Compositions comprising aflibercept and variants thereof and related methods and uses

Abstract

This application provides a composition comprising aflibercept and a variant thereof, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, and wherein the truncated variant is present in an amount of less than or equal to about 20% by mass. This application also provides a pharmaceutical formulation comprising the above composition and a method for preparing the same. This application also provides methods for detecting the above variant and their use in quality inspection or quality control of products containing aflibercept.

Description

[0001] Related applications

[0002] This application claims priority to Chinese patent application No. 202410533566.4 filed on April 29, 2024 and Chinese patent application No. 202510129375.6 filed on January 27, 2025, the entire contents of which are incorporated herein by reference for all purposes. Field of the Invention

[0003] The present application relates to the field of pharmaceutical preparations. Specifically, the present application provides compositions comprising aflibercept and variants thereof, as well as related methods and applications. Background of the Invention

[0004] Vascular endothelial growth factor (VEGF) is the most classic angiogenic factor in the body, promoting angiogenesis and enhancing vascular permeability. Abnormal VEGF production can lead to a variety of eye diseases, including wet age-related macular degeneration (wet-AMD) and diabetic retinopathy (DR).

[0005] Aflibercept is a VEGF inhibitor. It is a recombinant fusion protein composed of the Fc portion of human IgG1 immunoglobulin fused to the VEGF binding moiety, which is derived from the extracellular domains of human VEGF receptors 1 and 2. Aflibercept has been shown to be effective in preventing the formation of new blood vessels in CoNV. In the United States and Europe, aflibercept (trade name Eylea TM ) has been approved for the treatment of wet macular degeneration.

[0006] As a drug, aflibercept must maintain stability and efficacy. Quality control of pharmaceutical compositions primarily focuses on controlling the content of the active ingredient and related substances, such as variants or impurities. In particular, the content of related substances must meet pharmaceutical requirements. SUMMARY OF THE INVENTION

[0007] In a first aspect, the present application provides a composition comprising aflibercept and a variant thereof, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, comprising a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 1 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 2; and wherein the content of the truncated variant is less than or equal to about 20%, calculated as a percentage by mass.

[0008] In a specific embodiment, the content of the truncated variant is less than or equal to about 20% based on trypsin-cleaved peptide mass fingerprinting analysis.

[0009] In some embodiments, the content of aflibercept in the composition is greater than or equal to about 80% calculated as a percentage by mass, for example, based on trypsin-cleaved peptide mass fingerprint analysis.

[0010] In some embodiments, the content of the truncated variant in the composition is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, or greater than or equal to about 2%, calculated as a percentage by mass, for example, based on trypsin-cleaved peptide mass fingerprint analysis.

[0011] In some embodiments, the content of the truncated variant is less than or equal to about 19%, less than or equal to about 18%, less than or equal to about 17%, less than or equal to about 16%, less than or equal to about 15%, less than or equal to about 14%, less than or equal to about 13%, less than or equal to about 12%, less than or equal to about 11%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 10%, less than or equal to about 11%, less than or equal to about 10 ... Less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2.5%, less than or equal to about 2%, less than or equal to about 1.5%, less than or equal to about 1%, less than or equal to about 0.9%, or less than or equal to about 0.8%, less than or equal to about 0.7%, less than or equal to about 0.6%, less than or equal to about 0.5%, less than or equal to about 0.4%, less than or equal to about 0.3%, less than or equal to about 0.2%, or less than or equal to about 0.1%. In some embodiments, the content of the truncated variant can be any interval within the range defined by any two values ​​mentioned above or any value within the interval.

[0012] The truncated variant is also referred to herein as T100 and is described in detail below.

[0013] In some embodiments, the composition is prepared by a method comprising the steps of:

[0014] Cells capable of expressing aflibercept are cultured in a bioreactor, wherein the culture comprises a cell expansion stage and a protein production stage, wherein the culture temperature in the protein production stage is substantially maintained at 27.5° C.-37° C.

[0015] In some embodiments, the culture temperature is maintained between 27.5°C and 37°C for at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the time during the protein production phase.

[0016] In some embodiments, the composition is prepared by a method comprising the steps of:

[0017] Cells capable of expressing aflibercept are cultured in a bioreactor, wherein an average daily addition concentration of asparagine is 0.085-0.433 g / L / day, and / or an average daily addition concentration of methionine is 0.015-0.185 g / L / day during the culture period.

[0018] In a second aspect, the present application provides a pharmaceutical preparation comprising the composition described in the first aspect, and one or more pharmaceutically acceptable carriers.

[0019] In a third aspect, the present application provides a method for preparing the pharmaceutical preparation of the second aspect, comprising the following steps:

[0020] (1) preparing a composition comprising aflibercept and a variant thereof, wherein the variant is a truncated variant defined in the first aspect above, and

[0021] (2) evaluating the composition for the truncated variant and confirming that the truncated variant is present at a level of less than or equal to about 20%.

[0022] In some embodiments, the above step (2) includes performing peptide mass fingerprint analysis on the composition to obtain XIC spectra of truncated peptides and full-length peptides, and calculating the content of the truncated variants based on the corresponding peak areas.

[0023] In some embodiments, the method further comprises combining the composition after step (2) above with a pharmaceutically acceptable carrier.

[0024] In a fourth aspect, the present application provides a method for detecting an aflibercept variant in an aflibercept-containing composition or pharmaceutical formulation, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, comprising a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 1 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 2; and wherein the method comprises:

[0025] Digesting the composition or pharmaceutical preparation with IdeS enzyme and performing subunit molecular weight analysis, for example, using LC-MS to perform subunit molecular weight analysis, and determining the presence or absence of the variant based on the molecular weight; or

[0026] The composition or pharmaceutical preparation is digested with trypsin and then subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS method to perform peptide mass fingerprint analysis, and the presence of the variant is determined based on the primary and secondary mass spectra.

[0027] In some embodiments, the above method further includes obtaining XIC spectra of truncated peptides and full-length peptides after peptide mass fingerprint analysis when the presence of the variant is determined by performing peptide mass fingerprint analysis after trypsin cleavage, and calculating the content of the truncated variant based on the corresponding peak areas.

[0028] In a fifth aspect, the present application provides a method for quality inspection or quality control of an aflibercept-containing product, comprising detecting the content of an aflibercept variant in the aflibercept-containing product, wherein the variant is a truncated variant as defined in the first aspect. In some embodiments, the aflibercept-containing product is the composition described in the first aspect or the pharmaceutical preparation described in the second aspect.

[0029] In some embodiments, if the content of the variant is detected to be less than or equal to about 20%, it indicates that the aflibercept product meets the requirements for pharmaceutical use.

[0030] In a sixth aspect, the present application provides the use of an aflibercept variant in quality inspection or quality control of a product containing aflibercept, wherein the variant is a truncated variant as defined in the first aspect. In some embodiments, the aflibercept-containing product is the composition described in the first aspect or the pharmaceutical preparation described in the second aspect.

[0031] In a seventh aspect, the present application provides a method for preparing the composition of the first aspect, comprising:

[0032] Cells capable of expressing aflibercept are cultured in a bioreactor, wherein the culture comprises a cell expansion stage and a protein production stage, wherein the culture temperature in the protein production stage is substantially maintained at 27.5° C.-37° C.

[0033] In some embodiments, the culture temperature is maintained between 27.5°C and 37°C for at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the time during the protein production phase.

[0034] In an eighth aspect, the present application provides a method for preparing the composition of the first aspect, comprising:

[0035] Cells capable of expressing aflibercept are cultured in a bioreactor, wherein an average daily addition concentration of asparagine is 0.085-0.433 g / L / day, and / or an average daily addition concentration of methionine is 0.015-0.185 g / L / day during the culture period.

[0036] In a ninth aspect, the present application provides a method for preparing a composition comprising aflibercept, the method comprising the step of culturing cells capable of expressing aflibercept in a bioreactor, characterized in that: during the culture period, the average daily addition concentration of asparagine is 0.085-0.433 g / L / day and / or the average daily addition concentration of methionine is 0.015-0.185 g / L / day; and / or, the culture comprises a cell expansion stage and a protein production stage, wherein the culture temperature in the protein production stage is substantially maintained at 27.5°C-37°C.

[0037] In some embodiments, the culture temperature is maintained between 27.5°C and 37°C during at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the protein production phase.

[0038] In some embodiments, the composition comprises aflibercept and a variant thereof, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant comprising a first peptide chain with an amino acid sequence as shown in SEQ ID NO: 1 and a second peptide chain with an amino acid sequence as shown in SEQ ID NO: 2.

[0039] In some embodiments, the truncated variant is present in the composition at a mass percentage of less than or equal to about 20%, calculated based on trypsin-cleaved peptide mass fingerprinting. Preferably, the aflibercept content in the composition is greater than or equal to about 80%, calculated based on mass percentage.

[0040] In some embodiments, the content of the truncated variant is greater than or equal to about 0.01%, greater than or equal to about 0.05%, greater than or equal to about 0.1%, greater than or equal to about 0.5%, greater than or equal to about 1%, greater than or equal to about 1.5%, or greater than or equal to about 2%. Preferably, the content of the truncated variant is less than or equal to about 19%, less than or equal to about 18%, less than or equal to about 17%, less than or equal to about 16%, less than or equal to about 15%, less than or equal to about 14%, less than or equal to about 13%, less than or equal to about 12%, less than or equal to about 11%, less than or equal to about 10%, less than or equal to about 9%, less than or equal to about 8%, less than or equal to about 7%, less than or equal to about 6%, less than or equal to about 5%, less than or equal to about 10%, less than or equal to about 1 ... Less than or equal to about 4%, less than or equal to about 3%, less than or equal to about 2.5%, less than or equal to about 2%, less than or equal to about 1.5%, less than or equal to about 1%, less than or equal to about 0.9%, or less than or equal to about 0.8%, less than or equal to about 0.7%, less than or equal to about 0.6%, less than or equal to about 0.5%, less than or equal to about 0.4%, less than or equal to about 0.3%, less than or equal to about 0.2%, or less than or equal to about 0.1%. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The sequence and schematic structure of aflibercept are shown.

[0042] Figure 2 The TIC spectrum of the molecular weight analysis of the non-reducing subunit cleaved by IdeS is shown.

[0043] Figure 3 The peak A ( Figure 3 A), Peak B ( Figure 3 B) and peak C( Figure 3 C) of the deconvoluted spectrum, where Figure 3 The Lys-loss marked in A is the C-terminal lysine deletion, which is a common post-translational modification of antibodies.

[0044] Figure 4 The extracted ion current chromatogram of the primary mass spectrum of the truncated peptide fragment of T100 in the aflibercept sample is shown ( Figure 4 A), first-level mass spectrum ( Figure 4 B) and secondary mass spectra ( Figure 4 C).

[0045] Figure 5 Shown are the extracted ion chromatograms of truncated and full-length peptides in the aflibercept sample.

[0046] Sequence Description

[0047] SEQ ID NO: 1 is the amino acid sequence of two identical peptide chains of aflibercept, which is shown below (Note: the bold portion is the VEGFR-1 D2 domain, the underlined portion is the VEGFR-2 D3 domain, and the italicized portion is the Fc region):

[0048]

[0049] SEQ ID NO: 2 is a truncated sequence of SEQ ID NO: 1, which lacks the N-terminal 99 amino acid residues. The specific sequence is as follows (Note: the bold portion is the VEGFR-1 D2 domain, the underlined portion is the VEGFR-2 D3 domain, and the italic portion is the Fc segment):

[0050]

[0051] SEQ ID NO: 3 is the amino acid sequence of the long peptide chain in the T100 recombinant expression sequence, which is shown below (Note: the residues in italics are mutated amino acids):

[0052]

[0053]

[0054] SEQ ID NO: 4 is the amino acid sequence of the short peptide chain in the T100 recombinant expression sequence, which is shown below (Note: the residues in italics are mutated amino acids):

[0055] DETAILED DESCRIPTION

[0056] Angiogenesis is essential for normal embryonic and postnatal vascular development. Abnormal or pathological angiogenesis is a hallmark of cancer and several retinal diseases, in which upregulation of pro-angiogenic factors such as vascular endothelial growth factor (VEGF) leads to increased endothelium, altered vasculature, and increased vascular permeability. Elevated levels of VEGF are found in the vitreous humor and retinal vasculature of patients with a variety of eye diseases. Blocking VEGF activity has become the preferred treatment for eye diseases such as DME, wet AMD, CNV, and retinal vein occlusion.

[0057] Aflibercept is an anti-VEGF protein, a homodimer formed by two identical peptide chains linked by a disulfide bond. It contains a human amino acid sequence comprising the second Ig domain of human VEGFR-1 (VEGFR-1D2) and the third Ig domain of human VEGFR-2 (VEGFR-2D3). In a specific embodiment, the sequences of the two identical peptide chains of aflibercept are as shown in SEQ ID NO: 1.

[0058] The inventors of the present application unexpectedly discovered that variants of aflibercept, such as peptide truncations, are produced during the preparation process of aflibercept. A content of such variants of less than or equal to approximately 20% does not affect the binding activity and biological activity of an aflibercept sample. In other words, as long as the content of aflibercept variants does not exceed approximately 20%, the binding activity and biological activity of aflibercept for VEGF will not be reduced. Therefore, during the preparation of aflibercept, if the content of variants is assessed to be no greater than 20%, there is no need to remove variants or impurities, which simplifies the production process and reduces production costs.

[0059] In some embodiments, the commercially available Eylea TM As a positive control, the relevant activities of the composition comprising aflibercept and its variants were detected, including the biological activities of binding to VEGF and inhibiting the proliferation of HUVEC cells.

[0060] In some embodiments, the biological activity of aflibercept in the compositions disclosed herein is determined using the VEGF activity neutralization-HUVEC cell proliferation inhibition assay.

[0061] In a specific embodiment, the inventors identified the aforementioned variant and found that it differed from aflibercept only in that 99 amino acid residues were deleted from the N-terminus of one peptide chain, while the other peptide chain was identical to SEQ ID NO: 1. The sequence of this truncated peptide chain is shown in SEQ ID NO: 2. Because the truncated peptide chain begins at threonine 100 in SEQ ID NO: 1, this variant is designated "T100" herein.

[0062] In some embodiments, in the composition disclosed herein comprising aflibercept and variants thereof, the aflibercept variant is T100, wherein the content of variant T100 does not exceed 20%.

[0063] In some embodiments, the compositions described herein consist of aflibercept and a variant thereof (eg, T100).

[0064] Described herein is the use of cell culture medium to prepare aflibercept. In some embodiments, the cell culture medium is a chemically defined medium ("CDM").

[0065] In a specific embodiment, the cell culture medium used contains the following major components: 4-hydroxyethylpiperazineethanesulfonic acid, glucose, sodium pyruvate, sodium chloride, potassium chloride, sodium selenite, manganese sulfate, ethanolamine, ferric citrate, zinc sulfate, copper sulfate, glutathione, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, alanine, asparagine, arginine, aspartic acid, cystine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, biotin, calcium pantothenate, choline chloride, folic acid, inositol, nicotinamide, vitamin B6, vitamin B2, vitamin B1, vitamin B12, linoleic acid, block polyether F-68, and yeast extract.

[0066] In some embodiments, aflibercept is expressed in a suitable host cell. Non-limiting examples of such host cells include, but are not limited to, CHO, CHO K1, NS0, Sp2 / 0, embryonic kidney cells, BHK, etc.

[0067] In some embodiments, cells capable of expressing aflibercept are cultured in a bioreactor, wherein the culture comprises a cell expansion phase and a protein production phase. Preferably, the culture temperature during the protein production phase is substantially maintained at about 27.5°C to 37°C.

[0068] In specific embodiments, the culture temperature during the protein production stage is substantially maintained at, for example, about 28°C ± 0.5°C, about 30°C ± 0.5°C, about 32°C ± 0.5°C, about 34°C ± 0.5°C, or about 36.5°C ± 0.5°C, or any value between 27.5°C and 37°C.

[0069] In some embodiments, the culture temperature is maintained at about 27.5°C to about 37°C for at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the time during the protein production phase.

[0070] The inventors of the present application unexpectedly discovered that the T100 content in aflibercept samples is affected by the culture temperature during the protein production stage. In a specific embodiment, as the temperature gradually decreases, the T100 content gradually decreases.

[0071] In some embodiments, the average daily concentration of asparagine added to the culture medium during the culture period is 0.085-0.433 g / L / day. In specific embodiments, the average daily concentration of asparagine added can be 0.433 g / L / day, 0.166 g / L / day, or 0.085 g / L / day, or any other value between 0.085 and 0.433.

[0072] In some embodiments, the average daily concentration of methionine added to the culture medium during the culture period is 0.015-0.185 g / L / day. In specific embodiments, the average daily concentration of methionine added can be 0.185 g / L / day, or 0.015 g / L / day, or any other value between 0.015 and 0.185.

[0073] In a specific embodiment, the above-mentioned culture period is the entire culture period including the cell expansion stage and the protein production stage.

[0074] In some embodiments, the protein production phase is the time of the first feeding of the fed-batch culture ±0 days, ±0.5 days, ±1 day, ±1.5 days, ±2 days, 2.5 days, ±3 days, ±3.5 days, ±4 days, ±4.5 days, ±5 days, ±5.5 days, ±6 days, ±6.5 days, ±7 days, ±7.5 days, ±8 days, ±8.5 days, ±9 days, ±9.5 days, ±10 days, ±10.5 days, ±11 days, ±11.5 days, ±12 days, ±12.5 days, ±13 days, ±13.5 days, ±14 days, ±14.5 days, ±15 days, ±15.5 days, ±16 days, ±16.5 days, ±17 days, ±17.5 days, ±18 days, ±18.5 days, ±19 days, ±19.5 days, or ±20 days. The process to the end of culture, or the process from the time of start of perfusion culture of perfusion medium ±0 days, ±0.5 days, ±1 day, ±1.5 days, ±2 days, 2.5 days, ±3 days, ±3.5 days, ±4 days, ±4.5 days, ±5 days, ±5.5 days, ±6 days, ±6.5 days, ±7 days, ±7.5 days, ±8 days, ±8.5 days, ±9 days, ±9.5 days, ±10 days, ±10.5 days, ±11 days, ±11.5 days, ±12 days, ±12.5 days, ±13 days, ±13.5 days, ±14 days, ±14.5 days, ±15 days, ±15.5 days, ±16 days, ±16.5 days, ±17 days, ±17.5 days, ±18 days, ±18.5 days, ±19 days, ±19.5 days or ±20 days to the end of culture.

[0075] In a specific embodiment, the average daily added concentration of asparagine is calculated as follows:

[0076]

[0077] The total amount of asparagine added mentioned above also includes the asparagine content in the seed expansion medium.

[0078] In a specific embodiment, the average daily methionine addition concentration is calculated as follows:

[0079]

[0080] The total amount of methionine added mentioned above also includes the methionine content in the seed expansion medium.

[0081] In a specific embodiment, the average daily added concentration of asparagine is 0.433 g / L / day, and the average daily added concentration of methionine is 0.185 g / L / day.

[0082] In a specific embodiment, the average daily added concentration of asparagine is 0.433 g / L / day, and the average daily added concentration of methionine is 0.015 g / L / day.

[0083] In a specific embodiment, the average daily added concentration of asparagine is 0.166 g / L / day, and the average daily added concentration of methionine is 0.185 g / L / day.

[0084] In a specific embodiment, the average daily added concentration of asparagine is 0.166 g / L / day, and the average daily added concentration of methionine is 0.015 g / L / day.

[0085] In a specific embodiment, the average daily added concentration of asparagine is 0.085 g / L / day, and the average daily added concentration of methionine is 0.185 g / L / day.

[0086] In a specific embodiment, the average daily added concentration of asparagine is 0.085 g / L / day, and the average daily added concentration of methionine is 0.015 g / L / day.

[0087] The inventors of the present application unexpectedly discovered that the T100 content in aflibercept samples is affected by the average daily concentrations of asparagine and methionine added during the culture period. In a specific embodiment, the T100 content gradually decreases as the average daily concentrations of asparagine and methionine added during the culture period gradually decrease.

[0088] In some embodiments, the purification process of aflibercept comprises one or more steps selected from the group consisting of affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, desalting chromatography, virus removal nanofiltration, and ultrafiltration concentration.

[0089] In some embodiments, aflibercept samples are subjected to IdeS enzymatic digestion and non-reduced subunit molecular weight analysis. In specific embodiments, after IdeS digestion, subunit molecular weight analysis of the sample is performed using liquid chromatography-mass spectrometry (LC-MS).

[0090] In the present application, the inventors unexpectedly discovered variant T100 while performing subunit molecular weight analysis on aflibercept samples digested with IdeS using LC-MS.

[0091] In some embodiments, variant T100 in aflibercept products can be reduced or removed by methods such as ion exchange chromatography, hydrophobic chromatography, or complex chromatography.

[0092] In some embodiments, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis is used to detect variant T100 in aflibercept products. For example, LC-MS / MS is used to perform peptide mass fingerprinting analysis on the test sample.

[0093] In a specific embodiment, after obtaining mass spectrometry data, the extracted ion current chromatograms (XICs) of the two most strongly responsive charge forms of the truncated peptide and its corresponding full-length peptide are extracted, the peak areas of each are integrated, and the T100 content is calculated according to the following formula:

[0094]

[0095] In some embodiments, the inventors of the present application unexpectedly discovered that when the content of variant T100 in a prepared aflibercept sample is less than or equal to about 20%, the binding activity of aflibercept to VEGF is not reduced.

[0096] In some embodiments, the inventors of the present application unexpectedly discovered that a content of variant T100 in a prepared aflibercept sample of less than or equal to about 20% does not reduce the biological activity of aflibercept.

[0097] In some embodiments, the content of the variant (e.g., T100) in the compositions comprising aflibercept and variants thereof disclosed herein is about 0.01% to about 20%, about 0.05% to about 20%, about 0.1% to about 20%, about 0.2% to about 20%, about 0.3% to about 20%, about 0.4% to about 20%, about 0.5% to about 20%, about 0.6% to about 20%, about 0.7% to about 20%, about 0.8% to about 20%, about 0.9% to about 20%, about 1% to about 20%, about 1.5% to about 20%, about 2% to about 20%, about 2.5% to about 20%, about 3% to about 20%, about 3.5% to about 20%, about 4% to about 20%, about 5% to about 20%, about 6% to about 20%, about 7% to about 20%, about 8% to about 20%, about 9% to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, about 15% to about 20%, about 16% to about 20%, about 17% to about 20%, about 18% to about 20%, about 19% to about 20%, about 21% to about 20%, about 22% to about 20%, about 23% to about 20%, about 24% to about 20%, about 25% to about 20%, about 26% to about 20%, about 27% to about 20%, about 28% to about 20%, about 29% to about 20%, about 30% to About 4.5% to about 20%, about 5% to about 20%, about 5.5% to about 20%, about 6% to about 20%, about 6.5% to about 20%, about 7% to about 20%, about 7.5% to about 20%, about 8% to about 20%, about 8.5% to about 20%, about 9% to about 20%, about 9.5 to about 20%, about 10% to about 20%, about 11% to about 20%, about 12% to about 20%, about 13% to about 20%, about 14% to about 20%, about 15% to about 20%, about 16% to about 20%, about 17% to about 20%, about 18% to about 20%, about 19% to about 20%, or any interval in any of the above ranges or any value within the intervals.

[0098] In specific embodiments, the content of the variant (e.g., T100) in the compositions comprising aflibercept and variants thereof disclosed herein is about 0.5% to about 10%, about 0.5% to about 9%, about 0.5% to about 8%, about 0.5% to about 7%, about 0.5% to about 6%, about 0.5% to about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about 0.5% to about 2%, about 0.5% to about 1%, or any interval within any of the above ranges or any value within the intervals. For example, the content of the variant (e.g., T100) is 0.86% to 19.29%, preferably 0.86% to 2.59%.

[0099] Also disclosed herein are pharmaceutical preparations for preparing the compositions disclosed herein, i.e., the compositions are mixed with an optional pharmaceutically acceptable carrier and stored in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dosages and concentrations employed. In some embodiments, pharmaceutically acceptable carriers include, for example: water; buffers such as phosphates, citrates, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives; hydrophilic polymers such as polyvinylpyrrolidone; chelating agents such as EDTA, etc. In some embodiments, the formulations for in vivo administration must be sterile. This can be easily achieved by filtration using a sterile filter membrane.

[0100] Throughout this specification and claims, the term "about" or "substantially" refers to a range of values ​​that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result), for example, + / - 10% of the recited value.

[0101] In the present description and claims, the words “comprises,” “comprising,” and “containing” mean “including but not limited to,” and are not intended to exclude other parts, additives, components, or steps.

[0102] It will be appreciated that features, characteristics, components, or steps described in conjunction with a particular aspect, embodiment, or example of the present application may be applicable to any other aspect, embodiment, or example described herein unless incompatible therewith.

[0103] The above disclosure generally describes the present invention, which is further illustrated by the following examples. These examples are described only to illustrate the present invention and are not intended to limit the scope of the present invention. Although specific terms and values ​​are used herein, these terms and values ​​are also understood to be exemplary and do not limit the scope of the present invention. Unless otherwise specified, the experimental methods and techniques in this specification are methods and techniques well known to those skilled in the art.

[0104] Example

[0105] Example 1 Preparation of Aflibercept

[0106] Upstream cell culture process

[0107] The expression system used in this example is the Freedom TM The CHO-STM Kit contains a cGMP-prepared CHO-S host cell line and a pCHO1.0 expression plasmid. The expression vector is the pCHO1.0 plasmid, which enables efficient expression of exogenous recombinant proteins in mammalian cells. The production culture medium contains the following main components:

[0108] 4-Hydroxyethylpiperazineethanesulfonic Acid, Glucose, Sodium Pyruvate, Sodium Chloride, Potassium Chloride, Sodium Selenite, Manganese Sulfate, Ethanolamine, Ferric Citrate, Zinc Sulfate, Copper Sulfate, Glutathione, Magnesium Chloride, Sodium Dihydrogen Phosphate, Sodium Bicarbonate, Alanine, Asparagine, Arginine, Aspartic Acid, Cystine, Cysteine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine, Biotin, Calcium Pantothenate, Choline Chloride, Folic Acid, Inositol, Niacinamide, Vitamin B6, Vitamin B2, Vitamin B1, Vitamin B6 12 , linoleic acid, block polyether F-68, yeast extract.

[0109] With reference to the typical cell culture process of antibody drugs, the production of aflibercept bulk solution is carried out according to the process of cell recovery, step-by-step expansion in shake flasks and bioreactors, and production in production tanks. The production stage adopts the fed-batch culture process.

[0110] Take a cell cryopreservation tube, thaw it in a 37℃ water bath, culture it in a shake flask expansion culture medium, and after continuous expansion culture in shake flasks and bioreactors, inoculate the production tank for production.

[0111] Referring to the typical cell culture process of antibody drugs, appropriate process parameters such as culture temperature, pH, dissolved oxygen, stirring speed and ventilation should be set in the production stage. Appropriate feed culture medium, supplementary solution and glucose solution should be added according to the needs of cell growth. Defoamer solution or alkaline solution should be added according to the culture needs. After the culture is completed, the cell culture fluid is centrifuged and / or deep filtered and sterilized, and the supernatant is collected for purification.

[0112] Downstream purification process

[0113] This example refers to a typical purification process for antibody drugs, including affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, desalting chromatography, virus removal nanofiltration and ultrafiltration concentration, to finally obtain the target protein stock solution.

[0114] Affinity chromatography: After equilibrating the affinity chromatography column with a salt-containing buffer (the salt is selected from Na2SO4, NaCl, or (NH4)2SO4), the sample loading phase begins. After loading, the column is rinsed with a salt-containing buffer, then with a salt-free buffer, and eluted with a pH 3.0-4.5 buffer. The eluted protein solution is collected and then subjected to viral inactivation.

[0115] Anion exchange chromatography: pre-equilibrate the anion exchange chromatography column with a salt-containing buffer (the salt is selected from Na2SO4, NaCl or (NH4)2SO4), and then equilibrate with a salt-free buffer. Load the virus-inactivated protein solution, and after loading, continue to rinse with a salt-free equilibration buffer, and then rinse with a 0.02-0.06 mol / L salt equilibration buffer, and elute with a buffer containing 0.1-0.2 mol / L salt to elute the protein from the anion exchange chromatography column, and collect the eluted protein solution.

[0116] Cation exchange chromatography: Pre-equilibrate the cation exchange chromatography column with a salt-containing buffer (the salt is selected from Na2SO4, NaCl or (NH4)2SO4), and then equilibrate with a salt-free buffer. Load the protein solution collected by the anion exchange chromatography. After loading, continue to rinse with a salt-free equilibration buffer, and then elute with a buffer containing 0.1-0.2 mol / L salt to elute the protein from the cation exchange chromatography column and collect the eluted protein solution.

[0117] Hydrophobic chromatography: Equilibrate the hydrophobic chromatography column with a salt-containing buffer (the salt is selected from Na2SO4, NaCl or (NH4)2SO4), load the protein solution collected by cation exchange chromatography, and continue to rinse with a salt-containing equilibration buffer after loading. Then elute with a buffer containing 0.3-0.6 mol / L salt to elute the protein from the hydrophobic chromatography column and collect the eluted protein solution.

[0118] Desalting chromatography: The desalting chromatography column is balanced with an equilibrium buffer containing 0.02-0.06 mol / L salt. The eluted protein solution collected in the hydrophobic chromatography is loaded onto the desalting chromatography column for desalting. The desalted protein solution is collected and then concentrated through virus removal nanofiltration and ultrafiltration to obtain the target protein stock solution.

[0119] Example 2 Discovery and Identification of T100 in Aflibercept Samples

[0120] Discovery of T100 in Aflibercept Samples

[0121] The sequence and structure of Aflibercept prepared in Example 1 are shown in Figure 1 .

[0122] The sample prepared in Example 1 was subjected to IdeS-enzyme-cleaved non-reducing subunit molecular weight analysis. Because aflibercept contains five N-glycosylation modification sites, the heterogeneity caused by N-glycosylation is very severe and cannot be measured without cleaving the N-glycan chain. Therefore, PNGase F was used to cleave the N-glycan before subunit molecular weight analysis. The steps for IdeS-enzyme-cleaved non-reducing subunit molecular weight analysis are as follows:

[0123] 50 μg of sample was diluted to 2 μg / μL in IdeS digestion buffer (50 mM phosphate, 150 mM NaCl, pH 6.6). IdeS was added at a protein:enzyme ratio of 20:1 (mass ratio) and incubated at 37°C for 1 h.

[0124] Take 20 μg of sample digested with IdeS, add 4 μl of 5× Reaction buffer (non-reducing buffer), make up the volume to 20 μl with water, and incubate at 75°C for 5 minutes. After incubation, add 2 μl of Rapid PNGase F (non-reduction), mix well, and digest at 50°C for 30 minutes.

[0125] After the enzyme digestion, centrifuge at 12000 rpm for 3 min and transfer the supernatant to a sample injection bottle;

[0126] Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the subunit molecular weight of the samples. The chromatographic and mass spectrometric conditions were as follows:

[0127] Chromatographic conditions:

[0128]

[0129] Mass spectrometry conditions:

[0130] MS system Xevo G2-XS QTof, Waters Ionization method ESI positive Primary mass spectrometry scanning range 500-4000 m / z Capillary voltage 2.7kV Cone voltage 60V Ion source temperature 120℃ Desolvation temperature 400℃ Cone hole air flow rate 50L / h Desolvation gas flow rate 800L / h

[0131] Data were processed using Waters UNIFI 1.8 software. Figure 2 , the deconvoluted spectra of the main components are shown in Figure 3 .

[0132] After IdeS digestion, the sample fragments below the hinge region, forming the Fc / 2 and VEGFR regions. Non-reduced subunit molecular weight analysis revealed that the peak at 9.07 min (peak A) in the TIC spectrum represents the Fc / 2 region, and the peak at 10.55 min represents the VEGFR region (peak C). The difference between the measured and theoretical molecular weights after deconvolution was less than 5.0 Da, indicating that all major components after IdeS digestion were detectable. A shoulder (peak B) preceded peak C, and the measured molecular weight of the major component after deconvolution was 38141.0 Da. Peak B had a similar retention time to peak C and a molecular weight approximately 11,000 Da less than that of peak C, suggesting that this may be related to truncation of the VEGFR region. Combined with sequence analysis of aflibercept, the theoretical molecular weight of the remaining component after N-terminal truncation of one chain of the VEGFR region, S1-N99, is 38139.7 Da, similar to the measured molecular weight of peak B.

[0133] Identification of T100 in Aflibercept Samples

[0134] To confirm the cleavage site, peptide mass fingerprint analysis was performed on the sample prepared in Example 1. The analysis steps are as follows:

[0135] About 40 μg of sample was added with urea and 10 mM TCEP at a final concentration of 6 M and incubated at 37°C for 60 min for denaturation and reduction;

[0136] IAM was added at a final concentration of 20 mM and incubated at 37°C for 15 min to alkylate and block free sulfhydryl groups;

[0137] Add 50 mM ammonium bicarbonate buffer to dilute the urea concentration to 1 M, add trypsin at a protein:enzyme ratio of 20:1 (mass ratio), and digest at 37°C for 25 min;

[0138] After the enzyme digestion was completed, formic acid was added to a final concentration of 1% to terminate the digestion, and the mixture was centrifuged at 12000 rpm for 3 min. The supernatant was transferred to a sample injection bottle.

[0139] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to perform peptide mass fingerprint analysis on the samples. The chromatographic and mass spectrometric conditions were as follows:

[0140] Chromatographic conditions:

[0141]

[0142]

[0143] Mass spectrometry conditions:

[0144] MS system Q-Exactive Plus, Thermo Ionization method ESI positive Primary mass spectrometry scanning range 200-2000 m / z Capillary voltage 3.2kV Capillary Temp 320℃ First-level mass spectrometry resolution 70000 AGC Target <![CDATA[1e 6 ]]> Secondary mass spectrometry resolution 17500 AGC Target <![CDATA[1e 5 ]]> Isolation window 1.6m / z

[0145] Data processing was performed using pFind software. The extracted ion chromatogram (XIC), primary and secondary mass spectra of the truncated peptide are shown in Figure 4 .

[0146] The peptide mass fingerprint results showed that a short peptide segment QTN was found at a retention time of 44.3 min in the sample. 99 TIIDVVLSPSHGIELSVGEK (missing amino acid residues are marked with a strikethrough). The measured monoisotopic peak of this peptide is m / z 1047.0814, while the theoretical m / z is 1047.0781, with an error of only 3.15 ppm. Furthermore, the fragment ions in the secondary mass spectrum match well with the theoretical b and y ions of this peptide, thus confirming the amino acid sequence of this peptide.

[0147] Aflibercept is a homodimer formed by two identical peptide chains connected by disulfide bonds. Combined with molecular weight analysis of subunits cleaved by IdeS, Peak B is formed by a C-terminal peptide cleavage at position N99 of one peptide chain, resulting in a deletion of the S1-N99 portion of the VEGFR region. The measured molecular weight of Peak B is 38141.0 Da. The theoretical molecular weight of the remaining component after N-terminal truncation of one VEGFR chain from S1 to N99 is 38139.7 Da, further confirming that the cleavage site is at position N99 of one peptide chain.

[0148] Example 3 Content of T100 in Aflibercept Samples

[0149] Peptide mass fingerprint analysis was performed on six batches of samples prepared in Example 1 using LC-MS / MS. After obtaining the mass spectrometric data, the XIC spectra of the two most strongly responsive charge forms of the truncated peptide TIIDVVLSPSHGIELSVGEK and its corresponding full-length peptide QTNTIIDVVLSPSHGIELSVGEK were extracted. The peak areas were integrated to obtain the respective peaks, and the T100 content was calculated according to the following formula:

[0150]

[0151] The sample pretreatment method and LC-MS / MS detection method were as shown in Example 2, and data processing was performed using Xcalibur software Qualbrowser.

[0152] Taking the mass spectrometry data of batch 1 as an example, the XIC spectra of the truncated peptide and the full-length peptide are shown in Figure 5 .

[0153] The T100 content in the batch 1 sample was calculated according to the formula and was 2.59% as follows:

[0154]

[0155] The T100 content in multiple batches of samples was calculated in the same way. The results are shown in Table 1.

[0156] Table 1 T100 content in multiple batches of samples

[0157]

[0158]

[0159] The results showed that the T100 content in the aflibercept samples prepared in Example 1 was 0.86%-2.59%.

[0160] Example 4 Mass Range of T100 in Aflibercept

[0161] The samples prepared in Example 1 were compared with the commercially available Eylea TM Comparative studies of binding activity and biological activity were performed.

[0162] ELISA was used to determine the expression of VEGF in each sample. 165 The binding capacity of proteins. The determination method is as follows:

[0163] human VEGF 165 The protein was diluted to 0.15 μg / ml with coating solution (50 mM NaHCO3-Na2CO3, pH 9.6), 100 μl was added to each well of a 96-well plate, and the plate was coated overnight at 2-8°C;

[0164] After blocking with 2% BSA-PBS for 2 hours, 100 μl of samples serially diluted with 1% BSA-PBST were added to each well and incubated at 37°C for 2 hours;

[0165] Then, HRP-labeled goat anti-human IgG Fc antibody was added to the 96-well plate;

[0166] Wash thoroughly with PBST between the above steps;

[0167] Finally, TMB colorimetric solution and stop solution (2M H2SO4) were added, and the absorbance at 450 nm was read using a microplate reader (TECAN SPARK multifunctional microplate reader). The protein concentration was used as the X-axis and the absorbance was used as the Y-axis. The four-parameter method was used to fit the curve and calculate the results.

[0168] The samples of batch 6 prepared in Example 1 were used as reference (100%), and the comparison between the samples prepared in batch 6 and the commercially available Eylea TM VEGF 165 The results are shown in Table 2.

[0169] Table 2 Comparison of samples from each batch with commercially available EyleaTM VEGF 165 Binding activity results

[0170]

[0171] The results showed that each batch of samples had a significant difference from the commercially available Eylea TM VEGF 165 The binding activity was between 80% and 120% of that of the reference substance. Since the precision of the binding activity detection method is generally ≤20%, the binding activity measured to be 80% to 120% of the reference substance is within the method fluctuation, indicating that the aflibercept sample prepared in Example 1 is comparable to the commercially available Eylea TM VEGF 165 There was no difference in binding activity.

[0172] The biological activity of each sample was determined using the VEGF neutralization-HUVEC cell proliferation inhibition assay. This assay is based on the fact that VEGF can stimulate the proliferation of human umbilical vein endothelial cells (HUVEC). VEGFR:Fc fusion proteins bind to VEGF, neutralizing VEGF activity and thereby inhibiting HUVEC cell proliferation, thereby detecting biological activity.

[0173] First, the sample was diluted with assay medium in a gradient manner. VEGF protein was diluted to 210 ng / ml. The VEGF protein dilutions and the diluted samples of each concentration gradient were added to a 96-well cell culture plate in sequence, 50 μl / well, and mixed evenly with equal volumes. The cells were then incubated for 50-80 minutes.

[0174] Adjust the HUVEC cell density to 1.6 × 10 cells / mL using assay medium. 5 ~2.4×10 5 cells / ml, 50 μl / well was added to a 96-well cell culture plate and cultured in a 37°C, 5% CO2 incubator for 65-70 hours;

[0175] After the incubation, the 96-well culture plate was removed, 30 μl of Alamar Blue staining solution was added to each well, and the cells were incubated in a 37°C incubator for 6 h. The data were read using a fluorescence microplate reader (SpectraMax Gemini EM, Molecular Devices) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. The protein concentration was used as the X-axis and the fluorescence value as the Y-axis. The four-parameter method was used to fit the curve and calculate the results.

[0176] The samples of batch 6 prepared in Example 1 were used as reference (100%), and the comparison between the samples prepared in batch 6 and the commercially available Eylea TM The results are shown in Table 3.

[0177] Table 3 Comparison of samples from each batch and commercially available Eylea TM Biological activity results

[0178]

[0179] The results showed that each batch of samples had a significant difference from the commercially available Eylea TM The biological activities of the samples were between 70% and 130% of the reference substance. Since the precision of the cell experiment method is generally ≤30%, the measured binding activities of 70% to 130% of the reference substance are within the method fluctuation, indicating that the aflibercept sample prepared in Example 1 is comparable to the commercially available Eylea TM There was no difference in biological activity.

[0180] The above results show that the binding activity and biological activity of the aflibercept sample prepared in Example 1 are comparable to those of the commercially available Eylea TM are equivalent.

[0181] T100 was recombinantly expressed based on the identification results. If a wild-type Fc sequence were used for expression, a mixed protein sample of three components would be expected: both chains truncated, a single chain truncated (the target component), and neither chain truncated. To obtain a pure single-chain truncated component, T100 was recombinantly expressed using a knob-into-hole design. Sequence information is shown in Table 4.

[0182] Table 4 T100 recombinant expression sequence

[0183]

[0184] Note: The residues in italics are the amino acids that have been mutated.

[0185] The plasmid was transfected into ExpiCHO-S cells by transient electroporation. The cells were cultured in commercial culture medium according to the established culture process. The culture medium was harvested after 10 days, filtered through a 0.22 μm pore size filter membrane, and then subjected to Potein A affinity chromatography to obtain the desired T100.

[0186] The purity of the main peak of transiently expressed T100 sample by SEC was 98.9%, and the intact molecular weight analysis results of the N-glycans showed that the measured molecular weight was consistent with the theoretical one.

[0187] The prepared T100 gradient was added to the samples prepared in Example 1, and the T100 content in each sample was detected, and the relationship between each sample and VEGF was determined. 165 Protein binding capacity and biological activity.

[0188] The T100 content and activity of each sample are shown in Table 5.

[0189] Table 5 T100 content and activity of each sample

[0190] Sample name T100 content (%) <![CDATA[Binding activity (%) 1,2 > <![CDATA[Biological activity (%) 1,2 > Add sample 1 6.22 96 92 Add sample 2 9.68 95 95 Add sample 3 12.51 87 88 Add sample 4 16.19 84 83 Add sample 5 19.29 80 75 Add sample 6 23.63 73 69

[0191] Note: 1: The batch 1 sample prepared in Example 1 is the reference control (100%).

[0192] 2: The binding activity and biological activity results are the average of 3 measurements.

[0193] The results showed that as the T100 content gradually increased, the binding activity and biological activity of the aflibercept sample gradually decreased. Therefore, the T100 content needs to be controlled during the preparation of aflibercept.

[0194] In order to reduce the content of T100 in the finished product, the following article explores the impact of adjusting some process parameters in the upstream cell culture stage (before downstream purification) on the amount of T100 produced.

[0195] Example 5 Effect of culture temperature on T100 content during protein production

[0196] The cell culture process followed the method described in Example 1, with the culture process comprising a 5-day cell expansion phase and a 7-day protein production phase in a bioreactor. Different culture temperatures were tested during the protein production phase, as shown in Table 6. After the protein production phase, the culture fluid was collected once and subjected to a proA affinity chromatography step. T100 content was then directly measured as described in Example 3. The T100 contents obtained at different culture temperatures during the protein production phase are shown in Table 6:

[0197] Table 6 T100 content corresponding to culture temperature at different protein production stages

[0198] Culture temperature during protein production T100 content (%) 36.5℃±0.5℃ 19.8 34℃±0.5℃ 18.3 32℃±0.5℃ 15.3 30℃±0.5℃ 10.5 28℃±0.5℃ 6.9

[0199] Results showed that incubation temperature during protein production affected the T100 content in aflibercept samples, with T100 levels decreasing as the temperature gradually decreased. Using temperatures between 27.5°C and 37°C during protein production consistently kept T100 levels below 20%, and it is expected that T100 levels will be further significantly reduced after downstream purification processes.

[0200] Example 6 Effect of the Average Daily Addition Concentration of Asparagine and Methionine on T100 Content

[0201] In the preliminary screening exploration of the effect of amino acid component content in cell culture medium on T100 content, it was found that the amount of asparagine and methionine were potential influencing factors, so a content gradient experiment was further designed to verify this finding.

[0202] The cell culture process followed the methods described in Examples 1 and 5. The culture process included a 5-day cell expansion phase in a bioreactor and a 7-day protein production phase. The protein production phase was maintained at 32°C ± 0.5°C. After the protein production phase, the culture fluid was collected and subjected to a proA affinity chromatography step. T100 content was then directly measured as described in Example 3.

[0203] In this example, the average daily added concentration is used to characterize the dosage of asparagine and methionine.

[0204] The calculation formula for the average daily addition concentration of asparagine is:

[0205]

[0206] The calculation formula for the average daily methionine addition concentration is:

[0207]

[0208] The "total amount added" in the above formula simply refers to the cumulative amount of asparagine or methionine added to the bioreactor in any form throughout the bioreactor culture process. Specifically, the total amount includes the amount of asparagine or methionine already present in the cell culture at the time of inoculation into the bioreactor (which may also be zero), the amount of asparagine or methionine previously added to the bioreactor (which may also be zero), the amount of asparagine or methionine in the feed medium throughout the culture process, and the amount of asparagine or methionine added in other forms.

[0209] The effects of the average daily addition concentrations of asparagine and methionine on T100 content are shown in Table 7 below. The results show that the T100 content in aflibercept samples is affected by the average daily addition concentrations of asparagine and methionine. As the average daily addition concentrations of asparagine and / or methionine decrease during the culture period, the T100 content gradually decreases. The average daily addition concentrations of asparagine and methionine shown in Table 7 are all capable of controlling the T100 content below 10%, and it is expected that the T100 content can be further significantly reduced after downstream purification processes.

[0210] Table 7 Average daily added concentrations and T100 contents of asparagine and methionine

[0211]

[0212] Without departing from the essence and scope of the present disclosure, various changes and equivalent substitutions may be made to the various embodiments disclosed herein. Unless otherwise indicated in the context, any feature, step or embodiment of the embodiments of the present disclosure may be used in combination with any other feature or embodiment.

Claims

1. A composition comprising aflibercept and a variant thereof, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant consisting of a first peptide chain having an amino acid sequence as set forth in SEQ ID NO: 1 and a second peptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the content of the truncated variant in the composition is less than or equal to 20% by mass, as calculated based on trypsin-cleaved peptide mass fingerprinting analysis.

2. The composition according to claim 1, wherein the content of aflibercept in the composition is greater than or equal to 80% by mass.

3. The composition of claim 1, wherein the content of the truncated variant is greater than or equal to 0.01%.

4. The composition of claim 3, wherein the truncated variant is present in an amount greater than or equal to 2%.

5. The composition of claim 1, wherein the content of the truncated variant is less than or equal to 5%.

6. The composition of claim 1, wherein the composition is prepared by a method comprising the following steps: culturing cells capable of expressing aflibercept in a bioreactor, wherein the culturing comprises a cell expansion phase and a protein production phase, wherein the culture temperature during the protein production phase is maintained at 27.5°C-37°C.

7. The composition of claim 1, wherein the composition is prepared by a method comprising the following steps: culturing cells capable of expressing aflibercept in a bioreactor, wherein during the culture period, asparagine is added at an average daily concentration of 0.085-0.433 g / L / day, and / or methionine is added at an average daily concentration of 0.015-0.185 g / L / day.

8. The composition of claim 1, wherein the composition is prepared by a method comprising the following steps: culturing cells capable of expressing aflibercept in a bioreactor, wherein the average daily addition concentration of asparagine during the culture period is 0.085-0.433 g / L / day and / or the average daily addition concentration of methionine is 0.015-0.185 g / L / day; and wherein the culture comprises a cell expansion phase and a protein production phase, and the culture temperature during the protein production phase is maintained at 27.5°C-37°C.

9. The composition of claim 6 or 8, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 50% of the protein production phase.

10. The composition of claim 9, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 80% of the time during the protein production phase.

11. A method for preparing the composition according to any one of claims 1 to 8, comprising culturing cells capable of expressing aflibercept in a bioreactor, wherein the culturing comprises a cell expansion phase and a protein production phase, wherein the culture temperature during the protein production phase is maintained at 27.5° C. to 37° C.

12. The method of claim 11, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 50% of the protein production phase.

13. The method of claim 12, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 80% of the time during the protein production phase.

14. A method for preparing the composition of any one of claims 1 to 5, comprising culturing cells capable of expressing aflibercept in a bioreactor, wherein during the culture period, asparagine is added at an average daily concentration of 0.085-0.433 g / L / day and / or methionine is added at an average daily concentration of 0.015-0.185 g / L / day.

15. A method for preparing a composition comprising aflibercept, the method comprising the step of culturing cells capable of expressing aflibercept in a bioreactor, characterized in that: The composition comprises aflibercept and a variant thereof, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant consisting of a first peptide chain having an amino acid sequence as shown in SEQ ID NO: 1 and a second peptide chain having an amino acid sequence as shown in SEQ ID NO: 2, wherein the content of the truncated variant in the composition is less than or equal to 20% by mass, calculated based on trypsin-cleaved peptide mass fingerprint analysis; wherein the average daily addition concentration of asparagine during the culture period is 0.085-0.433 g / L / day and / or the average daily addition concentration of methionine is 0.015-0.185 g / L / day; and / or the culture comprises a cell expansion stage and a protein production stage, wherein the culture temperature in the protein production stage is maintained at 27.5° C.-37° C.

16. The method of claim 15, wherein the content of aflibercept in the composition is greater than or equal to 80% by mass.

17. The method of claim 15, wherein the content of the truncated variant is greater than or equal to 0.01%.

18. The method of claim 17, wherein the content of the truncated variant is greater than or equal to 2%.

19. The method of claim 15, wherein the content of the truncated variant is less than or equal to 5%.

20. The method of any one of claims 15-19, wherein the culture temperature is maintained at 27.5°C-37°C for at least 50% of the protein production phase.

21. The method of claim 20, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 80% of the time during the protein production phase.

22. A pharmaceutical preparation comprising the composition according to any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers.

23. A method for detecting an aflibercept variant in an aflibercept-containing composition or pharmaceutical formulation, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant consisting of a first peptide chain having an amino acid sequence as set forth in SEQ ID NO: 1 and a second peptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the content of the truncated variant in the composition or pharmaceutical formulation is less than or equal to 20% by mass, as calculated based on trypsin-cleaved peptide mass fingerprinting analysis, the method comprising: The composition or pharmaceutical preparation is digested with IdeS and then subjected to subunit molecular weight analysis, and the subunit molecular weight analysis is performed using LC-MS to determine the presence or absence of the variant based on the molecular weight; or The composition or pharmaceutical preparation is digested with trypsin and then subjected to peptide mass fingerprint analysis, which is then analyzed using LC-MS / MS to determine the presence or absence of the variant based on the primary and secondary mass spectra.

24. The method of claim 23, further comprising, when the presence of the variant is determined by peptide mass fingerprint analysis after trypsin cleavage, obtaining XIC spectra of the truncated peptide and the full-length peptide after the peptide mass fingerprint analysis, and calculating the content of the truncated variant based on the corresponding peak areas.

25. A method for quality inspection or quality control of an aflibercept-containing product, comprising detecting the content of an aflibercept variant in the aflibercept-containing product, wherein the aflibercept variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant consisting of a first peptide chain having an amino acid sequence as set forth in SEQ ID NO: 1 and a second peptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the content of the truncated variant is less than or equal to 20% by mass, as calculated based on trypsin-cleaved peptide mass fingerprinting analysis.

26. The method of claim 25, wherein if the detected aflibercept variant content is less than or equal to 20%, it indicates that the aflibercept product meets pharmaceutical requirements.

27. Use of an aflibercept variant in quality inspection or quality control of aflibercept-containing products, wherein the aflibercept variant is a truncated variant of the VEGF-binding portion of aflibercept, the truncated variant consisting of a first peptide chain having an amino acid sequence as set forth in SEQ ID NO: 1 and a second peptide chain having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the content of the truncated variant is less than or equal to 20% by mass, as calculated based on trypsin-cleaved peptide mass fingerprinting analysis.

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