Compositions comprising aflibercept and variants thereof and related methods and uses

Abstract

The present application provides compositions comprising aflibercept and variants thereof wherein the variants are truncated variants of a VEGF binding moiety of aflibercept, and wherein the content of the truncated variants is less than or equal to about 20%, calculated by mass percent. The invention further provides a pharmaceutical preparation containing the composition and a preparation method of the pharmaceutical preparation. In addition, the invention also provides a detection method of the variant and application of the variant in quality inspection or quality control of an aflibercept-containing product.

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 the above applications are incorporated herein by reference in their entirety for all purposes. Field of the Invention

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

[0005] Vascular endothelial growth factor (VEGF) is the most classical angiogenic factor in the body, which can promote angiogenesis and enhance vascular permeability. Abnormal VEGF can lead to various eye diseases including wet age-related macular degeneration (wet-AMD), diabetic retinopathy (DR), etc.

[0006] Aflibercept is an inhibitor of VEGF. It is a recombinant fusion protein formed by fusing the Fc portion of human IgG1 immunoglobulin with the VEGF-binding portion, and the VEGF-binding portion is derived from the extracellular domains of human VEGF receptors 1 and 2. Aflibercept has been shown to be effective in preventing neovascularization of CoNV. In the United States and Europe, aflibercept (trade name Eylea TM ) has been approved for the treatment of wet macular degeneration.

[0007] Aflibercept, as a drug, must maintain stability and effectiveness. The quality control of pharmaceutical compositions mainly controls the content of active ingredients and the content of related substances such as variants or impurities. In particular, the content of related substances needs to meet pharmaceutical requirements. Summary of the Invention

[0009] In a first aspect, 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, which comprises a first peptide chain having the amino acid sequence shown in SEQ ID NO: 1 and a second peptide chain having the amino acid sequence shown in SEQ ID NO: 2; and wherein, calculated by mass percentage, the content of the truncated variant is less than or equal to about 20%.

[0010] In a specific embodiment, based on the analysis method of trypsin digestion peptide mass fingerprinting, the content of the truncated variant is less than or equal to about 20%.

[0011] In some embodiments, calculated by mass percentage, for example, based on trypsin digested peptide mass fingerprint analysis, the content of aflibercept in the composition is greater than or equal to about 80%.

[0012] In some embodiments, calculated by mass percentage, for example, based on trypsin digested peptide mass fingerprint analysis, 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%.

[0013] In some embodiments, calculated by mass percentage, for example, based on trypsin digested peptide mass fingerprint analysis, 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 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 or any value within the range defined by any two of the above-mentioned values.

[0014] The truncated variant is also referred to as T100 herein and is described in detail below.

[0015] In some embodiments, the composition is prepared by a method comprising the following steps:

[0016] Culturing cells capable of expressing aflibercept in a bioreactor, the culturing including a cell expansion stage and a protein production stage, wherein the culturing temperature in the protein production stage is maintained substantially at 27.5°C - 37°C.

[0017] In some embodiments, the culture temperature is maintained at 27.5°C - 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.

[0018] In some embodiments, the composition is prepared by a method comprising the following steps:

[0019] Culturing cells capable of expressing aflibercept in a bioreactor, wherein the average daily addition concentration of asparagine during the culture 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.

[0020] 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.

[0021] In a third aspect, the present application provides a method for preparing the pharmaceutical preparation of the second aspect, which comprises the following steps:

[0022] (1) Preparing a composition comprising aflibercept and its variants, wherein the variants are truncated variants defined in the first aspect above, and

[0023] (2) Evaluating the truncated variants in the composition and confirming that their content is less than or equal to about 20%.

[0024] In some embodiments, the above step (2) comprises subjecting the composition to peptide mass fingerprinting analysis to obtain XIC spectra of truncated peptide segments and full-length peptide segments, and calculating the content of the truncated variants according to the corresponding peak areas.

[0025] In some embodiments, the method further comprises combining the composition after the above step (2) with a pharmaceutically acceptable carrier.

[0026] In a fourth aspect, the present application provides a method for detecting aflibercept variants in a composition or pharmaceutical preparation containing aflibercept, wherein the variants are truncated variants of the VEGF-binding portion of aflibercept, and comprise 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:

[0027] Subjecting the composition or pharmaceutical preparation to subunit molecular weight analysis after digestion with IdeS enzyme, for example, using LC-MS method for subunit molecular weight analysis, and determining the presence or absence of the variant based on the molecular weight; or

[0028] The composition or pharmaceutical preparation is digested with trypsin and then subjected to peptide mass fingerprint analysis, for example, peptide mass fingerprint analysis using LC-MS / MS method, and the presence or absence of the variant is determined based on the first-order and second-order mass spectra.

[0029] In some embodiments, the above method further includes, when it is determined that the variant exists by performing peptide mass fingerprint analysis after digestion with trypsin, obtaining the XIC spectra of the truncated peptide segments and full-length peptide segments after peptide mass fingerprint analysis, and calculating the content of the truncated variant according to the corresponding peak areas.

[0030] In a fifth aspect, the present application provides a method for quality inspection or quality control of an aflibercept-containing product, which includes detecting the content of aflibercept variants in the aflibercept-containing product, wherein the variant is the truncated variant 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 some embodiments, if the detected content of the variant is less than or equal to about 20%, it indicates that the aflibercept product meets the pharmaceutical requirements.

[0032] In a sixth aspect, the present application provides the use of an aflibercept variant in quality inspection or quality control of an aflibercept-containing product, wherein the variant is the truncated variant 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.

[0033] In a seventh aspect, the present application provides a method for preparing the composition described in the first aspect, which includes:

[0034] Culturing cells capable of expressing aflibercept in a bioreactor, the culturing including a cell expansion stage and a protein production stage, wherein the culturing temperature in the protein production stage is basically maintained at 27.5°C - 37°C.

[0035] In some embodiments, the culturing temperature is maintained at 27.5°C - 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 in the protein production stage.

[0036] In an eighth aspect, the present application provides a method for preparing the composition described in the first aspect, which includes:

[0037] Cultivate cells capable of expressing aflibercept in a bioreactor, wherein the average daily addition concentration of asparagine during cultivation 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.

[0038] In a ninth aspect, the present application provides a method for preparing a composition comprising aflibercept, the method comprising the step of cultivating cells capable of expressing aflibercept in a bioreactor, characterized in that: the average daily addition concentration of asparagine during cultivation 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 cultivation comprises a cell expansion stage and a protein production stage, wherein the cultivation temperature in the protein production stage is substantially maintained at 27.5°C - 37°C.

[0039] In some embodiments, the cultivation temperature is maintained at 27.5°C - 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 stage.

[0040] In some embodiments, the composition comprises aflibercept and its variants, wherein the variant is a truncated variant of the VEGF-binding portion of aflibercept, and the truncated variant comprises 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.

[0041] In some embodiments, based on the calculation by trypsin digestion peptide mass fingerprint analysis, the content of the truncated variant in the composition is less than or equal to about 20% by mass percentage. Preferably, the content of aflibercept in the composition is greater than or equal to about 80% by mass percentage.

[0042] 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 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

[0044] Figure 1 Shows the sequence and schematic structure of aflibercept.

[0045] Figure 2 Shows the TIC chromatogram of the molecular weight analysis of the non-reduced subunits digested by IdeS enzyme.

[0046] Figure 3 Shows the deconvoluted chromatograms of peaks A( Figure 3 A), B( Figure 3 B) and C( Figure 3 C) in the TIC chromatogram of the molecular weight analysis of the non-reduced subunits digested by IdeS enzyme, where Figure 3 The Lys-loss marked in A is the deletion of the C-terminal lysine, which is a common post-translational modification of antibodies.

[0047] Figure 4 Shows the extracted ion current chromatogram( Figure 4 A), the first-order mass spectrum( Figure 4 B) and the second-order mass spectrum( Figure 4 C) of the truncated peptide segment of T100 in the aflibercept sample.

[0048] Figure 5 Shows the extracted ion current chromatograms of the truncated peptide segment and the full-length peptide segment in the aflibercept sample.

[0049] Sequence Listing

[0050] SEQ ID NO:1 is the amino acid sequence of two identical peptide chains of aflibercept, which is shown as follows (note: the bold part is the VEGFR-1 D2 domain, the underlined part is the VEGFR-2 D3 domain, and the italic part is the Fc segment):

[0051]

[0052] SEQ ID NO:2 is a truncated sequence of SEQ ID NO:1, lacking the first 99 amino acid residues at the N-terminus, and the specific sequence is shown as follows (note: the bold part is the VEGFR-1 D2 domain, the underlined part is the VEGFR-2 D3 domain, and the italic part is the Fc segment):

[0053]

[0054] SEQ ID NO:3 is the amino acid sequence of the long peptide chain in the T100 recombinant expression sequence, which is shown as follows (note: the residues shown in italics are the mutated amino acids):

[0055]

[0056]

[0057] SEQ ID NO:4 is the amino acid sequence of the short peptide chain in the T100 recombinant expression sequence, which is shown as follows (note: the residues shown in italics are the mutated amino acids):

[0058] Detailed implementation mode

[0059] Angiogenesis is crucial for normal embryonic and postnatal blood vessel 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 morphology, and increased vascular permeability. Elevated levels of VEGF have been found in the vitreous humor and retinal vasculature of patients with various eye diseases. Blocking VEGF activity has become the preferred therapy for treating eye diseases such as DME, wet AMD, CNV, and retinal vein occlusion.

[0060] Aflibercept is an anti-VEGF protein, a homodimer formed by two identical peptide chains linked by disulfide bonds, which contains a human amino acid sequence that includes the second Ig domain of human VEGFR-1 (VEGFR-1 D2) and the third Ig domain of human VEGFR-2 (VEGFR-2 D3). In a specific embodiment, the sequences of the two identical peptide chains of aflibercept are as shown in SEQ ID NO:1.

[0061] The inventors of the present application unexpectedly found that aflibercept variants were generated during the preparation of aflibercept, such as truncated forms of the peptide chain. The content of the variant being less than or equal to about 20% does not affect the binding activity and biological activity of the aflibercept sample. In other words, as long as the content of the aflibercept variant does not exceed about 20%, it will not reduce the binding activity and biological activity of aflibercept to VEGF. Therefore, if the variant of aflibercept does not exceed 20% after evaluation during the preparation of aflibercept, there is no need to remove the variant or impurities, which simplifies the production process to a certain extent and saves production costs.

[0062] In some embodiments, commercially available Eylea TM is used as a positive control to detect the relevant activities of the composition containing aflibercept and its variants, including binding activity to VEGF, biological activities such as inhibiting the proliferation of HUVEC cells, etc.

[0063] In some embodiments, the VEGF activity neutralization - HUVEC cell proliferation inhibition method is used to determine the biological activity of aflibercept in the compositions disclosed herein.

[0064] In a specific embodiment, the inventors identified the above - mentioned variant and found that the difference between it and aflibercept was only that 99 amino acid residues were deleted from the N - terminus of one of the peptide chains, while the other peptide chain was the same as SEQ ID NO:1. The sequence of the truncated peptide chain is shown as SEQ ID NO:2. Since the truncated peptide chain starts from the 100th threonine of SEQ ID NO:1, this variant is named "T100" herein.

[0065] In some embodiments, in the compositions containing aflibercept and its variants disclosed herein, the aflibercept variant is T100, and the content of variant T100 does not exceed 20%.

[0066] In some embodiments, the compositions described herein are composed of aflibercept and its variants (such as T100).

[0067] This document describes the use of cell culture medium to prepare aflibercept. In some embodiments, the cell culture medium is a chemically defined medium ("CDM").

[0068] In a specific embodiment, the cell culture medium used contains the following main components: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic 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, yeast extract.

[0069] 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.

[0070] In some embodiments, cells capable of expressing aflibercept are cultured in a bioreactor, and the culture includes a cell expansion stage and a protein production stage. Preferably, the culture temperature in the protein production stage is maintained substantially at about 27.5°C - 37°C.

[0071] In a specific embodiment, the culture temperature in the protein production stage is maintained substantially 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 - 37°C.

[0072] In some embodiments, the culture temperature is maintained at about 27.5°C - 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 stage.

[0073] The inventors of the present application unexpectedly found that the content of T100 in the aflibercept sample is affected by the culture temperature in the protein production stage. In a specific embodiment, as the temperature gradually decreases, the content of T100 gradually decreases.

[0074] In some embodiments, the average daily addition concentration of asparagine in the culture medium during the culture is 0.085 - 0.433 g / L / day. In a specific embodiment, the average daily addition concentration of asparagine 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 - 0.433.

[0075] In some embodiments, the average daily addition concentration of methionine in the culture medium during the culture period is 0.015 - 0.185 g / L / day. In a specific embodiment, the average daily addition concentration of methionine can be 0.185 g / L / day, or 0.015 g / L / day, or any other value between 0.015 and 0.185.

[0076] In a specific embodiment, the above-mentioned culture period is the entire culture period including the cell expansion stage and the protein production stage.

[0077] In some embodiments, the protein production stage refers to the process from the time of the first feeding in 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 to the end of the culture, or the process from the time of starting to perfusion the culture medium in perfusion 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 to the end of the culture.

[0078] In a specific embodiment, the calculation formula for the average daily addition concentration of asparagine is:

[0079]

[0080] The total amount of asparagine added as described above also includes the content of asparagine in the seed expansion culture medium.

[0081] In a specific embodiment, the calculation formula for the average daily addition concentration of methionine is:

[0082]

[0083] The total amount of methionine added as described above also includes the methionine content in the seed culture medium.

[0084] In a specific embodiment, the average daily addition concentration of asparagine is 0.433 g / L / day, and the average daily addition concentration of methionine is 0.185 g / L / day.

[0085] In a specific embodiment, the average daily addition concentration of asparagine is 0.433 g / L / day, and the average daily addition concentration of methionine is 0.015 g / L / day.

[0086] In a specific embodiment, the average daily addition concentration of asparagine is 0.166 g / L / day, and the average daily addition concentration of methionine is 0.185 g / L / day.

[0087] In a specific embodiment, the average daily addition concentration of asparagine is 0.166 g / L / day, and the average daily addition concentration of methionine is 0.015 g / L / day.

[0088] In a specific embodiment, the average daily addition concentration of asparagine is 0.085 g / L / day, and the average daily addition concentration of methionine is 0.185 g / L / day.

[0089] In a specific embodiment, the average daily addition concentration of asparagine is 0.085 g / L / day, and the average daily addition concentration of methionine is 0.015 g / L / day.

[0090] The inventors of the present application unexpectedly found that the content of T100 in the aflibercept sample is affected by the average daily addition concentrations of asparagine and methionine during the culture. In a specific embodiment, as the average daily addition concentrations of asparagine and methionine during the culture gradually decrease, the content of T100 gradually decreases.

[0091] In some embodiments, the purification process of aflibercept includes one or more steps selected from the following: affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, desalting chromatography, virus removal nanofiltration, and ultrafiltration concentration.

[0092] In some embodiments, the aflibercept sample is subjected to IdeS enzymatic cleavage non-reducing subunit molecular weight analysis. In a specific embodiment, after the IdeS enzymatic cleavage is completed, a liquid chromatography-mass spectrometry (LC-MS) method is used to perform subunit molecular weight analysis on the sample to be tested.

[0093] In the present application, the inventors unexpectedly discovered variant T100 during the process of performing subunit molecular weight analysis on the IdeS-cleaved aflibercept sample using the LC-MS method.

[0094] In some embodiments, variant T100 in the aflibercept product can be reduced or removed by methods such as ion exchange chromatography, hydrophobic chromatography, or composite chromatography.

[0095] In some embodiments, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis is used to detect variant T100 in the aflibercept product. For example, peptide mass fingerprinting analysis is performed on the sample to be tested using the LC-MS / MS method.

[0096] In a specific embodiment, after obtaining the mass spectrometry data, the extracted ion chromatograms (XIC) of the two charge forms with the strongest response of the truncated peptide segment and its corresponding full-length peptide segment are extracted, the peak areas are integrated respectively, and the content of T100 is calculated according to the following formula:

[0097]

[0098] In some embodiments, the inventors of the present application unexpectedly found that when the content of variant T100 in the prepared aflibercept sample is less than or equal to about 20%, the binding activity of aflibercept to VEGF is not weakened.

[0099] In some embodiments, the inventors of the present application unexpectedly found that when the content of variant T100 in the prepared aflibercept sample is less than or equal to about 20%, the biological activity of aflibercept is not reduced.

[0100] In some embodiments, the content of the variant (such as T100) in the composition comprising aflibercept and its variants disclosed herein is about 0.01% - about 20%, about 0.05% - about 20%, about 0.1% - about 20%, about 0.2% - about 20%, about 0.3% - about 20%, about 0.4% - about 20%, about 0.5% - about 20%, about 0.6% - about 20%, about 0.7% - about 20%, about 0.8% - about 20%, about 0.9% - about 20%, about 1% - about 20%, about 1.5% - about 20%, about 2% - about 20%, about 2.5% - about 20%, about 3% - about 20%, about 3.5% - about 20%, about 4% - about 20%, about 4.5% - about 20%, about 5% - about 20%, about 5.5% - about 20%, about 6% - about 20%, about 6.5% - about 20%, about 7% - about 20%, about 7.5% - about 20%, about 8% - about 20%, about 8.5% - about 20%, about 9% - about 20%, about 9.5 - about 20%, about 10% - about 20%, about 11% - about 20%, about 12% - about 20%, about 13% - about 20%, about 14% - about 20%, about 15% - about 20%, about 16% - about 20%, about 17% - about 20%, about 18% - about 20%, about 19% - about 20%, or any interval within any of the above ranges or any value within the said interval.

[0101] In a specific embodiment, the content of the variant (such as T100) in the composition comprising aflibercept and its variants disclosed herein is about 0.5% - about 10%, about 0.5% - about 9%, about 0.5% - about 8%, about 0.5% - about 7%, about 0.5% - about 6%, about 0.5% - about 5%, about 0.5% - about 4%, about 0.5% - about 3%, about 0.5% - about 2%, about 0.5% - about 1%, or any interval within any of the above ranges or any value within the said interval. For example, the content of the variant (such as T100) is 0.86% - 19.29%, preferably 0.86% - 2.59%.

[0102] The present disclosure also discloses a pharmaceutical preparation of the composition disclosed herein, that is, mixing the composition with an optional pharmaceutically acceptable carrier and storing it in the form of a freeze-dried preparation or an aqueous solution. The pharmaceutically acceptable carrier is non-toxic to the recipient at the doses and concentrations employed. In some embodiments, the pharmaceutically acceptable carrier includes, 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 preparation for in vivo administration must be sterile. This can be easily achieved by filtering through a sterile filter membrane.

[0103] In this specification and the claims, the term "about" or "substantially" refers to a range of values that a person 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.

[0104] In this specification and the claims, the words "comprise", "include" and "contain" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

[0105] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application can be applied to any other aspect, embodiment or example described herein, unless there is a contradiction.

[0106] The above disclosure generally describes the present invention, and the present invention is further exemplified by the following examples. These examples are described only to illustrate the present invention and not 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.

[0107] Examples

[0108] Example 1 Preparation Process of Aflibercept

[0109] Upstream cell culture process

[0110] The expression system used in this example is the Freedom TM CHO-STM Kit of Thermo Fisher Scientific, which contains the CHO-S host cell line for cGMP library construction and the pCHO1.0 expression plasmid. The expression vector is the pCHO1.0 plasmid, which can achieve the high-efficiency expression of exogenous recombinant proteins in mammalian cells. The production medium contains the following main components:

[0111] 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic 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 B 6 , vitamin B 2 , vitamin B 1 , vitamin B 12 , vitamin B, linoleic acid, block copolymer polyether F-68, yeast extract.

[0112] Referring to the typical cell culture process of antibody drugs, the production of aflibercept bulk solution is carried out according to the process of cell resuscitation, step-by-step expansion culture in shake flasks and bioreactors, and production in production tanks. The fed-batch culture process is adopted in the production stage.

[0113] Take a cell cryopreservation tube, thaw it in a 37°C water bath, and culture it in the shake flask expansion medium. After continuous amplification culture in the shake flask and bioreactor, inoculate the production tank for production.

[0114] Referring to the typical cell culture process of antibody drugs, appropriate process parameters such as culture temperature, pH, dissolved oxygen, stirring speed, and ventilation are set in the production stage. According to the cell growth needs, appropriate feeding medium, supplement solution, and glucose solution are added. According to the culture needs, defoaming agent solution or alkali solution is added. After the culture is completed, the cell culture solution is centrifuged and / or deeply filtered and sterilized by filtration, and the supernatant is collected for purification.

[0115] Downstream purification process

[0116] This example refers to the typical purification process of antibody drugs, including affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobic chromatography, desalting chromatography, virus removal nanofiltration and ultrafiltration concentration, and finally obtaining the stock solution of the target protein.

[0117] Affinity chromatography: After equilibrating the affinity chromatography column with a buffer solution containing salt (the salt is selected from Na 2 SO 4 , NaCl or (NH 4 )) 2 SO 4 ), the sample loading stage begins. After the sample loading is completed, continue to wash with the buffer solution containing salt, then wash with the buffer solution without salt, elute with the buffer solution with a pH of 3.0 - 4.5, collect the eluted protein solution, and then inactivate the virus.

[0118] Anion exchange chromatography: Pre - equilibrate the anion exchange chromatography column with a buffer solution containing salt (the salt is selected from Na 2 SO 4 , NaCl or (NH 4 )) 2 SO 4 ), then equilibrate with the buffer solution without salt. Load the protein solution after virus inactivation. After the sample loading is completed, continue to wash with the balance buffer solution without salt, then wash with the balance buffer solution containing 0.02 - 0.06 mol / L salt, and elute with the buffer solution containing 0.1 - 0.2 mol / L salt to elute the protein from the anion exchange chromatography column, and collect the eluted protein solution.

[0119] Cation exchange chromatography: Pre - equilibrate the cation exchange chromatography column with a buffer solution containing salt (the salt is selected from Na 2 SO 4 , NaCl or (NH 4 )) 2 SO 4 ), then equilibrate with the buffer solution without salt. Load the protein solution collected from the anion exchange chromatography. After the sample loading is completed, continue to wash with the balance buffer solution without salt, and then elute with the buffer solution containing 0.1 - 0.2 mol / L salt to elute the protein from the cation exchange chromatography column, and collect the eluted protein solution.

[0120] Hydrophobic chromatography: Use a buffer solution containing salt (the salt is selected from Na 2 SO 4 , NaCl or (NH 4 )) 2 SO 4The hydrophobic chromatography column was equilibrated with the buffer solution, and the protein solution collected by cation exchange chromatography was loaded. After the loading was completed, the column was continuously rinsed with the equilibration buffer solution containing salt, and then eluted with a buffer solution containing 0.3 - 0.6 mol / L salt to elute the protein from the hydrophobic chromatography column, and the eluted protein solution was collected.

[0121] Desalting chromatography: The desalting chromatography column was equilibrated with an equilibration buffer solution containing 0.02 - 0.06 mol / L salt. The eluted protein solution collected from hydrophobic chromatography was loaded onto the desalting chromatography column for desalting, and the desalted protein solution was collected. Then, the target protein stock solution was obtained through virus removal nanofiltration and ultrafiltration concentration.

[0122] Example 2 Discovery and Identification of T100 in Aflibercept Samples

[0123] Discovery of T100 in aflibercept sample

[0124] For the sequence and structure of aflibercept prepared in Example 1, see Figure 1 .

[0125] The IdeS enzyme digestion non-reducing subunit molecular weight analysis was performed on the sample prepared in Example 1. Since aflibercept contains 5 N-glycosylation modification sites and the heterogeneity caused by N-glycosylation is very serious, it cannot be measured without cleaving the N-glycan chains. Therefore, before performing the subunit molecular weight analysis, the N-glycan cleavage treatment was carried out using PNGase F. The steps of the IdeS enzyme digestion non-reducing subunit molecular weight analysis method are as follows:

[0126] Take 50 μg of the sample and dilute it to 2 μg / μL with IdeS enzyme digestion buffer (50 mM phosphate, 150 mM NaCl solution, pH 6.6). Add IdeS according to the ratio of protein:enzyme = 20:1 (mass ratio) and incubate at 37 °C for 1 h;

[0127] Take 20 μg of the sample after IdeS enzyme digestion, 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 min. After the incubation, add 2 μl of Rapid PNGase F (Non-reduction) and mix well, then incubate at 50 °C for 30 min for enzymatic digestion;

[0128] After the enzymatic digestion, centrifuge at 12000 rpm for 3 min, and transfer the supernatant to the injection vial;

[0129] The subunit molecular weight analysis of the sample to be tested was performed using liquid chromatography - mass spectrometry (LC-MS). The chromatographic conditions and mass spectrometry conditions used are as follows:

[0130] Chromatographic conditions:

[0131]

[0132] Mass spectrometry conditions:

[0133] MS system Xevo G2-XS QTof, Waters Ionization mode ESI positive Full MS scan range 500 - 4000 m / z Capillary voltage 2.7 kV Cone voltage 60V Ion source temperature 120℃ Desolvation gas temperature 400℃ Cone gas flow rate 50 L / h Desolvation gas flow rate 800 L / h

[0134] Data processing was performed using Waters UNIFI 1.8 software. The total ion current chromatogram (TIC) of the molecular weight analysis of the non-reduced subunits after IdeS digestion is shown in Figure 2 and the deconvoluted spectra of the main components are shown in Figure 3 .

[0135] After the sample was digested with IdeS, it was cleaved below the hinge region, forming the Fc / 2 and VEGFR regions. The results of the non-reduced subunit molecular weight showed that the peak at 9.07 min (peak A) in the TIC chromatogram was Fc / 2, and the peak at 10.55 min was the VEGFR region (peak C). The error between the measured molecular weight and the theoretical molecular weight after deconvolution was less than 5.0 Da, indicating that all the main components after IdeS digestion could be detected. There was a shoulder peak (peak B) before peak C. The measured molecular weight of the main component after deconvolution was 38141.0 Da. The retention time of peak B was close to that of peak C, and the molecular weight was about 11000 Da less than that of peak C. It was speculated that it might be related to the truncation of the VEGFR region. Combining with the sequence analysis of aflibercept, the theoretical molecular weight of the remaining components after truncating S1-N99 at the N-terminus of one chain in the VEGFR region was 38139.7 Da, which was close to the measured molecular weight of peak B.

[0136] Identification of T100 in aflibercept sample

[0137] To confirm the cleavage site, peptide mass fingerprint analysis was performed on the sample prepared in Example 1. The steps of the analysis method are as follows:

[0138] Take about 40 μg of the sample and add urea with a final concentration of 6 M and TCEP with a final concentration of 10 mM, and incubate at 37 °C for 60 min for denaturation and reduction;

[0139] Add IAM with a final concentration of 20 mM and incubate at 37 °C for 15 min to alkylate and block free sulfhydryl groups;

[0140] Add 50 mM ammonium bicarbonate buffer to dilute the urea concentration to 1 M, add trypsin according to the ratio of protein:enzyme = 20:1 (mass ratio), and digest at 37 °C for 25 min;

[0141] After the digestion was completed, add formic acid with a final concentration of 1% to terminate the digestion, centrifuge at 12000 rpm for 3 min, and transfer the supernatant to an injection vial;

[0142] The peptide mass fingerprint analysis of the sample to be tested was carried out by liquid chromatography-tandem mass spectrometry (LC-MS / MS). The chromatographic conditions and mass spectrometry conditions used are as follows:

[0143] Chromatographic conditions:

[0144]

[0145]

[0146] Mass spectrometry conditions:

[0147] MS system Q-Exactive Plus, Thermo Ionization mode ESI positive Full MS scan range 200 - 2000 m / z Capillary voltage 3.2 kV Capillary Temp 320℃ Full MS resolution 70000 AGC Target <![CDATA[1e 6 > MS / MS resolution 17500 AGC Target <![CDATA[1e 5 > Isolation window 1.6 m / z

[0148] Data processing was performed using pFind software. The extracted ion chromatogram (XIC) of the first-order mass spectrum, the first-order and second-order mass spectra of the truncated peptide are shown in Figure 4 .

[0149] The results of the peptide mass fingerprint showed that a truncated peptide QTN 99 TIIDVVLSPSHGIELSVGEK (the deleted amino acid residues are marked with a strikethrough) was found at a retention time of 44.3 min in the sample. The measured monoisotopic peak m / z of this peptide was 1047.0814, and the theoretical m / z was 1047.0781, with an error of only 3.15 ppm. At the same time, the fragment ions in the second-order mass spectrum could be well matched with the theoretical b and y ions of this peptide, thus confirming the amino acid sequence of this peptide.

[0150] Aflibercept is a homodimer formed by the connection of two identical peptide chains through disulfide bonds. Combining with the analysis results of the molecular weight of the IdeS enzyme digestion subunit, peak B was formed by the cleavage of the C-terminal peptide chain at the N99 site of one peptide chain, resulting in the deletion of the S1-N99 sequence in the VEGFR region. The measured molecular weight of peak B was 38141.0 Da, and the theoretical molecular weight of the remaining components after truncating the N-terminus S1-N99 of one chain in the VEGFR region was 38139.7 Da, further proving that the cleavage site was the N99 site of one peptide chain.

[0151] Content of T100 in the aflibercept sample in Example 3

[0152] The peptide mass fingerprint analysis of 6 batches of the samples prepared in Example 1 was carried out by LC-MS / MS method. After obtaining the mass spectrometry data, the XIC spectra of the two charge forms with the strongest responses of the truncated peptide TIIDVVLSPSHGIELSVGEK and its corresponding full-length peptide QTNTIIDVVLSPSHGIELSVGEK were extracted, and the peak areas of each were integrated, and the content of T100 was calculated according to the following formula:

[0153]

[0154] The sample pretreatment method and the LC-MS / MS detection method are as shown in Example 2, and Xcalibur software Qualbrowser is used for data processing.

[0155] Taking the mass spectrometry data of the sample in Batch 1 as an example, the XIC spectra of the truncated peptide segments and the full-length peptide segments are shown in Figure 5 .

[0156] Calculate the content of T100 in the sample of Batch 1 according to the formula, and it is obtained as 2.59% through the following calculation:

[0157]

[0158] Calculate the content of T100 in multiple batches of samples in the same way, and the results are shown in Table 1.

[0159] Table 1 Content of T100 in Multiple Batches of Samples

[0160]

[0161]

[0162] The results show that the content of T100 in the aflibercept sample prepared in Example 1 is 0.86% - 2.59%.

[0163] Quality Range of T100 in Aflibercept of Example 4

[0164] A comparative study on the binding activity and biological activity was carried out between the sample prepared in Example 1 and the commercially available Eylea TM .

[0165] The ELISA method was used to determine the binding ability of each sample to VEGF 165 protein. The determination method is as follows:

[0166] Dilute human VEGF 165 protein with coating buffer (50 mM NaHCO 3 -Na 2 CO 3 , pH 9.6) to 0.15 μg / ml, add 100 μl to each well in a 96-well plate, and coat overnight at 2 - 8 °C;

[0167] Block with 2% BSA-PBS for 2 hours, then add 100 μl of the sample gradient-diluted with 1% BSA-PBST to each well and incubate at 37 °C for 2 hours;

[0168] Then add HRP-labeled goat anti-human IgG Fc antibody to the 96-well plate;

[0169] Wash thoroughly with PBST between the above steps;

[0170] Finally, add TMB chromogenic solution and stop solution (2M H 2 SO 4 ), and use a microplate reader (TECAN's SPARK multimode microplate reader) to read the absorbance at 450 nm. With the protein concentration on the X-axis and the absorbance on the Y-axis, use the four-parameter method to fit the curve and calculate the results.

[0171] Using the sample of batch 6 prepared in Example 1 as the reference control (100%), the VEGF TM binding activities of the samples prepared in 6 batches and commercially available Eylea 165 were determined. The results are shown in Table 2.

[0172] Table 2 VEGF TM binding activity results of samples from each batch and commercially available Eylea 165

[0173]

[0174] The results showed that the VEGF TM binding activities of the samples from each batch and commercially available Eylea 165 were all between 80% and 120% of the control. Since the precision of the detection method for binding activity is generally ≤20%, the measured binding activities between 80% and 120% of the control are all within the method fluctuations, indicating that there is no difference in the VEGF TM binding activities between the aflibercept samples prepared in Example 1 and commercially available Eylea 165 .

[0175] The VEGF activity neutralization-HUVEC cell proliferation inhibition method was used to determine the biological activities of each sample. This method is based on the fact that VEGF can stimulate the proliferation of human umbilical vein endothelial cells (HUVEC), and the VEGFR:Fc fusion protein binds to VEGF, which can neutralize the VEGF activity and thus inhibit the proliferation of HUVEC cells, so as to detect the biological activity.

[0176] First, dilute the samples with the assay medium in a gradient manner. At the same time, dilute the VEGF protein to 210 ng / ml. Add the diluted VEGF protein solution and the diluted samples at each concentration gradient to a 96-well cell culture plate in sequence, 50 μl / well, and mix evenly in equal volumes, and incubate for 50 - 80 minutes;

[0177] Adjust the density of HUVEC cells to 1.6×10 5 ~2.4×10 5cells / ml, add 50 μl per well into a 96-well cell culture plate, and culture in an incubator at 37 °C with 5% CO2 for 65 - 70 hours;

[0178] After the incubation, take out the 96-well culture plate, add 30 μl of Alamar Blue staining solution to each well, incubate in an incubator at 37 °C for 6 hours, read the data with a fluorescence microplate reader (SpectraMax Gemini EM from Molecular Devices Corporation) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Using the protein concentration as the X-axis and the fluorescence value as the Y-axis, calculate the results by fitting the curve with the four-parameter method.

[0179] Using the sample of batch 6 prepared in Example 1 as the reference control (100%), the biological activities of the samples prepared in 6 batches and the commercially available Eylea TM were determined. The results are shown in Table 3.

[0180] Table 3 Biological activity results of samples of each batch and commercially available Eylea TM Biological activity results

[0181]

[0182] The results showed that the biological activities of the samples of each batch and the commercially available Eylea TM were all between 70% and 130% of the reference product. Since the method precision of the cell experiment is generally ≤ 30%, the measured binding activity between 70% and 130% of the reference product is within the method fluctuation range, indicating that there is no difference in the biological activities between the aflibercept samples prepared in Example 1 and the commercially available Eylea TM Biological activity results

[0183] The above results prove that the binding activity and biological activity of the aflibercept samples prepared in Example 1 are equivalent to those of the commercially available Eylea TM Biological activity results

[0184] Recombinant expression of T100 was carried out according to the identification results. If the wild-type Fc sequence was used for expression, it was expected that a protein sample mixed with 3 components would appear: both double-strands truncated, single-strand truncated (the target component), and both double-strands not truncated. To obtain a relatively pure single-strand truncated component, Knob-into-hole design was used for the recombinant expression of T100, and the sequence information is shown in Table 4.

[0185] Table 4 Recombinant expression sequence of T100

[0186]

[0187] Note: The italicized residues are the mutated amino acids

[0188] The plasmid was transfected into ExpiCHO-S cells by transient electroporation. The cells were cultured in a commercial medium according to the established culture process. After 10 days, the culture broth was harvested, filtered through a 0.22 μm pore size filter membrane, and then subjected to Protein A affinity chromatography to obtain the desired T100.

[0189] The SEC purity of the main peak of the transiently expressed T100 sample was 98.9%. The analysis results of the intact molecular weight of the N-glycan showed that the measured molecular weight was consistent with the theoretical value.

[0190] The prepared T100 was added to the samples prepared in Example 1 in gradients. The T100 content in each sample was detected, and the binding ability and biological activity of each sample to VEGF 165 protein were determined.

[0191] The T100 content and its activity of each sample are shown in Table 5.

[0192] Table 5 T100 content and its activity of each sample

[0193] Sample name T100 content (%) <![CDATA[Binding activity (%) 1,2 > <![CDATA[Biological activity (%) 1,2 > Spiked sample 1 6.22 96 92 Spiked sample 2 9.68 95 95 Spiked sample 3 12.51 87 88 Spiked sample 4 16.19 84 83 Spiked sample 5 19.29 80 75 Spiked sample 6 23.63 73 69

[0194] Note: 1: The batch 1 sample prepared in Example 1 was used as a reference control (100%).

[0195] 2: The results of the binding activity and biological activity are the means after 3 measurements.

[0196] The results showed that as the T100 content gradually increased, the binding activity and biological activity of the aflibercept samples gradually decreased. Therefore, during the preparation of aflibercept, it is necessary to control the T100 content.

[0197] In order to reduce the T100 content in the final preparation, the effects of adjusting some process parameters in the upstream cell culture stage (before downstream purification) on the production amount of T100 were explored below.

[0198] Effect of culture temperature in the protein production stage of Example 5 on T100 content

[0199] The cell culture process was referred to the method described in Example 1. The culture process included a 5-day cell expansion stage and a 7-day protein production stage in a bioreactor. Different temperatures shown in Table 6 were used for culture during the protein production stage. After the protein production stage ended, the culture broth was collected at one time and subjected to one-step proA affinity chromatography. The T100 content was directly detected according to the method described in Example 3. The T100 content obtained at different culture temperatures in the protein production stage is shown in Table 6:

[0200] Table 6 T100 content corresponding to different culture temperatures in the protein production stage

[0201] Culture temperature during protein production stage 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

[0202] The results showed that the culture temperature during the protein production stage affected the content of T100 in the aflibercept sample. As the temperature gradually decreased, the content of T100 gradually decreased. Temperatures between 27.5°C and 37°C during the protein production stage could control the content of T100 below 20%, and it was expected that the content of T100 could be further significantly reduced after the downstream purification process.

[0203] Example 6 Effect of average daily addition concentrations of asparagine and methionine on the content of T100

[0204] In the preliminary screening exploration of the effect of the amino acid component content in the cell culture medium on the content of T100, it was found that the amounts of asparagine and methionine were potential influencing factors. Therefore, a content gradient experiment was further designed to verify this finding.

[0205] The cell culture process referred to the methods described in Examples 1 and 5, where the culture process included a 5-day cell expansion stage and a 7-day protein production stage in a bioreactor. The protein production stage was carried out at 32°C ± 0.5°C. After the protein production stage ended, the culture broth was collected at one time, and one-step proA affinity chromatography was performed, and the content of T100 was directly detected according to the method described in Example 3.

[0206] In this example, the average daily addition concentration was used to characterize the dosages of asparagine and methionine.

[0207] Calculation formula for the average daily addition concentration of asparagine:

[0208]

[0209] Calculation formula for the average daily addition concentration of methionine:

[0210]

[0211] The "total addition amount" in the above formula simply refers to the cumulative amount of asparagine or methionine added to the bioreactor in any form during the entire process of bioreactor culture. Specifically, the total amount includes the amount of asparagine or methionine already contained in the cell culture (which may be 0) when the cell culture is inoculated into the bioreactor, the amount of asparagine or methionine already added to the bioreactor in advance (which may be 0), the amount of asparagine or methionine in the feeding medium during the entire culture process, and the amount of asparagine or methionine added in other forms.

[0212] The influence of the average daily addition concentration of asparagine and methionine on the T100 content is shown in Table 7 below. The results show that the content of T100 in the aflibercept sample is affected by the average daily addition concentration of asparagine and methionine, and it shows that as the average daily addition concentration of asparagine and / or methionine gradually decreases during the cultivation period, the T100 content gradually decreases. Each average daily addition concentration of asparagine and methionine shown in Table 7 can control the T100 content below 10%, and it is expected that the T100 content can be further significantly reduced after the downstream purification process.

[0213] Table 7 Average daily addition concentration of asparagine and methionine and T100 content

[0214]

[0215] Without departing from the spirit and scope of the disclosure of the present application, various changes and equivalent substitutions can be made to the various embodiments disclosed in the present application. Unless otherwise specified in the context, any feature, step or embodiment of the embodiments of the present disclosure can be combined 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 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, wherein the content of the truncated variant in the composition is less than or equal to 20% by mass percentage based on trypsin-cleaved peptide mass fingerprint 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%. The composition of claim 3 , wherein the content of the truncated variant is 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 culturing temperature in 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 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.

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 stage and a protein production stage, and the culture temperature in the protein production stage 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-37°C for at least 50% of the time during the protein production phase.

10. The composition of claim 10, wherein the culture temperature is maintained at 27.5°C-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 culturing temperature in the protein production phase is maintained at 27.5°C-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 time during 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 a composition according to any one of claims 1 to 5, comprising 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.

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 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 phase and a protein production phase, wherein the culture temperature in the protein production phase is maintained at 27.5°C-37°C.

16. The method of claim 15, wherein 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 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.

17. The method of claim 16, 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.

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

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

20. The method of claim 19, wherein the content of the truncated variant is greater than or equal to 2%.

21. The method of claim 17, wherein the truncated variant is present at less than or equal to about 5%.

22. The method of any one of claims 15 to 21, wherein the culture temperature is maintained at 27.5°C to 37°C for at least 50% of the time during the protein production phase.

23. The method of claim 22, 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.

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

25. 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, wherein the truncated variant comprises 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, 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 method to determine whether the variant exists based on the molecular weight; or The composition or pharmaceutical preparation is digested with trypsin and then subjected to peptide mass fingerprint analysis, and peptide mass fingerprint analysis is performed using LC-MS / MS method to determine the presence or absence of the variant based on primary and secondary mass spectra.

26. The method of claim 25, further comprising, when the presence of the variant is determined by peptide mass fingerprint analysis after trypsin cleavage, obtaining XIC spectra of truncated peptides and full-length peptides after peptide mass fingerprint analysis, and calculating the content of the truncated variant based on the corresponding peak areas.

27. 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, and the truncated variant comprises 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.

28. The method of claim 27, wherein if the detected content of the aflibercept variant is less than or equal to 20%, it indicates that the aflibercept product meets the pharmaceutical requirements.

29. Use of an aflibercept variant in quality inspection or quality control of a product containing aflibercept, wherein the aflibercept variant is a truncated variant of the VEGF binding portion of aflibercept, and the truncated variant comprises 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.

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