Compositions of conjugate and non-conjugate proteins

Abstract

This specification provides pharmaceutical compositions and formulations of a conjugate comprising a first protein, such as an anti-VEGF antibody, conjugated to a phosphorylcholine-containing polymer, and a second protein not conjugated to the phosphorylcholine-containing polymer. The second protein may be the same as or different from the first protein. In some embodiments, the pH of the formulation is different from the isoelectric point (pI) of the second protein in order to prevent or reduce turbidity of the formulation. Also provided are a method for preparing the formulation, a method for using the formulation, and a kit including a pre-filled syringe containing the formulation.

Description

[Technical Field]

[0001] Reference to related applications This application claims priority under U.S. Provisional Patent Application No. 63 / 506781, filed on 7 June 2023. The contents of each of the aforementioned related applications are incorporated herein by reference in their entirety.

[0002] Sequence listing reference This application is submitted together with an electronic sequence listing. The sequence listing is provided as a file named KDIAK210WO_SEQLIST.xml, created on May 29, 2024, with a size of 196,681 bytes. The electronic information of the sequence listing is incorporated herein by reference in its entirety.

[0003] field The present invention relates to a composition comprising a mixture of non-conjugate and conjugate proteins (e.g., antibodies and their conjugates), and to a method for using and producing the above composition. [Background technology]

[0004] Diabetic retinopathy is a leading cause of blindness in people approximately 20–64 years of age. Engelgau M, Geiss L, Saaddine J, Boyle J, et al. al. 2004. The Evolving Diabetes Burden in the United States. Ann of Int Med. 140 (11): 945-951. In the United States, diabetic retinopathy accounts for approximately 12% of new cases of blindness. Typically, in cases of diabetic retinopathy, retinal blood vessels swell and leak fluid into the posterior part of the eye. Hyperglycemia induces intrawalled and thickened basement membranes, resulting in leaky or permeable vessels.

[0005] In diabetic retinopathy, changes in blood glucose levels cause changes in retinal blood vessels. Everyone with diabetes is at risk. The longer a person has had diabetes, the higher their risk of developing some form of eye problem. 40–45 percent of Americans diagnosed with diabetes have some stage of diabetic retinopathy. (Causes and Risk Factors. Diabetic Retinopathy. United States National Library of Medicine. 15 September 2009.)

[0006] Diabetic retinopathy first manifests as the formation of microaneurysms in the retina. Microaneurysms occur when there is swelling of the capillaries (very small blood vessels) that supply nutrients to the retina. The presence of a relatively small number of microaneurysms usually does not cause vision problems. However, if the retinopathy progresses to a later stage, vision loss is highly likely. Such early-stage retinopathy is called background diabetic retinopathy or non-proliferative diabetic retinopathy (NPDR). Although NPDR patients are generally asymptomatic, early detection of retinopathy is extremely important because there is a very high probability of significant vision loss if the disease progresses to a later stage.

[0007] The next stage of diabetic retinopathy is the formation of new blood vessels in the posterior part of the eye (proliferative diabetic retinopathy). These neovascularizations are prone to leakage, and rupture can cause bleeding, resulting in blurred or unclear vision. Oxygen deficiency within the eye leads to further neovascularization. The blood vessels grow along the retina in the vitreous fluid. When these vessels rupture, further bleeding occurs. Yes, the retina can be severely damaged or destroyed. Fluid accumulation in the macula due to leaking blood vessels is called diabetic macular edema. Many patients with diabetic retinopathy develop diabetic macular edema.

[0008] There are generally three treatment routes for patients with diabetic retinopathy: laser surgery, corticosteroid injections, and injections of biological agents (e.g., Avastin® (bevacizumab), Lucentis® (ranibizumab), Eylea® (aflibercept), Beovu® (brolucizumab), and Babismo® (falicimab)). Laser surgery is generally effective in treating diabetic retinopathy, but laser-induced retinal damage is a frequent side effect. Steroid preparations such as triamcinolone acetonide are administered via intravitreal injection to treat diabetic retinopathy. However, frequent administration of steroid solutions is necessary to treat diabetic retinopathy. Furthermore, intravitreal treatment with steroids is associated with cataracts, steroid-induced glaucoma, and endophthalmitis.

[0009] Another method for treating diabetic retinopathy is intravitreal injection of anti-VEGF agents. In this regard, anti-VEGF therapies such as Lucentis® (ranibizumab) and Eylea® (aflibercept) are approved for the treatment of diabetic retinopathy in patients with diabetic macular edema. VEGF-targeted therapy is effective not only for diabetic retinopathy but also for retinal vascular diseases such as age-related macular degeneration (AMD), neovascular (wet) AMD, and retinal vein occlusion (RVO).

[0010] To treat diseases such as retinal diseases, it may be useful to use biological agents such as antibodies or antibody fragments. These proteins should be formulated as clear solutions. Such formulations may have a defined buffer system, and several excipients may be added to further enhance protein stability. Proteins may be conjugated to other parts to impart enhanced properties or a set of properties to the protein. For example, conjugation of proteins to potent toxins to create antibody-drug conjugates that target toxins to specific receptor-containing cell types for enhanced potency or safety. This disclosure is not limited to diabetic retinopathy and can be applied to a variety of indications as will be understood by those skilled in the art. [Overview of the project]

[0011] A formulation is provided herein, comprising: a first molar amount of a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first and second molar amounts, and the formulation having a pH at least 0.5 pH units away from the isoelectric point (pI) of the second protein. A therapeutically acceptable composition is also provided, comprising a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a therapeutically acceptable carrier, wherein the composition contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the molar amounts of the first and second proteins, and the composition having a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein. A therapeutically acceptable composition is also provided, comprising a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the total protein mass weight concentration of the first and second proteins in the composition is Provided herein are therapeutically acceptable compositions in which the compositional percentage of the second protein is about 1% or more, and the composition has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein. Also provided herein are formulations comprising a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation contains the second protein in about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first and second molar amounts, the formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein, and the formulation has reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in total molar amount.

[0012] A therapeutically acceptable composition is provided, comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the composition percentage of the second protein is about 1% or more, with the remainder being the first protein; the composition has a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein; the composition has reduced viscosity and / or improved injectability compared to a reference composition containing the conjugate; and the first protein of the conjugate is present in the reference composition at a total mass weight concentration of the first and second proteins in the composition. Also provided is a low-viscosity formulation of a protein conjugate comprising: a first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation has a pH about 0.5 pH units or more away from the isoelectric point (pI) of the protein, and the formulation has reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in a total molar amount which is the sum of the first and second molar amounts. A therapeutically acceptable low-viscosity protein conjugate composition is provided, comprising: a first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the composition has a pH about 0.5 pH units or more away from the isoelectric point (pI) of the protein, and the composition has reduced viscosity and / or improved injectability compared to a reference composition comprising a total molar amount of conjugate which is the sum of the first and second molar amounts.

[0013] A formulation is also provided comprising: a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the second protein is present in the formulation in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first and second molar amounts; the formulation having a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein; and the formulation having reduced turbidity compared to a reference formulation containing a first molar amount (e.g., the same first molar amount) of conjugate and a second molar amount (e.g., the same second molar amount) of the second protein, at a pH within 0.5 pH units of the pI of the second protein. In some embodiments, the formulation comprises a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the second protein is present in the formulation in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first and second molar amounts, and the formulation is located at a pH of about 0.5 units or more away from the isoelectric point (pI) of the second protein. The formulation has a pH that is approximately the same as that of the second protein (e.g., within 0.05, 0.1, 0.15, 0.2, 0.2, 0.3, 0.4, or 0.5 pH units), and exhibits reduced turbidity compared to a reference formulation containing the same molar amount of the first conjugate and the same molar amount of the second protein. A therapeutically acceptable composition is provided, comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the composition percentage of the second protein is about 1% or more, with the remainder being the first protein, and the composition has a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein, and within 0.5 pH units of the pI of the second protein, and having reduced turbidity compared to a reference composition comprising the above-mentioned percentage (e.g., the same percentage) of the second protein, with the remainder being the first protein. In some embodiments, a therapeutically acceptable composition comprises a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the composition percentage of the second protein is about 1% or more, with the remainder being the first protein; the composition has a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein; and the composition has reduced turbidity compared to a reference composition containing the same composition percentage of the second protein and the remainder being the first protein at a pH approximately the same as the pI of the second protein (e.g., within 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 pH units).A pharmaceutical formulation is provided herein, comprising: a first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation contains the protein not conjugated to the phosphorylcholine-containing polymer in an amount of about 1% or more of the total molar amount of the conjugated and unconjugated proteins, the total molar amount being the sum of the first and second molar amounts, the formulation having a pH about 0.5 pH units away from the isoelectric point (pI) of the protein, and the formulation being substantially free of turbidity. The Specified Publication also provides a formulation comprising a phosphorylcholine-containing polymer present in a concentration of 100 mg / mL or more, and a protein not conjugated to the phosphorylcholine-containing polymer, wherein the protein is present in the formulation in a second molar amount, the protein is present in the formulation in a concentration of about 1% or more of the total molar amount of the polymer and protein, the total molar amount is the sum of the first and second molar amounts, and the formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the protein.

[0014] A formulation is provided herein, comprising a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the difference between the pI of the second protein in an acidic or basic direction and the pH of the formulation is selected to be greater than the minimum difference between the pI of the second protein in the corresponding acidic or basic direction and the pH of a reference formulation comprising a third molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a fourth molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the first total molar amount, including the sum of the first and second molar amounts, and the second total molar amount, including the sum of the third and fourth molar amounts, are substantially the same, the second molar amount is greater than the fourth molar amount, and the reference formulation is substantially clear. A therapeutically acceptable composition comprising: a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the difference between the pI of the second protein and the pH of the formulation is the pI of the second protein and the conjugate containing the first protein conjugated to the phosphorylcholine-containing polymer. Also provided herein are therapeutically acceptable compositions selected such that the pH difference between the reference formulation and a jugate, a second protein not conjugated to a phosphorylcholine-containing polymer, and a pharmaceutically acceptable carrier is greater than the minimum difference between the two compositions and the reference formulation, the percentage of the second protein in the composition being greater than the percentage of the second protein in the reference composition, and the reference composition being substantially free of turbidity.

[0015] A formulation is also provided comprising a first molar amount of conjugate containing a first protein conjugated to a polymer; and a second molar amount of a second protein not conjugated to a polymer, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the first and second proteins, and the total molar amount is the sum of the first and second molar amounts. A therapeutically acceptable composition is also provided herein comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the composition percentage of the second protein is about 1% or more, and the remainder is the first protein. Also provided is a therapeutically acceptable composition comprising a first molar amount of conjugate containing a first protein conjugated to a polymer; and a second molar amount of a second protein not conjugated to a polymer, wherein the composition comprises the second protein in an amount of about 1% or more of the total molar amount of the first and second proteins, and the total molar amount comprises the sum of the first and second molar amounts. Further provided herein is a formulation comprising a first molar amount of a first protein conjugated to a polymer; and a second molar amount of a second protein not conjugated to a polymer, further improved by: the formulation comprising the second protein in an amount of about 1% or more of the total molar amount of the conjugate and second protein, and the total molar amount comprises the sum of the first and second molar amounts. A therapeutically acceptable composition comprising a first protein conjugated to a polymer and a second protein not conjugated to a polymer is provided herein, further improved by: the composition percentage of the second protein being about 1% or more, with the remainder being the first protein.

[0016] A formulation is also provided comprising a conjugate containing a first protein conjugated to a polymer; and a second protein not conjugated to a polymer, wherein the first molar amount of the conjugate and the second molar amount of the second protein are combined in the formulation such that the second molar amount is about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount is the sum of the first and second molar amounts. A therapeutically acceptable composition is also provided comprising a conjugate containing a first protein conjugated to a polymer; and a second protein not conjugated to a polymer, wherein the first molar amount of the conjugate and the second molar amount of the second protein are combined in the composition such that the second molar amount is about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount is the sum of the first and second molar amounts. A therapeutically acceptable composition is provided comprising a conjugate containing a first protein conjugated to a polymer; and a second protein not conjugated to a polymer, wherein the second protein is combined with the conjugate at a compositional percentage of about 1% or more (e.g., about 5-90%, 15-25%, 25-35%, etc.) relative to the total protein mass weight concentration of the first and second proteins in the composition, and the remainder of the total protein mass weight concentration is the first protein.

[0017] A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer, wherein the polymer has nine arms and a molecular weight of 600,000 to 1,000,000 Da, and the polymer is present in the formulation at a concentration of approximately 100 mg / mL or more. A formulation is provided that includes a second protein, which is not conjugated to a polymer and is present in the formulation at a concentration of 5 to 15 mg / mL. An intraocular therapeutic composition is provided which contains an anti-VEGF-A antibody at a concentration of approximately 50 mg / mL of protein, wherein the anti-VEGF-A antibody is located in complementarity-determining region 1 (CDR). H 1): GYDFTHYGMN (SEQ ID NO: 9), CDRH 2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: Heavy chain containing YPYYYGTSHWYFDV (SEQ ID NO: 11); and CDR L 1:SASQDISNYLN (Sequence ID 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: A composition for intraocular treatment is also provided, comprising a light chain containing QQYSTVPWT (SEQ ID NO: 14), wherein the anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, with the unconjugated antibody making up about 10% to about 30% of the total molar amount of the antibody conjugate and unconjugated antibody, the antibody conjugate comprising an anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer at a non-natural cysteine ​​outside the variable region of the antibody, the phosphorylcholine-containing polymer being present in the composition at about 100 mg / mL or more, the phosphorylcholine-containing polymer having 9 arms and a molecular weight of 600,000 to 1,000,000 Da, and the pH of the composition being about 5.5 or less.

[0018] An intraocular therapeutic composition comprising approximately 50 mg / mL of protein containing an anti-VEGF-A antibody, wherein the anti-VEGF-A antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 (with or without C-terminal lysine) and a light chain containing the amino acid sequence of SEQ ID NO: 2, the anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, the unconjugated antibody is present in the formulation at approximately 10% to approximately 30% of the total molar amount of the antibody conjugate and unconjugated antibody (the sum of the molar amounts of the antibody conjugate and unconjugated antibody), and the antibody conjugate comprises the following structure: [ka] In the formula, each heavy chain of the conjugate is represented by the letter H, and each light chain of the conjugate is represented by the letter L; the polymer is via sulfhydryl C443 (EU numbering). It is bonded to the heavy chain of the conjugate, and this bond is shown on one of the heavy chains; PC is [ka] A composition for intraocular treatment is also provided, wherein the wavy line in the formula indicates a bond point to the remainder of the polymer, and X is a) -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H, c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS; 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%, the phosphorylcholine-containing polymer is present in the composition at a concentration of about 100 mg / mL or more, and the pH of the composition is about 5.5 or less.

[0019] An intraocular therapeutic composition is also provided, comprising a protein fusion construct at approximately 53 mg / mL, wherein the fusion construct comprises a VEGF trap fused to an anti-IL-6 antibody, and the fusion construct comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 105 (with or without C-terminal lysine); and a light chain containing the amino acid sequence of SEQ ID NO: 106, and the fusion construct is present in the composition as either a conjugate or an unconjugated fusion construct, with the unconjugated fusion construct accounting for approximately 20% to approximately 40% of the total molar amount of the conjugate and unconjugated fusion constructs, which is the sum of the molar amounts of the conjugate and the unconjugated fusion construct, and the conjugate comprises a fusion construct conjugated to a phosphorylcholine-containing polymer, with the phosphorylcholine-containing polymer present in the composition at approximately 100 mg / mL or more, and the pH of the composition being approximately 5 or less.

[0020] An intravitreal therapeutic composition containing a fusion construct with a protein at about 50 mg / mL, wherein the fusion construct comprises a VEGF trap fused to an anti-IL-6 antibody, and the fusion construct has complementarity-determining region 1 (CDR H 1): PFAMH (SEQ ID NO: 134), CDR H 2: KISPGGSWTYYSDTVTD (SEQ ID NO: 135), and CDR H 3: QAWGYYALDI (SEQ ID NO: 136) in the heavy chain; and CDR L 1: SASISVSYLY (SEQ ID NO: 137), CDR L 2: DDSSLAS (SEQ ID NO: 138), and CDR L 3: QQWSGYPYT (SEQ ID NO: 139) in the light chain, and the fusion construct is present in the composition as either a conjugate or a non-conjugate fusion construct, and the non-conjugate fusion construct is present in the formulation at about 20% to about 40% of the total molar amount of the conjugate and non-conjugate fusion constructs, which is the sum of the molar amounts of the conjugate and the non-conjugate fusion constructs, and the conjugate has the following structure:

Chemical formula

Chemical formula

[0021] A method for preparing a formulation is provided herein, comprising combining in the formulation a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first and second molar amounts, and the formulation having a pH about 0.5 pH units away from the isoelectric point (pI) of the second protein. A method for preparing a therapeutically acceptable composition is provided herein, comprising combining in the formulation a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer, wherein the formulation is therapeutically acceptable A method is also provided for preparing a therapeutically acceptable composition, the method comprising combining a conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer and a second protein not conjugated to a phosphorylcholine-containing polymer in a therapeutically acceptable composition, wherein the composition comprises a percentage of the composition of the second protein comprising about 1% or more of the total molar amount of the conjugate and the second protein, the remainder comprising the first protein, and the composition having a pH at least 0.5 pH units away from the isoelectric point (pI) of the second protein. A method for preparing a formulation is also provided, comprising adjusting the pH of the formulation to be at least 0.5 pH units away from the isoelectric point (pI) of a non-conjugated protein contained in the formulation, wherein the formulation comprises a first molar amount of conjugate, comprising a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of non-conjugated protein, wherein the non-conjugated protein is not conjugated to the phosphorylcholine-containing polymer, and the formulation contains non-conjugated protein in an amount of at least 1% of the total molar amount of conjugate and non-conjugated proteins, the total molar amount comprising the sum of the first and second molar amounts.

[0022] A method for preparing a therapeutically acceptable composition is provided, comprising adjusting the pH of the composition to be at least about 0.5 pH units away from the isoelectric point (pI) of a non-conjugated protein contained in the composition, wherein the composition comprises a first molar amount of conjugate, comprising a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of non-conjugated protein, wherein the non-conjugated protein is not conjugated to the phosphorylcholine-containing polymer, and the composition contains non-conjugated protein in an amount of at least 0.1% of the total molar amount of conjugated and non-conjugated proteins, the total molar amount comprising the sum of the first and second molar amounts. A method for preparing a therapeutically acceptable composition is also provided, comprising adjusting the pH of the therapeutically acceptable composition so that it is at least about 0.5 pH units away from the isoelectric point (pI) of a non-conjugated protein contained in the composition, wherein the composition comprises a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; and a second protein not conjugated to a phosphorylcholine-containing polymer, wherein the compositional percentage of the second protein is at least about 1%, with the remainder being the first protein.

[0023] A method for preparing a low-viscosity formulation of a protein conjugated to a phosphorylcholine-containing polymer is provided herein, the method comprising combining in the formulation a first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer, wherein the formulation contains the protein not conjugated to the phosphorylcholine-containing polymer in an amount of about 1% or more of the total molar amount of the conjugated and unconjugated proteins, the total molar amount comprising the sum of the first and second molar amounts, the formulation having a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein, and the formulation having reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in total molar amount. A method for preparing a therapeutically acceptable composition of low viscosity protein conjugated to a phosphorylcholine-containing polymer, comprising combining a conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer and a second protein not conjugated to a phosphorylcholine-containing polymer in a therapeutically acceptable composition, wherein the composition percentage of the second protein is about 1% or more, and the remainder comprises the first protein, and the composition has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein, and the composition is conjugated A method is also provided in which the first protein of the conjugate is present in the reference composition at a total mass weight concentration of the first and second proteins in the composition, having reduced viscosity and / or improved injectability compared to a reference composition containing the conjugate.

[0024] A method for preparing a formulation is also provided, comprising combining a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer, in the formulation, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount is the sum of the first and second molar amounts. A method for preparing a therapeutically acceptable composition is also provided, comprising combining a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; and a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer, in the therapeutically acceptable composition, wherein the composition contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount is the sum of the first and second molar amounts. A method for preparing a therapeutically acceptable composition is also provided herein, comprising combining a conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer and a second protein not conjugated to a phosphorylcholine-containing polymer in the therapeutically acceptable composition, wherein the compositional percentage of the second protein is about 1% or more, and the remainder comprises the first protein.

[0025] Formulations or compositions prepared by any one of the methods described herein are provided herein.

[0026] A method for treating a subject is also provided, comprising intraocular administration of a therapeutically effective amount of one of the formulations or compositions described herein to a subject in need of treatment. A method for treating a subject is also provided, comprising intraocular administration of a therapeutically effective amount of a low-viscosity formulation to a subject in need of treatment, wherein the formulation comprises a first concentration of a conjugate containing a first anti-VEGF antibody conjugated to a phosphorylcholine-containing polymer; a second concentration of an anti-VEGF agent not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation has a pH at least 0.5 pH units away from the isoelectric point (pI) of the anti-VEGF agent.

[0027] A kit is provided herein, comprising a pre-filled syringe containing a low-viscosity formulation comprising a conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and a second protein not conjugated to a phosphorylcholine-containing polymer; and a syringe needle for injecting the low-viscosity formulation, the needle having a gauge of 27 or greater.

[0028] A formulation is also provided comprising approximately 40–60 mM sodium acetate, approximately 0.01%–0.04% polysorbate 20, and a mixture of OG1950 and OG1953 in approximately 40–60 mg / mL (total protein concentration), wherein the mixture contains approximately 15%–25% OG1950 and approximately 75%–85% OG1953 in molar amounts, and the pH is approximately 4.5–5.5. A formulation is also provided herein comprising, consisting of, or essentially consisting of, a mixture of approximately 50 mM sodium acetate, approximately 0.025% polysorbate 20, and approximately 50 mg / mL (total protein concentration) OG1950 and OG1953, wherein the mixture contains approximately 20% OG1950 and approximately 80% OG1953 in molar amounts, and the pH is approximately 5.

[0029] A method for storing proteins, wherein the method is for a period of at least two months to two years. A method is provided herein that involves maintaining a protein in a formulation, comprising: a formulation comprising a first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount comprising the sum of the first and second molar amounts, the formulation having a pH at least about 0.5 pH units away from the isoelectric point (pI) of the second protein, and the protein comprising an antibody or a fusion construct. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows compound L. [Figure 2] Figure 2 shows compound K. [Figure 3] Figure 3 shows the synthesis of OG1802 from R3707. [Figure 4] Figure 4 shows OG1786. [Figure 5] Figure 5 shows the synthesis of OG1546 from OG1550. [Figure 6] Figure 6 shows the synthesis of OG1784 from OG1546 and OG1563. [Figure 7] Figure 7 shows the synthesis of OG1405 from OG1784. [Figure 8] Figure 8 shows the synthesis of OG1785 from OG1405. [Figure 9] Figure 9 shows the synthesis of OG1786 from OG1785. [Figure 10] Figure 10 shows OG1802. [Figure 11] Figure 11 shows compound E. [Figure 12]Figure 12 illustrates several embodiments of the anti-VEGF-A heavy chain having specific effector functional mutations and L443C (EU numbering, position 449 in SEQ ID NO: 1). [Figure 13] Figure 13 illustrates several embodiments of the anti-VEGF-A light chain (SEQ ID NO: 2). [Figure 14] Figure 14 illustrates several embodiments of the bevacizumab heavy chain (SEQ ID NO: 3). [Figure 15] Figure 15 illustrates several embodiments of the bevacizumab light chain (SEQ ID NO: 4). [Figure 16] Figure 16 illustrates several embodiments of the ranibizumab heavy chain (SEQ ID NO: 5). [Figure 17] Figure 17 illustrates several embodiments of the ranibizumab light chain (SEQ ID NO: 6). [Figure 18] Figure 18 illustrates several embodiments of a method for preparing antibody conjugates. [Figure 19] Figure 19 shows the ion exchange analysis (A280 absorbance) of reactions A through G. [Figure 20] Figure 20 illustrates the effects of various anti-VEGF molecules on the binding of biotin-VEGF to plate-bound VEGFR ECD-Fc protein, as well as their IC50 values. [Figure 21] Figure 21 illustrates the binding affinity of OG1950 to VEGF as measured by BIAcore single-cycle kinetics. [Figure 22] Figure 22 illustrates the binding of OG1950 to Fcγ receptor I. [Figure 23] Figure 23 illustrates the binding of OG1950 to the Fcγ receptor IIIa. [Figure 24] Figure 24 illustrates the binding of QG1950 to the human complement protein C1q. [Figure 25] Figure 25 shows the results of the proliferation assay (including the IC50 value). [Figure 26]Figure 26 illustrates the results of single-cycle kinetics of VEGF binding to anti-VEGF agents. [Figure 27] Figure 27 illustrates several embodiments of nucleic acid sequences encoding heavy chain variable regions and light chain variable regions. [Figure 28] Figure 28 is a collection of images and tables showing the results of mixing free antibodies with either a polymer or an OG1953 composition. [Figure 29] Figure 29 is a collection of images showing the visual appearance of 30 different formulation conditions in two Design of Experiments (DOE) screenings. [Figure 30] Figure 30 is a graph showing the turbidity measurements of antibody preparations containing the OG1801 polymer (samples #1 to #30) in the presence of different excipients. [Figure 31] Figure 31 is a plot created using JMP SAS software showing the change in turbidity over time for one embodiment of a DOE experiment in which the OG1950 antibody was mixed with the OG1801 polymer in the presence of different excipients. [Figure 32] Figure 32 is an image and table showing the effects of histidine and pH on the turbidity of the formulation. [Figure 33] Figure 33 is an image showing several embodiments of the formulation at different pH levels and the corresponding turbidity results. [Figure 34] Figure 34 is a graph showing several embodiments of the formulation at different pH levels and the corresponding turbidity results. [Figure 35A]Figure 35A is a collection of images showing the appearance of four different preparations of 50 mg / ml conjugated antibody with added non-conjugated antibody at different pH settings. From left to right: A, the preparation at pH 6.5 yields a cloudy mixture; B-D, the preparations at 5.0-5.2 yield clear solutions regardless of the percentage of formulation buffer and non-conjugated antibody. The mixture is prepared by first concentrating the purified conjugated antibody to approximately 6 mg / ml, adding the desired amount of non-conjugated antibody, exchanging the antibody solution with formulation buffer, and then concentrating it to 20 mg / ml. Polysorbate 20 is added to the protein solution, and then it is concentrated to the final target total concentration of 50 mg / ml. [Figure 35B] Figure 35B is a collection of chromatograms and tables showing cation exchange chromatography (CEX) and analytical size exclusion chromatography (SEC) analyses of several embodiments of the OG1950 / OG1953 formulation. [Figure 35C] Figure 35C is a collection of chromatograms and tables showing cation exchange chromatography (CEX) and analytical size exclusion chromatography (SEC) analyses of several embodiments of the OG1950 / OG1953 formulation. [Figure 36] Figure 36 is a table showing the turbidity results for one embodiment in which the OG2072 fusion protein is mixed with its conjugate form, OG2074. [Figure 37A] Figure 37A is a collection of tables, images, and graphs showing the screening results of various proteins other than the OG1950 antibody mixed with fixed concentrations of the OG1801 polymer. This serves as a control to assess whether turbidity is a more general phenomenon associated with the presence of the polymer. [Figure 37B] Figure 37B is a collection of tables, images, and graphs showing the screening results of various proteins other than the OG1950 antibody mixed with fixed concentrations of the OG1801 polymer. This serves as a control to assess whether turbidity is a more general phenomenon associated with the presence of the polymer. [Figure 38A] Figure 38A is a collection of images and plots showing the effect of pH adjustment on the turbidity of the formulation. The results demonstrate that turbidity is reversible or reproducible depending on the protein pI. [Figure 38B] Figure 38B is a collection of images and plots showing the effect of pH adjustment on the turbidity of the formulation. The results demonstrate that turbidity is reversible or reproducible depending on the protein pI. [Figure 39] Figure 39 is a table showing sample settings for formulations having different proportions of free protein relative to the conjugate protein. [Figure 40A] Figure 40A is a collection of images showing the appearance of formulations with different proportions of free protein relative to the conjugate protein. [Figure 40B] Figure 40B is a collection of traces showing the analysis of different distributions during tandem analysis of the formulation. [Figure 40C] Figure 40C is a collection of plots showing the analysis of different distributions during tandem analysis of formulations. [Figure 40D] Figure 40D is a table showing a comparison of peak areas between the CEX-bound (free protein) and the unbound fraction (conjugate). [Figure 40E] Figure 40E is a collection of traces showing the analysis of different distributions during tandem analysis of the formulation. [Figure 40F] Figure 40F is a collection of plots showing the analysis of different distributions during tandem analysis of formulations. [Figure 40G] Figure 40G is a table showing a comparison of peak areas between the CEX-bound (free protein) and the unbound fraction (conjugate). [Figure 41] Figure 41 is a table showing sample settings for long-term stability planning for various formulations of OG1953 conjugates containing 7.5-20% free protein with a total protein concentration of 50-65 mg / ml. [Figure 42]Figure 42 is a collection of tables and plots showing the results of ELISA assays measuring the potency of various formulations of OG1953 conjugates containing 7.5–20% free protein at a total protein concentration of 50–65 mg / ml after storage. [Figure 43] Figure 43 is a collection of tables and plots showing the results of ELISA assays measuring the potency of various formulations of OG1953 conjugates containing 7.5–20% free protein at a total protein concentration of 50–65 mg / ml after storage. [Figure 44-1] Figure 44 is a collection of tables and graphs showing the protein concentrations of the formulations measured by the SoloVPE OD280nm method. [Figure 44-2] Same as above. [Figure 45-1] Figure 45 is a data table showing an overview of the results of the stability test of the formulation. [Figure 45-2] Same as above. [Figure 45-3] Same as above. [Figure 46A] Figure 46A shows a collection of SEC-HPLC traces of formulation #4 (20% OG1950, 80% OG1953, 50 mM sodium acetate, 0.025% Tween 20, pH 5.0) after 6 months at a heightened temperature. [Figure 46B] Figure 46B provides tables and graphs showing the size exclusion chromatography analysis of the formulation for the aggregation and degradation levels of the OG1953 conjugate. [Figure 47A] Figure 47A is a schematic diagram illustrating the tandem HPLC method, which combines CEX-HPLC with an SEC-HPLC column in tandem. [Figure 47B-1] Figure 47B is a collection of traces, tables, and graphs showing the tandem analysis of OG1950 free protein and its aggregated forms (P1 and P2). [Figure 47B-2] Same as above. [Figure 47C] Figure 47C is a collection of traces and tables showing the tandem analysis of the formulation. [Figure 47D]Figure 47D is a collection of traces and tables showing the tandem analysis of the formulation. [Figure 48A] Figure 48A is a collection of tables, images, and graphs showing the formulation design matrix and visual results. [Figure 48B] Figure 48B is a collection of tables, images, and graphs showing the formulation design matrix and visual results. [Figure 48C] Figure 48C is a collection of tables, images, and graphs showing the formulation design matrix and its visual results. [Figure 49A] Figure 49A is a collection of plots showing the analysis of turbidity results against polymer, free protein, and pH. [Figure 49B] Figure 49B is a collection of plots showing the analysis of turbidity results against polymer, free protein, and pH. [Figure 49C] Figure 49C is a collection of plots and schematic diagrams showing the turbidity of formulations at various pH levels for different levels of OG1801 polymer and free protein. [Figure 49D] Figure 49D is a collection of plots and schematic diagrams showing the turbidity of formulations at various pH levels for different levels of OG1801 polymer and free protein. [Figure 49E] Figure 49E is a plot showing the turbidity measurement results against polymer, free protein concentration, and pH. [Figure 49F] Figure 49F is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1801 polymers and free proteins only. [Figure 49G] Figure 49G is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1801 polymers and free proteins only. [Figure 49H] Figure 49H is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1801 polymers and free proteins only. [Figure 49I]Figure 49I is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1801 polymers and free proteins only. [Figure 49J] Figure 49J is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1953 conjugate solutions and free protein only. [Figure 49K] Figure 49K is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1953 conjugate solutions and free protein only. [Figure 49L] Figure 49L is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1953 conjugate solutions and free protein only. [Figure 49M] Figure 49M is a collection of plots showing the pH boundary curve plot superimposed with experimental data for other OG1953 conjugate solutions and free protein only. [Figure 49N] Figure 49N is a plot showing the analysis of turbidity measurements in an OG1801 polymer solution containing free protein against polymer, free protein concentration, and pH. [Figure 49O] Figure 49O is a plot showing the analysis of turbidity measurements in an OG1953 conjugate solution containing free protein against polymer, free protein concentration, and pH. [Figure 50A] Figure 50A is a collection of graphs and schematic diagrams showing the viscosity of the formulation at 25°C. [Figure 50B] Figure 50B is a collection of graphs and tables showing the effect of increasing the percentage of free protein composition on the viscosity of the formulation at 25°C. [Figure 50C] Figure 50C is a collection of graphs and tables showing the effect of increasing the percentage of free protein composition on the viscosity of the formulation at 25°C. [Figure 50D] Figure 50D is a collection of plots and tables showing the viscosity of formulations containing OG1801 polymer at various concentrations. [Figure 51] Figure 51 is a collection of images showing the movement of air inside syringes filled with different formulations. [Figure 52A] Figure 52A is a collection of plots and tables showing a comparison of the potency of various OG1953 formulations using ELISA or cell-based assays. [Figure 52B] Figure 52B is a collection of plots and tables showing a comparison of the potency of various OG1953 formulations using ELISA or cell-based assays. [Figure 52C] Figure 52C is a collection of plots and tables showing a comparison of the potency of various OG1953 formulations using ELISA or cell-based assays. [Figure 53A] Figure 53A is a collection of graphs, charts, and tables showing the improvement in visual acuity in patients with wet-type AMD treated with OG1953, aflibercept, or other anti-VEGF agents. [Figure 53B] Figure 53B is a collection of graphs, charts, and tables showing the improvement in visual acuity in patients with wet-type AMD treated with OG1953, aflibercept, or other anti-VEGF agents. [Figure 53C] Figure 53C is a collection of graphs, charts, and tables showing the improvement in visual acuity in patients with wet-type AMD treated with OG1953, aflibercept, or other anti-VEGF agents. [Figure 53D] Figure 53D is a collection of graphs, charts, and tables showing the improvement in visual acuity in patients with wet-type AMD treated with OG1953, aflibercept, or other anti-VEGF agents. [Figure 54] Figure 54 is a set of arrays showing several non-limiting embodiments of the light and heavy chains of the fusion construct. [Figure 55A] Figure 55A is a set of arrays showing several non-limiting embodiments of the heavy and light chains of the fusion construct. [Figure 55B]Figure 55B is a set of arrays showing several non-limiting embodiments of the heavy and light chains of the fusion construct. [Figure 56A] Figure 56A is a collection of plots showing the levels of impurities (e.g., aggregation and / or degradation levels) in the OG1953 conjugate formulation over time. [Figure 56B] Figure 56B is a collection of plots showing the levels of impurities (e.g., aggregation and / or degradation levels) in the OG1953 conjugate formulation over time. [Figure 56C] Figure 56C is a collection of plots showing the levels of impurities (e.g., aggregation and / or decomposition levels) in the OG1953 conjugate formulation over time. [Figure 57A] Figure 57A is a collection of plots and tables showing a comparison of the potency of various OG1953 formulations using ELISA or cell-based assays. [Figure 57B] Figure 57B is a collection of plots and tables showing a comparison of the potency of various OG1953 formulations using ELISA or cell-based assays. [Figure 58A] Figure 58A is a set of schematic diagrams showing the components of non-limiting examples of the formulations of the present disclosure. [Figure 58B] Figure 58B is a set of schematic diagrams showing the components of non-limiting examples of the formulations of the present disclosure. [Figure 58C] Figure 58C is a set of schematic diagrams showing the components of non-limiting examples of the formulations of the present disclosure. [Figure 59-1] Figures 59A–59D illustrate continuous 80-minute tandem separation using a photodiode array (PDA) detection set to 200–350 nm. Figure 59A shows a 2D contour plot of elution time versus wavelength; Figure 59B illustrates the extraction wavelength profile at 280 nm and peak identification of various eluted fractions collected for further characterization using SDS-PAGE analysis following silver staining, with results shown in Figure 59C as the non-reducing gel and in Figure 59D as the reducing gel. [Figure 59-2] Same as above. [Figure 60A] Figure 60A illustrates KSI-301 stability data up to 9 months under different temperature conditions; -20±5℃ (Figure 60A). [Figure 60B] Figure 60B illustrates KSI-301 stability data up to 9 months under different temperature conditions; 5±3℃ (Figure 60B). [Figure 60C] Figure 60C illustrates KSI-301 stability data for up to 9 months under different temperature conditions; 25±2℃ / 60±5%RH (relative humidity) (Figure 60C). [Figure 61] Figure 61 illustrates the lot release data for KSI-501DS batches 1-3. [Figure 62] Figure 62 shows a comparison of injection force between OG1953 (100%) conjugate and KSI-301 mixed formulation using a 27G or 29G injection needle (Panel A); and a comparison of injection force between OG2074 (100%) conjugate and KSI-501 Batch 2 mixed formulation using a 27G or 29G injection needle (Panel B). [Figure 63] Figure 63 illustrates the viscosity comparison at ambient temperature for various batches of (Panel A) OG1953 (100%) conjugate versus KSI-301 mixed formulation and (Panel B) OG2074 (100%) conjugate versus KSI-501 mixed formulation. [Modes for carrying out the invention]

[0031] This specification provides formulations and compositions comprising unconjugated proteins (e.g., unconjugated anti-VEGF-A antibodies) and mixtures thereof with conjugates. In some embodiments, the conjugate may comprise a protein (which may be the same as or different from the unconjugated protein) conjugated to a phosphorylcholine-containing polymer. In some embodiments, the pH of the formulation may differ from the isoelectric point (pI) of the unconjugated protein in the formulation, thereby preventing turbidity or reducing turbidity. In some embodiments, the formulation has a reduced viscosity compared to a reference formulation of the conjugated protein (which does not contain the unconjugated protein). The reduced viscosity may improve the injectability (e.g., by syringe) of the formulation and / or handling of the formulation during manufacturing. In some embodiments, the compositions may be used for the treatment of specific conditions, such as ocular disorders, including retinal vascular disorders. The formulations, compositions, and methods of this disclosure may be provided for the treatment of any disease or disorder described herein, and are not necessarily limited to diabetic retinopathy or diabetic macular edema.

[0032] In some embodiments of this specification, drug compositions (formulations) are provided, which are mixtures of proteins and their conjugates, and which are stable solutions. While not limited by theory, reducing the final concentration of a biopolymer while maintaining the same amount of bioactive antibody can result in: (i) a decrease in solution viscosity, which has many advantages for the manufacture of the active pharmaceutical ingredient and drug; (ii) better handling of the drug for dose preparation and in clinical settings; and (iii) easier injection into the patient, for example, in the retina, where the needle is inserted into the vitreous humor of the eye and the syringe plunger is pressed with maximum force, including shorter injection times, reduced force required to push down the plunger to expel the drug, and finer needles such as 30G or 29G, and higher dose levels in smaller volumes. These properties (i), (ii), and (iii) can consequently reduce the possibility of undesirable side effects of intravitreal injection, including, but not limited to, bacterial contamination of the eye. In some cases, side effects include cataracts. While not limited by theory, mixtures of unconjugated and conjugated proteins may benefit from both the direct activity of the proteins and the modified activity of the proteins modified by conjugation to polymers. For example, the immediate effect of unconjugated proteins in a defined ratio (e.g., 20% of the administered antibody) and the modified sustained effect of conjugated proteins in a defined ratio (e.g., the remaining 80% of the antibody is in conjugated form) can be combined to provide a basal effect driven by the conjugated protein and a bolus effect driven by the unconjugated antibody. In some embodiments, the formulation is a clear solution at a specific pH comprising a free (unconjugated) antibody, an antibody conjugated to a phosphorylcholine polymer, and a buffer system, and the formulation is stable and suitable for drug development, manufacturing, and / or storage.

[0033] In some embodiments, an anti-VEGF antibody (OG1950) conjugated to a phosphorylcholine-containing biopolymer (OG1802) (this conjugate is called OG1953) can be formulated at 50 mg / mL by antibody weight and formulated in clear and stable sodium phosphate (pH 6.5). Turbidity may occur if a desired amount of free unconjugated anti-VEGF antibody (OG1950) is added to prepare a solution of 40 mg / mL conjugate (OG1953) and 10 mg / mL OG1950 without adjusting the formulation system as provided herein. In some embodiments, the formulations and methods provided herein can provide clear solutions of unconjugated and conjugated antibody co-formulations.

[0034] Provided in some embodiments of this specification are transparent and stable "formulation systems" comprising a protein and a phosphorylcholine-biopolymer conjugate of that protein, wherein the protein is soluble, transparent, and stable, and avoids turbidity formation. In some embodiments, methods for reducing turbidity are also provided.

[0035] In some embodiments, applying a soluble switch to OG1953 allows for the production of OG1953 80% + OG1950 20% at pH 5.0, thereby preventing the turbidity formed at pH 7.5, which is the protein pI or close to it. In some embodiments, the soluble switch involves the application of pH to control this switch. In some embodiments, this soluble switch is used for ophthalmic (intravitreal) injection or systemic disease, producing OG2074 70% + OG2072 at pH 5.0. It is applied to OG2074 to produce 30%.

[0036] In some embodiments, in ophthalmology where the dose volume is approximately 100 microliters, the concentration of the dose formulation is, for example, 50 mg / mL (measured by the antibody portion) in the case of OG1953, and the bioconjugate has persistence due to the conjugated biopolymer. On the other hand, to improve manufacturability, the relative amount of the unconjugated protein is increased while maintaining the same amount of bioactive protein (5.0 mg in a 100 microliter dose, i.e., 50 mg / mL in the formulation by antibody weight). For example, in some embodiments, OG1953 is 80% and OG1950 is 20%; or OG1953 is 75% and OG1950 is 25%; or OG1953 is 70% and OG1950 is 30%; or OG1953 is 65% and OG1950 is 35%; or OG1953 is 60% and OG1950 is 40%; or OG1953 is 50% and OG1950 is 50%. In some embodiments, the pH is 5.0 (without the addition of histidine, sucrose, or trehalose) by shifting the pH to 5.0 and adjusting the formulation buffering component from phosphate to acetate. In some embodiments, high-dose formulations with improved manufacturability are achieved for the following reasons: reduced viscosity in the active pharmaceutical ingredient and drug (vials, pre-filled syringes); improved usability and dose administration (due to reduced viscosity); and improved clinical immediacy (thus an improved balance of, for example, the clinical immediacy of 20% mAb while maintaining the clinical persistence of 80% mAb conjugate). In some embodiments, the composition may be adjusted to achieve an optimal viscosity to enable large-scale manufacturing (of the active pharmaceutical ingredient, drug, and pre-filled syringes), to enable safer dose handling and preparation (e.g., by physicians and patients), and to enable safer dose administration, such as injectability requiring lower injection force by physicians. In some embodiments, the injection force is less than 10 Newtons or less than 5 Newtons, the injection time is 10 seconds or less or 5 seconds or less, and the injection needle has a bore size of 30 gauge to 27 gauge, including 28 gauge or 29 gauge. In some embodiments, the composition The formulation can be adjusted to achieve an optimal balance between the duration of clinical effect (basal activity) and the immediate clinical effect (bolus activity). In some embodiments, the therapeutic agent is formulated into a clear solution with long-term stability.

[0037] This specification further provides methods for preparing antibody (any type of antibody and / or protein) conjugate compositions. In some embodiments, these methods enable the reduction of aggregate formation or the increased efficiency of the formation of desired compositions of antibodies and antibody conjugates.

[0038] These and additional embodiments are provided below, following the definition section.

[0039] term In light of this disclosure, all terms may have their conventional and ordinary meanings to those skilled in the art. “Angiogenic disorder” is a disorder or condition characterized by altered, dysregulated, or unregulated angiogenesis. Examples of angiogenic disorders include malignant transformations (e.g., cancer), as well as ocular angiogenic disorders, including diabetic retinopathy and age-related macular degeneration.

[0040] Ocular neovascularization disorders are characterized by altered, dysaccadic, or unaccommodative neovascularization in a patient's eye. Such disorders include 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.

[0041] The term "composition percentage" refers to the percentage amount (in units of mass or concentration) of a component present in a composition. Composition percentage is calculated by determining the amount of a component in units of mass (e.g., μg) or concentration (e.g., mg / mL), dividing that amount by the total amount of all components in the composition in the corresponding unit, and multiplying by 100. For the conjugate and unconjugate protein compositions and formulations described herein, the composition percentage can be obtained by dividing the amount of unconjugate protein by the total amount of protein components in the solution (excluding the contribution of the polymer component of the conjugate to the mass of the conjugate).

[0042] As used herein, "% total molar weight" represents the ratio (percentage) of the amount (moles or molar concentration) of one component of a composition to the amount (moles or molar concentration) of one or more other components of the composition that together constitute the whole (100%). It is understood that compositional percentages and % total molar weight are convertible to each other when the molecular weights of all the relevant components are known.

[0043] The term "antibody" includes intact antibodies and their binding fragments. A binding fragment is a molecule distinct from the intact antibody that contains a portion of the 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. The scFv antibody is described in Houston JS. 1991. Methods in Enzymol. 203:46-96. Furthermore, antibody fragments include single-chain polypeptides that possess the characteristics of a VH domain, i.e., the ability to assemble with the VL domain to form a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody, or the characteristics of a VL domain, i.e., the ability to assemble with the VH domain to form a functional antigen-binding site, thereby providing the antigen-binding properties of a full-length antibody.

[0044] The fact that an antibody specifically binds to its target antigen means that at least 10 6 M -1 , at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , or at least 10 10 M -1 This means that there is affinity. Specific binding is detectably high in magnitude and distinguishable from nonspecific binding, which occurs to at least one unrelated target. Specific binding can result from bond formation between specific functional groups or specific spatial fit (e.g., lock-and-key type), while nonspecific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that the antibody or fusion protein will bind to only one target.

[0045] The basic structural unit of an antibody is a tetramer of subunits. Each tetramer contains two identical polypeptide chain pairs, each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa). The amino-terminus of each chain contains a variable region consisting of approximately 100-110 or more amino acids, which is most significantly involved in antigen recognition. This variable region is initially expressed ligated to a cleavable signal peptide. A variable region that does not contain a signal peptide is sometimes called a mature variable region. Therefore, for example, the light chain mature variable region means a light chain variable region that does not contain a light chain signal peptide. However, the term "variable region" does not necessarily mean that a signal sequence is present; in fact, when an antibody or fusion protein is expressed and secreted, the signal sequence is cleaved. The pair of heavy chain variable regions and light chain variable regions defines the antibody binding region. The carboxyl-terminuses of the light and heavy chains define the light chain constant region and the heavy chain constant region, respectively. The heavy chain constant region primarily performs effector functions. In IgG antibodies, the heavy chain constant region is divided into the CH1 region, hinge region, CH2 region, and CH3 region. The CH1 region binds to the light chain constant region via disulfide bonds and non-covalent bonds. The hinge region provides flexibility between the antibody binding region and the effector region, and also provides a site for intermolecular disulfide bonding between the two heavy chain constant regions within the tetrameric subunit. The CH2 and CH3 regions are the main sites for effector functions and binding to FcR.

[0046] The light chain is classified as either kappa or lambda. The heavy chain is classified as gamma, mu, alpha, delta, or epsilon, defining the antibody isotypes as IgG, IgM, IgA, IgD, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by "J" segments consisting of approximately 12 or more amino acids, and the heavy chain also contains "D" segments consisting of approximately 10 or more amino acids. (For an overview, see Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, NY, 1989), Ch. 7) (The entire text is used by reference for all purposes).

[0047] The maturation variable region of each light / heavy chain pair forms an antibody binding site. Therefore, an intact antibody has two binding sites, i.e., it is bivalent. In naturally occurring antibodies, the binding sites are identical. However, bispecific antibodies with two distinct binding sites can also be produced (e.g., 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 See Immunol. 148: 1547-1553). All variable regions exhibit the same overall structure, with a relatively conserved framework region (FR) linked by three hypervariable regions (also called complementarity-determining regions or CDRs). The CDRs, derived from the two strands of each pair, are aligned by the framework region, enabling binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains are divided into the FR1 domain, CDR1 domain, FR2 domain, CDR2 domain, FR3 domain, CDR3 domain, and Includes the FR4 domain. For convenience, variable heavy chain CDR is referred to as CDR. H 1. CDR H 2, and CDR H It is sometimes referred to as 3, and variable light chain CDR is CDR L 1. CDR L 2, and CDR L It is sometimes referred to as 3. The amino acid assignments to each domain are described in Kabat EA, et al. 1987 and 1991. Sequences of Proteins. The definition follows that of Immunological Interest (National Institutes of Health, Bethesda, Maryland) 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 system (Kabat numbering) in which the same number is assigned to corresponding residues between different heavy chain variable regions or between different light chain variable regions. Kabat numbering can be used for antibody constant regions, but more commonly, EU numbering is used, as is the case in this application. While specific sequences are provided for exemplary antibodies disclosed herein, it will be understood that after the expression of the protein chain, one to several amino acids at the amino or carboxyl termini of the light and / or heavy chain, particularly the lysine residue at the C-terminus of the heavy chain, may be deleted or derivatized in part or all of the molecule.

[0048] The term "epitope" refers to a site on an antigen to which an antibody or extracellular trap segment binds. Epitopes on proteins can be formed from a continuous sequence of amino acids or from discontinuous amino acids juxtaposed by the tertiary structural folding of one or more proteins. Epitopes formed from continuous amino acids (also known as linear epitopes) are typically retained even when exposed to denaturing solvents, while epitopes formed by tertiary structural folding (also known as conformational epitopes) are typically lost when treated with denaturing solvents. Epitopes typically contain at least three, more commonly at least five or eight to ten, amino acids within a specific spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, X-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0049] Antibodies that recognize the same or overlapping epitopes can be identified by a simple immunoassay that demonstrates the ability of one antibody to compete for the binding of another antibody to its target antigen. Antibody epitopes can also be defined by identifying contact residues by X-ray crystallography of the antibody (or Fab fragment) bound to its antigen.

[0050] Alternatively, if all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have the same epitope. If several amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other antibody, then the two antibodies have overlapping epitopes.

[0051] Antibody competition is measured by assays that inhibit the specific binding of the test antibody to a common antigen of the reference antibody (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). An excess of the test antibody (e.g., at least 2x, 5x, 10x, 20x, or 100x) inhibits the binding of the reference antibody by at least 50% if it competes with the reference antibody. In some embodiments, the test antibody inhibits the binding of the reference antibody by 75%, 90%, or 90% as measured by competitive binding assays. It inhibits by 9%. Antibodies identified by competitive assays (competitive antibodies) include antibodies that bind to the same epitope as the reference antibody, and antibodies that bind to adjacent epitopes that are sufficiently close to the epitope to which the reference antibody binds to cause steric hindrance.

[0052] The term "conjugate" refers to a protein covalently bonded to a polymer. In some embodiments, the protein is an antibody.

[0053] As used herein, the terms “unconjugated” and “free” with respect to proteins or antibodies are used synonymously to refer to proteins or antibodies that are not conjugated to a polymer (for example, not conjugated to a phosphorylcholine-containing polymer).

[0054] The term "isotype" refers to a distinct class of antibodies that can be identified by the structure of their heavy chain, with each class differing in (1) the structure of the antibody hinge, (2) the sequence (and therefore the domain), and (3) the titer.

[0055] As used herein, “VEGF trap” or similar terms refer to a VEGF-binding domain (e.g., VEGFR1 domain 2, VEGFR2 domain 3). This fragment allows the protein to function as a trap for VEGF, preventing VEGF from binding to cellularly expressed VEGF receptors. Examples of this sequence can be found in Table 0.5. In some embodiments, the VEGF trap comprises only VEGFR1 domain 2 and VEGFR2 domain 3. Various embodiments of the trap protein are known in the art, for example, can be found in U.S. Patent Application Publication No. 20150376271, which in whole is incorporated herein by reference with respect to various trap embodiments and their fusions. In some embodiments, the term “VEGF trap” or similar terms refer to a full-length extracellular domain or any portion thereof, or a combination of portions derived from different VEGF receptors, which can antagonize signaling between at least one VEGF and VEGFR.

[0056] The term "patient" includes human and other mammalian subjects receiving prophylactic or therapeutic treatment. In some embodiments, the patient is a human patient.

[0057] For the purpose of classifying amino acid substitutions as conserved or non-conserved, amino acids are grouped as follows: 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, gin, his, lys, arg; Group V (residues affecting chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids within the same class. Non-conservative substitutions are equivalent to exchanging a member of one of these classes for a member of another class.

[0058] Sequence identity (%) is determined by maximizing the alignment of the antibody sequence using Kabat numbering rules for variable regions and EU numbering rules for constant regions. After alignment, when comparing the test antibody region (e.g., the full-length mature variable region of the heavy or light chain) with the same region of the reference antibody, the sequence identity % between the test antibody region and the reference antibody region is calculated by dividing the number of positions occupied by the same amino acid in both the test antibody region and the reference antibody region by the total number of positions after alignment of these two regions (gaps are not counted), and multiplying by 100 to convert it to a percentage. Sequence identity of other sequences is determined using Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, W). Sequence identity can be determined using algorithms such as BESTFIT, FASTA, and TFASTA in Disconsin, by using default gap parameters, by examination, and by the best alignment (i.e., the one that yields the highest sequence similarity %) across the comparison window. The percentage of sequence identity is calculated by comparing two optimally aligned sequences across the comparison window, determining the number of positions where the same residue appears in both sequences to obtain 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.

[0059] A composition or method "containing" one or more of the listed elements may contain other elements not specifically listed. For example, a composition containing an antibody may contain the antibody alone or in combination with other components such as an antibody conjugate. The composition may contain both conjugated and unconjugated antibodies.

[0060] The term "antibody-dependent cell-mediated cytotoxicity," or ADCC, refers to a mechanism for inducing cell death that relies on the interaction between antibody-coated target cells (i.e., cells to which antibodies are bound) and immune cells with lytic activity (also called effector cells). Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. ADCC is caused by the interaction between the Fc region of an antibody bound to a cell and Fcγ receptors, particularly FcγRI and FcγRIII, on immune effector cells such as neutrophils, macrophages, and natural killer cells. The target cell is eliminated by phagocytosis or lysis, depending on the type of effector cell present. The death of the antibody-coated target cell occurs as a result of the effector cell's activity.

[0061] The term "opsonization" (also known as "antibody-dependent cell phagocytosis" or ADCP) refers to the process by which antibody-coated cells are taken up, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the immunoglobulin Fc region.

[0062] The term "complement-dependent cell injury" or CDC refers to a mechanism by which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions, ultimately inducing cell death by creating pores in the target cell membrane. Typically, an antigen-antibody complex, such as one on an antibody-coated target cell, binds to and activates complement component C1q, which in turn activates the complement cascade, leading to target cell death. Complement activation can also result in the deposition of complement components on the target cell surface, promoting ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0063] Humanized antibodies are genetically engineered antibodies in which a CDR derived from a non-human "donor" antibody is transplanted into a human "acceptor" antibody sequence (see, for example, Queen, U.S. Patent Nos. 5,530,101 and 5,585,089; Winter, U.S. Patent No. 5,225,539; Carter, U.S. Patent No. 6,407,213; Adair, U.S. Patent Nos. 5,859,205 and 6,881,557; and Foote, U.S. Patent No. 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of a human antibody sequence, or a germline region sequence. Thus, a humanized antibody is an antibody that has some or all of the CDR derived from the donor antibody completely or substantially, and has the variable region framework sequence and constant region (if present) derived from the human antibody sequence completely or substantially. Similarly, the humanized heavy chain has all or substantially all of the CDRs derived from the donor antibody heavy chain, at least one, two, and usually three, and substantially the heavy chain variable region framework sequence and the heavy chain constant region (if present) derived from the human heavy chain variable region framework and constant region sequence. Similarly, a humanized light chain has all or substantially all of the CDRs derived from the donor antibody light chain, at least one, two, and usually three, and substantially the light chain variable region framework sequence and the light chain constant region (if present) derived from the human light chain variable region framework and constant region sequence. Except for nanobodies and dAbs, a humanized antibody includes a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially derived from the corresponding CDR in a non-human antibody if at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. A variable region framework sequence or constant region of an antibody chain is substantially derived from the human variable region framework sequence or human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical.

[0064] Humanized antibodies often incorporate all six CDRs (which may be defined by Kabat) derived from mouse antibodies, but humanized antibodies can also be produced using fewer than all CDRs (e.g., at least three, four, or five CDRs derived from mouse antibodies) (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).

[0065] Chimeric antibodies are antibodies in which the maturation variable regions of the light and heavy chains of a non-human antibody (e.g., mouse) are combined with the constant regions of the light and heavy chains of a human antibody. Such antibodies substantially or completely retain the binding specificity of the mouse antibody, and approximately two-thirds of the sequence is human.

[0066] A veneered antibody is a type of humanized antibody that retains some, usually all, of the CDRs and some non-human variable region framework residues of a non-human antibody, but replaces other variable region framework residues that can contribute to B cell or T cell epitopes, such as exposed residues, with residues derived from the corresponding positions in the human antibody sequence (Padlan EA. 1991. A possible procedural). This is for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. (Mol Immunol. 28:489-98). As a result, antibodies are obtained in which the CDR is entirely or substantially derived from a non-human antibody, and the variable region framework of the non-human antibody is made more human-like by substitution. Human antibodies can be isolated from humans or 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: Ostberg L, Pursch E. 1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367; Ostberg, U.S. Patent No. 4,634,664; and Engleman et al., U.S. Patent No. 4,634,666, the trioma method, and the use of transgenic mice containing human immunoglobulin genes (see, e.g., Lonberg et al.). al., International Publication No. 93 / 12227 (1993); 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, 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, International Publication No. 91 / 10741 (1991), and phage display methods (e.g., Dower et al., International Publication No. 91 / 17271, and McCafferty et al.) See also al., International Publication No. 92 / 01047, U.S. Patent Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332.

[0067] A "polymer" refers to a series of monomer groups linked together. A polymer can be composed of multiple units made of a single monomer (homopolymer) or multiple units made of different monomers (heteropolymer). High MW polymers are prepared from monomers including, but not limited to, vinyl esters such as acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinylpyridines, vinylpyrrolidones, and vinyl acetates. Additional monomers are useful in high MW polymers. When two different monomers are used, the two monomers are called "comonomers," meaning that different monomers copolymerize to form a single polymer. In some embodiments, one monomer is a phosphorylcholine-containing monomer, and the second comonomer is a different comonomer with different chemical properties of its side groups (e.g., it becomes a reactive / receptor in a chemical reaction conjugated to a low molecular weight bioactive substance or chemical linker, e.g., click chemistry). Polymers can be linear or branched. If a polymer is branched, each polymer chain is called a "polymer arm." The end of the polymer arm connected to the initiator portion is the proximal end, and the growth chain end of the polymer arm is the distal end. At the growth chain end of the polymer arm, the polymer arm end group may be a radical scavenger or another group.

[0068] An "initiator" refers to a compound that can initiate polymerization using a monomer or comonomer. Polymerization can be conventional free radical polymerization, or controlled / "living" radical polymerization such as atom transfer radical polymerization (ATRP), reversible addition-fragmentation-termination (RAFT) polymerization, or nitroxide-mediated polymerization (NMP). Polymerization can also be "pseudo" controlled polymerization, such as degenerative transfer. If the agent is suitable for ATRP, it may contain an unstable bond that, upon homolytic cleavage, forms an initiator fragment I, which is a radical capable of initiating radical polymerization, and a radical scavenger I', which reacts with the radical in the growing polymer chain to reversibly terminate 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 contains one or more 2-bromoisobutyrate groups as sites for polymerization by ATRP.

[0069] A "chemical linker" refers to a chemical moiety that links two groups, such as a half-life extension portion and a protein. Linkers may be cleavable or non-cleavable. Cleavable linkers may, among others, be hydrolyzable linkers, enzymatically cleavable linkers, pH-sensitive linkers, photosensitive linkers, or disulfide linkers. Other linkers include homobifunctional and heterobifunctional linkers. A "linking group" is a functional group that can form a covalent bond consisting of one or more bonds to a physiologically active agent. Non-limiting examples are shown in Table 1 of International Publication No. 2013059137 (as referenced).

[0070] The term "reactive group" refers to a group that can react with another chemical group to form a covalent bond; that is, a group that exhibits covalent reactivity under suitable reaction conditions and generally represents a bonding site to another substance. Reactive groups are moieties of maleimides or succinimidyl esters, for example, that can chemically react with functional groups on different moieties to form covalent bonds. Reactive groups generally include nucleophiles, electrophiles, and photoactivatable groups.

[0071] "Phosphorylcholine" (also written as "PC") refers to the following: [ka] In the formula, * represents a bond site. Phosphorylcholine is a zwitterionic group and includes salts (such as intramolecular salts), as well as its protonated and deprotonated forms.

[0072] A "phosphorylcholine-containing polymer" is a polymer that contains phosphorylcholine. A "zwitterion-containing polymer" refers to a polymer that contains zwitterions.

[0073] A poly(acryloyloxyethyl phosphorylcholine)-containing polymer refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC shown in Example 6 below) as a monomer.

[0074] A poly(methacryloyloxyethyl phosphorylcholine)-containing polymer refers to a polymer containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEMA-PC or MPC) as a monomer (see below). [ka]

[0075] When used in this specification, "MPC" and "HEMA-PC" are interchangeable.

[0076] In the context of polymers, "molecular weight" may be expressed as number-average molecular weight, weight-average molecular weight, or peak molecular weight. Unless otherwise indicated, all instances of "molecular weight" in this specification 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 for measuring molecular weight, such as the use of end-group analysis to determine the number-average molecular weight or measurement of colligative properties (e.g., freezing point depression, boiling point elevation, or osmotic pressure), or the use of light scattering techniques, ultracentrifugation, or viscometric methods to determine the weight-average molecular weight, may also be used. In some embodiments, molecular weight is measured by SEC-MALS (size exclusion chromatography-multiangle light scattering). In some embodiments, the multiangle light scattering method includes 18-angle MALS. In some embodiments, the multiangle light scattering method includes 3-angle MALS and 18-angle MALS. In some embodiments, the polymer reagent is typically polydisperse (i.e., the number-average molecular weight and weight-average molecular weight of the polymer are not equal) and may have a low polydispersity value, for example, less than about 1.5, as determined by the PDI value obtained from SEC-MALS measurement. In some embodiments, the polydispersity (PDI) is in the range of about 1.4 to about 1.2. In some embodiments, the PDI is less than about 1.15, less than about 1.10, less than about 1.05, or less than about 1.03.

[0077] The term “a” or “an” refers to one or more such entities; for example, “a compound” refers to one or more compounds or at least one compound. Therefore, the terms “a” (or “an”), “one or more,” and “at least one” may be used synonymously in this specification.

[0078] "Approximately" refers to the variability that may be observed in measurements performed using different instruments, samples, and sample preparations.

[0079] The terms "protected," "protected form," "protecting group," and "protective group" refer to the presence of a group (i.e., a protecting group) that inhibits or blocks the reaction of a specific chemically reactive functional group in a molecule under specific reaction conditions. The protecting group varies depending on the type of chemically reactive group being protected, the reaction conditions used, and, if any, the presence of additional reactive or protecting groups in the molecule. Suitable protecting groups are discussed in Greene et al., “Protective Groups In Organic Synthesis,” 3. rd This includes things like those found in Edition, John Wiley and Sons, Inc., New York, 1999.

[0080] "Alkyl" refers to a saturated, aliphatic free radical, either linear or branched, having the indicated number of carbon atoms. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, and hexyl. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, and decyl. Alkyl groups can contain any number of carbon atoms, 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, and 5-6. Alkyl groups are typically monovalent, but can also be divalent, for example, when an alkyl group is linked to two other parts.

[0081] When the term "lower" is used above and below in conjunction with an organic radical or compound, it defines a compound or radical that may be branched or unbranched, having up to seven carbon atoms and containing seven, or up to four and containing four, and (as unbranched) one or two carbon atoms.

[0082] "Alkylene" refers to an alkyl group as defined above, i.e., a divalent hydrocarbon radical, which is linked to at least two other groups. The two groups linked to the alkylene can be linked to the same atom or different atoms of the alkylene. For example, a linear alkylene is -(CH2) n The alkylene group may be a divalent radical (wherein n is 1, 2, 3, 4, 5, or 6). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, and hexylene.

[0083] Substituents on alkyl radicals and heteroalkyl radicals (including groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from the following, in numbers ranging from 0 to (2m'+1) (where m' is the total number of carbon atoms in the radical): -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', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -CN, and -NO2. R', R'', and R''' each independently refer to hydrogen, an unsubstituted (C1-C8) alkyl or heteroalkyl group, an unsubstituted aryl group, an aryl group substituted with 1-3 halogens, an unsubstituted alkyl group, an alkoxy group, or a thioalkoxy group, or an aryl-(C1-C4) alkyl group. When R' and R'' are bonded to the same nitrogen atom, they can combine with that nitrogen atom to form a 5-membered, 6-membered, or 7-membered ring. For example, -NR'R'' is intended to include 1-pyrrolidinyl and 4-morpholinyl. The term "alkyl" includes groups such as haloalkyls (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.). In some embodiments, substituted alkyl groups and substituted heteroalkyl groups have 1-4 substituents. In some embodiments, substituted alkyl groups and substituted heteroalkyl groups have 1, 2, or 3 substituents. An exception is perhaloalkyl groups (e.g., pentafluoroethyl).

[0084] substituents on alkyl radicals and heteroalkyl radicals (often alkylenes) A group (including groups referred to as alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of various groups selected from, but not limited to, the following, in a number ranging from 0 to (2m'+1) (where m' is the total number of carbon atoms in the radical): -OR', =O, =NR', =N-OR', -NR'R'', -SR', -H Rogen, -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', -NR-C(NR'R''R''')=NR'''', -NR-C(NR'R'')=NR''', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NRSO2R', -CN, and -NO2. R', R'', R''', and R'''' each independently refer to hydrogen, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group, for example, an aryl group substituted with 1 to 3 halogens, a substituted or unsubstituted alkyl group, an alkoxy group or thioalkoxy group, or an arylalkyl group. If a compound contains, for example, two or more R groups, each R group is selected independently of the others, and the same applies to each of the R', R'', R''', and R'''' groups if there are two or more of these groups. If R' and R'' are bonded to the same nitrogen atom, they can combine with that nitrogen atom to form a 5-membered, 6-membered, or 7-membered ring. For example, -NR'R'' includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, those skilled in the art will understand that the term "alkyl" is intended to include groups containing carbon atoms bonded to groups other than hydrogen groups, such as haloalkyls (e.g., -CF3 and -CH2CF3) and acyls (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, etc.).

[0085] "Alkoxy" refers to an alkyl group having an oxygen atom, either linked to the alkoxy group at a bonding site or bonded to two carbon atoms of the alkoxy group. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, and hexoxy. The alkoxy group may be further substituted with various substituents described herein. For example, the alkoxy group may be substituted with a halogen to form a "halo-alkoxy" group.

[0086] "Carboxyalkyl" means an alkyl group (as defined herein) substituted with a carboxyl group. "Carboxycycloalkyl" means a cycloalkyl group (as defined herein) substituted with a carboxyl group. "Alkoxyalkyl" means an alkyl group (as defined herein) substituted with an alkoxy group. As used herein, "carboxy" refers to carboxylic acids and their esters.

[0087] A "haloalkyl" refers to an alkyl group as defined above, in which some or all of its hydrogen atoms are replaced by halogen atoms. The halogen (halo) represents chloro or fluoro, but may also be bromo or iodine. Examples of haloalkyls include trifluoromethyl, fluoromethyl, and 1,2,3,4,5-pentafluorophenyl. The term "perfluoro" defines a compound or radical in which all available hydrogens are replaced by 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.

[0088] A "fluorosubstituted alkyl group" refers to an alkyl group in which one, some, or all of its hydrogen atoms are replaced by fluorine.

[0089] "Cytokine" is a member of protein signal transduction molecules susceptible to participate in intercellular communication in immune response and inflammatory response. Cytokine is typically a low molecular weight water-soluble glycoprotein having a mass of about 8 kDa to about 35 kDa.

[0090] "Cycloalkyl" refers to a cyclic hydrocarbon group containing approximately 3 to 12, 3 to 10, or 3 to 7 carbon atoms in the ring. Cycloalkyl groups include fused structures, crosslinked structures, and spiro ring structures.

[0091] "Intra-ring" refers to an atom or atomic group that constitutes a part of a cyclic ring structure.

[0092] "Exo-ring" refers to an atom or atomic group that is bonded but does not define a cyclic ring structure.

[0093] "Cyclic alkyl ether" refers to a 4- or 5-member cyclic alkyl group having 3 or 4 carbon atoms in the ring and 1 oxygen or sulfur atom in the ring (e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6- to 7-member cyclic alkyl group having 1 or 2 oxygen or sulfur atoms in the ring (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

[0094] An "alkenyl" refers to a linear or branched hydrocarbon with 2 to 6 carbon atoms and 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, or 1,3,5-hexadienyl. Alkenyl groups can also have 2-3, 2-4, 2-5, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbon atoms. Alkenyl groups are typically monovalent, but they can also be divalent, for example, when they are linked to two moieties.

[0095] "Alkenylene" refers to an alkenyl group, as defined above, that is linked to at least two other groups, i.e., a divalent hydrocarbon radical. The two groups linked to the alkenylene may be linked to the same atom or different atoms of the alkenylene. Examples of alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene, and hexenylene.

[0096] "Alkynyl" refers to a linear or branched hydrocarbon with 2 to 6 carbon atoms and at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiinyl, 1-pentynyl, 2-pentynyl, isopentinyl, 1,3-pentadinyl, 1,4-pentadinyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadinyl, 1,4-hexadinyl, 1,5-hexadinyl, 2,4-hexadinyl, or 1,3,5-hexatriinyl. Alkynyl groups can also have 2-3, 2-4, 2-5, 3-4, 3-5, 3-6, 4-5, 4-6, and 5-6 carbon atoms. Alkynyl groups are typically monovalent, but they can also be divalent, for example, when an alkynyl group is linked to two moieties.

[0097] "Alkynylene" refers to an alkynyl group, as defined above, that is, a divalent hydrocarbon radical, which is linked to at least two other groups. The two groups linked to the alkynylene may be linked to the same or different atoms of the alkynylene. Examples of alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene, and hexynylene.

[0098] "Cycloalkyl" refers to a ring assembly containing 3 to 12 ring atoms or the indicated number of atoms, which may be saturated or partially unsaturated, monocyclic, fused bicyclic, or bridged polycyclic. Examples of monocyclic rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Examples of bicyclic and polycyclic rings include norbornane, decahydronaphthalene, and adamantane. For example, C3-C8 cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0099] A "cycloalkylene" refers to a cycloalkyl group, as defined above, that is, a divalent hydrocarbon radical, which is linked to at least two other groups. The two groups linked to the cycloalkylene may be linked to the same or different atoms of the cycloalkylene. Examples of cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

[0100] A "heterocycloalkyl" refers to a ring system having 3 to approximately 20 ring members and 1 to approximately 5 heteroatoms such as N, O, and S. It includes, but is not limited to, B, Al, Si, and P, and further heteroatoms may be useful. The heteroatoms may be oxidized, for example, -S(O)- and -S(O)2-, but is not limited to these. Examples of heterocycles include, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolidinyl, piperadinyl, piperidinyl, indolinyl, quinuclidinyl, and 1,4-dioxa-8-aza-spiro[4.5]decane-8-yl.

[0101] A "heterocycloalkylene" refers to a heterocycloalkyl group as defined above, which is linked to at least two other groups. The two groups linked to the heterocycloalkylene may be linked to the same or different atoms of the heterocycloalkylene.

[0102] "Aryl" refers to an aromatic ring assembly containing 6 to 16 carbon atoms, either monocyclic or condensed bicyclic, tricyclic, or more. For example, aryls can be phenyl, benzyl, or naphthyl. "Arylene" refers to a divalent radical derived from an aryl group. The aryl group may be monosubstituted, disubstituted, or trisubstituted by one, two, or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy, and C2-C3 oxyalkylenes (all of these substituents may optionally be further substituted as defined above); or it may be 1-naphthyl or 2-naphthyl; or it may be 1-phenantrenyl or 2-phenantrenyl. Alkylenedioxy is a compound in which a divalent substituent is bonded to two adjacent carbon atoms of phenyl, for example, methylenedioxy or ethylenedioxy. C2-C3 oxyalkylenes are also compounds in which a divalent substituent is bonded to two adjacent carbon atoms of a phenyl molecule, such as oxyethylene or oxypropylene. An example of a C2-C3 oxyalkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0103] In some embodiments, the aryl is naphthyl, phenyl, or phenyl monosubstituted or disubstituted with alkoxy, phenyl, halogen, alkyl, or trifluoromethyl, and in particular is phenyl, or phenyl monosubstituted or disubstituted with alkoxy, halogen, or trifluoromethyl, and in particular is phenyl.

[0104] Examples of substituted phenyl groups as R include, for example, 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-(I These are midazole-1-yl)phenyl, 4-(imidazole-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-(pyrroridine-1-ylmethyl)phen-1-yl, 4-(thiophenyl)phen-1-yl, 4-(3-thiophenyl)phen-1-yl, 4-(4-methylpiperazine-1-yl)phen-1-yl, and 4-(piperidinyl)phenyl and optionally 4-(pyridinyl)phenyl having a heterocyclic substitution.

[0105] An "arylene" refers to an aryl group as defined above, which is linked to at least two other groups. The two groups linked to the arylene are linked to different atoms of the arylene. Phenylene is an example of an arylene group, but it is not limited to these.

[0106] "Arylene-oxy" refers to an arylene group as defined above, in which one of the parts linked to the arylene is linked via an oxygen atom. Phenylene-oxy is an example of an arylene-oxy group, but it is not limited to these.

[0107] Similarly, substituents on aryl and heteroaryl groups are diverse, selected from halogens, -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, with free valencies (open) on the aromatic ring system ranging from 0 to 0. A number in the range up to the total number of valences; where R', R'', and R''' are independently selected from hydrogen, alkyl and heteroalkyl (C1-C8), unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4) alkyl, and (unsubstituted aryl)oxy-(C1-C4) alkyl.

[0108] Two substituents on adjacent atoms of an aryl ring or heteroaryl ring are optionally of the formula -TC(O)-(CH2) q It may be replaced by a substituent of -U-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer between 0 and 2. Alternatively, substitution on adjacent atoms of an aryl ring or heteroaryl ring. The two bases, as desired, are given by formula -A-(CH2) r -B- may be replaced by a substituent, 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 by a double bond. Alternatively, two substituents on adjacent atoms of the aryl ring or heteroaryl ring may optionally be of the formula -(CH2) s -X-(CH2) tThe substituents may be replaced by -, where s and t are integers from 0 to 3, independently of each other, 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 an unsubstituted (C1-C6) alkyl group.

[0109] A "heteroaryl" refers to an aromatic ring aggregate, either monocyclic or fused bicyclic or tricyclic, containing 5 to 16 ring atoms, of which 1 to 4 are heteroatoms, each being N, O, or S. Examples of heteroaryls include pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radical, particularly monosubstituted or disubstituted, such as alkyl, nitro, or halogen. Pyridyl is 2-pyridyl, 3-pyridyl, or 4-pyridyl, with 2-pyridyl or 3-pyridyl being preferred. Thienyl is 2-thienyl or 3-thienyl. In some embodiments, quinolinyl is 2-quinolinyl, 3-quinolinyl, or 4-quinolinyl. In some embodiments, isoquinolinyl is 1-isoquinolinyl, 3-isoquinolinyl, or 4-isoquinolinyl. In some embodiments, benzopyranil and benzothiopyranil may be 3-benzopyranil or 3-benzothiopyranil, respectively. In some embodiments, thiazolyl may be 2-thiazolyl or 4-thiazolyl. In some embodiments, triazolyl may be 1-(1,2,4-triazolyl), 2-(1,2,4-triazolyl), or 5-(1,2,4-triazolyl). In some embodiments, tetrazolyl may be 5-tetrazolyl.

[0110] In some embodiments, the heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the above radicals that are substituted, particularly mono- or di-substituted.

[0111] The term "heteroalkyl" refers to an alkyl group having 1 to 3 heteroatoms such as N, O, and S. Additional heteroatoms, including but not limited to B, Al, Si, and P, may be useful. The heteroatoms may be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyl can include ethers, thioethers, alkyl-amines, and alkyl-thiols.

[0112] The term "heteroalkylene" refers to a heteroalkyl group as defined above that links at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same or different atoms of the heteroalkylene.

[0113] An "electrophile" refers to an ion or an atom or a collection of atoms that can be ionic and has an electrophilic center (i.e., a center that seeks electrons) capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond to its reaction partner (the nucleophile) by receiving both bonding electrons from that reaction partner.

[0114] A "nucleophile" refers to an ion, atom, or group of atoms that may be ionic and possess a nucleophilic center (i.e., the center from which the electrophile is sought) or can react with an electrophile. A nucleophile (or nucleophilic reagent) is a reagent that forms a bond to its reaction partner (electrophile) by donating both bonding electrons. A "nucleophilic group" refers to the nucleophile after it has reacted with a reactive group. Non-limiting examples include amino, hydroxyl, alkoxy, and haloalkoxy groups.

[0115] "Maleimide" refers to a pyrrole-2,5-dione-1-yl group having the following structure: [ka] When this group reacts with a sulfhydryl (e.g., a thioalkyl group), it forms an -S-maleimide group having the following structure: [ka] In the formula, "·" indicates a bond point to the maleimide group, [ka] The symbol indicates the bonding site of the sulfur atom of the thiol to the remainder of the original group which has sulfhydryl.

[0116] For the purposes of this disclosure, “naturally occurring amino acids” found 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. “Non-naturally occurring amino acids” found in proteins are any amino acids other than those listed as naturally occurring amino acids. Non-naturally occurring amino acids include, but are not limited to, D isomers of naturally occurring amino acids, and mixtures of D and L isomers of naturally occurring amino acids. Other amino acids, such as N-α-methyl amino acids (e.g., sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, ε-N-methyllysine, and 3-methylhistidine, are found in naturally occurring proteins, but are generally introduced by means other than ribosome translation of mRNA. For the purposes of this disclosure, they are considered to be amino acids that do not exist naturally in proteins.

[0117] In relation to the shape, architecture, or overall structure of a polymer, "linear" refers to a polymer having a single polymer arm.

[0118] The term "branched" in relation to the shape, architecture, or overall structure of a polymer refers to a polymer having two or more polymer "arms" extending from a core structure contained within an initiator. Initiators can be used in atom transfer radical polymerization (ATRP) reactions. Branched polymers 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. Each polymer arm extends from a polymer initiation site. Each polymer initiation site can be a site for polymer chain growth through monomer addition. For example, but not limited to, when using ATRP, the polymer initiation sites on the initiator are typically organic halides undergoing a reversible redox process catalyzed by a transition metal compound such as cuprous halide. In some embodiments, the halide is bromine.

[0119] "Pharmacopoeia-acceptable excipients" or "pharmacopoeia-acceptable carriers" refer to excipients that can be included in a composition, do not cause significant adverse toxic effects in the patient, and are approved or may be approved by the FDA for therapeutic use, particularly for therapeutic use in humans. Non-limiting examples of pharmacopoeia-acceptable excipients or carriers include water, NaCl, isotonic (normal) saline, Ringer's lactate solution, isotonic (normal) sucrose, and isotonic (normal) glucose. In any embodiment, a pharmacopoeia-acceptable carrier may be acceptable for direct administration to the patient's eye (e.g., acceptable for intravitreal administration).

[0120] Therapeutic proteins are administered in an effective regimen, meaning that the dosage, route of administration, and frequency of administration delay the onset of the disorder, reduce the severity of the disorder, inhibit further progression of the disorder, and / or improve at least one sign or symptom of the disorder. If a patient already has the disorder, the regimen may be called a therapeutically effective regimen. If a patient is at higher risk of the disorder compared to the general population but has not yet experienced symptoms, the regimen may be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic effectiveness may be observed in individual patients compared to historical controls or past experience in the same patient. In other cases, therapeutic or prophylactic effectiveness may be demonstrated in preclinical or clinical trials in the treated patient population compared to the control or untreated patient population.

[0121] The "biological half-life" of a substance is a pharmacokinetic parameter that defines the time it takes for half of the substance to be removed from tissues or organisms after its introduction.

[0122] "OG1786" is a nine-arm initiator used in polymer synthesis having the structure shown in Figure 4, which illustrates the salt form of OG1786 with trifluoroacetic acid. OG1786 can be used as a free base, as with other salts.

[0123] "OG1801" is a polymer of approximately (±25%) 800 kDa (depending on either Mn or Mp) prepared using the monomer HEMA-PC and OG1786 as an initiator for ATRP synthesis.

[0124] "OG1802" is OG1801 with a maleimide functional group added, as shown in Figure 10, where n1, n2, n3, n4, n5, n6, n7, n8, and n9 are arranged such that the total molecular weight of the polymer is (Mw) 800,000 ± 20% Daltons. It is a positive number (ranging from 0 to approximately 3000).

[0125] Multi-angle light scattering (MALS) is a technique for analyzing polymers. In this technique, when laser light collides with a molecule, the oscillating electric field of the light induces an oscillating dipole within the molecule. 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 size of the dipole induced within the polymer, which is further proportional to the polarizability of the polymer; the larger the induced dipole, the greater the intensity of the scattered light. Therefore, to analyze the scattering of such polymers from a solution, it is necessary to know the polarizability of those polymers with respect to the surrounding medium (e.g., solvent). This can be determined by measuring the dn / dc (=Δn / Δc) value using a Wyatt Optilab T-rEX differential refractometer, and measuring the change in refractive index n of the solution with respect to the change in molecular concentration Δc (Δn). The two molar weight parameters employed in MALS measurements are the number-average molecular weight (Mn) and the weight-average molecular weight (Mw), where the polydispersity index (PDI) is equal to Mw divided by Mn. SEC also allows for another average molecular weight measurement of the peak molecular weight Mp, which is defined as the molecular weight of the highest peak in SEC.

[0126] PDI is used as a measure of the broad molecular weight distribution of polymers and bioconjugates, where bioconjugates are obtained by conjugating a single (discrete) protein (e.g., OG1950) with a polydisperse biopolymer (e.g., OG1802). For protein samples, the polydispersity is close to 1.0, which is because proteins are translation products and all protein molecules in solution are expected to have approximately the same length and molar mass. In contrast, due to the polydispersity of biopolymers, in which polymer chains of various lengths are synthesized during the polymerization process, determining the PDI of a sample is very important as one of the quality attributes related to the narrowness of the molecular weight distribution.

[0127] Size exclusion chromatography (SEC) is a chromatographic technique that separates molecules in solution based on their size. Typically, an aqueous solution is used to transport the sample through a column packed with resins of varying pore sizes. The resin is expected to be inert to the analytes as it passes through the column, and the analytes separate from one another based on their specific size and the pore size characteristics of the selected column.

[0128] Combining SEC and MALS, or SEC / MALS, provides an accurate distribution of molecular mass and size (root mean square radius), in contrast to relying on a set of SEC calibration standards. This type of configuration offers many advantages over conventional column calibration methods. Since light scattering and concentration are measured for each elution fraction, molecular mass and size can be determined independently of the elution position. This is particularly relevant to molecular species with non-spherical polymers, such as biopolymers (OG1802) or bioconjugates (OG1953), which typically do not elute in a manner that can be described based on a set of column calibration standards.

[0129] In some embodiments, SEC / MALS analysis includes a Waters HPLC system equipped with an Alliance 2695 solvent delivery module and a Waters 2996 photodiode array detector, and a Shodex SEC-HPLC column (7.8 × 300 mm). This is connected online with a Wyatt miniDawn TREOS and a Wyatt 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 from Wyatt miniDawn TREOS to M ALS data can be used to obtain dn / dc data from a T-rEX detector, and mass recovery data using the A280 absorbance signal from a Waters 2996 photodiode array detector. SEC can be performed at 1 ml / min in 1×PBS (pH 7.4), and after sample injection, the MALS and RI signals can be analyzed using ASTRA software to determine the absolute molecular weight (Mp, Mw, Mn) and polydispersity index (PDI). Furthermore, this calculation requires the dn / dc values ​​of the polymer and protein (0.142 and 0.183, respectively) as input values. For the OG1953 bioconjugate, the dn / dc value is calculated based on the weighted MW of the polymer and protein, and is approximately 0.148 using the following formula: The dn / dc ratio of the conjugate is 0.142 × [MW polymer / (MW polymer + MW protein)] + 0.183 × [MW protein / (MW polymer + MW protein)]. In the formula, the MW polymer of OG1802 is 800 kDa, and the MW protein of OG1950 is 150 kDa.

[0130] General Protein and antibody compositions, and methods for preparing them. In some embodiments, formulations or compositions (e.g., therapeutically acceptable compositions) are provided, comprising a first protein conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) and a second protein that is not conjugated. In some embodiments, the first protein is an antibody, and the second protein is an antibody. Both antibodies may be therapeutic antibodies. In some embodiments, the composition is for treating an eye disorder in a subject. In some embodiments, the formulation comprises at least a polymer (e.g., a phosphorylcholine-containing polymer) and an unconjugated protein (e.g., an unconjugated antibody). As used herein, formulations and compositions (e.g., therapeutically acceptable compositions, pharmaceutical compositions, or therapeutic compositions) may be used synonymously. In some embodiments, the formulations or therapeutically acceptable compositions are safe for use in humans (e.g., administration to humans). In some embodiments, the formulations or therapeutically acceptable compositions are not intermediate products generated during the manufacture of the final product (e.g., one that may be suitable for use in humans).

[0131] In some embodiments, this specification provides compositions (e.g., therapeutically acceptable compositions) comprising any two proteins that may be functionally identical or different, one of which is conjugated to a polymer and the other is not conjugated to a polymer (or conjugated to any polymer, or conjugated to any effective amount of polymer). The composition may be for the treatment of an eye disorder. In some embodiments, both proteins are therapeutic proteins for the treatment of an eye disorder. In some embodiments, one or both of the proteins are therapeutic agents, antibodies, and / or therapeutic antibodies. In some embodiments, one antibody is conjugated to a polymer and the other antibody is not conjugated to a polymer. In some embodiments, the antibody may be synthesized. In some embodiments, the antibody may be a native sequence antibody. In some embodiments, the antibody may be a Fab fragment. In some embodiments, the antibody may be a trap fragment. In some embodiments, the antibody may be a fusion protein such as a trap-antibody fusion protein. In some embodiments, the antibody may be a peptide fragment. In some embodiments, a non-antibody scaffold protein may be used instead of the antibody.

[0132] A formulation comprising a first molar amount of conjugate ("conjugated protein") containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer ("unconjugated protein"); and a pharmaceutically acceptable carrier (or therapeutically acceptable) Provided herein are formulations (or therapeutically acceptable compositions) comprising the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount comprising the sum of the first molar amount and the second molar amount, and the formulation having a pH that is about 0.5 pH units or more away (e.g., high or low) from the isoelectric point (pI) of the second protein. As used herein, “molar amount” refers to a measure of the number of moles of a molecule. In some embodiments, molar amount is molar concentration (e.g., M, mM, μM, nM, etc.). In some embodiments, molar amount is expressed in units of moles (e.g., mole, millimoles, micromoles, etc.). As used herein, the isoelectric point (pI) of a protein has its conventional and ordinary meaning to those skilled in the art in view of this disclosure. pI indicates the pH at which the protein has no net charge. pI may be a known value for the same or similar proteins, or it may be determined based on a model or experimentally. In some embodiments, pI is a theoretically determined pI. In some embodiments, pI is an experimentally determined pI.

[0133] Low-viscosity formulations of protein conjugates (e.g., proteins conjugated to phosphorylcholine-containing polymers) are also provided. High concentrations of protein conjugate in the formulation can increase the viscosity of the formulation. In some embodiments, reducing the viscosity of the formulation (while maintaining the total amount of active protein) improves one or more of the manufacturability, handling, storability, and injectability when the formulation is delivered to the treatment site by syringe (e.g., intraocular administration).

[0134] The formulations (or therapeutically acceptable compositions) of the present disclosure (e.g., having polymers or polymer-conjugated proteins and unconjugated proteins) may contain unconjugated proteins (e.g., proteins not conjugated to a phosphorylcholine-containing polymer) in any preferred % molar amount of the total molar amount of polymer / polymer-conjugated and unconjugated proteins. In some embodiments, the formulation (or therapeutically acceptable composition) contains a second protein (unconjugated protein) in about 1% or more of the total molar amount of the conjugate and second protein, including the sum of a first molar amount of the conjugate and a second molar amount of the second protein. For example, if the total concentration of conjugated and unconjugated proteins is 100 μM, then 1% of the total molar amount of unconjugated protein is 1 μM and the conjugate is 99 μM. In some embodiments, the formulation (or therapeutically acceptable composition) comprises a second protein (non-conjugated protein) in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, the total molar amount being the sum of the first molar amount of the conjugate and the second molar amount of the second protein.In some embodiments, the formulation is 1% or about 1% or more of the total molar amount of the conjugate and the second protein, for example, about 2% or more, about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 35% or more, about 40% or more, about 45% or more, about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, or about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, The formulation contains a second protein (non-conjugate protein) in an amount of 45% or less, approximately 40% or less, approximately 35% or less, or approximately 30% or less, or optionally, the formulation contains the second protein in a percentage within the range defined by any two of the aforementioned values ​​of the total molar amount of the conjugate and the second protein (e.g., approximately 1-95%, 5-90%, 10-80%, 5-50%, 10-40%, 15-35%, 15-25%, 25-35%, 25-40%, 40-95%, 50-80%, etc.), where the total molar amount includes the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, the formulation contains the second protein (non-conjugate) in an amount of approximately 5% to approximately 50%, or approximately 15% to approximately 30%, of the total molar amount of the conjugate and the second protein, where the total molar amount includes the sum of the first molar amount of the conjugate and the second molar amount of the second protein. The formulation contains a conjugate protein. In some embodiments, the formulation contains a second protein (non-conjugate protein) in an amount of about 15% to about 25% of the total molar amount of the conjugate and the second protein, with the total molar amount including the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, the formulation contains a second protein (non-conjugate protein) in an amount of about 25% to about 35% of the total molar amount of the conjugate and the second protein, with the total molar amount including the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, the formulation contains a second protein (non-conjugate protein) in an amount of about 20% of the total molar amount of the conjugate and the second protein, with the total molar amount including the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, the formulation comprises the second protein (unconjugated protein) in about 30% of the total molar amount of the conjugate and the second protein, with the total molar amount comprising the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, any formulation or composition provided herein comprises the second protein (unconjugated protein) in more than 5% of the total molar amount of the conjugate and the second protein, with the total molar amount comprising the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, any formulation or composition provided herein comprises the second protein (unconjugated protein) in more than 10% of the total molar amount of the conjugate and the second protein, with the total molar amount comprising the sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, any composition or formulation described herein comprises two or more (e.g., 2, 3, 4, 5, or more) different second proteins (or non-conjugate proteins), where the second molar amount is the sum of the molar amounts of the two or more different second proteins.

[0135] A formulation (or therapeutically acceptable composition) is provided herein, comprising: a first molar amount of a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation (or therapeutically acceptable composition) contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.), the total molar amount being the sum of the first and second molar amounts, the formulation (or therapeutically acceptable composition) having a pH about 0.5 pH units away from the isoelectric point (pI) of the second protein (e.g., high or low), and the formulation (or therapeutically acceptable composition) having reduced viscosity and / or improved injectability compared to a reference formulation (or reference composition) containing the conjugate in total molar amount. In some embodiments, the reference formulation or reference composition contains the conjugate in total molar amount and substantially does not contain the second protein (or contains the second protein in less than 1% of total molar amount), but is otherwise identical to the formulation or composition acting as a reference. In some embodiments, the reference formulation or reference composition is a therapeutically acceptable reference composition.

[0136] Also provided are low-viscosity formulations (or therapeutically acceptable compositions) of protein conjugates comprising: a first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation (or therapeutically acceptable composition) has a pH that is at least about 0.5 pH units away from the isoelectric point (pI) of the protein (e.g., high or low), and the formulation (or therapeutically acceptable composition) has reduced viscosity and / or improved injectability compared to a reference formulation (or reference composition) containing the conjugate in a total molar amount which is the sum of the first and second molar amounts. In some embodiments, the reference formulation or reference composition contains the conjugate in a total molar amount, The second protein is substantially absent (or present in less than 1% of the total molar amount) but otherwise identical to the formulation or composition acting as the reference. In some embodiments, the reference formulation or reference composition is a therapeutically acceptable reference composition.

[0137] The formulations (or therapeutically acceptable compositions) of this disclosure may have any preferred viscosity. In some embodiments, the formulations may have a viscosity of 1000 mPa·s or less or about 1000 mPa·s or less, for example, about 900 mPa·s or less, about 800 mPa·s or less, about 700 mPa·s or less, about 600 mPa·s or less, about 500 mPa·s or less, about 400 mPa·s or less, about 300 mPa·s or less, about 200 mPa·s or less, about 100 mPa·s or less, or about 200 mPa·s or more, 300 mPa·s or more, about 400 The viscosity is defined as mPa·s or greater, approximately 500 mPa·s or greater, approximately 600 mPa·s or greater, approximately 700 mPa·s or greater, approximately 800 mPa·s or greater, approximately 900 mPa·s or greater, or within a range defined by any two of the aforementioned values ​​(e.g., 100-1000 mPa·s, 200-1000 mPa·s, 200-500 mPa·s, 300-800 mPa·s, 300-600 mPa·s, 200-300 mPa·s, etc.). In some embodiments, the formulation has a viscosity of approximately 100-300 mPa·s or approximately 200-500 mPa·s. In some embodiments, the formulation has a viscosity of approximately 300-400 mPa·s. Viscosity can be measured using any suitable option, such as a rotational rheometer, e.g., a DHR-20 from TA Instruments.

[0138] In some embodiments, the formulation (or therapeutically acceptable composition) has a reduced viscosity compared to a reference formulation having the same molar amount of conjugate as the total amount of conjugate and unconjugate proteins. In some embodiments, the viscosity is reduced by about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 90% or less, about 80% or more, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., about 10-90%, about 20-80%, about 50-80%, about 30-70%, about 40-90%, etc.). In some embodiments, the viscosity is reduced by about 50-80%. In some embodiments, the viscosity is reduced by about 70-80%.

[0139] In some embodiments, the reference formulation or reference composition (having only the conjugate type in total molar amount) may contain a conjugate (e.g., the polymer portion of the conjugate) in a molar amount high enough to result in a high-viscosity formulation. In some embodiments, the reference formulation has a viscosity of 700 mPa·s or more or about 700 mPa·s or more, for example, about 800 mPa·s or more, about 900 mPa·s or more, about 1000 mPa·s or more, about 1000 mPa·s or more, or about 1500 mPa·s or less, about 1400 mPa·s or less, about 1300 mPa·s or less, about 1200 mPa·s or less, about 1100 mPa·s or less, about 1000 mPa·s or less, or a viscosity within the range defined by any two of the aforementioned values ​​(for example, about 700 to 1500 mPa·s, about 800 to 1300 mPa·s, about 900 to 1200 mPa·s, about 1000 to 1300 mPa·s, etc.). In some embodiments, when the first protein and the second protein are the same or substantially the same protein (having substantially the same activity), a formulation with lower viscosity and the same total protein content as the reference formulation is provided by replacing a portion of the conjugated protein with a non-conjugated protein while maintaining the same total molar amount.

[0140] The formulations (or therapeutically acceptable compositions) of this disclosure may have low turbidity (e.g., a visually clear solution). In some embodiments, adding a non-conjugated protein to a formulation of conjugates (e.g., a protein conjugated to a phosphorylcholine-containing polymer provided herein) results in a turbid (e.g., cloudy appearance) mixed formulation. This can be obtained. In some embodiments, the turbidity of a formulation containing a mixture of unconjugated and conjugated proteins is reduced when the pH of the formulation is not at or near the isoelectric point (pI) of the unconjugated protein (e.g., at least 0.5 pH units away). When clarity is desired in the formulation (e.g., when the formulation is for intraocular administration), a low-turbidity formulation of a mixture of unconjugated and conjugated proteins can be obtained when the pH of the formulation is different from the pI of the unconjugated protein (e.g., at least 0.5 pH units higher or lower).

[0141] A formulation (or therapeutically acceptable composition) comprising: a first molar amount of a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the second protein is present in the formulation in an amount of about 1% or more of the total molar amount of the conjugate and the second protein (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.). A formulation (or therapeutically acceptable composition) is provided in which the total molar amount includes the sum of the first and second molar amounts, the formulation has a pH that is at least about 0.5 pH units away from the isoelectric point (pI) of the second protein, and the formulation has reduced turbidity compared to a reference formulation containing a first molar amount of conjugate and a second molar amount of the second protein at a pH approximately the same as the pI of the second protein (e.g., within 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 pH units). In some embodiments, the formulation has reduced turbidity compared to a reference formulation containing a first molar amount of conjugate and a second molar amount of the second protein at a pH within 0.5 pH units of the pI of the second protein. Turbidity can be measured using any preferred option. For example, turbidity can be measured at OD600nm using a plate reader, and the OD value can be calibrated against a suitable standard (e.g., a 4000 NTU formazin calibration standard). In some embodiments, turbidity is reduced by about 10% or more, for example, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 100%, or within a percentage range defined by any two of the aforementioned values ​​(e.g., 10-100%, 10-50%, 30-70%, 50-100%, 80-100%, etc.).

[0142] In some embodiments, the formulation (or therapeutically acceptable composition) is substantially free of turbidity. In some embodiments, the formulation is free of turbidity based on visual inspection. In some embodiments, a formulation or composition that is substantially free of turbidity is free of turbidity based on visual inspection. In some embodiments, the prepared formulation has a turbidity of 500 Nephelometric Turbidity Units (NTU) or less, expressed as an NTU value measured at OD600nm and calibrated against a 4000 NTU Formazin Calibration Standard, for example, about 400 NTU or less, about 300 NTU or less, about 200 NTU or less, about 100 NTU or less, or about -50 NTU or more, about 0 NTU or more, about 100 NTU or more, about 200 NTU or more, about 300 NTU or more, about 400 NTU or more, or a turbidity measurement within the range defined by any two of the aforementioned values ​​(e.g., about -50 to 500 NTU, about 0 to 400 NTU, about 100 to 300 NTU, or about 50 to 400 NTU) (or corresponding to such values). In some embodiments, the prepared formulation has a turbidity of about 300 NTU or less (or a corresponding value) when measured at OD600 nm and expressed as an NTU value calibrated against a 4000 NTU formazin calibration standard. In some embodiments, the prepared formulation has a turbidity of about 200 NTU or less (or a corresponding value) when measured at OD600 nm and expressed as an NTU value calibrated against a 4000 NTU formazin calibration standard. In some embodiments, the prepared formulation has a turbidity of about 100 NTU or less (or a corresponding value) when measured at OD600 nm and expressed as an NTU value calibrated against a 4000 NTU formazin calibration standard. In some embodiments, the formulation or composition Based on visual inspection, there is no turbidity if the turbidity, measured at OD600nm and expressed as an NTU value calibrated against a 4000 NTU Formazin calibration standard, is approximately or at most one of the following NTU values: 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 NTU.

[0143] Also provided herein are pharmaceutical formulations comprising: a first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the formulation contains the protein not conjugated to a phosphorylcholine-containing polymer in an amount of about 1% or more of the total molar amount of the conjugated and unconjugated proteins (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.), the total molar amount being the sum of the first and second molar amounts, the formulation having a pH at least about 0.5 pH units away from the isoelectric point (pI) of the protein, and the formulation being substantially free of turbidity.

[0144] A formulation is provided herein comprising a phosphorylcholine-containing polymer present in a concentration of approximately 100 mg / mL or more; and a protein not conjugated to the phosphorylcholine-containing polymer, wherein the protein is present in a second molar amount in the formulation, and the protein is present in the formulation in a concentration of approximately 1% or more of the total molar amount of the polymer and protein (e.g., approximately 5-90%, 15-25%, 25-35%, 25-40%, etc.), the total molar amount being the sum of the first and second molar amounts, and the formulation having a pH at least 0.5 pH units away from the isoelectric point (pI) of the protein. In some embodiments, the polymer is conjugated to a protein (e.g., an antibody, a fusion construct, etc.).

[0145] A formulation (or therapeutically acceptable composition) is also provided comprising a first molar amount of conjugate containing a first protein conjugated to a polymer; and a second molar amount of a second protein not conjugated to a polymer, wherein the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the first and second proteins (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.), and the total molar amount is the sum of the first and second molar amounts. In some embodiments, the formulation contains the second protein in an amount of about 1-90%, about 5-80%, about 10-95%, about 15-30%, about 5-50%, or about 10-40% of the total molar amount of the conjugate and the second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer. In some embodiments, the formulation contains the second protein in an amount of about 5-50% or about 15-30% of the total molar amount of the conjugate and the second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer.

[0146] Also provided are formulations (or therapeutically acceptable compositions) comprising a conjugate containing a first protein conjugated to a polymer; and a second protein not conjugated to a polymer, wherein the first molar amount of the conjugate and the second molar amount of the second protein are combined in the formulation such that the second molar amount is about 1% or more of the total molar amount of the conjugate and the second protein (e.g., about 5-90%, 15-25%, 25-35%, 25-40%), and the total molar amount is the sum of the first and second molar amounts. In some embodiments, formulations are prepared by combining a first molar amount of the conjugate and a second molar amount of the second protein, not conjugated to a polymer, such that the second molar amount is a specific percentage of the sum of the first and second molar amounts (e.g., a specific percentage of the total molar amount). In some embodiments, the second molar amount is about 1-90%, 5-90%, 5-80%, and 10-9% of the total molar amount of the conjugate and the second protein. The amount is 5%, approximately 15-30%, approximately 5-50%, or approximately 10-40%. In some embodiments, the second molar amount is approximately 5-50% of the total molar amount of the conjugate and the second protein. In some embodiments, the second molar amount is approximately 15-30% of the total molar amount of the conjugate and the second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer.

[0147] In some embodiments, the formulation or composition is prepared by combining a conjugate with a second protein whose compositional percentage is about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%) relative to the total protein mass weight concentration of the first and second proteins, where the remainder of the total protein mass weight concentration is the first protein. For example, when the total mass weight concentration is 50 mg / mL, a therapeutically acceptable composition can be prepared by combining an amount of the second protein equivalent to 10 mg / mL (20% compositional percentage) in the final composition with an amount of the first protein equivalent to 40 mg / mL (as a conjugate, excluding the contribution of the polymer to the mass weight concentration calculation) in the final composition.

[0148] In some embodiments, the conjugate comprises a first protein conjugated to a polymer, and the polymer comprises one or more of the following: 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-maleic anhydride copolymer, polystyrene-maleic anhydride copolymer, poly(1-hydroxymethylethylene hydroxymethylformal) Hydroxymethylformal (PHF), zwitterionic polymer, phosphorylcholine-containing polymer and polymer containing MPC, poly(Gly x -Ser y ), hyaluronic acid (HA), heparosan polymer (HEP), Fleximer, dextran, and polysialic acid (PSA).

[0149] A formulation (or therapeutically acceptable composition) is also provided, comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer, wherein the polymer has nine arms and a molecular weight of 600,000 to 1,000,000 Da, and the polymer is present in the formulation at a concentration of about 100 mg / mL or more; and a second protein not conjugated to the polymer and present in the formulation at a concentration of about 5 to 15 mg / mL. In some embodiments, the first and second proteins are therapeutic proteins. In some embodiments, the first and second proteins are identical (e.g., at least 85%, 90%, 95%, 97%, 98%, 99%, or at least about 85%, 90%, 95%, 97%, 98%, 99%, or about 100% identical in their amino acid sequences). In some embodiments, the first and second proteins are different proteins.

[0150] In some embodiments, the phosphorylcholine-containing polymer is concentrated in concentrations of 100 mg / mL or more, for example, about 150 mg / mL or more, about 200 mg / mL or more, about 250 mg / mL or more, about 300 mg / mL or more, about 350 mg / mL or more, about 400 mg / mL or more, about 450 mg / mL or more, or within the range defined by any two of the aforementioned values ​​(for example, 100-450 mg / mL, 150-400 mg / mL, 200-400 mg / mL). It is present in the formulation at concentrations of 250-450 mg / mL, 300-450 mg / mL, etc. In some embodiments, the phosphorylcholine-containing polymer is present in the formulation at approximately 150-400 mg / mL. In some embodiments, the phosphorylcholine-containing polymer is present in the formulation at approximately 200-300 mg / mL.

[0151] In some embodiments, the polymer has a density of approximately 100,000 Da or more, for example, approximately 150,000 Da or more, approximately 200,000 Da or more, approximately 350,000 Da or more, approximately 400,000 Da or more, approximately 450,000 Da or more, approximately 500,000 Da or more, approximately 550,000 Da or more, approximately 600,000 Da or more, approximately 650,000 Da or more, approximately 700,000 The molecular weights are approximately 750,000 Da or more, approximately 800,000 Da or more, approximately 850,000 Da or more, approximately 900,000 Da or more, approximately 950,000 Da or more, approximately 1,000,000 Da or more, or within the range defined by any two of the aforementioned values ​​(e.g., 100,000 to 1,000,000 Da, 300,000 to 950,000 Da, 400,000 to 800,000 Da, 500,000 to 750,000 Da, 600,000 to 700,000 Da, 600,000 to 1,000,000 Da, etc.). In some embodiments, the polymer has a molecular weight in the range of approximately 700,000 to approximately 800,000 Da. In some embodiments, the polymer is one of the polymers disclosed herein. In some embodiments, the polymer is OG1801 or OG1802.

[0152] In some embodiments, the formulations (or therapeutically acceptable compositions) of the Disclosure (e.g., having polymers or polymer-conjugated proteins and unconjugated proteins) have a pH different from (e.g., higher or lower than) the pI of the unconjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer). In some embodiments, the pH of the formulation is about 0.5 pH units or more away from the pI of the unconjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer).In some embodiments, the pH of the formulation is about 0.5 pH units or more away from the pI of a non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer), for example, about 0.6 pH units or more, about 0.7 pH units or more, about 0.8 pH units or more, about 0.9 pH units or more, about 1.0 pH unit or more, about 1.1 pH units or more, about 1.2 pH units or more, about 1.3 pH units or more, about 1.4 pH units or more, about 1.5 pH units or more, about 1.6 pH units or more, about 1.7 pH units or more, about 1.8 pH units or more, about 1.9 pH units or more, about 2.0 pH units or more, about 2.1 pH units or more, about 2.2 pH units or more, about 2.3 pH units or more. Distant by more than 1, approximately 2.4 pH units or more, approximately 2.5 pH units or more, approximately 2.6 pH units or more, approximately 2.7 pH units or more, approximately 2.8 pH units or more, approximately 2.9 pH units or more, approximately 3.0 pH units or more, approximately 3.2 pH units or more, approximately 3.4 pH units or more, approximately 3.6 pH units or more, approximately 3.8 pH units or more, approximately 4.0 pH units or more, approximately 4.5 pH units or more, approximately 5.0 pH units or more, or a pH unit distance from the pI of a non-conjugate protein within the range defined by any two of the aforementioned values ​​(e.g., 0.5-5.0 pH units, 1.0-4.0 pH units, 1.5-3.0 pH units, 2.0-3.0 pH units, 1.5-3.6 pH units, etc.). In some embodiments, the pH of the formulation is about 2.0 to 3.0 pH units away from the pI of a non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer).

[0153] In some embodiments, the formulations (or therapeutically acceptable compositions) of the present disclosure (e.g., having polymers or polymer-conjugated proteins and unconjugated proteins) have a pH that is more acidic than the pI of the unconjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer). In some embodiments, the pH of the formulation is about 0.5 pH units less or lower than the pI of a non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer), for example, about 0.6 pH units less or lower, about 0.7 pH units less or lower, about 0.8 pH units less or lower, about 0.9 pH units less or lower, about 1.0 pH unit less or lower, about 1.1 pH units less or lower, about 1.2 pH units less or lower, about 1.3 pH units less or lower, about 1.4 pH units less or lower, about 1.5 pH units less or lower, about 1.6 pH units less or lower, about 1.7 pH units less or lower, about 1.8 pH units less or lower, about 1.9 pH units less or lower, about 2.0 pH units less or lower, about 2.1 pH units less or lower, about 2.2 pH units less or lower. It is low, approximately 2.3 pH units less or lower, approximately 2.4 pH units less or lower, approximately 2.5 pH units less or lower, approximately 2.6 pH units less or lower, approximately 2.7 pH units less or lower, approximately 2.8 pH units less or lower, approximately 2.9 pH units less or lower, approximately 3.0 pH units less or lower, approximately 3.2 pH units less or lower, approximately 3.4 pH units less or lower, approximately 3.6 pH units less or lower, approximately 3.8 pH units less or lower, approximately 4.0 pH units less or lower, approximately 4.5 pH units less or lower, approximately 5.0 pH units less or lower, or pH units lower than the pI of a non-conjugate protein within the range defined by any two of the aforementioned values ​​(e.g., 0.5 to 5.0 pH units lower, 1.0 to 4.0 pH units lower, 1.5 to 3.0 pH units lower, 2.0 to 3.0 pH units lower, 1.5 to 3.6 pH units lower, etc.).In some embodiments, the pH of the formulation is about 2.0 to 3.0 pH units lower than the pI of a non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer).

[0154] In some embodiments, the formulations (or therapeutically acceptable compositions) of the present disclosure (e.g., having polymers or polymer-conjugated proteins and unconjugated proteins) have a pH that is more basic than the pI of the unconjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer). In some embodiments, the pH of the formulation is about 0.5 pH units more or higher than the pI of a non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer), for example, about 0.6 pH units more or higher, about 0.7 pH units more or higher, about 0.8 pH units more or higher, about 0.9 pH units more or higher, about 1.0 pH unit more or higher, about 1.1 pH units more or higher, about 1.2 pH units more or higher, about 1.3 pH units more or higher, about 1.4 pH units more or higher, about 1.5 pH units more or higher, about 1.6 pH units more or higher, about 1.7 pH units more or higher, about 1.8 pH units more or higher, about 1.9 pH units more or higher, about 2.0 pH units more or higher, about 2 0.1 pH unit greater or higher, approximately 2.2 pH units greater or higher, approximately 2.3 pH units greater or higher, approximately 2.4 pH units greater or higher, approximately 2.5 pH units greater or higher, approximately 2.6 pH units greater or higher, approximately 2.7 pH units greater or higher, approximately 2.8 pH units greater or higher, approximately 2.9 pH units greater or higher, approximately 3.0 pH units greater or higher, approximately 3.2 pH units greater or higher, approximately 3.4 pH units greater or higher, approximately 3.6 pH units greater or higher, approximately 3.8 pH units greater or higher, approximately 4.0 pH units greater or higher, approximately 4.5 pH units greater or higher, approximately 5.0 pH units greater or higher, or pH units greater than the pI of a non-conjugate protein within the range defined by any two of the aforementioned values ​​(e.g., 0.5 to 4.0 pH units greater). (e.g., 1.0-3.0 pH units larger, 1.5-3.0 pH units larger, 2.0-3.0 pH units larger, 1.5-3.6 pH units larger). In some embodiments, the pH of the formulation is about 2.0-3.0 pH units larger than the pI of the non-conjugated protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer).

[0155] In some embodiments, the difference between the pH of a formulation (or therapeutically acceptable composition) containing a mixture of polymers or polymer-conjugated proteins and unconjugated proteins (e.g., proteins not conjugated to phosphorylcholine-containing polymers) provided herein and the pI of the unconjugated proteins depends on the relative amounts (e.g., molar amounts) of polymers / polymer conjugates and unconjugated proteins in the formulation. While not limited to theory, generally, the greater the relative amount (e.g., molar amount) of unconjugated proteins compared to polymers / polymer conjugates, the further the pH deviates from the pI of the unconjugated proteins (for a given concentration (e.g., molar amount) of polymer or conjugate exceeding a threshold in the formulation). While not limited to theory, if the pH is too close to the pI of the unconjugated proteins, the formulation may become turbid. In some embodiments, formulations with a pH of about 4.0 or less are substantially turbid, regardless of the concentration of polymers / polymer conjugates or unconjugated proteins.

[0156] A formulation (or therapeutically acceptable composition) comprising: a first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the difference between the pI of the second protein in an acidic or basic direction and the pH of the formulation is such that the pI of the second protein in the corresponding acidic or basic direction and the first protein conjugated to a phosphorylcholine-containing polymer are equal. Formulations (or therapeutically acceptable compositions) are also provided herein, selected to be greater than the minimum difference between the pH of a reference formulation comprising a third molar amount of conjugate; a fourth molar amount of a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the first total molar amount, including the sum of the first and second molar amounts, and the second total molar amount, including the sum of the third and fourth molar amounts, are substantially the same, the second molar amount is greater than the fourth molar amount, and the reference formulation is substantially clear. While not limited to theory, in order to maintain a substantially clear formulation between two formulations having a non-conjugate protein (e.g., an antibody or fusion construct) and a polymer conjugate, and having the same total molar amount of protein (non-conjugate and conjugate), respectively, and with a sufficiently high polymer concentration, the difference (in either the acidic or basic direction) between the pI of the non-conjugate protein and the pH of the formulation with a high proportion of total molar amount of non-conjugate protein is generally greater than the minimum difference (in the corresponding acidic or basic direction) between the pI of the non-conjugate protein and the pH of the formulation with a low proportion of total molar amount of non-conjugate protein.For example, but not limited to, in the case of two formulations having the same non-conjugate / conjugate protein pair (the protein component of the conjugate may or may not be the same as the non-conjugate protein), the same total molar amount of non-conjugate and conjugate proteins, and a sufficiently high concentration of a polymer derived from the conjugate, if the pH falls below the pI of the non-conjugate protein, the pH of the formulation may be lower in the formulation with a higher proportion of non-conjugate protein to conjugate protein in order to maintain a clear solution. A formulation containing non-conjugate protein (with a pI of about 7.4) at about 5% of the total molar amount of non-conjugate protein and conjugate, and at least about 200 mg / mL of polymer may be clear down to about pH 6, with 10% or 15% of the total molar amount of non-conjugate protein, and a small amount of polymer. Another formulation containing at least approximately 200 mg / mL of polymer may be clear down to approximately pH 5, but may become turbid at approximately pH 6. In some embodiments, with respect to this tendency, the pH of the formulation and the reference formulation are at least approximately 4.5, respectively. In some embodiments, the pH of the formulation is selected to be lower than the maximum pH of the reference formulation (e.g., to maintain a clear formulation), and the pH of the formulation and the maximum pH of the reference formulation are lower than the pI of the second protein. In some embodiments, the pH of the formulation is selected to be higher than the minimum pH of the reference formulation (e.g., to maintain a clear formulation), and the pH of the formulation and the minimum pH of the reference formulation are higher than the pI of the second protein. In some embodiments, with respect to this tendency, the pH of the formulation and the reference formulation are at most approximately 8.5, respectively. In some embodiments, with respect to this tendency, the pH of the formulation and the reference formulation are approximately 4.5 to 8.5, respectively. In some embodiments, the concentration of the polymer component of the conjugate in the formulation and the reference formulation is at least about 100 mg / mL (for example, about 150 mg / mL, about 200 mg / mL, about 250 mg / mL, or about 300 mg / mL, or in the range of 150 to 300 mg / mL, for example, 200 to 300 mg / mL or 250 to 300 mg / mL).

[0157] The formulation (or therapeutically acceptable composition) may have any preferred pH. In some embodiments, the pH of the formulation may be about 3.0 or higher, about 3.5 or higher, about 4.0 or higher, about 4.5 or higher, about 5.0 or higher, about 5.5 or higher, about 6.0 or higher, about 6.5 or higher, about 7.0 or higher, about 7.5 or higher, about 8.0 or higher, about 8.5 or higher, about 9.0 or higher, about 9.5 or higher, about 10.0 or higher, or about 10.5 or higher, or about 12.0 or lower, about 11.5 or lower, about 11.0 or lower, about 10.5 or lower. The pH is within the range defined by approximately 10.0 or less, approximately 9.5 or less, approximately 9.0 or less, approximately 8.5 or less, approximately 8.0 or less, approximately 7.5 or less, approximately 7.0 or less, approximately 6.5 or less, approximately 6.0 or less, approximately 5.5 or less, approximately 5.0 or less, approximately 4.5 or less, approximately 4.0 or less, or any two of the aforementioned values ​​(e.g., 3.0 to 12.0, 3.5 to 10.0, 4.0 to 6.0, 8 to 12, 5.0 to 8.0, 9 to 10, etc.). In some embodiments, the pH of the formulation is in the range of approximately pH 4 to approximately pH 5.5. In some embodiments, the pH of the formulation is in the range of approximately pH 4.5 to approximately pH 5.3. In some embodiments, the pH of the formulation is approximately 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, or 5.5. In some embodiments, the pH of the formulation is approximately 5.0. In some embodiments, the pH of the formulation is approximately 4.5.

[0158] In some embodiments, the non-conjugated proteins in the formulation (e.g., proteins not conjugated to the phosphorylcholine-containing polymer) are 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6. The pH is 2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5 or higher, or approximately that value. In some embodiments, the pI of non-conjugated proteins (e.g., proteins not conjugated to a phosphorylcholine-containing polymer) in the formulation is approximately 4.0 to approximately 9.5, approximately 5.0 to approximately 9.5, approximately 5.5 to approximately 8.5, approximately 6 to approximately 8.5, approximately 7.0 to approximately 9.5, or approximately 5.0 to approximately 8.0. In some embodiments, the pI of non-conjugated proteins (e.g., proteins not conjugated to the phosphorylcholine-containing polymer) in the formulation is approximately 7.0 to approximately 8.5, the pH of the formulation is approximately 4.0 to approximately 5.5, and the phosphorylcholine-containing polymer is present in the formulation at a concentration of approximately 200 to approximately 300 mg / mL (e.g., as a protein conjugate). In some embodiments, the non-conjugated proteins (e.g., phosphorylcholine) in the formulation are present in the formulation. The pI of the protein (not conjugated with the choline-containing polymer) is approximately 7.0 to 8.5, the pH of the formulation is approximately 4.8 to 5.2, and the phosphorylcholine-containing polymer is present in the formulation at a concentration of approximately 200 to 300 mg / mL (e.g., as a protein conjugate).

[0159] In some embodiments, the formulations (or therapeutically acceptable compositions, pharmaceutical compositions, or therapeutic compositions) provided herein are storage stable. In some embodiments, the formulations (or therapeutically acceptable compositions, pharmaceutical compositions, or therapeutic compositions) provided herein exhibit long-term stability (e.g., when stored under standard storage conditions). In some embodiments, the components of the formulation (polymers / polymer conjugates and unconjugated proteins) are soluble, and the formulation is a clear solution. In some embodiments, the storage-stable formulation remains substantially clear. In some embodiments, the formulation remains stable long after a period of time following formulation at standard storage temperatures (e.g., clear solution, structurally stable in terms of protein and polymer components, and / or functionally stable in terms of protein activity). The formulations (or therapeutic compositions, pharmaceutical compositions, or therapeutically acceptable compositions) can be stored at any preferred temperature. In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at approximately 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 30°C, 35°C, 40°C, or at most these temperatures, or optionally at temperatures within a range defined by any two of the aforementioned values ​​(e.g., 0-40°C, 0-10°C, 10-20°C, 20-30°C, 30-40°C, etc.). In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at temperatures in the range of 0-10°C. In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at a temperature in the range of 10 to 20°C. In some embodiments, the formulation (or therapeutic composition, or therapeutically acceptable composition) is stored at 5°C or about 5°C. In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at 25°C or about 25°C.In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at room temperature. In some embodiments, the formulation (or therapeutically acceptable composition) is stored at ambient pressure. In some embodiments, the formulation (or therapeutic composition, pharmaceutical composition, or therapeutically acceptable composition) is stored at -5°C, -10°C, -15°C, -20°C, or -25°C, approximately or at least that temperature.

[0160] In some embodiments, a formulation is stable if the measured characteristics of the formulation (e.g., turbidity, structural integrity and concentration of protein and polymer components, protein activity) remain within at least about 20%, at least about 15%, at least about 10%, or at least about 5% of the levels initially measured when the formulation was first prepared. In some embodiments, a formulation (or therapeutically acceptable composition) is storage stable with respect to at least one of the following functional properties: turbidity, impurity percentage, concentration of intact protein, concentration of intact polymer, protein activity (e.g., inhibition of VEGF-A binding to VEGFR). In some embodiments, “impurities” refer to impurities associated with the product, such as degraded, aggregated, non-conjugated (e.g., if the product of interest is conjugated), or modified proteins. In some embodiments, host cell proteins, enzymes Impurities unrelated to the product, such as dotoxins and host cell DNA, are not considered “impurities” as defined herein. In some embodiments, the formulation is stable when stored at about 5°C for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 16 months, at least about 20 months, at least about 24 months, at least about 36 months, at least about 48 months, or longer (e.g., less than about 5% or less than 10% total impurities and / or IC of competitive binding to VEGF bound to VEGFR). 50 However, the decrease is only about 10% or 15% or less compared to the control formulation with the conjugate alone), or, if desired, the formulation is stable for a period defined by any two of the aforementioned values ​​(e.g., 1-48 months, 1-24 months, 1-12 months, 1-6 months, 3-9 months, 4-12 months, 12-24 months, 12-36 months, etc.). In some embodiments, the formulation is stable for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 16 months, at least about 20 months, at least about 24 months, or longer when stored at about 25°C (e.g., total impurities of less than about 5% or less than about 10%, and / or IC of competitive binding to VEGF bound to VEGFR). 50However, the IC50 is reduced by only about 10% or less than about 15% compared to the control formulation with the conjugate alone), or, if desired, stable for a period defined by any two of the aforementioned values ​​(e.g., 1–24 months, 1–20 months, 1–12 months, 1–6 months, 3–9 months, 4–12 months, 12–24 months, etc.). In some embodiments, when stored at room temperature, the formulation is stable for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 16 months, at least about 20 months, at least about 24 months, at least about 36 months, or longer (e.g., less than about 5% or less than 10% total impurities and / or IC50 of competitive binding to VEGF bound to VEGFR). 50However, the degradation is only about 10% or less than 15% compared to the control formulation with the conjugate alone), or, if desired, stable for a period defined by any two of the aforementioned values ​​(e.g., 1 to 12 months, 1 to 6 months, 3 to 9 months, 4 to 12 months, etc.). In some embodiments, the formulation is stable for 3 months, about 3 months, or at least 3 months when stored at about 5°C. In some embodiments, the formulation is stable for 6 months, about 6 months, or at least 6 months when stored at about 5°C. In some embodiments, the formulation is stable for 9 months, about 9 months, or at least 9 months when stored at about 5°C. In some embodiments, the formulation is stable for 12 months, about 12 months, or at least 12 months when stored at about 5°C. In some embodiments, the formulation is stable for 24 months, about 24 months, or at least 24 months when stored at about 5°C. In some embodiments, the formulation is stable for 3 months, about 3 months, or at least 3 months when stored at about 25°C. In some embodiments, the formulation is stable for 6 months, about 6 months, or at least 6 months when stored at about 25°C. In some embodiments, the formulation is stable for 9 months, about 9 months, or at least 9 months when stored at about 25°C. In some embodiments, the formulation is stable for 12 months, about 12 months, or at least 12 months when stored at about 25°C.

[0161] In some embodiments, the formulation (or therapeutically acceptable composition) is storage stability with respect to at least one of the following measures: color and clarity by visual inspection; and the stability of unconjugated proteins and / or conjugated proteins at different temperatures, as measured by size exclusion chromatography (SEC-HPLC), ion exchange chromatography (IEX-HPLC), SEC with multi-angle light scattering (MALS) detectors, or tandem HPLC, which allows for separate monitoring of the stability of conjugated or free protein populations. Aggregation or degradation of jugate proteins; titer; and maintenance of % non-conjugate proteins (e.g., % of composition or total molar amount) in the formulation.

[0162] In some embodiments, the formulation or composition comprises a mixture in which, by mass weight, 5% to 10% is a non-conjugate protein (e.g., a non-conjugate antibody or a non-conjugate fusion construct) and the remainder is a conjugate protein (e.g., a conjugate antibody or a conjugate fusion construct). In some embodiments, if the protein portions of the non-conjugate protein and the conjugate protein have the same or similar molecular weights (e.g., within about 10% of each other), the amount of non-conjugate protein relative to the total amount of non-conjugate and conjugate proteins in the formulation is expressed as a percentage by mass weight. In some embodiments, the composition comprises a mixture in which, by mass weight, 5% to 15% is a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins of the same molecular weight (e.g., an antibody and / or a fusion construct), wherein the mixture contains 15% to 25% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two antibodies of the same molecular weight, wherein the concentration of non-conjugate protein (e.g., a non-conjugate antibody) in the total protein concentration in the mixture is 15% to 25% by mass weight (e.g., grams / liter). In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 25% to 35% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein, by mass weight concentration, the mixture contains 35% to 45% of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder of the mixture contains a conjugate protein.In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies) wherein, by mass weight concentration, the mixture contains 5% to 20% non-conjugate protein (e.g., non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies) wherein, by mass weight concentration, the mixture contains 5% to 25% non-conjugate protein (e.g., non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct) wherein, by mass weight concentration, the mixture contains 5% to 30% non-conjugate protein (e.g., non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies), wherein the mixture contains 5% to 35% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 5% to 40% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies), wherein the mixture contains 5% to 50% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 5% to 55% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder of a conjugate protein.

[0163] In some embodiments, the composition or formulation comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 5% to 55% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody or a non-conjugate fusion construct) and the remainder is a conjugate protein (e.g., a conjugate antibody or a conjugate fusion construct). In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies), wherein the mixture contains 5% to 60% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 5% to 65% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies), wherein the mixture contains 5% to 70% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein the mixture contains 5% to 75% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., two antibodies), wherein the mixture contains 5% to 80% by mass weight of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder is a conjugate protein. In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein, by mass weight concentration, the mixture contains 5% to 85% of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder of the mixture contains a conjugate protein.In some embodiments, the composition comprises a mixture of two proteins (e.g., an antibody and / or a fusion construct), wherein, by mass weight concentration, the mixture contains 5% to 90% of a non-conjugate protein (e.g., a non-conjugate antibody) and the remainder of a conjugate protein.

[0164] In some embodiments, the percentage of conjugated protein relative to unconjugated protein (e.g., % total molar amount) is calculated by (1) measuring the conjugated and unconjugated proteins in mg / mL; (2) converting the mg / mL values ​​of the conjugated and unconjugated proteins to molecular weights measured in kDa; and (3) dividing the molecular weight of each conjugated and unconjugated protein by the total molecular weight of the conjugated and unconjugated proteins in the composition, multiplying by 100, and obtaining the percentage of each total molar amount.

[0165] In some embodiments, any formulations and compositions provided herein can be defined as the compositional percentage (e.g., in mass weight concentration) of one component relative to the total mass weight concentration of the protein in the composition (e.g., excluding any contributions of polymers that can be conjugated thereto). In some embodiments, a formulation or composition defined by the % total molar amount of a second protein (e.g., an unconjugated protein) can be defined as the compositional percentage (e.g., in mass weight concentration) of the second protein relative to the total mass weight concentration of the first and second proteins, given the relevant molecular weight of each protein. In some embodiments, the compositional percentage is measured in mass weight concentration (in other words, grams / liter or milligrams / milliliter) of the free protein relative to the total mass weight concentration of the protein in the mixture (e.g., excluding any contributions of polymers that can be conjugated thereto).

[0166] A conduit containing a first protein conjugated with a phosphorylcholine-containing polymer. A therapeutically acceptable composition is provided herein, comprising: a phosphorylcholine-containing polymer-conjugate; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.), and the composition has a pH about 0.5 pH units away from the isoelectric point (pI) of the second protein.

[0167] A therapeutically acceptable composition is also provided, comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.), the composition has a pH about 0.5 pH units away from the isoelectric point (pI) of the second protein, the composition has reduced viscosity and / or improved injectability compared to a reference composition containing the conjugate, and the first protein of the conjugate is present in the reference composition at the total mass weight concentration of the first and second proteins in the composition. In some embodiments, the reference composition contains the first protein of the conjugate in total mass weight concentration and substantially does not contain the second protein (or contains the second protein in a compositional percentage of less than 1% relative to the total mass weight concentration of the first and second proteins in the reference composition), but is otherwise identical to the composition that functions as the reference.

[0168] Also provided is a therapeutically acceptable composition comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.), the composition has a pH about 0.5 pH units away from the isoelectric point (pI) of the second protein, and the composition has reduced turbidity compared to a reference composition containing the second protein in a compositional percentage relative to the total protein mass weight concentration of the first and second proteins in the composition at a pH approximately the same as the pI of the second protein (e.g., within 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 pH units). In some embodiments, the composition has reduced turbidity compared to a reference composition containing the second protein in a percentage of compositional weight relative to the total protein mass weight concentration of the first and second proteins in the composition. In some embodiments, the reference composition comprises the first protein conjugated to a phosphorylcholine-containing polymer and the second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the percentage of compositional weight of the second protein in the reference composition relative to the total protein mass weight concentration of the first and second proteins is the same as the percentage of compositional weight of the second protein in a therapeutically acceptable composition, and the pH is approximately the same as the pI of the second protein (e.g., within 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 pH units). In some embodiments, the composition has reduced turbidity compared to a reference composition containing the second protein in a percentage of compositional weight relative to the total protein mass weight concentration of the first and second proteins in the composition.

[0169] A therapeutically acceptable composition comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the second protein The difference between the pI of the protein and the pH of the formulation is selected to be greater than the minimum difference between the pI of the second protein and the pH of a reference formulation comprising a conjugate containing the first protein conjugated to a phosphorylcholine-containing polymer; the second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, and the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is higher than the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition in the reference composition, and a therapeutically acceptable composition is provided herein, where the reference composition is substantially free of turbidity.

[0170] A therapeutically acceptable composition is provided herein, comprising a conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; a second protein not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is about 1% or more (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.).

[0171] Further, therapeutically acceptable compositions are provided, comprising a conjugate containing a first protein conjugated to a polymer; and a second protein not conjugated to a polymer, wherein the second protein is combined with the conjugate in a compositional percentage of about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.) relative to the total protein mass weight concentration of the first and second proteins in the composition, and the remainder of the total protein mass weight concentration is the first protein. In some embodiments, the composition is prepared by combining the second protein with the conjugate in a compositional percentage of about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.) relative to the total protein mass weight concentration of the first and second proteins, such that the first protein constitutes the remaining compositional percentage of the total protein mass weight concentration. For example, if the total mass weight concentration is 50 mg / mL, a therapeutically acceptable composition can be prepared by combining a second protein in an amount equivalent to 10 mg / mL (20% of the composition) in the final composition with a conjugate in an amount equivalent to 40 mg / mL of the first protein in the final composition (as a conjugate, excluding the contribution of the polymer to the mass weight concentration calculation).

[0172] In any embodiment of this specification, a second protein not conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) may be referred to as an unconjugated protein, and a first protein conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) may be referred to as a conjugated protein. In some embodiments, the compositional percentage of unconjugated proteins (e.g., unconjugated antibodies) relative to the total protein mass weight concentration of the first and second proteins in the composition is 5% to 6%, with the remainder being conjugated proteins. As used herein, “remaining” means the portion of the total protein mass weight concentration of the composition that is not unconjugated proteins (e.g., the second protein) (excluding the contribution of the polymer conjugated to the first protein), where the compositional percentages of unconjugated proteins (e.g., the second protein) and the remainder together constitute 100% of the total protein mass weight concentration. For example, if the composition percentage of non-conjugate proteins is 5% to 6% and the remainder consists of conjugate proteins, then 5% to 6% of the total mass weight concentration of the total protein in the mixture is non-conjugate proteins, and 94% to 95% of the total mass weight concentration of the protein in the mixture is conjugate proteins, where the percentages add up to 100%. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 1%. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 3% to 4%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 6% to 7%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 7% to 8%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 8% to 9%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 9% to 10%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 11%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 11% to 12%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12% to 13%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 13% to 14%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 14% to 15%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 16%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 16% to 17%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 17% to 18%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 18% to 19%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 19% to 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 20% to 21%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 21% to 22%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 22% to 23%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 23% to 24%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 24% to 25%.

[0173] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 25% to 26%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 26% to 27%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 27% to 28%, with the remainder being It contains conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 28% to 29%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 29% to 30%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 30% to 31%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 31% to 32%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 32% to 33%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 33% to 34%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 34% to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 35% to 36%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 36% to 37%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 37% to 38%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 38% to 39%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 39% to 40%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 40% to 41%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 41% to 42%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 42% to 43%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 43% to 44%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 44% to 45%.

[0174] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or fusion constructs) is 45% to 46%, with the remainder being conjugate proteins (e.g., conjugate antibodies or non-conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 46% to 47%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 47% to 48%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 48% to 49%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 49% to 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 50% to 51%, and the remainder The remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 51% to 52%, and the remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 52% to 53%, and the remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 53% to 54%, and the remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 54% to 55%, and the remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 55% to 56%, and the remainder includes conjugated proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 56% to 57%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 57% to 58%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 58% to 59%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 59% to 60%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 60% to 61%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 61% to 62%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 62% to 63%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 63% to 64%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 64% to 65%.

[0175] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 65% to 66%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 66% to 67%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 67% to 68%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 68% to 69%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 69% to 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 70% to 71%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 71% to 72%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 72% to 73%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 73% to 74%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 74% to 75%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 75% to 76%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 76% to 77%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 77% to 78%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 78% to 79%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 79% to 80%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 80% to 81%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 81% to 82%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 82% to 83%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 83% to 84%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 84% ​​to 85%.

[0176] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 85% to 86%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 86% to 87%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 87% to 88%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 88% to 89%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 89% to 90%, with the remainder being conjugate proteins.

[0177] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 5% to 25%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 1% to 5%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 12.5%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 15%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 17.5%. In some embodiments, non-conjugate proteins (e.g., non-conjugate antibodies) In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 25%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 45%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 55%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 60%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 65%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 75%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 80%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 85%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 90%.

[0178] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 10% to 12.5%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 15%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 17.5%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 25%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 25%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 45%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins (e.g., non-conjugate antibodies) In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 55%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 60%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 65%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 75%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 80%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 85%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10% to 90%.

[0179] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 12.5% ​​to 15%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 17.5%. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 17.5% to 22.5%. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 25%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 45%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 55%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 60%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 65%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 75%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins (e.g., non-conjugate antibodies) are present. The composition percentage of gated antibodies is 12.5% ​​to 80%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 12.5% ​​to 85%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 12.5% ​​to 90%.

[0180] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 15% to 17.5%, with the remainder being conjugate proteins (e.g., conjugate antibodies or fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 25%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 45%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 55%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 60%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 65%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 70%, with the remainder consisting of conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 75%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 80%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 85%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 90%.

[0181] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 20% to 25%, with the remainder being conjugate proteins (e.g., conjugate antibodies or fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 35%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 45%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 50%, with the remainder being conjugate proteins. It contains conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 25% to 55%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 25% to 60%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 25% to 65%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 25% to 70%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 25% to 75%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 80%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 85%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 90%, with the remainder being conjugate proteins.

[0182] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 1%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 5%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 6%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 7%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 8%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 9%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 10%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 11%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 13%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 14%, with the remainder being conjugate proteins.In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 16%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 17%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 18%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 19%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 20%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 21%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 22%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 23%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 24%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25%.

[0183] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 26%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 27%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 28%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 29%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 31%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 32%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 33%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 34%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 35%.

[0184] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 36%, with the remainder being conjugate proteins (e.g., conjugate antibodies or fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 37%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 38%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 39%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 40%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 41%, with the remainder consisting of conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 42%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 43%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 44%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 45%.

[0185] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 46%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 47%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 48%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 49%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 50%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 51%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 52%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 53%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 54%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 55%.

[0186] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 56%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 57%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 58%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 59%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 60%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 51%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 62%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 63%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 64%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 65%.

[0187] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 66%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 67%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 68%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 69%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 11%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 72%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 73%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 74%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 75%.

[0188] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate proteins) is 76%, with the remainder being conjugate proteins (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 77%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 78%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 79%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 80%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 81%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 82%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 83%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 84%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 85%.

[0189] In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 86%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins In some embodiments, the composition percentage of protein (e.g., non-conjugate antibodies) is 87%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 88%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 89%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 90%.

[0190] In some embodiments, the ratio of the molecular weight of the non-conjugated protein (e.g., a second protein not conjugated to the phosphorylcholine-containing polymer) to the polymer in the formulation can be any suitable ratio. In some embodiments, the ratio is at most about 1:2, for example, at most about 1:3, at most about 1:4, at most about 1:5, at most about 1:6, at most about 1:7, at most about 1:8, at most about 1:9, or at most about 1:10, or within the range defined by any two of the aforementioned values ​​(e.g., 1:2 to 1:10, 1:3 to 1:8, 1:4 to 1:6). In some embodiments, the ratio is about 1:4 to 1:6. In some embodiments, the ratio is about 1:5.33.

[0191] The protein conjugated to the phosphorylcholine-containing polymer and the unconjugated protein can each be any suitable protein. In some embodiments, the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the protein not conjugated to the phosphorylcholine-containing polymer) have the same activity or function (e.g., binding to the same epitope, inhibiting the same target, catalyzing the same reaction, etc.). In some embodiments, the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the protein not conjugated to the phosphorylcholine-containing polymer) are the same protein. In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., a protein not conjugated to a phosphorylcholine-containing polymer) are identical in their amino acid sequences by at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or about 100%, or by a percentage within the range defined by any two of the aforementioned values ​​(e.g., 85-100%, 90-99%, 90-95%, etc.). In some embodiments, if the first and second proteins each contain two or more polypeptide chains, each of the corresponding polypeptide chains may independently have any of the described sequence identities. In some embodiments, the second protein is a derivative of the first protein (e.g., a protein portion not containing a polymer) or contains the same. In some embodiments, the second protein is an alkylated form of the first protein (e.g., a protein portion not containing a polymer). For example, the second protein is an iodoacetamide (IAM) or N-ethylmaleimide (NEM) treated form of the first protein (e.g., the polymer-free protein portion).

[0192] In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) has a molecular weight (based on the protein portion) of approximately 5 kDa or more, approximately 10 kDa or more, approximately 15 kDa or more, approximately 25 kDa or more, approximately 50 kDa or more, approximately 75 kDa or more, approximately 100 kDa or more, approximately 125 kDa or more, approximately 150 kDa or more, approximately 175 kDa or more, approximately 200 kDa or more, approximately 250 kDa or more, approximately 300 kDa or more, or a molecular weight within the range defined by any two of the aforementioned values ​​(e.g., 50-300 kDa, 100-300 kDa, 100-200 kDa, 150-250 kDa, etc.). In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 150 kDa (based on the protein portion). In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 200 kDa. In some embodiments, the second protein (e.g., a non-conjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 50 kDa or more, for example, about 75 kDa or more, about 100 kDa or more, about 125 kDa or more, about 150 kDa or more, about 175 kDa or more, about 200 kDa or more, about 250 kDa or more, about 300 kDa or more, or a molecular weight within the range defined by any two of the aforementioned values ​​(e.g., 50-300 kDa, 100-300 kDa, 100-200 kDa, 150-250 kDa, etc.). In some embodiments, the second protein (e.g., a non-conjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 150 kDa. In some embodiments, the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) has a molecular weight of approximately 200 kDa.

[0193] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein, or a protein not conjugated to a phosphorylcholine-containing polymer) are therapeutic proteins. In some embodiments, at least one of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) is a therapeutic protein approved by the FDA as of May 2023. In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are antibodies (e.g., therapeutic antibodies). Any suitable antibody can be used in the formulation. In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are fusion constructs. Any suitable fusion construct can be used in formulations. Any antibody or fusion construct in any of the formulations or compositions herein may or may not contain C-terminal lysine. The terms "fusion protein" and "fusion construct" are used synonymously herein.

[0194] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are anti-VEGF-A antibodies, fusion constructs including a VEGF trap fused to the heavy chain of an anti-IL-6 antibody, anti-IL-6 antibodies, fusion constructs including a PDGFR extracellular trap fused to the heavy chain of an anti-VEGF-A antibody, VEGF trap-Fc fusion proteins, anti-HTRA1 antibodies, or anti-complement factor D (CFD) antibodies. In some embodiments, both the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are anti-VEGF-A antibodies. In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) Both (non-transferred proteins) are fusion constructs comprising a VEGF trap fused to the heavy chain of an anti-IL-6 antibody. In some embodiments, the anti-VEGF-A antibody is a full-length antibody. In some embodiments, the anti-VEGF-A antibody is or comprises an anti-VEGF-A Fab fragment. In some embodiments, the anti-VEGF-A antibody is OG1950, e.g., described herein and U.S. Patent Application Publication 2017 / 0190766 (the whole of which is incorporated herein by reference). In some embodiments, the anti-VEGF-A antibody is selected from bevacizumab, ranibizumab, brolucizumab, or falisimab. In some embodiments, the VEGF trap-Fc fusion protein is aflibercept. In some embodiments, the fusion construct is OG2072, e.g., described herein and U.S. Patent Application Publication 2019 / 0270806 (the whole of which is incorporated herein by reference). In some embodiments, the first protein comprises one or a combination thereof of the amino sequences described in Tables 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7, and Figures 12-17, 54, 55A, and 55B. In some embodiments, the second protein comprises one or a combination thereof of the amino sequences described in Tables 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7, and Figures 12-17, 54, 55A, and 55B. In some embodiments, the first protein comprises one or a combination thereof of the amino sequences described in Tables 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7, and Figures 12-17, 54, 55A, and 55B, and the second protein comprises one or a combination thereof of the amino sequences described in Tables 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7, and Figures 12-17, 54, 55A, and 55B.In some embodiments, the first protein includes one or a combination thereof of the amino sequences described in SEQ ID NOs: 1-6, 15-133, and 156-161. In some embodiments, the first protein includes one or a combination thereof of the amino sequences described in SEQ ID NOs: 1-6, 15-133, and 156-161, and the second protein includes one or a combination thereof of the amino sequences described in SEQ ID NOs: 1-6, 15-133, and 156-161. In some embodiments, the amino acid sequences of the first protein and the second protein are paired as shown in Tables 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7, and in Figures 12-17, 54, 55A, and 55B.

[0195] In some embodiments, the conjugated and unconjugated proteins are antibodies, and one or both antibodies are therapeutic. In some embodiments, one or both antibodies are identical or variants of each other, where one antibody is conjugated to a polymer at the cysteine ​​outside the variable region of the antibody, and the other protein is unconjugated. In some embodiments, both antibodies are therapeutic antibodies. In some embodiments, at least one of the antibodies is a therapeutic antibody approved by the FDA as of May 2023. In some embodiments, both antibodies are therapeutic antibodies for the treatment of eye disorders. In some embodiments, both antibodies (conjugated and unconjugated) share at least identical CDR sequences in their heavy and light chains. In some embodiments, both antibodies (conjugated and unconjugated) have at least one (e.g., 1, 2, 3, 4, 5, or 6) different CDRs in their heavy or light chain. The composition comprises (a) an antibody conjugate comprising an anti-VEGF-A antibody and a phosphorylcholine-containing polymer, wherein the polymer is covalently bound to the antibody at the cysteine ​​outside the variable region of the antibody; and (b) a non-conjugated anti-VEGF-A antibody.

[0196] In some embodiments, the anti-VEGF-A antibody in the antibody conjugate comprises a light chain and a heavy chain, the heavy chain comprising an Fc region. In some embodiments, the cysteine ​​in the antibody conjugate is located in the Fc region of the heavy chain. In some embodiments, the anti-VEGF-A antibody in the antibody conjugate is immunoglobulin G (IgG).

[0197] In some embodiments, the anti-VEGF-A antibody heavy chain of an antibody conjugate or unconjugate antibody is CDR H 1: GYDFTHYGMN (Sequence ID 9), CDR H 2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H3: Containing YPYYYGTSHWYFDV (SEQ ID NO: 11), the anti-VEGF-A light chain of an antibody conjugate or non-conjugate antibody is CDR L 1:SASQDISNYLN (Sequence ID 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: Includes QQYSTVPWT (SEQ ID NO: 14). In some embodiments, the anti-VEGFA heavy chain isotype of the antibody-conjugated or unconjugated antibody is human IgG1. In some embodiments, the heavy chain constant domain of the anti-VEGF-A antibody of the antibody-conjugated or unconjugated antibody has one or more mutations relative to the constant domain of human IgG1 to modulate effector function.

[0198] In some embodiments, mutations in the antibody conjugate (and / or unconjugated antibody) may be to one or more of the following amino acid positions (EU numbering): E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (where X is any natural or non-natural amino acid). In some embodiments, mutations in the antibody conjugate (and / or unconjugated antibody) are selected from the group consisting of (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, mutations in the antibody conjugate (and / or unconjugated antibody) include: L234A, L235A, and G237A (EU numbering). In some embodiments, the cysteine ​​of the antibody conjugate (and / or unconjugated antibody) is located in the anti-VEGF-A antibody heavy chain and is Q347C (EU numbering) or L443C (EU numbering).

[0199] In some embodiments, the sequence of the anti-VEGF-A antibody heavy chain of the antibody conjugate or unconjugate antibody is SEQ ID NO: 1 (with or without C-terminal lysine), and the sequence of the anti-VEGF-A light chain of the antibody conjugate or unconjugate antibody is SEQ ID NO: 2. In some embodiments, the sequence of the anti-VEGF-A antibody heavy chain of the antibody conjugate or unconjugate antibody is identical to SEQ ID NO: 1 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and the sequence of the anti-VEGF-A light chain of the antibody conjugate or unconjugate antibody is identical to SEQ ID NO: 2 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the cysteine ​​of the antibody-conjugated or unconjugated antibody is L443C (EU numbering).

[0200] In some embodiments, the solution pH of the composition depends on the isoelectric point of the desired protein, and the solution pH is at least 1-2 pH units away from the isoelectric point of the desired protein.

[0201] In some embodiments, compositions comprising a first antibody and a second antibody are provided. The first antibody is conjugated to a phosphorylcholine-containing polymer, the polymer covalently bonded to the first antibody at the cysteine ​​outside the variable region of the first antibody, and the second antibody is not conjugated to a phosphorylcholine-containing polymer.

[0202] In some embodiments, the first antibody is CDR H 1: GYDFTHYGMN (Sequence ID 9), CDR H2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3:YPYYYGTSHWYFDV (Sequence ID 11), CDR L 1:SASQDISNYLN (Sequence ID 12), CDR L 2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: Contains QQYSTVPWT (SEQ ID NO: 14). This is for one or more of the following amino acid positions (EU numbering): E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X (where X is any natural or non-natural amino acid). In some embodiments, the mutations of the first antibody are selected from the group consisting of: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, the following mutations of the first antibody are: L234A, L235A, and G237A (EU numbering). In some embodiments, the cysteine ​​of the first antibody is located in the anti-VEGF-A antibody heavy chain and is Q347C (EU numbering) or L443C (EU numbering). In some embodiments, the sequence of the anti-VEGF-A antibody heavy chain of the first antibody is SEQ ID NO: 1 (with or without C-terminal lysine), and the sequence of the anti-VEGF-A light chain of the first antibody is SEQ ID NO: 2. Any or all of the embodiments described in this paragraph may be applied to the second antibody. OG1950 represents an anti-VEGF-A antibody having a heavy chain of SEQ ID NO: 1 (with or without C-terminal lysine) and a light chain of SEQ ID NO: 2.

[0203] In some embodiments, the cysteine ​​of the first antibody is L443C (EU numbering).

[0204] In some embodiments, the first and second antibodies have the same CDR (1, 2, 3, 4, 5, or all 6), the same VH, VL, the same VH and the same VL, and / or the same HC and LC, the only difference being that one is conjugated to a polymer and the other is not.

[0205] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are anti-complement factor D (CFD) antibodies. In some embodiments, the anti-CFD antibody comprises a heavy chain having any three heavy chain CDRs from any one of SEQ ID NOs. 15-47 and a light chain having any three light chain CDRs from any one of SEQ ID NOs. 48-80. In some embodiments, the anti-CFD antibody is one of SEQ ID NOs. 15-47 and a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values. It includes a heavy chain variable region having the same amino acid sequence at a rate (e.g., 80-100%, 85-95%, 90-97%, etc.) and a light chain variable region having the same amino acid sequence as one of sequence numbers 48-80 at a rate (e.g., 80-100%, 85-95%, 90-97%, etc.) within a range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values. In some embodiments, the anti-CFD antibody comprises a heavy chain variable region having the same amino acid sequence as any one of SEQ ID NOs. 15-47 at a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and a light chain variable region having the same amino acid sequence as any one of SEQ ID NOs. 48-80 at a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), as they are paired in Table 0.1. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0206] In some embodiments, the anti-CFD antibody includes a heavy chain variable region having the same amino acid sequence as SEQ ID NO: 129 (or its heavy chain variable region) at a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and a light chain variable region having the same amino acid sequence as SEQ ID NO: 130 (or its light chain variable region) at a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the anti-CFD antibody heavy chain includes the sequence of SEQ ID NO: 129 (with or without C-terminal lysine). In some embodiments, the CFD antibody light chain may include the sequence of SEQ ID NO: 130.

[0207] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are anti-IL-6 antibodies. In some embodiments, the anti-IL-6 antibody comprises a heavy chain having any three heavy chain CDRs in any one of SEQ ID NOs. 81-89, and a light chain having any three light chain CDRs in any one of SEQ ID NOs. 90-92. In some embodiments, the anti-IL-6 antibody has a heavy chain variable region having the same amino acid sequence as any one of SEQ ID NOs. 81-89 in percentages within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% (e.g., 80-100%, 85-95%, 90-97%), and has the same amino acid sequence as any one of SEQ ID NOs. 90-92 in percentages within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% (e.g., 80-100%, 85-95%, 90-97%). It includes a light chain variable region. [Table 2] [Table 3]

[0208] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are fusion constructs comprising a VEGF trap fused to the heavy chain of an anti-IL-6 antibody. In some embodiments, the fusion construct includes a heavy chain variable region having any three heavy chain CDRs (or all three heavy chain CDRs) in any one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and a light chain variable region having any three light chain CDRs (or all three light chain CDRs) in any one of sequence numbers (ID NO) 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128. In some embodiments, the fusion construct is a heavy chain variable region having any three heavy chain CDRs (or all three heavy chain CDRs) in any one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, as well as sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, The fusion construct includes a light chain variable region having any three light chain CDRs (or all three light chain CDRs) in any one of 120, 122, 124, 126, and 128, as paired in Table 0.4. In some embodiments, the fusion construct has the same amino acid sequence as the heavy chain variable region in any one of SEQ ID NOs. 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, in a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.). The heavy chain variable region, and the light chain variable region in any one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, and the light chain variable region having the same amino acid sequence in a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.).In some embodiments, the fusion construct comprises a heavy chain variable region in any one of SEQ ID NOs: 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and a heavy chain variable region having the same amino acid sequence in percentages within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.), and The light chain variable region in any one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, and the light chain variable region having the same amino acid sequence in percentages defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), as paired in Table 0.4. In some embodiments, the fusion construct includes a heavy chain having any three heavy chain CDRs in any one of sequence numbers (ID NOs) 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and a light chain having any three light chain CDRs in any one of sequence numbers (ID NOs) 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128.In some embodiments, the fusion construct includes a heavy chain having any three heavy chain CDRs in any one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and a light chain having any three light chain CDRs in any one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, as paired in Table 0.4. In some embodiments, the fusion construct is one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any of the aforementioned values. A heavy chain variable region having the same amino acid sequence in percentages within the range defined by the following two factors (e.g., 80-100%, 85-95%, 90-97%, etc.), and one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, in amounts of at least 80%, at least 85%, at least 90%, and less. It includes light chain variable regions having the same amino acid sequence in at least 95%, at least 97%, at least 98%, at least 99%, or approximately 100%, or in percentages within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.). In some embodiments, the fusion construct has a heavy chain variable region having the same amino acid sequence as any one of SEQ ID NOs. 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127 in percentages within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and It includes light chain variable regions having the same amino acid sequence as one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, in percentages defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or approximately 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), as paired in Table 0.4.In some embodiments, the fusion construct comprises a heavy chain variable region in any one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127, and the same amino acid sequence in percentages within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), as well as sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, and 117 A heavy chain variable region having a heavy chain CDR among the three heavy chain CDRs in any one of 119, 121, 123, 125, and 127; and a light chain variable region having the same amino acid sequence as at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.) as the light chain variable region in any one of 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, and a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100% (e.g., 80-100%, 85-95%, 90-97%, etc.), and sequence number (ID The light chain variable region includes three light chain CDRs in any one of NO) 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, and the light chain and heavy chain sequence numbers are paired as shown in Table 0.4.In some embodiments, the fusion construct is composed of one of sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, and 127 (each with or without C-terminal lysine) and a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.) of the same amino acid composition The fusion construct comprises a heavy chain having a column, and a light chain having the same amino acid sequence as any one of sequence numbers 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, in a percentage within the range defined by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), as paired in Table 0.4. In some embodiments, the fusion construct includes sequence numbers 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, and 117. This includes heavy chains having one of the amino acid sequences 119, 121, 123, 125, and 127 (each with or without C-terminal lysine), and light chains having one of the amino acid sequences 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, and 128, as paired in Table 0.4. [Table 4-1] [Table 4-2] [Table 4-3] Table 4-4 Table 4-5 Table 4-6 Table 4-7 Table 4-8 Table 4-9 Table 4-10 Table 4-11 Table 4-12 Table 4-13 Table 4-14 Table 4-15 Table 4-16 Table 4-17 [Table 4-18]

[0209] In some embodiments, the VEGF trap includes a human VEGFR1 domain 2 and a human VEGFR2 domain 3. In some embodiments, the VEGF trap includes the amino acid sequence of SEQ ID NO: 133. In some embodiments, the VEGF trap includes an amino acid sequence identical to SEQ ID NO: 133 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.). [Table 5]

[0210] In some embodiments, the fusion construct is CDR in SEQ ID NO: 105 H CDR 1 H 1; CDR in Sequence ID No. 105 H CDR 2 H 2; CDR in Sequence ID No. 105 H CDR 3 H Heavy chain containing 3; CDR in SEQ ID NO: 106 L CDR 1 L 1; CDR in Sequence ID No. 106 L CDR 2 L 2; and CDR in Sequence ID No. 106 L CDR 3 L Includes 3. In some embodiments, the fusion construct includes complementarity determination region 1 (CDR). H 1): PFAMH (SEQ ID NO: 134), CDR H 2: KISPGGSWTYYSDTVTD (Sequence ID 135), and CDR H 3: Heavy chain containing QAWGYYALDI (SEQ ID NO: 136); and CDRL 1:SASISVSYLY (Sequence ID 137), CDR L 2:DDSSLAS (SEQ ID NO: 138), and CDR L 3: Includes a light chain containing QQWSGYPYT (SEQ ID NO: 139). In some embodiments, the heavy chain contains the same amino acid sequence as SEQ ID NO: 105 in terms of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and the light chain contains the same amino acid sequence as SEQ ID NO: 106 in terms of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO: 105 (with or without C-terminal lysine), and the light chain comprises the amino acid sequence of SEQ ID NO: 106. OG2072 represents a fusion construct comprising an anti-IL-6 antibody fused to a VEGF trap, and comprising the heavy chain of SEQ ID NO: 105 (with or without C-terminal lysine) and the light chain of SEQ ID NO: 106.

[0211] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are fusion constructs comprising a PDGFR extracellular trap fused to the heavy chain of an anti-VEGF-A antibody. In some embodiments, the fusion construct includes a heavy chain variable region having the same amino acid sequence as SEQ ID NO: 131 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and a light chain variable region having the same amino acid sequence as SEQ ID NO: 132 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the heavy chain includes the amino acid sequence of SEQ ID NO: 131 (with or without C-terminal lysine), and the light chain includes the amino acid sequence of SEQ ID NO: 132.

[0212] In some embodiments, one or both of the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) are anti-HTRA1 antibodies. In some embodiments, the anti-HTRA1 antibody is a complementation-determining region 1 CDR. H 1: FYHVH (Sequence ID: 140), CDR H 2:SIYTSGYTEYASALES (SEQ ID NO: 141), and CDR H 3: Heavy chains containing EGLQRVGVLDA (SEQ ID NO: 142), EGLQRVGVFDA (SEQ ID NO: 143), or EGLQRVGVMDA (SEQ ID NO: 144), and CDR L1: RSSQSLLDEAGETYLA (SEQ ID NO: 145), CDR L 2: EVSLLES (SEQ ID NO: 146), and CDR L 3: A light chain comprising QQATYFPYT (SEQ ID NO: 147). In some embodiments, the anti-HTRA1 antibody has a complementarity determining region 1 CDR H 1: GFSLTFYH (SEQ ID NO: 148), CDR H 2: IYTSGYT (SEQ ID NO: 149), and CDR H 3: A heavy chain comprising AREGLQRVGVFDA (SEQ ID NO: 150) or AREGLQRVGVMDA (SEQ ID NO: 151) or AREGLQRVGVLDA (SEQ ID NO: 152), and CDR L 1: QSLLDEAGETY (SEQ ID NO: 153), CDR L 2: EV, and CDR L 3: A light chain comprising QQATYFPYT (SEQ ID NO: 147). In some embodiments, the anti-HTRA1 antibody has a complementarity determining region 1 CDR H 1: GFSLTFY (SEQ ID NO: 154), CDR H 2: YTSGY (SEQ ID NO: 155), and CDR H 3: A heavy chain comprising EGLQRVGVLDA (SEQ ID NO: 142) or EGLQRVGVFDA (SEQ ID NO: 143) or EGLQRVGVMDA (SEQ ID NO: 144), and CDR L 1: RSSQSLLDEAGETYLA (SEQ ID NO: 145), CDR L 2: EVSLLES (SEQ ID NO: 146), and CDR L3: Containing a light chain containing QQATYFPYT (SEQ ID NO: 147). In some embodiments, the heavy chain contains the same amino acid sequence as any one of the VH sequences listed in Table 0.6 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and the light chain contains the same amino acid sequence as the VL sequences listed in Table 0.7 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or within the range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the heavy chain comprises one of the VH sequences listed in Table 0.6, and the light chain comprises the VL sequences listed in Table 0.7. [Table 6] [Table 7]

[0213] In some embodiments, non-conjugated proteins (e.g., non-conjugated antibodies or non-conjugated fusion constructs) may have conjugated cysteine, or manipulated cysteine, or blocked (therefore preventing polymer conjugation) conjugated cysteine. In some embodiments, non-conjugated species in a composition are distinct from conjugated species by the fact that the composition is prepared by subjecting a conjugated species to a conjugation reaction and then adding a non-conjugated protein (e.g., non-conjugated antibody or non-conjugated fusion construct) to the mixture. In some embodiments, non-conjugated proteins do not have non-natural cysteine ​​that can be conjugated to a polymer. In some embodiments, the conjugate comprises a polymer conjugated with cysteine, or with a free amino group of the protein, for example, an N-hydroxysuccinimide (NHS) ester. In some embodiments, the conjugate is conjugated with the ε-amine group of lysine, the α-amine group of the N-terminal amino acid, and / or the δ-amine group of histidine. Contains jugated polymers.

[0214] In some embodiments, the formulation or therapeutically acceptable composition comprises KSI-301. In some embodiments, KSI-301 comprises OG1950 and OG1953, which is its conjugated form with OG1802. OG1950 represents an anti-VEGF-A antibody having a heavy chain of SEQ ID NO: 1 (with or without C-terminal lysine) and a light chain of SEQ ID NO: 2. OG1802 may be conjugated to the heavy chain of OG1950 at C443 (EU numbering) to form OG1953. In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) is or comprises OG1950 conjugated with OG1802 (thereby forming OG1953), and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) is or comprises OG1950. In some embodiments, the formulation or therapeutically acceptable composition comprises about 40 to about 60 mM sodium acetate, about 0.01% to about 0.04% polysorbate 20, and a mixture of OG1950 and OG1953 at about 40 to about 60 mg / mL (total protein concentration), wherein the mixture contains about 15% to about 25% OG1950 and about 75% to about 85% OG1953 (e.g., in molar amounts or by total protein mass weight concentration), and the pH is about 4.5 to about 5.5. In some embodiments, the formulation or therapeutically acceptable composition comprises about 50 mM sodium acetate, about 0.025% polysorbate 20, and a mixture of OG1950 and OG1953 at about 50 mg / mL (total protein concentration), the mixture containing about 20% OG1950 and about 80% OG1953 (e.g., in molar amounts or total protein mass weight concentration) and having a pH of about 5.In some embodiments, the formulation or therapeutically acceptable composition consists of, or essentially consists of, a mixture of OG1950 and OG1953 at about 50 mM sodium acetate, about 0.025% polysorbate 20, and about 50 mg / mL (total protein concentration), the mixture containing about 20% OG1950 and about 80% OG1953 (e.g., in molar amounts or total protein mass weight concentration) and having a pH of about 5. In some embodiments, the second protein is or comprises OG1950IAM (e.g., OG1950 treated with iodoacetamide). Figure 58A illustrates components of non-limiting examples of formulations of the present disclosure, which may include, among other components, a mixture of anti-VEGF-A antibody and an anti-VEGF-A antibody conjugate, as described herein.

[0215] In some embodiments, the formulation or therapeutically acceptable composition comprises KSI-501. In some embodiments, KSI-501 comprises the fusion protein OG2072 and its conjugate form with OG1802, OG2074. OG2072 represents a fusion construct comprising an anti-IL-6 antibody fused to a VEGF trap and comprising the heavy chain of SEQ ID NO: 105 (with or without C-terminal lysine) and the light chain of SEQ ID NO: 106. OG1802 may be bound to the heavy chain of OG2072 at C443 (EU numbering) to form OG2074. In some embodiments, the first protein (e.g., a protein conjugated to a phosphorylcholine-containing polymer) is or comprises OG2072 conjugated with OG1802 (thereby forming OG2074), and the second protein (e.g., an unconjugated protein or a protein not conjugated to a phosphorylcholine-containing polymer) is or comprises OG2072. In some embodiments, the formulation or therapeutically acceptable composition comprises about 50 to about 60 mM sodium acetate, polysorbate 20 (e.g., 0.025% polysorbate 20), sucrose (e.g., 4% sucrose), and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), wherein the mixture contains 20 to 40% OG2072 and 60 to 80% OG2074 (e.g., in molar amounts or as a percentage of composition by total protein mass weight concentration), and the pH is about 4 The pH is approximately 0.5 to 5.5. In some embodiments, the formulation or therapeutically acceptable composition comprises approximately 50 to approximately 60 mM sodium acetate, approximately 0.025% polysorbate 20, approximately 4% sucrose, and a mixture of OG2072 and OG2074 at approximately 50 mg / mL (total protein concentration), wherein the mixture contains 30% OG2072 and 70% OG2074 (e.g., in molar amounts or by total protein mass weight concentration) and has a pH of approximately 5 (e.g., approximately pH 4.8 to approximately pH 5). In some embodiments, the formulation or therapeutically acceptable composition consists of, or essentially consists of, about 50 to about 60 mM sodium acetate, about 0.025% polysorbate 20, about 4% sucrose, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), the mixture containing about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or by total protein mass weight concentration), and has a pH of about 5 (e.g., about pH 4.8 to about pH 5). In some embodiments, the formulation or therapeutically acceptable composition comprises about 50 mM sodium acetate, about 0.025% polysorbate 20, about 4% sucrose, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), the mixture containing about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or total protein mass weight concentration) and having a pH of about 5. In some embodiments, the formulation or therapeutically acceptable composition consists of, or essentially consists of, about 50 mM sodium acetate, about 0.025% polysorbate 20, about 4% sucrose, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), the mixture containing about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or total protein mass weight concentration) and has a pH of about 5. In some embodiments, the formulation or therapeutically acceptable composition does not contain sucrose. In some embodiments, the formulation or therapeutically acceptable composition does not contain sugar.In some embodiments, the formulation or therapeutically acceptable composition comprises about 50 to about 60 mM sodium acetate, about 0.025% polysorbate 20, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), wherein the mixture contains about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or by total protein mass weight concentration), and has a pH of about 5 (e.g., about pH 4.8 to about pH 5), and optionally, the formulation or therapeutically acceptable composition is sucrose-free. In some embodiments, the formulation or therapeutically acceptable composition comprises or essentially consists of 50–60 mM sodium acetate, about 0.025% polysorbate 20, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), the mixture containing about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or by total protein mass weight concentration), and having a pH of about 5 (e.g., about pH 4.8 to about pH 5), and optionally, the formulation or therapeutically acceptable composition is sucrose-free. In some embodiments, the formulation or therapeutically acceptable composition comprises a histidine acetate buffer solution, about 0.025% polysorbate 20, about 4% sucrose, and a mixture of OG2072 and OG2074 at about 50 mg / mL (total protein concentration), the mixture containing about 30% OG2072 and about 70% OG2074 (e.g., in molar amounts or total protein mass weight percentages), and having a pH of about 5.2 to about 6.2. Figures 58B and 58C illustrate components of non-limiting examples of formulations of the present disclosure, which may include, among other components, mixtures of fusion constructs and fusion construct conjugates as described herein.

[0216] In some embodiments, the polymer component of the conjugate protein is approximately 100,000 Da or more, for example, approximately 150,000 Da or more, approximately 200,000 Da or more, approximately 350,000 Da or more, approximately 400,000 Da or more, approximately 450,000 Da or more, approximately 500,000 Da or more, approximately 550,000 Da or more, approximately 600,000 Da or more, approximately 650,000 Da or more, approximately 700,000 Da or more, approximately 750,000 Da or more, approximately 800,000 Da or more, approximately 850,000 Da or more, approximately 900,000 Da or more, approximately 950, The polymer has a molecular weight of 000 Da or more, about 1,000,000 Da or more, or a molecular weight within the range defined by any two of the aforementioned values ​​(e.g., 100,000 to 1,000,000 Da, 300,000 to 950,000 Da, 400,000 to 800,000 Da, 500,000 to 750,000 Da, 600,000 to 700,000 Da, 600,000 to 1,000,000 Da, etc.). In some embodiments, the polymer has a molecular weight of about 700,000 to about 800,000 Da. In some embodiments, the polymer component of the conjugate protein is one of the polymers disclosed herein. In some embodiments, the polymer component of the conjugate protein is OG1801.

[0217] In some embodiments, the phosphorylcholine-containing polymer conjugate comprises a 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomer, as shown below. [ka]

[0218] In some embodiments, the conjugate polymer has three or more arms, or is synthesized using an initiator containing three or more polymer initiation sites. In some embodiments, the conjugate polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms, or is synthesized using an initiator containing two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. In some embodiments, the conjugate polymer has nine arms, or is synthesized using an initiator containing nine polymer initiation sites. In some embodiments, the conjugate polymer has a polydispersity index (PDI) of less than approximately 1.2.

[0219] In some embodiments, the polymer of the first protein (e.g., the first antibody and / or conjugated antibody) is of the conjugate type and has three or more arms, or is synthesized using an initiator containing three or more polymer start sites. In some embodiments, the polymer of the first protein (e.g., antibody) is of the conjugate type and has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms, or is synthesized using an initiator containing two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer start sites. In some embodiments, the polymer of the first antibody has a polydispersity index (PDI) of less than about 1.2. In some embodiments, the polymer comprises an MPC monomer, the first antibody comprises the amino acid sequence of SEQ ID NO: 1 (with or without C-terminal lysine); the amino acid sequence of SEQ ID NO: 2; and the antibody comprises C4 in SEQ ID NO: 1. At 49, it is bonded to the polymer. In some embodiments, the polymer has nine arms, and the polymer has a molecular weight of about 600,000 to about 900,000 Da.

[0220] In some embodiments, the first protein or conjugate protein (e.g., an antibody conjugated to a polymer) is an antibody or fusion construct having the following structure: [ka] In the formula, each heavy chain of the antibody or fusion construct is represented by the letter H, and each light chain of the antibody or fusion construct is represented by the letter L; the polymer is bonded to the antibody or fusion construct via sulfhydryl C443 (EU numbering), and this bond is shown on one of the heavy chains; PC is [ka] Here, the dashed line indicates the binding point to the remainder of the polymer; X is a) -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H, c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS; 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%. In some embodiments, the first protein or conjugate protein (e.g., an antibody conjugated to a polymer) is an antibody or fusion construct having the following structure: [ka] In the formula, each heavy chain of the antibody or fusion construct is represented by the letter H, and each light chain of the antibody or fusion construct is represented by the letter L; the polymer is bonded to the antibody or fusion construct via sulfhydryl C443 (EU numbering), and this bond is shown on one of the heavy chains; PC is [ka] where the wavy line indicates the point of attachment to the remainder of the polymer; X is any halogen including a) -OR where R is -H, methyl, ethyl, propyl, isopropyl, b) -H, c) -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%.

[0221] In some embodiments, the second (and / or non-conjugated) antibody comprises the CDRs in: CDR H 1 in SEQ ID NO: 1 H 1; CDR H 2 in SEQ ID NO: 1 H 2; CDR H 3 in SEQ ID NO: 1 H 3; CDR L 1 in SEQ ID NO: 2 L 1; CDR L 2 in SEQ ID NO: 2 L 2; CDR L 3 in SEQ ID NO: 2 L 3; 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.

[0222] The formulations and compositions provided herein can include a suitable pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier includes water or a buffer. In some embodiments, the pharmaceutically acceptable carrier includes a buffer having a pKa within 2, 1, or 0.5 pH units of the pH of the formulation, or within a pH unit of approximately that value. The formulation includes a buffer. In some embodiments, the formulation includes a buffer selected from acetate buffer, phosphate buffer, citrate buffer, glycine buffer, histidine buffer, HEPES buffer, and Tris buffer as a pharmaceutically acceptable carrier. In some embodiments, the formulation includes sodium acetate buffer. In some embodiments, the formulation includes sodium acetate in a concentration of approximately 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 mM or more, or at least that concentration, or optionally, the formulation includes sodium acetate in a concentration within the range defined by any two of the aforementioned values ​​(e.g., 10-100 mM, 30-80 mM, 40-60 mM, 40-70 mM, etc.). In some embodiments, the formulation includes sodium acetate in a concentration in the range of 40-60 mM. In some embodiments, the formulation includes sodium acetate in a concentration in the range of 30-80 mM. In some embodiments, the formulation contains histidine-acetic acid buffer. In some embodiments, the formulation contains histidine-acetic acid in a concentration of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100 mM or more, or at least that concentration, or optionally, the formulation contains sodium acetate in a concentration within the range defined by any two of the aforementioned values ​​(e.g., 5-100 mM, 20-80 mM, 10-60 mM, 15-70 mM, 10-30 mM, 15-25 mM, etc.). In some embodiments, the formulation contains histidine-acetic acid in a concentration in the range of 10-60 mM. In some embodiments, the formulation contains histidine-acetic acid in a concentration in the range of 10-30 mM. In some embodiments, the formulation contains histidine-acetic acid at a concentration in the range of 15–25 mM.

[0223] In any of the compositions or formulations described herein, in some embodiments, the composition or formulation includes a surfactant, such as, but not limited to, polysorbate (e.g., polysorbate 20). The composition or formulation may contain any suitable amount of surfactant. In some embodiments, the composition or formulation contains a surfactant in approximately that percentage (w / w) or at least 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1% (w / w) or more, or at least that percentage (w / w), or optionally, the composition or formulation contains a surfactant in a percentage (w / w) within the range defined by any two of the aforementioned values ​​(e.g., 0.005-0.1%, 0.01-0.05%, 0.02-0.03%, 0.015-0.08%, etc.). In some embodiments, the composition or formulation contains a surfactant in approximately 0.01% (w / w) to approximately 0.04% (w / w). In some embodiments, the composition or formulation contains about 0.02% (w / w) to about 0.03% (w / w) of the surfactant. In some embodiments, the composition or formulation contains about 0.025% (w / w) of the surfactant. In some embodiments, the surfactant is or contains polysorbate 20, polysorbate 80, or poloxamer 188. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the composition or formulation contains about 0.01 to 0.05% (w / w) of polysorbate 20. In some embodiments, the composition or formulation contains about 0.02 to 0.03% (w / w) of polysorbate 20. In some embodiments, the composition or formulation contains about 0.025% (w / w) of polysorbate 20. In some embodiments, the surfactant is poloxamer 188.

[0224] In some embodiments of any of the compositions or formulations described herein, the composition or formulation comprises an isotonic agent. In some embodiments, the isotonic agent is or comprises a sugar. In some embodiments, the isotonic agent is or comprises sucrose or trehalose. The composition or formulation may contain any suitable amount of the isotonic agent. In some embodiments, the composition or formulation contains 2.0%, 2.5%, The composition or formulation contains an isotonic agent in approximately that percentage (w / v), or at least that percentage (w / v), or optionally, an isotonic agent in a percentage (w / v) within the range defined by any two of the aforementioned values ​​(e.g., 2-10%, 2.5-6%, 3-5%, 3.5-8.5%, etc.). In some embodiments, the composition or formulation contains an isotonic agent in about 2% (w / v) to about 10% (w / v). In some embodiments, the composition or formulation contains an isotonic agent in about 3% (w / v) to about 5% (w / v). In some embodiments, the composition or formulation contains about 4% (w / w) of an isotonic agent. In some embodiments, the composition or formulation contains about 2% (w / v) to about 10% (w / v) of sucrose. In some embodiments, the composition or formulation contains about 3% (w / v) to about 5% (w / v) of sucrose. In some embodiments, the composition or formulation contains about 4% (w / v) of sucrose.

[0225] An intraocular therapeutic composition containing approximately 50 mg / mL of anti-VEGF-A protein, wherein the anti-VEGF-A antibody is located in complementarity-determining region 1 (CDR). H 1): GYDFTHYGMN (SEQ ID NO: 9), CDR H 2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR H 3: Heavy chain containing YPYYYGTSHWYFDV (SEQ ID NO: 11); and CDR L 1:SASQDISNYLN (Sequence ID 12), CDR L2: FTSSLHS (SEQ ID NO: 13), and CDR L 3: A composition for intraocular treatment is also provided, comprising a light chain containing QQYSTVPWT (SEQ ID NO: 14), wherein the anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, the unconjugated antibody is present in the formulation at a concentration of approximately 10% to 30% of the total molar amount of the antibody conjugate and unconjugated antibody, the total molar amount being the sum of the molar amounts of the antibody conjugate and the unconjugated antibody, the antibody conjugate comprising an anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer at a non-natural cysteine ​​outside the variable region of the antibody, the phosphorylcholine-containing polymer being present in the composition at a concentration of approximately 100 mg / mL or more, the phosphorylcholine-containing polymer having a molecular weight of 300,000 to 1,200,000 Da, and the pH of the composition being approximately 5.5 or less.

[0226] An intraocular therapeutic composition comprising approximately 50 mg / mL of anti-VEGF-A protein, wherein the anti-VEGF-A antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 1 (with or without C-terminal lysine); and a light chain containing the amino acid sequence of SEQ ID NO: 2, and the anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, with the unconjugated antibody present in the formulation at approximately 10% to 30% of the total molar amount of the antibody conjugate and unconjugated antibodies, the total molar amount being the sum of the molar amounts of the antibody conjugate and the unconjugated antibody, and the antibody conjugate comprises the following structure: [ka] In the formula, each heavy chain of the conjugate is represented by the letter H, and each light chain of the conjugate is represented by the letter L; the polymer is bonded to the heavy chain of the conjugate via sulfhydryl C443 (EU numbering), and this bond is shown on one of the heavy chains; PC is, [ka] A composition for intraocular treatment is also provided, wherein the wavy line in the formula indicates a bond point to the remainder of the polymer, and X is a) -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H, c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS; 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%, the phosphorylcholine-containing polymer is present in the composition at a concentration of about 100 mg / mL or more, and the pH of the composition is about 5.5 or less.

[0227] An intraocular therapeutic composition comprising a protein fusion construct at approximately 53 mg / mL, wherein the fusion construct comprises a VEGF trap fused to an anti-IL-6 antibody, and the fusion construct comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 105 (with or without C-termi...

Claims

1. A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A formulation containing, The formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein. formulation.

2. A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Therapeutically acceptable carriers, A therapeutically acceptable composition comprising, The composition comprises the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The composition has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. A therapeutically acceptable composition.

3. A conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second protein not conjugated with a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A therapeutically acceptable composition comprising, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. The composition has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. A therapeutically acceptable composition.

4. A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A formulation containing, The formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. The formulation has reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in the total molar amount. formulation.

5. A conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second protein not conjugated with a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A therapeutically acceptable composition comprising, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. The composition has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein. The composition has reduced viscosity and / or improved injectability compared to the reference composition containing the conjugate. The first protein of the conjugate is present in the reference composition at a total mass weight concentration of the first protein and the second protein in the composition. A therapeutically acceptable composition.

6. A first molar amount of conjugate containing a protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A low viscosity formulation of protein conjugates, The formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the protein. The formulation has reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in a total molar amount which is the sum of the first molar amount and the second molar amount. Low viscosity formulation of protein conjugate.

7. A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A formulation containing, The second protein is present in the formulation in an amount of approximately 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. The formulation has a pH approximately the same as the pI of the second protein (for example, within 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, or 0.5 pH units) and has reduced turbidity compared to a reference formulation containing the first molar amount of the conjugate and the second molar amount of the second protein. formulation.

8. A first mole containing a protein conjugated to a phosphorylcholine-containing polymer. Quantity conjugate; A second molar amount of protein not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A pharmaceutical preparation containing, The formulation contains the protein not conjugated to the phosphorylcholine-containing polymer in an amount of about 1% or more of the total molar amount of the conjugated and unconjugated proteins, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the protein. The aforementioned formulation is substantially free of turbidity. Pharmaceutical preparations.

9. A phosphorylcholine-containing polymer present in the formulation at a concentration of 100 mg / mL or more; and A protein that is not conjugated to a phosphorylcholine-containing polymer, wherein the protein is present in the formulation in a second molar amount, A formulation containing, The protein is present in the formulation in an amount of about 1% or more of the total molar amount of the polymer and the protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the protein. formulation.

10. A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A formulation containing, The difference between the pI of the second protein in the acidic or basic direction and the pH of the formulation is selected to be greater than the minimum difference between the pI of the second protein in the corresponding acidic or basic direction and the pH of a reference formulation including: A third molar amount of the conjugate comprising the first protein conjugated to the phosphorylcholine-containing polymer; A fourth molar amount of the second protein, which is not conjugated to the phosphorylcholine-containing polymer; and The aforementioned pharmaceutically acceptable carrier, The first total molar amount, which includes the sum of the first and second molar amounts, and the second total molar amount, which includes the sum of the third and fourth molar amounts, are substantially the same, and the second molar amount is greater than the fourth molar amount. The aforementioned reference formulation is substantially free of turbidity. formulation.

11. A conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second protein not conjugated with a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A therapeutically acceptable composition comprising, The difference between the pI of the second protein and the pH of the formulation is selected to be greater than the minimum difference between the pI of the second protein and the pH of a reference formulation including: The conjugate comprising the first protein conjugated to the phosphorylcholine-containing polymer; The second protein not conjugated to the phosphorylcholine-containing polymer; and The aforementioned pharmaceutically acceptable carrier, The percentage of the second protein in the composition relative to the total protein mass weight concentration of the first and second proteins is higher than the percentage of the second protein in the reference composition relative to the total protein mass weight concentration of the first and second proteins. The aforementioned reference composition is substantially free of turbidity. A therapeutically acceptable composition.

12. A first molar amount of conjugate comprising a first protein conjugated to a polymer; and A second molar amount of the second protein, which is not conjugated to the polymer. A formulation containing, The formulation contains the second protein in an amount of approximately 1% or more of the total molar amount of the first protein and the second protein, and the total molar amount includes the sum of the molar amounts of the first and second proteins. formulation.

13. A conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second protein not conjugated with a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, A therapeutically acceptable composition comprising, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. A therapeutically acceptable composition.

14. A first molar amount of conjugate comprising a first protein conjugated to a polymer; and A second molar amount of the second protein, which is not conjugated to the polymer. A therapeutically acceptable composition comprising, The composition contains the second protein in an amount of about 1% or more of the total molar amount of the first protein and the second protein, and the total molar amount includes the sum of the molar amounts of the first and second proteins. A therapeutically acceptable composition.

15. A first molar amount of a first protein conjugated to a polymer; and A second molar amount of the second protein, which is not conjugated to the polymer. A formulation containing, Further improvements include: the formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. formulation.

16. A conjugate containing a first protein conjugated to a polymer; and A second protein not conjugated to the polymer, A formulation containing, The first molar amount of the conjugate and the second molar amount of the second protein are combined in the formulation such that the second molar amount is about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. formulation.

17. A conjugate containing a first protein conjugated to a polymer; and A second protein not conjugated to the polymer, A therapeutically acceptable composition comprising, The second protein in the composition is combined with the conjugate at a compositional percentage of about 1% or more relative to the total protein mass weight concentration of the first protein and the second protein, and the remainder of the total protein mass weight concentration is the first protein. A therapeutically acceptable composition.

18. A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer, wherein the polymer has nine arms and a molecular weight of 600,000 to 1,000,000 Da, and the polymer is present in the formulation at a concentration of approximately 100 mg / mL or more; and A second protein that is not conjugated to a polymer, wherein the second protein is present in the formulation at a concentration of 5 to 15 mg / mL. A preparation containing the above ingredients.

19. An intraocular therapeutic composition comprising an anti-VEGF-A antibody at a protein concentration of approximately 50 mg / mL, wherein the anti-VEGF-A antibody comprises the following: Complementarity Determination Region 1 (CDR) Ｈ 1): GYDFTHYGMN (SEQ ID NO: 9), CDR Ｈ 2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDR Ｈ 3: Heavy chain containing YPYYYGTSHWYFDV (SEQ ID NO: 11); and CD-R Ｌ 1: SASQDISNYLN (Sequence ID 12), CDR Ｌ 2: FTSSLHS (SEQ ID NO: 13), and CDR Ｌ 3: Light chain containing QQYSTVPWT (Sequence ID 14), The anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, the unconjugated antibody is present in the formulation in an amount between approximately 10% and approximately 30% of the total molar amount of the antibody conjugate and the unconjugated antibody, and the total molar amount is the sum of the molar amount of the antibody conjugate and the molar amount of the unconjugated antibody. The antibody conjugate comprises the anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer at a non-natural cysteine ​​outside the variable region of the antibody, the phosphorylcholine-containing polymer present in the composition at a concentration of approximately 100 mg / mL or more, and the phosphorylcholine-containing polymer has nine arms and a molecular weight of 600,000 to 1,000,000 Da. The pH of the aforementioned composition is approximately 5.5 or less. Composition for intraocular treatment.

20. An intraocular therapeutic composition comprising an anti-VEGF-A antibody at a protein concentration of approximately 50 mg / mL, wherein the anti-VEGF-A antibody comprises the following: Heavy chains containing the amino acid sequence of SEQ ID NO: 1 (with or without C-terminal lysine); and Light chain containing the amino acid sequence of SEQ ID NO: 2, The anti-VEGF-A antibody is present in the composition as either an antibody conjugate or an unconjugated antibody, and the unconjugated antibody is the antibody conjugate The antibody conjugate and the non-conjugate antibody are present in the formulation in amounts ranging from approximately 10% to approximately 30% of the total molar amount, and the total molar amount is the sum of the molar amount of the antibody conjugate and the molar amount of the non-conjugate antibody. The antibody conjugate described above includes the following structure: 【Chemistry 1】 During the ceremony, Each heavy chain of the aforementioned conjugate is represented by the letter H, and each light chain of the aforementioned conjugate is represented by the letter L; The polymer is bonded to the heavy chain of the conjugate via a sulfhydryl of C443 (EU numbering), and this bond is shown on one of the heavy chains; The PC is as follows: 【Chemistry 2】 In the formula, the dashed line indicates a bond to the remainder of the polymer, where X is a) -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H; c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS; n1, n2, n3, n4, n5, n6, n7, n8, and n9 may be the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500 ± 15%. The phosphorylcholine-containing polymer is present in the composition at a concentration of approximately 100 mg / mL or more. The pH of the aforementioned composition is approximately 5.5 or less. Composition for intraocular treatment.

21. An intraocular therapeutic composition comprising a fusion construct at a protein concentration of approximately 53 mg / mL, wherein the fusion construct comprises a VEGF trap fused to an anti-IL-6 antibody, and the fusion construct comprises the following: Heavy chains containing the amino acid sequence of SEQ ID NO: 105 (with or without C-terminal lysine); and Light chain containing the amino acid sequence of SEQ ID NO: 106, The fusion construct exists in the composition as either a conjugate or a non-conjugate fusion construct, wherein the non-conjugate fusion construct is present in the formulation at a concentration of approximately 20% to approximately 40% of the total molar amount of the conjugate and the non-conjugate fusion construct, and the total molar amount is the sum of the molar amount of the conjugate and the molar amount of the non-conjugate fusion construct. The conjugate comprises the fusion construct conjugated to a phosphorylcholine-containing polymer, the phosphorylcholine-containing polymer is present in the composition at a concentration of approximately 100 mg / mL or more, and the pH of the composition is approximately 5 or less. Composition for intraocular treatment.

22. An intraocular therapeutic composition comprising a fusion construct at a protein concentration of approximately 50 mg / mL, wherein the fusion construct comprises a VEGF trap fused to an anti-IL-6 antibody, and the fusion construct comprises the following: Complementary determining region 1 (CDR Ｈ 1): PFAMH (SEQ ID NO: 134), CDR Ｈ 2: KISPGGSWTYYSDTVTD (SEQ ID NO: 135), and CDR Ｈ 3: QAWGYYALD I (SEQ ID NO: 136) containing heavy chain; and CD-R Ｌ 1: SASISVSYLY (Sequence ID 137), CDR Ｌ 2: DDSSLAS (Sequence ID 138), and CDR Ｌ 3: Light chain containing QQWSGYPYT (Sequence ID 139), The fusion construct exists in the composition as either a conjugate or a non-conjugate fusion construct, wherein the non-conjugate fusion construct is present in the formulation at a concentration of approximately 20% to approximately 40% of the total molar amount of the conjugate and the non-conjugate fusion construct, and the total molar amount is the sum of the molar amount of the conjugate and the molar amount of the non-conjugate fusion construct. The aforementioned conjugate includes the following structure: 【Transformation 3】 During the ceremony, Each heavy chain of the aforementioned conjugate is represented by the letter H, and each light chain of the aforementioned conjugate is represented by the letter L; The polymer is bonded to the heavy chain of the conjugate via a sulfhydryl of C443 (EU numbering), and this bond is shown on one of the heavy chains; The PC is as follows: 【Chemistry 4】 In the formula, the dashed line indicates a bond to the remainder of the polymer, where X is a) -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H; c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS; n1, n2, n3, n4, n5, n6, n7, n8, and n9 may be the same or different, such that the sum of n1, n2, n3, n4, n5, n6, n7, n8, and n9 is 2500 ± 15%. The phosphorylcholine-containing polymer is present in the composition at a concentration of approximately 100 mg / mL or more, and the pH of the composition is approximately 5 or less. Composition for intraocular treatment.

23. A method for preparing a pharmaceutical product, comprising combining the following in the product: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer, The formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is about 0.5 pH units or more away from the isoelectric point (pI) of the second protein. method.

24. A method for preparing a therapeutically acceptable composition, comprising combining the following in the therapeutically acceptable composition: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer, The composition comprises the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The composition has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. method.

25. A method for preparing a therapeutically acceptable composition, comprising combining the following in the therapeutically acceptable composition: A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second protein not conjugated with a phosphorylcholine-containing polymer, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. The composition has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. method.

26. A method for preparing a pharmaceutical preparation, comprising adjusting the pH of the preparation so that it is at least 0.5 pH units away from the isoelectric point (pI) of a non-conjugate protein contained in the preparation, wherein the preparation includes: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of unconjugated protein that is not conjugated to a phosphorylcholine-containing polymer, The formulation contains the unconjugated protein in an amount of approximately 1% or more of the total molar amount of the conjugated and unconjugated proteins, and the total molar amount includes the sum of the first molar amount and the second molar amount. method.

27. A method for preparing a therapeutically acceptable composition, comprising adjusting the pH of the composition so that it is at least 0.5 pH units away from the isoelectric point (pI) of a non-conjugate protein contained in the composition, wherein the composition comprises: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of unconjugated protein that is not conjugated to a phosphorylcholine-containing polymer, The composition contains the unconjugate protein in an amount of about 0.1% or more of the total molar amount of the conjugate and unconjugate proteins, and the total molar amount includes the sum of the first molar amount and the second molar amount. method.

28. A method for preparing a therapeutically acceptable composition, comprising adjusting the pH of the therapeutically acceptable composition so that it is at least 0.5 pH units away from the isoelectric point (pI) of a non-conjugate protein contained in the composition, wherein the composition comprises: A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second protein not conjugated with a phosphorylcholine-containing polymer, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. The composition has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. method.

29. A method for preparing a low-viscosity formulation of a protein conjugated to a phosphorylcholine-containing polymer, comprising combining the following in the formulation: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of protein that is not conjugated to a phosphorylcholine-containing polymer, The formulation contains the protein not conjugated to the phosphorylcholine-containing polymer in an amount of about 1% or more of the total molar amount of the conjugated and unconjugated proteins, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. The formulation has reduced viscosity and / or improved injectability compared to a reference formulation containing the conjugate in the total molar amount. method.

30. A method for preparing a pharmaceutical product, comprising combining the following in the product: A first molar amount of conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer, The formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. method.

31. A method for preparing a therapeutically acceptable composition, comprising combining the following in the therapeutically acceptable composition: A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; and A second protein not conjugated with a phosphorylcholine-containing polymer, The compositional percentage of the second protein relative to the total protein mass weight concentration of the first and second proteins in the composition is approximately 1% or more. method.

32. A method of treating the subject, A therapeutically effective formulation or composition of any one of claims 1 to 22 for a subject requiring treatment. Administer the amount intraocularly. Methods that include...

33. The kit includes the following: A pre-filled syringe containing a low-viscosity formulation, wherein the low-viscosity formulation is A conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer, and A pre-filled syringe containing a low-viscosity formulation comprising a second protein not conjugated to a phosphorylcholine-containing polymer; and A syringe needle for injecting the low-viscosity formulation, wherein the gauge of the needle is 27 or greater. A kit that includes this.

34. A formulation comprising approximately 40 to 60 mM sodium acetate, approximately 0.01% to 0.04% polysorbate 20, and a mixture of OG1950 and OG1953 in a total protein concentration of approximately 40 to 60 mg / mL, wherein the mixture contains approximately 15% to 25% OG1950 and approximately 75% to 85% OG1953 in molar amounts, and the pH is approximately 4.5 to 5.

5.

35. A formulation comprising, consisting of, or essentially consisting of, approximately 50 mM sodium acetate, approximately 0.025% polysorbate 20, and a mixture of approximately 50 mg / mL (total protein concentration) of OG1950 and OG1953, wherein the mixture contains approximately 20% OG1950 and approximately 80% OG1953 in molar amounts, and has a pH of approximately 5.

36. A method for storing protein, comprising maintaining the protein in a formulation for at least two months to two years, wherein the formulation comprises the following: A first molar amount of conjugate containing a first protein conjugated to a phosphorylcholine-containing polymer; A second molar amount of a second protein that is not conjugated to a phosphorylcholine-containing polymer; and Pharmaceutically acceptable carriers, The formulation contains the second protein in an amount of about 1% or more of the total molar amount of the conjugate and the second protein, and the total molar amount includes the sum of the first molar amount and the second molar amount. The formulation has a pH that is at least 0.5 pH units away from the isoelectric point (pI) of the second protein. The protein comprises an antibody or a fusion construct. method.

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