Methods for analyzing mixed compositions of protein and protein conjugates

Abstract

Provided herein are methods and apparatuses for analyzing mixed formulations of proteins and protein conjugates thereof. Some embodiments of the protein can be conjugated to a moiety, such as a HEMA-PC polymer. Some embodiments of the antibody conjugates can retain or enhance antibody activity. The antibody and conjugate thereof can be particularly useful for treating diabetic retinopathy.

Description

KDIAK.211WO PATENT APPLICATION METHODS FOR ANALYZING MIXED COMPOSITIONS OF PROTEIN AND PROTEIN CONJUGATES INCORPORATION BY REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Ser. No. 63 / 506764, filed June 7, 2023, which is hereby incorporated by reference in its entirety. SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled KDIAK211WO_SEQLIST.xml, created June 4, 2024, which is 196,690 bytes in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety. FIELD

[0003] The present disclosure relates to methods and apparatuses for analyzing mixed formulations and compositions comprising a mixture of unconjugated and conjugated proteins (e.g., antibodies and conjugates thereof). BACKGROUND

[0004] There are a variety of systems available for people to purify proteins in the biopharmaceutical context. Often, the various systems have specific pros and cons. SUMMARY

[0005] Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein theprotein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

[0006] Also provided is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents.

[0007] Also provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation,wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents. Also provided is an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed formulation sample comprising 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation. Further provided herein is an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed therapeutic composition sample comprising 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition. Further provided herein is an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed therapeutically effective formulation sample comprising a) a first antibody that is an anti-VEGF-A antibody conjugatedto a polymer and b) a second antibody that is an anti-VEGF-A antibody that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation. Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; and wherein the method reduces the presence of antibody aggregates in the formulation; and wherein the polymer of the first antibody comprises a phosphorylcholine containing polymer, wherein the polymer is covalently bonded to the first antibody at a cysteine outside a variable region of the first antibody, and wherein said cysteine replaces a non-cysteine amino acid that occurs in a same position in a sequence, wherein the first antibody comprises a light chain and heavy chain, said heavy chain comprising an Fc region, wherein the cysteine is in the Fc region of the heavy chain, wherein the sequence of the heavy chain comprises SEQ ID NO: 1 (with or without the C-terminal lysine), and wherein the sequence of the light chain comprises SEQ ID NO: 2; wherein the antibody conjugate has the following structure:where X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, or c) a halide; (or optionally where X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, c) any halogen, including –Br, –Cl, or –I, d) –SCN, or e) –NCS;) wherein: each heavy chain of the first antibody is denoted by the letter H, and each light chain of the first antibody is denoted by the letter L; the polymer is bonded to the first antibody through the sulfhydryl of a cysteine at position 449, as numbered in SEQ ID NO: 1, which bond is depicted on one of the heavychains; PC is , where the curvy line indicates the point of attachment to the rest of the polymer; and n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%; and wherein, the second antibody comprises a light chain and heavy chain, said heavy chaincomprising an Fc region, wherein a cysteine is in the Fc region of the heavy chain, wherein the sequence of the heavy chain comprises SEQ ID NO: 1, and wherein the sequence of the light chain comprises SEQ ID NO: 2; and wherein the mixed formulation sample travels sequentially through a prefilter, the CEX column, and the SEC HPLC column, wherein the tandem CEX and SEC HPLC columns are a Shodex SP825 column with internal diameter (i.d.) x length dimensions of 9.0x75mm and a TSKgel G3000SWxl column with i.d. x length dimensions of 7.8x300mm column arrangement; wherein the isocratic running conditions comprise a flow rate of 0.5ml / min, and the buffer comprises 20mM sodium acetate, pH 5 any amount between 50mM NaCl and 5M NaCl; wherein the percentage of the second antibody is any percentage between 0% and 20%; wherein the mixed formulation sample comprises any amount between 25ug and 1340ug of protein; wherein the method further comprises assessment for high molecular weight aggregates within the purified mixed formulation by methods comprising SEC profile analysis and SDS PAGE gel; and wherein the method further comprises assessment of the percentage of the second antibody in the purified mixed formulation by SEC profile analysis; wherein the first run separates the first antibody from the second antibody and antibody aggregates, wherein the step for elution of CEX bound free protein during the second run comprises a pulse of high salt at 1M NaCl, wherein during the second run, the fourth filtered mixed formulation is run through a prefiltration step to provide a fifth filtered mixed formulation, wherein the fifth filtered mixed formulation is run through a CEX column to provide a six filtered mixed formulation, wherein the sixth mixed formulation is run through a SEC HPLC column; and wherein running the filtered mixed sample through a size exclusion exchange SEC HPLC column separates the second antibody from antibody aggregates.

[0008] Also provided is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusionexchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Also provided herein is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises: a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents. Further provided herein is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatographycolumn to provide a fourth filtered mixed therapeutically effective formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the methodallows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0009] Also provided is an apparatus comprising a tandem series 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation. Provided herein is an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in purifying a mixed therapeutic composition sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition. Also provided is an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in purifying a mixed therapeutically effective formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; and ii. a second run, wherein theprotein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation.

[0010] Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Also provided is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; iii.a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of theconstituents. Further provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0011] Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed formulation is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Also provided is a method of analyzing a mixed therapeutic composition sample, the methodcomprising: i. providing a mixed therapeutic composition that comprises: 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 that is not conjugated to the polymer; ii.loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down- stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed therapeutic composition is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents. Further provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: 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 that is not conjugated to the polymer; ii. loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed therapeutically effective formulation is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0012] Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer; ii.loading the sample into an HPLCsystem where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column; iii.a first run, wherein the mixed formulation is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Also provided is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer; ii. loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed therapeutic composition is injected and run through the system of ii. iv.a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents. Further provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i.providing a mixed therapeutically effective formulation that comprises a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer; ii.loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down- stream of two tandem serially connected columns, wherein the first column is a cation- exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed therapeuticallyeffective formulation is injected and run through the system of ii. iv.a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; v. wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0013] Also provided is a method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Provided herein is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto- injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents. Further provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the methodallows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0014] Provided herein is an apparatus comprising a tandem HPLC system for use in analyzing a mixed formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents. Also provided is an apparatus comprising a tandem HPLC system for use in analyzing a mixed therapeutic composition sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents. Further provided is an apparatus comprising a tandem HPLC system for use in analyzing a mixed therapeutically effective formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

[0015] Provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valveenabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run. Also provided is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run. Provided herein is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run.

[0016] Also provided is an apparatus comprising a tandem HPLC system for use in purifying a mixed formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein theapparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run. Provided herein is an apparatus comprising a tandem HPLC system for use in purifying a mixed therapeutic composition sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run. Also provided is an apparatus comprising a tandem HPLC system for use in purifying a mixed therapeutically effective formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run.

[0017] Also provided herein is a method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column. Alsoprovided is a method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column. Further provided is a method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation- exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

[0018] Provided herein is an apparatus comprising a tandem series of HPLC system for use in analyzing a mixed formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation- exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) abypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column. Also provided is an apparatus comprising a tandem series of HPLC system for use in analyzing a mixed therapeutic composition sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column. Further provided is an apparatus comprising a tandem series of HPLC system for use in analyzing a mixed therapeutically effective formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 depicts a schematic of mixed sample preparation and run 1 and run 2 of the tandem HPLC system.

[0020] FIGS. 2A-2B depict schematics of options for the HPLC and column systems. FIG 2A depicts an arrangement including an IEX column and a single SEC column, whereas FIG. 2B depicts a schematic including an IEX with a switch valve downstream that allows for selection of one of two different SEC columns with 1 SEC column for run 1 and 1 SEC column for run 2.

[0021] FIG. 3 depicts the operation of the tandem HPLC column system in run 1 and run 2.

[0022] FIGS. 4A-4C depict SEC profiles of the CEX-SEC tandem series analysis for loading known amounts of purified OG1953 (conjugated) and OG1950 (unconjugated) separately, where OG1953 and OG1950 were not combined in a preformulated mixture. FIG. 4A depicts SEC profiles in stack plots for samples 1-6. FIG. 4B depicts SEC profile data organized into overlay plots, and FIG. 4C depicts a summary of the sample information.

[0023] FIGS. 5A-5C depicts SEC profiles of the CEX-SEC tandem series analysis of OG1953+7.5%OG1950 (OG1953_R50037) where OG1953 and OG1950 were combined in a preformulated mixture. FIG.5A depicts SEC profiles in stack plots for samples 1-6. FIG.5B depicts SEC profile data organized into overlay plots, and FIG. 5C depicts a summary of the sample information.

[0024] FIGS. 6A-6C depict SEC profiles showing identification of OG1950 peak and quantitative recovery of OG1950 from R50037 (7.5%). FIG. 6A depicts SEC profiles in stack plots for samples 1-5. FIG.6B depicts SEC profile data organized into overlay plots, and FIG.6C depicts a summary of the sample information.

[0025] FIGS. 7A-7B depict evaluation of the SEC calibration standard using 2D method (QC9661, QC9729B, & QC9742). FIG. 7A depicts SEC profiles in stack plots for samples 1-5. FIG.7B depicts SEC profile data organized into overlay plots.

[0026] FIG. 8A-8C depicts SEC profiles of an analysis of the impact of running buffer with different NaCl concentration on OG1953 and OG1950 partitioning (R50037 or 7.5%OG1950) in the Tandem series of CEX and SEC column.

[0027] FIGS. 9A-9C depict SEC profiles showing identification of a potential histidine formulation peak in R50037 sample using the 2D method with 214nm detection. FIG. 9A depicts SEC profiles in stack plots for samples 1-5. FIG. 9B depicts SEC profile data organized into overlay plots, and FIG. 9C depicts a summary of the sample information.

[0028] FIGS. 10A-10B depict SEC profiles showing carry over evaluation after the system was stripped with various buffers and the residual bound material was assessed.

[0029] FIGS. 11A-11E depict a summary of the sensitivity limit of detection (LOD), linearity and recovery data of from run 1 and run 2 of sets of OG1950 and R50037 samples with varying protein amounts loaded. FIG. 11A-B depict SEC profiles for OG1950PUR samples where 25-250ug of protein were injected. FIG.11C depicts SEC profiles for R50035 (7.5%) samples where 100-1340ug of protein was injected. FIG. 11D depicts details for the samples in FIG.11A-B and graphs plotting OG1950 monomer or aggregate peak against load. FIG.11E depicts details for the samples in FIG.11C and additional SEC profiles for the samples in run 2.

[0030] FIG.12A (Top left) #1 mixture was first cloudy but becomes cleared upon addition of 20μl buffer. (Top right) Mild precipitation was observed upon microfuge centrifugation at 5K rpm for 5min. (Bottom) All other formulation setups (#2-#5) show no cloudiness at all. Buffer B143-1 is 50mM Sodium Acetate pH 5, 0.025% PS20.

[0031] FIG. 12B depicts 2D method analysis showing elution peak(s) of free protein at the expected retention time of 21.8min, whereas the conjugate is eluted as a broad peak at ~15.6min. This was confirmed by (a) SEC calibration standard; (b) 15% mix formulation standard (R50031); and (c) injection OG1950 free protein standard injection. The percentage on the right of each chromatogram represents the partition of CEX-unbound (conjugate) to CEX-bound fraction (free IgG), they are in agreement with the expected percent free protein as shown in FIG.12D.

[0032] FIG.12C (Top) depicts overlay of all curves from FIG.12B to show overlap of eluted IgG peaks which were aligned with both the standard IgG and IgG standard from the SEC calibration standard. (Bottom left) Offset overlay of various injections for clear illustration of successful separation between unbound CEX conjugate fraction and the CEX bound IgG fraction. (Bottom right) is a close-up view to show the aggregated form of IgG that is clearly detected as shown in the arrows.

[0033] FIG. 12D depicts a peak area comparison of CEX bound (free protein) to unbound fraction (conjugate) showing agreement for the detected values in (X) as compared to the expected values in (Y). X shows the detected values from the Tandem method peak analysis, where the values can be calculated either using the detected peak area or the peakarea can be converted to protein mass with Beer’s law. Converting the detected peak area into protein mass allows for calculating the injection recovery, which can be compared to the Y values. The Y values are the input based on protein mass during the formulation preparation. Comparison of column X and Y determines recovery.

[0034] FIG.12E depicts method analysis showing elution peak(s) of free protein at the expected retention time of about 25.3min whereas the conjugate eluted at 15-15.2min as a broad peak. This was confirmed by (a) OG468 Fab standard injection; and (b) SEC calibration standard.

[0035] FIG. 12F (Top) depicts overlay of all curves from FIG. 12E showing overlap of eluted OG468 Fab peaks which are aligned with the standard OG468 standard that was eluted later than the IgG standard though elutes later than the 44 kDa standard of the SEC calibration standard. (Bottom) Offset overlay of various injections for clear illustration of separation between unbound CEX conjugate fraction and the CEX bound Fab fraction.

[0036] FIG. 12G depicts peak area comparison of CEX bound (free protein) to unbound fraction (conjugate) showing agreement to the expected ratios, see column (X) as compared to column (Y). X and Y can be calculated and compared as described in the description of FIG. 12D above. For sample #5, there was only 6M GuHCl elution as OG468 Fab was found to bind tightly to CEX column and unable to be eluted with 1M NaCl and only recovered by 6M GuHCl elution; which was later repeated for #3&4 with 2M NaCl as elution buffer but was only about 60% and 75% eluted for samples #3 and #4, respectively. However, the accumulated total eluted CEX bound fraction was in agreement with the expected number as shown in column (Y).

[0037] FIG. 13A depicts an HPLC system configuration with a multiple column combination comprising a 3-way valve, where an additional optional switch valve may be present before or after the IEX column.

[0038] FIG.13B depicts additional details regarding the configuration of the 3-way valve where the effluent of the IEX column can be directed to 1) a bypass loop; 2) a Shodex- 806M SEC column; or 3) a TSK G3000 SEC column.

[0039] FIG.13C depicts additional detail regarding the flow path options for each of 1-3 of FIG. 13B.

[0040] FIG.14A depicts a summary of the sample information for the samples used to test HPLC system configuration with a multiple column combination described in FIG.14. Samples include SEC calibration standards, OG1950 antibody, and two representative OG1953 drug product formulation prototypes, one with 20% OG1950 +80% OG1953, and the other with 15% OG1950 +75% OG1953.

[0041] FIG.14B depicts the column flow path matrix for samples described in FIG. 14A, where each unique column combination is designated with a different SOP391 version (v1-v6).

[0042] FIGS. 15A-15C depict analysis of the R50032 formulation (20% OG1950+80% OG1953) via the SOP391_v1 featuring the tandem CEX+SEC HPLC method without the 3-way switch valve. FIG.15A depicts stack plots at 280nm for the blank, OG1950 standard, and CEX bound and unbound fractions. FIG.15B depicts an overlay plot of the peaks shown in FIG.15A. FIG.15C depicts mass recovery from the CEX-unbound fraction and mass recovery from the individual peaks of the CEX-bound fraction, as well as total recovery.

[0043] FIGS. 16A-16F depict analysis of the R50032 and R50037 (7.5% OG1950+92.5% OG1953) formulations using the SOP391_v2 operation mode using the 3- way switch valve. FIG. 16A depicts the flow path through the 3-way valve in SOP391_v2 where the CEX-bound (E) and CEX-unbound (L) fractions pass through the TSK G3000 SEC column. FIG. 16B depicts the individual peaks observed in the E and L fractions. FIGS. 16C and 16D depict overlay graphs of the individual peaks shown in FIG.16B. The arrows in FIG. 16C-16D indicate a peak at 16.966 minutes which accounts for 5.2% of the total CEX-bound fraction. FIG. 16E depicts a summary of the injection queue including run time (minutes), injection volume (^l), and acquisition method for the E and L fractions of the R50032 and R50037 formulations. FIG. 16F depicts a summary of the peak data including retention time (minutes) area, % area, height for the E and L fractions of the R50032 and R50037 formulations.

[0044] FIGS. 17A-17D depict analysis of the R50032 formulations using the SOP391_v3 operation mode using the 3-way switch valve, where the CEX-unbound fraction (L) was directed via bypass instead of flow through SEC column. FIG. 17A depicts the flow path through the 3-way valve in SOP391_v3 where the CEX-unbound (L) fraction is directed through the bypass and the CEX-bound (E) fraction is analyzed using the TSK G3000 SECcolumn. FIG.17B depicts the individual peaks observed in the E and L fractions and compares the peaks of the E and L fractions between the SOP391_v2 and SOP391_v3 operation modes. FIG.17C depicts overlay graphs of the individual peaks shown in FIG.17B. FIG.17D depicts a summary of the injection queue including run time (minutes), injection volume (^l), and acquisition method for the E and L fractions of the R50032 formulations.

[0045] FIGS. 18A-18D depict analysis of the R50032 formulations using the SOP391_v4 operation mode using the 3-way switch valve. FIG. 18A depicts the flow path through the 3-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. FIG. 18B depicts the individual peaks observed in the E and L fractions. FIG. 18C depicts overlay graphs of the individual peaks shown in FIG.18B. FIG.18D depicts a summary of the injection queue including run time (minutes), injection volume (^l), and acquisition method for the E and L fractions of the R0032 formulations.

[0046] FIGS. 19A-19B depict evaluation of the overloading issue seen in R50032 using the SOP391_v4 operation mode. FIG. 19A depicts individual peaks seen in X: the tandem HPLC method using a CEX column and Shodex 806M_HQ SEC column where the SEC column was overloaded; and Y: where the R50032 formulation was analyzed on the Shodex 806M_HQ SEC column without CEX column operation. FIG. 19B depict an overlay graph of the individual peaks shown in FIG.19A.

[0047] FIGS. 20A-20C depict analysis of the R50032 formulations using the SOP391_v5 and SOP391_v6 operation modes using the 3-way switch valve. FIG.20A depicts the flow path through the 3-way valve in SOP391_v5 where the CEX-unbound fraction (L) passes through the bypass loop and the CEX-bound (E) fraction is analyzed by a Shodex 806M_HQ column. FIG. 20A also depicts the flow path through the 3-way valve in SOP391_v6 where both the CEX-unbound fraction (L) and the CEX-bound (E) fraction are analyzed using a Shodex 806M_HQ column. FIG. 20B depicts the individual peaks observed in the E and L fractions and compares the peaks of the E and L fractions between the SOP391_v5 and SOP391_v6 operation modes. FIG. 20C depicts overlay graphs of the individual peaks shown in FIG. 20B. The red arrows seen in FIG. 20B-20C indicate an equivalent to the fraction of SOP391_v1 seen at the 16 minute peak here eluted at approximately 20 minutes.

[0048] FIG. 21A depicts a comparison of the individual peak and total recovery data between the SOP391_v1-v6 operational modes for the R50032 formulation.

[0049] FIG. 21B depicts a comparison between the 16 minute peak fraction observed in SOP391_v1 compared to; X: SOP391_v2-v4 where the 16 minute peak was further separated into two populations by the TSK G3000 SEC column; and Y: SOP391_v5-v6 where the 16 min peak coeluted with the bulk of OG1953 conjugate.

[0050] FIGS. 22A-22B depict a study using the tandem HPLC method of SOP391_v3 evaluating stability of OG1950PUR and R50032 samples. FIG. 22A depicts the individual peaks observed in the CEX-unbound (L) and CEX-bound (E) fractions of an OG1950PUR sample stored at 37°C for 7 months, an R50032 formulation stored at 5°C for 3 months, and an R50032 formulation stored at 25°C for 3 months. FIG. 22B depicts overlay graphs of the individual peaks of FIG. 22A, where X indicates the peaks for the R50032 formulations stored at 5°C and 5°C for 3 months, and Y indicates the OG1950PUR sample stored at 37°C for 7 months.

[0051] FIG. 23 depicts an overview of the principle and operation of the tandem CEX-SEC method where the KSI-301DP formulation can comprise OG1953, OG1953’, OG1950, aggregated OG1950, and insoluble components within a buffer. The prefilter step removes the insoluble components. The OG1950 and aggregates bind the CEX column and the OG1953 is not bound to the column and can be analyzed by SEC. A pulse of 1M NaCl can elute the OG1950 and aggregates from the CEX column and the OG1950, aggregated OG1950, and any OG1953 can be distinguished by SEC.

[0052] FIG.24 depicts a plan for a program to evaluate the stability of the OG1953 bioconjugate containing 7.5, 10, 15, and 20% free OG1950 protein at different temperatures over time. The study evaluates the tandem analytical HPLC method performance in terms of recovery, robustness, specificity, reproducibility, and accuracy.

[0053] FIG.25 depicts a visual assessment of the samples at 1, 2, 3, and 6 months at 5, 25, and 37 °C. Arrows indicate samples with cloudy appearance.

[0054] FIG.26 depicts an assessment of protein concentration of samples stored at 5, 25, and 37 °C for 1, 2, and 6 months with an analysis of changes in protein concentration over time.

[0055] FIGS. 27A-27C depict an overview of all sample recovery and elution profiles. FIG. 27A depicts total mass recovery over time and temperature (top and bottom panel) for CEX-bound and CEX-unbound samples where close to 100% mass recovery was achieved for all samples. FIG. 27B depicts chromatograms for the CEX-unbound showing OG1953 conjugate peak (LP1) with instability population designated LP2. FIG. 27C depicts chromatograms for the CEX-bound fractions showing a 16 minute peak that splits into A and B and a P1, P1, and M which were OG1950 free protein peaks.

[0056] FIGS. 28A-28D depict a comparison of LP2, 16 minute peak and monomeric free protein for sample # 4 and a representative clinical formulation. FIG. 28A depicts a summary of the results obtained for sample #4, OG1953 KSI-301DP 103A, OG1953 KSI-301DS 101 samples stored under different storage conditions including mass recovery and percent 280nm signal for the individual peaks identified, where X highlights the 16 minute A and B peaks. FIG.28B depicts a chromatogram of the CEX-unbound fraction comparing the LP1 and LP2 peaks of the OG1953 KSI-301DP 103A frozen and 6M 25°C samples. FIG.28C- 28D depict chromatograms of the CEX-bound fraction indicating the 16 minute peak and Z describing monomer form protein (M) peak.

[0057] FIGS.29A-29D depict kinetic analysis of various peak fractions of sample #4 at different times and temperatures. FIG. 29A depicts a summary of the analysis of the all the peaks detected in the CEX-bound and CEX-unbound fractions of sample #4. FIG. 29B depicts a summary of the degradation and / or aggregation rates for all peaks at 25 °C. FIG.29C depicts the kinetic curve for all peaks at each sampling including CEX-unbound run peaks (LP1, and LP2) and CEX-bound run peaks (16 minute peak, P2 (dimer), P1, M (monomer). FIG.29D depicts the accompanying chromatogram for the CEX-bound run peaks.

[0058] FIGS. 30A-30D depict kinetic analysis of various peal fractions of sample #3 (15%) and #9 (20% iodoacetamide (IAM)) at different time and temperatures. FIG. 30A depicts a summary of the peak analysis of all the peaks detected in the CEX-bound and CEX- unbound fractions of sample #3, and a summary of the degradation and / or aggregation rates for all peaks at 25 °C. FIG. 30B depicts the kinetic curve for all peaks at each sampling of the #3 sample including CEX-unbound run peaks (LP1, and LP2) and CEX-bound run peaks (16 minute peak, P2 (dimer), P1, M (monomer). FIG.30C depicts a summary of the peak analysis of all the peaks detected in the CEX-bound and CEX-unbound fractions of sample #9, and asummary of the degradation and / or aggregation rates for all peaks at 25 °C. FIG. 30D depicts the kinetic curve for all peaks at each sampling of sample #9 including CEX-unbound run peaks (LP1, and LP2) and CEX-bound run peaks (16 minute peak, P2 (dimer), P1, M (monomer).

[0059] FIGS. 31A-31B depict a projection of two year stability of samples # 3, 4, and 9 at different temperatures. FIG.31A depicts a summary of the peak percent of all detected peaks in the CEX-inbound and CEX-bound fractions for samples #3, 4, and 9. FIG.31B depicts projections for the percent of total impurities (e.g., aggregates and / or degradation products) over a two year period for samples # 3, 4, and 9.

[0060] FIG. 32 depicts a summary of the mass recovery data for all injections of samples #3, 4, and 9 at different times and temperatures.

[0061] FIGS. 33A-33B depict graphs for 100%B buffer pulse elution profiles of the 20% antibody with 80% bioconjugate using pulse elution profiles #3-7. FIG. 33A depicts individual peaks and FIG. 33B depicts overlay plots.

[0062] FIGS. 34A-34B depict data analysis summary (FIG. 34A) and chromatogram comparisons of various pulse elution profiles (FIG.34B).

[0063] FIG.35 depicts OG1786.

[0064] FIG.36 depicts OG1802.

[0065] FIGS. 37A-37C depict updates to the projections of FIG. 37A-37C, where additional samplings were performed at 12 and 15 months for 5°C and 25°C, respectively.

[0066] FIGS. 38A-38D depict some embodiments of continuous 80min tandem separation method with PhotoDiol Array (PDA) detection set at 200-350nm. FIG.38A depicts the 2D contour view of elution time versus wavelength; FIG. 38B depicts the extracted wavelength profile at 280nm and the peak identification of the various eluted fractions collected for further characterization using SDS-PAGE analysis followed by Silver Staining, and results were shown in FIG.38C as a non-reducing gel and FIG. 38D as a reducing gel.

[0067] FIGS.39A-39C depict KSI-301 stability data up to 6 months under different temperature conditions; -20 ± 5°C (FIG.39A), 5 ± 3°C (FIG.39B), and 25 ± 2°C / 60±5% RH (relative humidity) (FIG.39C).

[0068] FIG.40 depicts KSI-501DS Batches 1-3 Lot Release Data.DETAILED DESCRIPTION

[0069] Some embodiments provided herein are methods and apparatuses for tandem HPLC. In some embodiments, the methods and apparatuses for tandem HPLC allow for one to analyze a mixed protein formulation.

[0070] Some embodiments provided herein are methods and apparatuses for tandem HPLC combining ion exchange chromatography (IEX), such as cation exchange chromatography (CEX) and size exclusion chromatography (SEC). The methods and apparatuses for tandem HPLC allow for one to analyze a formulation comprising a protein conjugated to a polymer, and a protein not conjugated to a polymer.

[0071] Provided herein are methods and apparatuses for analyzing a mixed formulation comprising a fast-acting component comprising an unconjugated protein and a longer-term acting component comprising protein conjugated to a polymer. In some embodiments, the methods and apparatuses provided herein are part of any suitable process for producing and purifying a mixed composition of some conjugated and unconjugated protein.

[0072] Several surprises have been discovered when mixing the ABCTMconjugate with free protein formulation in general. Firstly, there was 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 was a surprise finding that such mixture resulted in white turbid solution under certain condition such as (1) the solution pH was at or close to the isoelectric point (pI) of the free unconjugated protein, (2) concentration of the conjugate and / or free protein was too high, or (3) presence of certain excipients such as histidine. Turbid solution is not desirable for injectable pharmaceutical in general, especially for intravitreal injectable medicines. In some embodiments, the methods and apparatuses described herein contribute to the development of a stable and clear solution with up to 6 months stability and projected to have at least 12 months long term stability at 2-8°C.

[0073] OG1953 bioconjugate can be polydisperse due to the covalently conjugated polymer moiety, it can contain small population of molecules with molecular weights that overlap with the free unconjugated protein fraction, especially the aggregates of the free protein. Furthermore, the protein moiety can also possess unique charge variation. Both the size and charge variation pose complex analytical challenges for the existing analyticalmethods such as CEX-HPLC and SEC-HPLC method for monitoring the molecules based on charge and size, respectively. The situation can be further exacerbated over time and stress temperature as the impurities such as degradants or aggregates of the conjugate and protein overlap with each other rendering it technically impossible to use either method alone with good resolution for such analysis.

[0074] In some embodiments, the methods and apparatuses described herein can provide one or more of the following advantages: (1) Leverage the existing CEX-HPLC and SEC-HPLC analytical methods for either the conjugate or free unconjugated protein; (2) it can be used for both batch release and stability indicating; (3) it can be performed under non- destructive and native buffer condition to prevent in-process modification and artifact; (4) other than simple sample dilution, there should be no further sample manipulation nor purification prior the sample analysis (5) the method can meet the requirements for GMP validation such as accuracy, robustness, reproducibility, and specificity; (6) the method can be implemented and set up in an analytical laboratory equipped with common instrumentation and trained technical experts in the art; (7) the method can provide good resolution and quantitative information for various degradation products and / or aggregates (e.g., impurities), (8) the method can provide kinetic recovery (100% recovery) for the injected sample, which also means the method should have minimal carry-over issue; (9) the method can also have good dynamic range of operation such as tolerance to running buffer pH, salt concentration, sample loading, flow rate, operation temperature, etc.; (10) the method can be further integrated seamlessly with other analytical instrumentation and methods, for example the MALS detector, icIEF, Mass spectrometry, fluorescent detector, etc.; (11) buffer running condition that minimizes the disulfide shuffling, therefore the running buffer can be at the acidic range such as pH 4-6.5.

[0075] In some embodiments, the methods and apparatuses described herein can provide one or more of the following advantages: (1) isocratic chromatographic running condition to take advantage of the existing gradient elution characteristic of CEX-HPLC method, which the bulk of OG1953 conjugate is eluted with minimal to no premature leaching of bound OG1950 free protein and impurities such as degradants, aggregates, or fragments. Without being limited by theory, OG1953 conjugate binds weaker to the CEX column through the protein moiety due to the charge shielding effect of the large polymer, whereas the freeunconjugated protein and its protein aggregates elutes later, it should also noted that smaller polymers that exert less shielding effect on conjugated protein moiety could binds tighter to the CEX column and delays elution as compared to the bulk of the conjugate, this phenomena can be seen as the prolonged trailing of CEX or SEC elution after the bulk conjugate elution peak; (2) rapid synchronous and mass transfer of the CEX-bound fraction for SEC separation based on size, the general principal of SEC chromatography is based on the synchronous sample application with a sample volume no more than 5% of total SEC column volume, hence a sufficiently high salt elution pulse that elutes all CEX bound fraction at smallest possible volume can be used for efficient SEC separation. In the event of asynchronous elution such as insufficient salt concentration or too shallow elution speed, it could compromise the SEC resolution and separation efficiency.

[0076] In some embodiments, an advantageous feature of the current tandem chromatography method is the instant finger-printing ability. In some embodiments, the first dimension separation is a group specific chromatography such as ion exchanger chromatography (IEX) for charge specific separation (e.g. CEX or AEX), affinity chromatography based on specific group (e.g. protein A, protein G, protein L, M2 resin for flag-tag proteins, metal chelating resins such as Nickle-NTA for His-tag protein, dye-affinity resin, lectin-affinity resin for specific carbohydrate separation, hydrophobic interaction resin, ligand specific antibody-affinity capturing resin, etc.). In some embodiments, the second dimensional separation can be by size with SEC, which the SEC provides instant identity finger print profile for both the unbound and thereafter bound fraction of the group specific column in a single operation. In some embodiments, this is conceptually different from the conventional concept of two dimensional chromatography which aim to simply separate a complex mixture of analytes for a unique finger-print. In some embodiments, the current tandem HPLC method aims to provide convenient, simple and yet efficient instant finger printing of the first dimensional separation, when combining the effluent of the SEC with other detection instrumentations and techniques, it can be widely used in other areas of application.

[0077] In some embodiments, the method can also be operated under interruptive and uninterrupted (continuous) mode. In some embodiments, interruptive mode can have separate chromatographic runs for the sample injection run and pulse elution of the first dimensional bound fraction, therefore it produces two separate chromatograms for a singlesample analysis. In some embodiments, the major disadvantage of such operation can include one or more of: (1) undesirable baseline ramping signal in the beginning of each run, (2) unnecessary regulatory justification for integrating peaks from completely different chromatographic runs, (3) potential chromatographic signal discrepancy alignment issue. In some embodiments, there is also advantage when only the first dimensional column bound fraction is needed, one can design a significantly shortened method with the aid of a multi- column valve with a bypass loop as shown in FIG.13A, with this operation mode, the unbound fraction can be discarded using the bypass loop and not pass through the SEC column. In some embodiments, for uninterrupted mode, a more sophisticated instrument can require to equip with two pumps and able to operate with small residual volume to deliver a salt pulse for synchronous elution of first dimensional bound fraction, failure to meet this requirement can result in undesirable baseline shift. In some embodiments, the advantage of operating in uninterrupted mode is that all elution peak of unbound and bound fractions of the first dimensional column can be displayed in a single chromatogram.

[0078] In some embodiments, understanding of the molecules in the mix formulation is useful for the success of using the current tandem HPLC method. In some embodiments, the first dimensional column allows the bulk of interference or matrix effect to be removed. In some embodiments, the unbound fraction contains undesirable contaminants such as sample formulation buffer and free unconjugated polymer (OG1802). In some embodiments, selection of the salt concentration and pH of the mobile phase confers specificity of separation. In some embodiments, the salt concentration and pH of the mobile phase can be manipulated to control how thorough the interference is to be removed. In some embodiments, too aggressive salt and pH condition can result in premature elution of the bound fraction and can result in compromised method performance.

[0079] Some embodiments provided herein can include one or more of the following: (1) Simple, effective and seamless integration of the existing chromatography methods that resulted in high resolution separation of various peaks in a complex mixture formulation that is previously impossible with individual methods alone; (2) Real time online identity and purity method that allows instant identification of the various fractions prior and post addition to the combined formulation; (3) Its sensitivity, robustness, accuracy and reproducibility can be adapted for GMP validation of pharmaceutical manufacturing; (4) Itprovides a single integrated method that can allow defining, detection and quantification of various degradation products and / or aggregates under native running condition similar to both the large scale purification process and the final drug substance and drug product formulation buffer; (5) It can be used for both lot release and stability indicating; (6) it is versatile and is able to integrate seamlessly with other analytical techniques that complement and expand the current power of resolution to gain even more insight into the property of the mixed population in the pharmaceutical formulation.

[0080] In some embodiments, when used in the context of a pharmaceutical composition (e.g. OG1953 drug substance or drug product), the following advantages can be achieved: (1) under optimized chromatography condition, it can allow kinetic recovery of all injected samples with high resolution for all major products peaks including OG1953 conjugate (LP1) and monomeric IgG of OG1950 (M), and their degradation products and / or aggregates (e.g., impurities), which can include OG1953 related degradation products such as LP2 and ‘16min peak’, OG1950 related aggregates such as dimer (P2) and an alternative aggregated form of P1; (2) it is broadly applicable to a panel of different formulation with 1%, 7.5%, 10%, 15%, 20%, 49% and 89% free antibody mixed with various percent of OG1953 conjugate; furthermore, it can also be applicable to other molecules such as Fab with similar performance; (3) it allows defining the degradation products and / or aggregates, and formation kinetic over time and temperature in a long term stability study, which allows setting specification and prediction model to project the long term shelf-life.

[0081] Provided herein are methods and apparatuses mixed formulations of antibodies and conjugates thereof and other proteins and protein conjugates by tandem HPLC. In some embodiments, the conjugate can be used for the treatment of certain conditions, such as diabetic retinopathy and / or age-related macular degeneration.

[0082] In some embodiments, a method of analyzing a mixed formulation sample is provided. In some embodiments, the method comprises providing a mixed formulation that comprises a combination of two protein moieties. In some embodiments, the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer. In some embodiments, the method comprises loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein thefirst column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column. In some embodiments, the method comprises a first run. In some embodiments, during the first run, the mixed formulation is injected and run through the tandem CEX-SEC system. In some embodiments, the method comprises a second run. In some embodiments, during the second run, a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column. In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column. In some embodiments, the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

[0083] These and additional embodiments are provided below, following the definition section. DEFINITIONS

[0084] All terms can have their customary and ordinary meaning to one of ordinary skill int the art, in view of the present disclosure.

[0085] The term “percent composition” refers to the percent amount (in mass or concentration units) of a component present in a composition. Percent composition is calculated by determining the amount of a component in mass units (e.g., μg) or in concentration units (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 compositions and formulations of a conjugate and an unconjugated protein described herein, the amount of the unconjugated protein can be divided by the total amount of the protein mass in the solution (excluding the contribution from the polymer component of the conjugate to the mass of the conjugate) to obtain a percent composition.

[0086] As used herein, “% total molar amount” denotes the proportion (in percent) of the amount (in moles or a molar concentration) of one component of a composition relative to the amount(s) (in moles or a molar concentration) of one or more other component of the composition, that together make up the whole (100%). It is understood that percent composition and % total molar amount is effectively interchangeable where the molecular weight of all of the relevant components are known.

[0087] “Molar ratio” refers to the ratio of the amount (in moles) of free protein (e.g. OG1950) to conjugated protein (e.g. OG1953) in the formulation (based on the protein portion of the conjugate).

[0088] As used herein “unconjugated” and “free” with reference to a protein, protein moiety, or antibody are used interchangeably to denote the protein or antibody that is not conjugated to a polymer (e.g., not conjugated to a phosphorylcholine-containing polymer).

[0089] A “neovascular disorder” is a disorder or disease state characterized by altered, dysregulated or unregulated angiogenesis. Examples of neovascular disorders include neoplastic transformation (e.g. cancer) and ocular neovascular disorders including diabetic retinopathy and age-related macular degeneration.

[0090] In some embodiments, “impurities” refer to product related impurities such as degraded, aggregated, unconjugated (e.g., where the product of interest is conjugated), or modified proteins. In some embodiments, product unrelated impurities such as host cell protein, endotoxin, host cell DNA, are not considered “impurities” as defined herein.

[0091] An “ocular neovascular” disorder is a disorder characterized by altered, dysregulated or unregulated angiogenesis in the eye of a patient. Such disorders include optic disc neovascularization, iris neovascularization, retinal neovascularization, choroidal neovascularization, corneal neovascularization, vitreal neovascularization, glaucoma, pannus, pterygium, macular edema, diabetic retinopathy, diabetic macular edema, vascular retinopathy, retinal degeneration, uveitis, inflammatory diseases of the retina, and proliferative vitreoretinopathy.

[0092] The term antibody includes intact antibodies and binding fragments thereof. A binding fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of binding fragments include Fv, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments. scFv antibodies are described in Houston JS. 1991. Methods in Enzymol. 203:46-96. In addition, antibody fragments comprise single chain polypeptides having the characteristics of a VH domain, namely being able to assemble together with a VL domain, or of a VL domain, namely being able to assemble together with a VH domain to a functional antigen binding site and thereby providing the antigen binding property of full length antibodies.

[0093] Specific binding of an antibody to its target antigen(s) means an affinity of at least 106, 107, 108, 109, or 1010M-1. Specific binding is detectably higher in magnitude and distinguishable from non-specific binding occurring to at least one unrelated target. Specific binding can be the result of formation of bonds between particular functional groups or particular spatial fit (e.g., lock and key type) whereas nonspecific binding is usually the result of van der Waals forces. Specific binding does not however necessarily imply that an antibody or fusion protein binds one and only one target.

[0094] A basic antibody structural unit is a tetramer of subunits. Each tetramer includes two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. This variable region is initially expressed linked to a cleavable signal peptide. The variable region without the signal peptide is sometimes referred to as a mature variable region. Thus, for example, a light chain mature variable region means a light chain variable region without the light chain signal peptide. However, reference to a variable region does not mean that a signal sequence is necessarily present; and in fact signal sequences are cleaved once the antibodies or fusion proteins have been expressed and secreted. A pair of heavy and light chain variable regions defines a binding region of an antibody. The carboxy-terminal portion of the light and heavy chains respectively defines light and heavy chain constant regions. The heavy chain constant region is primarily responsible for effector function. In IgG antibodies, the heavy chain constant region is divided into CH1, hinge, CH2, and CH3 regions. The CH1 region binds to the light chain constant region by disulfide and noncovalent bonding. The hinge region provides flexibility between the binding and effector regions of an antibody and also provides sites for intermolecular disulfide bonding between the two heavy chain constant regions in a tetramer subunit. The CH2 and CH3 regions are the primary site of effector functions and FcR binding.

[0095] Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and define the antibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" segment of about 12 or more amino acids, with the heavy chain also including a "D" segment of about 10 or more amino acids. (See generally, FundamentalImmunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989), Ch. 7) (incorporated by reference in its entirety for all purposes).

[0096] The mature variable regions of each light / heavy chain pair form the antibody binding site. Thus, an intact antibody has two binding sites, i.e., is divalent. In natural antibodies, the binding sites are the same. However, bispecific antibodies can be made in which the two binding sites are different (see, 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 Immunol.148: 1547-1553). The variable regions all exhibit the same general structure of relatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminal to C-terminal, both light and heavy chains comprise the domains FRl, CDRl, FR2, CDR2, FR3, CDR3 and FR4. For convenience, the variable heavy CDRs can be referred to as CDRH1, CDRH2 and CDRH3; the variable light chain CDRs can be referred to as CDRL1, CDRL2 and CDRL3. The assignment of amino acids to each domain is in accordance with the definitions of Kabat EA, et al.1987 and 1991. Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD) or Chothia C, Lesk AM. 1987. Canonical Structures for the Hypervariable Regions of Immunoglobulins. J Mol Biol 196:901-917; Chothia C, et al. 1989. Conformations of Immunoglobulin Hypervariable Regions. Nature 342:877-883. Kabat also provides a widely used numbering convention (Kabat numbering) in which corresponding residues between different heavy chain variable regions or between different light chain variable regions are assigned the same number. Although Kabat numbering can be used for antibody constant regions, EU numbering is more commonly used, as is the case in this application. Although specific sequences are provided for exemplary antibodies disclosed herein, it will be appreciated that after expression of protein chains one to several amino acids at the amino or carboxy terminus of the light and / or heavy chain, particularly a heavy chain C-terminal lysine residue, can be missing or derivatized in a proportion or all of the molecules.

[0097] The term "epitope" refers to a site on an antigen to which an antibody or extracellular trap segment binds. An epitope on a protein can be formed from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

[0098] Antibodies that recognize the same or overlapping epitopes can be identified in a simple immunoassay showing the ability of one antibody to compete with the binding of another antibody to a target antigen. The epitope of an antibody can also be defined by X-ray crystallography of the antibody (or Fab fragment) bound to its antigen to identify contact residues.

[0099] Alternatively, two antibodies have the same epitope if all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0100] Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, 10x, 20x or l00x) inhibits binding of the reference antibody by at least 50%. In some embodiments the test antibody inhibits binding of the reference antibody by 75%, 90%, or 99% as measured in a competitive binding assay. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.

[0101] As used herein, “VEGF Trap” or similar term denotes the VEGF binding domains (e.g., VEGFR1 domain 2, VEGFR2 domain 3). This fragment allows for the protein to work as a VEGF trap, preventing VEGF from binding to cellularly expressed VEGFreceptors. An example of this sequence can be found in Table 3. In some embodiments, the VEGF Trap only includes VEGFR1 domain 2, VEGFR2 domain 3. Various embodiments of Trap proteins are known in the art and can be found, for example in U.S. Pub. No. 20150376271, the entirety of which, with respect to various VEGF Trap embodiments (which are VEGFR proteins or fragments thereof) and fusions thereof, is incorporated herein by reference. In some embodiments, the term “VEGF Trap” or similar term refers to a full length extracellular region or any portion thereof, or combination of portions from different VEGF receptors that can antagonize signaling between at least one VEGF and VEGFR.

[0102] In some embodiments, Size exclusion chromatography (SEC) can also be called gel permeation chromatography (GPC). In some embodiments, affinity chromatography column can also be called group specific affinity column.

[0103] The term "patient" includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment.

[0104] For purposes of classifying amino acids substitutions as conservative or nonconservative, 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 influencing chain orientation): gly, pro; and Group VI (aromatic side chains): trp, tyr, phe. Conservative substitutions involve substitutions between amino acids in the same class. Non- conservative substitutions constitute exchanging a member of one of these classes for a member of another.

[0105] Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention for a variable region or EU numbering for a constant region. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. Sequence identities of other sequences can be determined by aligning sequences using algorithms, such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Dr., Madison, WI, using default gap parameters, or by inspection, and the best alignment (i.e., resulting in the highest percentage of sequence similarity over a comparison window). Percentage of sequence identity is calculated by comparing two optimally aligned sequences over a window of comparison, determining the number of positions at which the identical residues occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0106] Compositions or methods "comprising" one or more recited elements can include other elements not specifically recited. For example, a composition that comprises antibody can contain the antibody alone or in combination with other ingredients.

[0107] The term "antibody-dependent cellular cytotoxicity", or ADCC, is a mechanism for inducing cell death that depends upon the interaction of antibody-coated target cells (i.e., cells with bound antibody) with immune cells possessing lytic activity (also referred to as effector cells). Such effector cells include natural killer cells, monocytes / macrophages and neutrophils. ADCC is triggered by interactions between the Fc region of an antibody bound to a cell and Fcy receptors, particularly FcȖRI and FcȖRIII, on immune effector cells such as neutrophils, macrophages and natural killer cells. The target cell is eliminated by phagocytosis or lysis, depending on the type of mediating effector cell. Death of the antibody-coated target cell occurs as a result of effector cell activity.

[0108] The term opsonization also known as "antibody-dependent cellular phagocytosis", or ADCP, refers to the process by which antibody-coated cells are internalized, either in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils and dendritic cells) that bind to an immunoglobulin Fc region.

[0109] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism for inducing cell death in which an Fc effector domain(s) of a target-bound antibody activates a series of enzymatic reactions culminating in the formation of holes in the target cell membrane. Typically, antigen-antibody complexes such as those on antibody- coated target cells bind and activate complement component Clq which in turn activates the complement cascade leading to target cell death. Activation of complement can also result indeposition of complement components on the target cell surface that facilitate ADCC by binding complement receptors (e.g., CR3) on leukocytes.

[0110] A humanized antibody is a genetically engineered antibody in which the CDRs from a non-human "donor" antibody are grafted into human "acceptor" antibody sequences (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539, Carter, US 6,407,213, Adair, US 5,859,205 6,881,557, Foote, US 6,881,557). The acceptor antibody sequences can be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Thus, a humanized antibody is an antibody having some or all CDRs entirely or substantially from a donor antibody and variable region framework sequences and constant regions, if present, entirely or substantially from human antibody sequences. Similarly a humanized heavy chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody heavy chain, and a heavy chain variable region framework sequence and heavy chain constant region, if present, substantially from human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two and usually all three CDRs entirely or substantially from a donor antibody light chain, and a light chain variable region framework sequence and light chain constant region, if present, substantially from human light chain variable region framework and constant region sequences. Other than nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is substantially from a corresponding CDR in a non-human antibody when at least 85%, 90%, 95% or 100% of corresponding residues (as defined by Kabat) are identical between the respective CDRs. The variable region framework sequences of an antibody chain or the constant region of an antibody chain are substantially from a human variable region framework sequence or human constant region respectively when at least 85, 90, 95 or 100% of corresponding residues defined by Kabat are identical.

[0111] Although humanized antibodies often incorporate all six CDRs (which can be as defined by Kabat) from a mouse antibody, they can also be made with less than all CDRs (e.g., at least 3, 4, or 5 CDRs from a mouse antibody) (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 LessImmunogenic 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).

[0112] A chimeric antibody is an antibody in which the mature variable regions of light and heavy chains of a non-human antibody (e.g., a mouse) are combined with human light and heavy chain constant regions. Such antibodies substantially or entirely retain the binding specificity of the mouse antibody, and are about two-thirds human sequence.

[0113] A veneered antibody is a type of humanized antibody that retains some and usually all of the CDRs and some of the non-human variable region framework residues of a non-human antibody but replaces other variable region framework residues that can contribute to B- or T-cell epitopes, for example exposed residues (Padlan EA.1991. A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand- binding properties. Mol Immunol. 28:489-98) with residues from the corresponding positions of a human antibody sequence. The result is an antibody in which the CDRs are entirely or substantially from a non-human antibody and the variable region frameworks of the non- human antibody are made more human-like by the substitutions. A human antibody can be isolated from a human, or otherwise result from expression of human immunoglobulin genes (e.g., in a transgenic mouse, in vitro or by phage display). Methods for producing human antibodies include the trioma method of Östberg L, Pursch E.1983. Human x (mouse x human) hybridomas stably producing human antibodies. Hybridoma 2:361-367; Östberg, U.S. Patent No. 4,634,664; and Engleman et al., US Patent 4,634,666, use of transgenic mice including human immunoglobulin genes (see, e.g., Lonberg et al., W093 / 12227 (1993); US 5,877,397, US 5,874,299, US 5,814,318, US 5,789,650, US 5,770,429, US 5,661,016, US 5,633,425, US 5,625,126, US 5,569,825, US 5,545,806, Nature 148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996), Kucherlapati, WO 91 / 10741 (1991) and phage display methods(see, .e.g. Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, US 5,877,218, US 5,871,907, US 5,858,657, US 5,837,242, US 5,733,743 and US 5,565,332.

[0114] “Polymer” refers to a series of monomer groups linked together. A polymer is composed of multiple units of a single monomer (a homopolymer) or different monomers (a heteropolymer). High MW polymers are prepared from monomers that include, but are not limited to, acrylates, methacrylates, acrylamides, methacrylamides, styrenes, vinyl-pyridine, vinyl-pyrrolidone 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 the different monomers are copolymerized to form a single polymer. The polymer can be linear or branched. When the polymer is branched, each polymer chain is referred to as a “polymer arm.” The end of the polymer arm linked to the initiator moiety is the proximal end, and the growing-chain end of the polymer arm is the distal end. On the growing chain-end of the polymer arm, the polymer arm end group can be the radical scavenger, or another group.

[0115] “Initiator” refers to a compound capable of initiating a polymerization using monomers or comonomers. The polymerization can be a conventional free radical polymerization or a controlled / ”living” radical polymerization, such as Atom Transfer Radical Polymerization (ATRP), Reversible Addition-Fragmentation-Termination (RAFT) polymerization or nitroxide mediated polymerization (NMP). The polymerization can be a “pseudo” controlled polymerization, such as degenerative transfer. When the initiator is suitable for ATRP, it contains a labile bond which can be homolytically cleaved to form an initiator fragment, I, being a radical capable of initiating a radical polymerization, and a radical scavenger, I’, which reacts with the radical of the growing polymer chain to reversibly terminate the polymerization. The radical scavenger I’ is typically a halogen, but can also be an organic moiety, such as a nitrile. In some embodiments, the initiator contains one of more 2-bromoisobutyrate groups as sites for polymerization via ATRP.

[0116] A “chemical linker” refers to a chemical moiety that links two groups together, such as a half-life extending moiety and a protein. The linker can be cleavable or non-cleavable. Cleavable linkers can be hydrolyzable, enzymatically cleavable, pH sensitive, photolabile, or disulfide linkers, among others. Other linkers include homobifunctional and heterobifunctional linkers. A “linking group” is a functional group capable of forming acovalent linkage comprising one or more bonds to a bioactive agent. Non-limiting examples include those illustrated in Table 1 of WO2013059137 (incorporated by reference).

[0117] The term "reactive group" refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e. is covalently reactive under suitable reaction conditions, and generally represents a point of attachment for another substance. The reactive group is a moiety, such as maleimide or succinimidyl ester, is capable of chemically reacting with a functional group on a different moiety to form a covalent linkage. Reactive groups generally include nucleophiles, electrophiles and photoactivatable groups.

[0118] “Phosphorylcholine,” also denoted as “PC,” refers to the following:where * denotes the point of attachment. The phosphorylcholine is a zwitterionic group and includes salts (such as inner salts), and protonated and deprotonated forms thereof.

[0119] “Phosphorylcholine containing polymer” is a polymer that contains phosphorylcholine. “Zwitterion containing polymer” refers to a polymer that contains a zwitterion.

[0120] Poly(acryloyloxyethyl phosphorylcholine) containing polymer refers to a polymer containing 2-(acryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEA-PC shown below in Example 6) as monomer.

[0121] Poly(methacryloyloxyethyl phosphorylcholine) containing polymer refers to a polymer containing 2-(methacryloyloxy)ethyl-2-(trimethylammonium)ethyl phosphate (HEMA-PC or MPC) as monomer (see below):

[0122] As used herein, “MPC” and “HEMA-PC” are interchangeable.

[0123] “Molecular weight” in the context of the polymer can be expressed as either a number average molecular weight, or a weight average molecular weight or a peak molecular weight. Unless otherwise indicated, all references to molecular weight herein refer to the peak molecular weight. These molecular weight determinations, 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 values can also be used, such as the use of end-group analysis or the measurement of colligative properties (e.g., freezing-point depression, boiling-point elevation, or osmotic pressure) to determine number average molecular weight, or the use of light scattering techniques, ultracentrifugation or viscometry to determine weight average molecular weight. In some embodiments, the molecular weight is measured by SEC-MALS (size exclusion chromatography – multi angle light scattering). In some embodiments, the multi-angle light scattering includes 18-angle MALS. In some embodiments, the multi-angle light scattering includes 3-angle and 18-angle MALS. In some embodiments, the polymeric reagents are typically polydisperse (i.e., number average molecular weight and weight average molecular weight of the polymers are not equal), and can possess low polydispersity values of, for example, less than about 1.5, as judged, for example, by the PDI value derived from the SEC-MALS measurement. In some embodiments, the polydispersities (PDI) are in the range of about 1.4 to about 1.2. In some embodiments the PDI is less than about 1.15, 1.10, 1.05, or 1.03.

[0124] The phrase “a” or “an” entity refers to one or more of that entity; for example, a compound refers to one or more compounds or at least one compound. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0125] “About” means variation one might see in measurements taken among different instruments, samples, and sample preparations.

[0126] “Protected,” “protected form,” “protecting group” and “protective group” refer to the presence of a group (i.e., the protecting group) that prevents or blocks reaction of a particular chemically reactive functional group in a molecule under certain reaction conditions. Protecting groups vary depending upon the type of chemically reactive group being protected as well as the reaction conditions to be employed and the presence of additional reactive or protecting groups in the molecule, if any. Suitable protecting groups include those such as found in the treatise by Greene et al., “Protective Groups In Organic Synthesis,” 3rd Edition, John Wiley and Sons, Inc., New York, 1999.

[0127] “Alkyl” refers to a straight or branched, saturated, aliphatic radical having the number of carbon atoms indicated. For example, C1-C6 alkyl includes, but is 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 include any number of carbons, such as 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 3-4, 3-5, 3-6, 4-5, 4-6 and 5-6. The alkyl group is typically monovalent, but can be divalent, such as when the alkyl group links two moieties together.

[0128] The term “lower” referred to above and hereinafter in connection with organic radicals or compounds respectively defines a compound or radical which can be branched or unbranched with up to and including 7 or up to and including 4 and (as unbranched) one or two carbon atoms.

[0129] “Alkylene” refers to an alkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene. For instance, a straight chain alkylene can be the bivalent radical of -(CH2)n, where n is 1, 2, 3, 4, 5 or 6. Alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene and hexylene.

[0130] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R”’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, - OC(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R”’, -NR”C(O)2R’, -NH-C(NH2)=NH, -NR’C( NH2)=NH, -NH-C(NH2)=NR’, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -CN and -NO2in a number ranging from zero to (2m’+1), where m’ is the total number of carbon atoms in such radical. R’, R” and R”’ each independently refer to hydrogen, unsubstituted (C1-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C1-C4)alkyl groups. When R’ and R” are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR’R” is meant to include 1-pyrrolidinyl and 4-morpholinyl. The term “alkyl” includes groups such as haloalkyl (e.g., -CF3and -CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like). In some embodiments, the substituted alkyl and heteroalkyl groups have from 1 to 4 substituents. In some embodiments, the substituted alkyl and heteroalkyl groups have 1, 2 or 3 substituents. Exceptions are those perhalo alkyl groups (e.g., pentafluoroethyl and the like).

[0131] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR’, =O, =NR’, =N-OR’, -NR’R”, -SR’, -halogen, -SiR’R”R”’, -OC(O)R’, -C(O)R’, -CO2R’, -CONR’R”, -O C(O)NR’R”, -NR”C(O)R’, -NR’-C(O)NR”R”’, -NR”C(O)2R’, -NR-C(NR’R”R’”)=NR””, -N R-C(NR’R”)=NR’”, -S(O)R’, -S(O)2R’, -S(O)2NR’R”, -NRSO2R’, -CN and –NO2in a number ranging from zero to (2m’+1), where m’ is the total number of carbon atoms in such radical. R’, R”, R”’ and R”” each independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups. When a compound includes more than one R group, for example, each of the R groups is independently selected as are each R’, R”, R’” and R”” groups when more than one of these groups is present.When R’ and R” are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR’R” is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF3and –CH2CF3) and acyl (e.g., -C(O)CH3, -C(O)CF3, -C(O)CH2OCH3, and the like).

[0132] “Alkoxy” refers to alkyl group having an oxygen atom that either connects the alkoxy group to the point of attachment or is linked to two carbons of the alkoxy group. Alkoxy groups include, for example, methoxy, ethoxy, propoxy, iso-propoxy, butoxy, 2-butoxy, iso-butoxy, sec-butoxy, tert-butoxy, pentoxy, hexoxy, etc. The alkoxy groups can be further substituted with a variety of substituents described within. For example, the alkoxy groups can be substituted with halogens to form a “halo-alkoxy” group.

[0133] “Carboxyalkyl” means an alkyl group (as defined herein) substituted with a carboxy group. The term “carboxycycloalkyl” means a cycloalkyl group (as defined herein) substituted with a carboxy group. The term alkoxyalkyl means an alkyl group (as defined herein) substituted with an alkoxy group. The term “carboxy” employed herein refers to carboxylic acids and their esters.

[0134] “Haloalkyl” refers to alkyl as defined above where some or all of the hydrogen atoms are substituted with halogen atoms. Halogen (halo) represents chloro or fluoro, but can also be bromo or iodo. For example, haloalkyl includes trifluoromethyl, fluoromethyl, 1,2,3,4,5-pentafluoro-phenyl, etc. The term “perfluoro” defines a compound or radical which has all available hydrogens that are replaced with fluorine. For example, perfluorophenyl refers to 1,2,3,4,5-pentafluorophenyl, perfluoromethyl refers to 1,1,1-trifluoromethyl, and perfluoromethoxy refers to 1,1,1-trifluoromethoxy.

[0135] “Fluoro-substituted alkyl” refers to an alkyl group where one, some, or all hydrogen atoms have been replaced by fluorine.

[0136] “Cytokine” is a member of a group of protein signaling molecules that can participate in cell-cell communication in immune and inflammatory responses. Cytokines are typically small, water-soluble glycoproteins that have a mass of about 8-35 kDa.

[0137] “Cycloalkyl” refers to a cyclic hydrocarbon group that contains from about 3 to 12, from 3 to 10, or from 3 to 7 endocyclic carbon atoms. Cycloalkyl groups include fused, bridged and spiro ring structures.

[0138] “Endocyclic” refers to an atom or group of atoms which comprise part of a cyclic ring structure.

[0139] “Exocyclic” refers to an atom or group of atoms which are attached but do not define the cyclic ring structure.

[0140] “Cyclic alkyl ether” refers to a 4 or 5 member cyclic alkyl group having 3 or 4 endocyclic carbon atoms and 1 endocyclic oxygen or sulfur atom (e.g., oxetane, thietane, tetrahydrofuran, tetrahydrothiophene); or a 6 to 7 member cyclic alkyl group having 1 or 2 endocyclic oxygen or sulfur atoms (e.g., tetrahydropyran, 1,3-dioxane, 1,4-dioxane, tetrahydrothiopyran, 1,3-dithiane, 1,4-dithiane, 1,4-oxathiane).

[0141] “Alkenyl” refers to either a straight chain or branched hydrocarbon of 2 to 6 carbon atoms, having at least one double bond. Examples of alkenyl groups include, but are not limited to, vinyl, propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl, butadienyl, 1-pentenyl, 2-pentenyl, isopentenyl, 1,3-pentadienyl, 1,4-pentadienyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,5-hexadienyl, 2,4-hexadienyl, or 1,3,5-hexatrienyl. Alkenyl groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to 6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkenyl group is typically monovalent, but can be divalent, such as when the alkenyl group links two moieties together.

[0142] “Alkenylene” refers to an alkenyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkenylene can be linked to the same atom or different atoms of the alkenylene. Alkenylene groups include, but are not limited to, ethenylene, propenylene, isopropenylene, butenylene, isobutenylene, sec-butenylene, pentenylene and hexenylene.

[0143] “Alkynyl” refers to either a straight chain or branched hydrocarbon of 2 to 6 carbon atoms, having at least one triple bond. Examples of alkynyl groups include, but are not limited to, acetylenyl, propynyl, 1-butynyl, 2-butynyl, isobutynyl, sec-butynyl, butadiynyl, 1-pentynyl, 2-pentynyl, isopentynyl, 1,3-pentadiynyl, 1,4-pentadiynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 1,3-hexadiynyl, 1,4-hexadiynyl, 1,5-hexadiynyl, 2,4-hexadiynyl, or 1,3,5-hexatriynyl. Alkynyl groups can also have from 2 to 3, 2 to 4, 2 to 5, 3 to 4, 3 to 5, 3 to6, 4 to 5, 4 to 6 and 5 to 6 carbons. The alkynyl group is typically monovalent, but can be divalent, such as when the alkynyl group links two moieties together.

[0144] “Alkynylene” refers to an alkynyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the alkynylene can be linked to the same atom or different atoms of the alkynylene. Alkynylene groups include, but are not limited to, ethynylene, propynylene, butynylene, sec-butynylene, pentynylene and hexynylene.

[0145] “Cycloalkyl” refers to a saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from 3 to 12 ring atoms, or the number of atoms indicated. Monocyclic rings include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cyclooctyl. Bicyclic and polycyclic rings include, for example, norbornane, decahydronaphthalene and adamantane. For example, C3-8cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclooctyl, and norbornane.

[0146] “Cycloalkylene” refers to a cycloalkyl group, as defined above, linking at least two other groups, i.e., a divalent hydrocarbon radical. The two moieties linked to the cycloalkylene can be linked to the same atom or different atoms of the cycloalkylene. Cycloalkylene groups include, but are not limited to, cyclopropylene, cyclobutylene, cyclopentylene, cyclohexylene, and cyclooctylene.

[0147] “Heterocycloalkyl” refers to a ring system having from 3 ring members to about 20 ring members and from 1 to about 5 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heterocycle includes, but is not limited to, tetrahydrofuranyl, tetrahydrothiophenyl, morpholino, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl, piperazinyl, piperidinyl, indolinyl, quinuclidinyl and 1,4-dioxa-8-aza-spiro[4.5]dec-8-yl.

[0148] “Heterocycloalkylene” refers to a heterocyclalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heterocycloalkylene can be linked to the same atom or different atoms of the heterocycloalkylene.

[0149] “Aryl” refers to a monocyclic or fused bicyclic, tricyclic or greater, aromatic ring assembly containing 6 to 16 ring carbon atoms. For example, aryl can be phenyl, benzyl or naphthyl. “Arylene” means a divalent radical derived from an aryl group. Arylgroups can be mono-, di- or tri-substituted by one, two or three radicals selected from alkyl, alkoxy, aryl, hydroxy, halogen, cyano, amino, amino-alkyl, trifluoromethyl, alkylenedioxy and oxy-C2-C3-alkylene; all of which are optionally further substituted, for instance as hereinbefore defined; or 1- or 2-naphthyl; or 1- or 2-phenanthrenyl. Alkylenedioxy is a divalent substitute attached to two adjacent carbon atoms of phenyl, e.g. methylenedioxy or ethylenedioxy. Oxy-C2-C3-alkylene is also a divalent substituent attached to two adjacent carbon atoms of phenyl, e.g. oxyethylene or oxypropylene. An example for oxy- C2-C3-alkylene-phenyl is 2,3-dihydrobenzofuran-5-yl.

[0150] In some embodiments the aryl is naphthyl, phenyl or phenyl mono- or disubstituted by alkoxy, phenyl, halogen, alkyl or trifluoromethyl, especially phenyl or phenyl-mono- or disubstituted by alkoxy, halogen or trifluoromethyl, and in particular phenyl.

[0151] Examples of substituted phenyl groups as R are, e.g. 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-phenyl-phen-1-yl, 4-(imidazol-1-yl)-phenyl, 4-(imidazol-1-ylmethyl)-phen-1-yl, 4-(morpholin-1-yl)-phen-1-yl, 4-(morpholin-1-ylmethyl)-phen-1-yl, 4-(2-methoxyethylaminomethyl)-phen-1-yl and 4-(pyrrolidin-1-ylmethyl)-phen-1-yl, 4-(thiophenyl)-phen-1-yl, 4-(3-thiophenyl)-phen-1-yl, 4-(4-methylpiperazin-1-yl)-phen-1-yl, and 4-(piperidinyl)-phenyl and 4-(pyridinyl)-phenyl optionally substituted in the heterocyclic ring.

[0152] “Arylene” refers to an aryl group, as defined above, linking at least two other groups. The two moieties linked to the arylene are linked to different atoms of the arylene. Arylene groups include, but are not limited to, phenylene.

[0153] “Arylene-oxy” refers to an arylene group, as defined above, where one of the moieties linked to the arylene is linked through an oxygen atom. Arylene-oxy groups include, but are not limited to, phenylene-oxy.

[0154] Similarly, substituents for the aryl and heteroaryl groups are varied and are selected from: -halogen, -OR’, -OC(O)R’, -NR’R”, -SR’, -R’, -CN, -NO2, -CO2R’, -CONR’R”, -C(O)R’, -OC(O)NR’R”, -NR”C(O)R’, -NR”C(O)2R’, ,-NR’-C(O)NR”R”’, -NH-C(NH2)=NH, -NR’C(NH2)=NH, -NH-C(NH2)=NR’, -S(O)R’, -S( O)2R’, -S(O)2NR’R”, -N3, -CH(Ph)2, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and 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.

[0155] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituent of the formula -T-C(O)-(CH2)q-U-, wherein T and U are independently -NH-, -O-, -CH2- or a single bond, and q is an integer of from 0 to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituent of the formula -A-(CH2)r-B-, wherein 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 of from 1 to 3. One of the single bonds of the new ring so formed can optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring can optionally be replaced with a substituent of the formula -(CH2)s-X-(CH2)t-, where s and t are independently integers of 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 unsubstituted (C1-C6)alkyl.

[0156] “Heteroaryl” refers to a monocyclic or fused bicyclic or tricyclic aromatic ring assembly containing 5 to 16 ring atoms, where from 1 to 4 of the ring atoms are a heteroatom each N, O or S. For example, heteroaryl includes pyridyl, indolyl, indazolyl, quinoxalinyl, quinolinyl, isoquinolinyl, benzothienyl, benzofuranyl, furanyl, pyrrolyl, thiazolyl, benzothiazolyl, oxazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, or any other radicals substituted, especially mono- or di-substituted, by e.g. alkyl, nitro or halogen. Pyridyl represents 2-, 3- or 4-pyridyl, advantageously 2- or 3-pyridyl. Thienyl represents 2- or 3-thienyl. In some embodiments, quinolinyl represents 2-, 3- or 4-quinolinyl. In some embodiments, isoquinolinyl represents 1-, 3- or 4-isoquinolinyl. In some embodiments, benzopyranyl, benzothiopyranyl can represent 3-benzopyranyl or 3-benzothiopyranyl, respectively. In some embodiments, thiazolyl can represent 2- or4-thiazolyl. In some embodiments, triazolyl can be 1-, 2- or 5-(1,2,4-triazolyl). In some embodiments, tetrazolyl can be 5-tetrazolyl.

[0157] In some embodiments, heteroaryl is pyridyl, indolyl, quinolinyl, pyrrolyl, thiazolyl, isoxazolyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, thienyl, furanyl, benzothiazolyl, benzofuranyl, isoquinolinyl, benzothienyl, oxazolyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.

[0158] The term “heteroalkyl” refers to an alkyl group having from 1 to 3 heteroatoms such as N, O and S. Additional heteroatoms can also be useful, including, but not limited to, B, Al, Si and P. The heteroatoms can also be oxidized, such as, but not limited to, -S(O)- and -S(O)2-. For example, heteroalkyl can include ethers, thioethers, alkyl-amines and alkyl-thiols.

[0159] The term “heteroalkylene” refers to a heteroalkyl group, as defined above, linking at least two other groups. The two moieties linked to the heteroalkylene can be linked to the same atom or different atoms of the heteroalkylene.

[0160] “Electrophile” refers to an ion or atom or collection of atoms, which can be ionic, having an electrophilic center, i.e., a center that is electron seeking, capable of reacting with a nucleophile. An electrophile (or electrophilic reagent) is a reagent that forms a bond to its reaction partner (the nucleophile) by accepting both bonding electrons from that reaction partner.

[0161] “Nucleophile” refers to an ion or atom or collection of atoms, which can be ionic, having a nucleophilic center, i.e., a center that is seeking an electrophilic center or capable of reacting with an electrophile. A nucleophile (or nucleophilic reagent) is a reagent that forms a bond to its reaction partner (the electrophile) by donating both bonding electrons. A “nucleophilic group” refers to a nucleophile after it has reacted with a reactive group. Non limiting examples include amino, hydroxyl, alkoxy, haloalkoxy and the like.

[0162] “Maleimido” refers to a pyrrole-2,5-dione-1-yl group having the structure:

[0163] which upon reaction with a sulfhydryl (e.g., a thio alkyl) forms an -S-maleimido group having the structureindicates the point of attachment for the maleimido group and “ “indicates the point of attachment of the sulfur atom the thiol to the remainder of the original sulfhydryl bearing group.

[0164] For the purpose 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 anyamino acid other than those recited as naturally occurring amino acids. Non-naturally occurring amino acids include, without limitation, the D isomers of the naturally occurring amino acids, and mixtures of D and L isomers of the naturally occurring amino acids. Other amino acids, such as N-alpha- methyl amino acids (e.g. sarcosine), 4-hydroxyproline, desmosine, isodesmosine, 5-hydroxylysine, epsilon-N-methyllysine, 3-methylhistidine, although found in naturally occurring proteins, are considered to be non-naturally occurring amino acids found in proteins for the purpose of this disclosure as they are generally introduced by means other than ribosomal translation of mRNA.

[0165] “Linear” in reference to the geometry, architecture or overall structure of a polymer, refers to polymer having a single polymer arm.

[0166] “Branched,” in reference to the geometry, architecture or overall structure of a polymer, refers to a polymer having 2 or more polymer “arms” extending from a core structure contained within an initiator. The initiator can be employed in an atom transfer radical polymerization (ATRP) reaction. A branched polymer can possess 2 polymer chains (arms), 3 polymer arms, 4 polymer arms, 5 polymer arms, 6 polymer arms, 7 polymer arms, 8 polymer arms, 9 polymer arms or more. Each polymer arm extends from a polymer initiation site. Each polymer initiation site is capable of being a site for the growth of a polymer chain by the addition of monomers. For example and not by way of limitation, using ATRP, the site of polymer initiation on an initiator is typically an organic halide undergoing a reversible redox process catalyzed by a transition metal compound such as cuprous halide. In some embodiments, the halide is a bromine.

[0167] “Pharmaceutically acceptable excipient” refers to an excipient that can be included in compositions and that causes no significant adverse toxicological effect on the patient and is approved or approvable by the FDA for therapeutic use, particularly in humans. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose and the like. In any embodiment, a pharmaceutically acceptable carrier can be acceptable for administering directly into the eye of a patient (e.g., acceptable for intravitreal administration).

[0168] Therapeutic proteins are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and / or ameliorates at least one sign or symptom ofa disorder. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients.

[0169] The “biological half-life” of a substance is a pharmacokinetic parameter which specifies the time required for one half of the substance to be removed from a tissue or an organism following introduction of the substance.

[0170] “OG1786” is a 9-arm initiator used for polymer synthesis with the structure shown in FIG.35, which depicts that salt form of OG1786 with trifluororacetic acid. OG1786 can be used as other salts are used or as the free base.

[0171] “OG1801” is an approximately (+ / - 15%) 750 kDa polymer (either by Mn or Mp) made using OG1786 as an intiator for ATRP synthesis using the monomer HEMA-PC.

[0172] “OG1802” is OG1801 with a maleimide functionality added and is shown in FIG.36, wherein each of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is an integer (positive) (from 0 up to about 3000) such that the total molecular weight of the polymer is (Mw) 750,000 ± 15% daltons.

[0173] Multi-angle light scattering (MALS) is a technique of analyzing macromolecules where the laser light impinges on the molecule, the oscillating electric field of the light induces an oscillating dipole within it. This oscillating dipole will re-radiate light and can be measured using a MALS detector such as Wyatt miniDawn TREOS. The intensity of the radiated light depends on the magnitude of the dipole induced in the macromolecule which in turn is proportional to the polarizability of the macromolecule, the larger the induced dipole, and hence, the greater the intensity of the scattered light. Therefore, in order to analyze the scattering from a solution of such macromolecules, one should know their polarizability relative to the surrounding medium (e.g., the solvent). This can be determined from a measurement of the change, ǻn, of the solution's refractive index n with the molecular concentration change, ǻc, by measuring the dn / dc (=ǻn / ǻc) value using a Wyatt Optilab T-rEX differential refractometer. Two molar weight parameters that MALS determination employ are number average molecular weight (Mn) and weight average molecular weight (Mw) where the polydispersity index (PDI) equals Mw divided by Mn. SEC also allows another average molecular weight determination of the peak molecular weight Mp which is defined as the molecular weight of the highest peak at the SEC.

[0174] The PDI is used as a measure of the broadness of a molecular weight distribution of a polymer and bioconjugate which is derived from conjugation of a discrete protein (e.g. OG1950) to a polydisperse biopolymer (e.g., OG1802). For a protein sample, its polydispersity is close to 1.0 due to the fact that it is a product of translation where every protein molecule in a solution is expected to have almost the same length and molar mass. In contrast, due to the polydisperse nature of the biopolymer where the various length of polymer chains are synthesized during the polymerization process, it is very important to determine the PDI of the sample as one of its quality attribute for narrow distribution of molecular weight.

[0175] Size exclusion chromatography (SEC) is a chromatography technique in which molecules in solution are separated by their size. Typically, an aqueous solution is applied to transport the sample through the column which is packed with resins of various pore sizes. The resin is expected to be inert to the analyte when passing through the column and the analytes separate from each other based on their unique size and the pore size characteristics of the selected column.

[0176] Coupling the SEC with MALS or SEC / MALS provides accurate distribution of molar mass and size (root mean square radius) as opposed to relying on a set of SEC calibration standards. This type of arrangement has many advantages over traditional column calibration methods. Since the light scattering and concentration are measured for each eluting fraction, the molar mass and size can be determined independently of the elution position. This is particularly relevant for species with non-globular shaped macromolecules such as the biopolymers (OG1802) or bioconjugates (OG1953); such species typically do not elute in a manner that might be described by a set of column calibration standards.

[0177] In some embodiments, a SEC / MALS analysis includes a Waters HPLC system with Alliance 2695 solvent delivery module and Waters 2996 Photodiole Array Detector equipped with a Shodex SEC-HPLC column (7.8x300mm). This is connected online with a Wyatt miniDawn TREOS and Wyatt Optilab T-rEX differential refractometer. TheEmpower software from Waters can be used to control the Waters HPLC system and the ASTRA V 6.1.7.16 software from Wyatt can be used to acquire the MALS data from the Wyatt miniDawn TREOS, dn / dc data from the T-rEX detector and the mass recovery data using the A280 absorbance signal from the Waters 2996 Photodiole Array detector. SEC can be carried out at 1ml / min in 1xPBS pH 7.4, upon sample injection, the MALS and RI signals can be analyzed by the ASTRA software for determination of absolute molar mass (Mp, Mw, Mn) and polydisperse index (PDI). In addition, the calculation also involves the input dn / dc values for polymer and protein as 0.142 and 0.183, respectively. For OG1953 bioconjugates dn / dc value, the dn / dc is calculated based on the weighted MW of the polymer and the protein to be about 0.148 using the formula below: Conjugate dn / dc = 0.142 x [ MWpolymer / (MWpolymer+MWprotein)]+ 0.183 x [MWprotein / (MWpolymer+MWprotein)] where MWpolymer for OG1802 is 800 kDa and the MWprotein for OG1950 is 146 kDa.

[0178] “Isocratic” refers to a mobile phase kept constant and uniform during a chromatography run. Antibody Compositions and Methods

[0179] In some embodiments, a formulation (or therapeutically acceptable composition) that is a first protein that includes 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 is provided. In some embodiments, the first protein is an antibody and the second protein is an antibody. Both antibodies can be therapeutic antibodies. In some embodiments, the formulation or composition is for treating an eye disorder in a subject. In some embodiments, a formulation or composition sample includes at least a polymer (e.g., a phosphorylcholine-containing polymer) and an unconjugated protein (e.g., an unconjugated antibody). As used herein, formulation (e.g., mixed formulation) and composition (e.g., mixed therapeutically acceptable composition) can be used interchangeably. In some embodiments, a formulation or therapeutically acceptable composition is safe for human use (e.g., administering to a human). In some embodiments, a formulation or therapeutically acceptable composition is not an intermediate product generated during manufacture of a final product (e.g., that may be suitable for use in a human). In someembodiments, the formulation is a mixed formulation sample. In some embodiments the terms formulation and mixed formulation sample are used interchangeably. As used herein, “protein” and “protein moiety” can be used interchangeably.

[0180] In some embodiments, provided herein is a formulation (or therapeutically acceptable composition) comprising any two proteins that can be the same or different in function, wherein one is conjugated to a polymer and the other is not conjugated to the polymer (or conjugated to any polymer or conjugated to any effective amount of a polymer). The formulation (or therapeutically acceptable composition) can be for the treatment of an eye disorder. In some embodiments, both of the proteins are therapeutic proteins for the treatment of an eye disorder. In some embodiments, one or both of the proteins are therapeutics, 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 can be used instead of an antibody.

[0181] Provided herein is a formulation (or therapeutically acceptable composition) comprising: a first molar amount of a conjugate comprising a first protein or protein moiety conjugated to a phosphorylcholine-containing polymer (the “conjugated protein”); a second molar amount of a second protein or protein moiety that is not conjugated to a phosphorylcholine-containing polymer (the “unconjugated protein”); and a pharmaceutically acceptable carrier, wherein the formulation, or composition, comprises the second protein at about 1% or more of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a sum of the first molar amount and the second molar amount, wherein the formulation, or composition, has a pH that is about 0.5 pH units away (e.g., above or below) or more from the isoelectric point (pI) of the second protein. As used herein, “molar amount” denotes a measure of the molar quantity of a molecule. In some embodiments, molar amount is a molar concentration (e.g., M, mM, μM, nM, etc.). In some embodiments, molar amount is expressed in units of moles (e.g., moles, millimoles,micromoles, etc.). As used herein, the isoelectric point (pI) of a protein has its customary and ordinary meaning to one of ordinary skill in the art, in view of the present disclosure. The pI denotes the pH at which the protein carries no net charge. The pI can be a previously known value for the same or similar protein, or be determined based on a model, or empirically. In some embodiments, the pI is a theoretically determined pI. In some embodiments, the pI is an empirically determined pI.

[0182] Low-viscosity formulation (or therapeutically acceptable composition) of a protein conjugate (e.g., a protein conjugated to a phosphorylcholine-containing polymer) are also provided. A high concentration of the protein conjugate in the formulation, or composition, can raise the viscosity of the formulation, or composition. In some embodiments, lowering the viscosity of the formulation or composition (while maintaining the total amount of active protein) improves one or more of manufacturability, handling, and injectability, for example when delivering the formulation or composition with a syringe to a site of treatment (e.g., intraocular administration).

[0183] In any of the methods and apparatuses of the present disclosure, mixed formulations (or therapeutically acceptable compositions) (e.g., having a polymer or polymer- conjugated protein and unconjugated protein) can include the unconjugated protein or protein moiety (e.g., protein that is not conjugated to the phosphorylcholine-containing polymer) at any suitable % molar amount of the total molar amount of the polymer / polymer conjugate and the unconjugated protein or protein moiety. In some embodiments, the formulations (or therapeutically acceptable compositions) comprise the second protein or protein moiety (the unconjugated protein) at about 1% or more of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a sum of the first molar amount of the conjugate and the second molar amount of the second protein. For example, where the combined concentration of the conjugate and the unconjugated protein is 100 μM, the unconjugated protein at 1% of the total molar amount is at 1 μM, and the conjugate is at 99 μM. In some embodiments, the formulations (or therapeutically acceptable composition) comprise the second protein (the unconjugated protein) at, or at 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, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, or a percentage in a range defined by any two of the preceding values (e.g., about 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.) of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, the formulations (or therapeutically acceptable composition) comprise the second protein (the unconjugated protein) at between about 5% and about 50%, or between about 15% and about 30% of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a 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 (the unconjugated protein) at about 20% of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a 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 (the unconjugated protein) at about 30% of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, any formulation or composition provided herein comprises the second protein (the unconjugated protein) at more than 5% of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a sum of the first molar amount of the conjugate and the second molar amount of the second protein. In some embodiments, any formulation or composition provided herein comprises the second protein (the unconjugated protein) at more than 10% of a total molar amount of the conjugate and the second protein, wherein the total molar amount comprises a 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 herein includes two or more (e.g., 2, 3, 4, 5 or more) different second proteins (or unconjugated proteins),where the second molar amount is the sum of the molar amounts of the two or more different second proteins.

[0184] In some embodiments a formulation (or therapeutically acceptable composition) includes: a conjugate comprising a first protein or protein moiety conjugated to a phosphorylcholine-containing polymer, wherein the polymer has 9 arms and a molecular weight of between 600,000 and 1,000,000 Da, wherein the polymer is present in the formulation at about 100 mg / mL or more; and a second protein or protein moiety that is not conjugated to a polymer, wherein the second protein or protein moiety is present in the formulation at 5-15 mg / mL. In some embodiments, the first and second proteins are a therapeutic protein. In some embodiments, the first protein and second protein are the same (e.g., are at least, or at least about 85%, 90%, 95%, 97%, 99%, or are about 100% identical in amino acid sequence). In some embodiments, the first protein and second protein are different proteins.

[0185] In some embodiments, a mixed formulation (or therapeutically acceptable composition) includes: a first molar amount of a conjugate comprising a first protein or protein moiety conjugated to a polymer; and a second molar amount of a second protein or protein moiety that is not conjugated to a polymer, wherein the formulation comprises the second protein at about 1% or more of a total molar amount of the first protein and the second protein, wherein the total molar amount comprises a sum of the first molar amount and the second molar amount. In some embodiments, the formulation comprises the second protein at 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 the second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer. In some embodiments, the formulation comprises the second protein at about 5-50%, or about 15-30% of the total molar amount of the conjugate and the second protein. In some embodiments, the polymer is a phosphorylcholine-containing polymer.

[0186] In some embodiments, a mixed formulation (or therapeutically acceptable composition) includes: a conjugate comprising a first protein or protein moiety conjugated to a polymer; and a second protein that is not conjugated to a polymer, wherein a first molar amount of the conjugate and a second molar amount of the second protein or protein moiety has been combined in the formulation such that the second molar amount is about 1% or more(e.g., about 5-90%, 15-25%, 25-35%, 25-40%, etc.) of a total molar amount of the conjugate and the second protein or protein moiety, wherein the total molar amount comprises a sum of the first molar amount and the second molar amount. In some embodiments, the mixed formulation or composition is prepared by combining the first molar amount of the conjugate with the second molar amount of the second protein that is not conjugated to a polymer, such that the second molar amount is at the specified percentage of the sum of the first molar amount and the second molar amount (e.g., specified percentage of the total molar amount). In some embodiments, the second molar amount is 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 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.

[0187] In some embodiments, the mixed formulation or composition has been prepared by combining the conjugate at a percent composition of about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.) of the second protein relative to the total protein mass weight concentration of the first protein and the second protein, where the remainder of the total protein mass weight concentration includes the first protein. For example, for a total mass weight concentration of 50 mg / mL, the mixed therapeutically acceptable composition can be prepared by combining an amount of the second protein that corresponds to 10 mg / mL in the final composition (at percent composition of 20%) with an amount of the conjugate that corresponds to 40 mg / mL of the first protein (as the conjugate, excluding any contribution of the polymer to the mass weight concentration calculation) in the final composition.

[0188] In some embodiments, the conjugate includes a first protein or protein moiety conjugated to a polymer, wherein the polymer includes one or more of: polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxylethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinylalcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anyhydride, polystyrene-co-maleic acid anhydride, poly(1- hydroxymethyethylene hydroxymethylformal) (PHF), a zwitterionic polymer, a phosphorylcholine containing polymer and a polymer comprising MPC, Poly (Glyx-Sery), Hyaluronic acid (HA), Heparosan polymers (HEP), Fleximers, Dextran, and Poly-sialic acids (PSA). General

[0189] Provided herein are methods and apparatus for analyzing mixed formulations (or therapeutically acceptable compositions) of protein and protein conjugated to polymer by tandem HPLC. In some embodiments, the proteins are antibodies. In some embodiments, the antibodies are anti-VEGF antibodies and conjugates thereof. In some embodiments, the antibodies themselves are different from other anti-VEGF agents and provide superior results over 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 other antibody conjugates.

[0190] Historically, conjugating a molecule to a protein often resulted in a decrease in the protein’s binding interaction to its intended target. In some embodiments of the present disclosure, when conjugating to a location that is outside of the active site, the same level of decrease as might have been expected is not necessarily observed. The evidence provided herein shows the opposite effect as to what may have been expected. In some embodiments, and without intending to be limited by theory, the conjugate can be superior to the antibody alone. For example, the interaction of a ligand and its specific receptor is often driven through the stereospecific interaction of the ligand and the receptor, as directed by the interactions of the hydrophilic amino acids on the ligand with the hydrophilic amino acids on the receptor, and water molecules are front and center in those interactions. At the same time, this hydrophilic stereospecificity is further enhanced by de-emphasizing and / or suppressing non- specific hydrophobic interactions that might generally be mediated / created by hydrophobic- to-hydrophobic amino acids.

[0191] In some embodiments, an anti-VEGF antibody conjugate is provided that is capable of blocking at least 90% of an interaction between a VEGF ligand (“VEGFL”) and aVEGF-receptor (“VEGFR”). For example, it can block at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or effectively all of the interaction between VEGFR and VEGFL. In some embodiments, the noted blocking occurs at saturating concentrations. In some embodiments, an anti-VEGF antibody conjugate is provided that blocks at least 95% of an interaction between a VEGF ligand and a VEGF-receptor. Indeed, this result was unexpected in that while the addition of a polymer to an antibody (to form an antibody conjugate), could be expected to have some or no detrimental impact on binding / activity of the antibody, it was unexpected that it would actually improve the blocking ability of the antibody in this manner.

[0192] In some embodiments, the antibodies or conjugates thereof inhibit 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 can 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 preceding values. In some embodiments, the KD can be 2*10^-13, 1*10^-13, 1*10^-12, 1*10^-11, 1*10- ^10M or less than any one of the preceding values. In some embodiments, the IC50 value can be 1, 5, 10, 20, 30, 40, 50, 60, 7080, 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 preceding values.

[0193] In some embodiments, an anti-VEGF antibody is provided that blocks at least 90% of an interaction between a VEGF ligand and a VEGF-receptor. For example, it can block at least 91, 92, 93, 94, 95, 96, 97, 98, 99, or effectively all of the interaction between VEGFR and VEGFL.

[0194] In some embodiments, other antibodies, such as Lucentis®(ranibizumab) or Avastin®(bevacizumab) can be conjugated to one or more of the polymers as described herein, by one or more of the processes described herein. In some embodiments, any antibody, or fragment thereof, can be conjugated to one or more of the polymers as described herein, by one or more of the processes described herein.

[0195] In some embodiments the antibody comprises a heavy chain amino acid variable region that comprises SEQ ID NO: 1 (with or without the C-terminal lysine) and a light chain amino acid variable region that comprises SEQ ID NO: 2. In some embodiments, the antibody is conjugated to one or more of the polymers provided herein. In some embodiments, the conjugated antibody is at least 90% identical to SEQ ID NO: 1 and / or 2. In some embodiments, the antibody contains the 6 CDRs within SEQ ID NO:1 and SEQ ID NO:2, as well as a point mutation of 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 2 and includes the following mutations: L234A, L235A, and G237A (EU numbering), and at least one of the following mutations: Q347C (EU numbering) or L443C (EU numbering).

[0196] In some embodiments an antibody that binds to VEGF-A is provided. The antibody comprises: a CDRH1 that is the CDRH1 in SEQ ID NO: 1, a CDRH2 that is the CDRH2 in SEQ ID NO: 1, a CDRH3 that is the CDRH3 in SEQ ID NO: 1, a CDRL1 that is the CDRL1 in SEQ ID NO: 2, a CDRL2 that is the CDRL2 in SEQ ID NO: 2, a CDRL3 that is the 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).

[0197] As will be appreciated by one of skill in the art, in light of the present specification, any of the antibodies provided herein can be conjugated to any of the polymers provided herein and / or any antibody provided herein can have a cysteine added such that it allows for site specific conjugation to a polymer.

[0198] “VEGF” or “vascular endothelial growth factor” is a human vascular endothelial growth factor that affects angiogenesis or an angiogenic process. In particular, the term VEGF means any member of the class of growth factors that (i) bind to a VEGF receptor such as VEGFR-1 (Flt-1), VEGFR-2 (KDR / Flk-1), or VEGFR-3 (FLT-4); (ii) activates a tyrosine kinase activity associated with the VEGF receptor; and (iii) thereby affects angiogenesis or an angiogenic process.

[0199] The VEGF family of factors is made up of five related glycoproteins: VEGF-A (also known as VPE), -B, -C, -D and PGF (placental growth factor). Of these, VEGF- A is the most well studied and is the target of anti-angiogenic therapy. Ferrara et al, (2003) Nat. Med. 9:669-676. VEGF-A exists as a number of different isotypes which are generated both by alternative splicing and proteolysis: VEGF-A206, VEGF-A189, VEGF-A165, and VEGF-A121. The isoforms differ in their ability to bind heparin and non-signaling binding proteins called neuropilins. The isoforms are all biologically active as dimers.

[0200] The various effects of VEGF are mediated by the binding of a VEGF, e.g., VEGF-A (P15692), -B (P49766), -C (P49767) and –D (Q43915), to receptor tyrosine kinases (RTKs). The VEGF family receptors belong to class V RTKs and each carry seven Ig-likedomains in the 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 apparent from the context reference to a VEGF means any of VEGF-A, -B, -C , –D, and PGF, in any of the natural isoforms or natural variants or induced variants having at least 90, 95, 98 or 99% or 100% sequence identity to a natural form. In some embodiments, such VEGFs are human VEGFs. Likewise reference to a VEGFR means any of VEGR-1, R-2 or R-3, including any natural isoform or natural variant, or an induced variant having at least 90, 95, 98 or 99% or 100% sequence identity to a natural sequences.

[0201] VEGF antagonist therapies have been 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, in particular to all isoforms of VEGF-A and to bioactive proteolytic fragments of VEGF-A. See, e.g., Ferrara N, Hillan KJ, Gerber HP, Novotny W.2004. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 3(5):391-400. Bevacizumab has been approved for the treatment of certain cancers. The protein sequence of the heavy and light chains of bevacizumab (DrugBank DB00112) are set forth in SEQ ID NO: 3 (heavy) and SEQ ID NO: 4 (light).

[0202] Bevacizumab variable light chain CDRs are CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13) and CDRL3: QQYSTVPWT (SEQ ID NO: 14). Bevacizumab variable heavy chain CDRs are CDRH1: GYTFTNYGMN, CDRH2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3: YPHYYGSSHWYFDV. CDRs are defined by Kabat except CDRH1 uses the composite Kabat / Chothia definition. In some embodiments, a cysteine can be added to the Bevacizumab sequence and the antibody (and / or a variant that includes the 6 CDRs of Bevacizumab) can be conjugated to any one or more of the polymers provided herein.

[0203] Another anti-VEGF molecule, derived from the same mouse monoclonal antibody as bevacizumab has been approved as a treatment for wet AMD: ranibizumab (LUCENTIS®(ranibizumab), Genentech / Roche). Ranibizumab is an antibody fragment or Fab. Ranibizumab was produced by affinity maturation of the variable heavy and light chains of bevacizumab. The sequence of the heavy and light chains of ranibizumab (as published by Novartis) is set forth in SEQ ID NO: 5 and 6 respectively. In some embodiments, a cysteinecan be added to the ranibizumab sequence and the antibody (and / or a variant that includes the 6 CDRs of ranibizumab) can be conjugated to any one or more of the polymers provided herein.

[0204] The Ranibizumab CDRS are the same as Bevacizumab except where an improvement was added after affinity maturation: Ranibizumab variable light chain CDRs are CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13) and CDRL3: QQYSTVPWT (SEQ ID NO: 14). Ranibizumab variable heavy chain CDRs are CDRH1: GYDFTHYGMN (SEQ ID NO: 9), CDRH2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3: YPYYYGTSHWYFDV (SEQ ID NO: 11).

[0205] In some embodiments, an antibody conjugate is presented having an anti- VEGF-A antibody bonded at a cysteine outside a variable region of the antibody to a phosphorylcholine containing polymer, wherein the cysteine has been added via recombinant DNA technology. In some embodiments, the polymer is bonded to a single cysteine. In some embodiments, “added by recombinant DNA technology” means that the cysteine residue replaces a non-cysteine amino acid that occurs in the same position in a known or existing antibody or in a consensus antibody sequence. Thus, for example where the antibody is an IgG1 and the heavy chain possess a leucine at EU position 443, the leucine is replaced via recombinant DNA technology with a cysteine (L443C, EU numbering, or 449C in SEQ ID NO: 1). Correspondingly, the native IgG1 sequence at EU position 347 is Q (glutamine) and the Q is replaced with cysteine via recombinant DNA technology to yield Q347C.

[0206] In some embodiments, the anti-VEGF-A antibody comprises a light chain and a heavy chain where the heavy chain has an Fc region. In some embodiments, the cysteine is in the Fc region and the anti-VEGF-A antibody is an immunoglobulin G (IgG). In some embodiments, the anti-VEGF-A heavy chain has CDRH1: GYDFTHYGMN (SEQ ID NO: 9), CDRH2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3: YPYYYGTSHWYFDV (SEQ ID NO: 11), and position 221 (via sequential counting as in SEQ ID NO: 3) is T, and the anti-VEGF-A light chain has CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13), and CDRL3: QQYSTVPWT (SEQ ID NO: 14), and Kabat position 4 is L.

[0207] In some embodiments, the anti-VEGF-A heavy chain isotype is IgG1. In some embodiments, the IgG1 constant domain has one or more mutations relative to an IgG1 constant domain (e.g. constant region of SEQ ID NO: 3) to modulate effector function. Insome embodiments, the effector function mutations are one or more of the following: (EU numbering) E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X wherein X is any natural or unnatural amino acid. In some embodiments, the mutations are selected from the group comprising (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, antibody conjugate has the following mutations (EU numbering): L234A, L235A, and G237A.

[0208] In some embodiments, the cysteine residue is in 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 the C-terminal lysine) and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2.

[0209] In some embodiments, one or both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) is an anti-HTRA1 antibody. In some embodiments, the anti-HTRA1 antibody includes a heavy chain comprising a complementarity determining region 1 CDRH1: FYHVH (SEQ ID NO: 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 comprising a 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 includes a heavy chain comprising a complementarity determining region 1 CDRH1: GFSLTFYH (SEQ ID NO: 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 comprising a CDRL1: QSLLDEAGETY (SEQ ID NO:153), CDRL2: EV, and CDRL3: QQATYFPYT (SEQ ID NO:147). In some embodiments, the anti-HTRA1 antibody includes a heavy chain comprising a complementarity determining region 1 CDRH1: GFSLTFY (SEQ ID NO: 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), and a light chain comprising a CDRL1: RSSQSLLDEAGETYLA (SEQ ID NO:145), CDRL2: EVSLLES(SEQ ID NO:146), and CDRL3: QQATYFPYT (SEQ ID NO:147). In some embodiments, the heavy chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-95%, 90-97%, etc.) identical to any one of the VH sequences set forth in TABLE 1, and the light chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97% or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-95%, 90-97%, etc.) identical to the VL sequence set forth in TABLE 2. In some embodiments, the heavy chain comprises any one of the VH sequences set forth in TABLE 1, and the light chain comprises the VL sequence set forth in TABLE 2. TABLE 1TABLE 2

[0210] In some embodiments, the first or second antibody of the mixed formulation (or therapeutically acceptable composition) comprises a Complement Factor D (CFD) antibody. In some embodiments, the CFD antibody includes a heavy chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any twoof the preceding values (e.g., 80-100%, 85-95%, 90-97%, etc.) identical to SEQ ID NO: 129 (or a heavy chain variable region thereof), and a light chain variable region having an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-95%, 90-97%, etc.) identical to SEQ ID NO: 130 (or a light chain variable region thereof). In some embodiments, the CFD antibody heavy chain can comprise the sequence of SEQ ID NO: 129 (with or without the C-terminal lysine). In some embodiments, the CFD antibody light chain can comprise the sequence of SEQ ID NO: 130.

[0211] In some embodiments, the first or second antibody of the mixed formulation (or therapeutically acceptable composition) comprises a fusion protein that is a VEGF-A trap fused to an IL-6 antibody. In some embodiments, the VEGF-A trap fused to an IL-6 antibody can comprise a heavy chain comprising SEQ ID NO: 131 (with or without the C-terminal lysine). In some embodiments, the VEGF-A trap fused to an IL-6 antibody can comprise a light chain comprising SEQ ID NO: 132.

[0212] In some embodiments, the VEGF Trap comprises 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 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-95%, 90-97%, etc.) identical to SEQ ID NO:133 form in TABLE 3. ^ TABLE 3

[0213] In some embodiments a fusion construct includes a heavy chain that includes a CDRH1 that is the CDRH1 in SEQ ID NO: 105; a CDRH2 that is the CDRH2 in SEQ ID NO: 105; a CDRH3 that is the CDRH3 in SEQ ID NO: 105; a CDRL1 that is the CDRL1 in SEQ ID NO: 106; a CDRL2 that is the CDRL2 in SEQ ID NO: 106; and a CDRL3 that is the CDRL3 in SEQ ID NO: 106. In some embodiments, the fusion construct includes a heavy chain comprising a complementarity determining region 1 (CDRH1): PFAMH (SEQID 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 comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97% at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80-100%, 85-95%, 90-97%, etc.) identical to SEQ ID NO:105, and the light chain comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97% at least 99%, or about 100%, or a percentage in a range defined by any two of the preceding values (e.g., 80- 100%, 85-95%, 90-97%, etc.) identical to SEQ ID NO:106. In some embodiments, the heavy chain comprises the amino acid sequence of SEQ ID NO:105 (with or without the C-terminal lysine), and the light chain comprises the amino acid sequence of SEQ ID NO:106.

[0214] In some embodiments, the phosphorylcholine containing polymer comprises 2-(methacryloyloxyethyl)-2'-(trimethylammonium)ethyl phosphate (MPC) monomers as set forth below:.

[0215] Such that the polymer comprises the following repeating units:where n is an integer from 1 to 3000 and the wavy lines indicate the points of attachment between monomer units in the polymer.

[0216] In some embodiments, the polymer has three or more arms, or is synthesized with an initiator comprising 3 or more polymer initiation sites. In some embodiments, the polymer has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms, or is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer initiation sites. More preferably, the polymer has 3, 6, or 9 arms, or is synthesized with an initiator comprising 3, 6, or 9 polymer initiation sites. In some embodiments, the polymer has 9 arms, or is synthesized with an initiator comprising 9 polymer initiation sites.

[0217] In some embodiments, the polymer that is added has a molecular weight between about 300,000 and about 1,750,000 Da (SEC-MALs). In some embodiments, the polymer has a molecular weight between about 500,000 and about 1,000,000 Da. In some embodiments, the polymer has a molecular weight of between about 600,000 to about 1,000,000 Da. In some embodiments, the polymer has a molecular weight of between about 750,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of between about 800,000 to about 850,000 Da. In some embodiments, the polymer has a molecular weight of between about 750,000 to about 800,000 Da. In some embodiments, the polymer has a molecular weight in the range of about 700,000 to about 800,000 Da.

[0218] In some embodiments, any of the antibodies described herein can be further conjugated to a polymer to form a bioconjugate. The molecular weight of the bioconjugate (in total, SEC-MALs) can be between about 350,000 and 2,000,000 Daltons, for example, betweenabout 450,000 and 1,900,000 Daltons, between about 550,000 and 1,800,000 Daltons, between about 650,000 and 1,700,000 Daltons, between about 750,000 and 1,600,000 Daltons, between about 850,000 and 1,500,000 Daltons, between about 900,000 and 1,400,000 Daltons, between about 950,000 and 1,300,000 Daltons, between about 900,000 and 1,000,000 Daltons, between about 1,000,000 and 1,300,000 Daltons, between about 850,000 and 1,300,000 Daltons, between about 850,000 and 1,000,000 Daltons, and between about 1,000,000 and 1,200,000 Daltons.

[0219] In some embodiments, the antibody conjugate is purified. In some embodiments, the polymer is aspect of the antibody conjugate 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.

[0220] In some embodiments, the antibody conjugate has an anti-VEGF-A immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 (with or without the C-terminal lysine), and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2, and wherein the antibody is bonded only at C449 in SEQ ID NO: 1 to the polymer. In some embodiments, the polymer has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.

[0221] In some embodiments, the antibody conjugate has an anti-VEGF-A immunoglobulin G (IgG) bonded to a polymer, which polymer comprises MPC monomers, wherein the sequence of the anti-VEGF-A heavy chain is SEQ ID NO: 1 (with or without the C-terminal lysine), and the sequence of the anti-VEGF-A light chain is SEQ ID NO: 2, and wherein the antibody is bonded only at C443 (EU numbering, or 449C in SEQ ID NO: 1) to the polymer. In some embodiments, the polymer has 9 arms and has a molecular weight of between about 600,000 to about 1,000,000 Da.

[0222] In some embodiments, the antibody conjugate has the following structure:where X is a) –OR where R is H, methyl, ethyl, propyl, or isopropyl, b) –H, c) any halogen, including –Br, –Cl, or –I, d) –SCN, or e) –NCS; wherein: each heavy chain of the first antibody is denoted by the letter H, and each light chain of the first antibody is denoted by the letter L; the polymer is bonded to the first antibody through the sulfhydryl of a cysteine at position 449, as numbered in SEQ ID NO: 1, which bond is depicted on one of the heavy chains; PC is, where the curvy line indicates the point of attachment to the rest of the polymer; and n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%. In some embodiments, X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, or c) a halide. In someembodiments, the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is about 1500 to about 3500 plus or minus about 10% to about 20%.

[0223] 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.

[0224] In some embodiments, an anti-VEGF-A antibody is 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 those CDRs and in addition has threonine (T) at position 221 (via sequential counting as in 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 those CDRs and in addition has leucine (L) at Kabat position 4. In some embodiments, the isotype of the anti-VEGF-A antibody heavy chain, is IgG1 and has a CH1, hinge, CH2 and CH3 domains. In some embodiments the light chain isotype is kappa.

[0225] In some embodiments, the IgG1 domain of the anti-VEGF-A antibody has one or more mutations to modulate effector function, such as ADCC, ADCP, and CDC. In some embodiments, the IgG1 mutations reduce effector function. In some embodiments the amino acids to use for effector function mutations include (EU numbering) E233X, L234X, L235X, G236X, G237X, G236X, D270X, K322X, A327X, P329X, A330X, A330X, P331X, and P331X, in which 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 function mutations relative to a natural human IgG1 sequence is no more than 10. In some embodiments the number of effector function mutations relatative to a natural human IgG1 sequence is no more than 5, 4, 3, 2 or 1. In some embodiments, the antibody has decreased Fc gamma binding and / or complement C1q binding,such that the antibody’s ability to result in an effector function is decreased. This can be especially advantageous for ophthalmic indications / disorders.

[0226] 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).

[0227] In some embodiments, the anti-VEGF-A antibody is or is part of a human immunoglobulin G (IgG1).

[0228] In some embodiments, the VEGF-A antibody comprises a heavy chain constant domain that comprises one or more mutations that reduce an immune-mediated effector function.

[0229] In some embodiments an anti-VEGF-A antibody is provided. The anti- VEGF-antibody comprises a heavy chain that comprises a CDRH1 comprising the sequence GYDFTHYGMN (SEQ ID NO: 9), a CDRH2 comprising the sequence WINTYTGEPTYAADFKR (SEQ ID NO: 10), a CDRH3 comprising the sequence YPYYYGTSHWYFDV (SEQ ID NO: 11), a CDRL1 comprising the sequence SASQDISNYLN (SEQ ID NO: 12), a CDRL2 comprising the sequence FTSSLHS (SEQ ID NO: 13), and a CDRL3 comprising the sequence QQYSTVPWT (SEQ ID NO: 14).

[0230] Alternatively, the IgG domain can be IgG2, IgG3 or IgG4 or a composite in which a constant regions is formed from more than one of these isotypes (e.g., CH1 region from IgG2 or IgG4, hinge, CH2 and CH3 regions from IgG1). Such domains can contain mutations to reduce and / or modulate effector function at one or more of the EU position mentioned for IgG1. Human IgG2 and IgG4 have reduced effector functions relative to human IgG1 and IgG3.

[0231] The anti-VEGF-A heavy chain has a cysteine residue added as a mutation by recombinant DNA technology which can be used to conjugate a half-life extending moiety. 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 stoichiometry of antibody to polymer is 1:1; in other words, a conjugate has one molecule of antibody conjugated to one molecule of polymer.

[0232] The half-life of the anti-VEGF-A antibodies can be extended by attachment of a “half-life (“half life”) extending moieties” or “half-life (“half life”) extending groups”. Half-life extending moieties include peptides and proteins which can be expressed in frame with the biological drug of issue (or conjugated chemically depending on the situation) and various polymers which can be attached or conjugated to one or more amino acid side chain or end functionalities such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N- and / or O- glycan structures. Half-life extending moieties generally act to increase the in vivo circulatory half-life of biologic drugs.

[0233] Examples of peptide / protein half-life extending moieties include Fc fusion (Capon DJ, Chamow SM, Mordenti J, et al. Designing CD4 immunoadhesions for AIDS therapy. Nature. 1989. 337:525-31), human serum albumin (HAS) fusion (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) fusion (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), genetic fusion of non-exact repeat peptide sequence (XTEN) fusion (Schellenberger V, Wang CW, Geething NC, et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat Biotechnol.2009.27:1186-90), elastin like peptide (ELPylation) (MCpherson DT, Morrow C, Minehan DS, et al. Production and purification of a recombinant elastomeric polypeptide, G(VPGVG19-VPGV, from Escheriachia coli. Biotechnol Prog. 1992. 8:347-52), human transferrin fusion (Prior CP, Lai C-H, Sadehghi H et al. Modified transferrin fusion proteins. Patent WO2004 / 020405. 2004), proline-alanine-serine (PASylation) (Skerra A, Theobald I, Schlapsky M. Biological active proteins having increased in vivo and / or vitro stability. Patent WO2008 / 155134 A1. 2008), homo-amino acid polymer (HAPylation) (Schlapschy M, Theobald I, Mack H, et al. Fusion of a recombinant antibody fragment with a homo-amino acid polymer: effects on biophysical properties and prolonged plasma half-life. Protein Eng Des Sel. 2007. 20:273-84) and gelatin like protein (GLK) fusion (Huang Y-S, Wen X-F, 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).

[0234] Examples of polymer half-life extending moieties include polyethylene glycol (PEG), branched PEG, PolyPEG® (Warwick Effect Polymers; Coventry, UK), polysialic acid (PSA), starch, hydroxylethyl starch (HES), hydroxyalkyl starch (HAS), carbohydrate, polysaccharides, pullulane, chitosan, hyaluronic acid, chondroitin sulfate, dermatan sulfate, dextran, carboxymethyl-dextran, polyalkylene oxide (PAO), polyalkylene glycol (PAG), polypropylene glycol (PPG), polyoxazoline, polyacryloylmorpholine, polyvinyl alcohol (PVA), polycarboxylate, polyvinylpyrrolidone, polyphosphazene, polyoxazoline, polyethylene-co-maleic acid anyhydride, polystyrene-co-maleic acid anhydride, poly(1- hydroxymethyethylene hydroxymethylformal) (PHF), a zwitterionic polymer, a phosphorylcholine containing polymer and a polymer comprising MPC, Poly (Glyx-Sery), Hyaluronic acid (HA), Heparosan polymers (HEP), Fleximers, Dextran, and Poly-sialic acids (PSA).

[0235] In one embodiment a half-life extending moiety can be conjugated to an antibody via free amino groups of the protein using N-hydroxysuccinimide (NHS) esters. Reagents targeting conjugation to amine groups can randomly react to ^-amine group of lysines, Į-amine group of N-terminal amino acids, and į-amine group of histidines. In some embodiments, the conjugate includes a polymer conjugated to a cysteine, or free amino groups of the protein, e.g., using N-hydroxysuccinimide (NHS) esters. In some embodiments, the conjugate includes a polymer conjugated to ^-amine group of lysines, Į-amine group of N- terminal amino acids, and / or į-amine group of histidines.

[0236] However, the anti-VEGF-A antibodies disclosed herein have many amine groups available for polymer conjugation. Conjugation of polymers to free amino groups, thus, might negatively impact the ability of the antibody proteins to bind to VEGF.

[0237] In some embodiments, a half-life extending moiety is coupled to one or more free SH groups using any appropriate thiol-reactive chemistry including, without limitation, maleimide chemistry, or the coupling of polymer hydrazides or polymer amines to carbohydrate moieties of the antibody after prior oxidation. In some embodiments maleimide coupling is used In some embodiments, coupling occurs at cysteines naturally present or introduced via genetic engineering.

[0238] In some embodiments, polymers are covalently attached to cysteine residues introduced into anti-VEGF-A antibodies by site directed mutagenesis. In someembodiments, the cysteine residues are employed in the Fc portion of the antibody. In some embodiments, the sites to introduce cysteine residues into an Fc region are provided in WO 2013 / 093809, US 7,521,541, WO 2008 / 020827, US 8,008,453, US 8,455,622 and US2012 / 0213705, incorporated herein by reference for all purposes. In some embodiments, the cysteine mutations are Q347C (EU numbering) and L443C referring to the human IgG heavy chain by EU numbering.

[0239] In some embodiments, conjugates of antibody and high MW polymers serving as half-life extenders are provided. In some embodiments, a conjugate comprises an antibody that is coupled to a zwitterionic polymer wherein the polymer is formed from one or more monomer units and wherein at least one monomer unit has a zwitterionic group is provided. In some embodiments, the zwitterionic group is phosphorylcholine.

[0240] In some embodiments, one of the monomer units is HEMA-PC. In some embodiments, a polymer is synthesized from a single monomer which is HEMA-PC.

[0241] In some embodiments, some antibody conjugates have 2, 3, or more polymer arms wherein the monomer is HEMA-PC. In some embodiments, the conjugates have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 polymer arms wherein the monomer is HEMA-PC. In some embodiments, the conjugates have 3, 6 or 9 arms. In some embodiments, the conjugate has 9 arms.

[0242] In some embodiments, polymer-antibody conjugates have a polymer portion with a molecular weight of between 100,000 and 1,500,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 500,000 and 1,000,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 600,000 to 800,000 Da. In some embodiments, the conjugate has a polymer portion with a molecular weight between 600,000 and 850,000 Da and has 9 arms. When a molecular weight is given for an antibody conjugated to a polymer, the molecular weight will be the addition of the molecular weight of the protein, including any carbohydrate moieties associated therewith, and the molecular weight of the polymer.

[0243] In some embodiments, the polymer component of the conjugated protein has a molecular weight of about 100,000 Da or more, e.g., about 150,000 Da or more, about 200,000 Da or more, about 350,000 Da or more, about 400,000 Da or more, about 450,000 Da or more, about 500,000 Da or more, about 550,000 Da or more, about 600,000 Da or more,about 650,000 Da or more, about 700,000 or more, about 750,000 Da or more, about 800,000 Da or more, about 850,000 Da or more, about 900,000 Da or more, about 950,000 Da or more, about 1,000,000 Da or more, or a molecular weight in a range defined by any two of the preceding 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 conjugated protein is any of the polymers disclosed herein. In some embodiments, the polymer component of the conjugated protein is OG1801 or OG1802.

[0244] In some embodiments, an anti-VEGF-A antibody has a HEMA-PC polymer which has a molecular weight measured by Mw of between about 100 kDa and 1650 kDa is provided. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 500 kDa and 1000 kDa. In some embodiments, the molecular weight of the polymer as measured by Mw is between about 600 kDa to about 900 kDa. In some embodiments, the polymer molecular weight as measured by Mw is 750 kDa plus or minus 15%.

[0245] In some embodiments, the polymer is made from an initiator suitable for ATRP having one or more polymer initiation sites. In some embodiments, the polymer initiation site has a 2-bromoisobutyrate site. In some embodiments, the initiator has 3 or more polymer initiation sites. In some embodiments, the initiator has 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polymer initiation sites. In some embodiments, the initiator has 3, 6 or 9 polymer initiation sites. In some embodiments, the initiator has 9 polymer initiation sites. In some embodiments, the initiator is OG1786.

[0246] The anti-VEGF-A antibodies can be produced by recombinant expression including (i) the production of recombinant DNA by genetic engineering, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by, for example and without limitation, transfection, electroporation or microinjection, (iii) cultivating the transformed cells, (iv) expressing antibody, e.g. constitutively or on induction, and (v) isolating the antibody, e.g. from the culture medium or by harvesting the transformed cells, in order to (vi) obtain purified antibody.

[0247] The anti-VEGF-A antibodies can be produced by expression in a suitable prokaryotic or eukaryotic host system characterized by producing a pharmacologically acceptable antibody molecule. Examples of eukaryotic cells are mammalian cells, such asCHO, COS, HEK 293, BHK, SK-Hip, and HepG2. Other suitable expression systems are prokaryotic (e.g., E. coli with pET / BL21 expression system), yeast (Saccharomyces cerevisiae and / or Pichia pastoris systems), and insect cells.

[0248] A wide variety of vectors can be used for the preparation of the antibodies disclosed herein and are selected from eukaryotic and prokaryotic expression vectors. Examples of vectors for prokaryotic expression include plasmids such as, and without limitation, preset, pet, and pad, wherein the promoters used in prokaryotic expression vectors include one or more of, and without limitation, lac, trc, trp, recA, or araBAD. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as, and without limitation, pAO, pPIC, pYES, or pMET, using promoters such as, and without limitation, AOX1, GAP, GAL1, or AUG1; (ii) for expression in insect cells, vectors such as and without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoters such as and without limitation PH, p10, MT, Ac5, OpIE2, gp64, or polh, and (iii) for expression in mammalian cells, vectors such as, and without limitation, pSVL, pCMV, pRc / RSV, pcDNA3, or pBPV, and vectors derived from, in one aspect, viral systems such as and without limitation vaccinia virus, adeno-associated viruses, herpes viruses, or retroviruses, using promoters such as and without limitation CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and beta-actin. Analysis of mixed formulation and aggregates

[0249] In some embodiments, a method is presented for analyzing a mixed formulation sample (or therapeutically acceptable composition). In some embodiments, the method can include providing a mixed formulation sample (or therapeutically acceptable composition sample). In some embodiments, the mixed formulation sample (or therapeutically acceptable composition sample) includes a first antibody that is an anti-VEGF-A antibody conjugated to a polymer and a second antibody that is an anti-VEGF-A antibody that is not conjugated to the 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 run through a prefiltration step to provide a first filtered mixed formulation. In some embodiments, the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation. In some embodiments, the method further comprises a second run. In some embodiments, during the second run, the protein bound to the CEX column is eluted to provide a third filtered mixedformulation. In some embodiments, the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation. In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0250] In some embodiments the mixed formulation (or therapeutically acceptable composition) comprises a first antibody conjugated to a polymer and a second antibody that is not conjugated to a polymer, where one or both antibodies are not anti-VEGF-A antibodies. In some embodiments, the first and second antibody of the mixed formulation (or therapeutically acceptable composition) are the same antibody. In some embodiments, the first and second antibody of the mixed formulation are different antibodies. In some embodiments, the mixed formulation (or therapeutically acceptable composition) includes a first protein moiety conjugated to a polymer and a second protein moiety not conjugated to a polymer. In some embodiments the protein moieties are not antibodies. In some embodiments, the mixed formulation (or therapeutically acceptable composition) includes a mix of antibody and other protein moieties. In some embodiments, the first and second protein or antibody moieties are both 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 a polymer. In some embodiments, the mixed formulation (or therapeutically acceptable composition) comprises more than two antibodies or protein moieties.

[0251] In some embodiments, the proteins or antibodies of the mixed formulation (or therapeutically acceptable composition) are present in a desired percent composition. In some embodiments, the percent composition is calculated by determining the amount of the one component of the mixed formulation (or therapeutically acceptable composition) in mass units (e.g., ug) and dividing that amount by the total amount of all components in the composition in mass units, and multiplying by 100.

[0252] In some embodiments, the proteins or antibodies of the mixed formulation (or therapeutically acceptable composition) are present in a desired molar ratio of unconjugated to conjugated protein or antibody.

[0253] In some embodiments, the percent of the conjugated to unconjugated protein (e.g., % total molar amount) is calculated by (1) measuring the conjugated protein moiety and unconjugated protein moiety in mg / mL; (2) converting the mg / mL values of the conjugated protein moiety and unconjugated protein moiety into a molecular weight measured in kDa; and (3) dividing the molecular weight of each of the conjugated protein moiety and unconjugated protein moiety by the total molecular weight of the conjugated protein moiety and unconjugated protein moiety in the composition, and multiplied by 100 to achieve a percent of the total molar amount for each.

[0254] Any of the formulations and compositions provided herein, in some embodiments, can be defined as a percent composition (e.g., in mass weight concentration) of one component relative to the total mass weight concentration of the proteins (e.g., excluding any contribution of a polymer that may be conjugated thereto) in the composition. In some embodiments, a formulation or composition defined in % total molar amount of the second protein (e.g., the unconjugated protein) can be defined in percent composition (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, percent composition is measured in mass weight concentration (in other words, gram per liter or milligram per milliliter) of the free protein relative to the total mass weight concentration of the proteins (e.g., excluding any contribution of a polymer that may be conjugated thereto) in the mixture.

[0255] In some embodiments, a mixed therapeutically acceptable composition includes: a conjugate comprising a first protein conjugated to a phosphorylcholine-containing polymer; a second protein that is not conjugated to a phosphorylcholine-containing polymer; and a pharmaceutically acceptable carrier, wherein the percent composition of the second protein relative to the total protein mass weight concentration of the first protein and the second protein in the composition is about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.).

[0256] In some embodiments, a mixed therapeutically acceptable composition includes: a conjugate comprising a first protein conjugated to a polymer; and a second protein that is not conjugated to a polymer, wherein the second protein at a percent composition relative to the total protein mass weight concentration of the first protein and the second protein in the composition of about 1% or more (e.g., about 5-93%, 15-25%, 25-35%, 25-40%, etc.) has been combined with the conjugate, wherein the remainder of the total protein mass weight concentration comprises the first protein. In some embodiments, the mixed composition has been prepared by combining the second protein at a percent composition 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 protein and the second protein, with the conjugate such that the first protein at a percent composition of at a remainder of the total protein mass weight concentration. For example, for a total mass weight concentration of 50 mg / mL, the mixed therapeutically acceptable composition can be prepared by combining an amount of the second protein that corresponds to 10 mg / mL in the final composition (percent composition of 20%) with an amount of the conjugate that corresponds to 40 mg / mL of the first protein (as the conjugate, excluding any contribution of the polymer to the mass weight concentration calculation) in the final composition.

[0257] In any embodiment herein, the second protein that is not conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) can be referred to as the unconjugated protein or protein moiety, and the first protein that is conjugated to a polymer (e.g., a phosphorylcholine-containing polymer) can be referred to as the conjugated protein or protein moiety.

[0258] In some embodiments, the percent 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, “the remainder” denotes the portion of the total protein mass weight concentration of the composition (excluding any contribution of a polymer conjugated to the first protein to the mass weight concentration) that is not the unconjugated protein (e.g., the second protein), where the percent composition of the unconjugated protein (e.g., the second protein) and the remainder adds up to 100% of the total protein mass weight concentration. For example, between 5% and 6% of the total mass weight concentration of the total protein concentration in the mixture is theunconjugated protein, and between 94% and 95% of the total mass weight concentration of the proteins in the mixture is the conjugated protein, where the percentages add up to 100%. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 6% and 7%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 7% and 8%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 8% and 9%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 9% and 10%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 11%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 11% and 12%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12% and 13%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 13% and 14%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 14% and 15%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 16%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 16% and 17%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 17% and 18%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 18% and 19%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 19% and 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugatedantibody) is between 20% and 21%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 21% and 22%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 22% and 23%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 23% and 24%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 24% and 25%.

[0259] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 25% and 26%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 26% and 27%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 27% and 28%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 28% and 29%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 29% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 30% and 31%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 31% and 32%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 32% and 33%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 33% and 34%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 34% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percentcomposition of the unconjugated protein (e.g., unconjugated antibody) is between 35% and 36%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 36% and 37%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 37% and 38%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 38% and 39%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 39% and 40%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 40% and 41%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 41% and 42%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 42% and 43%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 43% and 44%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 44% and 45%.

[0260] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or fusion construct) is between 45% and 46%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or unconjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 46% and 47%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 47% and 48%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 48% and 49%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 49% and 50%, with theremainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 50% and 51%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 51% and 52%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 52% and 53%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 53% and 54%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 54% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 55% and 56%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 56% and 57%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 57% and 58%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 58% and 59%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 59% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 60% and 61%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 61% and 62%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 62% and 63%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 63% and 64%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 64% and 65%.

[0261] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 65% and 66%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugate fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 66% and 67%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 67% and 68%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 68% and 69%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 69% and 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 70% and 71%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 71% and 72%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 72% and 73%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 73% and 74%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 74% and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 75% and 76%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 76% and 77%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 77% and 78%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 78% and 79%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 79% and80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 80% and 81%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 81% and 82%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 82% and 83%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 83% and 84%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 84% and 85%.

[0262] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 85% and 86%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 86% and 87%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 87% and 88%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 88% and 89%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 89% and 90%, with the remainder comprising the conjugated protein.

[0263] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 5% and 25%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 10%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 12.5%, with the remainder comprising the conjugated protein. In some embodiments, thepercent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 15%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 17.5%. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 25%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 45%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 65%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 85%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 5% and 90%.

[0264] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 10% and 12.5%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 15%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 17.5%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 25%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 25%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 45%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 65%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10%and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 85%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 10% and 90%.

[0265] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 12.5% and 15%, with the remainder comprising the conjugated protein or protein moiety (e.g., the conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 17.5%. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 17.5% and 22.5%. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 25%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 45%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and65%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 85%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 12.5% and 90%.

[0266] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 15% and 17.5%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 25%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 45%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percentcomposition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 65%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 85%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 15% and 90%.

[0267] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is between 20% and 25%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 30%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 35%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 45%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 55%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 65%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 70%, with theremainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 75%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 85%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is between 25% and 90%, with the remainder comprising the conjugated protein.

[0268] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 5%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 6%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 7%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 8%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 9%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 10%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 11%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 12%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 13%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 14%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 15%, with the remainder comprising the conjugated protein. In someembodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 16%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 17%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 18%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 19%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 20%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 21%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 22%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 23%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 24%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 25%.

[0269] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 26%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 27%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 28%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 29%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 30%, with the remainder comprising the conjugated protein. In someembodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 31%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 32%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 33%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 34%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 35%.

[0270] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 36%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 37%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 38%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 39%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 40%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 41%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 42%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 43%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 44%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 45%.

[0271] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 46%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 47%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 48%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 49%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 50%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 51%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 52%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 53%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 54%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 55%.

[0272] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 56%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 57%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 58%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 59%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugatedantibody) is 60%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 51%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 62%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 63%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 64%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 65%.

[0273] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 66%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 67%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 68%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 69%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 70%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 11%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 72%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 73%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 74%, with the remainder comprising the conjugated protein. In someembodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 75%.

[0274] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated) is 76%, with the remainder comprising the conjugated protein or protein moiety (e.g., conjugated antibody or conjugated fusion construct). In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 77%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 78%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 79%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 80%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 81%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 82%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 83%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 84%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 85%.

[0275] In some embodiments, the percent composition of the unconjugated protein or protein moiety (e.g., unconjugated antibody or unconjugated fusion construct) is 86%, with the remainder comprising the conjugated protein or protein moiety. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 87%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 88%, with the remainder comprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 89%, with the remaindercomprising the conjugated protein. In some embodiments, the percent composition of the unconjugated protein (e.g., unconjugated antibody) is 90%.

[0276] In some embodiments, the ratio of the molecular weight of the unconjugated protein or protein moiety (e.g., second protein or protein moiety that is not conjugated to a 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 a ratio in a range defined by any two of the preceding values (e.g., 1:2-1:10, 1:3-1:8, 1:4-1:6). In some embodiments, the ratio is between about 1:4 and 1:6. In some embodiments, the ratio is about 1:5.33.

[0277] 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 that is not conjugated to a phosphorylcholine-containing polymer) have the same activity or function (e.g., bind the same epitope, inhibit the same target, catalyze 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 that is not conjugated to a phosphorylcholine-containing polymer) are the same 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 that is not conjugated to a phosphorylcholine-containing polymer) are at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 99%, or about 100%, or by a percentage in a range defined by any two of the preceding values (e.g., 85-100%, 90-99%, 90-95%, etc.) identical to each other in amino acid sequence. In some embodiments, where the first and second proteins each include two or more polypeptide chains, each of the corresponding polypeptide chains can independently have any of the noted sequence identity.

[0278] In some embodiments, the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) has a molecular weight (based on the protein portion) of about 50 kDa or more, e.g., 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 250kDa or more, about 300 kDa or more, or a molecular weight in a range defined by any two of the preceding values (e.g., 50-300 kDa, 100-300 kDa, 100-200 kDa, 150-250 kDa, etc.). In some embodiments, the first protein (e.g., the protein conjugated to the phosphorylcholine- containing polymer) has a molecular weight (based on the protein portion) of about 150 kDa. In some embodiments, first protein (e.g., the protein conjugated to the phosphorylcholine- containing polymer) has a molecular weight of about 200 kDa. In some embodiments, the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 50 kDa or more, e.g., 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 in a range defined by any two of the preceding values (e.g., 50- 300 kDa, 100-300 kDa, 100-200 kDa, 150-250 kDa, etc.). In some embodiments, the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine- containing polymer) has a molecular weight of about 150 kDa. In some embodiments, the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) has a molecular weight of about 200 kDa.

[0279] In some embodiments, one or both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) is a therapeutic protein. In some embodiments, both the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) are therapeutic proteins that are FDA approved as of May 2023. In some embodiments, one or both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) is an antibody (e.g., therapeutic antibody). Any suitable antibody can be used in the formulations (or therapeutically acceptable compositions). In some embodiments, one or both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) is a fusion construct. Any suitable fusion constructcan be used in the formulations (or therapeutically acceptable compositions). The antibody or fusion construct of any of the formulation or composition herein may or may not include a C-terminal lysine.

[0280] In some embodiments, sample analysis comprises evaluating samples with methods including but not limited to: SEC, SDS PAGE gel, multi angle light scattering (MALS), and imaged capillary isoelectric focusing (icIEF).

[0281] In some embodiments, prefiltration involves removal and discarding of insoluble particles such as dust, fiber and insoluble aggregates from the sample. In some embodiments, aggregates comprise insoluble clumps of antibody that have precipitated out of solution.

[0282] In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0283] In some embodiments, the second unconjugated antibody or protein moiety remains bound to the CEX column after run 1. In some embodiments, the first conjugated antibody or protein moiety flows through the CEX column. In some embodiments, the first conjugated antibody or protein moiety is separated by the SEC column. In some embodiments, the second unconjugated antibody or protein moiety is eluted from the CEX column with a NaCl solution. In some embodiments, 100uL of 1M NaCl is used to elute the second antibody or protein from the CEX column. In some embodiments, following elution, the second antibody is analyzed by a SEC column. In some embodiments, differences in charge and size between the first and second antibody or protein moiety permit differential partitioning in the CEX and SEC columns. In some embodiments, the differential partitioning of the first and second antibody or protein moiety in the CEX and SEC columns permits separation of the first and second antibody from the mixed formulation (or therapeutically acceptable composition).

[0284] In some embodiments, the polymer of the first antibody comprises a phosphorylcholine containing polymer. In some embodiments, the polymer is covalently bonded to the first antibody at a cysteine outside a variable region of the first antibody. In some embodiments, said cysteine replaces a non-cysteine amino acid that occurs in a same positionin a sequence. In some embodiments, the first antibody comprises a light chain and heavy chain, said heavy chain comprising an Fc region. In some embodiments, the cysteine is in the Fc region of the heavy chain. In some embodiments, the sequence of the heavy chain comprises SEQ ID NO: 1 (with or without the C-terminal lysine). In some embodiment the sequence of the light chain comprises SEQ ID NO: 2;

[0285] In some embodiments, the first antibody conjugated to a phosphorylcholine containing polymer has the following structure:where X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, or c) a halide. In some embodiments, 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.

[0286] In some embodiments, the heavy chain of the first antibody is denoted by the letter H, and each light chain of the first antibody is denoted by the letter L. In someembodiments, the polymer is bonded to the first antibody through the sulfhydryl of a cysteine at position 449, as numbered in SEQ ID NO: 1, which bond is depicted on one of the heavy chains;

[0287] In some embodiments, PC is , where the curvy line indicates the point of attachment to the rest of the polymer. In some embodiments, n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%. In some embodiments, the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is about 1500 to about 3500 plus or minus about 10% to about 20%.

[0288] In some embodiments, the second antibody comprises a light chain and heavy chain, said heavy chain comprising an Fc region. In some embodiments, a cysteine is in the Fc region of the heavy chain. In some embodiments, the sequence of the heavy chain comprises SEQ ID NO: 1 (with or without the C-terminal lysine), and wherein the sequence of the light chain comprises SEQ ID NO: 2. In some embodiments, the heavy chain of the first and second antibodies comprises CDRH1: GYDFTHYGMN, CDRH2: WINTYTGEPTYAADFKR, and CDRH3: YPYYYGTSHWYFDV, and position 231 is T (via sequential counting as in SEQ ID NO: 1), and the light chain of the first and second antibodies comprises CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13), and CDRL3: QQYSTVPWT (SEQ ID NO: 14), and Kabat position 4 is L.

[0289] In some embodiments, the mixed formulation (or therapeutically acceptable composition) sample is loaded into a tandem CEX and SEC HPLC column arrangement. In some embodiments, the formulation (or therapeutically acceptable composition) sample travels sequentially through a prefilter, a CEX column, and a SEC HPLC column. In some embodiments, the CEX column comprises a Shodex SP825 column with internal diameter (i.d.) x length dimensions of 9.0x75mm. In some embodiments, the SEC HPLC is a TSKgel G3000SWxl column with i.d. x length dimensions of 7.8x300mm.

[0290] In some embodiments, the mixed formulation (or therapeutically acceptable composition) sample is loaded into a tandem CEX and SEC HPLC column arrangement. In some embodiments, the formulation (or therapeutically acceptable composition) sample travels sequentially through a prefilter, a CEX column, and a SEC HPLC column. In someembodiments, the CE column comprises an IEC ProPac WCX-10 column with internal diameter (i.d.) x length dimensions of 4.0 mm (about 0.16 in) x 250 mm (about 9.84 in). In some embodiments, the SEC HPLC comprises a TSKgel G4000SWXL column with i.d. x length dimensions of 7.8 mm (about 0.31 in) ID x 30 cm (about 11.81 in), 8 μm.

[0291] In some embodiments, the column arrangement comprises an ion exchange column (IEX), hydrophobic interaction (HIC), or affinity column and a SEC column. In some embodiments, the IEX column can be a CEX or anion exchange (AEX) column.

[0292] In some embodiments, the isocratic running conditions comprise a flow rate of 0.1-5ml / min. In some embodiments, the isocratic running conditions comprise a flow rate of at 0.5-1ml / min. In some embodiments, the flow rate can be any number between 0.1- 5ml / min. In some embodiments, the flowrate can be any number between 0.5-1ml / min. In some embodiments, the buffer comprises 20mM sodium acetate, pH 3-6. In some embodiments, the isocratic conditions are kept constant and uniform during a chromatography run.

[0293] In some embodiments, the buffer comprises 20mM sodium acetate, pH 5. In some embodiments, the buffer comprises any amount between 50-500mM NaCl. In some embodiments, the buffer comprises any amount between 90-120mM NaCl. In some embodiments, the buffer comprises a conductivity of, or of about 2-32 mS / cm. In some embodiments, the buffer comprises a conductivity of, of about, or of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 mS / cm, or of, of about, or of at most 32, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, or 8 mS / cm, or a conductivity in range defined by any two of the preceding values (e.g., 2-32 mS / cm, 4-30 mS / cm, 5-15 mS / cm, 7-10 mS / cm, 8-10 mS / cm, etc.). In some embodiments, the buffer comprises a conductivity of, or of about 8.9 mS / cm.

[0294] In some embodiments, the method further comprises assessment for high molecular weight aggregates within the purified mixed formulation (or therapeutically acceptable composition) by methods comprising SEC profile analysis and SDS PAGE gel.

[0295] In some embodiments, the percent composition of the second antibody relative to the total protein mass weight concentration of the first antibody and the second antibody in the composition is any percentage between 0-93%, preferably between 0% and 50%, or between 5% and 93%. In some embodiments, the percent composition of the second antibody relative to the total protein mass weight concentration of the first antibody and thesecond antibody in the composition is any percentage between 0% and 50%, or between 5% and 50%. In some embodiments, the percent composition of the second antibody relative to the total protein mass weight concentration of the first antibody and the second antibody in the composition is any percentage between 0% and 25%, or between 5-25%.

[0296] In some embodiments, the mixed formulation (or therapeutically acceptable composition) sample comprises any amount between 50^g and 1340^g of protein.

[0297] In some embodiments, the method further comprises assessment of the percentage of the second antibody in the purified formulation (or therapeutically acceptable composition) by SEC profile analysis.

[0298] In some embodiments, the first run separates the first antibody from the second antibody and antibody aggregates.

[0299] In some embodiments, the tandem HPLC system further comprises SEC analysis on the second filtered mixed formulation (or therapeutically acceptable composition) of the first run performed by a second SEC HPLC column down-stream of the CEX column, wherein the second SEC HPLC column runs in parallel to the first SEC HPLC column. In some embodiments, the second HPLC column can be a Shodex SB-806 HQ or Shodex 806M-HQ column. In some embodiments, the second SEC HPLC column runs subsequent to the first SEC HPLC column instead of in parallel. In some embodiments, the first SEC HPLC column runs subsequent to the second SEC HPLC column instead of in parallel. In some embodiments, a switch valve allows for selection of the first or second SEC HPLC column.

[0300] In some embodiments, the step for elution of CEX bound free protein during the second run comprises a pulse of high salt at 0.5-5M NaCl. In some embodiments, the step for elution of CEX bound free protein during the second run comprises a pulse of high salt at 1M NaCl.

[0301] In some embodiments, during the second run, the fourth filtered mixed formulation (or therapeutically acceptable composition) is run through a prefiltration step to provide a fifth filtered mixed formulation (or therapeutically acceptable composition), wherein the fifth filtered mixed formulation (or therapeutically acceptable composition) is run through a CEX column to provide a six filtered mixed formulation (or therapeutically acceptable composition), wherein the sixth mixed formulation (or therapeutically acceptable composition) is run through a SEC HPLC column. In some embodiments, running the filtered mixed samplethrough a size exclusion exchange SEC HPLC column separates the second antibody from antibody aggregates.

[0302] In some embodiments, the effluent of the first and / or second SEC HPLC columns is connected to a to a multi angle light scattering (MALS) detector.

[0303] In some embodiments, additional valve is installed downstream of the first and / or second SEC column and the eluted free protein peaks are collected using a fraction collector for icIEF analysis. In some embodiments, the eluted free protein peaks are analyzed using any suitable analytical techniques, including without limitation, icIEF, mass spectrometry, fluorescence detection, photodiode array (PDA) detector.

[0304] In some embodiments, an apparatus is provided comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed formulation (or therapeutically acceptable composition) sample. In some embodiments, the mixed formulation (or therapeutically acceptable composition) sample comprises a first antibody that is an anti- VEGF-A antibody conjugated to a polymer and a second antibody that is an anti-VEGF-A antibody that is not conjugated to the polymer. In some embodiments, the analysis comprises a first run. In some embodiments, the mixed formulation (or therapeutically acceptable composition) is run through a prefiltration step to provide a first filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the first filtered mixed formulation (or therapeutically acceptable composition) is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the analysis comprises a second run. In some embodiments, during the second run, the protein bound to the CEX column is eluted to provide a third filtered mixed formulation. (or therapeutically acceptable composition) In some embodiments, the third filtered mixed formulation (or therapeutically acceptable composition) is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation (or therapeutically acceptable composition).

[0305] In some embodiments, the first, second, or first and second antibody comprises a light chain and a heavy chain, said heavy chain comprising an Fc region. In some embodiments, a cysteine of the first, second, or first and second antibody is in the Fc region of the heavy chain.

[0306] In some embodiments, the first, second, or first and second antibody is an immunoglobulin G (IgG).

[0307] In some embodiments, the heavy chain of the first, second, or first and second antibody comprises: CDRH1: GYDFTHYGMN (SEQ ID NO: 9), CDRH2: WINTYTGEPTYAADFKR (SEQ ID NO: 10), and CDRH3: YPYYYGTSHWYFDV (SEQ ID NO: 11), and the light chain of the first, second, or first and second antibody comprises CDRL1: SASQDISNYLN (SEQ ID NO: 12), CDRL2: FTSSLHS (SEQ ID NO: 13), and CDRL3: QQYSTVPWT (SEQ ID NO: 14).

[0308] In some embodiments, the heavy chain isotype of the first, second, or first and second antibody is human IgG1. In some embodiments, the heavy chain constant domain of the first, second, or first and second antibody has one or more mutations relative to the constant domain of human IgG1 to modulate effector function.

[0309] In some embodiments, the mutations of the first, second, or first and second antibody are to one or more of the following amino acid positions (EU numbering): E233X, L234X, L235X, G236X, G237X, A327X, A330X, and P331X wherein X is any natural or unnatural amino acid. In some embodiments, the mutations of the first, second, or first and second antibody are selected from the group comprising (EU numbering): E233P, L234V, L234A, L235A, G237A, A327G, A330S, and P331S. In some embodiments, the mutations of the first, second, or first and second antibody comprise the following mutations: L234A, L235A, and G237A (EU numbering).

[0310] In some embodiments, the cysteine of the first, second, or first and second antibody is in the antibody heavy chain and is Q347C (EU numbering) or L443C (EU numbering).

[0311] In some embodiments, the sequence of the antibody heavy chain of the first, second, or first and second antibody is SEQ ID NO: 1 (with or without the C-terminal lysine) and the sequence of the light chain of the first, second, or first and second antibody is SEQ ID NO: 2.

[0312] In some embodiments, the cysteine of the first, second, or first and second antibody is L443C (EU numbering).

[0313] In some embodiments, the polymer of the antibody conjugate has three or more arms or is synthesized with an initiator comprising 3 or more polymer initiation sites.

[0314] In some embodiments, the polymer of the antibody conjugate has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 arms or is synthesized with an initiator comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 polymer initiation sites.

[0315] In some embodiments, the polymer of the antibody conjugate has a polydispersity value (PDI) of less than about 1.2.

[0316] In some embodiments, the percent composition of the second antibody relative to the total protein mass weight concentration of the first protein and the second protein in the composition is any of the following: about 7% second antibody, about 10% second antibody; about 12.5% second antibody; about 15% second antibody; about 17.5% second antibody; about 20% second antibody; and about 25% (B).

[0317] In some embodiments, provided herein is a composition comprising any two proteins that can be the same or different in function, wherein one is conjugated to a polymer and the other is not conjugated to the polymer (or conjugated to any polymer or conjugated to any effective amount of a polymer). The composition can be for the treatment of an eye disorder. In some embodiments, both of the proteins are therapeutics proteins for the treatment of an eye disorder. In some embodiments, one or both of the proteins are therapeutics, 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 can be synthesized. In some embodiments, the antibody can be a native sequence antibody. In some embodiments, the antibody can be a Fab fragment. In some embodiments, the antibody can be a Trap fragment. In some embodiments, the antibody can be a fusion protein such as a Trap-antibody fusion protein. In some embodiments, the antibody can be a peptide fragment. In some embodiments, a non-antibody scaffold protein can be used instead of an antibody.

[0318] In some embodiments, the composition comprises a mixture of two antibodies by mass weight concentration of 0.1% to 1% unconjugated antibody with the remainder comprising the conjugated antibody. In some embodiments, the composition comprises a mixture of two antibodies by mass weight concentration of 1% to 5% unconjugated antibody with the remainder comprising the conjugated antibody. In some embodiments, the composition comprises a mixture of two antibodies by mass weight concentration of 5% to 10% unconjugated antibody with the remainder comprising theconjugated antibody. In some embodiments, the composition comprises a mixture of the two antibodies by mass weight concentration of 5% to 15% unconjugated antibody with the remainder comprising the conjugated antibody. In some embodiments, the composition comprises a mixture of the two antibodies of the same mass weight concentration, of which 15% to 25% unconjugated antibody with the remainder comprising the conjugated antibody represents 15-25% of unconjugated antibody concentration in mass weight concentration (e.g. gram / liter) of the total protein concentration in the mixture. In some embodiments, the composition comprises a mixture of the two antibodies by mass weight concentration of 25% to 35% unconjugated antibody with the remainder comprising the conjugated antibody. In some embodiments, the composition comprises a mixture of the two antibodies by mass weight concentration of 35% to 45% unconjugated antibody with the remainder comprising the conjugated antibody. In some embodiments, the composition comprises a mixture of the two antibodies by mass weight concentration (mg / mL) of 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to 70%, 70% to 75%, 75% to 80%, 80% to 85%, 85% to 90%, and 90% to 95%.

[0319] In some embodiments, a method of analyzing a mixed formulation (or therapeutically acceptable composition) sample is provided. In some embodiments, the method comprises providing a mixed formulation (or therapeutically acceptable composition) that comprises a fusion protein conjugated to a polymer a second protein that is not conjugated to the 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 run through a prefiltration step to provide a first filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the first filtered mixed formulation (or therapeutically acceptable composition) is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the method further comprises a second run. In some embodiments, during the second run, the protein bound to the CEX column is eluted to provide a third filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the third filtered mixed formulation (or therapeutically acceptable composition) is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation (ortherapeutically acceptable composition). In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0320] In some embodiments, an apparatus comprising a tandem series of CEX and SEC HPLC columns for use in purifying a mixed formulation (or therapeutically acceptable composition) sample is provided. In some embodiments, the sample comprises a first fusion protein conjugated to a polymer and a second protein that is not conjugated to the 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 run through a prefiltration step to provide a first filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the first filtered mixed formulation (or therapeutically acceptable composition) is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the method further comprises a second run. In some embodiments, during the second run, the protein bound to the CEX column is eluted to provide a third filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the third filtered mixed formulation (or therapeutically acceptable composition) is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation (or therapeutically acceptable composition).

[0321] In some embodiments, a method of analyzing a mixed formulation (or therapeutically acceptable composition) sample is provided. In some embodiments, the method comprises providing a mixed formulation (or therapeutically acceptable composition) that comprises a first antibody that is an anti-VEGF-A antibody conjugated to a polymer; and a second antibody that is an anti-VEGF-A antibody that is not conjugated to the 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 run through a prefiltration step to provide a first filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the first filtered mixed formulation (or therapeutically acceptable composition) is run through a cation exchange chromatography (CEX) column toprovide a second filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the method further comprises a second run. In some embodiments, during the second run, the protein bound to the CEX column is eluted to provide a third filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, the third filtered mixed formulation (or therapeutically acceptable composition) is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation (or therapeutically acceptable composition). In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0322] In some embodiments, a method of analyzing a mixed formulation sample is provided. In some embodiments, the method comprises providing a mixed formulation that comprises a first antibody that is an anti-VEGF-A antibody conjugated to a polymer and a second antibody that is an anti-VEGF-A antibody that is not conjugated to the polymer. In some embodiments, the method further comprises loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns. In some embodiments, the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column. In some embodiments, the method comprises a first run. In some embodiments, during the first run, the mixed formulation (or therapeutically acceptable composition) is injected and run through the tandem CEX-SEC system. In some embodiments, during the second run, a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column. In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0323] In some embodiments, a method of analyzing a mixed formulation (or therapeutically acceptable composition) sample is provided. In some embodiments, the method comprises providing a mixed formulation (or therapeutically acceptable composition) thatcomprises a combination of two protein moieties. In some embodiments, the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer. In some embodiments, the method comprises loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down-stream of the CEX column is a size exclusion chromatography (SEC) column. In some embodiments, the method comprises a first run. In some embodiments, during the first run, the mixed formulation (or therapeutically acceptable composition) is injected and run through the tandem CEX-SEC system. In some embodiments, the method comprises a second run. In some embodiments, during the second run, a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column. In some embodiments, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation (or therapeutically acceptable composition) in the CEX column. In some embodiments, the method allows analysis of the mixed formulation (or therapeutically acceptable composition) based on the differential charge and size variant of the constituents.

[0324] In some embodiments, the mixed formulation (or therapeutically acceptable composition) sample comprises protein moiety A and protein moiety B.

[0325] In some embodiments, A and B can be the same protein moiety.

[0326] In some embodiments, A and B are different protein moieties.

[0327] In some embodiments, A and B are antibodies.

[0328] In some embodiments, A and / or B can be non-antibody proteins. In some embodiments, non-antibody proteins can include, but are not limited to cytokines, interferons, enzymes, regulatory proteins, growth factors, scaffolds, bone morphogenic proteins, blood factors, anticoagulants, hormones, cell signaling proteins, cell adhesion proteins, cell cycle proteins. In some embodiments, the non-antibody proteins are recombinantly produced.

[0329] In some embodiment the MW of protein A is greater or equal to the MW of protein B.

[0330] In some embodiments, the MW of protein B is greater or equal to the MW of protein A.

[0331] In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 55. In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 0.05 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 1 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 2 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 3 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 5 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 10 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 15. In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 20 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 25 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 30 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 35 In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 40. In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 45 In some embodiments, the molar ratio range of the second protein orantibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 50.

[0332] In some embodiments, the molar ratio range of the second protein or antibody not conjugated to a polymer to the first protein or antibody conjugated to a polymer is any number between 0.01 and 16.

[0333] In some embodiments, the second protein or antibody is added directly to the drug product of the first antibody or formulation to generate the mixed formulation (or therapeutically acceptable composition).

[0334] In some embodiment, the second protein or antibody is added to the CEX pool with the first protein or antibody and undergoes ultrafiltration and diafiltration to generate the mixed formulation (or therapeutically acceptable composition).

[0335] In some embodiments, the second protein or antibody undergoes decapping and refolding prior to being added to the CEX pool.

[0336] In some embodiments, the decapped and refolded second protein or antibody undergoes further treatment with alkylation agents such as iodoacetamide (IAM) or N-ethylmaleimide (NEM) before being added to the CEX pool.

[0337] In some embodiments, the decapping process includes reducing one or more cysteines in a protein to form a decapped protein in a solution. In some embodiments, after reducing the one or more cysteines the decapped protein is reoxidized to restore at least one disulfide linkage in the reduced protein while ensuring that an engineered cysteine residue in the protein remains in a free thiol form to form a reoxidized decapped protein in the solution. In some embodiments, at least one excipient is then added to the solution. In some embodiments, the excipient reduces a polymer induced protein precipitation. In some embodiments, after the excipient is added, a polymer is added to the solution, which is conjugated to the reoxidized decapped protein at the engineered cysteine residue to form a conjugated protein.

[0338] In some embodiments, the mixed formulation (or therapeutically acceptable composition) further comprises an additional protein or antibody conjugated to a polymer.

[0339] In some embodiments, the polymer has 9 arms. In some embodiments, the polymer has a molecular weight of between about 600,000 to about 1,000,000 Da.

[0340] In some embodiments, the antibody is a complement factor D antibody. In some embodiments, the complement factor D antibody comprises a heavy chain and a light chain. In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 15-47; and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 48-80.

[0341] In some embodiments, the antibody comprises an antagonist IL-6 antibody fused to a VEGF trap. In some embodiments, the antibody comprises a heavy chain and a light chain. In some embodiments, the heavy chain variable region (VH) comprises the amino acid sequence of any one of SEQ ID NOs: 81-89; and the light chain variable region (VL) comprises the amino acid sequence of any one of SEQ ID NOs: 90-92.

[0342] In some embodiments, the heavy and light chains are selected from the group comprising: (A) the heavy chain of SEQ ID NO:93 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:94; (B) the heavy chain of SEQ ID NO:95 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:96; (C) the heavy chain of SEQ ID NO:97 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:98; (D) the heavy chain of SEQ ID NO:99 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:100; (E) the heavy chain of SEQ ID NO:101 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:102; (F) the heavy chain of SEQ ID NO:103 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:104; (G) the heavy chain of SEQ ID NO:105 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:106; (H) the heavy chain of SEQ ID NO:107 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:108; (I) the heavy chain of SEQ ID NO:109 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:110;(J) the heavy chain of SEQ ID NO:111 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:112; (K) the heavy chain of SEQ ID NO:113 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:114; (L) the heavy chain of SEQ ID NO:115 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:116; (M) the heavy chain of SEQ ID NO:117 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:118; (N) the heavy chain of SEQ ID NO:119 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:120; (O) the heavy chain of SEQ ID NO:121 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:122; (P) the heavy chain of SEQ ID NO:123 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:124; (Q) the heavy chain of SEQ ID NO:125 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:126; and (R) the heavy chain of SEQ ID NO:127 (with or without the C-terminal lysine), and the light chain of SEQ ID NO:128.

[0343] In some embodiments, the composition comprises OG1953 with variable amounts of unconjugated mAb and conjugated mAb. In some embodiments, one or more of the proteins of the composition is KSI-501, an antibody conjugate in which the protein is the trap antibody fusion of anti-VEGF and anti-IL-6 unconjugated protein (OG2072) and the conjugated protein (OG2074). In some embodiments, all the monomer pendant groups of the biopolymer of the composition are phosphorylcholine. In some embodiments, a quantity of desired small molecules is conjugated into the biopolymer of the composition. In some embodiments, the biopolymer of the compositions is conjugated and comprises copolymer characteristics that are distinct from the HEMA-PC monomer, in a desired ratio of unconjugated mAb to conjugated mAb.

[0344] In some embodiments, one or both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containingpolymer) is an anti-VEGF-A antibody, a fusion construct comprising a VEGF Trap fused to the heavy chain of an anti-IL-6 antibody, an anti-IL-6 antibody, a fusion construct comprising a PDGFR extracellular trap fused to the heavy chain of an anti-VEGF-A antibody, a VEGF trap-Fc fusion protein, an anti-HTRA1 antibody, or an anti-complement factor D (CFD) antibody. In some embodiments, both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) are an anti- VEGF-A antibody. In some embodiments, both of the first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) and the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) are a fusion construct comprising a VEGF Trap fused to the heavy chain of an anti-IL-6 antibody. In some embodiments, the anti-VEGF-A antibody is a full-length antibody. In some embodiments, the anti-VEGF-A antibody is or includes an anti-VEGF-A Fab fragment. In some embodiments, the anti-VEGF-A antibody is OG1950, e.g., as described herein and in US patent publication no.2017 / 0190766, the entirety of which is incorporated herein by reference.

[0345] In some embodiments, the anti-VEGF-A antibody is selected from bevacizumab, ranibizumab, brolucizumab, or faricimab. In some embodiments, the VEGF trap-Fc fusion protein is aflibercept. In some embodiments, the fusion construct is OG2072, e.g., as described herein and in US patent publication no.2019 / 0270806, the entirety of which is incorporated herein by reference.

[0346] In some embodiments, the mixed formulation comprises KSI-301. In some embodiments, KSI-301 comprises fusion protein OG1950 and its conjugated form with OG1802, OG1953.

[0347] In some embodiments, the KSI-301 Drug Product (DP) comprises 50mM sodium acetate, 0.025% polysorbate 20, pH 4.5-5.6), 50 mg / mL (total protein concentration) containing 15.0-25.0% OG1950, 75.0-85.0% OG1953.

[0348] In some embodiments, the mixed formulation comprises KSI-501. In some embodiments, KSI-501 comprises fusion protein OG2072 and its conjugated form with OG1802, OG2074.

[0349] In some embodiments, the KSI-501 Drug Substance (DS) comprises 50- 60mM sodium acetate, polysorbate 20, sucrose, pH 4.5-5.6, 50 mg / mL (total protein concentration) containing 20-40% OG2072, 60-80% OG2074.

[0350] In some embodiments, first protein (e.g., the protein conjugated to the phosphorylcholine-containing polymer) can be any of the antibodies or fusion proteins described herein. In some embodiments, the second protein (e.g., the unconjugated protein or protein that is not conjugated to a phosphorylcholine-containing polymer) can be any of the antibodies or fusion proteins described herein.

[0351] In some embodiments, any of the methods or apparatuses described herein can be used to analyze, purify, and / or characterize a mixed formulation sample comprising (A) a first protein conjugated to a polymer, wherein the first protein can be any of the antibodies or fusion proteins described herein, and (B) a second protein that is not conjugated to the polymer, wherein, the second protein can be any of the antibodies or fusion proteins described herein.

[0352] In some embodiments, provided herein is a composition comprising any two proteins that can be the same or different in function, wherein one is conjugated to a polymer and the other is not conjugated to the polymer (or conjugated to any polymer or conjugated to any effective amount of a polymer). The composition can be for the treatment of an eye disorder. In some embodiments, both of the proteins are therapeutics proteins for the treatment of an eye disorder. In some embodiments, one or both of the proteins are therapeutics, 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 can be synthesized. In some embodiments, the antibody can be a native sequence antibody. In some embodiments, the antibody can be a Fab fragment. In some embodiments, the antibody can be a Trap fragment. In some embodiments, the antibody can be a fusion protein such as a Trap-antibody fusion protein. In some embodiments, the antibody can be a peptide fragment. In some embodiments, a non-antibody scaffold protein can be used instead of an antibody. Method of Treatment

[0353] In some embodiments, a method is presented for the treatment or prophylaxis of an ocular disease having the step of administering a therapeutic protein selectedfrom the group comprising a protein, an antibody, or an anti-VEGF-A antibody (and conjugates thereof). In some embodiments, any one or more of the antibodies or antibody conjugates provided herein can be used as treatment and / or prophylaxis for an ocular disease. The method includes administering to the subject any one or more of the antibodies or antibody conjugates provided herein.

[0354] In some embodiments a method for treatment or prophylaxis of an ocular disease is provided. The method comprises administering an effective dose of any of the antibody and / or antibody conjugates described herein to a subject in need thereof. In some embodiments, the disease can be age-related macular degeneration (AMD) or diabetic macular edema (DME). In some embodiments, the disease can be wet AMD.

[0355] In some embodiments, the ocular disease is selected from one or more of the group comprising diabetic retinopathy, choroidal neovascularization (CNV), age-related macular degeneration (AMD), diabetic macular edema (DME), pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, central retinal vein occlusion (CRVO), branched central retinal vein occlusion (BRVO), corneal neovascularization, retinal neovascularization, retinopathy of prematurity (ROP), subconjunctival hemorrhage, and hypertensive retinopathy. In some embodiments, the ocular disease is diabetic retinopathy.

[0356] In some embodiments, the antibody or antibody conjugate is administered no more frequently than once a month. In some embodiments, the antibody or conjugate thereof is administered two times per month or weekly. In some embodiments, the antibody or conjugate thereof is administered once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, or once every twelve months.

[0357] In some embodiments, one or more of the compositions provided herein can allow for a reduction in the consequences of high treatment burdens from the use of intravitreal injection of anti-VEGF agents for the treatment of the wet (proliferative) form of age related macular degeneration (AMD). Real world outcomes for patients with wet AMD lag behind the clinical outcomes demonstrated in the phase 3 clinical studies such as the MARINA and ANCHOR studies with Lucentis®(ranibizumab) and the VIEW 1 and VIEW 2 studies with Eylea®(aflibercept). An anti-VEGF therapeutic with a longer ocular residence time such thatit can be administered less frequently and therefore with a more patient-tolerable profile can bring real world outcomes closer to phase 3 clinical outcomes for more patients.

[0358] In some embodiments, compounds, including antibody conjugates and anti- VEGF-A antibodies described herein are used to treat patients who have background or nonproliferative diabetic retinopathy but have little or no vision impairment. In some embodiments, such patients are dosed less than once a month via intravitreal injection. In some embodiments, such patients are dosed six times a year. In some embodiments, such pateints are dosed no more than four times a year. In some embodiments, the patients are dose no more than three times a year. In some embodiments, the patients are dosed no more than twice a year. In some embodiments, the patients are dosed no more than once a year. In some embodiments, the subject receives the antibody or antibody conjugate via intravitreal injection.

[0359] The therapeutic proteins (e.g., proteins, protein conjugates, antibodies and antibody conjugates) described herein can be employed by expression of such polypeptides in vivo in a patient, i.e., gene therapy.

[0360] There are two major approaches to getting the nucleic acid (optionally contained in a vector) into the patient's cells: in vivo and ex vivo. For in vivo delivery the nucleic acid is injected directly into the patient, usually at the sites where the therapeutic protein is required, i.e., where biological activity of the therapeutic protein is needed. For ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells, and the modified cells are administered to the patient either directly or, for example, encapsulated within porous membranes that are implanted into the patient (see, e.g , U.S. Pat. Nos. 4,892,538 and 5,283,187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or transferred in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, transduction, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. Transduction involves the association of a replication-defective, recombinant viral (including retroviral) particle with a cellular receptor, followed by introduction of the nucleic acids contained by the particle into the cell. A commonly used vector fo...

Claims

WHAT IS CLAIMED IS:

1. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

2. A method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition;iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents.

3. A method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

4. An apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed formulation sample comprising 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation.

5. An apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed therapeutic composition sample comprising 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition.

6. An apparatus comprising a tandem series of CEX and SEC HPLC columns for use in analyzing a mixed therapeutically effective formulation sample comprising 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 that is not conjugated to the polymer,wherein the purification comprises; i. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation.

7. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; and wherein the method reduces the presence of antibody aggregates in the formulation; and wherein the polymer of the first antibody comprises a phosphorylcholine containing polymer, wherein the polymer is covalently bonded to the first antibody at a cysteine outside a variable region of the first antibody, and wherein said cysteine replaces a non-cysteine amino acid that occurs in a same position in a sequence,wherein the first antibody comprises a light chain and heavy chain, said heavy chain comprising an Fc region, wherein the cysteine is in the Fc region of the heavy chain, wherein the sequence of the heavy chain comprises SEQ ID NO: 1 (with or without the C-terminal lysine), and wherein the sequence of the light chain comprises SEQ ID NO: 2; wherein the antibody conjugate has the following structure:where X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, or c) a halide; (or optionally where X is a) –OR where R is –H, Methyl, ethyl, propyl, or isopropyl, b) –H, c) any halogen, including –Br, –Cl, or –I, d) –SCN, or e) –NCS;) wherein: each heavy chain of the first antibody is denoted by the letter H, and each light chain of the first antibody is denoted by the letter L;the polymer is bonded to the first antibody through the sulfhydryl of a cysteine at position 449, as numbered in SEQ ID NO: 1, which bond is depicted on one of the heavy chains;PC is , where the curvy line indicates the point of attachment to the rest of the polymer; and n1, n2, n3, n4, n5, n6, n7, n8 and n9 are the same or different such that the sum of n1, n2, n3, n4, n5, n6, n7, n8 and n9 is 2500 plus or minus 15%; and wherein, the second antibody comprises a light chain and heavy chain, said heavy chain comprising an Fc region, wherein a cysteine is in the Fc region of the heavy chain, wherein the sequence of the heavy chain comprises SEQ ID NO: 1 (with or without the C-terminal lysine), and wherein the sequence of the light chain comprises SEQ ID NO: 2; and wherein the mixed formulation sample travels sequentially through a prefilter, the CEX column, and the SEC HPLC column, wherein the tandem CEX and SEC HPLC columns are a Shodex SP825 column with internal diameter (i.d.) x length dimensions of 9.0x75mm and a TSKgel G3000SWxl column with i.d. x length dimensions of 7.8x300mm column arrangement; wherein the isocratic running conditions comprise a flow rate of 0.5ml / min, and the buffer comprises 20mM sodium acetate, pH 5 any amount between 50mM NaCl and 5M NaCl; wherein the percentage of the second antibody is any percentage between 0% and 20%; wherein the mixed formulation sample comprises any amount between 25ug and 1340ug of protein; wherein the method further comprises assessment for high molecular weight aggregates within the purified mixed formulation by methods comprising SEC profile analysis and SDS PAGE gel; and wherein the method further comprises assessment of the percentage of the second antibody in the purified mixed formulation by SEC profile analysis;wherein the first run separates the first antibody from the second antibody and antibody aggregates, wherein the step for elution of CEX bound free protein during the second run comprises a pulse of high salt at 1M NaCl, wherein during the second run, the fourth filtered mixed formulation is run through a prefiltration step to provide a fifth filtered mixed formulation, wherein the fifth filtered mixed formulation is run through a CEX column to provide a six filtered mixed formulation, wherein the sixth mixed formulation is run through a SEC HPLC column; and wherein running the filtered mixed sample through a size exclusion exchange SEC HPLC column separates the second antibody from antibody aggregates.

8. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

9. A method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing a mixed therapeutic composition that comprises:a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutic composition in the CEX column; and wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents.

10. A method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective formulation that comprises: a. a fusion protein conjugated to a polymer; and b. a second protein that is not conjugated to the polymer; ii. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation;iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed therapeutically effective formulation in the CEX column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

11. An apparatus comprising a tandem series 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 that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation.

12. An apparatus comprising a tandem series of CEX and SEC HPLC columns for use in purifying a mixed therapeutic composition sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutic composition is run through a prefiltration step to provide a first filtered mixed therapeutic composition, wherein the first filtered mixed therapeutic composition is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutic composition; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutic composition, wherein the third filtered mixed therapeutic composition is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutic composition.

13. An apparatus comprising a tandem series of CEX and SEC HPLC columns for use in purifying a mixed therapeutically effective formulation sample comprising a) a first fusion protein conjugated to a polymer and b) a second protein that is not conjugated to the polymer, wherein the purification comprises; i. a first run, wherein the mixed therapeutically effective formulation is run through a prefiltration step to provide a first filtered mixed therapeutically effective formulation, wherein the first filtered mixed therapeutically effective formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed therapeutically effective formulation; and ii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed therapeutically effective formulation, wherein the third filtered mixed therapeutically effective formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed therapeutically effective formulation.

14. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. a first run, wherein the mixed formulation is run through a prefiltration step to provide a first filtered mixed formulation, wherein the first filtered mixed formulation is run through a cation exchange chromatography (CEX) column to provide a second filtered mixed formulation; iii. a second run, wherein the protein bound to the CEX column is eluted to provide a third filtered mixed formulation, wherein the third filtered mixed formulation is run through a size exclusion exchange (SEC) high performance liquid chromatography (HPLC) column to provide a fourth filtered mixed formulation; iv. a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; andwherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

15. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises: 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 that is not conjugated to the polymer; ii. loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down- stream of the CEX column is a size exclusion chromatography (SEC) column; iii. a first run, wherein the mixed formulation is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

16. A method of analyzing a mixed formulation sample, the method comprising: i. providing a mixed formulation that comprises a combination of two protein moieties, wherein the first protein moiety (A) is conjugated to a polymer, and the second protein (B) moiety is not conjugated to a polymer; ii. loading the sample into an HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed down-stream of two tandem serially connected columns, wherein the first column is a cation-exchanger column (CEX) and the second column down- stream of the CEX column is a size exclusion chromatography (SEC) column;iii. a first run, wherein the mixed formulation is injected and run through the system of ii. iv. a second run, wherein a concentrated salt is injected to elute the bound fraction of the CEX column to be separated by the SEC column; wherein, a specific ionic strength of the solvent is used to allow partitioning of the mixed formulation in the CEX column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

17. A method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

18. A method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents.

19. A method of analyzing a mixed therapeutically effective formulation sample, the method comprising:i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

20. An apparatus comprising a tandem HPLC system for use in analyzing a mixed formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents.

21. An apparatus comprising a tandem HPLC system for use in analyzing a mixed therapeutic composition sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets,wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents.

22. An apparatus comprising a tandem HPLC system for use in analyzing a mixed therapeutically effective formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents.

23. A method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run.

24. A method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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; andii. loading the sample into a tandem HPLC system where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run.

25. A method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein an additional pump is used to pulse elute the CEX-bound fraction upon wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents; and wherein the sample is analyzed via a single continuous chromatography run.

26. An apparatus comprising a tandem HPLC system for use in purifying a mixed formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX);d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run.

27. An apparatus comprising a tandem HPLC system for use in purifying a mixed therapeutic composition sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run.

28. An apparatus comprising a tandem HPLC system for use in purifying a mixed therapeutically effective formulation sample 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, wherein the apparatus comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents; wherein the sample is analyzed via a single continuous chromatography run.

29. A method of analyzing a mixed formulation sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the method allows analysis of the mixed formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

30. A method of analyzing a mixed therapeutic composition sample, the method comprising: i. providing 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; andwherein the method allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

31. A method of analyzing a mixed therapeutically effective formulation sample, the method comprising: i. providing a mixed therapeutically effective 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 where the HPLC pump is first connected to an auto-injector followed by a prefilter, which is then followed by a cation-exchanger column (CEX) and a switch valve enabling the effluent of the CEX to be directed to multiple possible targets; wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the method allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run, and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

32. An apparatus comprising a tandem series of HPLC system for use in analyzing a mixed formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter;c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

33. An apparatus comprising a tandem series of HPLC system for use in analyzing a mixed therapeutic composition sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed therapeutic composition based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

34. An apparatus comprising a tandem series of HPLC system for use in analyzing a mixed therapeutically effective formulation sample comprising (A) a first protein conjugated to a polymer and (B) a second protein that is not conjugated to the polymer, wherein, the HPLC system comprises: a) a HPLC pump connected to an auto-injector; b) a prefilter; c) cation-exchanger column (CEX); d) a switch valve enabling the effluent of the CEX to be directed to multiple possible targets, wherein the switch valve can direct the CEX effluent to: 1) a bypass loop; 2) a first SEC column; or 3) a second SEC column; and wherein the apparatus allows analysis of the mixed therapeutically effective formulation based on the differential charge and size variant of the constituents, wherein the sample is analyzed via a single continuous chromatography run; and wherein the conjugated sample is directed to the bypass loop and the unconjugated sample is directed to the first or second SEC column.

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