiments, the protein of interest is an antibody complex. In some embodiments, the affinity chromatography matrix is ​​a Protein A chromatography matrix. In some embodiments, the chaotropic agent in the buffer solution comprises one or more of lithium, a lithium salt, magnesium, a magnesium salt, calcium, a calcium salt, guanidinium, and / or a guanidinium salt. In some embodiments, the concentration of one or more of lithium, lithium salts, magnesium, magnesium salts, calcium, calcium salts, guanidinium, and / or guanidinium salts is 0.05-3.5M each. In some embodiments, the buffer solution further comprises Tris. In some embodiments, the concentration of Tris in the buffer solution is at least 5 mM. In some embodiments, the pH of the buffer solution is greater than 5.5. In some embodiments, the elution solution further comprises viral impurities. In some embodiments, the method further comprises removing the viral impurities. In some embodiments, the method further comprises inactivating the viral impurities. In some embodiments, the method further comprises washing the affinity chromatography matrix loaded with the load solution with a pre-wash buffer solution prior to washing with the buffer solution.In some embodiments, the method further comprises, after washing with the buffer solution, washing the affinity chromatography matrix loaded with the eluent with a post-wash buffer solution. In some embodiments, the pre-wash buffer solution comprises sodium phosphate. In some embodiments, the pre-wash buffer solution comprises Tris and salt. In some embodiments, the antibody complex has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, the antibody complex comprises an anti-VEGF antibody complex comprising an anti-VEGF-A light chain and an anti-VEGF-A heavy chain, wherein the anti-VEGF-A antibody heavy chain comprises CDRH1 which is CDRH1 of SEQ ID NO: 172, CDRH2 which is CDRH2 of SEQ ID NO: 173, and CDRH3 which is CDRH3 of SEQ ID NO: 174, and the anti-VEGF-A antibody light chain comprises CDRL1 which is CDRL1 of SEQ ID NO: 199, CDRL2 which is CDRL2 of SEQ ID NO: 200, and CDRL3 which is CDRL3 of SEQ ID NO: 201. In some embodiments, the anti-VEGF antibody conjugate comprises an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30, and the antibody is conjugated to the polymer at C449.

[0056] In some embodiments, the target protein of interest is produced by a cell culture. In some embodiments, the cell culture comprises CHO cells. In some embodiments, the method further comprises washing the affinity chromatography matrix loaded with the eluent with a post-wash buffer solution after washing with the buffer solution. In some embodiments, washing the affinity chromatography matrix with a buffer solution removes nucleic acids, endotoxins, antifoaming agents, or other small molecules other than the target protein of interest. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes impurities while leaving the target protein of interest bound to the affinity chromatography matrix. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes host cell proteins other than the target protein of interest. In some embodiments, the addition of a chaotropic agent to the buffer solution does not elute the target protein of interest. In some embodiments, the method further comprises one or more of viral inactivation, tangential flow filtration, diafiltration, ultrafiltration, ion exchange chromatography, or viral reduction filtration. In some embodiments, the eluate is produced in a bioreactor using an animal component-free cell culture. In some embodiments, the product is a protein of interest. In some embodiments, the impurities comprise host cell protein impurities. In some embodiments, the first wash buffer comprises 10 mM sodium phosphate. In some embodiments, the first wash buffer comprises a phosphate-based species. In some embodiments, the first wash buffer further comprises 50 mM sodium chloride.

[0057] In some embodiments, the method is for processing a product. In some embodiments, the method further comprises loading the eluent onto an affinity chromatography matrix and washing with a wash buffer comprising a chaotropic salt to collect the eluate, wherein the concentration of the chaotropic salt is increased from a first concentration to a second concentration, the eluate is collected in at least one fraction, and the at least one fraction comprises the product of interest. In some embodiments, the concentration of the chaotropic salt at the first concentration is 0M and the concentration of the chaotropic salt at the second concentration is 4.0M. In some embodiments, the chaotropic salt comprises one or more of lithium, a lithium salt, magnesium, a magnesium salt, calcium, a calcium salt, guanidinium, and / or a guanidinium salt. In some embodiments, the chaotropic salt is selected from magnesium chloride, calcium chloride, lithium chloride, and guanidinium hydrochloride.

[0058] In some embodiments, the buffer solution further comprises one or more of acetate, citrate, ACES, BES, bicine, HEPES, MES, MOPS, MOPSO, TAPS, tricine, bis-tris, bis-trispropane, cacodylate, CAPS, CAPSO, CHES, glycine, glycylglycine, imidazole, PIPES, TEA, and TES.

[0059] In some embodiments, unwanted molecules are removed from the solution using a method that includes a chaotropic agent to break up non-specific interactions. In some embodiments, a chaotropic agent is used as part of an affinity chromatography step. In some embodiments, a chaotropic agent is used as part of an ion exchange chromatography step. In some embodiments, the chaotropic agent used as part of ion exchange chromatography has low conductivity, such as urea, alcohols, and detergents. In some embodiments, unwanted molecules are removed from the solution using a method that includes a hydrophobic interaction resin, where the protein of interest binds to the resin with a kosmotropic agent and is eluted while applying a gradient to a chaotropic salt.

[0060] When harvested from cell cultures, desirable molecular species may be present in significant concentrations, while undesirable molecular species may also be present, such as host cell proteins, cell debris, nucleic acids, endotoxins, or other molecules that may compromise the purity of the desirable molecular species. Different supernatant compositions may be sorted and purified based on differences related to binding, shape, size, charge, and other physical properties. In some embodiments, purification of the desirable molecular species may result in better patient outcomes and fewer complications as a result of administration of the desirable molecular species. In some embodiments, the methods of the invention may allow for improved efficacy or reduced dosage of the desirable molecular species.

[0061] In some embodiments, the desired molecular species is collected from blood. In some embodiments, the desired molecular species is collected from plasma. In some embodiments, the desired molecular species is collected from cell culture. In some embodiments, the desired molecular species is collected from animal component-free cell culture. In some embodiments, the desired molecular species is collected from clarified cell culture fluid (CCCF). In some embodiments, the desired molecular species is collected from cell supernatant.

[0062] In some embodiments, the cell culture is bacteria. In some embodiments, the cell culture is Escherichia coli. In some embodiments, the cell culture is eukaryotic. In some embodiments, the cell culture is S. cerevisiae. In some embodiments, the cell culture is mammalian cells. In some embodiments, the cell culture is from human cells. In some embodiments, the cell culture is primary tissue. In some embodiments, the cell culture is an established cell line. Non-limiting examples of established cell lines include CHO cells, HeLa cells, mouse 3T3 fibroblasts, HEK293 cells, and KT-3 cells. In some embodiments, the cell culture comprises a mixture of cell types. In some embodiments, the cell culture comprises a single cell type. In some embodiments, the cell culture is immune cells. In some embodiments, the cell culture is lymphocytes. In some embodiments, the cell culture is T cells.

[0063] In some embodiments, the desired species may include anti-VEGF antibody conjugates (e.g., KSI-301, KSI-501) that include a polymer moiety that extends the half-life of the antibody when administered to a subject. In some embodiments, the undesired species may include host cell proteins (HCPs). In some embodiments, chromatography may be accomplished using a Protein A matrix, and administration of a specific chaotropic agent may allow desirable species to remain bound to the matrix while undesired species are efficiently eluted. Thus, in some embodiments, the disclosed methods and systems may provide a method for purification of cell products harvested from cell cultures. In some embodiments, the methods of the invention may achieve higher purity of desirable species while experiencing lower loss rates as cell processing progresses. In some embodiments, the methods of the invention may reduce levels of HCPs to reduce patient complications when administering the final pharmaceutical product. In some embodiments, the methods of the invention may reduce costs and time associated with purification of cell products.

[0064] Harvesting and processing of cell cultures can require significant purification and collection efforts, as whole cell extracts often contain undesired impurities as well as desirable molecular species. Multi-step processing of harvested cell cultures can efficiently remove impurities, including host cell proteins (HCPs), high molecular weight (HMW) species, and product-related impurities such as low molecular weight (LMW) species. Some processes use affinity chromatography as the first step in a multi-step purification process for a protein of interest (e.g., an antibody), and the efficiency and purity of the resulting protein of interest influences all downstream purification steps. Additionally, affinity chromatography during downstream processing can be useful in reducing costs and time spent during processing, as it helps to concentrate the product, allowing the use of proportionately smaller equipment in later processing steps. Therefore, there is an advantage to optimizing the removal of impurities during affinity chromatography without losing or compromising the concentration yield of subsequent processing steps.

[0065] In some embodiments, the method includes purifying an antibody and / or protein. In some embodiments, the method uses an optimized wash buffer. In some embodiments, the optimized wash buffer is pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, pH 9.0, or any integer between pH 6.0 and pH 9.0. In some embodiments, the optimized wash buffer is pH 7.0. In some embodiments, the optimized wash buffer is pH 7.2. In some embodiments, the optimized wash buffer is pH 6.0.

[0066] In some embodiments, the optimized wash buffer comprises 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any integer between 5 mM and 200 mM sodium phosphate. In some embodiments, the optimized wash buffer comprises 5 mM sodium phosphate. In some embodiments, the optimized wash buffer comprises 50 mM sodium phosphate.

[0067] In some embodiments, the optimized wash buffer comprises 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any integer between 5 mM and 200 mM Tris. In some embodiments, the optimized wash buffer comprises 5 mM Tris. In some embodiments, the optimized wash buffer comprises 50 mM Tris.

[0068] In some embodiments, the optimized wash buffer comprises 5 mM, 10 mM, 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any integer between 5 mM and 200 mM BisTris. In some embodiments, the optimized wash buffer comprises 5 mM BisTris. In some embodiments, the optimized wash buffer comprises 50 mM BisTris.

[0069] In some embodiments, the optimized wash buffer comprises 50 mM, 100 mM, 250 mM, 500 mM, 750 mM, 1 M, 1.5 M, 2 M, 3 M, or any integer between 50 mM and 3 M sodium chloride. In some embodiments, the optimized wash buffer comprises 2 M sodium chloride. In some embodiments, the optimized wash buffer comprises 50 mM sodium chloride.

[0070] In some embodiments, the optimized wash buffer comprises magnesium chloride at 1M, 1.5M, 1.65M, 2M, 2.8M, 3M, 4M, 5M, or any integer between 1M and 5M. In some embodiments, the optimized wash buffer comprises 2.8M magnesium chloride. In some embodiments, the optimized wash buffer comprises 2M magnesium chloride.

[0071] In some embodiments, the optimized wash buffer comprises a chaotrope. In some embodiments, the optimized wash buffer comprises a chaotrope salt. In some embodiments, the optimized wash buffer comprises a chaotropic cation. In some embodiments, the optimized wash buffer comprises a chaotropic anion. Non-limiting examples of chaotropic cations include guanidinium, magnesium, calcium, sodium, and lithium. Non-limiting examples of chaotropic anions include chloride, sulfate, acetate, citrate, nitrate, and nitrite. In some embodiments, the chaotrope salt is calcium chloride, guanidinium chloride, lithium acetate, lithium chloride, magnesium chloride, magnesium sulfate, sodium nitrate, or sodium chloride. In some embodiments, the optimized wash buffer comprises two or more chaotrope salts. In some embodiments, the chaotrope is present in the optimized wash buffer at 0.05M, 0.1M, 0.2M, 0.5M, 1M, 1.5M, 1.65M, 2M, 2.8M, 3M, 4M, 5M, 6M, 7M, 10M, or any integer between 0.05M and 10M. In some embodiments, the chaotrope is present in the optimized wash buffer at 1M. In some embodiments, the chaotrope is present in the optimized wash buffer at 2.8M. In some embodiments, the concentration of the chaotrope present in the optimized wash buffer is optimized for the protein and / or antibody being purified.

[0072] Related As described herein, a washing procedure was established that reduces HCP levels in the eluate of an affinity chromatography step. The buffer strength, conductivity, and pH range within the buffer were also defined. Salts capable of reducing HCP levels in the eluate were identified. The efficiency of salts to reduce HCP levels was measured using the Hofmeister series. It was found that the chaotropic strength correlates with the chaotropic strength according to the ELISA (Figure 1) series. Additionally, this approach was found to work for different antibody types and different Fc fusion proteins, and therefore the inventive concepts disclosed herein are generally applicable to any antibody and / or protein. This technique was also shown to work with two different Protein A affinity chromatography resins.

[0073] The studies described herein used various antibodies and antibody-like constructs derived from Chinese Hamster Ovary (CHO) cells. The CHO expression system is the most commonly used system in the industry for the production of biopharmaceuticals, due to its ability to produce complex proteins with post-translational modifications similar to those produced in humans. To date, more than 6,000 CHO HCPs have been identified.

[0074] Currently, downstream processing of biopharmaceutical products from mammalian cell cultures represents a large portion of the total production costs. A major challenge in downstream processing is the removal of host cell proteins (HCPs). HCPs are process-related impurities that may be co-purified with the biopharmaceutical product. Downstream processing typically involves a Protein A affinity chromatography step followed by an additional polishing step to remove aggregates, product variants, HCPs, and host cell DNA. Many of the same HCPs are found after the Protein A chromatography step throughout the biopharmaceutical industry. This is a consequence of the near-universal use of CHO cells in the industry for the production of antibodies and antibody-like constructs, and the use of very similar conditions for the Protein A affinity chromatography step. In the latter case, biopharmaceuticals are produced under physiological conditions (neutral pH and physiological salt conditions). After applying to the resin and washing the resin with physiological conditions and high sodium chloride buffer, the product is collected in an elution step. As a result of this platform approach, the affinity chromatography eluate contains many of the same HCPs in similar amounts. There are some differences depending on the type of biopharmaceutical product, but they are minor. In a survey, 69% of companies said they have experienced issues with individual HCPs during drug manufacturing, which is considered one of the biggest challenges in biomanufacturing. Some HCPs are high risk, including those that have immunogenic, biological, or enzymatic activity and can degrade product molecules or excipients used in the formulation. In process development, the need to remove HCPs is easily recognized, but it is difficult but important to reduce HCP levels as much as possible. Some of these high-risk HCPs are lipases, enzymes that break down fats and lipids. Lipases can also break down polysorbates, which are often used in formulations, affecting the stability of therapeutic drugs. Similarly, hydrolases have been found to be the underlying cause of polysorbate degradation. Such HCPs can compromise the stability of drug products and reduce their shelf life. Degradation of polysorbates may result in the formation of particles in the formulation, raising safety concerns. In turn, proteases such as serine proteases, cathepsins, and metalloproteases degrade antibodies, antibody-like constructs, and other proteins. In conclusion, various HCP species may affect both the active ingredients and excipients of biologics. Importantly, HCPs may also be immunogenic in humans. Unwanted immune responses are the most serious harm caused by HCPs, which may be fatal. Lebrikizumab contains phospholipase B-like 2 protein, which has been shown to cause immune responses in approximately 90% of subjects based on clinical trial data. A summary of high-risk HCPs and their potential impacts is provided in Table 5A. For all these reasons, it is important to reduce HCP levels as much as possible. Some HCPs bind nonspecifically to the product and are subsequently co-purified. These HCPs are the most difficult to remove.In this study, we focused on breaking down non-specific interactions between the product and HCPs, thereby reducing HCP levels. Characterization of the HCP types removed by this approach was outside the scope of this study. We show that the levels of HCPs can be significantly reduced by applying a washing step containing chaotropic salts. It is not yet known what type of HCPs are removed.

[0075] definition The term "cell" includes prokaryotic and eukaryotic cells, and may also include bacterial cells, tuberculosis cells, fungal cells, yeast cells, plant cells, insect cells, non-human animal cells, human cells, or cell fusions, e.g., hybridomas. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is derived from a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from the following cells: CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT 060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived therefrom. In some embodiments, the cell comprises one or more viral genes.

[0076] As used herein, "affinity chromatography" refers to a method of chromatographic separation that utilizes specific and reversible interactions between biomolecules.

[0077] As used herein, "Protein A chromatography" refers to the chromatography of IgG with Protein A. It refers to a specific affinity chromatography method that relies on the affinity of the binding domain with the Fc portion of an immunoglobulin molecule. In an immunoglobulin, the Fc portion includes the immunoglobulin constant domains CH2 and CH3, or immunoglobulin domains substantially similar thereto. Protein A is derived from a natural protein in the cell wall of Staphylococcus aureus, and recombinant or synthetically produced Protein A and variants of Protein A can retain the ability to bind to the Fc region. Protein A chromatography is often immobilized on a solid support, such as in a Protein A column. Protein G and Protein L can be used in a similar manner. In some embodiments, the solid support is a matrix attached to Protein A.

[0078] The term "affinity chromatography matrix" or "AC matrix" as used herein is intended to refer to a solid phase medium (usually a gel or resin) that allows for the separation of biochemical mixtures based on highly specific binding interactions between proteins of interest and the AC matrix, such as between receptor and ligand, between enzyme and substrate, or between antigen and antibody. Thus, the solid phase medium comprises a target to which a protein of interest can reversibly attach, depending on the buffer conditions. Non-limiting examples of immobilization or solid phase media that may comprise an AC matrix include gel matrices such as agarose beads (such as commercially available Sepharose matrix), and glass matrices such as porous glass beads (such as commercially available ProSep matrix).

[0079] In some embodiments, the method for separating the protein of interest includes column chromatography. In this process, an AC matrix is ​​formed in a column, and a biochemical mixture containing the protein of interest is applied to the column. The protein of interest binds to the AC matrix. Next, a washing solution is applied to the column to wash the column, and then an elution buffer is applied to the column to elute the protein of interest from the column.

[0080] In some embodiments, the method of isolating the protein of interest comprises membrane chromatography. Membrane chromatography relies on AC matrix formatted to fit a membrane sheet, and the biochemical mixture containing the protein of interest passes through the membrane. A non-limiting example of membrane chromatography includes Sartorius' Sartobind Rapid A. In some embodiments, when the protein of interest is at least about 500 kDa, membrane chromatography is the preferred chromatography method.

[0081] Additional affinity chromatography systems that can be used in the present invention include, for example, Protein G, Protein A / G, and Protein L columns, each of which is an immunoglobulin-binding bacterial protein with art-established binding properties. Thus, AC matrices that are Protein G matrices, Protein A / G matrices, or Protein L matrices can be used to purify antibodies, antibody fragments, or proteins that contain an Fc region (e.g., Fc fusion proteins).

[0082] Although the present disclosure is specifically described with respect to purifying antibodies using Protein A, so long as the technology is known for selectively binding any protein (including fusion proteins) to a particular AC matrix, that protein is suitable for purification using the washing methods described herein.

[0083] As used herein, a "chaotropic agent" is a molecule that weakens or otherwise interferes with non-covalent binding forces and increases entropy within a biomolecular system. In some embodiments, lithium chloride, magnesium chloride, calcium chloride, and / or guanidinium chloride are used. are chaotropic agents. Non-limiting examples of chaotropic agents include butanol, calcium chloride, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, and urea. Chaotropic agents include salts that affect the solubility of proteins. More chaotropic anions include chloride, nitrate, bromide, chlorate, iodide, perchlorate, and thiocyanate. More chaotropic cations include lithium, magnesium, calcium, and guanidinium.

[0084] As used herein, "eluent" or "eluent" refers to the carrier portion of the mobile phase in chromatography. In liquid chromatography, the eluent is the liquid solvent that enters the column, while in gas chromatography it is the carrier gas. In some embodiments, the eluent refers to the carrier liquid solvent that contains antibodies, host cell proteins (HCPs), and other molecules of interest.

[0085] As used herein, "eluate" refers to the analyte that emerges from a chromatography step, and includes both the analyte and solute that passes through the solid phase. In some embodiments, eluate specifically refers to the analyte that is collected for further processing. In some embodiments, eluate refers to the antibodies collected from the harvested cells, also known as "product eluate." In some embodiments, eluate refers to the HCPs collected from the harvested cells, also known as "wash eluate."

[0086] A "neovascular disorder" is a disorder or disease characterized by altered, dysregulated, or unregulated angiogenesis. Examples of neovascular diseases include neovascular changes (e.g., cancer), and ocular neovascular diseases such as diabetic retinopathy, age-related macular degeneration, and retinal vein occlusion.

[0087] "Ocular neovascular" disorders are disorders characterized by altered, dysregulated, or incapable of regulating vascularization in the eye of a patient. Such disorders include retinal vein occlusion, optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreous neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, diabetic macular edema, vascular retinopathy, retinal degeneration, uveitis, inflammatory diseases of the retina, and proliferative vitreoretinopathy.

[0088] The term antibody includes intact antibodies and binding fragments thereof. A binding fragment refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments. scFv antibodies are described in Houston JS. 1991. Methods in Enzymol. 203:46-96. Furthermore, antibody fragments include single-chain polypeptides that have the properties of a VH domain, i.e., the ability to assemble with a VL domain into a functional antigen-binding site, or the properties of a VL domain, i.e., the ability to assemble with a VH domain into a functional antigen-binding site, thereby displaying the antigen-binding properties of a full-length antibody.

[0089] An antibody specifically binds to its target antigen by at least 10 6 , 10 7 , 10 8 , 10 9 , or 10 10 M -1 Specific binding refers to the affinity of an antibody or fusion protein to only one target. Specific binding will be strong enough to be detectable and distinguishable from non-specific binding that occurs to at least one unrelated target. Specific binding can be the result of the formation of bonds between specific functional groups or a specific spatial fit (e.g., lock and key type), whereas non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody or fusion protein binds only to one target. That doesn't mean.

[0090] The basic antibody structural unit is a tetramer of subunits. Each tetramer contains two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain contains a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. A variable region without a signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without a light chain signal peptide. However, reference to a variable region does not necessarily imply the presence of a signal sequence, and in practice, the signal sequence is cleaved when the antibody or fusion protein is expressed and secreted. The pair of heavy and light chain variable regions defines the binding region of the antibody. The carboxy-terminal portions of the light and heavy chains define the light chain constant region and the heavy chain constant region, respectively. The heavy chain constant region is primarily responsible for effector functions. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. The CH1 region is linked to the light chain constant region by disulfide bonds and non-covalent bonds. The hinge region confers flexibility between the binding and effector regions of the antibody and also provides a site for intermolecular disulfide bonds between the two heavy chain constant regions in the tetrameric subunit. The CH2 and CH3 regions are the main sites of effector function and FcR binding.

[0091] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" segment of about 12 or more amino acids, with the heavy chain also including a "D" segment of about 10 more amino acids (see generally Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (incorporated by reference for all purposes).

[0092] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, i.e., it is bivalent. In a natural antibody, the binding sites are the same. However, bispecific antibodies can be produced, in which the two binding sites are different (see, for example, Songsivilai S, Lachmann PC. 1990. Bispecific antibody: a tool for diagnosis and treatment of disease. Clin Exp Immunol. 79:315-321; Kostelny SA, Cole MS, Tso JY. 1992. Formation of bispecific antibody by the use of leucine zippers. J Immunol. 148: 1547-1553). All variable regions exhibit the same overall structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs of the two chains of each pair are aligned by the framework regions, allowing binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. For convenience, the variable heavy chain CDRs are referred to as CDRs. H 1. CDR H 2, and CDR H 3, and the variable light chain CDRs are referred to as CDR L 1. CDR L 2, and CDR LThe domains may be referred to as 3. The amino acid assignment to each domain follows: Kabat EA, et al. 1987 and 1991.Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) or Chothia C, Lesk AM. 1987. Canonical Structures for the Hypervariable Regions of Immunoglobulins. J Mol Biol 196:901-917; Chothia C, et al. 1989. Conformations of Immunoglobulin Hypervariable Regions. Nature 342:877-883. Kabat also provides a widely used numbering scheme (Kabat numbering) that assigns the same numbers to corresponding residues between different heavy chain variable regions or different light chain variable regions. Kabat numbering can be used for antibody constant regions, but EU numbering is more widely used, as in the present application. Although specific sequences are provided for the exemplary antibodies disclosed herein, it will be understood that following expression of the protein chains, one or several amino acids at the amino or carboxy termini of the light and / or heavy chains, particularly the C-terminal lysine residue of the heavy chain, may be missing or derivatized in some or all of the molecules.

[0093] The term "epitope" refers to a site on an antigen to which an antibody or extracellular trap segment binds. Epitopes on proteins are formed from contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are usually retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding (also known as conformational epitopes) are usually lost on treatment with denaturing solvents. An epitope usually contains at least 3, usually at least 5 or 8-10 amino acids in a unique spatial configuration. Methods for determining the spatial conformation of an epitope include, for example, X-ray crystallography and 2-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed.(1996).

[0094] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by identifying contact residues by X-ray crystallography of the antibody (or Fab fragment) bound to the antigen.

[0095] Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if all amino acid mutations that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody.

[0096] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess (e.g., at least 2-fold, 5-fold, 10-fold, 20-fold, or 100-fold) of the test antibody inhibits binding of the reference antibody by at least 50%. In some embodiments, the test antibody inhibits binding of the reference antibody by 75%, 90%, or 99%, as measured by a competitive binding assay. Antibodies identified by competitive assays (competitor antibodies) include antibodies that bind to the same epitope as the reference antibody and antibodies that bind to adjacent epitopes in sufficient proximity to the epitope bound by the reference antibody that steric hindrance occurs.

[0097] The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment.

[0098] To classify amino acid substitutions as conservative or non-conservative, amino acids are classified into the following groups: The loops are divided into: Group I (hydrophobic side chains): met, ala, val, leu, ile; Group II (neutral hydrophilic side chains): cys, ser, thr; Group III (acidic side chains): asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V (residues that affect chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids of the same class. Non-conservative substitutions involve exchanging a member of one of these classes for a member of another class.

[0099] Percentage sequence identity is determined for maximally aligned antibody sequences using Kabat numbering conventions for variable regions and EU numbering for constant regions. After alignment, when a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is compared to the same region of a reference antibody, the percentage of sequence identity between the subject antibody region and the reference antibody region is converted to a percentage by dividing the number of positions occupied by the same amino acid in both the subject antibody region and the reference antibody region by the total number of aligned positions of the two regions, not counting gaps, and multiplying by 100. Sequence identity for other sequences can be determined using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, Inc., 575 Science Dr., Madison, Wis.) by aligning the sequences using default gap parameters or by inspection and optimal alignment (i.e., the one that results in the highest percentage of sequence similarity over the entire comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences over a comparison window, determining the number of positions where identical residues occur in both sequences to calculate the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity.

[0100] A composition or method "comprising" one or more recited elements may include other elements not specifically recited, for example, a composition that includes an antibody may include the antibody alone or in combination with other components.

[0101] The term "antibody-dependent cellular cytotoxicity" (or ADCC) is a mechanism that induces cell death depending on the interaction of antibody-coated target cells (i.e., antibody-bound cells) with immune cells (also called effector cells) that have lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is triggered by the interaction of the Fc region of a cell-bound antibody with FcγRI and FcγRIII on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cells are removed by phagocytosis or lysis, depending on the type of mediating effector cell. Death of the antibody-coated target cells occurs as a result of the activity of the effector cells.

[0102] The term opsonization, also known as "antibody-dependent cellular phagocytosis" (or ADCP), refers to the process by which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0103] The term "complement-dependent cytotoxicity" (or CDC) refers to the mechanism by which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions that ultimately induce cell death by forming holes in the target cell membrane. Antigen-antibody complexes, such as those found on antibody-coated target cells, bind and activate the complement component Clq, which then activates the complement cascade leading to the death of the target cell. Complement activation leads to the deposition of complement components on the target cell surface, which may then bind to complement receptors (e.g., CR3) on white blood cells to promote ADCC.

[0104] Humanized antibodies are genetically engineered antibodies in which the CDRs of a non-human "donor" antibody are grafted onto human "acceptor" antibody sequences (e.g., U.S. Patent Nos. 5,530,101 and 5,585,089 by Queen, 5,225,539 by Winter, 6,407,213 by Carter, 5,859,205 and 6,881,557 by Adair, and 6,881,557 by Foote). The acceptor antibody sequences can be, for example, mature human antibody sequences, a mixture of such sequences, a consensus sequence of human antibody sequences, or germline region sequences. Thus, a humanized antibody is an antibody having some or all of the CDRs from a donor antibody and variable region framework sequences and constant regions, if present, that are derived completely or substantially from human antibody sequences. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs that are entirely or substantially derived from a donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region, if present, that are substantially derived from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs that are entirely or substantially derived from a donor antibody light chain, and a light chain variable region framework sequence and a light chain constant region, if present, that are substantially derived from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, humanized antibodies include humanized heavy chains and humanized light chains. The CDRs of a humanized antibody are substantially identical to the corresponding CDRs of a non-human antibody when at least 85%, 90%, 95%, or 100% (according to the Kabat definition) of the corresponding residues are identical between the respective CDRs. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues defined by Kabat are identical.

[0105] Humanized antibodies often incorporate all six CDRs (as defined by Kabat) from a murine antibody, but may be made with fewer than all of the CDRs (e.g., at least three, four, or five CDRs from a murine antibody) (see, e.g., De Pascalis R, Iwahashi M, Tamura M, et al. 2002. Grafting "Abbreviated" Complementary-Determining Regions Containing Specificity-Determining Residues Essential for Ligand Contact to Engineer a Less Immunogenic Humanized Monoclonal Antibody. J Immunol. 169:3076-3084; Vajdos FF, Adams CW, Breece TN, Presta LG, de Vos AM, Sidhu, SS. 2002. Comprehensive functional maps of the antigen-binding site of an anti-ErbB2 antibody obtained with shotgun scanning mutagenesis. J Mol Biol. 320: 415-428; Iwahashi M, Milenic DE, Padlan EA, et al. 1999. CDR substitutions of a humanized monoclonal antibody(CC49): Contributions of individual CDRs to antigen binding and immunogenicity. Mol Immunol. 36:1079-1091; Tamura M, Milenic DE, Iwahashi M, et al. 2000.Structural correlates of an anticarcinoma antibody: Identification of specificity-determining regions(SDRs) and development of a minimally immunogenic antibody variant by retention of SDRs only. J Immunol. 164:1432-1441). .

[0106] Chimeric antibodies are antibodies in which the mature variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or completely retain the binding specificity of the mouse antibody and contain approximately two-thirds human sequences.

[0107] A veneered antibody is a type of humanized antibody that retains some (usually all) of the CDRs and some of the non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that may contribute to B-cell or T-cell epitopes, such as exposed residues (Padlan EA. 1991. A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol Immunol. 28:489-98), with residues at the corresponding positions in a human antibody sequence. The result is an antibody whose CDRs are entirely or substantially derived from a non-human antibody, and whose variable region framework has been made more human-like by the replacement. Human antibodies are isolated from humans or are otherwise obtained from the expression of human immunoglobulin genes (e.g., in transgenic mice, in vitro, or by phage display). Methods for producing human antibodies include the trioma method (Ostberg L, Pursch E. 1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367; U.S. Patent No. 4,634,664 by Ostberg, and U.S. Patent No. 4,634,666 by Engleman et al.), the use of transgenic mice containing human immunoglobulin genes (e.g., WO 93 / 12227 (1993) by Lonberg et al.; U.S. Patent Nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, and 5,545,806; Nature 148, 1547-1553 (1994), and Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991)), and phage display methods (see, e.g., Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047; U.S. Pat. Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332).

[0108] A "polymer" refers to a series of linked monomer groups. A polymer is composed of multiple units of a single monomer (homopolymer) or different monomers (heteropolymer). High molecular weight (MW) polymers are prepared from monomers including, but not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinylpyridines, vinylpyrrolidones, and vinyl esters such as vinyl acetate. Additional monomers are useful for high MW polymers. When two different monomers are used, the two monomers are called "comonomers," meaning that the different monomers are copolymerized to form a single polymer. A polymer can be linear or branched. When a polymer is branched, each polymer chain is referred to as a "polymer arm." The end of the polymer arm connected to the initiator moiety is the proximal end, and the growing chain end of the polymer arm is the distal end. At the growing chain end of the polymer arm, the polymer arm end group can be a radical scavenger or another group.

[0109] "Initiator" refers to a compound capable of initiating polymerization using a monomer or comonomer. The polymerization may be a conventional free radical polymerization or a controlled / "living" radical polymerization such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation termination (RAFT) polymerization, or nitroxide-mediated polymerization (NMP). The polymerization may be a "quasi" controlled polymerization such as a denaturing transformation. When the initiator is suitable for ATRP, it includes an initiator fragment I, which is a radical that can be homolytically cleaved to initiate radical polymerization, and a labile bond that can react with a radical of a growing polymer chain to form a radical scavenger I', which reversibly terminates the polymerization. The radical scavenger I' is typically a halogen, but may also be an organic moiety such as a nitrile. In some embodiments, the initiator includes one or more 2-bromoisobutyric acid groups as sites for polymerization by ATRP.

[0110] "Chemical linker" refers to a chemical moiety that connects two groups, such as a half-life extending moiety and a protein. The linker may be cleavable or non-cleavable. Cleavable linkers can be hydrolytically cleavable, enzyme-cleavable, pH-sensitive, photolabile, or disulfide linkers, among others. Other linkers include homobifunctional linkers and heterobifunctional linkers. A "linking group" is a functional group that can form a covalent bond of one or more bonds with a bioactive agent. Non-limiting examples include those shown in Table 1 of WO2013059137 (incorporated by reference).

[0111] The term "reactive group" refers to a group that can react with another chemical group to form a covalent bond, i.e., a group that reacts covalently under appropriate reaction conditions and typically represents a point of attachment for another substance. Reactive groups are moieties such as maleimides or succinimidyl esters that can chemically react with a functional group on a different moiety to form a covalent bond. Reactive groups typically include nucleophiles, electrophiles, and photoactivatable groups.

[0112] "Phosphorylcholine" is also known as "PC" and refers to: [ka] During the ceremony, * indicates a connection point. Phosphorylcholine is a zwitterionic group, including salts (such as inner salts) and its protonated and deprotonated forms.

[0113] A "phosphorylcholine-containing polymer" is a polymer that contains phosphorylcholine. A "zwitterion-containing polymer" refers to a polymer that contains a zwitterion.

[0114] A poly(acryloyloxyethyl phosphorylcholine)-containing polymer refers to a polymer that contains 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC shown in Example 6 below) as a monomer.

[0115] Poly(methacryloyloxyethyl phosphorylcholine)-containing polymers refer to polymers that contain 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEMA-PC or MPC) as a monomer (see below). [ka]

[0116] In this document, "MPC" and "HEMA-PC" are interchangeable.

[0117] "Molecular weight" in the context of a polymer can be expressed as either number average molecular weight, weight average molecular weight, or peak molecular weight. Unless otherwise specified, all references to molecular weight herein refer to peak molecular weight. These molecular weight measurements, number average (Mn), weight average (Mw), and peak (Mp), can be measured using size exclusion chromatography or other liquid chromatography techniques. Other methods of measuring molecular weight values ​​can also be used. Number average molecular weights can be determined using end group analysis or measurements of aggregation properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure), and weight average molecular weights can be determined using light scattering techniques, ultracentrifugation, or viscometry. In some embodiments, the molecular weight is measured by SEC-MALS (size exclusion chromatography-multi-angle light scattering). In some embodiments, the polymeric reagent is typically polydisperse (i.e., the number average and weight average molecular weights of the polymer are not equal) and can have low polydispersity values, e.g., less than about 1.5, as judged by the PDI value obtained from a SEC-MALS measurement. In some embodiments, the polydispersity index (PDI) is in the range of about 1.4 to about 1.2. In some embodiments, the PDI is less than about 1.15, 1.10, 1.05, or 1.03.

[0118] The phrase "a / an" refers to one or more of the whole. For example, a compound can refer to one or more compounds, or at least one. As such, the terms "a / an," "one or more," and "at least one" can be used interchangeably herein.

[0119] By "about" it is meant the variation found in measurements taken among different instruments, samples, and sample preparations.

[0120] "Protected," "protected form," "protecting group," and "protective group" refer to the presence of a group (i.e., a protecting group) that prevents or inhibits reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups vary depending on the type of chemically reactive group being protected, the reaction conditions used, and the presence (if any) of additional reactive or protecting groups in the molecule. Suitable protecting groups include those described in Greene et al., "Protective Groups In Organic Synthesis," 3rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0121] "Alkyl" refers to a straight or branched saturated aliphatic radical having the indicated number of carbon atoms. For example, C1-C6 alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, and the like. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, and the like. Alkyl can contain any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6, 5-6, and the like. Alkyl groups are usually monovalent, but can be divalent, such as when an alkyl group joins two moieties.

[0122] The term "lower" referred to above and below in connection with organic radicals or compounds defines branched or unbranched compounds or radicals having up to 7 or up to 4 carbon atoms and (if unbranched) 1 or 2 carbon atoms, respectively.

[0123] "Alkylene" refers to an alkyl group, as defined above, that is linked to at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene. For example, a straight chain alkylene is -(CH2) n where n is 1, 2, 3, 4, 5, or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene.

[0124] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: -OR', ═O, ═NR', ═N-OR', -NR'R'', -SR', -halogen, -SiR'R''R''', -OC(O) R', -C(O)R', CO2R', CONR'R'', OC(O)NR'R'', NR''C(O)R', NR'C(O)NR''R''', NR''C(O)2R', NHC(NH2)=NH, NR'C(NH2)=NH, NHC(NH2)=NR', S(O)R', S(O)2R', S(O)2NR'R'', CN, and NO2, a number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical. R', R'', and R''' each independently represent hydrogen, unsubstituted (C1-C8) alkyl and heteroalkyl groups, unsubstituted aryl groups, aryl groups substituted with one to three halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, "-NR'R" includes 1-pyrrolidinyl and 4-morpholinyl. The term "alkyl" includes groups such as haloalkyl (e.g., -CF3, -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.). In some embodiments, substituted alkyl and heteroalkyl groups have 1-4 substituents. In some embodiments, substituted alkyl and heteroalkyl groups have 1, 2, or 3 substituents. An exception is perhaloalkyl groups (e.g., pentafluoroethyl, etc.).

[0125] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, OR', ═O, ═NR', ═NOR', NR'R'', SR', halogen, SiR'R''R''', OC(O) R', C(O)R', CO2R', CONR'R'', OC(O)NR'R'', NR''C(O)R', NR'C(O)NR''R''', NR''C(O)2R', NRC(NR'R''R'')=NR'''', NRC(NR'R'')=NR''', S(O)R', S(O)2R', S(O)2NR'R'', NRS02R', CN, and -NO2, a number ranging from 0 to (2m'+1), where m' is the total number of carbon atoms in such radical. R', R'', R''', and R'''' each independently represent hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1 to 3 halogens), a substituted or unsubstituted alkyl group, an alkoxy or thioalkoxy group, or an arylalkyl group. For example, when a compound contains multiple R groups, each R group is independently selected, and when multiple of these groups are present, each R', R'', R''', and R'''' group is also independently selected. When R' and R'' are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, "-NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the discussion of the substituents above, one of skill in the art will understand that the term "alkyl" is meant to include groups that contain carbon atoms bonded to groups other than hydrogen groups. Included are groups such as haloalkyl (e.g., -CF3, -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CHOCH3, etc.).

[0126] "Alkoxy" refers to an alkyl group having an oxygen atom connected to the connection point or to two carbons of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, and the like. Alkoxy groups can be further substituted with a variety of substituents described herein. For example, alkoxy groups can be substituted with halogens to form "haloalkoxy" groups.

[0127] The term "carboxyalkyl" refers to an alkyl group (as defined herein) substituted with a carboxy group. The term "carboxycycloalkyl" refers to a cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl refers to an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, the term "carboxy" refers to carboxylic acids and esters thereof.

[0128] "Haloalkyl" refers to an alkyl, as defined above, in which some or all of the hydrogen atoms have been replaced with halogen atoms. Halogen (halo) refers to chloro or fluoro, but may also refer to bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluorophenyl, and the like. The term "perfluoro" defines a compound or radical in which all available hydrogens have been replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0129] The term "fluorine-substituted alkyl" refers to an alkyl group in which one, some, or all of the hydrogen atoms are replaced with fluorine.

[0130] "Cytokines" are involved in intercellular communication in immune and inflammatory responses. Cytokines belong to a group of protein signaling molecules that may play important roles in the regulation of cytokine production. Cytokines are typically small, water-soluble glycoproteins with masses of approximately 8–35 kDa.

[0131] "Cycloalkyl" refers to a cyclic hydrocarbon group containing about 3 to 12, 3 to 10, or 3 to 7 ring carbon atoms. Cycloalkyl groups include fused, bridged, and spiro ring structures.

[0132] "Intracyclic" refers to an atom or group of atoms that form part of a cyclic ring structure.

[0133] "Exocyclic" refers to an atom or group of atoms that is attached but does not define a ring structure.

[0134] The term "cyclic alkyl ether" refers to a 4- or 5-membered cyclic alkyl group having 3 or 4 ring carbon atoms and 1 ring oxygen atom or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene), or a 6- to 7-membered cyclic alkyl group having 1 or 2 ring oxygen atoms or sulfur atoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

[0135] "Alkenyl" refers to a straight or branched chain hydrocarbon of 2 to 6 carbon atoms having at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, and 1,3,5-hexatrienyl. The alkenyl group may have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, or 5 to 6 carbon atoms. Alkenyl groups are usually monovalent, but can be divalent, such as when an alkenyl group links two moieties.

[0136] "Alkenylene" refers to an alkenyl group, as defined above, linked to at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene, hexenylene, and the like.

[0137] "Alkynyl" refers to a straight or branched chain hydrocarbon of 2 to 6 carbon atoms having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, and 1,3,5-hexatriynyl. The alkynyl group may have 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, or 5 to 6 carbon atoms. Alkynyl groups are usually monovalent, but can be divalent, such as when an alkynyl group links two moieties.

[0138] "Alkynylene" refers to an alkynyl group, as defined above, that is linked to at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. An alkynylene group can include any one of the following: Examples of aryl groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene, and hexynylene.

[0139] "Cycloalkyl" refers to a saturated or partially unsaturated monocyclic, fused bicyclic, or bridged polycyclic ring assembly containing 3 to 12 ring atoms, or the number of atoms indicated. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene, and adamantane. For example, C3-C8 cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0140] "Cycloalkylene" refers to a cycloalkyl group, as defined above, that is linked to at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the cycloalkylene can be linked to the same atom or different atoms of the cycloalkylene. Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

[0141] "Heterocycloalkyl" refers to a ring system containing 3 to about 20 ring members and 1 to about 5 heteroatoms, such as N, O, and S. Additional heteroatoms are also useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heterocycles include, but are not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl, and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

[0142] "Heterocycloalkylene" refers to a heterocycloalkyl group, as defined above, that is linked to at least two other groups. The two moieties linked to the heterocycloalkylene can be linked to the same atom or to different atoms of the heterocycloalkylene.

[0143] "Aryl" refers to a monocyclic or fused bicyclic, tricyclic or higher aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl may be phenyl, benzyl, or naphthyl. "Arylene" means a divalent radical derived from an aryl group. The aryl group may be mono-, di-, or tri-substituted with one, two, or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, aminoalkyl, trifluoromethyl, alkylenedioxy, and oxy-C2-C3-alkylene, all of which may be optionally further substituted, for example as defined above, or 1- or 2-naphthyl, or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substituent attached to two adjacent carbon atoms of phenyl (e.g., methylenedioxy, ethylenedioxy). Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, for example, oxyethylene or oxypropylene. Examples of oxy-C2-C3-alkylene-phenyl include 2,3-dihydrobenzofuran-5-yl.

[0144] In some embodiments, the aryl is naphthyl, phenyl, or phenyl mono- or di-substituted with alkoxy, phenyl, halogen, alkyl, or trifluoromethyl, especially phenyl, or phenyl mono- or di-substituted with alkoxy, halogen, or trifluoromethyl, especially phenyl.

[0145] Examples of substituted phenyl groups for R are 4-chlorophen-1-yl, 3,4-dichlorophen-1-yl, 4-methoxyphen-1-yl, 4-methylphen-1-yl, 4-aminomethylphen-1-yl, 4-methoxyethylaminomethylphen-1-yl, 4-hydroxyethylaminomethylphen-1-yl, 4-hydroxyethyl-(methyl)-aminomethylphen-1-yl, 3-aminomethylphen-1-yl, 4-N-acetylaminomethylphen-1-yl, 4-aminophen-1-yl, 3-aminophen-1-yl, 2-aminophen-1-yl, 4-phenylphen-1-yl, 4-(isopropyl)aminomethylphen-1-yl, 4-phenyl ... 4-(imidazol-1-yl)-phenyl, 4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl, 4-(morpholin-1-ylmethyl)-phen-1-yl, 4-(2-methoxyethylaminomethyl)-phen-1-yl and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl, 4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, as well as 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substituted in the heterocycle.

[0146] "Arylene" refers to an aryl group, as defined above, that is linked to at least two other groups. The two moieties linked to the arylene are linked to different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

[0147] "Aryleneoxy" refers to an arylene group, as defined above, in which one of the moieties linked to the arylene is linked through an oxygen atom. Aryleneoxy groups include, but are not limited to, phenyleneoxy groups.

[0148] Similarly, the substituents on the aryl and heteroaryl groups are varied and include: -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S (O)2NR'R'', -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, the number ranging from 0 to the total number of open valences on the aromatic ring system, where R', R'', and R''' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0149] Two of the substituents on adjacent atoms of an aryl or heteroaryl ring are of the formula -TC(O)-(CH2) q and optionally substituted with a substituent of the formula -U-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring are of the formula -A-(CH2) r A and B are optionally substituted with a substituent of the formula -B-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may be optionally substituted with a substituent of the formula -(CH2) s -X-(CH2) twhere s and t are independently integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' of NR' and S(O)2NR' is selected from hydrogen or unsubstituted (C1-C6) alkyl.

[0150] "Heteroaryl" means a monocyclic, fused bicyclic, or tricyclic ring system containing 5 to 16 ring atoms. where 1 to 4 of the ring atoms are heteroatoms, N, O, or S, respectively. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or other radicals, particularly mono- or di-substituted, for example, with alkyl, nitro, or halogen. Pyridyl represents 2-, 3-, or 4-pyridyl, with 2- or 3-pyridyl being preferred. Thienyl represents 2- or 3-thienyl. In some embodiments, quinolinyl represents 2-, 3-, or 4-quinolinyl. In some embodiments, isoquinolinyl represents 1-, 3-, or 4-isoquinolinyl. In some embodiments, benzopyranyl and benzothiopyranyl can represent 3-benzopyranyl and 3-benzothiopyranyl, respectively. In some embodiments, thiazolyl can represent 2- or 4-thiazolyl. In some embodiments, triazolyl can be 1-, 2-, or 5-(1,2,4-triazolyl). In some embodiments, tetrazolyl can be 5-tetrazolyl.

[0151] In some embodiments, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any radical that is substituted, particularly mono- or di-substituted.

[0152] The term "heteroalkyl" refers to an alkyl group having 1-3 heteroatoms, such as N, O, and S. Additional heteroatoms are also useful, including, but not limited to, B, Al, Si, and P. The heteroatoms may be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyls include ethers, thioethers, alkylamines, and alkylthiols.

[0153] The term "heteroalkylene" refers to a heteroalkyl group, as defined above, that is linked to at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same atom or to different atoms of the heteroalkylene.

[0154] "Electrophile" refers to an ion, atom, or collection of atoms, which may be ionic, that has an electrophilic center, i.e., a center that seeks electrons and can react with a nucleophile. An electrophile (or electrophile reagent) is a reagent that forms a bond with a reaction partner (nucleophile) by accepting both bonding electrons from the reaction partner.

[0155] "Nucleophile" refers to an ion, atom, or collection of atoms that may be ionic and that has a nucleophilic center, i.e., a center that seeks an electrophilic center or can react with an electrophile. A nucleophile (or nucleophile reagent) is a reagent that forms a bond with a reaction partner (electrophile) by donating both bonding electrons. "Nucleophilic group" refers to a nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, and haloalkoxy.

[0156] "Maleimide" refers to a pyrrole-2,5-dione-1-yl group having the following structure: [ka] (Reacts with sulfhydryls (e.g., thioalkyls) to form an -S-maleimide group having the structure: [ka] (In the formula, "·" represents the attachment point of the maleimide group, [ka] indicates the point at which the sulfur atom of the thiol is attached to the remainder of the original sulfhydryl group).

[0157] For purposes of this disclosure, "naturally occurring amino acids" in proteins and polypeptides are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and / or L-valine. "Unnaturally occurring amino acids" in proteins refer to amino acids other than those referred to as natural amino acids. Unnatural amino acids include, but are not limited to, D-isomers of the natural amino acids and mixtures of D and L-isomers of the natural amino acids. Other amino acids, such as N-alpha methyl amino acids (e.g., sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, ε-N-methyllysine, 3-methylhistidine, etc., occur in naturally occurring proteins but are typically introduced by means other than ribosomal translation of mRNA and are therefore considered, for purposes of this disclosure, to be unnatural amino acids found in proteins.

[0158] "Linear" in reference to the shape, configuration, or overall structure of a polymer refers to a polymer having a single polymer arm.

[0159] "Branched" refers to a polymer having two or more polymer "arms" extending from a core structure contained within the initiator, in terms of the shape, architecture, or overall structure of the polymer. The initiator may be used in an atom transfer radical polymerization (ATRP) reaction. A branched polymer may have two polymer chains (arms), three polymer arms, four polymer arms, five polymer arms, six polymer arms, seven polymer arms, eight polymer arms, nine polymer arms, or more. The polymeric initiator may have two or more polymer arms, each extending from a polymer initiation site. Each polymer initiation site can be a site for polymer chain growth by addition of monomer. For example, and without limitation, when using ATRP, the polymer initiation site on the initiator is typically an organic halide that undergoes a reversible oxidation-reduction process catalyzed by a transition metal compound such as copper halide. In some embodiments, the halide is bromine.

[0160] A "pharmaceutical acceptable excipient" refers to an excipient that may be included in a composition, does not cause significant adverse toxic effects in a patient, and is approved or may be approved by the FDA, particularly for human therapeutic use. Non-limiting examples of pharmaceutical acceptable excipients include water, sodium chloride, saline, lactated Ringer's solution, normal sucrose, normal dextrose, and the like.

[0161] The therapeutic protein is administered in an effective regimen, meaning a dosage, route of administration, and frequency of administration that delays onset, reduces severity, inhibits further deterioration, and / or ameliorates at least one sign or symptom of the disorder. If the patient already suffers from a disorder, the regimen is said to be therapeutically effective. If the patient is at high risk for disease compared to the general population, but has not yet developed symptoms, the regimen is said to be prophylactically effective. In some cases, a therapeutic or prophylactic effect may be observed in an individual patient compared to historical controls or the past experience of the same patient. In other cases, a therapeutic or prophylactic effect may be demonstrated in preclinical or clinical studies comparing a population of treated patients with a control population of untreated patients.

[0162] The "biological half-life" of a substance is a pharmacokinetic parameter that specifies the time required for half of the substance to be cleared from a tissue or organism following its introduction.

[0163] "OG1786" is a 9-arm initiator used in polymer synthesis, and together with OG1786 represents the salt form of trifluoroacetic acid. OG1786 can be used as other salts or as a free base.

[0164] "OG1801" is a polymer of approximately (±15%) 750 kDa (depending on Mn or Mp) made using OG1786 as the initiator for ATRP synthesis with the monomer HEMA-PC.

[0165] "OG1802" is OG1801 with a maleimide functional group added, where n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each positive integers (0 to about 3000), and the total molecular weight of the polymer is (Mw) 750,000±15% Daltons.

[0166] Multi-angle light scattering (MALS) is a technique for analyzing macromolecules where the oscillating electric field of the light induces an oscillating dipole in the molecule when a laser beam strikes it. This oscillating dipole re-emits light, which can be measured using a MALS detector such as the Wyatt miniDawn® TREOS®. The intensity of the emitted light depends on the magnitude of the dipole induced in the macromolecule, which is proportional to the polarizability of the macromolecule: the larger the induced dipole, the greater the intensity of the scattered light. Therefore, to analyze scattering from a solution of such macromolecules, it is necessary to know the polarizability with respect to the surrounding medium (e.g., solvent). This may be determined from the measurement of the change in the refractive index n of the solution, Δn, with a change in molecular concentration, Δc, by measuring the dn / dc (=Δn / Δc) value using a Wyatt Optilab® T-rEX differential refractometer. The two molar weight parameters used in MALS measurements are the number average molecular weight (Mn) and the weight average molecular weight (Mw). The polydispersity index (PDI) is equal to Mw divided by Mn. SEC also allows the measurement of the average molecular weight of the peak molecular weight, Mp, defined as the molecular weight of the highest peak in SEC.

[0167] PDI is used as a measure of the broadness of the molecular weight distribution of polymers and bioconjugates derived from the conjugation of individual proteins (e.g., OG1950) with polydisperse biopolymers (e.g., OG1802). For protein samples, the polydispersity is close to 1.0, since all protein molecules in solution are translation products that are expected to be approximately the same in length and molar mass. In contrast, due to the polydispersity of biopolymers, where polymer chains of various lengths are synthesized during the polymerization process, it is very important to determine the PDI of a sample as one of the quality characteristics of a narrow distribution of molecular weights.

[0168] Size Exclusion Chromatography (SEC) is a chromatographic technique that separates molecules in a solution by size. Typically, an aqueous solution is applied to pass the sample through a column packed with resins of various pore sizes. The resins are expected to be inert to the analytes as they pass through the column, and the analytes are separated from one another based on their inherent size and the pore size characteristics of the selected column.

[0169] Combining SEC with MALS or SEC / MALS provides accurate distributions of molar mass and size (root mean square radius) as opposed to relying on a set of SEC calibration standards. This type of arrangement has many advantages over previous column calibration methods. Light scattering and concentration are measured for each elution fraction, so molar mass and size can be determined regardless of elution position. This is especially true for species with non-spherical macromolecules such as biopolymers (OG1802) or bioconjugates (e.g., KSI-301, KSI-501). Such species do not typically elute in a way that can be accounted for by a set of column calibration standards.

[0170] In some embodiments, the SEC / MALS analysis includes a Waters HPLC system with an Alliance 2695 solvent delivery module and a Waters 2996 photodiode array detector with a Shodex SEC-HPLC column (7.8 x 300 mm), which is connected online with a Wyatt miniDawn TREOS and an Optilab T-rEX differential refractometer. Waters Empower™ software can be used to control the Waters HPLC system, and Wyatt ASTRA V 6.1.7.16 software can be used to acquire MALS data from the miniDawn TREOS, dn / dc data from the T-rEX detector, and mass recovery data using the A280 absorbance signal from the Waters 2996 photodiode array detector. SEC can be performed at 1 mL / min in 1x PBS pH 7.4, and upon sample injection, the MALS and RI signals can be analyzed by ASTRA software to determine the absolute molar mass (Mp, Mw, Mn) and polydispersity index (PDI). Additionally, the calculation also includes the input dn / dc values ​​of the polymer and protein (0.142 and 0.183, respectively). For the dn / dc value of KSI-301, dn / dc is calculated to be approximately 0.148 based on the weighted MW of the polymer and protein with the following formula: Complex dn / dc=0.142×[polymer Mw / (polymer Mw+protein Mw)]+0.183×[protein Mw / (polymer Mw+protein Mw)] (wherein the polymer Mw of OG1802 is 800 kDa and the protein Mw of OG1950 is 146 kDa).

[0171] "KSI-301" is a bioconjugate of a recombinant mammalian cell expressed full-length humanized anti-VEGF monoclonal antibody covalently linked to a branched, high molecular weight phosphorylcholine-based biopolymer. In some embodiments, KSI-301 is provided in single-use glass vials at a concentration of 50 mg / mL (based on antibody mass) as a preservative-free sterile aqueous solution. The antibody is supplied to NIH. Figure 8 shows the amino acid sequence of the antibody portion of KSI-301. KSI-301 is an anti-vascular endothelial growth factor (VEGF) biopharmaceutical with a long intraocular half-life. KSI-301 is a bioconjugate of two intermediates: (1) OG1950 antibody intermediate, a recombinant full-length humanized anti-huVEGFA monoclonal antibody, and (2) OG1802 biopolymer intermediate, a phosphorylcholine biopolymer. The addition of OG1802, an inert biopolymer, increases the size of the biologic and extends the ocular pharmacokinetics (PK) of KSI-301 beyond the PK of currently approved anti-huVEGF-A therapeutics. Preclinical studies of KSI-301 have shown that KSI-301 binds appropriately with high affinity to huVEGF-A and inhibits binding to huVEGF receptor 1 and receptor 2 (huVEGFR). This abolishes huVEGF-A-mediated function.

[0172] In some embodiments, the molecule administered in any one or more of the methods provided herein is any one of the molecules disclosed in U.S. Patent Publication No. 2017 / 0190766, the entire contents of which are incorporated by reference herein.

[0173] FIG. 8 shows the amino acid sequence of the antibody portion of KSI-301 with the terminal lysine removed. Removal of the terminal lysine is a post-translational modification in antibodies. The lysine residue at the C-terminus of the heavy chain of recombinant IgG is removed (often in large part) by carboxypeptidases endogenous to the CHO host cell during cell culture. For all antibody sequences described herein, although specific sequences (which may be longer) are provided for the exemplary antibodies disclosed herein, it will be understood that after expression of the protein chains, one to several amino acids at the amino or carboxy termini of the light and / or heavy chains, particularly the C-terminal lysine residue of the heavy chain, may be missing or derivatized in some or all of the molecule. Thus, in some embodiments, any of the antibody sequences provided herein may be modified by this lysine clip, which may include the modified version shown in FIG. 8 (where one or more of the underlined residues are removed). In some embodiments, the lysine in the heavy chain of FIG. 8 is removed. In some embodiments, the GK is removed. In some embodiments, the PGK is removed. In some embodiments, the SPGK is removed. As will be appreciated by those skilled in the art, across a population of molecules, the lysine clip may vary and not be complete. Thus, for example, 90%, 95%, 98%, 99%, or 100% of the molecules may have one or more clipped versions, while up to 10%, 5%, 2%, 1%, or 0% may be full length (including ranges defined between any two of the aforementioned values). Thus, as used herein, KSI-301 includes any one and all options of the heavy chain variants outlined in Figure 8.

[0174] In the present specification, whenever "anti-VEGF antibody" or "anti-VEGF antibody conjugate" is referred to, anti-VEGF protein, such as anti-VEGF fusion protein, such as Aflibercept, is also considered. Thus, as disclosed herein, whenever "anti-VEGF antibody conjugate" is referred to, anti-VEGF protein (e.g., Aflibercept) covalently bound to phosphorylcholine-containing biopolymer (e.g., OG1802) as disclosed herein is also considered. In various embodiments disclosed herein, reference to anti-VEGF antibody conjugate therapy also considers anti-VEGF protein, such as Aflibercept conjugate therapy. In various embodiments of the method of treating ocular disease disclosed herein, reference to anti-VEGF antibody conjugate also considers Aflibercept biopolymer conjugate.

[0175] Purification method In some embodiments, a method of purifying a product is provided, the method comprising washing the bound protein product with a chaotropic agent and then collecting the bound protein. The method may further include additional upstream and downstream purification processes.

[0176] In some embodiments, a method of purifying a product using affinity chromatography is provided, the method comprising loading an eluent onto an affinity chromatography matrix, which binds a protein of interest, and washing the affinity chromatography matrix with a buffer solution that includes a chaotropic agent.

[0177] In some embodiments, the target protein of interest is produced by a cell culture. In some embodiments, the cell culture is CHO cells. In some embodiments, the protein of interest is a bispecific antibody. In some embodiments, the bispecific antibody is specific for VEGF and IL-6. In some embodiments, the bispecific antibody is OG2072. In some embodiments, the protein of interest is an antibody complex. In some embodiments, the affinity chromatography matrix is ​​a Protein A chromatography matrix.

[0178] In some embodiments, the chaotropic agent in the buffered solution comprises one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0179] In some embodiments, the concentration of the magnesium salt is between 2 and 3.5 M. In some embodiments, the concentration of the magnesium chloride is about 2.8 M.

[0180] In some embodiments, the concentration of the magnesium salt is 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, or 3.5 M, or any value within the aforementioned range.

[0181] In some embodiments, the concentration of the calcium salt is 1-3 M. In some embodiments, the concentration of calcium chloride is about 2.0 M.

[0182] In some embodiments, the concentration of the calcium salt is 1, 1.5, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.7, or 3M, or any value within the aforementioned ranges.

[0183] In some embodiments, the concentration of the guanidinium salt is 0.05 to 3 M. In some embodiments, the concentration of the guanidinium hydrochloride is about 1.0 M.

[0184] In some embodiments, the concentration of the guanidinium salt is 0.05, 0.075, 0.1, 0.2, 0.25, 0.5, 0.75, 1, 1.5, 1.75, 2, 2.5, 2.75, or 3M, or any value within the aforementioned ranges.

[0185] In some embodiments, the buffer solution further comprises Tris.

[0186] In some embodiments, the concentration of Tris in the buffer solution is at least 5 mM.

[0187] In some embodiments, the concentration of Tris in the buffer solution is at least 10 mM.

[0188] In some embodiments, the concentration of Tris in the buffer solution is at least 25 mM, 30 mM M, 35 mM, 40 mM, 50 mM, or greater than 50 mM.

[0189] In some embodiments, the pH of the buffered solution is greater than 5.5.

[0190] In some embodiments, provided herein are methods for purifying a product from a load solution containing a protein and one or more impurities to reduce impurities, the method comprising passing the load solution through an affinity chromatography matrix, followed by at least one wash solution comprising a chaotropic salt, and collecting the protein using an elution solution.

[0191] In some embodiments, provided herein is a method for separating impurities in an eluate containing a protein of interest, comprising loading an eluate containing the protein of interest onto an affinity chromatography matrix, and washing the affinity chromatography matrix with one or more buffer solutions comprising one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0192] In some embodiments, provided herein is a method for separating impurities in an eluate comprising a protein of interest. The method includes loading an eluate comprising a protein of interest onto an affinity chromatography matrix, and washing the affinity chromatography matrix with one or more buffer solutions comprising one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts. In some embodiments, the method further includes washing the affinity chromatography matrix loaded with the eluate with a post-wash buffer solution after washing with the buffer solution. In some embodiments, washing the affinity chromatography matrix with a buffer solution removes nucleic acids, endotoxins, antifoam agents, other molecules, or other small molecules other than the target protein of interest. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes impurities while leaving the target protein of interest bound to the affinity chromatography matrix. In some embodiments, washing the affinity chromatography matrix with the buffer solution removes host cell proteins other than the target protein of interest. In some embodiments, the addition of a chaotropic agent to the buffer solution does not elute the target protein of interest. In some embodiments, the method further comprises one or more of viral inactivation, tangential flow filtration, diafiltration, ultrafiltration, ion exchange chromatography, or viral reduction filtration. In some embodiments, the eluate is produced in a bioreactor using an animal component-free cell culture. In some embodiments, the product is a purified protein of interest. In some embodiments, the impurities comprise host cell protein (HCP) impurities. In some embodiments, the eluate further comprises viral impurities. In some embodiments, the method further comprises removing viral impurities.In some embodiments, the method further comprises washing the loaded affinity chromatography matrix with a pre-wash buffer solution prior to washing with the buffer solution. In some embodiments, the method further comprises washing the loaded affinity chromatography matrix with a post-wash buffer solution after washing with the buffer solution. In some embodiments, the pre-wash buffer solution comprises sodium phosphate. In some embodiments, the pre-wash buffer solution comprises Tris and salt. In some embodiments of the method, the antibody complex comprises an anti-VEGF antibody complex comprising an anti-VEGF-A light chain and an anti-VEGF-A heavy chain, wherein the anti-VEGF-A antibody heavy chain comprises CDRH1 which is CDRH1 of SEQ ID NO: 172, CDRH2 which is CDRH2 of SEQ ID NO: 173, and CDRH3 which is CDRH3 of SEQ ID NO: 174, and the anti-VEGF-A antibody light chain comprises CDRL1 which is CDRL1 of SEQ ID NO: 199, CDRL2 which is CDRL2 of SEQ ID NO: 200, and CDRL3 which is CDRL3 of SEQ ID NO: 201. In some embodiments of the methods provided herein, the anti-VEGF antibody conjugate comprises an antibody conjugate comprising anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30, and the antibody is conjugated to the polymer at C449.

[0193] In some embodiments, a method of producing a product using affinity chromatography is provided, the method comprising loading an eluent containing a protein of interest onto an affinity chromatography matrix, and washing the affinity chromatography matrix with one or more buffer solutions containing one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0194] In some embodiments, a method for processing a product is provided. The method includes loading an eluent onto an affinity chromatography matrix. In some embodiments, the method further includes washing with a wash buffer containing a chaotropic salt and collecting an eluate, wherein the concentration of the chaotropic salt is increased from a first concentration to a second concentration, the eluate is collected in at least one fraction, and the at least one fraction includes the product of interest. In some embodiments, the method includes a concentration of the chaotropic salt at the first concentration of 0M and a concentration of the chaotropic salt at the second concentration of 4.0M. In some embodiments, the chaotropic salt is magnesium-based. In some embodiments, the chaotropic salt is magnesium chloride.

[0195] In some embodiments, a method of producing a product using affinity chromatography is provided, the method comprising loading an eluent containing a protein of interest onto an affinity chromatography matrix, first washing the affinity chromatography matrix with a first buffer comprising sodium phosphate and a salt, and second washing the affinity chromatography matrix with a second buffer comprising a chaotropic agent.

[0196] In some embodiments, a method of producing a product using affinity chromatography is provided, the method comprising loading an eluent containing a protein of interest onto an affinity chromatography matrix, performing a first wash with a first buffer comprising Tris and a salt, and performing a second wash with a second buffer comprising Tris and a chaotropic agent, wherein the chaotropic agent of the second buffer is not the same as the salt contained in the first buffer.

[0197] In some embodiments, a method of producing a product is provided, the method comprising collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, then washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0198] In some embodiments, a method of producing a product is provided, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, applying a buffer solution comprising a chaotropic salt to the affinity chromatography matrix, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0199] In some embodiments, a method of producing a product is provided, the method comprising: collecting a complex protein, the complex protein comprising an antibody bound to a complex polymer, loading the complex protein onto an affinity chromatography matrix, the affinity chromatography matrix binding to the complex protein, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the complex protein, and collecting an eluate, the eluate comprising the complex protein.

[0200] In some embodiments, a method of producing a product is provided, the method comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, eluting and collecting an eluate, the eluate containing the target protein of interest, and removing viral contaminants from the eluate.

[0201] In some embodiments, removing viral contaminants from the eluate comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration, ultrafiltration, and / or diafiltration.

[0202] In some embodiments, a method of producing a product is provided, the method comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, removing the chaotropic salt, and eluting and collecting an eluate, the eluate containing the target protein of interest.

[0203] In some embodiments, the eluate is further combined with an acceptable pharmaceutical excipient to form a pharmaceutical composition. In some embodiments, a buffer solution is added to the pharmaceutical composition. In some embodiments, a preservative solution is added to the pharmaceutical composition. In some embodiments, the pharmaceutical composition is further purified for intravitreal injection.

[0204] In some embodiments, a method of producing a product is provided, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting and collecting an eluate, the eluate comprising the target protein of interest, and removing viral contaminants from the eluate.

[0205] In some embodiments, a method of producing a product is provided, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0206] In some embodiments, a method of producing a product is provided, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, applying a buffer solution comprising a chaotropic salt to the affinity chromatography matrix, eluting the bound protein of interest, and collecting an eluate, the eluate containing the protein of interest.

[0207] In some embodiments, a method of producing a product is provided, the method comprising: collecting a complex protein, the complex protein comprising an antibody bound to a complex polymer, loading the complex protein onto an affinity chromatography matrix, the affinity chromatography matrix binding to the complex protein, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting the complex protein, and collecting an eluate, the eluate comprising the complex protein.

[0208] In some embodiments, a method of producing a product is provided, the method comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, eluting and collecting an eluate, the eluate containing the target protein of interest, and removing viral contaminants from the eluate.

[0209] The method may further include removing viral contaminants from the eluate comprising one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and / or diafiltration (DF). The method may further include further combining the eluate with an acceptable pharmaceutical excipient to form a pharmaceutical composition. The method may further include adding a buffer solution to the pharmaceutical composition. The method may further include adding a preservative solution to the pharmaceutical composition. The method may further include further purifying the pharmaceutical composition for intravitreal injection.

[0210] In some embodiments, a method of producing a product is provided, the method comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, removing the chaotropic salt, and eluting and collecting an eluate, the eluate containing the target protein of interest.

[0211] In some embodiments, a method of producing a product is provided, the method comprising: collecting a load solution, the load solution comprising a protein of interest, loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest, washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt, eluting and collecting an eluate, the eluate comprising the target protein of interest, and removing viral contaminants from the eluate.

[0212] In some embodiments, a method of producing a product is provided, comprising loading an eluate onto an affinity chromatography matrix, washing with a first wash buffer, washing with a second wash buffer comprising a chaotropic salt, washing with a third wash buffer, where the third wash buffer removes the chaotropic salt, and eluting with an elution buffer, collecting an eluate, where the eluate comprises the protein product.

[0213] The method may further include the first wash buffer comprising 50 mM sodium phosphate. The method may further include the first wash buffer further comprising about 250 mM sodium chloride. The method may further include the first wash buffer comprising Tris and salt. The method may further include removing viral contaminants from the eluate. The method may further include removing viral contaminants comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and diafiltration (DF). The method may further include the eluate comprising a protein of interest. The method may further include the protein of interest being an antibody. The method may further include the antibody being further conjugated to a polymer to form an antibody complex. The method may further include the antibody complex comprising a bispecific antibody. The method may further include the bispecific antibody comprising an anti-VEGF binding moiety and an anti-IL-6 binding moiety.

[0214] The method may further include the first wash buffer comprising 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any integer between 10 and 200 mM sodium phosphate. The method may further include the first wash buffer comprising a phosphate-based species. The method may further include the first wash buffer comprising 25 mM, 50 mM, 100 mM, 150 mM, 200 mM, or any integer between 25 and 200 mM sodium chloride. The method may further include a concentration of the chaotropic salt at the first concentration of 0 M and a concentration of the chaotropic salt at the second concentration of 4.0 M. The method may further include the chaotropic salt being magnesium-based. The method may further include the chaotropic salt being magnesium chloride.

[0215] In some embodiments, the antibody conjugate comprises a protein construct comprising an antagonist IL-6 antibody fused to VEGF Trap, the antibody comprising an isolated antagonist IL-6 antibody or fragment thereof. In some embodiments, the bispecific antibody comprises a VEGF-anti-IL-6 dual inhibitor, the VEGFR-anti-IL-6 dual inhibitor comprising a trap antibody fusion of an anti-IL-6 antibody or fragment thereof with an anti-VEGF Trap (VEGFR1 / 2), the dual inhibitor comprising at least one point mutation within the VEGFR sequence to reduce cleavage of the VEGFR protein, the antibody comprising a fragment antigen binding (Fab) region, a hinge region, and a fragment crystallizable (Fc) region, the anti-VEGF Trap being located at the N-terminus of the heavy chain of the antibody, the heavy chain comprising an IL-6 VH, or between the Fab region and the hinge region. In some embodiments, the antibody conjugate comprises an antibody conjugate comprising (1) an anti-VEGF-A antibody and (2) a phosphorylcholine-containing polymer, wherein the polymer is covalently attached to the antibody at a cysteine ​​outside the variable region of the antibody, wherein the cysteine ​​is added by recombinant DNA techniques. In some embodiments, the antibody conjugate comprises an isolated antagonist antibody that specifically binds complement factor D (CFD) and directly inhibits the proteolytic activity of CFD.

[0216] In some embodiments, the antibody conjugate has the structure of formula (I): [ka] wherein each heavy chain of the antibody is designated with the letter H and each light chain of the anti-CFD antibody is designated with the letter L, and the polymer is attached to the antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains; PC [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X=a) OR (R=H, methyl, ethyl, propyl, isopropyl), b) H, or c) any halide, including Br, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is about 1500 to about 3500±about 10% to about 20%.

[0217] Some embodiments include those shown in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. In some embodiments, the compositions disclosed herein comprise an anti-CFD antibody or variant thereof having a partial light chain sequence and a partial heavy chain sequence as set forth in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. In some embodiments, the antibodies (or binding fragments thereof) may comprise one or more of the CDRs set forth in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. In some embodiments, the antibodies (or binding fragments thereof) may comprise three or more of the CDRs set forth in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. In some embodiments, the antibodies (or binding fragments thereof) may include any and all of the six CDRs shown in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. The CDR sequences of various constructs are also set forth in Table 1A, Table 1B, Table 1C, Table 1D, and / or Table 1E. [Table 1-1]

Table 1-2

Table 1-3

Table 2-1

Table 2-2

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

[0218] The methods described herein may further include, wherein the anti-VEGF-A antibody conjugate has the structure: [ka]

[0219] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X=a) OR (R=H, methyl, ethyl, propyl, isopropyl), b) H, or c) any halide including Br; and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0220] In some embodiments, the anti-VEGF-A heavy chain comprises CDRH1 which is CDRH1 of SEQ ID NO: 172, CDRH2 which is CDRH2 of SEQ ID NO: 173, CDRH3 which is CDRH3 of SEQ ID NO: 174, and T at position 221; the anti-VEGF-A light chain comprises CDRL1 which is CDRL1 of SEQ ID NO: 199, CDRL2 which is CDRL2 of SEQ ID NO: 200, and CDRL3 which is CDRL3 of SEQ ID NO: 201, and Kabat position 4 is L. Optionally, the anti-VEGF-A heavy chain isotype is human IgG1. Optionally, the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, and the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30. In some embodiments, the antibody that binds to VEGF-A comprises a CDRH1 that is CDRH1 of SEQ ID NO: 172, a CDRH2 that is CDRH2 of SEQ ID NO: 173, a CDRH3 that is CDRH3 of SEQ ID NO: 174, a CDRL1 that is CDRL1 of SEQ ID NO: 199, a CDRL2 that is CDRL2 of SEQ ID NO: 200, and a CDRL3 that is CDRL3 of SEQ ID NO: 201, at least one of the following mutations (EU numbering): L234A, L235A, and G237A, and at least one of the following mutations (EU numbering): Q347C or L443C.

[0221] The methods described herein may further include, wherein the anti-IL-6 antibody conjugate has the structure: [ka] where each heavy chain of an anti-IL-6 antibody is designated with the letter H and each light chain of an anti-IL-6 antibody is designated with the letter L, the polymer is attached to the anti-IL-6 antibody (and / or Ab-Trap) via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X=a) OR (R=H, methyl, ethyl, propyl, isopropyl), b) H, or c) any halide including Br, and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X=OR, and R is a sugar, an aminoalkyl, or a mono-, poly-, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl-CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n -NR8R9, ester, alkoxycarbonyl, or aryloxycarbonyl, n is an integer from 1 to 6, and R7, R8, and R9 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, and a mono-substituted, poly-substituted, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and polyhalogenated alkyls, 5-membered rings, and 6-membered alkyl halides.

[0222] In some embodiments, anti-IL-6 antibody, or as otherwise used herein, "IL-6 antibody-VEGF Trap fusion", "IL-6 antibody-VEGF Trap", "Ab IL-6-VEGF Trap", "anti-IL-6-VEGF Trap", "VEGFR-anti-IL6", "VEGFR-anti-IL-6", "VEGF Trap-anti-IL6 antibody fusion (TAF)", "VEGF Trap-IL6", "VEGFR IL-6", "IL6-VEGFR", or synonyms or antonyms (e.g., "VEGF Trap-IL-6 Ab", "VEGF Trap-IL-6 antibody fusion"), refers to a fusion of an IL-6 antibody with VEGF Trap. An embodiment is shown in FIG. 9. When normally used, the order of the two terms may be interchanged. When specifically used, the order of the two terms indicates the relative positions of the components in the construct. "Ab-Trap", "IL-6 Ab-VEGF Trap", "Ab IL-6 VEGF Trap" or "Ab IL-6-Trap" or "anti-IL-6 VEGF Trap", "Trap-Ab", anti-IL-6-VEGFR, anti-IL6-VEGFR, or other synonyms or antonyms (e.g., "VEGF Trap-IL-6 Ab", "VEGF Trap-IL-6 antibody fusion") refer to the arrangement of an Ab fused to the relevant domain of a VEGF binding protein to provide a VEGF Trap. As discussed above, this section of the VEGF binding protein prevents VEGF from binding to the VEGF receptor. As explained herein, the arrangement (order) of the trap section and the antibody section can be varied. Thus, unless otherwise indicated explicitly or by context, the phraseology used herein with respect to an Ab-Trap (or Il-6 / VEGF Trap, etc.) fusion refers to all disclosed embodiments of the antibody and trap arrangement. Thus, unless otherwise stated, the phrase Ab-Trap (or Il-6 / VEGF Trap, etc.) refers to the embodiment on the left side of Figure 9, the embodiment on the right side of Figure 9, and both embodiments of Figure 9. Thus, for convenience, general language is presented as disclosing all three options.When an orientation is specifically indicated, it can be indicated, for example, by indicating that the "configuration" can be either Trap-Ab, Trap IL-6 Ab, VEGF Trap Ab IL-6, or VEGF Trap Ab IL6. Similarly, it will be understood that the contents of some of the examples of the present invention indicate a particular orientation or configuration of the molecules indicated by the contents of the examples. Both configurations (alternatives and combinations) are explicitly contemplated in all descriptions of fusion proteins provided herein. Also, by order, it can be seen that the phrase IL-6Ab when used in the context of a fusion protein includes both the option where the antibodies are contiguous (left side of FIG. 9) and the option where the TRAP is placed "within" the Ab (right side of FIG. 9). When the term "Ab" or "antibody" is used in the context of a fusion protein (or other synonyms), all three options (left side of FIG. 9, right side of FIG. 9, and both options) are encompassed unless otherwise indicated. In some embodiments, VEGF Trap is fused to IL-6 either at the N-terminus of the heavy chain containing IL-6 VH or between the hinge region and the CH1 domain of the heavy chain containing IL-6 VH. When used in designations to designate antibodies or fragments thereof, there is no difference between the designations Ab, antibody, "anti-", or other synonyms. There is no difference between the designations "IL-6", "IL6" and "IL-6". In this specification, when referring to fusion constructs with IL-6, the terms "VEGF", "VEGFR", "VEGF Trap", and "VEGFR Trap" are used interchangeably. These terms may have different meanings when used separately from the IL-6 fusion sequence, and the meanings vary depending on the context of the terms.

[0223] In some embodiments, the antibody may be linked or fused to a VEGF Trap sequence. In some embodiments, the trap sequence is as shown in Table 2A. In some embodiments, the sequence is at least 80% identical to that shown in Table 2A, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to that shown in 2A. In some embodiments, any of the VEGF Trap molecules of US Patent Publication No. 2015 / 0376271 may be used herein. In some embodiments, the VEGF Trap sequence is fused to IL-6 either at the N-terminus of the heavy chain containing the IL-6 VH (left side of FIG. 9) or between the hinge region and the CH1 domain of the heavy chain containing the IL-6 VH (right side of FIG. 9). Unless otherwise specified, in the embodiments provided herein where Ab-Trap fusions are discussed, both options are considered, alternately and simultaneously. In some embodiments, the term "Trap" refers to the full-length extracellular domain or any portion thereof, or a combination of portions of different VEGF receptors that can antagonize signaling between at least one VEGF and VEGFR. Preferably, the extracellular trap segment includes at least one domain from any one of VEGFR-1, -2, or -3, and more preferably includes at least two consecutive domains, such as D2 and D3. Optionally, the extracellular domain includes at least one domain from at least two different VEGFRs. A preferred extracellular domain includes or consists essentially of D2 of VEGFR-1 and D3 of VEGFR-2. [Table 7]

[0224] In some embodiments, the IL-6 Ab VEGF Trap construct may have any of the sequences shown in Table 2B or Table 2B-1. In some embodiments, the construct may be at least identical to the sequences of Table 2B or Table 2B-1 (e.g., 80, 85, 90, 95, 96, 97, 98, 99 or more). In some embodiments, the fusion protein may correspond to these percentages, except that the IL-6 domain of the antibody does not include one or more CDRs of Table 1E. In some embodiments, the fusion protein includes one or more of the sequences identified in Figure 5, e.g., one or more of the CDRs (including 2, 3, 4, 5, or 6 boxed CDRs) and / or the entire heavy and light chain variable regions with a VEGF Trap sequence (e.g., Table 2C). In some embodiments, the sequences may be fused directly to each other. In some embodiments, one or more flexible linking sequences or moieties may be used. A linking sequence may be placed between the Ab sequence and the VEGF Trap sequence. These sequences can be 5-30 amino acids in length. In some embodiments, the linking sequence may include G and S in a ratio of about 4:1. In some embodiments, the linker includes the following sequence: GGGGSGGGGS (SEQ ID NO: 748). In some embodiments, any flexible linker can be used. In some embodiments, the Fc portion of the IL-6Ab is IgG1. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 9] [Table 10] [Table 11]

[0225] In some embodiments, the antibody has one or more (or any) CDRs of Table 2E (including Table 2E1, Table 2E2, and / or Table 2E3). [Table 12] [Table 13] [Table 14]

[0226] In some embodiments, the IL-6 antagonist antibody comprises three CDRs from any one of the heavy chain variable regions set forth in Tables 2C, 2D, 2E1, 2E2, and / or 2E3. In some embodiments, the antibody comprises three CDRs from any one of the light chain variable regions set forth in Tables 2C, 2D, 2E1, 2E2, and / or 2E3. In some embodiments, the antibody comprises three CDRs from any one of the heavy chain variable regions set forth in Table 2C and three CDRs from any one of the light chain variable regions set forth in Table 2D. In some embodiments, the CDRs are one or more of those specified in Table 3 (including Tables 3A and / or 3B) or Table 4 (including Tables 4A and / or 4B) below. [Table 15] [Table 16] [Table 17] [Table 18]

[0227] In some embodiments, the antibody used to bind IL-6 may be an antibody that comprises one or more of the sequences in Tables 2C, 2D, 2E1, 2E2, 2E3, 3A, 3B, 4A, and / or 4B. In some embodiments, the antibody used to bind IL-6 may be an antibody that comprises three or more of the sequences in Tables 2C, 2D, 2E1, 2E2, 2E3, 3A, 3B, 4A, and / or 4B. In some embodiments, the antibody used to bind IL-6 may be an antibody that comprises six of the sequences in any one of Tables 2C, 2D, 2E1, 2E2, 2E3, 3A, 3B, 4A, and / or 4B. In some embodiments, the antibody that binds IL-6 may compete for binding with an antibody that comprises six of the CDRs specified in any one of Tables 2C, 2D, 2E1, 2E2, 2E3, 3A, 3B, 4A, and / or 4B.

[0228] The methods described herein may further include, wherein the antibody conjugate has a structure of formula (I): [ka] wherein each heavy chain of the anti-VEGF-A antibody is designated with the letter H and each light chain of the anti-VEGF-A antibody is designated with the letter L; The polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains; PC [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0229] In some embodiments, a method for producing a product is provided, comprising: harvesting a cell culture supernatant, the cell culture supernatant comprising a protein of interest; treating the cell culture supernatant with an eluent, the eluent comprising the protein of interest; loading the eluent onto an affinity chromatography matrix; washing with a first wash buffer comprising Tris or sodium phosphate; washing with a second wash buffer comprising a chaotropic salt; eluting with an elution buffer, the eluate comprising the protein product; inactivating viral contaminants present in the eluate with a low pH viral buffer to produce a viral inactivated eluate; filtering the viral inactivated eluate; performing at least one round of ion exchange chromatography on the viral inactivated eluate; and filtering the viral inactivated eluate to obtain a retentate, the retentate comprising the protein of interest.

[0230] In some embodiments, the method may further comprise the cell culture supernatant being produced in a bioreactor using an animal component-free cell culture. The method may further comprise processing the cell culture supernatant harvesting a cell product from the cell culture. The method may further comprise clarifying the cell culture to remove cells and cell debris. The method may further comprise the elution solution comprising the clarified cell culture supernatant. A method of purifying a protein using affinity chromatography comprising contacting a load solution with a medium, the medium being an affinity chromatography matrix that binds a protein of interest, washing the medium with a buffer solution comprising a chaotropic agent, the chaotropic agent being a salt, and contacting the washed medium with an elution solution under conditions suitable for eluting the protein of interest.

[0231] In some embodiments, a method of producing a product is provided, the method comprising applying a solution comprising a protein of interest to an affinity chromatography matrix, washing the affinity chromatography matrix with a first buffer, washing the affinity chromatography matrix with a second buffer comprising a chaotropic agent, washing the affinity chromatography matrix with a third buffer to remove the chaotropic agent, and eluting with an elution buffer, wherein the eluate is collected, the eluate comprising the protein product.

[0232] In some embodiments, a system for protein purification is provided that includes a column to which a first antigen binding protein is bound, a phosphate wash buffer comprising sodium phosphate and a salt, an intermediate wash buffer comprising Tris, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate.

[0233] In some embodiments, a system for protein purification is provided that includes a column having a first antigen binding protein bound thereto, a first Tris wash buffer comprising Tris and a salt, an intermediate Tris wash buffer, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate.

[0234] In some embodiments, the system may further include the column comprising a ligand for affinity chromatography. The system may further include the ligand comprising Protein A or Protein G. The system may further include the first wash buffer comprising sodium phosphate and a salt having a pH of 5.5 to 9.5. The system may further include the phosphate wash buffer comprising sodium phosphate and a salt comprising about 50 mM sodium phosphate. The system may further include the phosphate wash buffer comprising sodium phosphate and salts comprising about 250 mM sodium chloride. The system may further include the first Tris wash buffer comprising about 50 mM Tris. The system may further include the first Tris wash buffer comprising about 250 mM sodium chloride. The system may further include the intermediate Tris wash buffer comprising about 50 mM Tris. The system may further include the first Tris wash buffer having a pH of about 7.2. The system may further include the second wash buffer having a pH of about 7.8. The system may further include the magnesium chloride concentration in the second wash buffer being about 2.8 M. The system may further include the sodium formate concentration in the elution buffer being 10 mM.

[0235] In some embodiments, a system for protein purification is provided, the system comprising a column having a Protein A resin bound to an antibody, the antibody comprising the light and heavy chains of SEQ ID NO:2 and SEQ ID NO:1, respectively, a chaotropic wash buffer comprising a chaotropic salt, and an elution buffer comprising sodium formate.

[0236] The methods described herein may further include the protein of interest being a bispecific antibody. The methods described herein may further include the bispecific antibody being specific for VEGF and IL-6. The methods described herein may further include the protein of interest being an antibody complex. The methods described herein may further include the affinity chromatography matrix being a Protein A chromatography matrix. The methods described herein may further include the chaotropic agent in the buffer solution comprising a magnesium salt. The methods described herein may further include the magnesium salt having a concentration of 1.5-3.5M. The methods described herein may further include the chaotropic agent in the buffer solution comprising a calcium salt. The methods described herein may further include the calcium salt having a concentration of 1-3M. The methods described herein may further include the chaotropic agent in the buffer solution comprising a guanidinium salt. The methods described herein may further include the guanidinium salt having a concentration of 0.05-3M.

[0237] The methods described herein may further include the buffer solution further comprising Tris. The methods described herein may further include the concentration of Tris in the buffer solution being at least 5 mM. The methods described herein may further include the pH of the buffer solution being greater than 5.5. The methods described herein may further include the eluate further comprising a viral impurity. The methods described herein may further include removing the viral impurity. The methods described herein may further include inactivating the viral impurity. The methods described herein may further include washing the load-loaded affinity chromatography matrix with a pre-wash buffer solution prior to washing with the buffer solution. The methods described herein may further include washing the eluate-loaded affinity chromatography matrix with a post-wash buffer solution after washing with the buffer solution. The methods described herein may further include the pre-wash buffer solution comprising sodium phosphate. The methods described herein may further include the pre-wash buffer solution comprising Tris and a salt.

[0238] The methods described herein further comprise the step of: , may further include. [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0239] The methods described herein include comprising administering to the patient an antibody complex comprising an anti-VEGF-A light chain and an anti-VEGF light chain. and wherein the anti-VEGF-A antibody heavy chain comprises a CDRH1 that is CDRH1 of SEQ ID NO: 172, a CDRH2 that is CDRH2 of SEQ ID NO: 173, and a CDRH3 that is CDRH3 of SEQ ID NO: 174, and wherein the anti-VEGF-A antibody light chain comprises a CDRL1 that is CDRL1 of SEQ ID NO: 199, a CDRL2 that is CDRL2 of SEQ ID NO: 200, and a CDRL3 that is CDRL3 of SEQ ID NO: 201. The methods described herein may further include an anti-VEGF antibody conjugate comprising an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, and the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30, and the antibody is conjugated to the polymer with C449. The methods described herein may further include the target protein of interest being produced by a cell culture. The methods described herein may further include the cell culture comprising CHO cells. The methods described herein may further include, after washing with the buffer solution, washing the affinity chromatography matrix loaded with the eluent with a post-wash buffer solution. The methods described herein may further include washing the affinity chromatography matrix with a buffer solution to remove nucleic acids, endotoxins, antifoaming agents, or other small molecules other than the target protein of interest. The methods described herein may further include washing the affinity chromatography matrix with the buffer solution to remove impurities while leaving the target protein of interest bound to the affinity chromatography matrix. The methods described herein may further include washing the affinity chromatography matrix with the buffer solution to remove host cell proteins other than the target protein of interest.The methods described herein may further include, where the addition of a chaotropic agent to the buffer solution does not elute the target protein of interest. The methods described herein may further include one or more of viral inactivation, tangential flow filtration, diafiltration, ultrafiltration, ion exchange chromatography, or viral reduction filtration. The methods described herein may further include, where the eluate is produced in a bioreactor using an animal component-free cell culture. The methods described herein may further include, where the product is a protein of interest. The methods described herein may further include, where the impurities include host cell protein impurities.

[0240] As disclosed herein, the purification methods of the present disclosure are effective in removing impurities such as host cell proteins (HCPs). As detailed in the Examples, the methods are effective in reducing HCPs in the wash eluate while achieving a high percent yield of the protein of interest and a high concentration of the protein of interest. For example, in various embodiments, the methods described herein result in a percent yield of the protein of interest of greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0241] As disclosed herein, the purification methods of the present disclosure are effective in removing impurities from affinity chromatography-based purification methods. In some embodiments, the chromatography-based purification methods include methods based on specific macromolecular binding interactions between an analyte in the mobile phase and a ligand in the stationary phase. In some embodiments, the methods are effective in reducing impurities in the wash eluate while simultaneously achieving a high percent yield of the species of interest. In some embodiments, the methods are effective in achieving a high concentration of the species of interest while reducing impurities in the wash eluate. For example, in various embodiments, the methods described herein result in a percent yield of the species of interest that is greater than 80%, greater than 81%, greater than 82%, greater than 83%, greater than 84%, greater than 85%, greater than 86%, greater than 87%, greater than 88%, greater than 89%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, or greater than 99%.

[0242] As disclosed herein, the purification methods of the present disclosure are effective in removing impurities from samples comprising monoclonal antibodies (mAbs). In some embodiments, residues comprising mAbs may be altered or modified to change the overall shape or structure of the antibody. In some embodiments, residues comprising mAbs may be altered or modified to modulate binding efficiency to a binding partner. In some embodiments, residues comprising mAbs may be altered or modified to modulate binding efficiency at an antigen binding site. In some embodiments, residues comprising mAbs may be altered or modified to modulate binding efficiency. In some embodiments, the altered residue may be an engineered cysteine. In some embodiments, the monoclonal antibody comprises a specific binding domain comprising a domain specific for VEGF. In some embodiments, the specific binding domain comprises one or more extracellular components of one or more VEGF receptors. In some embodiments, the binding domain further comprises an Fc portion.

[0243] Table 5 lists common species of high-risk HCPs that may be present and that can be removed with current methods. The table also includes potential downstream impacts to formulated pharmaceutical products. Without current technology, some HCP species are difficult to remove due to physicochemical properties approaching those of other desirable molecular species. In some embodiments, any one or more of the embodiments provided herein can be used to remove any one or more of the species in Table 5. In some embodiments, the product of interest is selected from one or more of the species in Table 5. In some embodiments, the protein of interest is selected from one or more of the species in Table 5. HCPs that are difficult to remove can be classified as proteins with a molecular weight of greater than 15 kDa or a pI between 7.3 and 9.3. [Table 19-1] [Table 19-2]

[0244] With reference to FIG. 1, a process 100 for purifying OG2072 antibody from an animal component-free cell culture process is disclosed. The process includes three chromatography steps and two TFF (tangential flow filtration) steps, as well as low pH viral inactivation. First, clarified cell supernatant is harvested at 110. Next, affinity chromatography 120 is performed on the clarified cell supernatant. Low pH viral inactivation 130 followed by intermediate filtration 140 is performed on the output eluate collected during affinity chromatography. Further chromatography is performed, including anion exchange chromatography 150 followed by cation exchange chromatography 160. Virus reduction filtration 170 is performed, followed by final ultrafiltration / diafiltration 180.

[0245] Low pH viral inactivation may include holding the solution at pH 3.5 for 240 minutes followed by neutralization to pH 7. Low pH viral inactivation may alternatively include holding at pH 3.5 for 60 minutes followed by stepwise neutralization to pH 5.5, 6, or 6.5. MabSelect SuRe LX affinity chromatography (MSS LX) may be followed by a viral inactivation / neutralization step, followed by a first TFF (TFF1) to prepare the antibody for Sartobind Q anion exchange chromatography (AEX chromatography). This is followed by POROS XS cation exchange chromatography (CEX chromatography) and viral reduction filtration (Planova 20N). POROS XS involves binding with 10 mM sodium phosphate, pH 5, 40 mM sodium chloride, acetic acid as additive (<15 mS / cm), followed by gradient elution with 50 mM sodium acetate, pH 6, 10 mM sodium chloride to 50 mM sodium acetate, pH 6, 300 mM sodium chloride in 10 CV. During POROS XS chromatography, an additional wash with 50 mM sodium acetate, pH 5 and 150 mM sodium chloride can be performed, followed by a gradual increase in sodium chloride to 165, 180, 195, or 210 mM. A gradient may be applied to the POROS XS column step, providing a gradient from 150 mM sodium chloride at pH 5 to 400 mM sodium chloride at 30 cm bed height in 10 CV, or a gradient from 50 mM sodium chloride at pH 6 to 350 mM sodium chloride at 30 cm bed height in 12 CV. A wash step can be performed prior to the gradient elution. Washing steps include 2 CV of 50 mM sodium acetate, pH 5.0, 10 mM sodium chloride, followed by 5 CV of 18.8 mM sodium phosphate, pH 7.0, 22.5 mM sodium chloride, followed by 3 CV of 50 mM sodium acetate, pH 5.0, 10 mM sodium chloride, followed by 2 CV of 50 mM sodium acetate, pH 6.0, 10 mM sodium chloride. Finally, a second TFF (TFF2) is performed to formulate the antibody and obtain the antibody intermediate.

[0246] FIG. 2 illustrates a biomolecule purification process 200. First, a cell culture is grown and cultivated using a fermentation process 210. The cell culture is then collected at harvest 220 to obtain a clarified cell concentrate. The clarified cell concentrate is then purified using affinity chromatography and viral inactivation / neutralization 230. The output of the process is a product retentate. The collected product retentate is then subjected to tangential flow filtration 240, followed by anion exchange chromatography 250 and cation exchange chromatography 260, in sequence. The treated product retentate is then subjected to viral reduction filtration 270 and a second tangential flow filtration 280 to produce a purified product.

[0247] With reference to FIG. 3, a protocol was developed to assess the percent reduction of measurable HCP species to assess the yield and purity of desired molecular species. A process 300 for column purification of OG2072 antibody from an animal component-free cell culture process is disclosed. The process includes equilibration 310 followed by loading 320 of clarified cell culture fluid (CCCF) onto the column. The column is then treated with a series of washes including wash 1 330, followed by wash 2 340, wash 3 350, and wash 4 360. Wash 2 340 may include sub-washes 340A, 340B, and 340C. Wash 4 360 is followed by elution 370, whereby post-elution buffer 380 is flushed through the column to regenerate the resin. The process includes equilibration 310 of a Protein A column with 50 mM sodium phosphate and 250 mM sodium chloride at pH 7, followed by a load 320 step where the column is loaded with clarified cell culture fluid (CCCF), followed by a series of washes 330-360 including a first wash 330 with 50 mM sodium phosphate and 250 mM sodium chloride at pH 7. Wash 2 340 includes various wash buffers and conditions as described in Table 6C. Wash 3 350 includes a wash with 50 mM sodium phosphate and 2 M sodium chloride at pH 7, and Wash 4 360 includes a wash with 50 mM sodium phosphate and 250 mM sodium chloride at pH 7. Elution 370 is with 10 mM sodium formate at pH 3.5, followed by post-elution with 100 mM citric acid at pH 2.1 passed through the column.

[0248] In some embodiments, any one or more of the sequences of the designated amino acid sequences in any one or more of 11-16 of Figure 8 can be substituted for the corresponding structure of any of the other embodiments provided herein, or can be replaced with any of the other sequences provided herein. In some embodiments, the construct is a VEGFR-anti-IL-6 configuration, including any one of SEQ ID NO:1A-SEQ ID NO:1D (Figure 12), linked to a linker (e.g., Figure 12, SEQ ID NO:2A), linked to a heavy chain IL-6 sequence (e.g., Figure 13, SEQ ID NO:3A or SEQ ID NO:3B), or linked to a light chain sequence (e.g., Figure 15, SEQ ID NO:4A). In some embodiments, any one of SEQ ID NO:1A-SEQ ID NO:1D (Figure 12) can be combined with a linker (Figure 12), and can be combined with a heavy chain anti-IL-6 sequence (Figures 13-14, SEQ ID NO:3A-SEQ ID NO:3I) and a light chain anti-IL-6 sequence (Figure 15, SEQ ID NO:4A-SEQ ID NO:4C). In some embodiments, any other sequence that corresponds to a particular unit of the constructs provided herein can be substituted or exchanged for any of these units.

[0249] In some embodiments, any of the anti-IL-6 antibody constructs are contemplated for use as a composition, component, or therapeutic agent, including, for example, those contained in Tables 2C, 2D, 2E, 0.3, and / or 0.4, and Figures 8, 11-16 of the embodiments provided herein.

[0250] In some embodiments, the fusion protein comprises a mutation at position 94 of the VEGF Trap sequence, at position 95 of the VEGF Trap sequence, or at T94I and H95I of the VEGF Trap sequence.

[0251] In some embodiments, a VEGFR-anti-IL-6 dual inhibitor is provided. The VEGFR-anti-IL-6 dual inhibitor comprises a trap antibody fusion of an anti-IL-6 antibody and a VEGF (VEGFR1 / 2) Trap, and the dual inhibitor comprises at least one point mutation in the VEGFR sequence to reduce cleavage of the VEGFR sequence. In some embodiments, the molecular weight of the VEGFR-anti-IL-6 dual inhibitor is 1.0 MDa.

[0252] In some embodiments, the dual VEGR-anti-IL-6 inhibitor is used to treat an inflammatory retinal disease.

[0253] In some embodiments, the VEGFR-anti-IL-6 dual inhibitor comprises a constant heavy chain, a constant light chain, an antigen binding fragment, a fragment crystallizable (Fc), a vascular endothelial growth factor receptor (VEGFR), a variable heavy chain, a variable light chain, and a CDR region.

[0254] In some embodiments, the anti-IL-6 heavy chain variable region sequence can be selected from SEQ ID NOs: 7, 8, 9, 10, 11, 12, or 13 of FIG.

[0255] In some embodiments, the VEGF Trap sequence can be selected from at least one of SEQ ID NOs: 14, 15, 16, or 17 of FIG.

[0256] In some embodiments, in the case of a VEGR-anti-IL-6 dual inhibitor, the heavy chain sequence of the anti-IL-6 molecule can be selected from at least one of SEQ ID NOs: 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 in Figures 13-14.

[0257] In some embodiments, in the case of a VEGFR-anti-IL-6 dual inhibitor, the light chain sequence of the anti-IL-6 molecule comprises at least one, two, or three light chain CDRs from at least one of SEQ ID NOs: 28, 29, or 30 of FIG. 15.

[0258] In some embodiments, in the case of a VEGFR-anti-IL-6 dual inhibitor, the heavy chain sequence of the anti-IL-6 molecule comprises at least one, two, or three heavy chain CDRs from at least one of options 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, or 5I in Figure 16.

[0259] In some embodiments, the VEGFR-anti-IL-6 dual inhibitor comprises a VEGFR-Fc sequence from at least one of SEQ ID NOs: 85, 86, 87, or 88 of FIG.

[0260] In some embodiments, the dual VEGFR-anti-IL-6 inhibitor comprises one or more of the sequences in Figures 11-18.

[0261] In some embodiments, the VEGFR-anti-IL-6 dual inhibitor comprises an IL-6 VH, an IL-6 VL, an IL-6 Fc, a VEGF Trap, and a linker. In some embodiments, the IL-6 VH comprises a sequence from the IL6VH sequence of FIG. 16 or FIG. 13-FIG. 14. In some embodiments, the IL-6 VL comprises a sequence from the IL6VL sequence of FIG. 15. In some embodiments, the Fc comprises a sequence from the Fc sequence of FIG. 16. In some embodiments, the VEGF Trap comprises a sequence from the VEGF Trap sequence.

[0262] In some embodiments, a protein construct is provided that includes at least three heavy chain CDRs, at least three light chain CDRs, a VEGF Trap sequence, and a linker sequence, each sequence selected from the sequences in Figures 11-18.

[0263] In some embodiments, a fusion protein is provided that includes an IL-6 VH, an IL-6 VL, an IL-6 Fc, and a VEGF Trap, the fusion protein altering proliferation of HUVEC, in some embodiments, each sequence is selected from the sequences in Figures 11-18.

[0264] In other embodiments, the methods of the invention result in a percent reduction of HCP contaminants in the eluate by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold.

[0265] Thus, in one aspect, the disclosure provides a method of producing a purified protein (e.g., an antibody, an antibody fragment, or a protein that comprises an Fc region (e.g., an Fc fusion protein)) using an affinity chromatography (AC) matrix to which a protein of interest is bound, comprising washing the AC matrix with a wash solution comprising magnesium chloride or an equivalent chaotropic agent. In some embodiments, the wash solution comprising magnesium chloride elutes the protein of interest from the AC matrix. The previous pH is about 7.8, about 6, 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, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.9, about 8, 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. In some embodiments, the wash solution comprises about 1M, about 1.1M, about 1.2M, about 1.3M, about 1.4M, about 1.5M, about 1.6M, about 1.7M, about 1.8M, about 1.9M, about 2M, about 2.1M, about 2.2M, about 2.3M, about 2.4M, about 2.5M, about 2.6M, about 2.7M, about 2.8M, about 2.9M, about 3M, about 3.1M, about 3.2M, about 3.3M, about 3.4M, about 3.5M, about 3.6M, about 3.7M, about 3.8M, about 3.9M, about 4M, about 4.1M, about 4.2M, or greater than 4.2M magnesium chloride.

[0266] antibody complex Provided herein are anti-VEGF antibodies (including anti-VEGF proteins, e.g., aflibercept) and conjugates thereof. In some embodiments, the antibodies themselves are distinct from and provide superior results to other anti-VEGF agents. In some embodiments, the anti-VEGF antibody conjugates exhibit surprising advantages over the activity of other antibodies and / or other antibody conjugates.

[0267] In some embodiments, the anti-VEGF antibody conjugate is KSI-301; (1) Anti-VEGF-A antibody and (2) A phosphorylcholine-containing polymer covalently attached to an anti-VEGF-A antibody at a cysteine ​​outside a variable region of the anti-VEGF-A antibody, wherein the cysteine ​​replaces a non-cysteine ​​amino acid present at the same position in the sequence, the anti-VEGF-A antibody comprising a light chain and a heavy chain, the heavy chain comprising an Fc region, the cysteine ​​being within the Fc region of the heavy chain, the heavy chain comprising at least one sequence of SEQ ID NO: 270, and the light chain comprising at least one sequence of SEQ ID NO: 275, (or any of its variants in FIG. 8), wherein the antibody conjugate has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via a sulfhydryl having EU number C443, which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0268] Historically, conjugation of a molecule to a protein has often resulted in a decrease in the binding interaction between the protein and the intended target. In some embodiments, the same level of decrease as expected is not always observed when conjugated to a location outside the active site. Evidence presented herein shows the opposite effect to what was expected. In some embodiments, without being limited by theory, the complex may be superior to the antibody alone. For example, the interaction of a ligand with its particular receptor is often driven by stereospecific interactions of the ligand and receptor, dictated by the interaction of hydrophilic amino acids on the ligand with hydrophilic amino acids on the receptor, with water molecules being central to those interactions. At the same time, this hydrophilic stereospecificity is further enhanced by deemphasizing and / or suppressing nonspecific hydrophobic interactions that are usually mediated / generated by hydrophobic amino acids with each other.

[0269] In some embodiments, anti-VEGF antibody conjugates are provided that can inhibit the interaction between a VEGF ligand ("VEGFL") and a VEGF receptor ("VEGFR") by at least 90%. For example, at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or substantially all of the interaction between a VEGFR and a VEGFL can be inhibited. In some embodiments, the noted inhibition occurs at saturating concentrations. In some embodiments, anti-VEGF antibody conjugates are provided that inhibit the interaction between a VEGF ligand and a VEGF receptor by at least 95%. An example of the superiority of such inhibition includes the ability of an anti-VEGF antibody bioconjugate (antibody conjugate provided herein, e.g., KSI-301) to inhibit to a greater extent than LUCENTIS® (Ranibizumab) or AVASTIN® (Bevacizumab) or antibody OG1950 (unconjugated). Indeed, this result is unexpected: while the addition of a polymer to an antibody (to form an antibody complex) would be expected to have some or no adverse effect on antibody binding / activity, it is unexpected that this would actually enhance the inhibitory capacity of the antibody.

[0270] In some embodiments, the antibody or conjugate thereof inhibits at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity and / or interaction between VEGFR and VEGFL. In some embodiments, the IC50 value will be 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 nM, or less than one or more of the foregoing values. In some embodiments, the KD is 2×10 -13 , 1×10 -13 , 1×10 -12 , 1×10 -11 , or 1×10 -10In some embodiments, the IC50 value will be less than 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or any of the foregoing values.

[0271] In some embodiments, anti-VEGF antibodies are provided that inhibit the interaction between VEGF ligands and VEGF receptors by at least 90%. For example, they can inhibit at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or substantially all of the interaction between VEGFR and VEGFL. As an example of the superiority of such inhibition, OG1950 (and antibodies provided herein) is capable of inhibiting to a greater extent than LUCENTIS® (Ranibizumab) or AVASTIN® (Bevacizumab).

[0272] In some embodiments, other antibodies, such as Lucentis® (ranibizumab) or Avastin® (bevacizumab), can be conjugated to one or more polymers described herein by one or more processes described herein. In some embodiments, any antibody or fragment thereof can be conjugated to one or more polymers described herein by one or more processes described herein.

[0273] In some embodiments, the antibody comprises a heavy chain amino acid variable region comprising at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, and a heavy chain amino acid variable region comprising at least one of SEQ ID NO: 91. and a light chain amino acid variable region comprising at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262 and / or at least one of SEQ ID NOs: 91-93 and 28-30. In some embodiments, the antibody is conjugated to one or more of the polymers provided herein. In some embodiments, the conjugate antibody is at least 90% identical to at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262 and / or at least one of SEQ ID NOs: 91-93 and 28-30. In some embodiments, the antibody comprises six CDRs contained in at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262 and at least one of SEQ ID NOs: 91-93 and 28-30, and a point mutation at L443C (or 449C, EU numbering). In some embodiments, the complex antibody is at least 90% identical to at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262 and / or at least one of SEQ ID NOs: 91-93 and 28-30, and contains the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0274] In some embodiments, an antibody that binds to VEGF-A is provided, the antibody comprising a CDRH1 that is CDRH1 of SEQ ID NO: 172, a CDRH2 that is CDRH2 of SEQ ID NO: 173, a CDRH3 that is CDRH3 of SEQ ID NO: 174, a CDRL1 that is CDRL1 of SEQ ID NO: 199, a CDRL2 that is CDRL2 of SEQ ID NO: 200, a CDRL3 that is CDRL3 of SEQ ID NO: 201, at least one of the following mutations (EU numbering): L234A, L235A, and G237A, and at least one of the following mutations (EU numbering): Q347C or L443C.

[0275] As will be appreciated by one of skill in the art, in light of this specification, any of the antibodies provided herein can be conjugated to any of the polymers provided herein and / or any of the antibodies provided herein can have a cysteine ​​addition to allow for site-specific conjugation to a polymer.

[0276] "VEGF" or "vascular endothelial growth factor" refers to a human vascular endothelial growth factor that affects the angiogenesis or vasculogenesis process. In particular, the term VEGF refers to any member of a class of growth factors that (i) binds to a VEGF receptor, such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4), (ii) activates tyrosine kinase activity associated with the VEGF receptor, and (iii) thereby affects the angiogenesis or vasculogenesis process.

[0277] The VEGF factor family consists of five related glycoproteins, VEGF-A (also known as VPE), VEGF-B, VEGF-C, VEGF-D, and PGF (placental growth factor). Of these, VEGF-A is the most well-studied and is the target of antiangiogenic therapy (Ferrara et al, (2003) Nat. Med. 9:669-676). VEGF-A is generated by both alternative splicing and proteolysis. 206 , VEGF-A 189 , VEGF-A 165 , and VEGF-A 121 It exists as a variety of isotypes, including heparin and neuropilin. The isoforms differ in their ability to bind to non-signaling binding proteins called heparin and neuropilin. All isoforms are biologically active as dimers.

[0278] The various effects of VEGF are mediated by the binding of VEGF, such as VEGF-A (P15692), VEGF-B (P49766), VEGF-C (P49767), and VEGF-D (Q43915), to receptor tyrosine kinases (RTKs). VEGF family receptors belong to the class VRTKs, each of which has seven Ig-like domains in its extracellular domain (ECD). In humans, VEGF binds to three types of RTKs: VEGFR-1 (Flt-1) (P17948), VEGFR-2 (KDR, Flk-1) (P935968), and VEGFR-3 (Flt-4) (P35916). Unless the context requires otherwise, VEGF refers to any of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF, or any naturally occurring isoform, naturally occurring variant, or induced variant having at least 90%, 95%, 98%, 99%, or 100% sequence identity to the native form. In some embodiments, such VEGF is human VEGF. Similarly, reference to VEGFR refers to any of VEGR-1, VEGR-2, or VEGR-3, including any naturally occurring isoform or naturally occurring variant, or any induced variant having at least 90%, 95%, 98%, 99%, or 100% sequence identity to the native sequence.

[0279] VEGF antagonist therapy is approved for the treatment of certain cancers and wet AMD. Bevacizumab (AVASTIN, Genentech / Roche) is a humanized murine monoclonal antibody that binds to and neutralizes human VEGF, specifically all isoforms of VEGF-A and biologically active proteolytic fragments of VEGF-A. See, e.g., Ferrara N, Hillan KJ, Gerber HP, Novotny W. 2004. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 3(5):391-400. Bevacizumab is approved for the treatment of certain cancers.

[0280] The bevacizumab variable light chain CDRs are L 1: SASQDISNYLN (SEQ ID NO: 749), CDR L 2: FTSSLHS (SEQ ID NO: 750), and CDR L 3: QQYSTVPWT (SEQ ID NO: 751). The bevacizumab variable heavy chain CDRs are H 1: GYTFTNYGMN (SEQ ID NO: 752), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 753), and CDR H 3:YPHYYGSSHWYFDV (SEQ ID NO: 755). The CDRs are defined by Kabat, with the combined Kabat / Chothia definition being used for CDRH1. In some embodiments, cysteines can be added to the bevacizumab sequence, and the antibody (and / or variants containing the six CDRs of bevacizumab) can be attached to any one or more of the polymers described herein. In some embodiments, the CDRs of bevacizumab or ranibizumab can be used in the compositions and methods provided herein.

[0281] Another anti-VEGF molecule, ranibizumab (LUCENTIS®, Genentech / Roche), derived from the same murine monoclonal antibody as bevacizumab, has been approved for the treatment of wet AMD. Ranibizumab is an antibody fragment or Fab. Ranibizumab was generated by affinity maturation of the variable heavy and light chains of bevacizumab. In some embodiments, cysteines can be added to the ranibizumab sequence and the antibody (and / or variants containing the six CDRs of ranibizumab) can be attached to any one or more of the polymers described herein.

[0282] The CDRs of ranibizumab are the same as those of bevacizumab, except that they were refined after affinity maturation. The ranibizumab variable light chain CDRs are L 1: SASQDISNYLN (SEQ ID NO: 749), CDR L2: FTSSLHS (SEQ ID NO: 750), and CDR L 3: QQYSTVPWT (SEQ ID NO: 751). The ranibizumab variable heavy chain CDRs are H 1: GYDFTHYGMN (SEQ ID NO: 754), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 753), and CDR H 3: YPYYYGTSHWYFDV (sequence number 756).

[0283] In some embodiments, an antibody conjugate is provided in which an anti-VEGF-A antibody is attached to a phosphorylcholine-containing polymer at a cysteine ​​outside the variable region of the antibody, the cysteine ​​being recombinant. The polymer is attached by DNA techniques. In some embodiments, the polymer is linked to a single cysteine. In some embodiments, "added by recombinant DNA techniques" means that a cysteine ​​residue replaces a non-cysteine ​​amino acid present at the same position in a known or existing antibody or consensus antibody sequence. Thus, for example, if the antibody is an IgG1 and there is a leucine at EU position 443 of the heavy chain, the leucine is replaced by a cysteine ​​(L443C, EU numbering, or 449C) by recombinant DNA techniques. Similarly, the native IgG1 sequence at EU numbering position 347 is Q (glutamine), and Q is replaced by a cysteine ​​by recombinant DNA techniques to generate Q347C.

[0284] In some embodiments, the anti-VEGF-A antibody comprises a light chain and a heavy chain, and the heavy chain has an Fc region. In some embodiments, the cysteine ​​is in the Fc region and the anti-VEGF-A antibody is an immunoglobulin G (IgG). In some embodiments, the anti-VEGF-A heavy chain has a CDR H 1: GYDFTHYGMN (SEQ ID NO: 754), CDR H 2: WINTYTGEPTYAADFKR (SEQ ID NO: 753), and CDR H 3: YPYYYGTSHWYFDV (SEQ ID NO: 756), position 221 is T, and the anti-VEGF-A light chain has the CDR L1: SASQDISNYLN (SEQ ID NO: 749), CDR L 2: FTSSLHS (SEQ ID NO: 750), and CDR L 3: Has QQYSTVPWT (sequence number 751), with L at position 4 of Kabat.

[0285] In some embodiments, the anti-VEGF-A heavy chain isotype is IgG1. In some embodiments, the IgG1 constant domain comprises one or more mutations relative to the IgG1 constant domain to modulate effector function. In some embodiments, the effector function mutations are one or more of the following: E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (EU numbering), where X is any natural or unnatural amino acid. In some embodiments, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the antibody conjugate comprises the following mutations: L234A, L235A, and G237A (EU numbering).

[0286] In some embodiments, the cysteine ​​residue is in an anti-VEGF-A heavy chain and is Q347C (EU numbering) or L443C (EU numbering). In some embodiments, the cysteine ​​residue is L443C (or 449C, EU numbering). In some embodiments, the anti-VEGF-A heavy chain sequence is SEQ ID NO:7-13, 19-27, 89, 90, and 256-262, and the light chain sequence comprises at least one of SEQ ID NO:91-93 and 28-30.

[0287] In some embodiments, the phosphorylcholine-containing polymer comprises 2-(methacryloyloxyethyl)-2′-(trimethylammonium) ethyl phosphate (MPC) monomers as shown below: [ka] (The polymer contains the following repeat units: [ka] ; (n is an integer from 1 to 3000, and the wavy lines indicate the connection points between monomer units within the polymer).

[0288] In some embodiments, the polymer has three or more arms or is synthesized using an initiator that includes three or more polymer initiation sites. In some embodiments, the polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms or is synthesized using an initiator that includes two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. More preferably, the polymer has three, six, or nine arms or is synthesized using an initiator that includes three, six, or nine polymer initiation sites. In some embodiments, the polymer has nine arms or is synthesized using an initiator that includes nine polymer initiation sites.

[0289] In some embodiments, the molecular weight of the polymer added is about 300,000 to about 1,750,000 Da (SEC-MAL). In some embodiments, the molecular weight of the polymer is about 500,000 to about 1,000,000 Da. In some embodiments, the molecular weight of the polymer is about 600,000 to about 900,000 Da. In some embodiments, the molecular weight of the polymer is about 750,000 to about 850,000 Da. In some embodiments, the molecular weight of the polymer is about 800,000 to about 850,000 Da. In some embodiments, the molecular weight of the polymer is about 750,000 to about 800,000 Da.

[0290] In some embodiments, any of the antibodies described herein can be further conjugated with a polymer to form a bioconjugate. The molecular weight of the bioconjugate (SEC-MAL as a whole) is in the range of about 350,000 to 2,000,000 daltons, e.g., about 450,000 to 1,900,000 daltons, about 550,000 to 1,800,000 daltons, about 650,000 to 1,700,000 daltons, about 750,000 to 1,600,000 daltons, about 850,000 to 1,500,000 daltons. 1,000,000 Daltons, approximately 900,000 to 1,400,000 Daltons, approximately 950,000 to 1,300,000 Daltons, approximately 900,000 to 1,000,000 Daltons, approximately 1,000,000 to 1,300,000 Daltons, approximately 850,000 to 1,300,000 Daltons, approximately 850,000 to 1,000,000 Daltons, approximately 1,000,000 to 1,200,000 Daltons.

[0291] In some embodiments, the antibody conjugate is purified. In some embodiments, the polymer is polydisperse on the side of the antibody conjugate, i.e., the polymer PDI is not 1.0. In some embodiments, the PDI is less than 1.5. In some embodiments, the PDI is less than 1.4. In some embodiments, the PDI is less than 1.3. In some embodiments, the PDI is less than 1.2. In some embodiments, the PDI is less than 1.1.

[0292] In some embodiments, the antibody conjugate comprises an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the anti-VEGF-A heavy chain sequence comprises SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, the light chain sequence comprises at least one of SEQ ID NOs: 91-93 and 28-30, and the antibody is conjugated to the polymer only by C449. In some embodiments, the polymer has 9 arms and a molecular weight of about 600,000 to about 1,000,000 Da.

[0293] In some embodiments, the antibody conjugate comprises an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, the anti-VEGF-A antibody heavy chain sequences are SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, the anti-VEGF-A antibody light chain sequences are SEQ ID NOs: 91-93 and 28-30, and the antibody is conjugated to the polymer only at C443 (or 449C, EU numbering). In some embodiments, the polymer has 9 arms and a molecular weight of about 600,000 to about 1,000,000 Da.

[0294] In some embodiments, the antibody conjugate has the structure of formula (I): [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody through a sulfhydryl of C449, which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X is -OR and R is a sugar, an aminoalkyl, or a mono-, poly-, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl-CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n -NR8R9, ester, alkoxycarbonyl, or aryloxycarbonyl, n is an integer from 1 to 6, and R7, R8, and R9 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, and a mono-substituted, poly-substituted, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and polyhalogenated alkyls, 5-membered rings, and 6-membered alkyl halides.

[0295] In some embodiments, the antibody conjugate has the structure of Formula (I): [ka] wherein each heavy chain of the anti-VEGF-A antibody is designated by the letter H and each heavy chain of the anti-VEGF-A antibody is designated by the letter H. The light chain is represented by the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (or 449C, EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 500. In some embodiments, X is OR, and R is a sugar, an aminoalkyl, or a mono-, poly-, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24 Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyls, -CO-O-R7, carbonyl-CCO-R7, -CO-NR8R9, -(CH2) n -COOR7, -CO-(CH) n -COOR7, -(CH2) n -NR8R9, ester, alkoxycarbonyl, or aryloxycarbonyl, n is an integer from 1 to 6, and R7, R8, and R9 are each independently selected from the group consisting of a hydrogen atom, a halogen atom, and a mono-substituted, poly-substituted, or unsubstituted variant of the following residues: saturated C1-C 24 Alkyl, unsaturated C2-C 24 Alkenyl or C2-C 24Alkynyl, acyl, acyloxy, alkyloxycarbonyloxy, aryloxycarbonyloxy, cycloalkyl, cycloalkenyl, alkoxy, cycloalkoxy, aryl, heteroaryl, arylalkoxycarbonyl, alkoxycarbonylacyl, amino, aminocarbonyl, aminocarboyloxy, nitro, azido, phenyl, hydroxy, alkylthio, arylthio, oxysulfonyl, carboxy, cyano, and alkyl halides including polyhalogenated alkyl, 5-membered ring, and 6-membered ring. In some embodiments, this construct is designated KSI-301.

[0296] In some embodiments, the antibody conjugate is present as a liquid formulation. In some embodiments, the antibody conjugate is combined with a pharma- ceutically acceptable carrier. In some embodiments, any of the methods provided herein can use the following drug formulations: a) 12.5 mM sodium phosphate buffer, pH 6.5, containing 0.025% (w / w) polysorbate 20, at a concentration of 50 mg / mL (based on antibody mass), corresponding to a total antibody-biopolymer conjugate mass of 324 mg / mL, containing 50 mg / m LOG1950 antibody intermediate and 274 mg / m LOG1802 biopolymer intermediate; b) 10-15 mM sodium phosphate buffer, containing 0.01-0.5% (w / w) polysorbate 20, at a concentration of 50 mg / mL (based on antibody mass), corresponding to a total antibody-biopolymer conjugate mass of 324 mg / mL, containing 50 mg / m LOG1950 antibody intermediate and 274 mg / m LOG1802 biopolymer intermediate; Sodium phosphate buffer, pH 6.5, at a concentration of about 50 mg / mL (based on antibody mass), corresponding to about 324 mg / mL of total mass of antibody-biopolymer complex; c) 12.5 mM sodium phosphate buffer, optionally containing 0.025% (w / w) polysorbate 20, pH 6.5, at a concentration of 50 mg / mL (based on antibody mass), corresponding to 324 mg / mL of total mass of antibody-biopolymer complex, containing 50 mg / mLOG1950 antibody intermediate and 274 mg / mLOG1802 biopolymer intermediate; d) polysorbate 20 50 mg / mL (based on antibody mass), equivalent to about 324 mg / mL of total mass of antibody-biopolymer complex, containing about 50 mg / mL of OG1950 antibody intermediate and about 274 mg / mL of OG1802 biopolymer intermediate; e) 10-15 mM sodium phosphate buffer containing 0.01-0.5% (w / w) polysorbate 20, pH 6.5, and a concentration of about 40-55 mg / mL (based on antibody mass), equivalent to about 259-356 mg / mL of total mass of antibody-biopolymer complex. f) 10-15 mM sodium phosphate buffer containing 0.01-0.5% (w / w) polysorbate 20, pH 6.5, at a concentration of about 45-52.5 mg / mL (based on antibody mass), equivalent to about 292-340 mg / mL of total mass of antibody-biopolymer complex; or g) 10-15 mM sodium phosphate buffer containing 0.01-0.5% (w / w) polysorbate 20, pH 6.5, at a concentration of about 40-55 mg / mL (based on antibody mass), equivalent to about 259-356 mg / mL of total mass of antibody-biopolymer complex.In any of these formulations, the antibody can use the VH and VL of Figure 8 (or one, two, three, four, five, or all six CDRs within these VH and / or VL sequences), e.g., OG1950 (e.g., SEQ ID NOs: 1, 15-18, and 2).

[0297] In some embodiments, an anti-VEGF-A antibody is provided, wherein the anti-VEGF-A antibody heavy chain has at least the following CDR sequences: CDR H 1: CDRH1, CDR of SEQ ID NO: 172 H 2: CDRH2 and CDRs of SEQ ID NO: 173 H 3: CDRH3 of SEQ ID NO: 174. In some embodiments, the anti-VEGF-A heavy chain has these CDRs and further has a threonine (T) at position 221. In some embodiments, the anti-VEGF-A light chain has at least the following CDRs: CDR L 1: CDRL1, CDR of SEQ ID NO: 199 L 2: CDRL2 and CDR of SEQ ID NO: 200 L 3: CDRL3 of SEQ ID NO: 201. In some embodiments, the anti-VEGF-A antibody has these CDRs and further has a leucine (L) at Kabat position 4. In some embodiments, the anti-VEGF-A antibody heavy chain isotype is IgG1, having CH1, hinge, CH2, and CH3 domains. In some embodiments, the light chain isotype is kappa. In some embodiments, the anti-VEGF antibody conjugate (e.g., KSI-301) construct includes one or more of these CDRs.

[0298] In some embodiments, the IgG1 domain of the anti-VEGF-A antibody has one or more mutations that modulate effector function, such as ADCC, ADCP, CDC. In some embodiments, the IgG1 mutations reduce effector function. In some embodiments, amino acids used for effector function mutations include E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X (EU numbering), where X is any natural or unnatural amino acid. In some embodiments, mutations include one or more of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the anti-VEGF-A heavy chain has the following mutations: L234A, L235A, and G237A (EU numbering). In some embodiments, the number of effector function mutations relative to the native human IgG1 sequence is 10 or less. In some embodiments, the number of effector function mutations relative to the native human IgG1 sequence is 5, 4, 3, 2, or 1 or less. In some embodiments, the antibody has reduced Fcγ binding and / or complement C1q binding, thereby reducing the ability of the antibody to provide effector function. This is particularly advantageous for ophthalmic indications / diseases.

[0299] In some embodiments, the anti-VEGF-A antibody comprises one or more of the following amino acid mutations: L234A, L235A, G237A (EU numbering), and L443C (or 449C, EU numbering).

[0300] In some embodiments, the anti-VEGF-A antibody is a human immunoglobulin G (IgG1) or a portion thereof.

[0301] In some embodiments, the VEGF-A antibody comprises a heavy chain constant domain comprising one or more mutations that reduce immune-mediated effector function.

[0302] In some embodiments, an anti-VEGF-A antibody is provided. The anti-VEGF antibody comprises a CDR comprising a sequence that is CDRH1 of SEQ ID NO: 172. H 1. A CDR comprising a sequence which is CDRH2 of SEQ ID NO: 173 H 2. A CDR comprising a sequence which is CDRH3 of SEQ ID NO: 174 H 3. A CDR comprising a sequence which is CDRL1 of SEQ ID NO: 199 L 1. A CDR comprising a sequence which is CDRL2 of SEQ ID NO: 200 L 2, and a CDR comprising a sequence which is CDRL3 of SEQ ID NO: 201. L The heavy chain comprises:

[0303] Alternatively, the IgG domain may be IgG2, IgG3, or IgG4, or a mixture in which the constant region is formed from one or more of these isotypes (e.g., CH1 region from IgG2 or IgG4, hinge, CH2, and CH3 regions from IgG1). Such domains may contain mutations that reduce and / or modulate effector function of one or more of the EU positions mentioned for IgG1. Human IgG2 and IgG4 have reduced effector function compared to human IgG1 and IgG3.

[0304] The anti-VEGF-A heavy chain has a cysteine ​​residue added as a mutation by recombinant DNA technology that can be used to attach a half-life extending moiety. In some embodiments, the mutation is Q347C (EU numbering) and / or L443C (or 449C, EU numbering). In some embodiments, the mutation is L443C (or 449C, EU numbering). In some embodiments, the stoichiometry of antibody to polymer is 1:1. In other words, the conjugate is one molecule of antibody bound to one molecule of polymer.

[0305] The half-life of an anti-VEGF-A antibody can be extended by the addition of a "half-life ("half-life") extending moiety" or "half-life ("half-life") extending group". Half-life extending moieties include peptides and proteins that can be expressed in frame (or optionally chemically conjugated) with the biological agent of interest, as well as a variety of polymers that can be attached or conjugated to one or more amino acid side chain or terminal functional groups, such as -SH, -OH, -COOH, -CONH2, -NH2, or to one or more N- and / or O-glycan structures. Half-life extending moieties typically serve to extend the circulating half-life of a biological agent in vivo.

[0306] Moieties that extend the half-life of peptides / proteins include Fc fusions (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525-31), human serum albumin (HAS) fusions (Yeh P, Landais D, Lemaitre M, et al. Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc Natl Acad Sci USA. 1992. 89:1904-08), carboxy-terminal peptide (CTP) fusions (Fares FA, Suganuma N. Nishimori K, et al. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci USA. 1992. 89:4304-08), and gene fusions of non-exact repeat peptide sequences (XTEN) fusions (Schellenberger V, Wang CW, Geething NC, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009. 27:1186-90), elastin-like peptide (ELPylation) (MCpherson DT, Morrow C, Minehan DS, et al. Production and purification of a recombinant elastomeric polypeptide, G (VPGVG19-VPGV, from Escherichia coli. Biotechnol Prog. 1992. 8:347-52), human transferrin fusion (Prior CP, Lai CH, Sadehghi H et al.Modified transferrin fusion proteins. International Publication No. WO 2004 / 020405 (2004)), proline-alanine-serine (PASylation) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and / or vitro stability. International Publication No. WO 2008 / 155134 (2008)), homoamino acid polymer (HAPylation) (Schlapschy M, Theobald I, Mack H, et al. Fusion of a recombinant antibody fragment with a homo-amino acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007. 20:273-84), and gelatin-like protein (GLK) fusion (Huang YS, Wen XF, Zaro JL, et al. Engineering a pharmacologically superior form of granulocyte-colony-stimulating-factor by fusion with gelatin-like protein polymer. Eur J. Pharm Biopharm. 2010. 72:435-41).

[0307] Polymeric half-life extending moieties include polyethylene glycol (PEG), branched PEG, polyPEG® (Warwick Effect Polymers, Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylates, polyvinylpyrrolidone, polyphosphazenes, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF), zwitterionic polymers, phosphorylcholine-containing polymers, as well as MPC, poly(Gly x -Ser y ), hyaluronic acid (HA), heparosan polymer (HEP), fleximer, dextran, and polymers including polysialic acid.

[0308] In one embodiment, half-life extending moieties can be attached to antibodies through free amino groups on the protein using N-hydroxysuccinimide (NHS) esters. Reagents targeted for attachment to amine groups can react randomly with the ε-amine groups of lysines, the α-amine group of the N-terminal amino acid, and the δ-amine groups of histidines.

[0309] However, the anti-VEGF-A antibodies disclosed herein have numerous amine groups available for polymer attachment, and therefore, attachment of a polymer to the free amino groups may adversely affect the ability of the antibody protein to bind to VEGF.

[0310] In some embodiments, the half-life extending moiety is coupled to one or more free SH groups using any suitable thiol-reactive chemistry, including but not limited to maleimide chemistry, or coupling of a polymeric hydrazide or polymeric amine to the carbohydrate moiety of the antibody after prior oxidation. In some embodiments, maleimide coupling is used. In some embodiments, the coupling occurs at a naturally occurring cysteine ​​or a cysteine ​​introduced by genetic engineering.

[0311] In some embodiments, the polymer is covalently attached to a cysteine ​​residue introduced into the anti-VEGF-A antibody by site-directed mutagenesis. In some embodiments, the cysteine ​​residue is used in the Fc portion of the antibody. In some embodiments, the site for introducing a cysteine ​​residue into the Fc region is described in WO 2013 / 093809, U.S. Patent No. 7,521,541, WO 2008 / 020827, U.S. Patent No. 8,008,453, U.S. Patent No. 8,455,622, and U.S. Patent Application Publication No. 2012 / 0213705, which are incorporated herein by reference for all purposes. In some embodiments, the cysteine ​​mutations are Q347C (EU numbering) and L443C, which refer to human IgG heavy chain according to EU numbering.

[0312] In some embodiments, a conjugate of an antibody and a high molecular weight polymer that functions as a half-life extender is provided. In some embodiments, the conjugate comprises an antibody bound to a zwitterionic polymer, the polymer being formed from one or more monomeric units, at least one of the monomeric units having a zwitterionic group. In some embodiments, the zwitterionic group is phosphorylcholine.

[0313] In some embodiments, one of the monomer units is HEMA-PC, hi some embodiments, the polymer is synthesized from a single monomer, HEMA-PC.

[0314] In some embodiments, some antibody conjugates have 2, 3, or more polymer arms, where the monomer is HEMA-PC. In some embodiments, the conjugates have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms, where the monomer is HEMA-PC. In some embodiments, the conjugates have 3, 6, or 9 arms. In some embodiments, the conjugates have 9 arms.

[0315] In some embodiments, the polymer / antibody conjugate has a polymer portion with a molecular weight of 100,000-1,500,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight of 500,000-1,000,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight of 600,000-800,000 Da. In some embodiments, the conjugate comprises a polymer moiety having a molecular weight of 600,000-850,000 Da and has nine arms. When an antibody bound to a polymer has a given molecular weight, that molecular weight is the molecular weight of the protein, including its associated carbohydrate moiety, plus the molecular weight of the polymer.

[0316] In some embodiments, an anti-VEGF-A antibody is provided having a HEMA-PC polymer with a molecular weight, as measured by Mw, between about 100 kDa and 1650 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is between about 500 kDa and 1000 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is between about 600 kDa and about 900 kDa. In some embodiments, the molecular weight, as measured by Mw, of the polymer is 750 kDa ± 15%.

[0317] In some embodiments, the polymer is made from an initiator suitable for ATRP having one or more polymer initiation sites. In some embodiments, the polymer initiation site is a 2-bromoisobutyric acid site. In some embodiments, the initiator has 3 or more polymer initiation sites. In some embodiments, the initiator has 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. In some embodiments, the initiator has 3, 6, or 9 polymer initiation sites. In some embodiments, the initiator has 9 polymer initiation sites. In some embodiments, the initiator is OG1786.

[0318] The anti-VEGF-A antibody can be produced by recombinant expression, which includes: (i) producing recombinant DNA by genetic engineering, (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells, for example by transfection, electroporation, or microinjection, (iii) culturing the transformed cells, (iv) expressing the antibody (for example, constitutively or inducibly), and (v) isolating the antibody, for example from the culture medium or by harvesting the transformed cells, and (vi) obtaining a purified antibody.

[0319] Anti-VEGF-A antibodies can be produced by expression in suitable prokaryotic or eukaryotic host systems characterized by the production of pharmacologically acceptable antibody molecules. Eukaryotic cells include mammalian cells such as CHO, COS, HEK293, BHK, SK-Hip, and HepG2. Other suitable expression systems include prokaryotes (e.g., E. coli using the pET / BL21 expression system), yeast (Saccharomyces cerevisiae and / or Pichia pastoris systems), and insect cells.

[0320] A wide variety of vectors can be used to prepare the antibodies disclosed herein, selected from eukaryotic and prokaryotic expression vectors. Prokaryotic expression vectors include, but are not limited to, plasmids such as preset, pet, and pad, and promoters used in prokaryotic expression vectors include, but are not limited to, one or more of lac, trc, trp, recA, and araBAD. Vectors for eukaryotic expression include, but are not limited to, (i) for expression in yeast, vectors such as, but not limited to, pAO, pPIC, pYES, or pMET, using promoters such as, but not limited to, AOX1, GAP, GAL1, or AUG1; (ii) for expression in insect cells, vectors such as, but not limited to, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as, but not limited to, PH, p10, MT, Ac5, OpIE2, gp64, or polh; and (iii) for expression in mammalian cells, vectors such as, but not limited to, pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV, as well as vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, or retrovirus, using promoters such as, in one embodiment, CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and beta-actin.

[0321] Methods for conjugating proteins to polymers In some embodiments, a method is provided for preparing a therapeutic protein half-life extender conjugate, comprising the step of coupling a therapeutic protein to a half-life extender having a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinyl sulfones, orthopyridyl disulfides, and iodoacetamides to obtain a therapeutic protein half-life extender conjugate.

[0322] In some embodiments, a method is provided for preparing an anti-VEGF antibody conjugate, e.g., KSI-301, from OG1950. The method includes reducing OG1950 protein with a 30-fold molar excess of TCEP reducing agent. After reduction, the antibody is oxidized to produce a decapped OG1950 antibody in which disulfide bonds are formed between (and within) the light and heavy chains naturally present in the antibody. Excipients are then added, followed by a 3-10-fold molar excess of a maleimide biopolymer to conjugate OG1950. The biopolymer is linked to the OG1950 antibody via a covalent thiol ether bond. After conjugation, the anti-VEGF antibody conjugate (e.g., KSI-301) is purified to remove both unbound antibody and polymer.

[0323] The proteins and processes described above may be modified. Thus, in some embodiments, a process for preparing a binding protein (not necessarily an antibody or an anti-VEGF antibody) is provided. The process includes reducing one or more cysteines in a protein to form a decapped protein in solution. After reducing one or more cysteines, the decapped protein is reoxidized to restore at least one disulfide bond in the reduced protein while leaving the engineered cysteine ​​residue in the protein in the form of a free thiol, ensuring the formation of a reoxidized decapped protein in solution. Then, at least one excipient is added to the solution. The excipient reduces the precipitation of the protein by the polymer. After adding the excipient, a polymer is added to the solution to bind to the reoxidized decapped protein at the engineered cysteine ​​residue to form the binding protein.

[0324] In some embodiments, the molar excess of the reducing agent can be varied to any amount that works. In some embodiments, a 10, 20, 30, 40, 50, 60, 70, 80, or 90-fold molar excess of the reducing agent (not necessarily TCEP in all embodiments) can be used. In some embodiments, any antibody (therapeutic or otherwise) can be used. In some embodiments, a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15-fold molar excess of maleimide biopolymer can be used. In some embodiments, there is an excess of decapped protein relative to the polymer. In some embodiments, the amount of reoxidized decapped protein or decapped protein is less than the amount of polymer. In some embodiments, the amount of reoxidized decapped protein or decapped protein is 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the amount of polymer. In some embodiments, 2.5 to 4.5 times as much polymer as protein is used, measured in molar excess. In some embodiments, 10 to 20 times as much polymer as protein is used, measured by mass. In some embodiments, the amount of reduced antibody is greater than the amount of polymer. In some embodiments, the amount of polymer is greater than the amount of reduced antibody.

[0325] In some embodiments, a purification step is optional.

[0326] In some embodiments, a method for making an antibody conjugate comprises conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer. In some embodiments, the method comprises conjugating an anti-VEGF-A antibody to a phosphorylcholine-containing polymer. The anti-VEGF-A antibody comprises an amino acid residue added by recombinant DNA techniques. In some embodiments, the added amino acid residue is a cysteine ​​residue. In some embodiments, the cysteine ​​residue is added outside the variable region of the antibody. The cysteine ​​residue can be added to either the heavy or light chain of the antibody.

[0327] In some embodiments, the polymer comprises or consists of a phosphorylcholine-containing polymer. In some embodiments, the phosphorylcholine-containing polymer comprises a sulfhydryl-specific reactive group selected from the group consisting of maleimide, vinylsulfone, orthopyridyl disulfide, and iodoacetamide. In some embodiments, the sulfhydryl-specific reactive group on the phosphorylcholine-containing polymer reacts with a cysteine ​​residue on the anti-VEGF-A antibody to form an antibody conjugate.

[0328] In some embodiments, the bound protein may be an antibody, an antibody-protein fusion, or a binding fragment thereof. In some embodiments, the protein is not an antibody, but is an enzyme, ligand, receptor, or other protein, or a mutant or variant thereof. In some embodiments, the native protein contains at least one disulfide bond and at least one non-native cysteine.

[0329] In some embodiments, the excipient may be an acid or a base. In some embodiments, the excipient is a detergent, a sugar, or a charged amino acid. In some embodiments, the excipient helps to keep the protein in solution during binding to the polymer. In some embodiments, an excipient is added to a solution containing the protein prior to adding a polymer to the solution containing the protein.

[0330] In some embodiments, the reaction occurs under aqueous conditions between about pH 5 and about pH 9. In some embodiments, the reaction occurs at pH 6.0 to pH 8.5, pH 6.5 to pH 8.0, or pH 7.0 to pH 7.5. In some embodiments, the reaction occurs at pH 8 to pH 9.

[0331] In some embodiments, the polymer is conjugated to the protein at between 2° C. and 37° C. In some embodiments, the conjugation occurs at between 0° C. and 40° C., between 5° C. and 35° C., between 10° C. and 30° C., and between 15° C. and 25° C. In some embodiments, the polymer is conjugated at between 5° C. and 10° C.

[0332] In some embodiments, the binding proteins described herein can be contacted with an ion exchange medium or a hydrophobic interaction or affinity chromatography medium for purification (to remove complexed proteins from uncomplexed proteins). In some embodiments, the ion exchange medium, hydrophobic interaction chromatography, and / or affinity chromatography medium separates the binding proteins from free polymer and reoxidized decapped proteins.

[0333] In some embodiments, the polymers disclosed herein may comprise one or more of a zwitterion, a phosphorylcholine, or a PEG linker that crosslinks the center of the polymer branch point to a maleimide functional group. In some embodiments, any of the polymers provided herein can be added to a protein via the methods provided herein.

[0334] In some embodiments, any of the proteins provided herein can be conjugated to any of the polymers provided herein via one or more of the methods provided herein.

[0335] In some embodiments, the processes provided herein allow for larger scale processing to make and purify proteins and / or antibody conjugates. In some embodiments, the volumes used are at least 1 liter, e.g., 1, 10, 100, 1,000, 5,000, or 10,000 liters or more. In some embodiments, the amount of antibody conjugate produced and / or purified can be 0.1, 1, 10, 100, 1000, 2000, 2500, or 3000 grams or more.

[0336] In some embodiments, the therapeutic protein may be any of the anti-VEGF-A antibodies described herein to which a cysteine ​​residue has been added by recombinant DNA techniques. In some embodiments, the anti-VEGF-A antibody heavy chain has the following CDR sequences: H 1: CDRH1, CDR of SEQ ID NO: 172 H 2: CDRH2 and CDRs of SEQ ID NO: 173 H 3: CDRH3 of SEQ ID NO: 174. The heavy chain may also have a threonine (T) at position 221. In some embodiments, the anti-VEGF-A light chain has the following CDRs: L 1: CDRL1, CDR of SEQ ID NO: 199 L 2: CDRL2 and CDR of SEQ ID NO: 200 L 3: CDRL3 of SEQ ID NO: 201. The anti-VEGF-A light chain may also have a leucine (L) at position 4 of Kabat.

[0337] In some embodiments, the anti-VEGF-A antibody is an IgG1. In some embodiments, the heavy chain has one or more mutations to modulate effector function. In some embodiments, the mutations occur at one or more of the following amino acid positions (EU numbering): E233, L234, L235, G236, G237, A327, A330, and P331. In some embodiments, the mutations are selected from the group consisting of E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the mutations are L234A, L235A, and G237A (EU numbering).

[0338] In some embodiments, the cysteine ​​residues added to the therapeutic protein by the recombinant DNA technology will not participate in Cys-Cys disulfide bond pairing. In this regard, the therapeutic protein may be a dimer. For example, an intact anti-VEGF-A antibody has two light chains and two heavy chains. For example, when a Cys residue is introduced into the heavy chain, the intact antibody will have two cysteines introduced at the same position, which will form intrachain disulfide bonds. If the introduced cysteine ​​residue forms or has a tendency to form a Cys-Cys disulfide bond, the introduced Cys residue will not be useful for binding. Methods for avoiding positions that cause intrachain disulfide pairing in the heavy and light chains are known in the art. See, for example, U.S. Patent Application No. 2015 / 0158952.

[0339] In some embodiments, the cysteine ​​residue introduced by recombinant DNA techniques is selected from the group consisting of Q347C and L443C (EU numbering). In some embodiments, the cysteine ​​residue is L443C (or 449C, EU numbering). In some embodiments, the heavy chain of the antibody has an amino acid sequence set forth in at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, and the light chain has an amino acid sequence set forth in at least one of SEQ ID NOs: 91-93 and 28-30.

[0340] In some embodiments, the sulfhydryl-specific reactive group is a maleimide.

[0341] In some embodiments, the half-life extending moiety is polyethylene glycol (PEG), branched PEG, polyPEG® (Warwick Effect Polymers Ltd, Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrates, polysaccharides, pullulan, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic anhydride, polystyrene-co-maleic anhydride, poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF), zwitterionic polymers, phosphorylcholine containing polymers, and polymers comprising 2-methacryloyloxy-2'-ethyltrimethylammonium phosphate (MPC).

[0342] In some embodiments, the half-life extending moiety is a zwitterionic polymer. In some embodiments, the zwitterion is phosphorylcholine, i.e., a phosphorylcholine-containing polymer. In some embodiments, the polymer is composed of MPC units.

[0343] In some embodiments, the MPC polymer has 3 or more arms. In some embodiments, the MPC polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms. In some embodiments, the MPC polymer has 3, 6, or 9 arms. In some embodiments, the MPC polymer has 9 arms. In some embodiments, the polymer is synthesized using an initiator that includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more polymer initiation sites.

[0344] In some embodiments, the molecular weight of the MPC polymer is about 300,000 to 1,750,000 Da. In some embodiments, the molecular weight of the MPC polymer is about 500,000 to 1,000,000 Da or about 600,000 to 900,000 Da.

[0345] In some embodiments, the method of preparing a conjugate of a therapeutic protein with a half-life extending moiety includes the additional step of contacting the therapeutic protein with a thiol reducing agent under conditions that generate reduced cysteine ​​sulfhydryl groups. As mentioned above, it is preferred that the cysteine ​​residues added by recombinant DNA technology are unpaired, i.e., not involved in Cys-Cys intrachain disulfide bonds or not substantially involved in such bonds. However, such Cys residues that are not involved in Cys-Cys disulfide bonds and are free to bind are known to react with free cysteines in the culture medium to form disulfide adducts. See, for example, WO 2009 / 052249. Cysteines thus derivatized are not available for conjugation. To release the newly added cysteines from the disulfide adducts, the purified protein is treated with a reducing agent, e.g., dithiothreitol. However, such treatment with a reducing agent reduces all of the cysteine ​​residues in the therapeutic protein. This includes native cysteines involved in inter- and intra-chain Cys-Cys disulfide bonds, which are usually critical for protein stability and activity and therefore need to be reformed. In some embodiments, all native (e.g., inter- and intra-chain) Cys-Cys disulfides are reformed.

[0346] After removal and reduction of cysteine ​​disulfide adducts, the therapeutic protein is exposed to oxidizing conditions and / or an oxidizing agent for a period of time, e.g., overnight, to reform native inter- and intrachain disulfide residues. In some embodiments, overnight exposure to air can accomplish reformation of native disulfide bonds. In some embodiments, an oxidizing agent is used to restore native disulfides. In some embodiments, the oxidizing agent is selected from the group consisting of aqueous CuSO4 and dehydroascorbic acid (DHAA). In some embodiments, the oxidizing agent is DHAA. In some embodiments, the range of DHAA used is within the range of 5-30 equivalents. In some embodiments, the range is 10-20 equivalents. In some embodiments, the range is 15 equivalents.

[0347] In some embodiments, the thiol reducing agent is selected from the group consisting of tris[2-carboxyethyl]phosphine hydrochloride (TCEP), dithiothreitol (DTT), dithioerythritol (DTE), sodium borohydride (NaBH4), sodium cyanoborohydride (NaCNBH3), β-mercaptoethanol (BME), cysteine ​​hydrochloride, and cysteine. In some embodiments, the thiol reducing agent is TCEP.

[0348] In some embodiments, the concentration of the thiol reducing agent is in 1-100-fold molar excess over the concentration of the therapeutic protein. In some embodiments, the concentration of the thiol reducing agent is in 20-50-fold molar excess over the concentration of the therapeutic protein. In some embodiments, the thiol reducing agent is removed after incubation with the therapeutic protein and prior to oxidation of the therapeutic protein.

[0349] In some embodiments, the method of conjugating a therapeutic protein to a half-life extending moiety comprises the further step of purifying the therapeutic protein conjugate after conjugation, hi some embodiments, said therapeutic protein conjugate is purified using a technique selected from the group consisting of ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, and affinity chromatography, or a combination thereof.

[0350] In some embodiments, the therapeutic protein conjugate retains at least 20% of the biological activity compared to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 50% of the biological activity compared to the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate retains at least 90% of the biological activity compared to the native therapeutic protein.

[0351] In some embodiments, the therapeutic protein conjugate has a longer half-life than the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has a half-life that is at least 1.5 times longer than the unconjugated therapeutic protein. In some embodiments, the therapeutic protein conjugate has a half-life that is at least 5 times longer than the unconjugated therapeutic protein.

[0352] In some embodiments, the zwitterionic polymer of the method of conjugating a therapeutic protein to a half-life extending moiety is a radically polymerizable monomer having a zwitterionic group. The method comprises the further step of polymerizing the free radically polymerizable zwitterionic monomer in a polymerization medium to obtain a polymer, the medium comprising the radically polymerizable zwitterionic monomer, the transition metal catalyst M t (q-1)+ (In the formula, M t is a transition metal, q is a higher oxidation state of the metal, and q-1 is a lower oxidation state of the metal, and the metal catalyst is M t (q-1)+ X' (q-1)and X′ is a counter ion or group, or the transition metal catalyst is provided as a salt of the form t q+ X' q In situ, the inactive metal salt of the present invention may be provided by providing the inactive metal salt of the present invention together with a reducing agent capable of reducing the transition metal from an oxidized inactive state to a reduced active state. (used to be a catalyst), a ligand, and an initiator.

[0353] To function as an ATRP transition metal catalyst, the transition metal must have at least two readily accessible oxidation states, i.e., a higher and a lower oxidation state, separated by one electron. In ATRP, a reversible redox reaction causes the transition metal catalyst to cycle between the higher and lower oxidation states, and the polymer chain to cycle between the propagating and dormant chain ends. See, for example, U.S. Pat. No. 7,893,173.

[0354] In some embodiments, the radically polymerizable zwitterionic monomer is selected from the group consisting of: [ka] In the formula, R1 is H or C 1~6 ZW is a zwitterion, and n is an integer from 1 to 6.

[0355] In some embodiments, the radically polymerizable monomer is [ka] and In the formula, R1 is H or C 1~6 alkyl, R2, R3, R4 are the same or different and are H or C 1~4 and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R1, R2, R3, and R4 are each methyl and X and Y are each 2.

[0356] In some embodiments, the radically polymerizable monomer is [ka] and In the formula, R1 is H or C 1~6 alkyl, R2 and R3 are the same or different and are H or C 1~4 R1, R2, and R3 are alkyl, R4 is PO4-, SO3-, or CO2-, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R1, R2, and R3 are methyl, R4 is PO4-, and X and Y are each 2.

[0357] In some embodiments, the monomer is [ka] and In the formula, R1 is H or C 1~6 alkyl, and R2, R3, and R4 are the same or different. H or C 1~4 R1, R2, R3, and R4 are alkyl, R5 is PO4-, SO3-, or CO2-, and X and Y are the same or different and are integers from 1 to 6. In some embodiments, R1, R2, R3, and R4 are methyl, R5 is PO4-, and X and Y are 2.

[0358] In some embodiments, the transition metal Mt is selected from the group consisting of Cu, Fe, Ru, Cr, Mo, W, Mn, Rh, Re, Co, V, Zn, Au, and Ag. t (q-1)+ X' (q-1) It is supplied as a salt in the form of M t (q-1)+ Cu 1+ , Fe 2+ , Ru 2+ , Cr 2+ , Mo2+ , W 2+ , Mn 3+ , Rh 3+ , Re 2+ , Co + , V 2+ , Zn + , Au + , and Ag + and X' is selected from the group consisting of halogen, C 1~6 Alkoxy, (SO4) 1 / 2 , (PO4) 1 / 3 , (R7PO4) 1 / 2 , (R72PO4), triflate, hexafluorophosphate, methanesulfonate, arylsulfonate, CN, and R7CO2. (Wherein, R7 is a straight-chain or branched C 1~6 In some embodiments, M t (q-1)+ Cu 1+ and X' is Br.

[0359] In some embodiments, M t (q-1)+ In some embodiments, M is provided in situ. t q+ X q is CuBr2. In some embodiments, the reducing agent is an inorganic compound. In some embodiments, the reducing agent is selected from the group consisting of low oxidation level sulfur compounds, sodium bisulfite, inorganic salts containing metal ions, metals, hydrazine hydrate, and derivatives of such compounds. In some embodiments, the reducing agent is a metal. In some embodiments, the reducing agent is Cu 0 It is.

[0360] In some embodiments, the reducing agent is an organic compound selected from the group consisting of alkylthiols, mercaptoethanol, or carbonyl compounds that can be readily enolized, ascorbic acid, acetylacetonate, camphosulfonic acid, hydroxyacetone, reducing sugars, monosaccharides, glucose, aldehydes, and derivatives of such organic compounds.

[0361] In some embodiments, the ligand is 2,2'-bipyridine, 4,4'-di-5-nonyl-2,2'-bipyridine, 4,4-dinonyl-2,2'-dipyridyl, 4,4',4''-tris(5-nonyl)-2,2':6',2''-terpyridine, N,N,N',N',N''-pentamethyldiethylenetriamine, 1,1,4,7,10,10-hexamethyltriethylenetetramine, tris(2-dimethylaminoethyl)amine, N,N-bis(2-pyridylmethyl)o The ligand is selected from the group consisting of octadecylamine, N,N,N',N'-tetra[(2-pyridal)methyl]ethylenediamine, tris[(2-pyridyl)methyl]amine, tris(2-aminoethyl)amine, tris(2-bis(3-butoxy-3-oxopropyl)aminoethyl)amine, tris(2-bis(3-(2-ethylhexoxy)-3-oxopropyl)aminoethyl)amine, and tris(2-bis(3-dodecoxy-3-oxopropyl)aminoethyl)amine. In some embodiments, the ligand is 2,2'-bipyridine.

[0362] In some embodiments, the initiator has the structure: [ka] wherein R1 is a nucleophilic reactive group, R2 comprises a linker, and R3 comprises a polymer synthesis initiator moiety having the structure: [ka] wherein R4 and R5 are the same or different and are selected from the group consisting of alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, and any combination thereof; Z is halogen, -OR (wherein R is -H, methyl, ethyl, propyl, or isopropyl), -SCN, or -NCS; and s is an integer from 1 to 20.

[0363] In some embodiments, Z is Br and R4 and R5 are each methyl. In some embodiments, R1 is selected from the group consisting of -NH2, -OH, and -SH.

[0364] In some embodiments, R2 is alkyl, substituted alkyl, alkylene, alkoxy, carboxyalkyl, haloalkyl, cycloalkyl, cyclic alkyl ether, alkenyl, alkenylene, alkynyl, alkynylene, cycloalkylene, heterocycloalkyl, heterocycloalkylene, aryl, arylene, aryleneoxy, heteroaryl, amino, amido, or any combination thereof. [ka] (wherein X and Y are the same or different and are integers from 1 to 20.) In some embodiments, X and Y are each 4.

[0365] In some embodiments, R3 is [ka] and wherein R6, R7, and R8 are the same or different and are selected from the group consisting of: [ka] , [ka] , and [ka] In the formula, Z is -OR (R is -H, methyl, ethyl, propyl, or isopropyl), -SCN, -NCS, -F, -Cl, -Br, or -I. In some embodiments, Z is -Br, and R6, R7, and R8 are each [ka] It is.

[0366] In some embodiments, the initiator has the structure: [ka] wherein A and B, which may be the same or different, are integers from 2 to 12, and Z is any halide, such as Br. In some embodiments, A and B are each 4.

[0367] In some embodiments, the method further comprises reacting the polymer with a maleimide reagent to provide a polymer having a terminal maleimide. [ka] It is.

[0368] Pharmaceutical Compositions The therapeutic protein can be incorporated into a pharmaceutical composition together with a pharma- ceutically acceptable excipient. Pharmaceutical compositions suitable for oral administration may be provided as discrete units such as capsules, solutions, syrups, or suspensions (aqueous or non-aqueous liquids, or edible foams or whipped creams, or emulsions). Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or its salts. Suitable excipients for soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols. For the preparation of solutions and syrups, for example, water, polyol, and sugar excipients may be used. For the preparation of suspensions, oils (e.g., vegetable oils) may be used to provide oil-in-water or water-in-oil suspensions.

[0369] The pharmaceutical composition may be adapted for nasal administration, where the excipient is a solid, including a coarse powder, for example having a particle size in the range of 20-500 microns, and is administered by nasal inhalation, i.e., by rapid inhalation through the nasal passages from a container of powder held close to the nose. Suitable compositions in which the excipient is a liquid, for administration as a nasal spray or nasal drops, include aqueous or oil solutions of the active ingredient. Pharmaceutical compositions suitable for inhalation administration include fine particle dusts or mists that may be generated by various types of metered-dose pressurized aerosols, nebulizers, or inhalers.

[0370] Pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions containing antioxidants, buffers, bactericides, and solutes that render the formulation substantially isotonic with the subject's blood, as well as aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents. Excipients that can be used for injection solutions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. The compositions may be provided in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state by simply adding a sterile liquid, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets. The pharmaceutical compositions are substantially isotonic, with an osmolality of about 250-400 mOsm / kg water.

[0371] The pharmaceutical composition may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (the substances themselves may be provided in the form of pharma- ceutical acceptable salts), buffers, and the like. The pharmaceutical composition may include a medicament, a coating agent, or an antioxidant. In addition to the substance, a therapeutically active agent may also be included. The pharmaceutical composition may be used in combination with one or more pharma- ceutical acceptable excipients. Such excipients include, but are not limited to, saline, buffered saline (such as phosphate buffered saline), dextrose, liposomes, water, glycerol, ethanol, and combinations thereof.

[0372] The antibodies and pharmaceutical compositions comprising them can be administered in a manner effective for treating or preventing a disease in a patient, including, for example, by oral, intravitreal, intravenous, subcutaneous, intramuscular, intraosseous, intranasal, topical, intraperitoneal, and intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration or routes. For treatment or prevention, the active agent may be administered to an individual as an injectable composition, such as, for example, a sterile aqueous dispersion. In some embodiments, the agent is isotonic or substantially isotonic.

[0373] For administration to mammals, particularly humans, the dosage of active ingredient is expected to be about 0.01 mg per kg of body weight, typically about 1 mg / kg. A physician can determine the actual dosage that is most suitable for an individual, depending on factors such as the individual's age, weight, sex, and response, the disease or disorder being treated, and the age and condition of the individual being treated. The dosages listed above are exemplary of the average case. Of course, higher or lower dosages may be effective. In some embodiments, the dosage may be 0.5 to 20 mg / eye, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mg.

[0374] This dosage may be repeated at an appropriate frequency (e.g., weekly, biweekly, monthly, bimonthly, quarterly, twice a year, annually). If side effects occur, the dosage and / or frequency of administration may be reduced, in accordance with normal clinical practice. In one embodiment, the pharmaceutical composition may be administered once every 1-30 days. In one embodiment, the pharmaceutical composition may be administered twice every 30 days. In one embodiment, the pharmaceutical composition may be administered once a week.

[0375] The antibodies and pharmaceutical compositions can be used alone or in combination with other compounds, such as therapeutic compounds or molecules, such as anti-inflammatory drugs, analgesics, or antibiotics. Administration with other compounds can be simultaneous, separate, or sequential. The components can be provided in the form of a kit, optionally including instructions.

[0376] The antibodies and pharmaceutical compositions disclosed herein can be used in the treatment or prevention of diseases, particularly ocular diseases or conditions described herein.

[0377] Anti-VEGF antibody conjugates or anti-VEGF protein conjugates and pharmaceutical compositions comprising them can be formulated and administered in the form of eye drops and / or ointments, ocular, intraocular, and / or intravitreal injections, and / or juxtascleral injections, and / or subretinal injections, and / or sub-Tenon injections, and / or suprachoroidal injections, and / or subconjunctival and / or topical administration. Such antibodies and compositions can be delivered, for example, intravitreally, as devices and / or depots that allow sustained release of compounds into the vitreous, including those described in references such as Intraocular Drug Delivery, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006). In one example, the device can be in the form of a minipump, matrix, passive diffusion system, or encapsulated cells that release the compound over a long period of time (Intraocular Drug Delivery, Jaffe, Ashton, and Pearson, editors, Taylor & Francis (March 2006)).

[0378] Formulations for ocular, intraocular, or intravitreal administration can be prepared using methods and ingredients known in the art. The main requirement for effective treatment is adequate penetration into the eye. Unlike diseases of the front of the eye, where drugs can be administered locally, retinal diseases require a more site-specific approach. Eye drops and ointments rarely penetrate deep into the eye, and the blood-ocular barrier prevents systemically administered drugs from penetrating into ocular tissues. Therefore, direct intravitreal injection is usually the drug administration method of choice for treating retinal diseases such as AMD and CNV. Intravitreal injection is usually repeated at regular intervals depending on the patient's condition, the properties and half-life of the drug administered.

[0379] Therapeutic antibodies and related conjugates are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. Such compositions may also be supplied in the form of a prefilled syringe.

[0380] A "stable" formulation is one in which the protein or other half-life extending moiety polymer-bound protein essentially retains physical and / or chemical stability and / or biological activity upon storage. "Stable" also refers to a formulation that exhibits little or no signs of instability, including aggregation and / or deamidation. For example, the provided formulations can remain stable for at least two years when stored as specified at a temperature of 5-8°C. Suitable formulations for the anti-VEGF antibody conjugates of the present disclosure are described, for example, in WO 2017 / 117464 (PCT Publication No.), the entire contents of which are incorporated herein by reference.

[0381] A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301 (Vincent Lee ed., New York, NY, 1991) and Jones, 1993 Adv. Drug Delivery Rev. 10: 29-90. Stability can be measured at a selected temperature and over a selected period of time. In some embodiments, the formulation is stable for storage for at least 6 months, 12 months, 12-18 months, or 2 years or more.

[0382] A protein, such as an antibody or fragment thereof, is considered to "retain physical stability" in a pharmaceutical formulation if it shows no signs of aggregation, precipitation, deamidation, and / or denaturation as measured by visual inspection of color and / or clarity, or by ultraviolet scattering or size exclusion chromatography.

[0383] A protein "retains chemical stability" in a pharmaceutical formulation if the chemical stability at a particular time point is such that the protein is still considered to retain its biological activity. Chemical stability can be assessed by detecting and quantifying chemically altered proteins. Chemical alterations can include size changes (e.g., clipping), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., resulting from deamidation), which can be assessed, for example, by ion exchange chromatography. An antibody is considered to "retain biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a particular time point is within about 10% (within the error of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared, as measured, for example, in an antigen binding assay.

[0384] The protein-polymer conjugate is "chemically stable" in that the chemical bonds between the protein and the polymer remain intact, eg, are not hydrolyzed or broken. The protein portion of the complex maintains chemical stability as described above.

[0385] "Isotonic" means that the formulation has essentially the same osmotic pressure as human blood or the vitreous body for intravitreal injection. The osmotic pressure of an isotonic formulation is usually about 250-400 mOsm. Isotonicity can be measured, for example, using a vapor pressure osmometer or a freezing point depression osmometer.

[0386] As used herein, the term "buffer" refers to a buffer solution that resists pH changes due to the action of an acid-base complex component. In some embodiments, the pH of the buffer is about 3.0 to about 8.0, e.g., about 4.5 to 8, or about pH 6 to about 7.5, or about 6.0 to about 7.0, or about 6.5 to 7.0, or about pH 7.0 to about 7.5, or about 7.1 to about 7.4. Any pH within the above range is also contemplated.

[0387] In some embodiments, "PBS" phosphate buffered saline, Tris-based buffers, and histidine-based buffers are used. In some embodiments, acetate buffers are used.

[0388] In some embodiments, the PBS buffer is composed of at least disodium hydrogen phosphate, potassium dihydrogen phosphate, and sodium chloride adjusted to provide an appropriate pH, in some embodiments, the buffer may contain other pharmaceutical excipients such as potassium chloride and other salts, surfactants, and / or preservatives to provide a stable storage solution.

[0389] A "preservative" is a compound that can be included in a formulation to essentially reduce bacterial activity, for example, facilitating the manufacture of a multi-use formulation. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl alcohol, and benzyl alcohol, alkylparabens such as methylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0390] In some embodiments, the level of endotoxin must be sufficiently low for the formulation to be safe for human use and animal studies. "Endotoxin" is lipopolysaccharide (LPS) derived from the cell membrane of gram-negative bacteria. Endotoxins consist of a hydrophilic polysaccharide moiety covalently bound to a hydrophobic lipid moiety (lipid A) (Raetz CR, Ulevitch RJ, Wright SD, Sibley CH, Ding A, Nathan CF. 1991. Gram-negative endotoxin: an extraordinary lipid with profound effects on eukaryotic signal transduction. FASEB J. 5(12):2652-2660). Lipid A is responsible for most of the endotoxin's biological activity, i.e., its toxicity. Endotoxins are released in large quantities not only during bacterial cell death, but also during cell growth and division. They are highly heat stable and are not destroyed under normal sterilization conditions. Extreme heat or pH treatments must be used, such as 180-250°C and acids or bases above 0.1 M (Petsch D, Anspach F. 2000. Endotoxin removal from protein solutions. J Biotechnol. 76: 97-119). Of course, such conditions would be highly detrimental to biological agents.

[0391] In the biotechnology and pharmaceutical industries, endotoxins can be found both during the manufacturing process and in the final product. Bacteria can grow in nutrient-poor media such as water, saline, and buffer solutions, so endotoxins can become widespread if precautions are not taken. Injection of endotoxins into animals and humans can cause a variety of pathophysiological effects, including endotoxic shock, tissue damage, and even death (Ogikubo Y, Ogikubo Y, Norimatsu M, Noda K, Takahashi J, Inotsume M, Tsuchiya M, Tamura Y. 2004. Evaluation of the bacterial endotoxin test for quantifications of endotoxin contamination of porcine vaccines. Biologics 32:88-93).

[0392] Intravenous injection of low concentrations (1 ng / mL) of endotoxin into mammals induces a febrile response and shock (Fiske JM, Ross A, VanDerMeid RK, McMichael JC, Arumugham. 2001. Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations. J Chrom B. 753:269-278). The maximum endotoxin level for intravenous administration of pharmaceuticals and biological products is set by all pharmacopoeias at 5 endotoxin units (EU) per kg body weight per hour (Daneshiam M, Guenther A, Wendel A, Hartung T, Von Aulock S. 2006. In vitro pyrogen test for toxic or immunomodulatory drugs. J Immunol Method 313:169-175). EU is a measure of the biological activity of endotoxins. For example, 100 pg of standard endotoxin EC-5 and 120 pg of endotoxin from E. coli O111:B4 have an activity of 1 EU (Hirayama C, Sakata M. 2002. Chromatographic removal of endotoxin from protein solutions by polymer particles. J Chrom B 781:419-432). Meeting this threshold level has been a constant challenge in biological research and the pharmaceutical industry (Berthold W, Walter J. 1994. Protein Purification: Aspects of Processes for Pharmaceutical Products. Biologicals 22:135-150; Petsch D, Anspach FB. 2000. Endotoxin removal from protein solutions. J Biotech 76:97-119).

[0393] The presence of endotoxins in drugs administered by intravitreal injection is of particular concern. Intravitreal injection of a drug (penicillin) was first performed by Rycroft in 1945 (Rycroft BW. 1945. Penicillin and the control of deep intra-ocular infection. British J Ophthalmol 29(2): 57-87). The vitreous is a chamber in which high concentrations of drugs can be introduced and maintained for relatively long periods of time. Drug concentrations achievable by intravitreal injection far exceed those that can be produced by local or systemic administration (e.g., intravenous).

[0394] One of the most dangerous complications that can occur from intravitreal injections is endophthalmitis. Endophthalmitis is classified into two types: infectious and sterile. Infectious endophthalmitis is typically caused by bacteria, fungi, or parasites. Symptoms of infectious endophthalmitis include severe pain, loss of vision, and redness of the conjunctiva and underlying sclera. Infectious endophthalmitis requires urgent diagnosis and treatment. Possible treatments include intravitreal antibiotic injections and possibly vitrectomy. Enucleation may be necessary to remove the blinding and painful eye. See, for example, Christy NE, Sommer A. 1979. Antibiotic prophylaxis of postoperative endophthalmitis. Ann Ophthalmol 11(8): 1261-1265.

[0395] In contrast, sterile endophthalmitis can be defined as an acute intraocular inflammation of the vitreous cavity that is not associated with an infectious agent and that heals without the need for intravitreal antibiotics and / or vitreoretinal surgery. If a microbiological examination of the vitreous has been performed, cultures must prove negative to support the diagnosis of sterile endophthalmitis (Marticorena J, Romano V, Gomez-Ulla F. 2012 “Sterile Endophthalmitis after Intravitreal Injections” Med Inflam. 928123).

[0396] It has been observed that intravitreal injection of endotoxin-contaminated biologic agents can cause sterile endophthalmitis. According to Marticorena, et al., bevacizumab (Avastin) is approved by the U.S. Food and Drug Administration for the treatment of glioblastoma, metastatic colorectal cancer, advanced nonsquamous non-small cell lung cancer, and metastatic kidney cancer. Bevacizumab is also widely used off-label for the treatment of wet AMD. Bevacizumab is shipped from the manufacturer as 100 mg / 4 mL. This solution cannot be used directly for intravitreal injection and must be dispensed by a pharmacist. Clusters of sterile endophthalmitis have been observed, which are theorized to be caused by inadvertent contamination of bevacizumab with endotoxin by the dispensing pharmacist.

[0397] Considering the serious clinical consequences of intravitreal injection of endotoxin, the total amount of endotoxin that can be administered to a patient by intravitreal administration is very limited. In some embodiments, a solution comprising an antibody or antibody complex is provided that has an endotoxin concentration not exceeding 5.0 EU / mL. In some embodiments, the endotoxin concentration does not exceed 1.0 EU / mL. In some embodiments, the endotoxin concentration does not exceed 0.5 EU / mL. In some embodiments, the endotoxin concentration does not exceed 0.2 EU / mL. In some embodiments, the endotoxin concentration does not exceed 2, 1, 0.5, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, or 0.01 EU / mL.

[0398] Two widely used FDA-approved tests to test for the presence of endotoxin are the rabbit pyrogen test and the Limulus amebocyte lysate (LAL) test (Hoffman S, et al. 2005. International validation of novel pyrogen tests based on human monocytoid cells J. Immunol. Methods 298:161-173; Ding JL, Ho BA. 2001. New era in pyrogen testing. Biotech. 19:277-281). The rabbit pyrogen test was developed in the 1920s and involves monitoring the rise in body temperature in rabbits injected with a test solution. However, the rabbit pyrogen test has seen a significant decline in use over the years due to its expense and lengthy turnaround time. The LAL test is more common. LAL is extracted from the blood of horseshoe crabs and clots when exposed to endotoxins.

[0399] One of the simplest LAL assays is the LAL gel clot assay. Essentially, LA The LAL clotting assay is combined with a serial dilution of the sample. Gel formation is proportional to the amount of endotoxin in the sample. Serial dilutions are prepared from the sample and each dilution is tested for LAL gel-forming ability. At some point, the negative reaction is suppressed. The amount of endotoxin in the original sample can be estimated from the dilution assay.

[0400] Turbidimetric LAL Assay (Ong KG, Lelan JM, Zeng KF, Barrett G, Aourob M, Grimes CA. 2006. A rapid highly-sensitive endotoxin detection system. Biosensors and Bioelectronics 21:2270-2274) and chromogenic LAL assay (Haishima Other LAL tests have also been developed, including the turbidimetric and chromogenic assays (Y, Hasegawa C, Yagami T, Tsuchiya T, Matsuda R, Hayashi Y. 2003. Estimation of uncertainty in kinetic-colorimetric assay of bacterial endotoxins. J Pharm Biomed Analysis. 32:495-503). The turbidimetric and chromogenic assays are much more sensitive and quantitative than the simple gel clot dilution method.

[0401] In some embodiments, a method is provided for reducing the amount of endotoxin in a composition having an antibody disclosed herein, comprising contacting the composition with an affinity chromatography resin that binds the antibody, eluting the antibody from the affinity chromatography resin to form an affinity chromatography eluate comprising an antagonist, contacting the affinity chromatography eluate with an ion exchange resin that binds the antibody, and eluting the antibody from the ion exchange resin, wherein the antibody eluted from the ion exchange resin is substantially free of endotoxin.

[0402] The above method for reducing the amount of endotoxin, or any other method or process described herein, may be carried out in the order of steps described above, or may be carried out by changing the order of steps or repeating one or more steps. In one embodiment, the method for reducing the amount of endotoxin in the composition is carried out in the order of steps described. In some embodiments, the steps of contacting, washing, and eluting the affinity chromatography resin are repeated multiple times in the same order before contacting the affinity chromatography eluent with the ion exchange resin. The method may also include a filtration step, for example, using a 0.1 micron, 0.20 micron, or 0.45 micron filter, which may be carried out on one or more eluents removed after each resin binding step.

[0403] In certain instances, the steps of contacting the composition with an affinity chromatography resin, washing, and eluting the antibody from the affinity chromatography resin can be repeated multiple times before contacting the first eluent with an ion exchange resin. In one embodiment, the affinity chromatography resin comprises a recombinant Protein A ("rProteinA") resin. Examples of suitable recombinant Protein A resins include MabSelect Sure and Mabselect Sure LX (Cytiva). In another embodiment, a suitable affinity chromatography resin comprises a Protein G chromatography resin. In other embodiments, a suitable affinity chromatography resin comprises a Protein A / Protein G mixed resin. In some embodiments, a suitable affinity chromatography resin comprises a Protein L resin. In other embodiments, a suitable affinity chromatography resin comprises a hydrophobic charge-inducing resin that includes a 4-mercaptoethylpyridine ligand, such as MEP HyperCel® resin. (BioSepra, Cergy Saint-Christophe, Saint-Christophe, France).

[0404] In some embodiments, the ion exchange resin comprises an anion exchange resin. As known to those skilled in the art, ion exchangers can be based on various materials with respect to the matrix and the attached charged groups. For example, the following matrices can be used, in which the mentioned materials may be more or less crosslinked: POROS XS (ThermoFisher), POROS XQ (ThermoFisher), MacroCap Q (Cytiva, Piscataway, NJ), agarose-based (Sepharose CL-6B®, Sepharose Fast Flow®, Sepharose High Performance®, etc.), cellulose-based (DEAE Sephacel®, etc.), dextran-based (Sephadex®, etc.), silica-based, synthetic polymer-based. In the case of anion exchange resins, the charged groups covalently attached to the matrix may be, for example, diethylaminoethyl, quaternary aminoethyl, and / or quaternary ammonium. In some embodiments, the anion exchange resin comprises quaternary amine groups. An exemplary anion exchange resin having quaternary amine groups for binding to anti-M-CSF antibodies is Q Sepharose® resin (Amersham, Piscataway, NJ).

[0405] In other aspects, if endotoxin levels are higher than desired after subjecting the composition to the aforementioned anion exchange chromatography step, the composition may be subjected to a cation exchange resin instead. In some embodiments, the endotoxin in the composition must exhibit a different binding to the ion exchange resin than the protein, so that the protein can be purified from the endotoxin. In this regard, endotoxins are negatively charged and typically bind to anion exchange resins. If both proteins and endotoxins bind to anion exchange resins, one can be purified from the other by using a salt gradient to elute the two in different fractions. The relative binding of proteins to a particular resin can also be altered by changing the pH of the buffer relative to the pI of the protein. In some embodiments, cation exchange chromatography is the only ion exchange chromatography used.

[0406] In some embodiments, if the endotoxin concentration after the anion exchange resin is too high, the composition may be further subjected to a second ion exchange step, for example, by contacting the composition with a cation exchange resin, followed by a washing step, and then eluting from the ion exchange resin. In some embodiments, the cation exchange resin contains sulfonic acid groups for binding. Exemplary cation exchange resins include SP Sepharose® resin FF (Amersham, Piscataway, NJ) and POROS XS (CEX) (ThermoFisher). In some embodiments, endotoxin removal is performed using hydrophobic interaction chromatography. In some embodiments, hydrophobic interaction chromatography is used when both endotoxin and mAb have the same or similar charge. In some embodiments, hydrophobic interaction chromatography can be performed by an exemplary system such as Sartobind Phenyl (Sartorius, Gottingen, Germany).

[0407] In some embodiments, after a solution of antibody protein containing a particular level of endotoxin is produced, there are several steps before final formulation of the protein. In some embodiments, a half-life extending moiety is attached to the protein. The conjugate is then compounded into a final formulation and injected into a patient. In some embodiments, the conjugate is purified again on an ion exchange resin, which may be a cation exchange resin. In other embodiments, the protein is formulated. In either case, the endotoxin is added to the protein sample or to the protein-polymer complex. Routine laboratory procedures should be employed to prevent the introduction of doxorubicin contaminants.

[0408] In some embodiments, the present specification contemplates any of the following configurations:

[0409] A method for purifying a product using affinity chromatography, comprising loading an eluent onto an affinity chromatography matrix, which binds to a protein of interest, and washing the affinity chromatography matrix with a buffer solution containing a chaotropic agent.

[0410] A method for purifying a product from a load solution containing a protein and one or more impurities by passing the load solution through an affinity chromatography matrix, followed by at least one wash solution containing a chaotropic salt, and collecting the protein using an elution solution to reduce impurities.

[0411] 1. A method for separating impurities in an eluate containing a protein of interest, comprising loading the eluate containing the protein of interest onto an affinity chromatography matrix, and washing the affinity chromatography matrix with one or more buffer solutions comprising one or more of lithium and lithium salts, magnesium and magnesium salts, calcium and calcium salts, and guanidinium and guanidinium salts.

[0412] 1. A method for producing a product using affinity chromatography, comprising: loading an eluent containing a protein of interest onto an affinity chromatography matrix; performing a first wash of the affinity chromatography matrix with a first buffer comprising sodium phosphate and a salt; and performing a second wash of the affinity chromatography matrix with a second buffer comprising a chaotropic agent.

[0413] 1. A method for producing a product using affinity chromatography, comprising: loading an eluent containing a protein of interest onto an affinity chromatography matrix; performing a first wash with a first buffer containing Tris and a salt; and performing a second wash with a second buffer containing Tris and a chaotropic agent, wherein the chaotropic agent of the second buffer is not the same as the salt contained in the first buffer.

[0414] 1. A method for producing a product, comprising: collecting a load solution, the load solution comprising a protein of interest; loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest; washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt; eluting the bound protein of interest; and collecting an eluate, the eluate containing the protein of interest.

[0415] 1. A method for producing a product, comprising: collecting a load solution, the load solution comprising a protein of interest; loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest; applying a buffer solution comprising a chaotropic salt to the affinity chromatography matrix; eluting the bound protein of interest; and collecting an eluate, the eluate containing the protein of interest.

[0416] 1. A method for producing a product, comprising: collecting a complex protein, the complex protein comprising an antibody bound to a complex polymer; loading the complex protein onto an affinity chromatography matrix, the affinity chromatography matrix binding to the complex protein; washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt; eluting the complex protein; and collecting an eluate, the eluate containing the complex protein.

[0417] A method of producing a product, comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer comprising a chaotropic salt, eluting and collecting an eluate, the eluate containing the target protein of interest, and removing viral contaminants from the eluate. The method may further comprise removing viral contaminants from the eluate comprising one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and / or diafiltration (DF). The method may further comprise combining the eluate with an acceptable pharmaceutical excipient to form a pharmaceutical composition. The method may further comprise adding a buffer solution to the pharmaceutical composition. The method may further comprise adding a preservative solution to the pharmaceutical composition. The method may further comprise further purifying the pharmaceutical composition for intravitreal injection.

[0418] 1. A method for producing a product, comprising washing an affinity chromatography matrix bound to a target protein of interest with a buffer containing a chaotropic salt, removing the chaotropic salt, and eluting and collecting an eluate, wherein the eluate contains the target protein of interest.

[0419] 1. A method for producing a product, comprising: collecting a load solution, the load solution comprising a protein of interest; loading the load solution onto an affinity chromatography matrix, the affinity chromatography matrix binding to the protein of interest; washing the affinity chromatography matrix with a buffer solution comprising a chaotropic salt; eluting and collecting an eluate, the eluate comprising the target protein of interest; and removing viral contaminants from the eluate.

[0420] A method of producing a product comprising loading an eluate onto an affinity chromatography matrix, washing with a first wash buffer, washing with a second wash buffer comprising a chaotropic salt, washing with a third wash buffer, removing the chaotropic salt with the third wash buffer, and eluting with an elution buffer, collecting an eluate, the eluate comprising a protein product. The method may further comprise: the first wash buffer comprises 50 mM sodium phosphate. The method may further comprise: the first wash buffer further comprises about 250 mM sodium chloride. The method may further comprise: the first wash buffer comprises Tris and salt. The method may further comprise: removing viral contaminants from the eluate. The method may further comprise: removing viral contaminants comprises one or more of low pH inactivation, detergent inactivation, a polishing chromatography step, viral filtration (VF), ultrafiltration (UF), and diafiltration (DF). The method may further include the eluent comprising a protein of interest. The method may further include the protein of interest being an antibody. The method may further include the antibody being further conjugated to a polymer to form an antibody complex. The method may further include the antibody complex comprising a bispecific antibody. The method may further include the bispecific antibody comprising an anti-VEGF binding portion and an anti-IL-6 binding portion. The method may further include the antibody complex having the structure: [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7 n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0421] Provided herein is a method for producing a product, comprising: recovering a cell culture supernatant, the cell culture supernatant comprising a protein of interest; treating the cell culture supernatant with an eluent, the eluent comprising the protein of interest; loading the eluent onto an affinity chromatography matrix; washing with a first wash buffer comprising Tris or sodium phosphate; washing with a second wash buffer comprising a chaotropic salt; eluting with an elution buffer, the eluate comprising the protein product; inactivating viral contaminants present in the eluate with a low pH viral buffer to produce a viral inactivated eluate; filtering the viral inactivated eluate; subjecting the viral inactivated eluate to at least one round of ion exchange chromatography; and filtering the viral inactivated eluate to obtain a retentate, the retentate comprising the protein of interest. The method may further comprise the cell culture supernatant being produced in a bioreactor using an animal component-free cell culture. The method may further comprise processing the cell culture supernatant to harvest a cell product from the cell culture. The method may further comprise clarifying the cell culture to remove cells and cell debris. The method may further comprise the elution solution comprising the clarified cell culture supernatant. A method of purifying a protein using affinity chromatography comprising contacting a load solution with a medium, the medium being an affinity chromatography matrix that binds a protein of interest, washing the medium with a buffer solution comprising a chaotropic agent, the chaotropic agent being a salt, and contacting the washed medium with an elution solution under conditions suitable for eluting the protein of interest.

[0422] 1. A method for producing a product, comprising: applying a solution containing a protein of interest to an affinity chromatography matrix; washing the affinity chromatography matrix with a first buffer; washing the affinity chromatography matrix with a second buffer containing a chaotropic agent; washing the affinity chromatography matrix with a third buffer to remove the chaotropic agent; and eluting with an elution buffer; collecting an eluate, wherein the eluate comprises the protein product.

[0423] 1. A system for protein purification, comprising a column having a first antigen-binding protein bound thereto, a phosphate wash buffer comprising sodium phosphate and a salt, an intermediate wash buffer comprising Tris, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate.

[0424] A system for protein purification, comprising a column to which a first antigen-binding protein is bound, a first Tris wash buffer comprising Tris and a salt, an intermediate Tris wash buffer, a second wash buffer comprising magnesium chloride, and an elution buffer comprising sodium formate. The system may further comprise that the column comprises a ligand for affinity chromatography. The system may further comprise that the ligand comprises Protein A or Protein G. The system may further comprise that the first wash buffer comprising sodium phosphate and a salt has a pH of 5.5 to 9.5. The system may further comprise that the phosphate wash buffer comprising sodium phosphate and a salt comprises about 50 mM sodium phosphate. The system may further comprise that the phosphate wash buffer comprising sodium phosphate and a salt comprises about 250 mM sodium chloride. The system may further comprise that the first Tris wash buffer comprises about 50 mM Tris. The system may further comprise that the first Tris wash buffer further comprises about 250 mM sodium chloride. The system may further include the intermediate Tris wash buffer comprising about 50 mM Tris. The system may further include the first Tris wash buffer having a pH of about 7.2. The system may further include the second wash buffer having a pH of about 7.8. The system may further include the second wash buffer having a magnesium chloride concentration of about 2.8 M. The system may further include the elution buffer having a sodium formate concentration of 10 mM.

[0425] 1. A system for antibody purification, comprising: a column having a Protein A resin bound to an antibody, the antibody comprising a light chain and a heavy chain each having at least one of SEQ ID NOs: 91-93 and 28-30, and at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262; a chaotropic wash buffer comprising a chaotropic salt; and an elution buffer comprising sodium formate.

[0426] The methods described herein may further include the protein of interest being a bispecific antibody. The methods described herein may further include the bispecific antibody being specific for VEGF and IL-6. The methods described herein may further include the bispecific antibody being specific for VEGF and IL-6. The methods described herein may further include the protein of interest being an antibody complex. The methods described herein may further include the affinity chromatography matrix being a Protein A chromatography matrix. The methods described herein may further include the chaotropic agent in the buffer solution comprising a magnesium salt. The methods described herein may further include the magnesium salt having a concentration of 1.5-3.5M. The methods described herein may further include the chaotropic agent in the buffer solution comprising a calcium salt. The methods described herein may further include the calcium salt having a concentration of 1-3M. The methods described herein may further include the chaotropic agent in the buffer solution comprising a guanidinium salt. The methods described herein may further include a concentration of the guanidinium salt of 0.05-3M. The methods described herein may further include the buffer solution further comprising Tris. The methods described herein may further include a concentration of Tris in the buffer solution of at least 5 mM. The methods described herein may further include a pH of the buffer solution of greater than 5.5. The methods described herein may further include the eluate further comprising a viral impurity. The methods described herein may further include removing the viral impurity. The methods described herein may further include inactivating the viral impurity.The methods described herein may further include washing the loaded affinity chromatography matrix with a pre-wash buffer solution prior to washing with the buffer solution. The methods described herein may further include washing the loaded affinity chromatography matrix with a post-wash buffer solution after washing with the buffer solution. The methods described herein may further include the pre-wash buffer solution comprising sodium phosphate. The methods described herein may further include the pre-wash buffer solution comprising Tris and salt.

[0427] The methods described herein further comprise the step of: It may include. [ka] wherein each heavy chain of an anti-VEGF-A antibody is designated with the letter H and each light chain of an anti-VEGF-A antibody is designated with the letter L, the polymer is attached to the anti-VEGF-A antibody via the sulfhydryl of C443 (EU numbering), which is shown in one of the heavy chains, and PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer, X is a) -OR (R is -H, methyl, ethyl, propyl, or isopropyl), b) -H, c) any halogen including -Br, -Cl, or -I, d) -SCN, or e) -NCS, and i) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different and are integers from 0 to 3000, or ii) n1, n2, n3, n4, n5, n6, n7, n8, and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500±15%.

[0428] The methods described herein include comprising administering to the patient an antibody complex comprising an anti-VEGF-A light chain and an anti-VEGF light chain. and wherein the anti-VEGF-A antibody heavy chain comprises a CDRH1 that is CDRH1 of SEQ ID NO: 172, a CDRH2 that is CDRH2 of SEQ ID NO: 173, and a CDRH3 that is CDRH3 of SEQ ID NO: 174, and wherein the anti-VEGF-A antibody light chain comprises a CDRL1 that is CDRL1 of SEQ ID NO: 199, a CDRL2 that is CDRL2 of SEQ ID NO: 200, and a CDRL3 that is CDRL3 of SEQ ID NO: 201. The methods described herein may further include the anti-VEGF antibody conjugate comprising an antibody conjugate comprising an anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer comprising an MPC monomer, wherein the sequence of the anti-VEGF-A antibody heavy chain is at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262, and the sequence of the anti-VEGF-A antibody light chain is at least one of SEQ ID NOs: 91-93, and 28-30, and the antibody is conjugated to the polymer with C449. The methods described herein may further include the target protein of interest being produced by a cell culture. The methods described herein may further include the cell culture comprising CHO cells. The methods described herein may further include, after washing with the buffer solution, washing the affinity chromatography matrix loaded with the eluent with a post-wash buffer solution. The methods described herein may further include washing the affinity chromatography matrix with a buffer solution to remove nucleic acids, endotoxins, antifoaming agents, or other small molecules other than the target protein of interest. The methods described herein may further include washing the affinity chromatography matrix with the buffer solution to remove impurities while leaving the target protein of interest bound to the affinity chromatography matrix. The methods described herein may further include washing the affinity chromatography matrix with the buffer solution to remove host cell proteins other than the target protein of interest.The methods described herein may further include, where the addition of a chaotropic agent to the buffer solution does not elute the target protein of interest. The methods described herein may further include one or more of viral inactivation, tangential flow filtration, diafiltration, ultrafiltration, ion exchange chromatography, or viral reduction filtration. The methods described herein may further include, where the eluate is produced in a bioreactor using an animal component-free cell culture. The methods described herein may further include, where the product is a protein of interest. The methods described herein may further include, where the impurities include host cell protein impurities.

[0429] The methods described herein may further include the first wash buffer comprising a salt. The methods described herein may further include the first wash buffer comprising a phosphate-based species. The methods described herein may further include the first wash buffer comprising sodium phosphate. The methods described herein may further include that the first wash buffer comprises about 0.1 to about 250 mM phosphate, such as about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, or any integer between 0.1 and 250 mM. The methods described herein may further include that the first wash buffer comprises 10 mM sodium phosphate.

[0430] The methods described herein may further include wherein the second wash buffer comprises a salt. The methods described herein may further include wherein the second wash buffer comprises a phosphate buffer. The method described herein may further include that the second wash buffer comprises a species of sodium phosphate. The method described herein may further include that the second wash buffer comprises sodium phosphate. The method described herein may further include that the second wash buffer comprises about 0.1 to about 2500 mM phosphate, such as about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, or any integer between 0.1 and 250 mM. The methods described herein may further comprise, wherein the second wash buffer comprises 10 mM sodium phosphate.

[0431] The methods described herein may further include the wash buffer comprising a salt. The methods described herein may further include the wash buffer comprising a phosphate-based species. The methods described herein may further include the wash buffer comprising sodium phosphate. The methods described herein may further include that the wash buffer comprises about 0.1 to about 250 mM phosphate, such as about 0.1 mM, about 1 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, or any integer between 0.1 and 250 mM. The methods described herein may further include that the wash buffer comprises 10 mM sodium phosphate.

[0432] In some embodiments, the buffer system may employ a system other than Tris. In some embodiments, the buffer system may employ a system other than sodium phosphate. In some embodiments, the buffer solution may be one or more of acetate, citrate, ACES, BES, bicine, HEPES, MES, MOPS, MOPSO, TAPS, tricine, bis-tris, bis-tris propane, cacodylate, CAPS, CAPSO, CHES, glycine, glycylglycine, imidazole, PIPES, TEA, and TES. In some embodiments, the buffer system may use pH values ​​other than those presented. In some embodiments, the buffer system may employ pH values ​​between 2 and 13. In some embodiments, the elution buffer used is not sodium formate. In some embodiments, the elution buffer is basic. In some embo...

Claims

1. A method for purifying a product and reducing impurities from a load solution containing a protein and one or more impurities by passing the load solution through an affinity chromatography matrix, followed by at least one wash buffer containing a chaotropic salt, and collecting the protein in the eluate using an elution solution, comprising: The method, wherein the chaotropic salt comprises one or more of a guanidinium salt, a calcium salt, a magnesium salt, and a lithium salt.

2. The method described in claim 1, wherein the affinity chromatography matrix binds to the protein.

3. Washing the affinity chromatography matrix with a first buffer; washing the affinity chromatography matrix with a second buffer, wherein the second buffer is the wash buffer containing the chaotropic salt; washing the affinity chromatography matrix with a third buffer to remove the chaotropic salt; and eluting with the elution solution, collecting the eluate, wherein the eluate comprises the protein product; The method of claim 1 , comprising:

4. The method described in claim 1, wherein the affinity chromatography matrix comprises a ligand, and optionally the ligand comprises protein A or protein G.

5. The method of claim 1, wherein the wash buffer comprises 5 to 200 mM sodium phosphate, 5 to 200 mM Tris, 5 to 3000 mM sodium chloride, or any combination thereof.

6. The method described in claim 1, wherein the pH of the wash buffer is greater than 5.5, and optionally the wash buffer is pH 6.0, pH 6.5, pH 7.0, pH 7.5, pH 8.0, pH 8.5, or pH 9.

0.

7. The method described in claim 1, wherein the concentration of the chaotropic salt in the wash buffer is 0.05 to 3.5 M, and optionally the concentration of the chaotropic salt in the wash buffer is 0.05 M, 0.1 M, 0.2 M, 0.5 M, 1 M, 1.5 M, 1.65 M, 2 M, 2.8 M, or 3 M.

8. 2. The method of claim 1, wherein the protein is an antibody, optionally wherein the antibody comprises a bispecific antibody, optionally wherein the bispecific antibody comprises an anti-VEGF binding portion and an anti-IL-6 binding portion.

9. The antibody a heavy chain amino acid variable region comprising at least one of SEQ ID NOs: 7-13, 19-27, 89, 90, and 256-262; and a light chain amino acid variable region comprising at least one of SEQ ID NOs: 91-93 and 28-30; or a heavy chain amino acid variable region comprising any one of SEQ ID NOs: 270-274; and Light chain amino acid variable region comprising SEQ ID NO: 275 The method of claim 8, comprising:

10. The antibody comprising: i) a heavy chain comprising: CDRH1, which is CDRH1 of SEQ ID NO: 49; a CDRH2 which is the CDRH2 of SEQ ID NO: 50; a CDRH3 which is the CDRH3 of SEQ ID NO: 51; and Light chains, including: CDRL1, which is CDRL1 of SEQ ID NO: 76; CDRL2, which is CDRL2 of SEQ ID NO: 77; a CDRL3 which is the CDRL3 of SEQ ID NO: 78; or ii) a heavy chain comprising: CDRH1, which is CDRH1 of SEQ ID NO: 172; a CDRH2 which is the CDRH2 of SEQ ID NO: 173; a CDRH3 which is the CDRH3 of SEQ ID NO: 174; and Light chains, including: CDRL1, which is CDRL1 of SEQ ID NO: 199; CDRL2, which is CDRL2 of SEQ ID NO: 200; a CDRL3 which is the CDRL3 of SEQ ID NO: 201; or iii) a heavy chain comprising: CDRH1, which is CDRH1 of SEQ ID NO: 754; a CDRH2 which is the CDRH2 of SEQ ID NO: 753; a CDRH3 which is the CDRH3 of SEQ ID NO: 756; and Light chains, including: CDRL1, which is CDRL1 of SEQ ID NO: 749; CDRL2, which is CDRL2 of SEQ ID NO: 750; CDRL3 is CDRL3 of SEQ ID NO: 75 The method of claim 8, comprising:

11. The method of claim 1, wherein the protein is produced by a cell culture, optionally the cell culture is bacterial or eukaryotic, optionally the cell culture is mammalian cells, optionally the cell culture is CHO cells, yeast cells, E. coli, human cells, primary tissue, HeLa cells, mouse 3T3 fibroblasts, HEK293 cells, or KT-3 cells.

12. 12. The method of claim 11, wherein the cell culture supernatant is produced in a bioreactor using an animal component-free cell culture.

13. The method of claim 1, wherein the wash buffer removes host cell proteins, nucleic acids, endotoxins, antifoaming agents, or other small molecules other than the target protein of interest.

14. 10. The method of claim 1, wherein the wash buffer further comprises one or more of acetate, citrate, Bis-Tris, glycine, ACES, BES, bicine, HEPES, MES, MOPS, MOPSO, TAPS, tricine, Bis-Tris propane, cacodylate, CAPS, CAPSO, CHES, glycylglycine, imidazole, PIPES, TEA, and TES.

15. The method of any one of claims 1 to 14, wherein the eluate is further combined with an acceptable pharmaceutical excipient to form a pharmaceutical composition, optionally a preservative solution is added to the pharmaceutical composition, and optionally the pharmaceutical composition is further purified for intravitreal injection.