Attributes of Aflibercept, and methods for determining and modifying its characteristics.

Abstract

The present invention provides a composition containing aflibercept possessing the attributes of aflibercept. [Solution] A composition is provided comprising a mixture of aflibercept species, wherein the aflibercept species contains 1.0 to 12% afucosylation in the Fc domain.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 798,903, filed Jan. 30, 2019, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application is filed with a sequence listing in electronic format. The sequence listing is provided as a file named A-2287-WO-PCT_SeqList.txt, created on Jan. 29, 2020, with a size of 20.5 kb. The information in the sequence listing in electronic format is hereby incorporated by reference in its entirety.

[0003] The present disclosure relates to aflibercept, specifically to the attributes of aflibercept. Also provided herein are methods for characterizing and modifying the attributes of aflibercept, and compositions comprising aflibercept having specific attributes.

Background Art

[0004] Vascular endothelial growth factor (VEGF), also referred to as VEGF-A, is a signaling protein that promotes the growth of new blood vessels and binds to VEGFR-1 and VEGFR-2. VEGF has been found to be elevated in many tumors and has a role in angiogenesis. VEGF has also been found to have a role in intraocular angiogenesis such as choroidal neovascularization (CNV), a significant aspect of exudative age-related macular degeneration (AMD).

[0005] VEGF inhibitors, such as anti-VEGF antibodies and fragments, as well as decoy receptors or chimeric receptors, have been developed as therapeutic agents for treating various pathologies, such as cancer and eye disorders. For example, both anti-VEGF antibodies and anti-VEGF Fab are commercially available as bevacizumab and ranibizumab, respectively. Also, aflibercept, a VEGFR-Fc fusion protein or "VEGF-trap", is commercially available.

[0006] Aflibercept is a fusion protein composed of an IgG1 Fc domain fused to the Ig domain 2 of VEGFR-1 and the Ig domain 3 of VEGFR-2. Aflibercept is marketed as Eylea® (Regeneron, Tarrytown, NY) for the treatment of various ophthalmic conditions, including exudative AMD, and is formulated for intravitreal administration. The fusion protein is also marketed as Zaltrap® (ziv-aflibercept) (Regeneron, Tarrytown, NY) for the treatment of certain types of cancer and is formulated for intravenous administration.

[0007] The attributes of a protein can play a significant role, at least in the quality of the protein product. Therefore, this specification provides methods for characterizing and modifying the attributes of aflibercept, as well as compositions comprising aflibercept possessing these attributes, and related advantages. [Overview of the project] [Means for solving the problem]

[0008] This disclosure provides aflibercept compositions, including compositions containing a mixture of aflibercept species. Aflibercept species are also provided herein. In one embodiment, a species of aflibercept has one or more attributes that differ from another species of aflibercept. In some embodiments, the difference is the presence or absence of a certain attribute. In other embodiments, the difference is the level or amount of a certain attribute. Methods for characterizing one or more attributes of aflibercept, as well as methods for modifying one or more attributes of aflibercept, purifying aflibercept, and producing aflibercept compositions are also provided herein.

[0009] One aspect of this disclosure is an aflibercept species, which is a Y92L clipped species of aflibercept. The Y92L clipped species may include Sequence ID No. 3.

[0010] Another aspect of the present disclosure is a composition comprising a mixture of aflibercept species including the Y92L clipping species. In one embodiment, the amount of the Y92L clipping species in the composition is less than 5.0% as determined by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS). In one embodiment, the amount of the Y92L clipping species in the composition is less than 0.8% as determined by rCE-SDS. In some embodiments, the amount of the Y92L clipping species in the composition is less than 3.0%, 1.0% to 5.0%, or 1.0% to 3.0%, about 1.1%, about 3.0%, or about 4.7% of the composition as determined by rCE-SDS. In one embodiment, the amount of the Y92L clipping species in the composition is about 0.4% as determined by rCE-SDS.

[0011] In another aspect of this disclosure, a composition comprising a mixture of aflibercept species comprises N68-occupied species of aflibercept. In one embodiment, at least 30% of the aflibercept species are occupied at position N68, as determined by rCE-SDS. In some embodiments, at least 50%, 50-60%, about 39%, about 53%, about 54%, or about 55% of the aflibercept species are occupied at position N68. In some embodiments, the composition further comprises Y92L clipping species in amounts such as 1%-3%, less than 0.8%, about 1.1%, or about 0.4% of the composition.

[0012] This disclosure further provides compositions comprising a mixture of aflibercept species, wherein the total sialic acid content of the aflibercept species is 6.0–10.0 mol / mol protein as determined by LC-MS-based peptide mapping. In one embodiment, the total sialic acid content is about 6.8, about 8.5, and about 9.5 mol / mol protein. In some embodiments, the total sialic acid content is about 9.5 mol / mol protein, where 54–55% of the aflibercept species are occupied at position N68, and the amount of Y92L clipping species in the mixture is about 1.1%.

[0013] Another aspect of the present disclosure is a composition comprising a mixture of aflibercept species, wherein each aflibercept species contains less than 13% afucosylation in its Fc domain. The percentage of afucosylation can be determined by any method known in the art, such as LC-MS based peptide mapping. In one embodiment, the aflibercept species contains 1.0 to 12% afucosylation in its Fc domain. In another embodiment, the aflibercept species contains less than 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% afucosylation in its Fc domain. In one embodiment, the aflibercept species contains less than 10% afucosylation in its Fc domain. In another embodiment, the aflibercept species contains less than 5% afucosylation in its Fc domain. In another embodiment, the aflibercept species contains approximately 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain. In yet another embodiment, the aflibercept species contains approximately 4% afucosylation in the Fc domain.

[0014] Another aspect of the present disclosure is a composition comprising a mixture of aflibercept species, wherein the aflibercept species contains 0.1 to 2.0 (mol / mol protein) of sialic acid at N68, 1.0 to 25.0% O-glycosylation at T33, 70% to 99% N-glycosylation at N36, 20% to 75% N-glycosylation at N68, 0.1 to 1.0% high mannose in the Fc domain, about 26% galactosylation in the Fc domain, about 1% sialylation in the Fc domain, or any combination thereof. Aflibercept species may further contain less than 13% afucosylation in the Fc domain, for example 1.0–12% afucosylation in the Fc domain, or about 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain, for example less than 5% afucosylation in the Fc domain. In one embodiment, the aflibercept species contains about 4% afucosylation in the Fc domain.

[0015] The aflibercept species in the mixture may further contain approximately 0.7 (mol / mol protein) of sialic acid at N68, approximately 4% O-glycosylation at T33, approximately 95% N-glycosylation at N36, approximately 26% N-glycosylation at N68, approximately 1% high mannose in the Fc domain, approximately 26% galactosylation in the Fc domain, approximately 1% sialylation in the Fc domain, less than 13% afucosylation in the Fc domain, for example, 1.0-12% afucosylation in the Fc domain, or approximately 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain, or any combination thereof. In one embodiment, the aflibercept species in the mixture contains approximately 0.7 (mol / mol protein) of sialic acid at N68, approximately 4% O-glycosylation at T33, approximately 95% N-glycosylation at N36, approximately 26% N-glycosylation at N68, approximately 1% high mannose in the Fc domain, approximately 6% afucosylation in the Fc domain, approximately 26% galactosylation in the Fc domain, approximately 1% sialylation in the Fc domain, or any combination thereof.

[0016] The aflibercept species in the mixture may further include approximately 0.7 (mol / mol protein) of sialic acid at N68, approximately 4% O-glycosylation at T33, approximately 95% N-glycosylation at N36, approximately 26% N-glycosylation at N68, approximately 1% high mannose in the Fc domain, approximately 26% galactosylation in the Fc domain, approximately 1% sialylation in the Fc domain, less than 13% afucosylation in the Fc domain, for example 1.0-12% afucosylation in the Fc domain, or approximately 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain, less than 0.8%, for example about 0.4% of the Y92L clipping species, or any combination thereof.

[0017] The aflibercept species in the mixture may further contain approximately 0.8 (mol / mol protein) of sialic acid at N68, approximately 8.3% O-glycosylation at T33, approximately 90.9% N-glycosylation at N36, approximately 51.9% N-glycosylation at N68, approximately 0.4% high mannose in the Fc domain, approximately 24.4% galactosylation in the Fc domain, approximately 3.8% sialylation in the Fc domain, less than 13% afucosylation in the Fc domain, for example, 1.0-12% afucosylation in the Fc domain, or approximately 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain, or any combination thereof. In one embodiment, the aflibercept species in the mixture contains approximately 0.8 (mol / mol protein) of sialic acid at N68, approximately 8.3% O-glycosylation at T33, approximately 90.9% N-glycosylation at N36, approximately 51.9% N-glycosylation at N68, approximately 0.4% high mannose in the Fc domain, approximately 4% afucosylation in the Fc domain, approximately 24.4% galactosylation in the Fc domain, approximately 3.8% sialylation in the Fc domain, or any combination thereof.

[0018] The aflibercept species in the mixture may further include approximately 0.8 (mol / mol protein) of sialic acid in N68, approximately 8.3% O-glycosylation in T33, approximately 90.9% N-glycosylation in N36, approximately 51.9% N-glycosylation in N68, approximately 0.4% high mannose in the Fc domain, approximately 24.4% galactosylation in the Fc domain, approximately 3.8% sialylation in the Fc domain, less than 13% afucosylation in the Fc domain, for example 1.0-12% afucosylation in the Fc domain, or approximately 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% afucosylation in the Fc domain, less than 0.8%, for example about 0.4% of the Y92L clipping species, or any combination thereof. In one embodiment, the aflibercept species in the mixture contains approximately 0.8 (mol / mol protein) of sialic acid at N68, approximately 8.3% O-glycosylation at T33, approximately 90.9% N-glycosylation at N36, approximately 51.9% N-glycosylation at N68, approximately 0.4% high mannose in the Fc domain, approximately 4% afucosylation in the Fc domain, approximately 24.4% galactosylation in the Fc domain, approximately 3.8% sialylation in the Fc domain, approximately 0.4% Y92L clipping species, or any combination thereof. In one embodiment, the aflibercept species in the mixture contains approximately 0.8 (mol / mol protein) of sialic acid at N68, approximately 8.3% O-glycosylation at T33, approximately 90.9% N-glycosylation at N36, approximately 51.9% N-glycosylation at N68, approximately 0.4% high mannose in the Fc domain, approximately 4% afucosylation in the Fc domain, approximately 24.4% galactosylation in the Fc domain, approximately 3.8% sialylation in the Fc domain, and approximately 0.4% Y92L clipping species.

[0019] The disclosure further provides a method for increasing the binding of a composition comprising a mixture of aflibercept species to placental growth factor (PlGF) and / or VEGF-A by reducing the amount of Y92L clipping species in the mixture or by reducing the N68 occupancy of aflibercept. PlGF may be PlGF-1 or PlGF-2. In one embodiment, a method for reducing the amount of Y92L clipping species in the mixture comprises purifying aflibercept by an anion exchange chromatography step followed by a hydrophobic interaction chromatography step.

[0020] Another aspect of the present disclosure is a method for producing a composition comprising a mixture of aflibercept species. The method may include subjecting a cell culture medium comprising a mixture of aflibercept species to a purification process comprising an anion exchange chromatography (AEX) step followed by a hydrophobic interaction chromatography (HIC) step, thereby producing a smaller amount of Y92L clipping species compared to a purification process comprising an AEX step followed by a cation exchange chromatography (CEX) step. In some embodiments, the purification process does not include a CEX step.

[0021] Another method for producing a composition containing a mixture of aflibercept species comprises harvesting aflibercept-producing cells, subjecting the harvested cell culture to a protein A column, and performing anion exchange chromatography (AEX), followed by cation exchange chromatography (CEX) or hydrophobic interaction chromatography (HIC). In some embodiments, HIC follows AEX, and optionally, the method does not include CEX. In some embodiments, CEX follows AEX. In one embodiment, the cells are Chinese hamster ovary (CHO) cells that can be grown by perfusion or fed batch. The cells can be harvested by acid precipitation, centrifugation, microfiltration, deep filtration, or any combination thereof. The method may further include one or more viral inactivation and / or viral filtration steps, or ultrafiltration / dialysis filtration (UF / DF) steps. In some embodiments, the method does not include performing CEX before performing AEX, HIC, and / or size exclusion chromatography (SEC). [Brief explanation of the drawing]

[0022] [Figure 1] This shows the attributes of Aflibercept mapped onto that structure. [Figure 2] This graph shows the relative VEGF-A binding (%) as a function of the proportion of Y92L clipping species in aflibercept. [Figure 3] This is a graph of relative PlGF-1 binding (%) as a function of the proportion of N68 occupancy of aflibercept. [Figure 4] Two versions of the process for producing aflibercept are shown. In the first process, CEX chromatography follows AEX chromatography. In the second process, HIC chromatography follows AEX chromatography. [Figure 5]The results of reducing capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) of afucosylated afiberscept from hydrophobic interaction chromatography (HIC) fractions (F1-F5) are shown together with the afucosylated afiberscept species represented by each peak. [Figure 6] The results of reducing capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) of afiberscept (DS) produced by a process using anion exchange followed by cation exchange, and afiberscept produced by a process using anion exchange followed by hydrophobic interaction chromatography are shown. [Figure 7] The ratio of the Y92L clipped species of afiberscept grown in various culture conditions is shown. DETAILED DESCRIPTION OF THE INVENTION

[0023] The present disclosure provides compositions comprising mixtures of afiberscept species and various species of afiberscept. In some embodiments, one species of afiberscept has one or more attributes that are different from another species of afiberscept. Also provided herein are methods for characterizing one or more attributes of afiberscept and methods for modifying one or more attributes of afiberscept.

[0024] In one embodiment, afiberscept comprises the amino acid sequence of SEQ ID NO: 1 that does not have a C-terminal lysine. In another embodiment, afiberscept comprises the amino acid sequence of SEQ ID NO: 2 that has a C-terminal lysine. SEQ ID NO: 1 SEQ ID NO: 2

[0025] This specification also provides one or more attributes of aflibercept. Attributes may include deamidation, glycosylation (e.g., O-glycosylation, N-glycosylation), sialylation (e.g., NANA or NGNA sialylation), afucosylation, mannosylation (e.g., high mannose), galactosylation, or clipping (e.g., C-terminus or N-terminus). In one embodiment, the attributes of aflibercept are characterized by the level or amount of deamidation, glycosylation (e.g., O-glycosylation, N-glycosylation), sialylation (e.g., NANA or NGNA sialylation), afucosylation, mannosylation (e.g., high mannose), galactosylation, clipping (e.g., C-terminus or N-terminus), or any combination thereof, of a composition comprising a mixture of aflibercept species.

[0026] In some embodiments, the characterization of the attributes of aflibercept is determined by the level or amount of deamidation, glycosylation (e.g., O-glycosylation, N-glycosylation), sialylation (e.g., NANA or NGNA sialylation), afucosylation, mannosylation (e.g., high mannose), galactosylation, clipping (e.g., C-terminus or N-terminus), or any combination thereof, of the VEGFR domain of a composition containing a mixture of aflibercept species. In other embodiments, the characterization of the attributes of aflibercept is determined by the level or amount of deamidation, glycosylation (e.g., O-glycosylation, N-glycosylation), sialylation (e.g., NANA or NGNA sialylation), afucosylation, mannosylation (e.g., high mannose), galactosylation, clipping (e.g., C-terminus or N-terminus), or any combination thereof, of the Fc domain of a composition containing a mixture of aflibercept species. In one embodiment, the attribute is the level or amount of sialylation and / or glycosylation of the VEGFR domain. In another embodiment, the attribute is the level or amount of sialylation, N-glycosylation, O-glycosylation, or any combination thereof of the VEGFR domain. In yet another embodiment, the attribute is the level or amount of high mannose, sialylation, afucosylation, galactosylation, or any combination thereof of the Fc domain.

[0027] In another embodiment, the characterization of the attributes of aflibercept is determined by the proportions of deamidation, glycosylation (e.g., O-glycosylation, N-glycosylation), sialylation (e.g., NANA or NGNA sialylation), afucosylation, mannosylation (e.g., high mannose), galactosylation, clipping (e.g., C-terminus or N-terminus), or any combination thereof, of a composition containing a mixture of aflibercept species. In yet another embodiment, the characterization of the attributes of aflibercept is determined by the number of moles of glycan (e.g., sialic acid) per mole of aflibercept or aflibercept species in a composition containing a mixture of aflibercept species.

[0028] In some embodiments, the attribute is at a specific amino acid position of aflibercept. For example, the attribute may be deamidation at N84 and / or N99, sialic acid at N36, N88, N123, N196, or any combination thereof, O-glycosylation at T33, N-glycosylation at N36, N68, N123, N196, or any combination thereof, clipping at Y92L (e.g., resulting in an aflibercept species having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, lacking the first 92 amino acids of SEQ ID NO: 1 (SEQ ID NO: 5)), clipping at R153D (e.g., resulting in an aflibercept species having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, lacking the first 153 amino acids of SEQ ID NO: 1 (SEQ ID NO: 8)), or at a level or amount of any combination thereof. In some embodiments, the clipping at R153D results in a protein containing the amino acid sequence of SEQ ID NO: 1, where the first 153 amino acids clipped (SEQ ID NO: 8) are bound to the protein via disulfide bonds. Sequence ID 3 [ka] Sequence ID 4 [ka] Sequence ID 5 (amino acids 1-92 of Sequence ID 1) [ka] Sequence ID 6 [ka] Sequence ID 7 [ka] SEQ ID NO: 8 (Amino acids 1-153 of SEQ ID NO: 1) [ka]

[0029] Accordingly, the Specified Classification also provides aflibercepts having one or more of the attributes disclosed herein. In some embodiments, one species of aflibercept has one or more attributes that differ from another species of aflibercept. The difference may be the presence or absence of an attribute, or the level or amount of an attribute. In one embodiment, an aflibercept species is deamidated at N84 and / or N99, sialylated at N36, N88, N123, N196, or any combination thereof, O-glycosylated at T33, N-glycosylated at N36, N68, N123, N196, or any combination thereof, or undergoes any combination thereof.

[0030] In another embodiment, the aflibercept species is clipped at Y92L (for example, resulting in an aflibercept species having the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4, lacking the first 92 amino acids of SEQ ID NO: 1 (SEQ ID NO: 5)). In some embodiments, the clipped Y92L species undergoes deamidation at N99, sialylation at N123 and / or N196, N-glycosylation at N123 and / or N196, or any combination thereof.

[0031] In another embodiment, the aflibercept species is clipped at R153D (for example, resulting in an aflibercept species having the amino acid sequence of SEQ ID NO: 6 or SEQ ID NO: 7, lacking the first 153 amino acids of SEQ ID NO: 1 (SEQ ID NO: 8)). In some embodiments, the clipped R153D species is sialylated and / or N-glycosylated at N196.

[0032] This specification also provides compositions comprising a mixture of aflibercept species having specific attributes. In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.8% of the aflibercept species are clipping species (e.g., Y92L or R153D clipping species). The amount of clipping species can be determined by any method known in the art, such as rCE-SDS or trypsin peptide mapping. In one embodiment, the composition comprises a mixture of aflibercept species in which less than 5.0% of the aflibercept species are Y92L clipping species. In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.8% of the aflibercept species are Y92L clipping species. In one embodiment, the composition comprises a mixture of aflibercept species in which less than 0.8% of the aflibercept species are Y92L clipping species. In another embodiment, the composition comprises a mixture of aflibercept species, where less than 0.5% of the aflibercept species are Y92L clipping species. In some embodiments, the amount of Y92L clipping species in the composition is 1% to 10%, 1.0% to 5.0%, or 1.0% to 3.0%. In some embodiments, the amount of Y92L clipping species in the composition is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, or about 6.0% of the composition. In some embodiments, the amount of Y92L clipping species in the composition is about 1.1%, about 3.0%, or about 4.7% of the composition. In one embodiment, the composition comprises a mixture of aflibercept species, where about 0.4% of the aflibercept species are Y92L clipping species.

[0033] This specification also provides compositions comprising a mixture of aflibercept species in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the aflibercept species are occupied at position N68 (i.e., glycosylated). The amount of aflibercept species occupied at position N68 can be determined by any method known in the art, such as rCE-SDS or peptide mapping. In some embodiments, 10% to 90%, 20% to 70%, 30% to 60%, or 50% to 60% of the aflibercept species are occupied at position N68. In some embodiments, the amount of aflibercept species occupied at position N68 is about 39%, about 53%, about 54%, or about 55%.

[0034] This specification also provides compositions comprising a mixture of aflibercept species, wherein the total sialic acid content of the aflibercept species is 1.0–20.0 mol / mol protein, 2.0–15.0 mol / mol protein, 5.0–12.0 mol / mol protein, or 6.0–10.0 mol / mol protein. The amount of sialic acid can be determined by any method known in the art, such as any peptide mapping or glycan analysis method, such as liquid chromatography, combined with mass spectrometry (LC-MS) based peptide mapping, total sialic acid method, or total hydrophilic interaction liquid chromatography (HILIC) glycan mapping. In one embodiment, the total sialic acid content is about 6.8, about 8.5, or about 9.5 mol / mol protein.

[0035] In another embodiment, the composition comprises a mixture of aflibercept species, the glycan profile of the mixture comprising about 0.7 sialic acid (mol / mol protein) at N88, about 4% O-glycosylation at T33, about 95% N-glycosylation at N36, about 26% N-glycosylation at N68, about 1% high mannose at its Fc domain, about 6% afucosylation at its Fc domain, about 26% galactosylation at its Fc domain, about 1% sialation at its Fc domain, or any combination thereof. In some embodiments, the glycan profile may further include approximately 3.2 sialic acid (mol / mol protein) at N36, approximately 1.6 sialic acid (mol / mol protein) at N123, approximately 1.9 sialic acid (mol / mol protein) at N196, approximately 7.4 total sialic acid (mol / mol protein) in its VEGFR domain, approximately 100% N-glycosylation at N123, approximately 99% N-glycosylation at N196, or any combination thereof.

[0036] In one embodiment, the composition has a glycan profile in which the mixture contains approximately 3.2 sialic acid (mol / mol protein) in N36, approximately 0.7 sialic acid (mol / mol protein) in N88, approximately 1.6 sialic acid (mol / mol protein) in N123, approximately 1.9 sialic acid (mol / mol protein) in N196, approximately 7.4 total sialic acid (mol / mol protein) in its VEGFR domain, and approximately 4% O-glycoglycan in T33. The mixture contains aflibercept species, including sylation, approximately 95% N-glycosylation at N36, approximately 26% N-glycosylation at N68, approximately 100% N-glycosylation at N123, approximately 99% N-glycosylation at N196, approximately 1% high mannose in its Fc domain, approximately 6% afucosylation in its Fc domain, approximately 26% galactosylation in its Fc domain, approximately 1% sialylation in its Fc domain, or any combination thereof.

[0037] In some embodiments, the composition comprises a mixture of Aflibercept species, wherein the amount of Y92L clipping species in the composition is 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, less than 0.8%, 1% to 10%, 1.0% to 5.0%, or 1.0% to 3.0%, about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, or about 6.0%, or about 1.1%, about 3.0%, about 4.7%, or about 0.4%. The glycan profile of the Lucept mixture includes approximately 0.7 sialic acid (mol / mol protein) in N88, approximately 4% O-glycosylation in T33, approximately 95% N-glycosylation in N36, approximately 26% N-glycosylation in N68, approximately 1% high mannose in its Fc domain, approximately 6% afucosylation in its Fc domain, approximately 26% galactosylation in its Fc domain, approximately 1% sialylation in its Fc domain, or any combination thereof. In some embodiments, the glycan profile further comprises approximately 3.2 sialic acid (mol / mol protein) at N36, approximately 1.6 sialic acid (mol / mol protein) at N123, approximately 1.9 sialic acid (mol / mol protein) at N196, approximately 7.4 total sialic acid (mol / mol protein) in its VEGFR domain, approximately 100% N-glycosylation at N123, approximately 99% N-glycosylation at N196, or any combination thereof. In some embodiments, the composition further comprises a mixture of aflibercept species in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 10%-90%, 20%-70%, 30%-60%, or 50-60%, or about 39%, about 53%, about 54%, or about 55% are occupied at the N68 position (i.e., glycosylated). In some embodiments, the composition further comprises a total sialic acid content of aflibercept species of which is 1.0-20.0 mol / mol protein, 2.0-15.0 mol / mol protein, 5.0-12.0 mol / mol protein, 6.0-10.0 mol / mol protein, about 6.8%, about 8.5%, or about 9.5 mol / mol protein.

[0038] In one embodiment, the composition comprises a mixture of aflibercept species in which at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, 10%-90%, 20%-70%, 30%-60%, or 50-60%, or about 39%, about 53%, about 54%, or about 55% of the aflibercept species are occupied at the N68 position (i.e., glycosylated), and the aflibercept species in the composition The glycan profile includes approximately 0.7 sialic acid (mol / mol protein) in N88, approximately 4% O-glycosylation in T33, approximately 95% N-glycosylation in N36, approximately 26% N-glycosylation in N68, approximately 1% high mannose in its Fc domain, approximately 6% afucosylation in its Fc domain, approximately 26% galactosylation in its Fc domain, approximately 1% sialylation in its Fc domain, or any combination thereof. In some embodiments, the glycan profile further comprises about 3.2 sialic acid (mol / mol protein) at N36, about 1.6 sialic acid (mol / mol protein) at N123, about 1.9 sialic acid (mol / mol protein) at N196, about 7.4 total sialic acid (mol / mol protein) in its VEGFR domain, about 100% N-glycosylation at N123, about 99% N-glycosylation at N196, or any combination thereof. In some embodiments, the composition further comprises the total sialic acid content of the aflibercept species, which is 1.0–20.0 mol / mol protein, 2.0–15.0 mol / mol protein, 5.0–12.0 mol / mol protein, 6.0–10.0 mol / mol protein, about 6.8, about 8.5, or about 9.5 mol / mol protein.

[0039] In one embodiment, the composition comprises a mixture of aflibercept species in which the amount of Y92L clipping species in the mixture is 1% to 3% (e.g., about 1.1%, less than 0.8%, or about 0.4%), 53 to 55% (e.g., about 54 to 55%) of the aflibercept species are occupied at the N68 position, and the total sialic acid content is about 9.5 mol / mol protein.

[0040] This specification also provides a method for producing a composition comprising a mixture of aflibercept species. Aflibercept can be produced by host cells, such as mammalian host cells. Mammalian host cells may be Chinese hamster ovary (CHO) cells. In one embodiment, the method comprises culturing and harvesting host cells that produce aflibercept, and purifying the protein by protein A, followed by anion exchange chromatography (AEX), and then cation exchange chromatography (CEX). In another embodiment, the method comprises culturing host cells by fed batch, harvesting the cells, and purifying the protein by protein A, followed by anion exchange chromatography (AEX), and then hydrophobic interaction chromatography (HIC). In some embodiments, the method comprising AEX, followed by HIC, produces a smaller amount of Y92L clipping species compared to the same method, except that CEX is included instead of HIC. In some embodiments, the method comprising AEX, followed by HIC, produces less than half the amount of Y92L clipping species compared to the same method, except that CEX is included instead of HIC. In some embodiments, the method comprising AEX, followed by HIC, does not include the CEX step. In some embodiments, the method does not include any size exclusion chromatography (SEC) step. In some embodiments, the method does not include any CEX step before AEX.

[0041] In some embodiments, the method includes culturing host cells by fed batch or perfusion. In some embodiments, such as when culturing by a method including perfusion, cells can be harvested by soft agglutination, microfiltration, or any combination thereof. In some embodiments, such as when culturing by fed batch, cells can be harvested by precipitation, centrifugation, deep filtration, or any combination thereof. In one embodiment, cells are harvested by acid precipitation. The acid may be any known in the art, such as acetic acid or citric acid. In one embodiment, the acid precipitation is at a pH of about 5.5, about 5.0, about 4.5, about 4.0, about 3.5, or about 3.0. In one embodiment, the acid precipitation is at a pH of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. In one embodiment, the acid precipitation is at a pH of 3.0–4.0 or 3.5–4.0. In another embodiment, the pH is 5.5±0.2, 5.4±0.2, 5.3±0.2, 5.2±0.2, 5.1±0.2, 5.0±0.2, 4.9±0.2, 4.8±0.2, 4.7±0.2, 4.6±0.2, 4.5±0.2, 4.4±0.2, 4.3±0.2, 4.2±0.2, 4.1±0.2, 4.0±0.2, 4.0±0.2, 3.9±0.2, 3.8±0.2, 3.7±0.2, 3.6±0.2, 3.5±0.2, 3.4±0.2, 3.3±0.2, 3.2±0.2, 3.1±0.2, or 3.0±0.2. Acid precipitation may occur at temperatures of approximately 20°C, 15°C, 10°C, or 5°C. In one embodiment, the temperature is approximately 20±3°C, 19±3°C, 18±3°C, 17±3°C, 16±3°C, 15±3°C, 14±3°C, 13±3°C, 12±3°C, 11±3°C, 10±3°C, 9±3°C, 8±3°C, 7±3°C, 6±3°C, 5±3°C, or 4±3°C. The acid precipitation process may last approximately 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, or 105 minutes.

[0042] In another embodiment, cells are harvested by acid precipitation followed by deep filtration. The temperature during deep filtration can be approximately 20±3°C, 19±3°C, 18±3°C, 17±3°C, 16±3°C, 15±3°C, 14±3°C, 13±3°C, 12±3°C, 11±3°C, 10±3°C, 9±3°C, 8±3°C, 7±3°C, 6±3°C, 5±3°C, or 4±3°C. In one embodiment, pH neutralization follows deep filtration. Neutralization can be carried out using any base, such as a Tris base. Neutralization may be carried out at temperatures of approximately 20±3°C, 19±3°C, 18±3°C, 17±3°C, 16±3°C, 15±3°C, 14±3°C, 13±3°C, 12±3°C, 11±3°C, 10±3°C, 9±3°C, 8±3°C, 7±3°C, 6±3°C, 5±3°C, or 4±3°C. The target pH for neutralization may be approximately 8.0, approximately 7.5, approximately 7.0, approximately 6.5, or approximately 6.0. In one embodiment, the pH is approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, or approximately 7.9. In one embodiment, the pH is 6.0 to 8.0, 6.5 to 8.0, or 7.0 to 8.0. In another embodiment, the pH is 8.0±0.2, 7.9±0.2, 7.8±0.2, 7.7±0.2, 7.6±0.2, 7.5±0.2, 7.4±0.2, 7.3±0.2, 7.2±0.2, 7.1±0.2, 7.0±0.2, 6.9±0.2, 6.8±0.2, 6.7±0.2, 6.6±0.2, 6.5±0.2, 6.4±0.2, 6.3±0.2, 6.2±0.2, 6.1±0.2, or 6.0±0.2.

[0043] In another embodiment, deep filtration is followed by viral inactivation, such as pH neutralization and viral inactivation using a detergent (e.g., Triton). Viral inactivation may be carried out at 20±3°C, 19±3°C, 18±3°C, 17±3°C, 16±3°C, 15±3°C, 14±3°C, 13±3°C, 12±3°C, 11±3°C, 10±3°C, 9±3°C, 8±3°C, 7±3°C, 6±3°C, 5±3°C, or 4±3°C. In some embodiments, viral inactivation is not carried out after deep filtration.

[0044] Therefore, in one embodiment, host cells are cultured by fed batch or perfusion, followed by acid precipitation, then deep filtration and pH neutralization for harvesting. In another embodiment, host cells are cultured by fed batch or perfusion, followed by acid precipitation, then deep filtration, pH neutralization and virus inactivation for harvesting.

[0045] After harvesting, aflibercept may be purified from the cell lysate by protein A affinity chromatography, followed by anion exchange chromatography (AEX), and then cation exchange chromatography (CEX). In another embodiment, purification is performed by protein A affinity chromatography, followed by anion exchange chromatography (AEX), and then hydrophobic interaction chromatography (HIC). In some embodiments, a method comprising AEX, followed by HIC, produces a smaller amount of Y92L clipping species compared to the same method except that CEX is included instead of HIC. In some embodiments, a method comprising AEX, followed by HIC, produces less than half the amount of Y92L clipping species compared to the same method except that CEX is included instead of HIC. In some embodiments, a method comprising AEX, followed by HIC, does not include a CEX step. In some embodiments, the method does not include any size exclusion chromatography (SEC) step. In some embodiments, the method does not include any CEX step before AEX.

[0046] In some embodiments, the virus inactivation and / or filtration step is performed between the Protein A step and the AEX step. In some embodiments, the virus inactivation and / or virus filtration step is performed after the final column purification (e.g., CEX or HIC). In one embodiment, virus filtration is performed after the final column purification (e.g., CEX or HIC).

[0047] In some embodiments, viral inactivation, such as after protein A purification, is due to low pH. In one embodiment, viral inactivation occurs at pH values ​​of approximately 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, or 3.0. In one embodiment, acid precipitation occurs at pH values ​​of 3.0–4.0 or 3.5–4.0. In another embodiment, the pH values ​​are 4.0±0.2, 3.9±0.2, 3.8±0.2, 3.7±0.2, 3.6±0.2, 3.5±0.2, 3.4±0.2, 3.3±0.2, 3.2±0.2, 3.1±0.2, or 3.0±0.2. In some embodiments, low-pH virus inactivation is followed by neutralization, with target pH values ​​of approximately 5.5, 5.0, or 4.5. In one embodiment, the pH range is 4.5–6.0, 4.5–5.5, 5.0–6.0, or 5.0–5.5. In another embodiment, the pH range is 6.0±0.2, 5.9±0.2, 5.8±0.2, 5.7±0.2, 5.6±0.2, 5.5±0.2, 5.4±0.2, 5.3±0.2, 5.2±0.2, 5.1±0.2, 5.0±0.2, 4.9±0.2, 4.8±0.2, 4.7±0.2, 4.6±0.2, or 4.5±0.2. In some embodiments, deep filtration is performed after neutralization.

[0048] In some embodiments, ultrafiltration / dialysis (UF / DF) is performed after chromatography, and / or virus inactivation / filtration, and / or deep filtration. In some embodiments, excipients such as surfactants (e.g., polysorbate) are added to the UF / DF recovery pool and optionally filtered.

[0049] Chromatography, virus inactivation, virus filtration, and / or ultrafiltration / dialysis filtration steps may be carried out at 25±5℃, 24±5℃, 23±5℃, 22±5℃, 21±5℃, 20±5℃, 19±5℃, 18±5℃, 17±5℃, 16±5℃, 15±5℃, 14±5℃, 13±5℃, 12±5℃, 11±5℃, or 10±5℃.

[0050] Therefore, in one embodiment, aflibercept can be purified from harvested cells by protein A affinity chromatography, followed by viral inactivation (e.g., by low pH, followed by neutralization, and optionally by deep filtration), AEX, HIC, viral filtration, and UF / DF. In some embodiments, a surfactant (e.g., polysorbate) is added. In some embodiments, the purified aflibercept is filtered after the addition of the surfactant.

[0051] This specification also provides a method for increasing the binding of a composition comprising a mixture of aflibercept species to PlGF (e.g., PlGF-1 or PlGF-2) and / or VEGF-A, comprising reducing the amount of Y92L clipping species in the mixture. In some embodiments, the method comprises purifying the aflibercept by an anion exchange chromatography step followed by a hydrophobic interaction chromatography step. In some embodiments, the method is as described herein, such as the second process shown in Figure 4.

[0052] The disclosure also provides a method for increasing the binding of a composition comprising a mixture of aflibercept species to PlGF (e.g., PlGF-1 or PlGF-2), including reducing the N68 occupancy of aflibercept. In some embodiments, the method includes a chromatography step following the purification of protein A, such as the first or second process shown in Figure 4.

[0053] The detailed description and the following examples are illustrative of the present invention and should not be construed as limiting the present invention. Various modifications and alterations can be made by those skilled in the art based on the description of the present invention, and such modifications and alterations are included in the present invention. [Examples]

[0054] Example 1: The effect of aflibercept's attributes Various attributes of aflibercept were analyzed using the method shown in Table 1. Figure 1 shows these attributes mapped onto the aflibercept structure. The influence of various attributes on the ability of aflibercept to bind VEGF-A and PlGF-1 was determined, and the results are shown in Table 1.

[0055] In addition to assessing the potential presence of chemical and post-translational modifications, peptide mapping characterization or reduced peptide mapping was performed to confirm the amino acid sequence of aflibercept. Peptide mapping analysis was performed by enzymatic digestion with trypsin after reduction with dithiothreitol and alkylation with sodium iodoacetate. The resulting cleavage fragments were separated by reversed-phase ultrafast liquid chromatography (UHPLC) using an increasing gradient of acetonitrile in water, and the peptides were identified by online liquid chromatography-mass spectrometry (LC-MS / MS) using a high-resolution linear ion trap mass spectrometer.

[0056] The N-linked glycans of aflibercept were evaluated using hydrophilic interaction liquid chromatography (HILIC) glycan mapping. Aflibercept has five N-linked glycosylation sites, one of which is mainly a bifurcated complex on the conserved Fc region, and the other four sites are mainly bifurcated complexes containing sialylated glycan species on the VEGF receptor region. The N-linked glycans of aflibercept were evaluated by hydrophilic interaction liquid chromatography (HILIC) UHPLC glycan mapping analysis. This procedure involves reduction and denaturation of aflibercept, release of N-linked glycans using peptide N-glycosidase F (PNGase F), derivatization using fluorescent labeling, and fluorescence detection of labeled glycans separated by HILIC UHPLC using an increasing gradient of ammonium formate in water.

[0057] The total sialic acid method was used to determine the sialic acid content. Terminal sialic acids derived from the N-linked glycans of aflibercept were hydrolyzed under acidic conditions. The sialic acids in the hydrolysis solution were labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB). The released and labeled sialic acid preparation was diluted with water and subsequently analyzed by ultra-high-performance liquid chromatography (UHPLC) with fluorescence detection. A standard curve was generated using linear regression analysis with a series of injections of known amounts of NANA standards prepared in the same manner and corresponding peak areas. Subsequently, the sialic acid content was determined using the generated linear standard curve.

[0058] Capillary isoelectric focusing (cIEF) analysis of aflibercept was performed using a high-resolution capillary electrophoresis separation instrument equipped with a neutral-coated capillary. Aflibercept migrates via the pH gradient in the capillary until the pH value equals its pI. Subsequently, the aflibercept is chemically mobilized and detected by UV absorbance (280 nm) as it passes through the detection window in the capillary.

[0059] The purity of aflibercept was evaluated using reduced capillary electrophoresis-sodium dodecyl sulfate rCE-SDS. The sample was reduced with β-mercaptoethanol and denatured with SDS. The reduced and denatured proteins were separated based on their hydrodynamic size, where smaller proteins moved faster and larger proteins moved slower. The analytes were monitored by UV absorbance.

[0060] The binding of PlGF-1 or PlGF-2 was determined using a bead-based Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen®, PerkinElmer) for detecting biomolecular interactions. This assay includes two bead types: acceptor beads and donor beads. The donor beads are coated with a hydrogel containing phthalocyanine, a photosensitizer, and streptavidin. The acceptor beads are coated with a hydrogel containing a thioxene derivative and a mouse monoclonal anti-FITC antibody. The donor beads bind to biotinylated PlGF via an interaction between streptavidin and biotin, while the acceptor beads bind to FITC-labeled aflibercept. This FITC-labeled aflibercept acts as a competitor to the aflibercept test sample. When FITC-labeled aflibercept and biotinylated PlGF bind to each other, the acceptor beads and donor beads are brought into close proximity. When this complex is irradiated with a laser, the surrounding oxygen is converted to singlet oxygen by the donor beads. If the beads are in close proximity, energy transfer occurs to the acceptor beads, resulting in luminescence generation, which is measured with a microplate reader equipped with AlphaScreen® signal detection capability. If the unbound aflibercept test sample is present at a concentration sufficient to inhibit the binding of FITC-labeled aflibercept to biotinylated PlGF, a dose-dependent decrease in luminescence output occurs. The activity of the test sample is determined by comparing the response of the test sample to the response of a control standard, which represents relative potency or relative binding (e.g., relative binding %).

[0061] The binding of VEGFA was determined using a bead-based amplified luminescence proximity homogeneity assay screen (AlphaScreen®, PerkinElmer) that detects biomolecular interactions. This assay includes two bead types: acceptor beads and donor beads. The donor beads are coated with a hydrogel containing phthalocyanine, a photosensitizer, and streptavidin. The acceptor beads are coated with a hydrogel containing a thioxene derivative and a nickel chelate. The donor beads bind to biotinylated VEGFA-165 via an interaction between streptavidin and biotin, and the acceptor beads bind to histidine-labeled VEGFR2 due to an interaction between the nickel chelate and histidine. When VEGFR2-His and biotinylated VEGFA-165 bind to each other, the acceptor beads and donor beads are brought into close proximity. When this complex is irradiated with a laser, the donor beads convert ambient oxygen to singlet oxygen. When beads are in close proximity, energy transfer occurs to the acceptor beads, resulting in luminescence generation, which is measured using a microplate reader equipped with AlphaScreen® signal detection capabilities. Aflibercept binds to biotinylated VEGFA-165, preventing it from binding to VEGFR2-His, thereby reducing luminescence output in a dose-dependent manner. The activity of the test sample is determined by comparing the response of the test sample to the response of a control standard, which represents relative potency or relative binding (e.g., relative binding %).

[0062] [Table 1]

[0063] As shown in Table 1, in addition to sialylation, the attributes that affect the ability of aflibercept to bind to VEGF-A and PlGF-1 are Y92L clipping and N68 glycosylation occupancy.

[0064] As shown in Figure 2, Y92L clipping significantly affects aflibercept's ability to bind to VEGF-A. Furthermore, 1% Y92L clipping results in a 2% loss of VEGF-A binding, suggesting that both VEGFR regions of aflibercept play a role in VEGF-A binding. As shown in Figure 3, a 10% increase in N68 occupancy leads to a 4–5% decrease in aflibercept's PlGF-1 binding activity.

[0065] Example 2: Process steps affecting Y92L clipping and N68 occupancy of aflibercept Since increased Y92L clipping reduces the VEGF-A, PlGF-1, and PlGF-2 binding activity of aflibercept, and increased N68 occupancy reduces the PlGF-1 and PlGF-2 binding activity of aflibercept, the aflibercept purification process was investigated to determine whether specific steps in the process can affect the level of Y92L clipping species and the amount of N68 occupancy in aflibercept.

[0066] Aflibercept was expressed in Chinese hamster ovary (CHO) cells and produced by fed batch. The cells were then harvested by acid precipitation followed by deep filtration. The pH was then neutralized before the virus inactivation step. Subsequently, aflibercept was purified from the harvested cell culture medium using a protein A column and then subjected to the virus inactivation step.

[0067] Following this second virus inactivation step, one process used anion exchange chromatography (AEX) followed by cation exchange chromatography (CEX), and the second process used AEX followed by hydrophobic interaction chromatography (HIC) (Figure 4). In both processes, these chromatographic steps were followed by viral filtration and ultrafiltration / dialysis filtration (UF / DF) of the viral filtration pool. Polysorbate was then added to the UF / DF recovery pool and subsequently filtered into a container.

[0068] Approximately 60% of aflibercept meeting specific quality attributes was obtained from the fed-batch process. The first purification process (AEX, followed by CEX) yielded a higher yield than the second process (AEX, followed by HIC). Table 2 shows the percentage of Y92L clip species, percentage of aflibercept by N68 occupancy, total sialic acid concentration (mol / mol protein), and percentage of cIEF peak 1 (referring to the group of cIEF peaks, which are low-sialic acid-containing versions of aflibercept) of aflibercept before AEX, after the first process, and after the second process. Table 2 also includes the yield percentage of aflibercept across the column 2 and column 3 chromatography steps (AEX and CEX for the first process, and AEX and HIC for the second process).

[0069] [Table 2]

[0070] As shown in Table 2, the AEX process increases the N68 occupancy of aflibercept, while the HIC process significantly reduces the Y92L clipping species of aflibercept. AEX can reduce cIEF peak 1 and increase N68 occupancy, while HIC can reduce Y92L clipping and increase N68 occupancy.

[0071] Example 3: rCE-SDS analysis of aflibercept Aflibercept samples (DS) prepared by a process using anion exchange chromatography followed by cation exchange chromatography (CEX) were subjected to HIC analytical HPLC, and five fractions were recovered across the entire HIC elution peak. These fractions were analyzed by reduced capillary electrophoresis-sodium dodecyl sulfate (rCE-SDS) (Figure 5), which shows that HIC HPLC partially degrades the clipped aflibercept species and N68 glycosylated species.

[0072] Figure 6 shows the rCE-SDS of aflibercept produced by the first process compared to aflibercept produced by the second process, indicating that the yield of the Y92L clipping species was lower when using the second process compared to the first process, while the second process had a higher N68 occupancy rate compared to the first process.

[0073] Example 4: Influence of attributes on VEGF-A, PlGF-1, and PlGF-2-bound aflibercept The proportion of aflibercept purified from a process using Protein A, column, and cation exchange chromatography (CEX) cleaved with Y92L, and the proportion of aflibercept purified from a process using Protein A, column, and cation exchange chromatography (CEX) with N68 glycosylated (N68 occupancy) were determined for each of the analytical HPLC HIC fractions shown in Figure 5 using rCE-SDS. Each fraction was further subjected to VEGF-A, PlGF-1, and PlGF-2 binding assays. The results are shown in Table 3.

[0074] [Table 3]

[0075] As shown in Table 3, aflibercept clipping in Y92L affects VEGF-A and PlGF binding. Fractions with a higher proportion of Y92L clipping species showed lower binding to VEGF-A and PlGF-1 and PlGF-2. On the other hand, as shown in Table 3, N68 occupancy affects PlGF binding but not VEGF-A binding. Fractions with a higher proportion of species with glycosylated N68 showed lower binding to PlGF-1 and PlGF-2.

[0076] Example 5: Glycan Profile of Aflibercept The glycan profile of aflibercept purified by a process using protein A followed by cation exchange chromatography (CEX) was determined by rCE-SDS (N-glycosylation %) at N68 and LC-MS-based peptide mapping (e.g., other attributes listed in Table 4).

[0077] [Table 4]

[0078] The glycan profile of aflibercept purified by a process using protein A, followed by anion exchange chromatography (AEX) and hydrophobic interaction chromatography (HIC), was determined by rCE-SDS (N-glycosylation at N68 and Y92L clipping species %) and LC-MS-based peptide mapping (e.g., other attributes listed in Table 5).

[0079] [Table 5]

[0080] Example 6: Effects of temperature and pH shift on Y92L clipping Fed-batch bioreactor cultures of aflibercept-expressing CHO cells were subjected to temperature and / or pH shifts on day 6 of culture and compared to unshifted cultures. Two control cultures in different bioreactors (Figure 7, control A, control B) were grown at 36°C and pH 6.9 until harvest. Two cultures in different bioreactors (Figure 7, temperature shift A, temperature shift B) were grown at 36°C and pH 6.9, and on day 6 of culture, both temperatures were reduced to 32.5°C and maintained at 32.5°C and pH 6.9 until harvest. One culture (Figure 7, pH shift) was grown at 36°C and pH 6.9, and on day 6 of culture, the pH was reduced to 6.8 and maintained at 36°C and pH 6.8 until harvest. One culture medium (Figure 7, temperature shift and pH shift) was grown at 36°C and pH 6.9. On the 6th day of cultivation, both the temperature and pH were reduced to 32.5°C and pH 6.8, and maintained at 32.5°C and pH 6.8 until harvest.

[0081] Aflibercept was purified from the purified culture medium broth of each culture medium using protein A, and the proportion of Y92L clipping species was determined by rCE-SDS on days 10, 11, and 12 (Figure 7). In all culture mediums, the proportion of Y92L clipping species increased with incubation time. Shifts in culture medium temperature and / or pH to reduce the value on day 6 reduced the proportion of Y92L clipping species compared to controls (control A, control B). Temperature shifts were more effective than pH shifts in reducing the proportion of Y92L clipping species, and combinations of pH and temperature shifts did not increase the effect of further reducing the proportion of Y92L clipping species.

[0082] Although the present invention has been described in terms of various embodiments, it will be understood that those skilled in the art will conceive of variations and modifications. Therefore, the appended claims are intended to encompass all such equivalent variations that fall within the scope of the claimed invention. Furthermore, the headings used herein are for structural purposes only and should not be construed as limiting the subject matter described.

[0083] All references cited in this application are expressly incorporated herein by reference for any purpose.

Claims

1. A Y92L clipping variety of Aflibercept containing Sequence ID 3.

2. A composition comprising a mixture of aflibercept species, wherein the amount of Y92L clipping species in the composition is less than 5.0% as determined by rCE-SDS.

3. The composition according to claim 2, wherein the amount of Y92L clipping species in the composition is less than 0.8%.

4. The composition according to claim 3, wherein the amount of Y92L clipping species in the composition is about 0.4%.

5. A composition comprising a mixture of aflibercept species, wherein at least 30% of the aflibercept species are occupied at the N68 position as determined by rCE-SDS.

6. The composition according to claim 5, wherein at least 50% of the aflibercept species are occupied at the N68 position.

7. The composition according to claim 6, wherein 50 to 60% of the aflibercept species are occupied at the N68 position.

8. The composition according to claim 5, wherein about 39%, 53%, 54%, or 55% of the aflibercept species are occupied at the N68 position.

9. The composition according to claim 8, wherein the amount of Y92L clipping species in the mixture is 1% to 3%.

10. The composition according to claim 8, wherein the amount of Y92L clipping species in the mixture is less than 0.8%.

11. The composition according to claim 8, wherein the amount of Y92L clipping species in the mixture is about 0.4%.

12. The composition according to claim 9, wherein 54-55% of the aflibercept species are occupied at the N68 position, and the amount of the Y92L clipping species in the mixture is about 1.1%.

13. The composition according to claim 11, wherein 54-55% of the aflibercept species are occupied at the N68 position, and the amount of the Y92L clipping species in the mixture is about 0.4%.

14. A composition comprising a mixture of aflibercept species, wherein the total sialic acid content of the aflibercept species is 6.0 to 10.0 mol / mol of protein as determined by LC-MS-based peptide mapping.

15. The composition according to claim 14, wherein the total sialic acid content is approximately 6.8, 8.5, or 9.5 (mol / mol protein).

16. The composition according to claim 15, wherein the total sialic acid content is approximately 9.5 mol / mol protein, 54-55% of the aflibercept species are occupied at the N68 position, and the amount of the Y92L clipping species in the mixture is approximately 1.1%.

17. The composition according to claim 15, wherein the total sialic acid content is approximately 9.5 mol / mol protein, 54-55% of the aflibercept species are occupied at the N68 position, and the amount of the Y92L clipping species in the mixture is approximately 0.4%.

18. A composition comprising a mixture of aflibercept species, wherein the aflibercept species contains 1.0 to 12% afucosylation in its Fc domain.

19. The composition according to claim 18, wherein the aflibercept species contains less than 10% afucosylation in the Fc domain.

20. The composition according to claim 19, wherein the aflibercept species contains about 6% afucosylation in the Fc domain.

21. The composition according to claim 19, wherein the aflibercept species contains less than 5% afucosylation in the Fc domain.

22. The composition according to claim 19, wherein the aflibercept species contains about 4% afucosylation in the Fc domain.

23. The aforementioned Aflibercept species is a) N68 contains 0.1 to 2.0 (mol / mol protein) sialic acid, b) 1.0-25.0% O-glycosylation of T33, c) 70-99% N-glycosylation of N36, d) N68 with 20-75% N-glycosylation, e) 0.1 to 1.0% high mannose in the Fc domain, f) Approximately 26% galactosylation in the Fc domain, g) Approximately 1% sialylation in the Fc domain, or h) The composition according to any one of claims 18 to 22, comprising any combination thereof.

24. The aforementioned Aflibercept species is a) N68 contains approximately 0.7 (mol / mol protein) sialic acid, b) Approximately 4% O-glycosylation of T33, c) Approximately 95% N-glycosylation of N36, d) N68 has approximately 26% N-glycosylation. e) Approximately 1% high mannose in the Fc domain, f) Approximately 26% galactosylation in the Fc domain, g) Approximately 1% sialylation in the Fc domain, or h) The composition according to claim 23, comprising any combination thereof.

25. The aforementioned Aflibercept species is a) N68 contains approximately 0.8 (mol / mol protein) of sialic acid, b) Approximately 8.3% O-glycosylation of T33, c) N36 has approximately 90.9% N-glycosylation. d) N68 has approximately 51.9% N-glycosylation. e) Approximately 0.4% high mannose in the Fc domain, f) Approximately 24.4% galactosylation in the Fc domain, g) Approximately 3.8% sialylation in the Fc domain, or h) The composition according to claim 23, comprising any combination thereof.

26. The composition according to any one of claims 18 to 25, wherein the amount of Y92L clipping species in the mixture is about 0.4%.

27. A method for increasing the binding of a composition comprising a mixture of aflibercept species to PlGF and / or VEGF-A, comprising reducing the amount of Y92L clipping species in the mixture.

28. A method for increasing the binding of a composition containing a mixture of aflibercept species to PLGF, comprising reducing the N68 occupancy rate of aflibercept.

29. The method according to claim 27 or 28, wherein the PLGF is PLGF-1.

30. The method according to claim 27 or 28, wherein the PLGF is PLGF-2.

31. The method according to claim 27, comprising purifying aflibercept by an anion exchange chromatography step followed by a hydrophobic interaction chromatography step.

32. A method for producing a composition containing a mixture of Aflibercept species, The process involves subjecting a cell culture medium containing a mixture of aflibercept species to a purification process including an anion exchange chromatography (AEX) step followed by a hydrophobic interaction chromatography (HIC) step. A method that produces a smaller amount of Y92L clipping species compared to a purification process that includes an AEX step followed by a cation exchange chromatography (CEX) step.

33. The method according to claim 32, wherein the purification process does not include a CEX step.

34. A method for producing a composition containing a mixture of Aflibercept species, a) Harvesting cells that produce aflibercept, b) Applying the harvested cell culture medium to a protein A column, c) A method comprising performing anion exchange chromatography (AEX), followed by cation exchange chromatography (CEX) or hydrophobic interaction chromatography (HIC).

35. The method according to claim 34, wherein HIC follows AEX.

36. The method described above is the method of claim 35, wherein the method does not include CEX.

37. The method according to claim 36, wherein CEX follows AEX.

38. The method according to any one of claims 34 to 37, wherein the cells are Chinese hamster ovary (CHO) cells.

39. The method according to any one of claims 34 to 38, wherein the cells are grown by perfusion or fed batch.

40. The method according to any one of claims 34 to 39, wherein the cells are harvested by acid precipitation, centrifugation, deep filtration, or any combination thereof.

41. The method according to any one of claims 34 to 40, further comprising one or more virus inactivation and / or virus filtration steps.

42. The method according to any one of claims 34 to 41, further comprising an ultrafiltration / diafiltration (UF / DF) step.

43. The method according to any one of claims 34 to 42, wherein the method does not include performing CEX before performing AEX.

44. The method according to any one of claims 34 to 43, wherein the method does not involve performing size exclusion chromatography (SEC).

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