Method for analyzing mixed compositions of proteins and protein conjugates

Abstract

This specification provides methods and apparatus for analyzing mixed formulations of proteins and their protein conjugates. Some embodiments of proteins can be conjugated to a portion such as a HEMA-PC polymer. Some embodiments of antibody conjugates can retain or enhance antibody activity. Antibodies and their conjugates may be particularly useful for treating diabetic retinopathy.

Description

[Technical Field]

[0001] Reference to related applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 506764, filed on 7 June 2023, which is incorporated herein by reference in its entirety.

[0002] Sequence List This application is submitted together with an electronic sequence listing. The sequence listing is provided as a file named KDIAK211WO_SEQLIST.xml, created on June 4, 2024, with a size of 196,690 bytes. The electronic information of the sequence listing is incorporated herein by reference in its entirety.

[0003] field This disclosure relates to methods and apparatus for analyzing mixed formulations and compositions comprising mixtures of non-conjugate proteins and conjugate proteins (e.g., antibodies and their conjugates). [Background technology]

[0004] In the field of biopharmaceuticals, various systems are available for purifying proteins. In many cases, these different systems have their own unique advantages and disadvantages. [Overview of the project]

[0005] A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The mixed preparation is passed through a pre-filtering step to prepare a first filtered mixed preparation, and the first filtered mixed preparation is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed preparation, the first run; iii. A second run, the protein bound to the CEX column is eluted to prepare a third filtered mixed preparation, and the third filtered mixed preparation is passed through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to prepare a fourth filtered mixed preparation, the second run; EC) high performance liquid chromatography (HPLC) column to prepare a fourth filtered mixed preparation, the second run; iv. Using a solvent of a specific ionic strength to enable distribution of the mixed preparation in the CEX column; The method enables analysis of the mixed preparation based on differences in the charge and size variants of the components, A method is provided herein.

[0006] Also provided is a method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. Preparing a mixed therapeutic composition comprising: a. A first antibody that is an anti-VEGF-A antibody conjugated to a polymer; and[[ID=??]] b. A second antibody that is an anti-VEGF-A antibody not conjugated to the polymer antibody; ii. A first run, the mixed therapeutic composition is passed through a pre-filtering step to prepare a first filtered mixed therapeutic composition, and the first filtered mixed therapeutic composition is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition, the first run; iii. A second run, the protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is passed through a size exclusion exchange (size exclusion It seems there is a formatting issue in the original text where "size exclusion exchange)(S " might be incorrect. I've translated it as best as possible with the given text. If there are any corrections or clarifications needed, please let me know.passed through a strong cation exchange (CEX) column and a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to prepare a fourth filtered mixed therapeutic composition, a second run; iv. using a solvent of a specific ionic strength to enable partitioning of the mixed therapeutic composition in the CEX column; The method enables analysis of the mixed therapeutic composition based on differences in charge and size variants of the components. A method is provided. Also provided is a method of analyzing a mixed therapeutic active agent sample, the method comprising: i. preparing a mixed therapeutic active agent comprising:

[0007] a. a first antibody that is an anti-VEGF-A antibody conjugated to a polymer; and b. a second antibody that is an anti-VEGF-A antibody not conjugated to the polymer; ii. a first run in which the mixed therapeutic active agent is passed through a pre-filtering step to prepare a first filtered mixed therapeutic active agent, and the first filtered mixed therapeutic active agent is passed through a strong cation exchange (CEX) column to prepare a second filtered mixed therapeutic active agent; iii. a second run in which the protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic active agent, and the third filtered mixed therapeutic active agent is passed through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to prepare a fourth filtered mixed therapeutic active agent; iv. using a solvent of a specific ionic strength to enable partitioning of the mixed therapeutic active agent in the CEX column; The method enables analysis of the mixed therapeutic active agent based on differences in charge and size variants of the components. A method is provided. ​​​​​Furthermore, an apparatus including a tandem configuration of CEX and SEC HPLC columns for use when analyzing a mixed formulation sample containing a) a first antibody which is an anti-VEGF-A antibody conjugated to a polymer and b) a second antibody which is an anti-VEGF-A antibody not conjugated to the polymer, The apparatus provided for the purification includes the following: i. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation undergoes a size exclusion exchange (S The second run involves passing the mixture through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation. Furthermore, a) the first antibody is an anti-VEGF-A antibody conjugated to a polymer and b) An apparatus including a tandem configuration of CEX and SEC HPLC columns for use in analyzing a sample of a mixed therapeutic composition containing a second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer, The purification process includes the following: The apparatus is provided herein; i. The first run, The first run involves passing the mixed therapeutic composition through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and passing the first filtered mixed therapeutic composition through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared. Furthermore, the apparatus includes a tandem configuration of CEX and SEC HPLC columns for use when analyzing a mixed therapeutic active formulation sample comprising a) a first antibody which is an anti-VEGF-A antibody conjugated to a polymer and b) a second antibody which is an anti-VEGF-A antibody not conjugated to the polymer, The purification process includes the following: The apparatus is provided herein; i. The first run, The first run involves passing the mixed therapeutic agent through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and then passing the first filtered mixed therapeutic agent through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic formulations are prepared in the second run. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation undergoes a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; The method described above reduces the presence of antibody aggregates in the formulation; The polymer of the first antibody comprises a phosphorylcholine-containing polymer, the polymer covalently bonded to the first antibody at a cysteine ​​outside the variable region of the first antibody, the cysteine ​​being substituted with a non-cysteine ​​amino acid occurring at the same position in the sequence, the first antibody comprising a light chain and a heavy chain, the heavy chain comprising an Fc region, the cysteine ​​being within the Fc region of the heavy chain, and the sequence of the heavy chain being Sequence ID No. 1 (having a C-terminal lysine) (or not) the sequence of the light chain includes sequence number 2; The antibody conjugate has the following structure: [ka] In the formula, X is a) an -OR in which R is -H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) a halogen; (or optionally, X is a) an -OR in which R is -H, methyl, ethyl, propyl, or isopropyl; b) -H; c) any halogen including -Br, -Cl, or -I; d) -SCN; or e) -NCS;) During the ceremony: Each heavy chain of the first antibody is represented by the letter H, and each light chain of the first antibody is represented by the letter L; The polymer is bound to the first antibody via a cysteine ​​sulfhydryl at position 449, numbered in SEQ ID NO: 1, and this binding is shown on one of the heavy chains; PCs are [ka] In the formula, the dashed line indicates the bonding point to the remainder of the polymer; 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 second antibody comprises a light chain and a heavy chain, the heavy chain comprising an Fc region, cysteine ​​located within the Fc region of the heavy chain, the sequence of the heavy chain comprising SEQ ID NO: 1, and the sequence of the light chain comprising SEQ ID NO: 2; The mixed formulation sample passes sequentially through a prefilter, the CEX column, and the SEC HPLC column, wherein the tandem CEX and SEC HPLC columns are arranged as a Shodex SP825 column with an inner diameter (id) × length dimension of 9.0 × 75 mm and a TSKgel G3000SWxl column with an inner diameter (id) × length dimension of 7.8 × 300 mm; The isocratic run conditions include a flow rate of 0.5 ml / min, and the buffer solution includes 20 mM sodium acetate (pH 5) in any amount between 50 mM NaCl and 5 M NaCl; The proportion of the second antibody is any proportion between 0% and 20%; The aforementioned mixed formulation sample contains any amount of protein between 25 μg and 1340 μg; The method further includes evaluating high molecular weight aggregates in the purified mixed formulation by a method comprising SEC profile analysis and SDS PAGE gel; The method further includes evaluating the proportion of the second antibody in the purified mixed formulation by SEC profile analysis; The first run separates the first antibody from the second antibody and antibody aggregates. The step for eluting the CEX-bound free protein during the second run includes a high-salt concentration pulse with 1M NaCl, During the second run, the fourth filtered mixed formulation is passed through a pre-filtration step to prepare the fifth filtered mixed formulation, the fifth filtered mixed formulation is passed through a CEX column to prepare the sixth filtered mixed formulation, and the sixth mixed formulation is passed through an SEC HPLC column; The filtered mixed sample is subjected to size exclusion exchange (SEC HPL). Passing through a C column separates the second antibody from the antibody aggregates. A method is provided herein.

[0008] Furthermore, a method for analyzing a mixed formulation sample, the method comprising the following: i. Prepare a mixed formulation containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and then passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation undergoes a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; To enable the distribution of the mixed formulation in the CEX column, use a solvent with a specific ionic strength; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. A method is provided. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. Prepare a mixed therapeutic composition containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, The first run involves passing the mixed therapeutic composition through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and then passing the first filtered mixed therapeutic composition through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared; iv. To enable the distribution of the mixed therapeutic composition in the CEX column, use a solvent of a specific ionic strength; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. A method is provided herein. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. Prepare a mixed therapeutic formulation containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, The first run involves passing the mixed therapeutic agent through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and then passing the first filtered mixed therapeutic agent through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic active formulations are prepared in the second run; iv. To enable the distribution of the mixed therapeutic active formulation in the CEX column, use a solvent of a specific ionic strength; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. A method is provided herein.

[0009] Furthermore, an apparatus including a tandem configuration of CEX and SEC HPLC columns for use in purifying a mixed formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein not conjugated to the polymer, The purification method provided includes the following: i. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation undergoes a size exclusion exchange (S The second run involves passing the mixture through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation. a) A first fusion protein conjugated to the polymer and b) Conjugated to the polymer An apparatus comprising a tandem configuration of CEX and SEC HPLC columns for use in purifying a mixed therapeutic composition sample containing a second, unjugated protein, A method for the aforementioned purification, comprising the following, is provided herein: i. The first run, The first run involves passing the mixed therapeutic composition through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and passing the first filtered mixed therapeutic composition through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared. Furthermore, an apparatus including a tandem configuration of CEX and SEC HPLC columns for use in purifying a mixed therapeutic active formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein not conjugated to the polymer, The purification method provided includes the following: i. The first run, The first run involves passing the mixed therapeutic agent through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and then passing the first filtered mixed therapeutic agent through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic formulations are prepared in the second run.

[0010] A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and then passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation undergoes a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; iv. To enable the distribution of the mixed formulation in the CEX column, use a solvent of a specific ionic strength; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. A method is provided herein. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. Prepare a mixed therapeutic composition containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The first run involves passing the mixed therapeutic composition through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and then passing the first filtered mixed therapeutic composition through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared; iv. To enable the distribution of the mixed therapeutic composition in the CEX column, use a solvent of a specific ionic strength; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. A method is provided. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. Prepare a mixed therapeutic formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The first run involves passing the mixed therapeutic agent through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and then passing the first filtered mixed therapeutic agent through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic active formulations are prepared in the second run; iv. To enable the distribution of the mixed therapeutic active formulation in the CEX column, use a solvent of a specific ionic strength; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. A method is provided.

[0011] A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed formulation is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. A method is provided herein. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. Prepare a mixed therapeutic composition containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed therapeutic composition is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed therapeutic composition in the CEX column; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. A method is provided. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. Prepare a mixed therapeutic formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody, which is an anti-VEGF-A antibody not conjugated to the polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run, in which the mixed therapeutic active formulation is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed therapeutic agent in the CEX column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. A method is provided.

[0012] A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an auto-injector, and then a pre-filter is... Next, two tandem-connected columns follow downstream; the first column is a cation exchange (CEX) column, and the second column, downstream of the CEX column, is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed formulation is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. A method is provided herein. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed therapeutic composition is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed therapeutic composition in the CEX column; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. A method is provided. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into an HPLC system, wherein the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run, in which the mixed therapeutic active formulation is injected and passed through the system of ii; iv. A second run, wherein a concentrated salt is injected to elute the bound fraction from the CEX column, and the fraction is separated by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed therapeutic agent in the CEX column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. A method is provided.

[0013] Furthermore, a method for analyzing a mixed formulation sample, the method comprising the following: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. A method is provided. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. A method is provided herein. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. A method is provided.

[0014] An apparatus including a tandem HPLC system for use in analyzing a mixed formulation sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. Apparatus is provided herein. It can also be used when analyzing samples of mixed therapeutic compositions containing a combination of two protein moieties. Apparatus including a tandem HPLC system for use, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. The device is provided. Furthermore, the apparatus includes a tandem HPLC system for use in analyzing a mixed therapeutic active formulation sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. The device is provided.

[0015] A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; An additional pump is used to pulse-elute the CEX-bound fraction. The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. A method is provided herein. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; An additional pump is used to pulse-elute the CEX-bound fraction. The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. A method is provided. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; An additional pump is used to pulse-elute the CEX-bound fraction. The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. A method is provided herein.

[0016] Furthermore, the apparatus includes a tandem HPLC system for use in purifying a mixed formulation sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. The device is provided. An apparatus including a tandem HPLC system for use in purifying a sample of a mixed therapeutic composition containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. Apparatus is provided herein. Furthermore, the apparatus includes a tandem HPLC system for use in purifying a mixed therapeutic active ingredient sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second The protein portion (B) is not conjugated to the polymer. The aforementioned device includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components; The sample is analyzed by a single continuous chromatography run. The device is provided.

[0017] Furthermore, a method for analyzing a mixed formulation sample, the method comprising the following: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. A method is provided herein. Furthermore, a method for analyzing a sample of a mixed therapeutic composition, the method comprising the following: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. A method is provided. Furthermore, a method for analyzing a sample of an effective mixed therapeutic formulation, the method comprising the following: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent from the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. A method is provided.

[0018] Apparatus including a tandem configuration of an HPLC system for use in analyzing a mixed formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. Apparatus is provided herein. Furthermore, the apparatus includes a tandem configuration of an HPLC system for use in analyzing a sample of a mixed therapeutic composition comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. The device is provided. Furthermore, the apparatus includes a tandem configuration of an HPLC system for use in analyzing a mixed therapeutic formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes: a) An HPLC pump connected to an autoinjector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX effluent to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) First SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. The device is provided. [Brief explanation of the drawing]

[0019] [Figure 1] Figure 1 shows a schematic diagram of the mixed sample preparation and runs 1 and 2 of the tandem HPLC system. [Figure 2A] Figures 2A and 2B illustrate schematics of HPLC and column system options. Figure 2A illustrates a configuration including an IEX column and a single SEC column, while Figure 2B illustrates a schematic including an IEX with a switch valve downstream, allowing selection of one of two different SEC columns: one SEC column for run 1 and one SEC column for run 2. [Figure 2B]Figures 2A and 2B illustrate schematics of HPLC and column system options. Figure 2A illustrates a configuration including an IEX column and a single SEC column, while Figure 2B illustrates a schematic including an IEX with a switch valve downstream, allowing selection of one of two different SEC columns: one SEC column for run 1 and one SEC column for run 2. [Figure 3] Figure 3 illustrates the operation of the tandem HPLC column system during Run 1 and Run 2. [Figure 4A] Figures 4A to 4C illustrate the SEC profiles of CEX-SEC tandem composition analysis when purified OG1953 (conjugated) and OG1950 (unconjugated) are loaded separately in known amounts, and here OG1953 and OG1950 are not combined as a pre-formulated mixture. Figure 4A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 4B illustrates the SEC profile data organized into superimposed plots, and Figure 4C illustrates an overview of the sample information. [Figure 4B] Figures 4A to 4C illustrate the SEC profiles of CEX-SEC tandem composition analysis when purified OG1953 (conjugated) and OG1950 (unconjugated) are loaded separately in known amounts, and here OG1953 and OG1950 are not combined as a pre-formulated mixture. Figure 4A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 4B illustrates the SEC profile data organized into superimposed plots, and Figure 4C illustrates an overview of the sample information. [Figure 4C]Figures 4A to 4C illustrate the SEC profiles of CEX-SEC tandem composition analysis when purified OG1953 (conjugated) and OG1950 (unconjugated) are loaded separately in known amounts, and here OG1953 and OG1950 are not combined as a pre-formulated mixture. Figure 4A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 4B illustrates the SEC profile data organized into superimposed plots, and Figure 4C illustrates an overview of the sample information. [Figure 5A] Figures 5A to 5C illustrate the SEC profiles of the CEX-SEC tandem compositional analysis of OG1953 + 7.5% OG1950 (OG1953_R50037), where OG1953 and OG1950 were combined as a pre-formulated mixture. Figure 5A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 5B illustrates the SEC profile data organized into superimposed plots, and Figure 5C illustrates an overview of the sample information. [Figure 5B] Figures 5A to 5C illustrate the SEC profiles of the CEX-SEC tandem compositional analysis of OG1953 + 7.5% OG1950 (OG1953_R50037), where OG1953 and OG1950 were combined as a pre-formulated mixture. Figure 5A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 5B illustrates the SEC profile data organized into superimposed plots, and Figure 5C illustrates an overview of the sample information. [Figure 5C]Figures 5A to 5C illustrate the SEC profiles of the CEX-SEC tandem compositional analysis of OG1953 + 7.5% OG1950 (OG1953_R50037), where OG1953 and OG1950 were combined as a pre-formulated mixture. Figure 5A illustrates the SEC profiles as stacked plots for samples 1 to 6. Figure 5B illustrates the SEC profile data organized into superimposed plots, and Figure 5C illustrates an overview of the sample information. [Figure 6A] Figures 6A to 6C illustrate the SEC profiles showing the identification of the OG1950 peak and the quantitative recovery rate (7.5%) of OG1950 from R50037. Figure 6A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 6B illustrates the SEC profile data organized into an overlay plot, and Figure 6C illustrates an overview of the sample information. [Figure 6B] Figures 6A to 6C illustrate the SEC profiles showing the identification of the OG1950 peak and the quantitative recovery rate (7.5%) of OG1950 from R50037. Figure 6A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 6B illustrates the SEC profile data organized into an overlay plot, and Figure 6C illustrates an overview of the sample information. [Figure 6C] Figures 6A to 6C illustrate the SEC profiles showing the identification of the OG1950 peak and the quantitative recovery rate (7.5%) of OG1950 from R50037. Figure 6A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 6B illustrates the SEC profile data organized into an overlay plot, and Figure 6C illustrates an overview of the sample information. [Figure 7A]Figures 7A and 7B illustrate the evaluation of SEC calibration standards (QC9661, QC9729B, and QC9742) using the 2D method. Figure 7A shows the SEC profiles for samples 1-5 as stacked plots. Figure 7B shows the SEC profile data compiled into an overlaid plot. [Figure 7B] Figures 7A and 7B illustrate the evaluation of SEC calibration standards (QC9661, QC9729B, and QC9742) using the 2D method. Figure 7A shows the SEC profiles for samples 1-5 as stacked plots. Figure 7B shows the SEC profile data compiled into an overlaid plot. [Figure 8A] Figures 8A to 8C illustrate the SEC profiles of the analysis of the effect of running buffers with different NaCl concentrations on the partitioning of OG1953 and OG1950 (R50037 or 7.5% OG1950) in tandem-configured CEX and SEC columns. [Figure 8B] Figures 8A to 8C illustrate the SEC profiles of the analysis of the effect of running buffers with different NaCl concentrations on the partitioning of OG1953 and OG1950 (R50037 or 7.5% OG1950) in tandem-configured CEX and SEC columns. [Figure 8C] Figures 8A to 8C illustrate the SEC profiles of the analysis of the effect of running buffers with different NaCl concentrations on the partitioning of OG1953 and OG1950 (R50037 or 7.5% OG1950) in tandem-configured CEX and SEC columns. [Figure 9A] Figures 9A to 9C illustrate SEC profiles showing the identification of potential histidine formulation peaks in the R50037 sample using a 2D method with 214 nm detection. Figure 9A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 9B illustrates the SEC profile data organized into an overlay plot, and Figure 9C illustrates an overview of the sample information. [Figure 9B]Figures 9A to 9C illustrate SEC profiles showing the identification of potential histidine formulation peaks in the R50037 sample using a 2D method with 214 nm detection. Figure 9A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 9B illustrates the SEC profile data organized into an overlay plot, and Figure 9C illustrates an overview of the sample information. [Figure 9C] Figures 9A to 9C illustrate SEC profiles showing the identification of potential histidine formulation peaks in the R50037 sample using a 2D method with 214 nm detection. Figure 9A illustrates the SEC profiles as stacked plots for samples 1 to 5. Figure 9B illustrates the SEC profile data organized into an overlay plot, and Figure 9C illustrates an overview of the sample information. [Figure 10A] Figures 10A and 10B illustrate the SEC profiles showing the carryover evaluation after the system has been stripped with various buffers and residual binding substances have been evaluated. [Figure 10B] Figures 10A and 10B illustrate the SEC profiles showing the carryover evaluation after the system has been stripped with various buffers and residual binding substances have been evaluated. [Figure 11A] Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11B]Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11C] Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11D-1]Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11D-2] Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11D-3]Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 11E] Figures 11A–11E illustrate an overview of sensitivity limit of detection (LOD), linearity, and recovery data from Run 1 and Run 2 for sets of OG1950 and R50037 samples loaded with various protein amounts. Figures 11A–11B illustrate the SEC profiles for OG1950PUR samples injected with 25–250 ug of protein. Figure 11C illustrates the SEC profile for R50035 (7.5%) samples injected with 100–1340 ug of protein. Figure 11D illustrates sample details from Figures 11A–11B and a graph plotting the OG1950 monomer or aggregate peaks against the loading amount. Figure 11E illustrates sample details from Figure 11C and an additional SEC profile for the samples in Run 2. [Figure 12A] Figure 12A (top left) Mixture #1 was initially cloudy but became clear after the addition of 20 μl of buffer. (top right) A gentle precipitate was observed during microcentrifugation at 5K rpm for 5 minutes. (bottom) All other formulation setups (#2-#5) showed no cloudiness at all. Buffer B143-1 is 50 mM sodium acetate (pH 5), 0.025% PS20. [Figure 12B]Figure 12B illustrates that the 2D analysis shows a free protein elution peak at the predicted retention time of 21.8 minutes, while the conjugate elutes as a broad peak at approximately 15.6 minutes. This was confirmed by (a) SEC calibration standard solution; (b) 15% mixed formulation standard (R50031); and (c) injection OG1950 free protein standard injection. The percentages on the right of each chromatogram represent the distribution of CEX-unbound (conjugate) and CEX-bound fractions (free IgG), which are consistent with the expected percentage of free protein as shown in Figure 12D. [Figure 12C] Figure 12C (top) illustrates an enlarged superposition of all curves from Figure 12B, showing the overlap of eluted IgG peaks, which were consistent with both standard IgG and IgG standards from the SEC calibration standard. (bottom left) Offset superposition of various injections clearly illustrates the successful separation between the unbound CEX conjugate fraction and the CEX-bound IgG fraction. (bottom right) is a close-up view to show the aggregated morphology of the clearly detected IgG, as indicated by the arrows. [Figure 12D] Figure 12D illustrates a comparison of peak areas between the CEX-bound (free protein) and unbound (conjugate) fractions, showing that the detected values ​​in (X) agree with the predicted values ​​in (Y). X represents the detected values ​​from tandem peak analysis, and these values ​​can be calculated using the detected peak areas, or the peak areas can be converted to protein mass using Beer's Law. Converting the detected peak areas to protein mass allows for the calculation of the injection recovery rate, which can be compared with the value of Y. The value of Y is the input amount based on the protein mass during formulation preparation. The recovery rate is determined by comparing columns X and Y. [Figure 12E]Figure 12E illustrates that method analysis shows the elution peak of the free protein at approximately 25.3 minutes, the predicted retention time, while the conjugate elutes as a broad peak at 15–15.2 minutes. This was confirmed by (a) injection of the OG468 Fab standard; and (b) SEC calibration standard. [Figure 12F] Figure 12F (top) illustrates the superposition of all curves from Figure 12E, showing that the eluted OG468 Fab peak coincided with the standard OG468 standard, eluting later than the IgG standard but later than the 44kDa standard of the SEC calibration solution. (bottom) Offset superposition of various injections to clearly illustrate the separation between the unbound CEX conjugate fraction and the CEX-bound Fab fraction. [Figure 12G] Figure 12G illustrates a comparison of peak areas between the CEX-bound (free protein) and unbound fractions (conjugates), showing agreement with the predicted ratio. See the comparison between columns (X) and (Y). X and Y can be calculated and compared as described in the explanation of Figure 12D above. For sample #5, OG468 Fab was found to bind strongly to the CEX column and could not be eluted with 1M NaCl, and was only recovered by 6M GuHCl elution; therefore, only 6M GuHCl elution was performed. This was later repeated for samples #3 and #4 using 2M NaCl as the elution buffer, but only about 60% and 75% were eluted for samples #3 and #4, respectively. However, the cumulative total eluted CEX-bound fraction was in agreement with the expected number, as shown in column (Y). [Figure 13A] Figure 13A illustrates an HPLC system configuration with a multi-column combination including a three-way valve, and additional switch valves may be present before or after the IEX column, if desired. [Figure 13B] Figure 13B illustrates further details regarding the configuration of the three-way valve, in which the effluent from the IEX column can be directed to 1) a bypass loop; 2) a Shodex-806M SEC column; or 3) a TSK G3000 SEC column. [Figure 13C]Figure 13C illustrates further details regarding the flow path options for each of Figures 13B1-1-3. [Figure 14A] Figure 14A illustrates a summary of sample information for the samples used to test the HPLC system configuration with the multi-column combination described in Figure 14. The samples included SEC calibration standards, OG1950 antibody, and two representative OG1953 pharmaceutical formulation prototypes (one containing 20% ​​OG1950 + 80% OG1953, and the other containing 15% OG1950 + 75% OG1953). [Figure 14B] Figure 14B illustrates the column flow path matrix for the sample described in Figure 14A, where each unique column combination is specified by a different SOP391 version (v1 to v6). [Figure 15A] Figures 15A–15C illustrate the analysis of the R50032 formulation (20% OG1950 + 80% OG1953) via SOP391_v1, featuring a tandem CEX+SEC HPLC method without a three-way switch valve. Figure 15A shows stack plots at 280 nm for the blank, OG1950 standard, and CEX-bound and unbound fractions. Figure 15B shows an overlay plot of the peaks shown in Figure 15A. Figure 15C shows the mass recovery from the CEX-unbound fraction, the mass recovery from individual peaks in the CEX-bound fraction, and the total recovery. [Figure 15B] Figures 15A–15C illustrate the analysis of the R50032 formulation (20% OG1950 + 80% OG1953) via SOP391_v1, featuring a tandem CEX+SEC HPLC method without a three-way switch valve. Figure 15A shows stack plots at 280 nm for the blank, OG1950 standard, and CEX-bound and unbound fractions. Figure 15B shows an overlay plot of the peaks shown in Figure 15A. Figure 15C shows the mass recovery from the CEX-unbound fraction, the mass recovery from individual peaks in the CEX-bound fraction, and the total recovery. [Figure 15C]Figures 15A–15C illustrate the analysis of the R50032 formulation (20% OG1950 + 80% OG1953) via SOP391_v1, featuring a tandem CEX+SEC HPLC method without a three-way switch valve. Figure 15A shows stack plots at 280 nm for the blank, OG1950 standard, and CEX-bound and unbound fractions. Figure 15B shows an overlay plot of the peaks shown in Figure 15A. Figure 15C shows the mass recovery from the CEX-unbound fraction, the mass recovery from individual peaks in the CEX-bound fraction, and the total recovery. [Figure 16A] Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 16B]Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 16C] Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 16D]Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 16E] Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 16F]Figures 16A to 16F illustrate the analysis of the R50032 and R50037 (7.5% OG1950 + 92.5% OG1953) formulations using the SOP391_v2 operating mode with a three-way switch valve. Figure 16A illustrates the flow path through the three-way valve in SOP391_v2, through which the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. Figure 16B illustrates the individual peaks observed in the E and L fractions. Figures 16C and 16D illustrate superimposed graphs of the individual peaks shown in Figure 16B. The arrows in Figures 16C to 16D indicate the peak at 16.966 min, which accounts for 5.2% of the total CEX-bound fraction. Figure 16E illustrates a summary of the infusion queue for the E and L fractions of formulations R50032 and R50037, including execution time (minutes), infusion volume (μl), and acquisition method. Figure 16F illustrates a summary of peak data for the E and L fractions of formulations R50032 and R50037, including retention time (minutes), area, area %, and height. [Figure 17A] Figures 17A–17D illustrate the analysis of the R50032 formulation using the SOP391_v3 operating mode with a three-way switch valve, where the CEX-unbound fraction (L) was induced via a bypass instead of flowing through the SEC column. Figure 17A illustrates the flow path through the three-way valve in SOP391_v3, where the CEX-unbound (L) fraction is induced via the bypass, and the CEX-bound (E) fraction is analyzed using a TSK G3000 SEC column. Figure 17B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v2 and SOP391_v3 operating modes. Figure 17C illustrates an overlay graph of the individual peaks shown in Figure 17B. Figure 17D illustrates a summary of the injection queue for the E and L fractions of the R50032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 17B]Figures 17A–17D illustrate the analysis of the R50032 formulation using the SOP391_v3 operating mode with a three-way switch valve, where the CEX-unbound fraction (L) was induced via a bypass instead of flowing through the SEC column. Figure 17A illustrates the flow path through the three-way valve in SOP391_v3, where the CEX-unbound (L) fraction is induced via the bypass, and the CEX-bound (E) fraction is analyzed using a TSK G3000 SEC column. Figure 17B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v2 and SOP391_v3 operating modes. Figure 17C illustrates an overlay graph of the individual peaks shown in Figure 17B. Figure 17D illustrates a summary of the injection queue for the E and L fractions of the R50032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 17C] Figures 17A–17D illustrate the analysis of the R50032 formulation using the SOP391_v3 operating mode with a three-way switch valve, where the CEX-unbound fraction (L) was induced via a bypass instead of flowing through the SEC column. Figure 17A illustrates the flow path through the three-way valve in SOP391_v3, where the CEX-unbound (L) fraction is induced via the bypass, and the CEX-bound (E) fraction is analyzed using a TSK G3000 SEC column. Figure 17B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v2 and SOP391_v3 operating modes. Figure 17C illustrates an overlay graph of the individual peaks shown in Figure 17B. Figure 17D illustrates a summary of the injection queue for the E and L fractions of the R50032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 17D]Figures 17A–17D illustrate the analysis of the R50032 formulation using the SOP391_v3 operating mode with a three-way switch valve, where the CEX-unbound fraction (L) was induced via a bypass instead of flowing through the SEC column. Figure 17A illustrates the flow path through the three-way valve in SOP391_v3, where the CEX-unbound (L) fraction is induced via the bypass, and the CEX-bound (E) fraction is analyzed using a TSK G3000 SEC column. Figure 17B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v2 and SOP391_v3 operating modes. Figure 17C illustrates an overlay graph of the individual peaks shown in Figure 17B. Figure 17D illustrates a summary of the injection queue for the E and L fractions of the R50032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 18A] Figures 18A–18D illustrate the analysis of the R50032 formulation using the SOP391_v4 operating mode with a three-way switch valve. Figure 18A illustrates the flow path through the three-way valve in SOP391_v4, where the CEX-unbound fraction (L) passes through a Shodex 806M_HQ column and the CEX-bound (E) fraction is analyzed using a TSK G3000 column. Figure 18B illustrates the individual peaks observed in the E and L fractions. Figure 18C illustrates an overlay graph of the individual peaks shown in Figure 18B. Figure 18D illustrates a summary of the injection queue for the E and L fractions of the R0032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 18B]Figures 18A–18D illustrate the analysis of the R50032 formulation using the SOP391_v4 operating mode with a three-way switch valve. Figure 18A illustrates the flow path through the three-way valve in SOP391_v4, where the CEX-unbound fraction (L) passes through a Shodex 806M_HQ column and the CEX-bound (E) fraction is analyzed using a TSK G3000 column. Figure 18B illustrates the individual peaks observed in the E and L fractions. Figure 18C illustrates an overlay graph of the individual peaks shown in Figure 18B. Figure 18D illustrates a summary of the injection queue for the E and L fractions of the R0032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 18C] Figures 18A–18D illustrate the analysis of the R50032 formulation using the SOP391_v4 operating mode with a three-way switch valve. Figure 18A illustrates the flow path through the three-way valve in SOP391_v4, where the CEX-unbound fraction (L) passes through a Shodex 806M_HQ column and the CEX-bound (E) fraction is analyzed using a TSK G3000 column. Figure 18B illustrates the individual peaks observed in the E and L fractions. Figure 18C illustrates an overlay graph of the individual peaks shown in Figure 18B. Figure 18D illustrates a summary of the injection queue for the E and L fractions of the R0032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 18D] Figures 18A–18D illustrate the analysis of the R50032 formulation using the SOP391_v4 operating mode with a three-way switch valve. Figure 18A illustrates the flow path through the three-way valve in SOP391_v4, where the CEX-unbound fraction (L) passes through a Shodex 806M_HQ column and the CEX-bound (E) fraction is analyzed using a TSK G3000 column. Figure 18B illustrates the individual peaks observed in the E and L fractions. Figure 18C illustrates an overlay graph of the individual peaks shown in Figure 18B. Figure 18D illustrates a summary of the injection queue for the E and L fractions of the R0032 formulation, including run time (minutes), injection volume (μl), and acquisition method. [Figure 19A] Figures 19A and 19B illustrate the evaluation of the overload problem observed in R50032 using the SOP391_v4 operating mode. Figure 19A illustrates the individual peaks observed in X (tandem HPLC using a CEX column and a Shodex 806M_HQ SEC column with an overloaded SEC column) and Y (R50032 formulation analyzed on a Shodex 806M_HQ SEC column without CEX column operation). Figure 19B illustrates an overlay graph of the individual peaks shown in Figure 19A. [Figure 19B] Figures 19A and 19B illustrate the evaluation of the overload problem observed in R50032 using the SOP391_v4 operating mode. Figure 19A illustrates the individual peaks observed in X (tandem HPLC using a CEX column and a Shodex 806M_HQ SEC column with an overloaded SEC column) and Y (R50032 formulation analyzed on a Shodex 806M_HQ SEC column without CEX column operation). Figure 19B illustrates an overlay graph of the individual peaks shown in Figure 19A. [Figure 20A]Figures 20A–20C illustrate the analysis of the R50032 formulation using the SOP391_v5 and SOP391_v6 operating modes with a three-way switch valve. Figure 20A illustrates the flow path through the three-way valve in SOP391_v5, where the CEX-unbound fraction (L) passes through a bypass loop and the CEX-bound (E) fraction is analyzed by a Shodex 806M_HQ column. Figure 20A also illustrates the flow path through the three-way valve in SOP391_v6, where both the CEX-unbound (L) and CEX-bound (E) fractions are analyzed using a Shodex 806M_HQ column. Figure 20B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v5 and SOP391_v6 operating modes. Figure 20C shows an overlay graph of the individual peaks shown in Figure 20B. The red arrows in Figures 20B and 20C indicate the fraction observed at the 16-minute peak of SOP391_v1, where elution occurred at approximately 20 minutes. [Figure 20B] Figures 20A–20C illustrate the analysis of the R50032 formulation using the SOP391_v5 and SOP391_v6 operating modes with a three-way switch valve. Figure 20A illustrates the flow path through the three-way valve in SOP391_v5, where the CEX-unbound fraction (L) passes through a bypass loop and the CEX-bound (E) fraction is analyzed by a Shodex 806M_HQ column. Figure 20A also illustrates the flow path through the three-way valve in SOP391_v6, where both the CEX-unbound (L) and CEX-bound (E) fractions are analyzed using a Shodex 806M_HQ column. Figure 20B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v5 and SOP391_v6 operating modes. Figure 20C shows an overlay graph of the individual peaks shown in Figure 20B. The red arrows in Figures 20B and 20C indicate the fraction observed at the 16-minute peak of SOP391_v1, where elution occurred at approximately 20 minutes. [Figure 20C] Figures 20A–20C illustrate the analysis of the R50032 formulation using the SOP391_v5 and SOP391_v6 operating modes with a three-way switch valve. Figure 20A illustrates the flow path through the three-way valve in SOP391_v5, where the CEX-unbound fraction (L) passes through a bypass loop and the CEX-bound (E) fraction is analyzed by a Shodex 806M_HQ column. Figure 20A also illustrates the flow path through the three-way valve in SOP391_v6, where both the CEX-unbound (L) and CEX-bound (E) fractions are analyzed using a Shodex 806M_HQ column. Figure 20B illustrates the individual peaks observed in the E and L fractions, comparing the peaks of the E and L fractions between the SOP391_v5 and SOP391_v6 operating modes. Figure 20C shows an overlay graph of the individual peaks shown in Figure 20B. The red arrows in Figures 20B and 20C indicate the fraction observed at the 16-minute peak of SOP391_v1, where elution occurred at approximately 20 minutes. [Figure 21A-1] Figure 21A illustrates a comparison of individual peak and total recovery data for the R50032 formulation across SOP391_v1 to v6 operating modes. [Figure 21A-2] Figure 21A illustrates a comparison of individual peak and total recovery data for the R50032 formulation across SOP391_v1 to v6 operating modes. [Figure 21B] Figure 21B illustrates a comparison between the 16-minute peak fraction observed in SOP391_v1 and SOP391_v2~v4, where the X: 16-minute peak was further separated into two populations by the TSK G3000 SEC column, and SOP391_v5~v6, where the Y: 16-minute peak co-eluted with the majority of the OG1953 conjugate. [Figure 22A]Figures 22A and 22B illustrate the stability tests of OG1950PUR and R50032 samples using the tandem HPLC method according to SOP391_v3. Figure 22A shows the individual peaks observed in the CEX-unbound (L) and CEX-bound (E) fractions of the OG1950PUR sample stored at 37°C for 7 months, the R50032 formulation stored at 5°C for 3 months, and the R50032 formulation stored at 25°C for 3 months. Figure 22B shows an overlay graph of the individual peaks in Figure 22A, where X represents the peak of the R50032 formulation stored at 5°C and 5°C for 3 months, and Y represents the OG1950PUR sample stored at 37°C for 7 months. [Figure 22B] Figures 22A and 22B illustrate the stability tests of OG1950PUR and R50032 samples using the tandem HPLC method according to SOP391_v3. Figure 22A shows the individual peaks observed in the CEX-unbound (L) and CEX-bound (E) fractions of the OG1950PUR sample stored at 37°C for 7 months, the R50032 formulation stored at 5°C for 3 months, and the R50032 formulation stored at 25°C for 3 months. Figure 22B shows an overlay graph of the individual peaks in Figure 22A, where X represents the peak of the R50032 formulation stored at 5°C and 5°C for 3 months, and Y represents the OG1950PUR sample stored at 37°C for 7 months. [Figure 23] Figure 23 illustrates the principle and operation of the tandem CEX-SEC method, where the KSI-301DP formulation may contain OG1953, OG1953', OG1950, aggregated OG1950, and insoluble components in the buffer. The insoluble components are removed by a pre-filtering step. OG1950 and its aggregates bind to the CEX column, but OG1953 does not bind to the column and can be analyzed by SEC. A pulse of 1M NaCl can elute OG1950 and aggregates from the CEX column, and OG1950, aggregated OG1950, and any OG1953 can be distinguished by SEC. [Figure 24]Figure 24 illustrates a program plan to evaluate the stability of OG1953 biconjugates containing 7.5%, 10%, 15%, and 20% free OG1950 protein at different temperatures over time. This test evaluates the performance of the tandem analytical HPLC method in terms of recovery, robustness, specificity, reproducibility, and accuracy. [Figure 25] Figure 25 illustrates the visual evaluation of samples at 1 month, 2 months, 3 months, and 6 months at 5°C, 25°C, and 37°C. Arrows indicate samples with a cloudy appearance. [Figure 26] Figure 26 illustrates the evaluation of protein concentrations in samples stored at 5°C, 25°C, and 37°C for 1 month, 2 months, and 6 months, based on analysis of changes in protein concentration over time. [Figure 27A] Figures 27A to 27C illustrate an overview of all sample recovery and elution profiles. Figure 27A shows the total mass recovery rate (upper and lower panels) against time and temperature for CEX-bound and CEX-unbound samples, with near 100% mass recovery achieved for all samples. Figure 27B shows the chromatogram of the CEX-unbound sample, indicating the OG1953 conjugate peak (LP1), with the unstable population represented as LP2. Figure 27C shows the chromatogram of the CEX-bound fraction, indicating the 16-minute peaks separated into A and B, and the OG1950 free protein peaks P1, P1, and M. [Figure 27B] Figures 27A to 27C illustrate an overview of all sample recovery and elution profiles. Figure 27A shows the total mass recovery rate (upper and lower panels) against time and temperature for CEX-bound and CEX-unbound samples, with near 100% mass recovery achieved for all samples. Figure 27B shows the chromatogram of the CEX-unbound sample, indicating the OG1953 conjugate peak (LP1), with the unstable population represented as LP2. Figure 27C shows the chromatogram of the CEX-bound fraction, indicating the 16-minute peaks separated into A and B, and the OG1950 free protein peaks P1, P1, and M. [Figure 27C] Figures 27A to 27C illustrate an overview of all sample recovery and elution profiles. Figure 27A shows the total mass recovery rate (upper and lower panels) against time and temperature for CEX-bound and CEX-unbound samples, with near 100% mass recovery achieved for all samples. Figure 27B shows the chromatogram of the CEX-unbound sample, indicating the OG1953 conjugate peak (LP1), with the unstable population represented as LP2. Figure 27C shows the chromatogram of the CEX-bound fraction, indicating the 16-minute peaks separated into A and B, and the OG1950 free protein peaks P1, P1, and M. [Figure 28A-1] Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 28A-2]Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 28A-3] Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 28B]Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 28C] Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 28D]Figures 28A–28D illustrate a comparison of LP2, 16 min peaks, and monomeric free protein for sample #4 and a representative clinical formulation. Figure 28A shows an overview of the results obtained for sample #4, OG1953 KSI-301DP 103A, and OG1953 KSI-301DS 101 under different storage conditions, including the mass recovery and 280 nm signal percentage of each identified peak, where X highlights the 16 min A and B peaks. Figure 28B shows chromatograms of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6-month (6M) 25°C sample. Figures 28C–28D illustrate chromatograms of the CEX-bound fraction, with Z indicating the 16 min peak and monomeric protein (M) peak. [Figure 29A] Figures 29A to 29D illustrate the kinetic analysis of each peak fraction of sample #4 at different times and temperatures. Figure 29A shows an overview of the analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. Figure 29B shows an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 29C shows the kinetic curves for all peaks in each sample, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 29D shows the accompanying chromatogram for the CEX-bound run peak. [Figure 29B]Figures 29A to 29D illustrate the kinetic analysis of each peak fraction of sample #4 at different times and temperatures. Figure 29A shows an overview of the analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. Figure 29B shows an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 29C shows the kinetic curves for all peaks in each sample, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 29D shows the accompanying chromatogram for the CEX-bound run peak. [Figure 29C-1] Figures 29A to 29D illustrate the kinetic analysis of each peak fraction of sample #4 at different times and temperatures. Figure 29A shows an overview of the analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. Figure 29B shows an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 29C shows the kinetic curves for all peaks in each sample, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 29D shows the accompanying chromatogram for the CEX-bound run peak. [Figure 29C-2] Figures 29A to 29D illustrate the kinetic analysis of each peak fraction of sample #4 at different times and temperatures. Figure 29A shows an overview of the analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. Figure 29B shows an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 29C shows the kinetic curves for all peaks in each sample, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 29D shows the accompanying chromatogram for the CEX-bound run peak. [Figure 29D]Figures 29A to 29D illustrate the kinetic analysis of each peak fraction of sample #4 at different times and temperatures. Figure 29A shows an overview of the analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. Figure 29B shows an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 29C shows the kinetic curves for all peaks in each sample, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 29D shows the accompanying chromatogram for the CEX-bound run peak. [Figure 30A] Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 30B-1]Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 30B-2] Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 30C] Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 30D-1] Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 30D-2] Figures 30A to 30D illustrate the kinetic analysis of the peak fractions of samples #3 (15%) and #9 (20% iodoacetamide (IAM)) at different times and temperatures. Figure 30A shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #3, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30B shows the kinetic curves for all peaks in each sample of sample #3, including the CEX-unbound run peaks (LP1 and LP2) and the CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). Figure 30C shows an overview of the peak analysis of all peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, as well as an overview of the decomposition and / or aggregation rates for all peaks at 25°C. Figure 30D illustrates the kinetic curves for all peaks in each sample of sample #9, including CEX-unbound run peaks (LP1 and LP2) and CEX-bound run peaks (16-minute peaks, P2 (dimer), P1, M (monomer)). [Figure 31A-1] Figures 31A and 31B illustrate the predicted 2-year stability of samples #3, 4, and 9 at different temperatures. Figure 31A shows an overview of the peak percentages of all detected peaks in the CEX-unbound and CEX-bound fractions for samples #3, 4, and 9. Figure 31B shows the predicted percentage of total impurities (e.g., aggregates and / or decomposition products) over 2 years for samples #3, 4, and 9. [Figure 31A-2] Figures 31A and 31B illustrate the predicted 2-year stability of samples #3, 4, and 9 at different temperatures. Figure 31A shows an overview of the peak percentages of all detected peaks in the CEX-unbound and CEX-bound fractions for samples #3, 4, and 9. Figure 31B shows the predicted percentage of total impurities (e.g., aggregates and / or decomposition products) over 2 years for samples #3, 4, and 9. [Figure 31B-1]Figures 31A and 31B illustrate the predicted 2-year stability of samples #3, 4, and 9 at different temperatures. Figure 31A shows an overview of the peak percentages of all detected peaks in the CEX-unbound and CEX-bound fractions for samples #3, 4, and 9. Figure 31B shows the predicted percentage of total impurities (e.g., aggregates and / or decomposition products) over 2 years for samples #3, 4, and 9. [Figure 31B-2] Figures 31A and 31B illustrate the predicted 2-year stability of samples #3, 4, and 9 at different temperatures. Figure 31A shows an overview of the peak percentages of all detected peaks in the CEX-unbound and CEX-bound fractions for samples #3, 4, and 9. Figure 31B shows the predicted percentage of total impurities (e.g., aggregates and / or decomposition products) over 2 years for samples #3, 4, and 9. [Figure 32-1] Figure 32 illustrates an overview of the mass recovery data for all injections of samples #3, 4, and 9 at different times and temperatures. [Figure 32-2] Figure 32 illustrates an overview of the mass recovery data for all injections of samples #3, 4, and 9 at different times and temperatures. [Figure 32-3] Figure 32 illustrates an overview of the mass recovery data for all injections of samples #3, 4, and 9 at different times and temperatures. [Figure 33A] Figures 33A and 33B illustrate graphs of pulsed elution profiles of 20% antibody + 80% bioconjugate in 100% B buffer, using pulsed elution profiles #3 to #7. Figure 33A shows individual peaks, while Figure 33B shows an overlay plot. [Figure 33B] Figures 33A and 33B illustrate graphs of pulsed elution profiles of 20% antibody + 80% bioconjugate in 100% B buffer, using pulsed elution profiles #3 to #7. Figure 33A shows individual peaks, while Figure 33B shows an overlay plot. [Figure 34A]Figures 34A and 34B illustrate an overview of the data analysis (Figure 34A) and a comparison of chromatograms of various pulsed elution profiles (Figure 34B). [Figure 34B] Figures 34A and 34B illustrate an overview of the data analysis (Figure 34A) and a comparison of chromatograms of various pulsed elution profiles (Figure 34B). [Figure 35] Figure 35 illustrates OG1786. [Figure 36] Figure 36 illustrates OG1802. [Figure 37A] Figures 37A to 37C illustrate the updates to the forecasts in Figures 37A to 37C, with additional sampling conducted at 12 months and 15 months for 5°C and 25°C, respectively. [Figure 37B] Figures 37A to 37C illustrate the updates to the forecasts in Figures 37A to 37C, with additional sampling conducted at 12 months and 15 months for 5°C and 25°C, respectively. [Figure 37C] Figures 37A to 37C illustrate the updates to the forecasts in Figures 37A to 37C, with additional sampling conducted at 12 months and 15 months for 5°C and 25°C, respectively. [Figure 38AB] Figures 38A–38D illustrate several embodiments of a continuous 80-minute tandem separation method using photodiode array (PDA) detection set to 200–350 nm. Figure 38A shows a 2D contour plot of elution time versus wavelength; Figure 38B 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 the results shown in Figure 38C as a non-reducing gel and in Figure 38D as a reducing gel. [Figure 38C]Figures 38A–38D illustrate several embodiments of a continuous 80-minute tandem separation method using photodiode array (PDA) detection set to 200–350 nm. Figure 38A shows a 2D contour plot of elution time versus wavelength; Figure 38B 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 the results shown in Figure 38C as a non-reducing gel and in Figure 38D as a reducing gel. [Figure 38D] Figures 38A–38D illustrate several embodiments of a continuous 80-minute tandem separation method using photodiode array (PDA) detection set to 200–350 nm. Figure 38A shows a 2D contour plot of elution time versus wavelength; Figure 38B 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 the results shown in Figure 38C as a non-reducing gel and in Figure 38D as a reducing gel. [Figure 39A] Figures 39A to 39C illustrate KSI-301 stability data for up to 6 months under different temperature conditions: -20±5°C (Figure 39A), 5±3°C (Figure 39B), and 25±2°C / 60±5%RH (relative humidity) (Figure 39C). [Figure 39B] Figures 39A to 39C illustrate KSI-301 stability data for up to 6 months under different temperature conditions: -20±5°C (Figure 39A), 5±3°C (Figure 39B), and 25±2°C / 60±5%RH (relative humidity) (Figure 39C). [Figure 39C] Figures 39A to 39C illustrate KSI-301 stability data for up to 6 months under different temperature conditions: -20±5°C (Figure 39A), 5±3°C (Figure 39B), and 25±2°C / 60±5%RH (relative humidity) (Figure 39C). [Figure 40] Figure 40 illustrates the lot release data for KSI-501DS batches 1-3. [Modes for carrying out the invention]

[0020] Some embodiments provided herein are methods and apparatus for tandem HPLC. In some embodiments, the methods and apparatus for tandem HPLC enable the analysis of mixed protein formulations.

[0021] Some embodiments provided herein are methods and apparatus for tandem HPLC combining ion exchange chromatography (IEX), such as cation exchange chromatography (CEX), with size exclusion chromatography (SEC). These methods and apparatus for tandem HPLC enable the analysis of formulations containing proteins conjugated to polymers and proteins not conjugated to polymers.

[0022] Methods and apparatus for analyzing mixed formulations containing fast-acting components including unconjugated proteins and long-acting components including polymer-conjugated proteins are provided herein. In some embodiments, the methods and apparatus provided herein are part of any preferred process for producing and purifying mixed compositions of some conjugated proteins and unconjugated proteins.

[0023] In general, when ABC(trademark) conjugates were mixed with free protein formulations, several surprising findings were discovered. Firstly, there was the presence of unpaired, manipulated cysteine ​​residues in the protein moiety, which post the possibility of disulfide shuffling-mediated protein aggregation, particularly in the free and unconjugated protein fractions, or at high protein concentrations with bioconjugates. The presence of unpaired engineered cysteine ​​residues present in the protein moiety that posts potential disulfide shuffling mediated protein aggregation, especially between the free and unconjugated protein fraction or with the bioconjugate at high protein concentration. Secondly, there has been a surprising discovery that such mixtures result in a turbid solution under certain conditions, such as (1) when the pH of the solution is at or near the isoelectric point (pI) of the free unconjugated protein, (2) when the concentration of the conjugate and / or free protein is too high, or (3) when certain excipients such as histidine are present. Turbid solutions are generally undesirable for injectable pharmaceuticals, especially intravitreal injectable pharmaceuticals. In some embodiments, the methods and apparatus described herein contribute to the development of a stable and clear solution that has stability for up to 6 months and is expected to have long-term stability for at least 12 months at 2–8°C.

[0024] The OG1953 bioconjugate can be polydispersible due to the covalently conjugated polymer moiety and may contain a small number of molecules with overlapping molecular weights with the free, unconjugated protein fraction, particularly aggregates of free proteins. Furthermore, the protein moiety may also exhibit unique charge variations. Variations in both size and charge can be monitored using CEX-HPLC and SEC-HPLC methods, respectively, to observe the molecules based on charge and size. This presents a complex analytical challenge for any existing analytical method. The overlapping of conjugates and impurities such as protein degradation products and aggregates makes it technically impossible to achieve sufficient resolution for such analysis using any single method, and this situation can be further exacerbated by time-dependent and temperature stress.

[0025] In some embodiments, the methods and apparatus described herein may offer one or more of the following advantages: (1) utilizing existing CEX-HPLC and SEC-HPLC analytical methods for either conjugate or free unconjugate proteins; (2) usable for both batch release and stability indicators; (3) being able to be performed under non-destructive and native buffer conditions to prevent denaturation or artifacts during the process; (4) requiring no further sample handling or purification before sample analysis other than simple sample dilution; (5) being able to meet GMP validation requirements such as accuracy, robustness, reproducibility, and specificity; (6) being able to be implemented and set up in an analytical laboratory equipped with common equipment and trained technical experts in the art; and (7) being able to perform various (8) The method can provide good resolution and quantitative information for decomposition products and / or aggregates (e.g., impurities); (9) The method can provide kinetic recovery (100% recovery) for the injected sample, which also means that the method should have minimal carryover problems; (10) The method can also have a good operating dynamic range, such as tolerance to the pH of the running buffer, salt concentration, sample load, flow rate, and operating temperature; (11) The method can be further seamlessly integrated with the use and methods of other analytical instruments such as MALS detectors, icIEF, mass spectrometry, and fluorescence detectors; (12) Buffer running conditions that minimize disulfide shuffling, thus the running buffer can be in an acidic range such as pH 4-6.5.

[0026] In some embodiments, the methods and apparatus described herein can provide one or more of the following advantages: (1) isocratic chromatographic running conditions to take advantage of the existing gradient elution characteristic of the CEX-HPLC method, which allows the bulk of the OG1953 conjugate to be eluted with minimal to no premature leaching of bound OG1950 free protein and impurities such as degradants, aggregates, or fragments. Although not limited by theory, the OG1953 conjugate is large polypropylene (2) Rapid and synchronous mass transfer of CEX-bound fractions for size-based SEC separation. Since the general principle of SEC chromatography is based on synchronous sample application with a sample volume of 5% or less of the total SEC column volume, a sufficiently high salt elution pulse can be used to elute all CEX-bound fractions with the smallest possible volume for efficient SEC separation. In the case of asynchronous elution, such as when the salt concentration is insufficient or the elution rate is too slow, the resolution and separation efficiency of SEC may be impaired. This phenomenon can be seen as prolonged tailing of CEX or SEC elution after the bulk conjugate elution peak; (2) Rapid and synchronous mass transfer of CEX-bound fractions for size-based SEC separation. Since the general principle of SEC chromatography is based on synchronous sample application with a sample volume of 5% or less of the total SEC column volume, a sufficiently high salt elution pulse can be used to elute all CEX-bound fractions with the smallest possible volume for efficient SEC separation. In the case of asynchronous elution, such as when the salt concentration is insufficient or the elution rate is too slow, the resolution and separation efficiency of SEC may be impaired.

[0027] In some embodiments, a favorable feature of this tandem chromatography method is its immediate fingerprinting capability. In some embodiments, the first dimension of separation is electrochemical This includes group-specific chromatography such as ion exchange chromatography (IEX) for specific separation (e.g., CEX or AEX), and affinity chromatography based on specific groups (e.g., M2 resin for protein A, protein G, protein L, and flag-tagged proteins, metal chelate resins such as nickel-NTA for His-tagged proteins, dye affinity resins, lectin affinity resins for specific carbohydrate separation, hydrophobic interaction resins, ligand-specific antibody affinity capture resins, etc.). In some embodiments, the second dimension separation can be performed size by size with SEC, which provides immediate identity fingerprint profiles for both the unbound and subsequently bound fractions of the group-specific column in a single operation. In some embodiments, this is conceptually different from the concept of conventional two-dimensional chromatography, which is simply aimed at separating complex mixtures of analytes to obtain a unique fingerprint. In some embodiments, this tandem HPLC method aims to provide a convenient, simple, and efficient immediate fingerprint of the first dimension separation and can be widely used in other application areas when the SEC effluent is combined with the use and techniques of other detection instruments.

[0028] In some embodiments, the method can also be operated in interrupted and non-interrupted (continuous) modes. In some embodiments, the interrupted mode may have separate chromatographic runs for the sample injection run and the pulsed elution of the first-dimensional bound fraction, thus producing two separate chromatograms for a single sample analysis. In some embodiments, the main disadvantages of such operation may include one or more of the following: (1) an undesirable baseline ramp signal at the start of each run, (2) unnecessary justification to regulatory authorities for integrating peaks obtained from completely separate chromatographic runs, and (3) alignment issues due to potential chromatographic signal mismatches. In some embodiments, there are also advantages when only the first-dimensional column-bound fraction is required, and a significantly shortened method can be designed by using a multi-column valve with a bypass loop, as shown in Figure 13A. In this operating mode, the unbound fraction can be discarded using the bypass loop and does not pass through the SEC column. In some embodiments, for the non-interrupted mode, more sophisticated instruments may be required to be equipped with two pumps and be able to operate with a small residual volume so that salt pulses can be delivered for synchronous elution of the first-dimensional bound fraction. Failure to meet this requirement may result in undesirable baseline shifts. In some embodiments, the advantage of operating in non-interrupted mode is that all elution peaks of the unbound and bound fractions of the first-dimensional column can be displayed in a single chromatogram.

[0029] In some embodiments, understanding the molecules in the mixed formulation is useful for the successful use of this tandem HPLC method. In some embodiments, the first-dimensional column makes it possible to remove most of the interference or matrix effects. In some embodiments, the unbound fraction contains undesirable contaminants such as sample formulation buffer or free non-conjugated polymers (OG1802). In some embodiments, the selection of the mobile phase salt concentration and pH confers separation specificity. In some embodiments, the mobile phase salt concentration and pH can be manipulated to control how thoroughly interference is removed. In some embodiments, excessively strong salt and pH conditions may lead to premature elution of the bound fraction and impair the performance of the method.

[0030] Some embodiments provided herein may include one or more of the following: (1) a simple, effective, and seamless integration of existing chromatographic methods that results in high-resolution separation of various peaks in complex mixed formulations, which was previously impossible with individual methods alone; (2) a real-time online identity and purity method that enables immediate identification of various fractions before and after addition to a conjugated formulation; and (3) its sensitivity, robustness, accuracy, and (4) The reproducibility can be made compliant with GMP validation for pharmaceutical manufacturing; (5) It provides a single, integrated method that enables the definition, detection, and quantification of various degradation products and / or aggregates under native running conditions similar to both large-scale purification processes and the final active pharmaceutical ingredient and formulation buffers; (6) It can be used for both lot release and stability indicators; (7) It is highly versatile and can be seamlessly integrated with other analytical techniques that complement and extend current resolutions to gain further insights into the characterization of mixed populations in pharmaceutical formulations.

[0031] In some embodiments, when used in the context of pharmaceutical compositions (e.g., OG1953 active pharmaceutical ingredient or formulation), the following advantages may be achieved: (1) under optimized chromatographic conditions, kinetic recovery of all injected samples with high resolution for all major product peaks, including the OG1953 conjugate (LP1) and the monomeric IgG (M) of OG1950, as well as their degradation products and / or aggregates (e.g., impurities). This may include OG1953-related degradation products such as LP2 and the "16-minute peak," and OG1950-related aggregates such as the dimer (P2) and alternative aggregate forms of P1; (2) It is widely applicable to panels of different formulations containing 1%, 7.5%, 10%, 15%, 20%, 49%, and 89% free antibodies mixed with various proportions of OG1953 conjugate. Furthermore, it can be applied to other molecules such as Fab with similar performance; (3) In long-term stability tests, it is possible to define degradation products and / or aggregates over time and temperature, as well as their formation kinetics, thereby enabling the setting of standards and predictive models for predicting long-term storage periods (shelf life).

[0032] Methods and apparatus for analyzing antibody and its conjugates, as well as mixed formulations of other proteins and protein conjugates, by tandem HPLC are provided herein. In some embodiments, the conjugates can be used to treat certain conditions such as diabetic retinopathy and / or age-related macular degeneration.

[0033] In some embodiments, a method is provided for analyzing a mixed formulation sample. In some embodiments, the method includes providing a mixed formulation comprising a combination of two protein moieties. In some embodiments, the first protein moiety (A) is conjugated to a polymer, and the second protein moiety (B) is not conjugated to a polymer. In some embodiments, the method includes loading the sample into an HPLC system, in which the HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem-connected columns, the first column being a cation exchange column (CEX), and the second column downstream of the CEX column being a size exclusion chromatography (SEC) column. In some embodiments, the method includes a first run. In some embodiments, during the first run, the mixed formulation is injected and passed through a tandem CEX-SEC system. In some embodiments, the method includes a second run. In some embodiments, during the second run, a concentrated salt is injected, the bound fraction on the CEX column is eluted, and it is separated by the SEC column. In some embodiments, a solvent of a specific ionic strength is used to enable the distribution of the mixed formulation in a CEX column. In some embodiments, the method enables the analysis of the mixed formulation based on differences in the charge and size variants of its components.

[0034] These and additional embodiments are provided below, following the definition section.

[0035] definition In light of this disclosure, all terms may have their conventional and ordinary meanings to those skilled in the art.

[0036] 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 clumps in the solution (excluding the contribution of the polymer component of the conjugate to the mass of the conjugate).

[0037] 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, given that the molecular weights of all the components in question are known, compositional percentages and % total molar weight are substantially interchangeable.

[0038] "Molar ratio" refers to the ratio (in moles) of the amount of free protein (e.g., OG1950) in a formulation to the amount of conjugated protein (e.g., OG1953) (based on the protein portion of the conjugate).

[0039] As used herein, “unconjugated” and “free” with respect to proteins, protein moieties, 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).

[0040] "Angiogenic disorders" are disorders or conditions 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.

[0041] 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, impurities unrelated to the product, such as host cell proteins, endotoxins, and host cell DNA, are not considered “impurities” as defined herein.

[0042] 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.

[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 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. It includes a single-chain polypeptide having the characteristic of providing [a certain feature].

[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 means having an affinity. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding that occurs to at least one irrelevant target. Specific binding can be the result of bond formation between specific functional groups or specific spatial fit (e.g., lock and key type), whereas non-specific binding is usually the result of van der Waals forces. However, specific binding does not necessarily mean that an antibody or fusion protein binds only to a single 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, in which relatively conserved framework regions (FRs) are linked by three hypervariable regions (also called complementarity-determining regions or CDRs). The resulting CDRs are aligned by a framework region, enabling binding to specific epitopes. From the N-terminus to the C-terminus, both the light and heavy chains contain the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains. For convenience, variable heavy chain CDRs may be referred to as CDRH1, CDRH2, and CDRH3, and variable light chain CDRs may be referred to as CDRL1, CDRL2, and CDRL3. The amino acid assignments to each domain are from 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 the common antigen of the reference antibody (see, for example, Junghans et al., Cancer Res. 50: 1495, 1990). Excess test antibody (e.g., small amount) A test antibody competes with a reference antibody if it inhibits the binding of the reference antibody by at least 50% (at least 2x, 5x, 10x, 20x, or 100x). In some embodiments, the test antibody inhibits the binding of the reference antibody by 75%, 90%, or 99% when measured by a competitive binding assay. Antibodies identified by a competitive assay (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] 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 3. 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.

[0053] In some embodiments, size exclusion chromatography (SEC) may also be referred to as gel permeation chromatography (GPC). In some embodiments, affinity chromatography columns may also be referred to as group-specific affinity columns.

[0054] The term "patient" includes human and other mammalian subjects receiving preventive or therapeutic treatment.

[0055] 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.

[0056] 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 regions 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 can be determined using algorithms such as BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), using default gap parameters, or by testing, 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 This is calculated by comparing two optimally aligned sequences across a 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 to calculate the percentage of sequence identity.

[0057] 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.

[0058] The term "antibody-dependent cell-mediated cytotoxicity", or ADCC, is a mechanism for inducing cell death that depends on the interaction between antibody-coated target cells (i.e., cells to which the antibody has 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 the antibody bound to the cell and Fcγ receptors on immune effector cells such as neutrophils, macrophages, and natural killer cells, particularly FcγRI and FcγRIII. The target cells are eliminated by phagocytosis or lysis depending on the type of intervening effector cell. The death of antibody-coated target cells occurs as a result of the activity of effector cells.

[0059] The term "opsonization" (also known as "antibody-dependent cell phagocytosis", or ADCP) refers to the process by which cells coated with an antibody are taken up in whole or in part by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc region of the immunoglobulin.

[0060] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death in which the Fc effector domain of an antibody bound to a target activates a series of enzymatic reactions that ultimately result in the formation of pores in the target cell membrane. Typically, antigen-antibody complexes, such as those on target cells coated with an antibody, bind to and activate complement component C1q, which then activates the complement cascade leading to target cell death. Activation of the complement can also result in the deposition of complement components on the surface of the target cell, which promotes ADCC by binding to complement receptors on leukocytes (e.g., CR3).

[0061] A humanized antibody is a genetically engineered antibody in which the CDRs derived from a non-human "donor" antibody are transplanted into a human "acceptor" antibody sequence (see, e.g., 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, 6,881,557, Foote, U.S. Patent No. 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody has some or all of the CDRs derived from the donor antibody, either fully or substantially, and has the variable region framework sequence and constant region (if present) derived from the human antibody sequence, either fully or substantially. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs derived from the donor heavy chain, either fully or substantially, and has the heavy chain variable region framework sequence and heavy chain constant region (if present) derived from the human heavy chain variable region framework and constant region sequences, substantially. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived from the donor antibody light chain, either fully or substantially, and has the light chain variable region framework sequence and light chain constant region (if present) derived from the human light chain variable region framework and constant region sequences, substantially. Except for nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. The CDRs in a humanized antibody are substantially derived from those in the non-human antibody when at least 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region when at least 85%, 90%, 95%, or 100% of the corresponding residues as defined by Kabat are identical.

[0062] 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).

[0063] 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.

[0064] 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 procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol Immunol. 28:489-98). As a result, the CDRs are entirely or substantially derived from the non-human antibody, and the variable region framework of the non-human antibody is made more human-like through substitution. Antibodies can be obtained. 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, using trioma methods and 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.

[0065] A "polymer" refers to a series of monomer groups linked together. A polymer is 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, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridines, vinyl-pyrrolidones, and vinyl esters such as vinyl acetate. 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. Polymers can be linear or branched. If a polymer is branched, each polymer chain is called a "polymer arm." The end of a polymer arm linked to an initiator is the proximal end, and the growth chain end of a polymer arm is the distal end. At the growth chain end of a polymer arm, the polymer arm end group may be a radical scavenger or another group.

[0066] 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 initiator 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 a radical in the growing polymer chain to reversibly terminate the polymerization. The radical scavenger I' is typically a halogen, but may also be an organic moiety such as a nitrile. In some embodiments, the initiator contains one or more 2-bromoisobutyrate groups as sites for polymerization by ATRP.

[0067] A "chemical linker" refers to a chemical moiety that links two groups, such as a half-life extension moiety 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 containing one or more bonds to a physiologically active agent. Non-limiting examples are shown in Table 1 of International Publication No. 2013059137 (as referenced).

[0068] 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.

[0069] "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.

[0070] A "phosphorylcholine-containing polymer" is a polymer that contains phosphorylcholine. A "zwitterion-containing polymer" refers to a polymer that contains zwitterions.

[0071] 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.

[0072] 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]

[0073] When used in this specification, "MPC" and "HEMA-PC" are interchangeable.

[0074] 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.

[0075] 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.

[0076] "Approximately" is an indication that measurements are taken between different instruments, samples, and sample preparations. It means a certain fluctuation.

[0077] "Protected", "protection form", "protecting group", and "protective group" refer to the presence of a group (i.e., a protecting group) that suppresses 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 the chemically reactive group to be protected, the reaction conditions used, and, if any, the presence of additional reactive groups or protecting groups in the molecule. Suitable protecting groups include those as found in Greene et al., "Protective Groups In Organic Synthesis," 3rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0078] "Alkyl" refers to a straight-chain or branched-chain, saturated, aliphatic radical having the indicated number of carbon atoms. For example, alkyls having 1 to 6 carbon atoms include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Other alkyl groups include, but are not limited to, heptyl, octyl, nonyl, decyl, etc. Alkyl can contain any number of carbons, such as 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6, and 5 to 6. An alkyl group is typically monovalent, but can be divalent in cases where the alkyl group is connected to two moieties.

[0079] When the term "lower" is mentioned in connection with an organic radical or compound above and below, it defines a compound or radical that can be branched or unbranched and has up to 7, including 7, or up to 4, including 4, and (as unbranched) 1 or 2 carbon atoms.

[0080] "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 may be linked to the same or different atoms of the alkylene. For example, a linear alkylene may be a divalent radical of -(CH2)n (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.

[0081] 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)N R”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. R' and R'' refer to the same nitrogen source. When bonded to a child, it can combine with the 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 to 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, etc.).

[0082] 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 one or more of various groups selected from, but not limited to, the following, 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', -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 a hydrogen atom, a substituted or unsubstituted heteroalkyl group, a substituted or unsubstituted aryl group (e.g., an aryl group substituted with 1 to 3 halogens), a substituted or unsubstituted alkyl group, an alkoxy group or thioalkoxy group, or an arylalkyl group. If a compound contains, for example, two or more R groups, each R group is independently selected from 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 encompass 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.).

[0083] "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.

[0084] "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.

[0085] "Haloalkyl" refers to an alkyl group as defined above, which contains some or all water. This refers to compounds in which elementary 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 compounds or radicals 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.

[0086] A "fluorosubstituted alkyl group" refers to an alkyl group in which one, some, or all of its hydrogen atoms are replaced by fluorine.

[0087] Cytokines are a group of protein signaling molecules that can be involved in intercellular communication in immune and inflammatory responses. Cytokines are typically small, water-soluble glycoproteins with a mass of approximately 8 kDa to 35 kDa.

[0088] A "cycloalkyl" refers to a cyclic hydrocarbon group containing approximately 3 to 12, 3 to 10, or 3 to 7 intra-ring carbon atoms. Cycloalkyl groups include condensed structures, cross-linked structures, and spiro-ring structures.

[0089] "Within the ring" refers to an atom or group of atoms that constitute a part of a cyclic ring structure.

[0090] "Extra-ring" refers to atoms or groups of atoms that are bonded but do not define a cyclic ring structure.

[0091] "Cyclic alkyl ether" refers to a 4- or 5-membered cyclic alkyl group having 3 or 4 intraring carbon atoms and 1 intraring acid atom or sulfur atom (e.g., oxetane, thietan, tetrahydrofuran, tetrahydrothiophene); or a 6- to 7-membered cyclic alkyl group having 1 or 2 intraring oxygen atoms or sulfur atoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxatiane).

[0092] 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.

[0093] "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.

[0094] "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.

[0095] "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.

[0096] "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, cycloalkyls with 3 to 8 carbon atoms include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0097] 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.

[0098] 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.

[0099] A "heterocycloalkylene" refers to a heterocycloalkyl group, as defined above, that 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.

[0100] "Aryl" refers to an aromatic ring aggregate 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. Aryl groups include alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, The molecules may be monosubstituted, disubstituted, or trisubstituted by one, two, or three radicals selected from amino-alkyl, trifluoromethyl, alkylenedioxy, and C2-C3 oxyalkylenes (all of these substituents may optionally be further substituted as defined above); or they may be 1-naphthyl or 2-naphthyl; or they may be 1-phenantrenyl or 2-phenantrenyl. Alkylenedioxy molecules have a divalent substituent bonded to two adjacent carbon atoms of phenyl, for example, methylenedioxy or ethylenedioxy. C2-C3 oxyalkylenes also have a divalent substituent bonded to two adjacent carbon atoms of phenyl, for example, oxyethylene or oxypropylene. An example of a C2-C3 oxyalkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] "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.

[0105] Similarly, substituents on aryl and heteroaryl groups are diverse, including -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -CO2R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -N3, -C A number selected from H(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, ranging from 0 to the total number of open valences on the aromatic ring system; where R', R'', and R''' are independently selected from hydrogen, (C1-C8)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C1-C4)alkyl, and (unsubstituted aryl)oxy-(C1-C4)alkyl.

[0106] Two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with substituents of the formula -TC(O)-(CH2)qU-, where T and U are independently -NH-, -O-, -CH2-, or a single bond, and q is an integer from 0 to 2. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with substituents of the formula -A-(CH2)rB-, where A and B are independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond, and r is an integer from 1 to 3. One of the single bonds of the new ring thus formed may optionally be replaced with a double bond. Alternatively, two substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with substituents of the formula -(CH2)sX-(CH2)t-, where s and t are independent integers from 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is selected from hydrogen or an unsubstituted (C1-C6) alkyl group.

[0107] 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 preferable. 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.

[0108] In some embodiments, the heteroaryl is any of the following: 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 monosubstituted or disubstituted.

[0109] The term "heteroalkyl" refers to an alkyl group having 1 to 3 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 Examples include (O)- and -S(O)2-, but are not limited to these. For example, heteroalkyls may include ethers, thioethers, alkylamines, and alkylthiols.

[0110] The term "heteroalkylene" refers to a heteroalkyl group, as defined above, that is linked to at least two other groups. The two groups linked to the heteroalkylene may be linked to the same or different atoms of the heteroalkylene.

[0111] An "electrophile" refers to an ion, atom, or group of atoms that may be ionic and possess an electrophilic center (i.e., a center that seeks electrons) that can react with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond with its reaction partner (nucleophile) by accepting both bonding electrons from that partner.

[0112] 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.

[0113] "Maleimide" refers to a pyrrole-2,5-dione-1-yl group having the following structure: [ka]

[0114] When this group reacts with sulfhydryl (e.g., thioalkyl), 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 thiol sulfur atom to the remainder of the original group that has sulfhydryl.

[0115] 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.

[0116] In relation to the shape, architecture, or overall structure of a polymer, "linear" refers to a polymer having a single polymer arm.

[0117] 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.

[0118] "Pharmacopoeially acceptable excipients" 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 pharmacopoeially acceptable excipients include water, NaCl, isotonic (normal) saline, Ringer's lactate solution, isotonic (normal) sucrose, and isotonic (normal) glucose. In any embodiment, a pharmacopoeially acceptable carrier may be acceptable for direct administration to the patient's eye (e.g., acceptable for intravitreal administration).

[0119] Therapeutic proteins are administered in effective regimens, where the method of administration, route of administration, and frequency of administration are designed to delay the onset of the disorder, reduce its severity, and inhibit further deterioration of the disorder. This means that the regimen improves at least one sign or symptom of the disorder. If the patient already has the disorder, the regimen may be called a therapeutically effective regimen. If the patient is at higher risk of the disorder than 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.

[0120] 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 the tissue or organism after its introduction.

[0121] "OG1786" is a nine-arm initiator used in polymer synthesis having the structure shown in Figure 35, which illustrates the salt form of OG1786 with trifluoroacetic acid. OG1786 can be used as a free base, as with other salts.

[0122] "OG1801" is a polymer of approximately (±15%) 750 kDa (depending on either Mn or Mp) prepared using the monomer HEMA-PC and OG1786 as an initiator for ATRP synthesis.

[0123] "OG1802" is OG1801 with a maleimide functional group added, as shown in Figure 36, where n1, n2, n3, n4, n5, n6, n7, n8, and n9 are each positive integers (from 0 to approximately 3000) such that the total molecular weight of the polymer is (Mw) 750,000 ± 15% Daltons.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] Combining SEC with 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.

[0128] 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 The 6.1.7.16 software can be used to acquire mass recovery data using MALS data from Wyatt miniDawn TREOS, dn / dc data from T-rEX detectors, and A280 absorbance signals from Waters 2996 photodiode array detectors. SEC can be performed at 1 ml / min in 1×PBS (pH 7.4), and after sample injection, the absolute molecular weight (Mp, Mw, Mn) and polydispersity index (PDI) can be determined by analyzing the MALS and RI signals with ASTRA software. 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 146 kDa.

[0129] "Isocratic" refers to a mobile phase that is kept constant and uniform throughout a chromatography run.

[0130] Antibody composition and method In some embodiments, a formulation (or therapeutically acceptable composition) is provided, which is a first protein comprising a first protein or protein moiety conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) and a second protein or protein moiety that is not conjugated. therapeutically acceptable composition) that is a first protein that includes a A first protein or protein moiety that is conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) and a second protein or protein moiety that is unconjugated are provided. In some embodiments, the first protein is an antibody. Yes, the second protein is an antibody. Both antibodies can be therapeutic antibodies. In some embodiments, the formulation or composition is for the treatment of an eye disorder in a subject. In some embodiments, the formulation or composition sample comprises at least a polymer (e.g., a phosphorylcholine-containing polymer) and a non-conjugate protein (e.g., a non-conjugate antibody). As used herein, the terms formulation (e.g., a mixed formulation) and composition (e.g., a mixed therapeutically acceptable composition) may be used synonymously. In some embodiments, the formulation or therapeutically acceptable composition is safe for use in humans (e.g., administration to humans). In some embodiments, the formulation or therapeutically acceptable composition is not an intermediate product generated during the manufacture of the final product (e.g., one that may be suitable for use in humans). In some embodiments, the formulation is a mixed formulation sample. In some embodiments, the terms formulation and mixed formulation sample may be used synonymously. As used herein, the terms "protein" and "protein portion" may be used synonymously.

[0131] In some embodiments, the Specified Provisions provide a formulation (or therapeutically acceptable composition) comprising any two proteins that may be functionally identical or different, wherein one 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 formulation (or therapeutically acceptable 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 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 (or therapeutically acceptable composition) is provided herein, comprising: a first molar amount of a conjugate ("conjugated protein") containing a first protein or protein moiety conjugated to a phosphorylcholine-containing polymer; a second molar amount of a second protein or protein moiety not conjugated to a phosphorylcholine-containing polymer ("unconjugated protein"); and a pharmaceutically acceptable carrier, wherein the formulation or 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 first and second molar amounts, and the formulation or composition 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 quantities are expressed in moles (e.g., moles, millimoles, micromoles, etc.). As used herein, the isoelectric point (pI) of a protein has its conventional and ordinary meaning in view of this disclosure to those skilled in the art. pI indicates the pH at which the protein has no net charge. pI may be a known value for the same or similar protein, 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 (or therapeutically acceptable compositions) of protein conjugates (e.g., proteins conjugated to phosphorylcholine-containing polymers) are also provided. High concentrations of protein conjugates in the formulation or composition may increase the viscosity of the formulation or composition. In some embodiments, reducing the viscosity of the formulation or composition (while maintaining the total amount of active protein) improves one or more of the manufacturability, handling, and injectability when the formulation or composition is delivered to the treatment site by syringe (e.g., intraocular administration).

[0134] In any of the methods and apparatus of this disclosure, a mixed formulation (or therapeutically acceptable composition) (e.g., having a polymer or polymer-conjugate protein and an unconjugate protein) may contain an unconjugate protein or protein moiety (e.g., a protein not conjugated to a phosphorylcholine-containing polymer) in any preferred % molar amount of the total molar amount of the polymer / polymer conjugate and the unconjugate protein or protein moiety. In some embodiments, the formulation (or therapeutically acceptable composition) contains the second protein or protein moiety (unconjugate protein) in about 1% or more 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. For example, if the total concentration of the conjugate and unconjugate proteins is 100 μM, then 1% of the total molar amount of the unconjugate protein is 1 μM and the conjugate is 99 μM. In some embodiments, the formulation (or therapeutically acceptable composition) contains a second protein (non-conjugate protein) in an amount of 1% or more, or about 1% or more, 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. , including approximately 60% or less, approximately 55% or less, approximately 50% or less, approximately 45% or less, approximately 40% or less, approximately 35% or less, approximately 30% or less, or including in percentages within the range defined by any two of the aforementioned values ​​for the total molar amounts of the conjugate and the second protein (e.g., approximately 1-95%, 1-90%, 1-80%, 1-95%, 1-50%, 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 (or therapeutically acceptable composition) comprises the second protein (unconjugated protein) in an amount of about 5% to about 50%, or about 15% to about 30%, 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 (or therapeutically acceptable composition) comprises the second protein (unconjugated 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 (or therapeutically acceptable composition) comprises the second protein (unconjugated protein) in an amount of about 30%, 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, any formulation or composition provided herein comprises a conjugate and a second protein in a proportion of more than 5% of the second protein (unconjugated protein), and 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, any formulation or composition provided herein comprises a conjugate and a second protein in a proportion of more than 10% of the second protein (unconjugated protein), and 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, any composition or formulation provided herein comprises two or more (e.g., 2, 3, 4, 5, or more) different second proteins (or unconjugated proteins), and the second molar amount is the sum of the molar amounts of the two or more different second proteins.

[0135] In some embodiments, the formulation (or therapeutically acceptable composition) comprises a first protein or protein moiety conjugated to a phosphorylcholine-containing polymer, the polymer having nine arms and a molecular weight of 600,000 to 1,000,000 Da, and the polymer present in the formulation at a concentration of about 100 mg / mL or more; and a second protein or protein moiety 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%, 99%, or at least about 85%, 90%, 95%, 97%, 99%, or about 100% identical in their amino acid sequences). In some embodiments, the first and second proteins are different proteins.

[0136] In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises a first molar amount of conjugate containing a first protein or protein moiety conjugated to a polymer; and a second molar amount of a second protein or protein moiety 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, with the total molar amount being 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 5-90%, 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 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 second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer.

[0137] In some embodiments, a formulation (or therapeutically acceptable composition) comprises a conjugate containing a first protein or protein moiety conjugated to a polymer; and a second protein or protein moiety not conjugated to a polymer, wherein the first molar amount of the conjugate and the second molar amount of the second protein or protein moiety 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 or protein moiety (e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.), and the total molar amount includes the sum of the first and second molar amounts. In some embodiments, a mixed formulation or composition is prepared by combining a first molar amount of the conjugate and a second molar amount of a 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%, 10-95%, 15-30%, 5-50%, or 10-40% of the total molar amount of the conjugate and the second protein. In some embodiments, the second molar amount is about 5-50% of the total molar amount of the conjugate and the second protein. In some embodiments, the second molar amount is 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.

[0138] In some embodiments, the mixed formulation or composition is prepared by combining a conjugate such that the composition percentage of the second protein is about 1% or more relative to the total protein mass weight concentration of the first and second proteins (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.), where the remainder of the total protein mass weight concentration is the first protein. For example, a total mass weight concentration of 50 mg / m³ In the case of L, the mixed therapeutically acceptable composition can be prepared by combining a second protein in an amount equivalent to 10 mg / mL (20% of compositional percentage) 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).

[0139] In some embodiments, the conjugate comprises a first protein or protein moiety conjugated to a polymer, the polymer comprising 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, dextrose Trans, 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-hydroxymethyethylene hydroxymethylformal) (PHF), zwitterionic polymers, phosphorylcholine-containing polymers and polymers containing MPC, poly(Glyx-Sery), hyaluronic acid (HA), heparosan polymer (HEP), Fleximer, dextran, and polysialic acid (PSA).

[0140] General Methods and apparatus for analyzing mixed formulations (or therapeutically acceptable compositions) of proteins and proteins conjugated to polymers by tandem HPLC are provided herein. In some embodiments, the protein is an antibody. In some embodiments, the antibody is an anti-VEGF antibody and its conjugate. In some embodiments, the antibody itself differs from other anti-VEGF agents and yields superior results compared to other anti-VEGF agents. In some embodiments, the anti-VEGF antibody conjugate displays a surprising superiority over other antibodies and / or the expectation of the activity of other antibody conjugates.

[0141] Historically, conjugating molecules to proteins has often resulted in a reduction in the protein's binding interaction to its intended target. In some embodiments of this disclosure, when conjugation occurs at a location outside the active site, the same level of reduction as might be expected is not necessarily observed. The evidence provided herein shows an effect opposite to that expected. In some embodiments, though not intended to be limited by theory, the conjugate may be superior to the antibody alone. For example, the interaction between a ligand and its specific receptor is often driven through a stereospecific interaction between ligand and receptor, led by the interaction between hydrophilic amino acids on the ligand and hydrophilic amino acids on the receptor, with water molecules playing a central role in these interactions. Simultaneously, this hydrophilic stereospecificity is further enhanced by de-emphasizing and / or suppressing nonspecific hydrophobic interactions that can generally be mediated / generated by hydrophobic amino acids themselves.

[0142] In some embodiments, anti-VEGF antibody conjugates are provided that can block at least 90% of the interaction between VEGF ligands ("VEGFL") and VEGF receptors ("VEGFR"). For example, the anti-VEGF antibody conjugate is V It is possible to block at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or virtually all of the interaction between EGFR and VEGFL. In some embodiments, the blockade mentioned occurs at saturation concentration. In some embodiments, an anti-VEGF antibody conjugate is provided that blocks at least 95% of the interaction between a VEGF ligand and a VEGF receptor. In fact, this result was unexpected in the following respect: while it could be expected that the addition of a polymer to the antibody (and thereby the formation of an antibody conjugate) would have some adverse effect on the antibody's binding / activity, or not have any adverse effect, it was not expected that such an enhancement of the antibody's blocking ability would actually occur.

[0143] In some embodiments, the antibody or its conjugate inhibits at least 70, 80, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the activity and / or interaction between VEGFR and VEGFL. In some embodiments, the IC50 value may be 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 nM, or less than any one or more of the aforementioned values. In some embodiments, the KD is 2 × 10⁻¹⁶ -13 , 1 x 10 -13 , 1 x 10 -12 , 1 x 10 -11 , 1 x 10 -10The IC50 value may be M, or less than any one of the aforementioned values. In some embodiments, the IC50 value may be 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, or less than any one of the aforementioned values.

[0144] In some embodiments, anti-VEGF antibodies are provided that block at least 90% of the interaction between VEGF ligands and VEGF receptors. For example, an anti-VEGF antibody can block at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or virtually all, of the interaction between VEGFR and VEGFL.

[0145] In some embodiments, other antibodies such as Lucentis® (ranibizumab) or Avastin® (bevacizumab) may be conjugated to one or more polymers described herein by one or more processes described herein. In some embodiments, any antibody, or a fragment thereof, may be conjugated to one or more polymers described herein by one or more processes described herein.

[0146] In some embodiments, the antibody includes a heavy-chain amino acid variable region containing SEQ ID NO: 1 (with or without C-terminal lysine) and a light-chain amino acid variable region containing SEQ ID NO: 2. In some embodiments, the antibody is conjugated to one or more polymers provided herein. In some embodiments, the conjugated antibody is at least 90% identical to SEQ ID NO: 1 and / or SEQ ID NO: 2. In some embodiments, the antibody includes six CDRs in SEQ ID NO: 1 and SEQ ID NO: 2, as well as the point mutation L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the conjugated antibody is at least 90% identical to SEQ ID NO: 1 and / or SEQ ID NO: 2 and includes the following mutations: L234A, L235A, and G237A (EU numbering), as well as at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0147] In some embodiments, antibodies that bind to VEGF-A are included. The antibodies include: CDRH1, which is CDRH1 in SEQ ID NO: 1, and CDRH2 in SEQ ID NO: 1. CDRH2 is CDRH3 in SEQ ID NO: 1, CDRH3 is CDRH3 in SEQ ID NO: 2, CDRL1 is CDRL1 in SEQ ID NO: 2, CDRL2 is CDRL2 in SEQ ID NO: 2, CDRL3 is CDRL3 in SEQ ID NO: 2, at least one of the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0148] As will be understood by those skilled in the art, in light of this Spec, any antibody provided herein can be conjugated to any polymer provided herein, and / or any antibody provided herein can be conjugated with cysteine ​​to enable site-specific conjugation of the antibody to a polymer.

[0149] "VEGF" or "vascular endothelial growth factor" is a human vascular endothelial growth factor that influences angiogenesis or the process of angiogenesis. In particular, the term VEGF means any member of the growth factor class that (i) binds to VEGF receptors such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4); (ii) activates tyrosine kinase activity associated with VEGF receptors; and (iii) thereby influences angiogenesis or the process of angiogenesis.

[0150] The VEGF family of factors consists of five related glycoproteins: VEGF-A (also known as VPE), VEGF-B, VEGF-C, VEGF-D, and PGF (placental growth factor). Of these, VEGF-A is the most well-studied and is a target of anti-angiogenic therapies. Ferrara et al, (2003) Nat. Med. 9:669-676. VEGF-A exists as several different isotypes: VEGF-A206, VEGF-A189, VEGF-A165, and VEGF-A121, which are produced by both alternative splicing and proteolysis. The isoforms differ in their ability to bind to heparin and non-signaling-binding proteins called neuropilin. All isoforms are biologically active as dimers.

[0151] The various effects of VEGF are mediated by the binding of VEGF, such as VEGF-A (P15692), VEGF-B (P49766), VEGF-C (P49767), and VEGF-D (Q43915), to receptor tyrosine kinases (RTKs). The VEGF family receptors belong to class V RTKs, each possessing seven Ig-like domains in its extracellular domain (ECD). In humans, VEGF binds to three types of RTKs: VEGFR-1 (Flt-1) (P17948), VEGFR-2 (KDR, Flk-1) (P935968), and VEGFR-3 (Flt-4) (P35916). Unless otherwise stated in the context, when we refer to VEGF, we mean any of VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PGF, which are either natural isoforms or natural variants, or derived variants having at least 90, 95, 98, 99%, or 100% sequence identity with respect to the natural type. In some embodiments, such VEGF is human VEGF. Similarly, when we refer to VEGFR, we mean any of VEGFR-1, VEGFR-2, or VEGFR-3, which encompasses any natural isoform or natural variant, or derived variants having at least 90, 95, 98, 99%, or 100% sequence identity with respect to the natural sequence.

[0152] VEGF antagonist therapy is approved for the treatment of certain cancers and wet AMD. Bevacizumab (Avastin, Genentech / Roche) is a humanized mouse monoclonal antibody that binds to and neutralizes human VEGF, particularly all isoforms of VEGF-A, and the bioactive proteolytic fragments of VEGF-A. For example, Ferrara N, Hillan KJ, Gerber HP, Novotny See W. 2004. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 3(5):391-400. Bevacizumab is approved for the treatment of certain cancers. The heavy and light chain protein sequences of bevacizumab (DrugBank DB00112) are described in SEQ ID NO: 3 (heavy chain) and SEQ ID NO: 4 (light chain).

[0153] The CDRs for the light chain variable region of bevacizumab are CDRL1:SASQDISNYLN (SEQ ID NO: 12), CDRL2:FTSSLHS (SEQ ID NO: 13), and CDRL3:QQYSTVPWT (SEQ ID NO: 14). The CDRs for the heavy chain variable region of bevacizumab are CDRH1:GYTFTNYGMN, CDRH2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3:YPHYYGSSHWYFDV. The CDRs are defined by Kabat, except for CDRH1, where the Kabat / Chothia composite definition is used. In some embodiments, cysteine ​​may be added to the bevacizumab sequence, and the antibody (and / or a variant of bevacizumab containing the six CDRs) may be conjugated to one or more of the polymers provided herein.

[0154] Ranibizumab (Lucentis® (ranibizumab), Genentech / Roche), another anti-VEGF molecule derived from the same mouse monoclonal antibody as bevacizumab, is approved for the treatment of wet AMD. Ranibizumab is an antibody fragment or Fab. Ranibizumab was produced by affinity maturation of the heavy-chain and light-chain variable regions of bevacizumab. The heavy-chain and light-chain sequences of ranibizumab (as published by Novartis) are described in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. In some embodiments, cysteine ​​may be added to the ranibizumab sequence, and the antibody (and / or a variant of ranibizumab containing six CDRs) may be conjugated to one or more of the polymers provided herein.

[0155] Unless modified after affinity maturation, the CDRs for ranibizumab are the same as those for bevacizumab: the CDRs for the light chain variable region of ranibizumab are CDRL1:SASQDISNYLN (SEQ ID NO: 12), CDRL2:FTSSLHS (SEQ ID NO: 13), and CDRL3:QQYSTVPWT (SEQ ID NO: 14). The CDRs for the heavy chain variable region of ranibizumab are CDRH1:GYDFTHYGMN (SEQ ID NO: 9), CDRH2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3:YPYYYGTSHWYFDV (SEQ ID NO: 11).

[0156] In some embodiments, antibody conjugates are presented that have an anti-VEGF-A antibody conjugated to a phosphorylcholine-containing polymer at a cysteine ​​outside the variable region of the antibody, and the cysteine ​​is added by recombinant DNA technology. In some embodiments, the polymer is conjugated to a single cysteine. In some embodiments, "added by recombinant DNA technology" means the substitution of a non-cysteine ​​amino acid by a cysteine ​​residue at the same position in a known or existing antibody or consensus antibody sequence. For example, if the antibody is IgG1 and the heavy chain has leucine at position 443 of the EU numbering, the leucine is substituted by recombinant DNA technology with cysteine ​​(L443C, EU numbering, or 449C in SEQ ID NO: 1). Correspondingly, the natural IgG1 sequence at position 347 of the EU numbering is Q (glutamine), and Q is substituted by recombinant DNA technology with cysteine ​​to produce Q347C.

[0157] In some embodiments, the anti-VEGF-A antibody comprises a light chain and a heavy chain, the heavy chain having an Fc region. In some embodiments, cysteine ​​is located in the Fc region, and the anti-VEGF The FA antibody is immunoglobulin G (IgG). In some embodiments, the heavy chain of anti-VEGF-A has CDRH1:GYDFTHYGMN (SEQ ID NO: 9), CDRH2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3:YPYYYGTSHWYFDV (SEQ ID NO: 11), with T at position 221 (by sequential counting similar to SEQ ID NO: 3), and the light chain of anti-VEGF-A has CDRL1:SASQDISNYLN (SEQ ID NO: 12), CDRL2:FTSSLHS (SEQ ID NO: 13), and CDRL3:QQYSTVPWT (SEQ ID NO: 14), with L at position 4 according to Kabat numbering.

[0158] In some embodiments, the anti-VEGF-A heavy chain isotype is IgG1. In some embodiments, the IgG1 constant domain has one or more mutations in the IgG1 constant domain (e.g., the constant region of SEQ ID NO: 3) to modulate effector function. In some embodiments, the effector function mutations are one or more of the following: (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 are selected from the group including (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, the antibody conjugate has the following mutations (EU numbering): L234A, L235A, and G237A.

[0159] In some embodiments, the cysteine ​​residue is located within the anti-VEGF-A heavy chain and is Q347C (EU numbering) or L443C (EU numbering). In some embodiments, the cysteine ​​residue is L443C (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 (with or without C-terminal lysine), and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[0160] 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 comprises a heavy chain containing complementarity-determining region 1 CDRH1:FYHVH (SEQ ID NO: 140), CDRH2:SIYTSGYTEYASALES (SEQ ID NO: 141), and CDRH3:EGLQRVGVLDA (SEQ ID NO: 142) or EGLQRVGVFDA (SEQ ID NO: 143) or EGLQRVGVMDA (SEQ ID NO: 144), and a light chain containing CDRL1:RSSQSLLDEAGETYLA (SEQ ID NO: 145), CDRL2:EVSLLES (SEQ ID NO: 146), and CDRL3:QQATYFPYT (SEQ ID NO: 147). In some embodiments, the anti-HTRA1 antibody comprises a heavy chain containing complementarity-determining region 1 CDRH1:GFSLTFYH (SEQ ID NO: 148), CDRH2:IYTSGYT (SEQ ID NO: 149), and CDRH3:AREGLQRVGVFDA (SEQ ID NO: 150) or AREGLQRVGVMDA (SEQ ID NO: 151) or AREGLQRVGVLDA (SEQ ID NO: 152), and a light chain containing CDRL1:QSLLDEAGETY (SEQ ID NO: 153), CDRL2:EV, and CDRL3:QQATYFPYT (SEQ ID NO: 147). In some embodiments, the anti-HTRA1 antibody is a heavy chain containing complementarity-determining region 1 CDRH1:GFSLTFY (SEQ ID NO: 154), CDRH2:YTSGY (SEQ ID NO: 155), and CDRH3:EGLQRVGVLDA (SEQ ID NO: 142) or EGLQRVGVFDA (SEQ ID NO: 143) or EGLQRVGVMDA (SEQ ID NO: 144), as well as CDRL1:RSSQSLLDEAGETYLA (SEQ ID NO: 145), CDRL2:EVSLLES (SEQ ID NO: 146), and CDRL3:QQATYF The light chain contains PYT (SEQ ID NO: 147). In some embodiments, the heavy chain contains an amino acid sequence identical to one of the VH sequences listed in Table 1 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%), and the light chain contains an amino acid sequence identical to the VL sequences listed in Table 2 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or about 100%, or any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%). In some embodiments, the heavy chain contains one of the VH sequences listed in Table 1, and the light chain contains the VL sequences listed in Table 2. [Table 1] [Table 2]

[0161] In some embodiments, the first and second antibodies of the mixed formulation (or therapeutically acceptable composition) include a complement factor D (CFD) antibody. In some embodiments, the 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%, etc.), 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%, etc.). In some embodiments, the CFD antibody heavy chain includes SEQ ID NO: 129 (with or without C-terminal lysine). The sequence may include the sequence of ). In some embodiments, the CFD antibody light chain may include the sequence of SEQ ID NO: 130.

[0162] In some embodiments, the first and second antibodies of the mixed formulation (or therapeutically acceptable composition) comprise a fusion protein that is a VEGF-A trap fused to an IL-6 antibody. In some embodiments, the VEGF-A trap fused to the IL-6 antibody may comprise a heavy chain containing SEQ ID NO: 131 (with or without C-terminal lysine). In some embodiments, the VEGF-A trap fused to the IL-6 antibody may comprise a light chain containing SEQ ID NO: 132.

[0163] In some embodiments, the VEGF trap includes human VEGFR1 domain 2 and 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 the SEQ ID NO: 133 form in Table 3 by at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, 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 3]

[0164] In some embodiments, the fusion construct includes a heavy chain comprising CDRH1, which is CDRH1 in SEQ ID NO: 105; CDRH2, which is CDRH2 in SEQ ID NO: 105; CDRH3, which is CDRH3 in SEQ ID NO: 105; CDRL1, which is CDRL1 in SEQ ID NO: 106; CDRL2, which is CDRL2 in SEQ ID NO: 106; and CDRL3, which is CDRL3 in SEQ ID NO: 106. In some embodiments, the fusion construct includes a heavy chain comprising complementarity determination region 1 (CDRH1): PFAMH (SEQ ID NO: 134), CDRH2: KISPGGSWTYYSDTVTD (SEQ ID NO: 135), and CDRH3: QAWGYYALDI (SEQ ID NO: 136); and a light chain comprising CDRL1: SASISVSYLY (SEQ ID NO: 137), CDRL2: DDSSLAS (SEQ ID NO: 138), and CDRL3: QQWSGYPYT (SEQ ID NO: 139). In some embodiments, the heavy chain contains the same amino acid sequence as SEQ ID NO: 105 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or about 100%, or within a range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.), and the light chain contains the same amino acid sequence as SEQ ID NO: 106 in percentages of at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or about 100%, or within a range defined by any two of the aforementioned values ​​(e.g., 80-100%, 85-95%, 90-97%, etc.). In some embodiments, the heavy chain contains the amino acid sequence of SEQ ID NO: 105 (with or without C-terminal lysine), and the light chain contains the amino acid sequence of SEQ ID NO: 106.

[0165] In some embodiments, the phosphorylcholine-containing polymer is 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate as described below. Contains (MPC) monomer: [ka]

[0166] As a result, the polymer comes to contain the following repeating units: [ka] n is an integer between 1 and 3000, and the dashed lines represent the bond points between monomer units in the polymer.

[0167] In some embodiments, the polymer has three or more arms, Or, they are synthesized using an initiator containing three or more polymer initiation sites. In some embodiments, the polymer has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve arms, or is synthesized using an initiator containing two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer initiation sites. More preferably, the polymer has three, six, or nine arms, or is synthesized using an initiator containing three, six, or nine polymer initiation sites. In some embodiments, the polymer has nine arms, or is synthesized using an initiator containing nine polymer initiation sites.

[0168] In some embodiments, the polymer to be added has a molecular weight of approximately 300,000 Da to approximately 1,750,000 Da (SEC-MALs). In some embodiments, the polymer has a molecular weight of approximately 500,000 Da to approximately 1,000,000 Da. In some embodiments, the polymer has a molecular weight of approximately 600,000 to approximately 1,000,000 Da. In some embodiments, the polymer has a molecular weight of approximately 750,000 Da to approximately 850,000 Da. In some embodiments, the polymer has a molecular weight of approximately 800,000 Da to approximately 850,000 Da. In some embodiments, the polymer has a molecular weight of approximately 750,000 Da to approximately 800,000 Da. In some embodiments, the polymer has a molecular weight in the range of approximately 700,000 to approximately 800,000 Da.

[0169] In some embodiments, a bioconjugate can be formed by further conjugating one of the antibodies described herein to a polymer. The molecular weight (total, SEC-MALs) of the bioconjugate is approximately 350,000 daltons to approximately 2,000,000 daltons, for example, approximately 450,000 daltons to approximately 1,900,000 daltons, approximately 550,000 daltons to approximately 1,800,000 daltons, approximately 650,000 daltons to approximately 1,700,000 daltons, approximately 750,000 daltons to approximately 1,600,000 daltons, approximately 850,000 daltons to approximately 1,500,000 daltons, and approximately 90 Possible ranges include 0,000 Daltons to approximately 1,400,000 Daltons, approximately 950,000 Daltons to approximately 1,300,000 Daltons, approximately 900,000 Daltons to approximately 1,000,000 Daltons, approximately 1,000,000 Daltons to approximately 1,300,000 Daltons, approximately 850,000 Daltons to approximately 1,300,000 Daltons, approximately 850,000 Daltons to approximately 1,000,000 Daltons, and approximately 1,000,000 Daltons to approximately 1,200,000 Daltons.

[0170] In some embodiments, the antibody conjugate is purified. In some embodiments, the polymer is an aspect of the antibody conjugate and is polydisperse, i.e., the polymer PDI is not 1.0. In some embodiments, the PDI is less than 1.5. In some embodiments, the PDI is less than 1.4. In some embodiments, the PDI is less than 1.3. In some embodiments, the PDI is less than 1.2. In some embodiments, the PDI is less than 1.1.

[0171] In some embodiments, the antibody conjugate has anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, the polymer comprising an MPC monomer, the sequence of the anti-VEGF-A heavy chain being SEQ ID NO: 1 (with or without C-terminal lysine), the sequence of the anti-VEGF-A light chain being SEQ ID NO: 2, and the antibody being conjugated to the polymer only at C449 in SEQ ID NO: 1. In some embodiments, the polymer has nine arms and a molecular weight of approximately 600,000 to approximately 1,000,000 Da.

[0172] In some embodiments, the antibody conjugate has anti-VEGF-A immunoglobulin G (IgG) conjugated to a polymer, the polymer comprising an MPC monomer, the sequence of the anti-VEGF-A heavy chain being SEQ ID NO: 1 (with or without C-terminal lysine), the sequence of the anti-VEGF-A light chain being SEQ ID NO: 2, and the antibody being conjugated to the polymer only at C443 (EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the polymer has nine arms and a molecular weight of approximately 600,000 to approximately 1,000,000 Da.

[0173] In some embodiments, the antibody conjugate has the following structure: [ka] In the formula, 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; in the formula: each heavy chain of the first antibody is represented by the letter H; and each light chain of the first antibody is represented by the letter L; The polymer is bound to the first antibody via a sulfhydryl cysteine ​​at position 449, numbered in SEQ ID NO: 1, and this binding is shown on one of the heavy chains; PC is [ka] In the formula, the dashed line indicates the bond point to the remainder of the polymer; 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%. In some embodiments, X is a) an -OR where R is -H, methyl, ethyl, propyl, or isopropyl, b) -H, or c) a halide. 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%.

[0174] In some embodiments, the antibody conjugate is present in a liquid formulation. In some embodiments, the antibody conjugate is combined with a pharmaceutically acceptable carrier.

[0175] In some embodiments, anti-VEGF-A antibodies are presented. The anti-VEGF-A antibody heavy chain has at least the following CDR sequences: CDRH1:GYDFTHYGMN (SEQ ID NO: 9), CDRH2:WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3:YPYYYGTSHWYFDV (SEQ ID NO: 11). In some embodiments, the anti-VEGF-A heavy chain has these CDRs and also has threonine (T) at position 221 (by sequential counting, similar to SEQ ID NO: 3). In some embodiments, the anti-VEGF-A light chain has at least the following CDRs: CDRL1:SASQDISNYLN (SEQ ID NO: 12), CDRL2:FTSSLHS (SEQ ID NO: 13), and CDRL3:QQYSTVPWT (SEQ ID NO: 14). In some embodiments, the anti-VEGF-A antibody has these CDRs and also has leucine (L) at position 4 by Kabat numbering. In some embodiments, the isotype of the anti-VEGF-A antibody heavy chain is IgG1 and has a CH1 domain, a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the light chain isotype is kappa.

[0176] In some embodiments, the IgG1 domain of an anti-VEGF-A antibody has one or more mutations to modulate effector functions such as ADCC, ADCP, and CDC. In some embodiments, the IgG1 mutation reduces effector function. In some embodiments, the amino acids used for effector function mutations include (EU numbering) E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X, where X is any natural or non-natural amino acid. In some embodiments, the mutations include one or more of the following: E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S (EU numbering). In some embodiments, the anti-VEGF-A heavy chain has the following mutations (EU numbering): L234A, L235A, and G237A. In some embodiments, the number of effector functional mutations relative to the native human IgG1 sequence is 10 or less. In some embodiments, the number of effector functional mutations relative to the native human IgG1 sequence is 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less. In some embodiments, the antibody has reduced Fcγ binding and / or complement C1q binding, resulting in a reduced ability of the antibody to confer effector function. This may be particularly advantageous for ophthalmic indications / disorders.

[0177] In some embodiments, the anti-VEGF-A antibody comprises one or more of the following amino acid mutations: L234A, L235A, G237A (EU numbering), and L443C (EU numbering, or 449C in SEQ ID NO: 1).

[0178] In some embodiments, the anti-VEGF-A antibody is human immunoglobulin G (IgG1) or a portion thereof.

[0179] In some embodiments, the VEGF-A antibody includes a heavy chain constant domain containing one or more mutations that reduce its immune-mediated effector function.

[0180] In some embodiments, anti-VEGF-A antibodies are provided. The anti-VEGF antibody comprises a heavy chain including CDRH1 containing the sequence GYDFTHYGMN (SEQ ID NO: 9), CDRH2 containing the sequence WINTYTGEPTYAADFKR (SEQ ID NO: 10), CDRH3 containing the sequence YPYYYGTSHWYFDV (SEQ ID NO: 11), CDRL1 containing the sequence SASQDISNYLN (SEQ ID NO: 12), CDRL2 containing the sequence FTSSLHS (SEQ ID NO: 13), and CDRL3 containing the sequence QQYSTVPWT (SEQ ID NO: 14).

[0181] Alternatively, the IgG domain may be IgG2, IgG3, or IgG4, or a complex in which the constant region is formed from two or more of these isotypes (e.g., the CH1 region from IgG2 or IgG4, and the hinge region, CH2 region, and CH3 region from IgG1). Such a domain may contain mutations to reduce and / or modulate effector function at one or more EU numbering positions as mentioned in the case of IgG1. Human IgG2 and human IgG4 have reduced effector function compared to human IgG1 and human IgG3.

[0182] The anti-VEGF-A heavy chain has a cysteine ​​residue added as a mutation by recombinant DNA technology, which can be used to conjugate the half-life extension portion. In some embodiments, the mutation is (EU numbering)Q347C(EU numbering) and / or L443C(EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the mutation is L443C(EU numbering, or 449C in SEQ ID NO: 1). In some embodiments, the antibody:polymer stoichiometry is 1:1; in other words, the conjugate has one molecule of antibody conjugated to one molecule of polymer.

[0183] The half-life of an anti-VEGF-A antibody can be extended by attaching a "half-life extension moiety" or a "half-life extension group." Examples of half-life extension moieties include peptides and proteins that can be expressed in frame with the biological agent (or chemically conjugated as needed), as well as various polymers that can be attached to or conjugated to one or more amino acid side chains or terminal functional groups such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N-glycan and / or O-glycan structures. Half-life extension moieties typically function to extend the in vivo circulatory half-life of the biological agent.

[0184] Examples of peptide / protein half-life extension regions include: Fc fusions (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525-31), human serum albumin (HAS) fusions (Yeh P, Landais D, Lemaitre M , et al. Design of yeast-secreted albumin derivatives for human therapy: biological and antiviral properties of a serum albumin-CD4 genetic conjugate. Proc Natl Acad Sci USA. 1992. 89:1904-08), carboxy-terminal peptide (CTP) fusions (Fares FA, Suganuma N. Nishimori K, et al. Design of a long-acting follitropin agonist by fusing the C-terminal sequence of the chorionic gonadotropin beta subunit to the follitropin beta subunit. Proc Natl Acad Sci USA. 1992. 89:4304-08), fusions with gene fusion of non-accurate repeat peptide sequences (XTEN) (Schellenberger V, Wang CW, Geething NC, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol. 2009. 27:1186-90), elastin-like peptides (ELP-modified) (MCpherson DT, Morrow C, Minehan DS, et al.) al. Production and purification of a recombinant elastomeric polypeptide, G(VPGVG19-VPGV, from Escheriachia coli. Biotechnol Prog. 1992. 8:347-52), human transferrin fusion (Prior CP, Lai CH, Sadehghi H et al. Modified transferrin fusion proteins. Patent WO2004 / 020405. 2004), proline-alanine-serine (PAS-modified) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and / or vitro stability. Patent WO2008 / 155134 A1. 2008), homoamino acid polymer (HAP-modified) (Schlapsky M, Theobald I, Mack H, et al. al. Fusion of a recombinant antibody fragment with a homo-amino acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007. 20:273-84), and gelatin-like protein (GLK) fusion (Huang YS, Wen XF, Zaro JL, et al. Engineering a pharmacologically superior form of granulocyte-colony-stimulating-factor by fusion with gelatin-like protein polymer. Eur J. Pharm Biopharm. 2010. 72:435-41).

[0185] Examples of polymer half-life extension components include polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxyethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrates, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, Examples include polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene maleic anhydride copolymer, polystyrene maleic anhydride copolymer, poly(1-hydroxymethyethylene hydroxymethylformal) (PHF), zwitterionic polymers, phosphorylcholine-containing polymers, and polymers containing MPC, poly(Glyx-Sery), hyaluronic acid (HA), heparosan polymer (HEP), Fleximer, dextran, and polysialic acid (PSA).

[0186] In one embodiment, the half-life extension portion may be conjugated to the antibody via the free amino group of the protein using an N-hydroxysuccinimide (NHS) ester. Reagents intended for conjugation to amine groups may react randomly with the ε-amine group of lysine, the α-amine group of the N-terminal amino acid, and the δ-amine group of histidine. In some embodiments, the conjugate includes a polymer conjugated to cysteine ​​or the free amino group of the protein using, for example, an N-hydroxysuccinimide (NHS) ester. In some embodiments, the conjugate includes a polymer conjugated to the ε-amine group of lysine, the α-amine group of the N-terminal amino acid, and / or the δ-amine group of histidine.

[0187] However, the anti-VEGF-A antibodies disclosed herein have many amine groups that can be used for polymer conjugation. Therefore, polymer conjugation to free amino groups may adversely affect the ability of the antibody protein to bind to VEGF.

[0188] In some embodiments, the half-life extension portion is attached to one or more free SH groups by any suitable thiol-reactive chemical reaction (including, but not limited to, maleimide chemical reactions), or by attaching a polymer hydrazide or polymer amine to the antibody glycan after pre-oxidation. In some embodiments, maleimide coupling is used. In some embodiments, the coupling occurs with naturally occurring or genetically engineered cysteine.

[0189] In some embodiments, the polymer is covalently attached to a cysteine ​​residue introduced into an anti-VEGF-A antibody by site-directed mutagenesis. In some embodiments, the cysteine ​​residue is utilized in the Fc region of the antibody. In some embodiments, sites for introducing the cysteine ​​residue into the Fc region are provided in International Publication No. 2013 / 093809, U.S. Patent No. 7,521,541, International Publication No. 2008 / 020827, U.S. Patent No. 8,008,453, U.S. Patent No. 8,455,622, and U.S. Patent Application Publication No. 2012 / 0213705, which are incorporated herein by reference in all purposes. In some embodiments, the cysteine ​​mutations are Q347C (EU numbering) and L443C, which refer to the human IgG heavy chain in EU numbering.

[0190] In some embodiments, a conjugate is provided comprising an antibody and a high molecular weight polymer acting as a half-life extender. In some embodiments, the conjugate comprises an antibody conjugated to a zwitterionic polymer, the polymer being formed from one or more monomer units, at least one of which has a zwitterionic group. In some embodiments, the zwitterionic group is phosphorylcholine.

[0191] In some embodiments, one of the monomer units is HEMA-PC. In some embodiments, a polymer is synthesized from a single monomer that is HEMA-PC.

[0192] In some embodiments, some antibody conjugates have two, three, or more polymer arms, and the monomer is HEMA-PC. In some embodiments, the conjugate has two, three, four, five, six, seven, eight, nine, ten, eleven, or twelve polymer arms, and the monomer is HEMA-PC. In some embodiments, the conjugate has three, six, or nine arms. In some embodiments, the conjugate has nine arms.

[0193] In some embodiments, the polymer-antibody conjugate has a polymer moiety having a molecular weight of 100,000 Da to 1,500,000 Da. In some embodiments, the conjugate has a polymer moiety having a molecular weight of 500,000 Da to 1,000,000 Da. In some embodiments, the conjugate has a polymer moiety having a molecular weight of 600,000 Da to 800,000 Da. In some embodiments, the conjugate has a polymer moiety having a molecular weight of 600,000 Da to 850,000 Da and has nine arms. When indicating the molecular weight of an antibody conjugated to a polymer, the molecular weight is the sum of the molecular weight of the protein (including any glycans bound to it) and the molecular weight of the polymer.

[0194] In some embodiments, the polymer component of the conjugate protein 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., 100,000~1,000,000 Da, 300,000~950,000 Da, 400,000~800,000 Da, 500,000~750,000 Da, 600,000~700,000 Da, etc.). 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 or OG1802.

[0195] In some embodiments, an anti-VEGF-A antibody is provided having a HEMA-PC polymer with a molecular weight of approximately 100 kDa to approximately 1650 kDa, as measured by Mw. In some embodiments, the molecular weight of the polymer, as measured by Mw, is approximately 500 kDa to approximately 1000 kDa. In some embodiments, the molecular weight of the polymer, as measured by Mw, is approximately 600 kDa to approximately 900 kDa. In some embodiments, the molecular weight of the polymer, as measured by Mw, is 750 kDa ± 15%.

[0196] In some embodiments, the polymer is prepared from an initiator suitable for ATRP having one or more polymer initiation sites. In some embodiments, the polymer initiation site has a 2-bromoisobutyrate site. In some embodiments, the initiator has three or more polymer initiation sites. In some embodiments, the initiator has 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. In some embodiments, the initiator has 3, 6, or 9 polymer initiation sites. In some embodiments, the initiator has 9 polymer initiation sites. In some embodiments, the initiator is OG1786.

[0197] Anti-VEGF-A antibodies can be produced by recombinant expression, including: (i) genetic engineering of recombinant DNA, and (ii) transfection, for example, but not limited to (iii) introducing recombinant DNA into prokaryotic or eukaryotic cells by injection, electroporation, or microinjection; (iv) culturing transformed cells; (iv) expressing antibodies, for example by constitutive or inducible expression; (v) isolating antibodies, for example from culture medium or by recovery of transformed cells; and (vi) obtaining purified antibodies.

[0198] Anti-VEGF-A antibodies can be produced by expression in suitable prokaryotic or eukaryotic host systems, characterized by the production of pharmacologically acceptable antibody molecules. Examples of eukaryotic cells include mammalian cells such as CHO, COS, HEK293, BHK, SK-Hip, and HepG2. Other suitable expression systems include prokaryotes (e.g., Escherichia coli with the pET / BL21 expression system), yeast (Saccharomyces cerevisiae and / or Pichia pastoris systems), and insect cells.

[0199] A wide variety of vectors can be used for the preparation of the antibodies disclosed herein, selected from eukaryotic expression vectors and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include, but are not limited to, plasmids such as preset, pet, and pad, and promoters used in prokaryotic expression vectors include, but are not limited to, one or more of lac, trc, trp, recA, or araBAD. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, but are not limited to, vectors using promoters such as AOX1, GAP, GAL1, or AUG1, or, but are not limited to, vectors such as pAO, pPIC, pYES, or pMET; (ii) for expression in insect cells, but are not limited to, vectors using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, or polh, or, but are not limited to, pMT, pAc5, pIB, pMIB , or vectors such as pBAC, and (iii) in the case of expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV, but not limited to those using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin, and in one embodiment, vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpesvirus, or retrovirus.

[0200] Analysis of mixed formulations and aggregates In some embodiments, a method for analyzing a mixed formulation sample (or therapeutically acceptable composition) is presented. In some embodiments, the method may include providing a mixed formulation sample (or therapeutically acceptable composition sample). In some embodiments, the mixed formulation sample (or therapeutically acceptable composition sample) comprises a first antibody, which is an anti-VEGF-A antibody conjugated to a polymer, and a second antibody, which is an anti-VEGF-A antibody not conjugated to a polymer. In some embodiments, the method further comprises a first run. In some embodiments, during the first run, the mixed formulation (or therapeutically acceptable composition) is passed through a pre-filtration step to prepare a first filtered mixed formulation. In some embodiments, the first filtered mixed formulation is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation. In some embodiments, the method further comprises a second run. In some embodiments, during the second run, proteins bound to the CEX column are eluted to prepare a third filtered mixed formulation. In some embodiments, the third filtered mixed formulation is subjected to size exclusion exchange (SEC) high-performance liquid chromatography (HPLC). The mixture is passed through a column to prepare a fourth filtered mixed formulation. In some embodiments, a solvent of a specific ionic strength is used to enable the distribution of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method is used to distribute the mixed formulation (or therapeutically acceptable combination) based on differences in the charge and size variants of the components. It enables the analysis of the finished product.

[0201] In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises a first antibody conjugated to a polymer and a second antibody not conjugated to a polymer, and one or both antibodies are not anti-VEGF-A antibodies. In some embodiments, the first and second antibodies in the mixed formulation (or therapeutically acceptable composition) are the same antibody. In some embodiments, the first and second antibodies in the mixed formulation are different antibodies. In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises a first protein moiety conjugated to a polymer and a second protein moiety not conjugated to a polymer. In some embodiments, the protein moiety is not an antibody. In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises a mixture of an antibody and another protein moiety. In some embodiments, both the first and second protein or antibody moieties are conjugated to a polymer. In some embodiments, the first and second protein or antibody moieties are conjugated to the same polymer. In some embodiments, the first and second protein or antibody moieties are conjugated to different polymers. In some embodiments, the first and second antibody or protein moieties are not conjugated to the polymer. In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises three or more antibody or protein moieties.

[0202] In some embodiments, the proteins or antibodies in the mixed formulation (or therapeutically acceptable composition) are present in a desired compositional percentage. In some embodiments, the compositional percentage is calculated by determining the amount of one component of the mixed formulation (or therapeutically acceptable composition) in units of mass (e.g., μg), dividing that amount by the total amount of all components in the composition in units of mass, and multiplying by 100.

[0203] In some embodiments, the protein or antibody in the mixed formulation (or therapeutically acceptable composition) is present in a desired molar ratio of the unconjugated protein or antibody to the conjugated protein or antibody.

[0204] In some embodiments, the percentage of conjugated protein relative to unconjugated protein (e.g., % total molar amount) is calculated by (1) measuring the conjugated protein portion and the unconjugated protein portion in mg / mL; (2) converting the mg / mL values ​​of the conjugated protein portion and the unconjugated protein portion to molecular weight measured in kDa; and (3) dividing the molecular weight of each of the conjugated protein portion and the unconjugated protein portion by the total molecular weight of the conjugated protein portion and the unconjugated protein portion in the composition, multiplying by 100, and obtaining the percentage of the respective total molar amounts.

[0205] 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).

[0206] In some embodiments, the mixed 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 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.).

[0207] In some embodiments, the mixed therapeutically acceptable composition comprises 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 5-93%, 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 consists of the first protein. In some embodiments, the mixed composition is prepared by combining the second protein with the conjugate at 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 mixed 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).

[0208] 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 or protein moiety, and a first protein conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) may be referred to as a conjugated protein or protein moiety.

[0209] In some embodiments, the percentage composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody) is between 5% and 6%, with the remainder comprising the conjugated protein moiety or protein moiety. As used herein, “remainder” means the portion of the total protein mass weight concentration of the composition that is not the unconjugated protein (e.g., the second protein) (excluding the contribution of the polymer conjugated to the first protein to the mass weight concentration), where the percentages of the unconjugated protein (e.g., the second protein) and the remainder together constitute 100% of the total protein mass weight concentration. For example, 5% to 6% of the total mass weight of the total protein concentration in the mixture is non-conjugate protein, and 94% to 95% of the total mass weight of the protein concentration in the mixture is conjugate protein, where the percentages add up to 100%. In some embodiments, the composition percentage of non-conjugate protein (e.g., non-conjugate antibody) is 6% to 7%, with the remainder being conjugate protein. In some embodiments, the composition percentage of non-conjugate protein (e.g., non-conjugate antibody) is 7% to 8%, with the remainder being conjugate protein. In some embodiments, the composition percentage of non-conjugate protein (e.g., non-conjugate antibody) is 8% to 9%, with the remainder being conjugate protein. In some embodiments, the composition percentage of non-conjugate protein (e.g., non-conjugate antibody) The percentage is 9% to 10%, with the remainder being conjugated proteins. In some embodiments, the percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 10% to 11%, with the remainder being conjugated proteins. In some embodiments, the percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 11% to 12%, with the remainder being conjugated proteins. In some embodiments, the percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 12% to 13%, with the remainder being conjugated proteins. In some embodiments, the percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 13% to 14%, with the remainder being conjugated proteins. In some embodiments, the percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 14% to 15%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 15% to 16%, with the remainder being 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 consisting of 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%.

[0210] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 25% to 26%, with the remainder being conjugate proteins or protein moieties (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 conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 28% to 29%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 29% to 30%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 30% to 31%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 31% to 32%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins (e.g., In some embodiments, the composition percentage of non-conjugate antibodies is 32% to 33%, with the remainder being conjugate 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 being 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 consisting of conjugate proteins.In some embodiments, the percentage of non-conjugate proteins (e.g., non-conjugate antibodies) in the composition is 44% to 45%.

[0211] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or fusion constructs) is 45% to 46%, with the remainder being conjugate proteins or protein moieties (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%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 51% to 52%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 52% to 53%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 53% to 54%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 54% to 55%, with the remainder being conjugate proteins. In some embodiments, non-conjugate proteins (e.g., In some embodiments, the composition percentage of non-conjugate antibodies is 55% to 56%, with the remainder being conjugate 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 being 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%.

[0212] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 65% to 66%, with the remainder being conjugate proteins or protein moieties (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, 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 consisting of 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. Contains gate 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%.

[0213] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 85% to 86%, with the remainder being conjugate proteins or protein moieties (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.

[0214] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 5% to 25%, with the remainder being conjugate proteins or protein moieties (e.g., conjugate antibodies or conjugate fusion constructs). In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 5% to 10%, 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, 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, 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 being 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%.

[0215] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 10% to 12.5%, with the remainder being conjugate proteins or protein moieties (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, 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 consisting of 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%.

[0216] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 12.5% ​​to 15%, with the remainder being conjugate proteins or protein moieties (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, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 12.5% ​​to 80%, 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 85%, with the remainder being conjugate proteins. In some embodiments, the percentage of non-conjugate proteins (e.g., non-conjugate antibodies) in the composition is 12.5% ​​to 90%.

[0217] In some embodiments, non-conjugate proteins or protein moieties (e.g.) For example, the composition percentage of a non-conjugate antibody or non-conjugate fusion construct is 15% to 17.5%, with the remainder being a conjugate protein or protein moiety (e.g., a conjugate antibody or fusion construct). In some embodiments, the composition percentage of a non-conjugate protein (e.g., a non-conjugate antibody) is 15% to 20%, with the remainder being a conjugate protein. In some embodiments, the composition percentage of a non-conjugate protein (e.g., a non-conjugate antibody) is 15% to 25%, with the remainder being a conjugate protein. In some embodiments, the composition percentage of a non-conjugate protein (e.g., a non-conjugate antibody) is 15% to 30%, with the remainder being a conjugate protein. In some embodiments, the composition percentage of a non-conjugate protein (e.g., a non-conjugate antibody) is 15% to 35%, with the remainder being a conjugate protein. 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%.

[0218] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 20% to 25%, with the remainder being conjugate proteins or protein moieties (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. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 55%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 60%, 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 25% to 65%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 70%, with the remainder being conjugate proteins. In some embodiments, the composition percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 25% to 75%, with the remainder being conjugate 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.

[0219] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 5%, with the remainder being conjugate proteins or protein moieties (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. Includes. 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%.

[0220] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 26%, with the remainder being conjugate proteins or protein moieties (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%.

[0221] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 36%, with the remainder being conjugate proteins or protein moieties (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 being 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%.

[0222] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 46%, with the remainder being conjugate proteins or protein moieties (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%.

[0223] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 56%, with the remainder being conjugate proteins or protein moieties (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%.

[0224] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 66%, with the remainder consisting of conjugate proteins or protein moieties (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%. In some embodiments, the percentage composition of the unconjugated protein (e.g., unconjugated antibody) is 68%, with the remainder comprising the conjugated protein. In some embodiments, the percentage composition of the unconjugated protein (e.g., unconjugated antibody) is 69%, with the remainder comprising the conjugated protein. In some embodiments, the percentage composition of the unconjugated protein (e.g., unconjugated antibody) is 70%, with the remainder comprising the conjugated protein. In some embodiments, the percentage composition of the unconjugated protein (e.g., unconjugated antibody) is 11%, with the remainder comprising the conjugated protein. In some embodiments, the composition percentage of conjugated proteins (e.g., non-conjugated antibodies) is 72%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 73%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 74%, with the remainder being conjugated proteins. In some embodiments, the composition percentage of non-conjugated proteins (e.g., non-conjugated antibodies) is 75%.

[0225] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate ) is 76%, with the remainder being conjugate proteins or protein moieties (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%.

[0226] In some embodiments, the composition percentage of non-conjugate proteins or protein moieties (e.g., non-conjugate antibodies or non-conjugate fusion constructs) is 86%, with the remainder being conjugate proteins or protein moieties. In some embodiments, the composition percentage of non-conjugate proteins (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 this composition, the percentage of non-conjugate proteins (e.g., non-conjugate antibodies) is 90%.

[0227] In some embodiments, the ratio of the molecular weight of a non-conjugated protein or protein moiety (e.g., a second protein or protein moiety not conjugated to the phosphorylcholine-containing polymer) to the polymer in the formulation (or therapeutically acceptable composition) can be any suitable ratio. In some embodiments, the ratio is at most about 1:2, e.g., 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.

[0228] The protein conjugated to the phosphorylcholine-containing polymer and the unconjugated protein or protein moiety 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 a percentage within the range defined by at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or about 100%, or 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.

[0229] 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 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 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 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) The protein (not conjugated with a phosphorylcholine-containing polymer) has a molecular weight of approximately 150 kDa. In some embodiments, the second protein (e.g., an unconjugated protein or a protein not conjugated with a phosphorylcholine-containing polymer) has a molecular weight of approximately 200 kDa.

[0230] 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, 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 therapeutic proteins 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 (or therapeutically acceptable composition). 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 a formulation (or therapeutically acceptable composition). Any antibody or fusion construct of any formulation or composition described herein may or may not contain C-terminal lysine.

[0231] In some embodiments, sample analysis includes evaluation of the sample by methods including, but not...

Claims

1. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, In the first run, the mixed formulation is passed through a pre-filtration step to prepare a first filtered mixed formulation, and the first filtered mixed formulation is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

2. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. Prepare a mixed therapeutic composition containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, In the first run, the mixed therapeutic composition is passed through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and the first filtered mixed therapeutic composition is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed therapeutic composition in the CEX column; The method enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. method.

3. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. Prepare a mixed therapeutic formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The mixed therapeutic active formulation is passed through a pre-filtration process to prepare a first filtered mixed therapeutic active formulation, and the first filtered mixed therapeutic active formulation is subjected to cation exchange chromatography (C EX) The first run is passed through a column to prepare the second filtered mixed therapeutic agent; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic agents are prepared in the second run; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed therapeutic agent in the CEX column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. method.

4. An apparatus comprising a tandem configuration of CEX and SEC HPLC columns for use in analyzing a mixed formulation sample comprising a) a first antibody which is an anti-VEGF-A antibody conjugated to a polymer and b) a second antibody which is an anti-VEGF-A antibody not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run) is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation. A device including a device.

5. An apparatus comprising a tandem configuration of a CEX column and an SEC HPLC column for use in analyzing a sample of a mixed therapeutic composition comprising a) a first antibody which is an anti-VEGF-A antibody conjugated to a polymer and b) a second antibody which is an anti-VEGF-A antibody not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, A first run in which the mixed therapeutic composition is passed through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and the first filtered mixed therapeutic composition is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic compositions are prepared in the second run A device including a device.

6. An apparatus comprising a tandem configuration of a CEX column and an SEC HPLC column for use in analyzing a sample of a mixed therapeutic active formulation containing a) a first antibody which is an anti-VEGF-A antibody conjugated to a polymer and b) a second antibody which is an anti-VEGF-A antibody not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, In the first run, the mixed therapeutic agent is passed through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and the first filtered mixed therapeutic agent is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is passed through a size exclusion exchange (SEC) high-performance liquid chromatography (HPLC) column. A fourth filtered mixed therapeutic active formulation is prepared, second run A device including a device.

7. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; The method described above reduces the presence of antibody aggregates in the formulation; The polymer of the first antibody comprises a phosphorylcholine-containing polymer, the polymer covalently bonded to the first antibody at a cysteine ​​outside the variable region of the first antibody, the cysteine ​​being substituted with a non-cysteine ​​amino acid occurring at the same position in the sequence, the first antibody comprising a light chain and a heavy chain, the heavy chain comprising an Fc region, the cysteine ​​being within the Fc region of the heavy chain, the sequence of the heavy chain comprising SEQ ID NO: 1 (with or without C-terminal lysine), and the sequence of the light chain comprising SEQ ID NO: 2; The antibody conjugate has the following structure: 【Chemistry 1】 In the formula, X is a) an -OR where R is -H, methyl, ethyl, propyl, or isopropyl; b) -H; or c) a halogen; (or optionally, X is a) an -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;) During the ceremony: Each heavy chain of the first antibody is represented by the letter H, and each light chain of the first antibody is represented by the letter L; The polymer is bound to the first antibody via a cysteine ​​sulfhydryl at position 449, numbered in SEQ ID NO: 1, and this binding is shown on one of the heavy chains; PCs are 【Chemistry 2】 In the formula, the dashed line indicates the bonding point to the remainder of the polymer; n1, n2, n3, n4, n5, n6, n7, n8, and n9 are n1, n2, n3, n4, n5, n6, n7, n8 and n9 may be the same or different, such that the sum of n8 and n9 is 2500 ± 15%; The second antibody comprises a light chain and a heavy chain, the heavy chain comprising an Fc region, cysteine ​​located within the Fc region of the heavy chain, the sequence of the heavy chain comprising SEQ ID NO: 1 (with or without C-terminal lysine), and the sequence of the light chain comprising SEQ ID NO: 2; The mixed formulation sample passes sequentially through a prefilter, the CEX column, and the SEC HPLC column, and the tandem CEX and SEC HPLC columns consist of a Shodex SP825 column with an inner diameter (i.d.) × length dimension of 9.0 × 75 mm and a TSKgel G3000SWxl column with an inner diameter (i.d.) × length dimension of 7.8 × 300 mm; The isocratic operating conditions include a flow rate of 0.5 ml / min, and the buffer consists of 20 mM sodium acetate (pH 5) in any amount between 50 mM NaCl and 5 M NaCl; The proportion of the second antibody is any proportion between 0% and 20%; The aforementioned mixed formulation sample contains any amount of protein between 25 μg and 1340 μg; The method further includes evaluating high molecular weight aggregates in the purified mixed formulation by a method comprising SEC profile analysis and SDS PAGE gel; The method further includes evaluating the proportion of the second antibody in the purified mixed formulation by SEC profile analysis; The first run separates the first antibody from the second antibody and antibody aggregates. The step for eluting the CEX-bound free protein during the second run includes a high-salt concentration pulse with 1 M NaCl, During the second run, the fourth filtered mixed formulation is passed through a pre-filtration step to prepare a fifth filtered mixed formulation, the fifth filtered mixed formulation is passed through a CEX column to prepare a sixth filtered mixed formulation, and the sixth mixed formulation is passed through an SEC HPLC column; The filtered mixed sample is subjected to size exclusion exchange (SEC HPL). Passing through a C column separates the second antibody from the antibody aggregates. method.

8. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, In the first run, the mixed formulation is passed through a pre-filtration step to prepare a first filtered mixed formulation, and the first filtered mixed formulation is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; To enable the distribution of the mixed formulation in the CEX column, use a solvent with a specific ionic strength; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

9. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. Prepare a mixed therapeutic composition containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, In the first run, the mixed therapeutic composition is passed through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and the first filtered mixed therapeutic composition is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. In the second run, four filtered mixed therapeutic compositions are prepared; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed therapeutic composition in the CEX column; The method enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. method.

10. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. Prepare a mixed therapeutic formulation containing the following: a. Fusion proteins conjugated to polymers; and b. A second protein not conjugated to the polymer; ii. The first run, In the first run, the mixed therapeutic agent is passed through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and the first filtered mixed therapeutic agent is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic agents are prepared in the second run; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed therapeutic agent in the CEX column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. method.

11. An apparatus comprising a tandem configuration of a CEX column and an SEC HPLC column for use in purifying a mixed formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, The first run involves passing the mixed formulation through a pre-filtration step to prepare a first filtered mixed formulation, and passing the first filtered mixed formulation through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run) is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation. A device including a device.

12. An apparatus comprising a tandem configuration of a CEX column and an SEC HPLC column for use in purifying a mixed therapeutic composition sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, A first run in which the mixed therapeutic composition is passed through a pre-filtration step to prepare a first filtered mixed therapeutic composition, and the first filtered mixed therapeutic composition is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic composition; and ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic composition, and the third filtered mixed therapeutic composition is subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic compositions are prepared in the second run A device including a device.

13. An apparatus comprising a tandem configuration of a CEX column and an SEC HPLC column for use in purifying a mixed therapeutic active formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein not conjugated to the polymer, The aforementioned purification process is as follows: i. The first run, In the first run, the mixed therapeutic agent is passed through a pre-filtration step to prepare a first filtered mixed therapeutic agent, and the first filtered mixed therapeutic agent is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed therapeutic agent; ii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed therapeutic agent, and the third filtered mixed therapeutic agent is then subjected to size exclusion exchange. (SEC) exchange (high-performance liquid chromatography) is passed through a high-performance liquid chromatography (HPLC) column. Four filtered mixed therapeutic active formulations are prepared, second run A device including a device.

14. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. The first run, In the first run, the mixed formulation is passed through a pre-filtration step to prepare a first filtered mixed formulation, and the first filtered mixed formulation is passed through a cation exchange chromatography (CEX) column to prepare a second filtered mixed formulation; iii. The second run, The protein bound to the CEX column is eluted to prepare a third filtered mixed formulation, and the third filtered mixed formulation is subjected to a size exclusion exchange (S The second run is passed through a high-performance liquid chromatography (HPLC) column to prepare the fourth filtered mixed formulation; iv. Using a solvent of a specific ionic strength to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

15. A method for analyzing a mixed formulation sample, the method comprising: i. Prepare a mixed formulation containing the following: a. A first antibody which is an anti-VEGF-A antibody conjugated to a polymer; and b. A second antibody which is an anti-VEGF-A antibody not conjugated to the polymer; ii. Loading the sample into an HPLC system, wherein the HPLC system is configured such that an HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed formulation is injected and passed through the system of ii; iv. A second run in which a concentrated salt is injected to elute the bound fraction of the CEX column and separate it by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

16. A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into an HPLC system, wherein the HPLC system is configured such that an HPLC pump is first connected to an autoinjector, followed by a prefilter, and downstream thereafter two tandem columns, the first of which is a cation exchange column (CEX), and the second column downstream of the CEX column is a size exclusion chromatography (SEC) column; iii. A first run into which the mixed formulation is injected and passed through the system of ii; iv. A second run in which a concentrated salt is injected to elute the bound fraction of the CEX column and separate it by the SEC column; A solvent of a specific ionic strength is used to enable the distribution of the mixed formulation in the CEX column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

17. A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and the flow of the CEX A switch valve follows, allowing the discharge to be directed to multiple possible targets; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. method.

18. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; The method enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. method.

19. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; The method described above enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. method.

20. An apparatus including a tandem HPLC system for use in analyzing a mixed formulation sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. Device.

21. An apparatus including a tandem HPLC system for use in analyzing a sample of a mixed therapeutic composition containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. Device.

22. An apparatus including a tandem HPLC system for use in analyzing a sample of a mixed therapeutic active ingredient containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. Device.

23. A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; An additional pump is used to pulse elute the CEX-bound fraction upon The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. method.

24. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; An additional pump is used to pulse-elute the CEX-bound fraction. The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. method.

25. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; An additional pump is used to pulse-elute the CEX-bound fraction. The method described above enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. method.

26. An apparatus including a tandem HPLC system for use in purifying a mixed formulation sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. Device.

27. An apparatus including a tandem HPLC system for use in purifying a sample of a mixed therapeutic composition containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. Device.

28. An apparatus including a tandem HPLC system for use in purifying a mixed therapeutic active ingredient sample containing a combination of two protein moieties, The first protein portion (A) is conjugated to a polymer, and the second protein portion (B) is not conjugated to a polymer. The aforementioned device includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components; The aforementioned sample is analyzed by a single continuous chromatography run. Device.

29. A method for analyzing a mixed formulation sample, the method comprising: i. To prepare a mixed formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. method.

30. A method for analyzing a sample of a mixed therapeutic composition, the method comprising: i. To prepare a mixed therapeutic composition comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. method.

31. A method for analyzing a sample of an effective mixed therapeutic preparation, the method comprising: i. To prepare a mixed therapeutic formulation comprising a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer and the second protein moiety (B) is not conjugated to a polymer; and ii. Loading the sample into a tandem HPLC system, wherein the HPLC pump is first connected to an auto-injector, followed by a pre-filter, then a cation exchange column (CEX), and a switch valve that allows the effluent of the CEX to be directed to a number of possible targets; The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The method described above enables the analysis of the mixed therapeutic formulation based on differences in charge and size variants of its constituent components. The aforementioned sample was analyzed by a single continuous chromatography run. The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. method.

32. An apparatus including a tandem configuration of an HPLC system for use in analyzing a mixed formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed formulation based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. Device.

33. An apparatus including a tandem configuration of an HPLC system for use in analyzing a sample of a mixed therapeutic composition comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed therapeutic composition based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. Device.

34. An apparatus including a tandem configuration of an HPLC system for use in analyzing a mixed therapeutic agent sample comprising (A) a first protein conjugated to a polymer and (B) a second protein not conjugated to the polymer, The HPLC system includes the following: a) HPLC pump connected to an auto-injector; b) Pre-filter; c) Cation exchange column (CEX); d) A switch valve that enables the flow of the CEX to be directed to multiple possible targets. The switch valve can guide the CEX effluent to the following: 1) Bypass loop; 2) The first SEC column; or 3) Second SEC column; The apparatus enables the analysis of the mixed therapeutic formulation based on differences in the charge and size variants of its constituent components. The sample was analyzed by a single continuous chromatography run; The conjugated sample is guided to the bypass loop, and the unconjugated sample is guided to the first or second SEC column. Device.

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