Compositions containing sotexip and its variants, as well as related methods and applications

Abstract

This application provides compositions comprising sotexip and its variants, as well as related methods and applications, wherein the variants involve a peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or both monomeric peptide chains of sotexip, and the content of the variant is less than or equal to about 10% based on peptide mass fingerprinting analysis. This application also provides pharmaceutical formulations comprising the above compositions and methods for preparing the same. Furthermore, this application provides methods for detecting the above variants and their application in quality inspection or quality control of products containing sotexip.

Description

Technical Field

[0001] This application relates to the field of biopharmaceuticals. Specifically, this application provides compositions comprising sotexip and its variants, as well as related methods and applications. Background Technology

[0002] Bone diseases, including osteoporosis and fractures, represent a group of pathological conditions for which very few drug formulations are effective. Treatment focuses on physical and behavioral interventions, including stabilization, exercise, and dietary changes. Methods controlling osteoclast and osteoblast activity are effective in promoting the healing of fractures and other bone injuries, as well as in treating diseases such as osteoporosis, which is associated with bone loss and bone mineralization.

[0003] For osteoporosis, estrogen, calcitonin, osteocalcin, and vitamin K, or high doses of dietary calcium, are used as interventional treatments. Other treatment options for osteoporosis include bisphosphonates, parathyroid hormone, calcimimetic agents, statins, lanthanum and strontium salts, and sodium fluoride. However, these treatments often produce adverse side effects.

[0004] Pulmonary arterial hypertension (PAH) is a severe and progressive disease characterized by abnormal constriction, remodeling (thickening of the vessel wall, abnormal cell proliferation), and occlusion of pulmonary arterioles. This leads to increased pulmonary vascular resistance, increased right ventricular load, and ultimately right ventricular failure and death. In healthy pulmonary vessels, the BMPR-II signaling pathway (primarily via Smad1 / 5 / 8 phosphorylation) is crucial for maintaining the homeostasis of vascular endothelial cells and smooth muscle cells. It has anti-proliferative and pro-apoptotic effects, inhibiting excessive growth and migration of vascular cells. When BMPR-II signaling is weakened or absent (e.g., due to gene mutations or functional inhibition), this inhibition is diminished, leading to excessive proliferation and increased survival of pulmonary vascular cells, promoting vascular remodeling and PAH development. A key pathological mechanism is dysfunction of the bone morphogenetic protein receptor type II (BMPR-II) signaling pathway. A large number of hereditary and sporadic PAH patients have BMPR2 gene mutations or impaired downstream signaling.

[0005] The soluble form of ActRIIA (activin receptor IIA) acts as an inhibitor of activin-ActRIIA signaling and promotes increased bone density, bone growth, and bone strength in vivo. However, most pharmaceutical formulations that promote bone growth or inhibit bone loss are either anti-catabolic or anabolic agents. The soluble ActRIIA protein exhibits dual activity, possessing both catabolic and anabolic effects. Therefore, antagonists of the activin-ActRIIA signaling pathway can be used to increase bone density and promote bone growth.

[0006] On the other hand, given that impaired BMPR-II signaling is one of the core pathological mechanisms of PAH, and that overactive ligands such as activin / GDF can further suppress the already fragile BMP signaling, scientists have hypothesized that by capturing these inhibitory ligands, the balance of the BMP signaling pathway can be restored, thereby inhibiting the process of pulmonary vascular remodeling.

[0007] This mechanism of action differs from existing PAH-targeting drugs (such as endothelin receptor antagonists, phosphodiesterase-5 inhibitors, prostacyclin drugs, and guanylate cyclase stimulants), which primarily act on vasomotor tension (vasodilation). Sotecept, on the other hand, directly targets the cell proliferation signaling pathway that leads to abnormal vascular remodeling, possessing the potential for "disease modification."

[0008] Sortexip is an activin receptor IIA-Fc fusion protein. It works by targeting and blocking signaling pathways of the TGF-β superfamily, such as activins and growth differentiation factors. In PAH, this signaling pathway is overactive, leading to abnormal thickening and blockage of the vessel wall. Sortexip aims to restore the balance of vascular wall cell growth, thereby improving the health of pulmonary vessels.

[0009] Furthermore, sotexip can dually regulate bone metabolism: it has both anti-catabolic effects, inhibiting bone loss, and promoting anabolism, accelerating bone growth, significantly improving bone density, bone strength, and fracture healing ability. Its unique mechanism differs from traditional anti-osteoporosis drugs, such as bisphosphonates / PTH, and it does not increase muscle mass, making it suitable for the treatment of osteoporosis and bone repair.

[0010] As a therapeutic drug, the safety and efficacy of soteccept highly depend on maintaining its stability and effectiveness throughout its lifespan. Therefore, quality control of its pharmaceutical composition is crucial, focusing on ensuring that the content of the active ingredient meets the prescription specifications and strictly controlling the content of related substances such as variants or impurities to meet pharmaceutical regulatory requirements, thereby ultimately ensuring patient safety and treatment outcomes. Summary of the Invention

[0011] In a first aspect, this application provides a composition comprising sotexip and variants thereof, wherein the variants have a peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or two monomeric peptide chains of sotexip as shown in SEQ ID NO:2, and the percentage of monomeric peptide chains with peptide bond cleavage between Arg(4)-Ser(5) in the composition is less than or equal to about 10% of the total number of monomeric peptide chains in the composition, calculated based on peptide mass fingerprinting analysis, i.e., the peptide bond cleavage rate between Arg(4)-Ser(5) is less than or equal to about 10%.

[0012] In a specific implementation, the sequence of the truncated monomeric peptide chain generated after the peptide bond cleavage between Arg(4)-Ser(5) is shown in SEQ ID NO:1.

[0013] The truncated variant resulting from the peptide bond cleavage between Arg(4) and Ser(5) is referred to in this paper as R. 4 / S 5 Truncated variant.

[0014] In a specific implementation plan, based on peptide quality fingerprint analysis and measured by LC-MS / MS, the peptide bond breakage rate is less than or equal to about 10%.

[0015] In a specific implementation scheme, based on the peptide quality fingerprint analysis method, the percentage of monomeric peptide chains in the composition that have not undergone peptide bond cleavage between Arg(4) and Ser(5) is greater than or equal to about 90%, that is, the peptide bond cleavage rate between Arg(4) and Ser(5) is greater than or equal to about 90%.

[0016] In a specific implementation, the peptide bond cleavage rate between Arg(4) and Ser(5) 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%.

[0017] In specific embodiments, the peptide bond cleavage rate between Arg(4) and Ser(5) is 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 peptide bond cleavage rate between Arg(4) and Ser(5) can be any interval within the range defined by any two of the values ​​mentioned above, or any value within the interval.

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

[0019] The host cells containing a nucleic acid molecule or its vector encoding an amino acid sequence as shown in SEQ ID NO:2 are cultured in a bioreactor, the culture comprising a cell expansion phase and a protein production phase, wherein the culture temperature of the protein production phase is maintained substantially between 29.5°C and 37°C.

[0020] In some embodiments, the composition is prepared by a method comprising the following steps:

[0021] Host cells containing a nucleic acid molecule or its vector encoding an amino acid sequence as shown in SEQ ID NO:2 are cultured in a bioreactor, wherein the average daily concentration of iron ions (e.g., ferric citrate) added during culture is from 1.5 μmol / L / day to 125 μmol / L / day.

[0022] In a second aspect, this application provides a method for preparing the composition described in the first aspect, the method comprising culturing host cells in a bioreactor containing a nucleic acid molecule encoding an amino acid sequence as shown in SEQ ID NO:2 or a vector containing the nucleic acid molecule, the culturing comprising a cell expansion phase and a protein production phase, wherein the protein production phase is maintained at a temperature substantially between 29.5°C and 37°C, and / or using a culture medium supplemented with iron ions.

[0023] In a specific implementation, the sotexip variant is a truncated variant formed by breaking the Arg(4)-Ser(5) peptide bond at the N-terminus of at least one monomeric peptide chain of sotexip, and the amino acid sequence of the monomeric peptide chain formed by breaking the Arg(4)-Ser(5) peptide bond at the N-terminus is shown in SEQ ID NO: 1.

[0024] In a specific implementation plan, the iron ions are derived from one or more of the following: ferric nitrate, ferrous sulfate, ferric citrate, ferric ethylenediaminetetraacetate, and transferrin-bound iron.

[0025] In a specific implementation, the iron ions are derived from ferric citrate.

[0026] In a specific implementation plan, the average daily concentration of iron ions (e.g., ferric citrate) added during the cultivation period is from 1.5 μmol / L / day to 125 μmol / L / day.

[0027] In a specific implementation, the average daily concentration of iron ions (e.g., ferric citrate) is 1.50 μmol / L / day to 5.00 μmol / L / day, 5.01 μmol / L / day to 15.00 μmol / L / day, 15.01 μmol / L / day to 30.00 μmol / L / day, 30.01 μmol / L / day to 45.00 μmol / L / day, 45.01 μmol / L / day to 60.00 μmol / L / day, 60.01 μmol / L / day to 75.00 μmol / L / day, 75.01 μmol / L / day to 90.00 μmol / L / day, 90.01 μmol / L / day to 115.00 μmol / L / day, or 115.01 μmol / L / day to 125.00 μmol / L / day.

[0028] In a specific implementation, the average daily concentration of iron ions (e.g., ferric citrate) is selected from 1.77 μmol / L / day, 7.88 μmol / L / day, 62.85 μmol / L / day, 93.39 μmol / L / day, or 123.93 μmol / L / day.

[0029] In a specific implementation, the culture temperature is maintained at approximately 29.5°C or higher and below or equal to 37°C (i.e., 29.5°C-37°C).

[0030] In a specific implementation, the culture temperature is maintained at at least 29.5°C, at least 30°C, at least 30.5°C, at least 31°C, at least 31.5°C, at least 32°C, at least 32.5°C, at least 33°C, at least 33.5°C, at least 34°C, at least 34.5°C, at least 35°C, at least 35.5°C, at least 36°C, at least 36.5°C, or at least 37°C.

[0031] In a specific implementation plan, the protein production stage is maintained at 29.5°C-37°C for at least 50% to 100% of the time.

[0032] In a specific implementation plan, the culture temperature is maintained at 29.5°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.

[0033] In the specific implementation plan, the protein production stage is maintained at 29.5°C-37°C for approximately 95% of the time.

[0034] In a specific implementation, the host cell is a mammalian cell.

[0035] In a specific implementation plan, the mammalian cells are derived from mice or humans.

[0036] In specific implementation schemes, the human-derived cells include, but are not limited to: HEK 293 and PER.C6.

[0037] In specific implementations, the cells derived from mice include, but are not limited to: CHO, Hybridoma, YB2 / O, NSO, Sp2 / O, and BHK.

[0038] In a specific implementation scheme, the host cell is a CHO cell.

[0039] Thirdly, this application provides pharmaceutical formulations comprising the compositions described in the first aspect or the compositions prepared according to the methods described in the second aspect, and one or more pharmaceutically acceptable excipients.

[0040] Fourthly, this application provides the use of the composition described in the first aspect or the composition prepared according to the method described in the second aspect in the preparation of a medicament for treating pulmonary hypertension or bone diseases.

[0041] Fifthly, this application provides a method for treating pulmonary hypertension or bone disease, comprising administering to a subject or patient in need a therapeutically effective amount of the composition described in the first aspect, or a composition prepared according to the method described in the second aspect, or a pharmaceutical preparation described in the third aspect.

[0042] In a sixth aspect, this application provides the use of iron ions in reducing the content of sotexip variants in sotexip products, wherein the variant has a peptide bond break between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) in the amino acid sequence (SEQ ID NO:2) of one or two monomeric peptide chains of sotexip, and the sequence of at least one monomeric peptide chain of the variant is shown in SEQ ID NO:1.

[0043] In a preferred embodiment, the iron ions are derived from ferric citrate.

[0044] Seventhly, this application provides a method for detecting sotexip variants in products containing sotexip, wherein the variant has a peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of the amino acid sequence (SEQ ID NO:2) of one or two monomeric peptide chains of sotexip, and the sequence of at least one monomeric peptide chain of the variant is shown in SEQ ID NO:1, the method comprising:

[0045] The product was digested with lysyl endonuclease (Lys-C) to obtain the digested product; and

[0046] The enzyme digestion products are subjected to peptide mass fingerprint analysis, for example, using LC-MS / MS, to determine the presence or absence of the variant based on primary and secondary mass spectra.

[0047] In some embodiments, the above method further includes, when the presence of the variant is determined, obtaining XIC spectra of the truncated peptide and the full-length peptide after peptide mass fingerprinting analysis, and calculating the content of the truncated variant based on the corresponding peak areas.

[0048] Eighthly, this application provides a method for quality inspection or quality control of products containing sotexip, comprising detecting the content of a sotexip variant in the sotexip-containing product, wherein the variant has a peptide bond break between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) in the amino acid sequence (SEQ ID NO:2) of one or two monomeric peptide chains of sotexip, and the sequence of at least one monomeric peptide chain of the variant is shown in SEQ ID NO:1.

[0049] In some embodiments, the sotexip-containing product is the composition described in the first aspect or the pharmaceutical preparation described in the third aspect.

[0050] In some implementations, if the content of the variant is detected to be less than or equal to about 10%, it indicates that the sotexip product meets pharmaceutical requirements.

[0051] Ninthly, this application provides the application of sotexip variants in the quality inspection or quality control of sotexip-containing products, wherein the variant has a peptide bond break between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) in the amino acid sequence (SEQ ID NO:2) of one or two peptide chains of sotexip, and the sequence of at least one monomeric peptide chain of the variant is shown in SEQ ID NO:1.

[0052] In some embodiments, the sotexip-containing product is the composition described in the first aspect or the pharmaceutical preparation described in the third aspect. Attached Figure Description

[0053] Figure 1 A schematic diagram of the fracture site of the Sotschip and Sotschip variants.

[0054] Figure 2 The unconvolution spectrum of the main components in the total ion chromatogram for complete molecular weight analysis of sotecip.

[0055] Figure 3In the image, A is the extracted ion chromatogram of the primary mass spectrometry of the sotracyp variant, B is the primary mass spectrum of the sotracyp variant, and C is the secondary mass spectrum of the sotracyp variant.

[0056] Figure 4 This is a summary graph showing the effects of culture temperature and the average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride on the content of truncated variants during the protein production stage.

[0057] Figure 5 This is a trend graph showing the effects of culture temperature and the average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride on the content of truncated variants during the protein production stage.

[0058] Figure 6 R under different culture temperatures and average daily concentrations of ferric citrate during the protein production stage 4 / S 5 Graph showing the content variation of the truncated variant.

[0059] Figure 7 R under different culture temperature conditions during the protein production stage 4 / S 5 Graph showing the content variation of the truncated variant.

[0060] Figure 8 R under different average daily concentrations of ferric citrate 4 / S 5 Graph showing the content variation of the truncated variant.

[0061] Figure 9 Image A shows the affinity binding-dissociation curve fitting of the sotasia soteric sample with 0.33% R4 / S5 truncated variant to Activin A; image B shows the affinity binding-dissociation curve fitting of the sotasia soteric sample with 3.85% R4 / S5 truncated variant to Activin A; and image C shows the affinity binding-dissociation curve fitting of the sotasia soteric sample with 8.58% R4 / S5 truncated variant to Activin A.

[0062] Sequence Description

[0063] SEQ ID NO:1 is a truncated sequence of SEQ ID NO:2, in which an Arg(4)-Ser(5) peptide bond cleavage occurs at the N-terminus, resulting in the deletion of four amino acid residues: isoleucine, leucine, glycine, and arginine. The specific sequence is shown below:

[0064] SETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPV PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0065] SEQ ID NO:2 is the amino acid sequence of the monomeric peptide chain of sotexip, as shown below:

[0066] ILGR SETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTSNPVTPKPP TGGGTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Detailed Implementation

[0067] Sotatercept (CAS No. 1001080-50-7) is a homodimer formed by two identical monomeric peptide chains linked by disulfide bonds. Its monomeric peptide chain is a fusion peptide chain of the ActRIIA extracellular binding domain and the human Fc fragment. This fusion peptide comprises two parts: one is the extracellular domain of human activin receptor type IIA (ActRIIA), used to "capture" specific ligands; the other is the Fc fragment of human immunoglobulin G1 (IgG1), used to prolong the duration of drug action in vivo. The ActRIIA extracellular binding domain and the Fc fragment are fused to form the polypeptide sequence shown in SEQ ID NO:2. Specifically, sotatercept is a homodimer formed by two monomeric peptide chains shown in SEQ ID NO:2 linked by disulfide bonds between cysteine ​​residues of the Fc fragment.

[0068] The inventors of this application unexpectedly discovered that sotexip produces variants during its preparation, such as truncated variants formed by peptide bond cleavage. At least one polypeptide chain of these truncated variants exhibits peptide bond cleavage between the 4th and 5th amino acid residues (Arg(4)-Ser(5)) corresponding to the sequence shown in SEQ ID NO:2, i.e., R... 4 / S 5Truncated variants. The inventors unexpectedly discovered that a peptide bond breakage of less than or equal to approximately 10% between Arg(4) and Ser(5) did not affect the binding and biological activity of sotexip samples. In other words, as long as the peptide bond breakage between Arg(4) and Ser(5) does not exceed approximately 10%, the binding and biological activity of sotexip will not be reduced. Therefore, if the peptide bond breakage rate between Arg(4) and Ser(5) is assessed to be no higher than 10% during the preparation of sotexip, there is no need to perform a process to remove variants or impurities, which simplifies the production process and saves production costs to some extent.

[0069] In a specific implementation, this application identified the aforementioned sotexip variant and found that it differs from sotexip in that at least one of its polypeptide chains has an Arg(4)-Ser(5) peptide bond cleavage at the N-terminus, resulting in the loss of four amino acid residues: isoleucine, leucine, glycine, and arginine. The truncated peptide chain sequence is shown in SEQ ID NO:1. In this document, it is also referred to as R... 4 / S 5 Truncated variant.

[0070] In some embodiments, the composition described herein is composed of Sotexip and R 4 / S 5 The truncated variant composition has a peptide bond breakage rate of no more than 10% between Arg(4) and Ser(5).

[0071] In this article, the terms “peptide bond cleavage rate between Arg(4) and Ser(5),” “percentage of peptide bond cleavage between Arg(4) and Ser(5),” and “content of truncated variants” are used interchangeably and are calculated using the same method. They all refer to the percentage of monomeric peptide chains in the composition that have undergone peptide bond cleavage between Arg(4) and Ser(5) as a percentage of the total number of monomeric peptide chains in the composition, calculated based on peptide quality fingerprint analysis.

[0072] In some implementations, liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis is used to detect R in sotecip products. 4 / S 5 Truncated variants. For example, LC-MS / MS is used to perform peptide mass fingerprinting analysis on the test sample.

[0073] In a specific implementation plan, the method for calculating the peptide bond cleavage rate between Arg(4) and Ser(5) based on peptide mass fingerprint analysis includes: after enzyme digestion of the sample, centrifugation, collection of the supernatant, and peptide mass fingerprint analysis of the sample using liquid chromatography-tandem mass spectrometry (LC-MS / MS). After obtaining the mass spectrometry data, the extractable ion current chromatograms (XICs) of the two charge forms with the strongest response to the truncated peptide and its corresponding full-length peptide are extracted, the peak areas of each are integrated, and R is calculated according to the following formula. 4 / S 5 Content of truncated variants (i.e., peptide bond breakage rate between Arg(4) and Ser(5):

[0074]

[0075] In a more specific implementation scheme, the method for calculating the peptide bond cleavage rate between Arg(4) and Ser(5) based on peptide mass fingerprint analysis includes: the peptide bond cleavage rate between Arg(4) and Ser(5) is detected and calculated using the following method: the sample is denatured by urea and reduced by TCEP, then alkylated by IAM, and after adding ammonium bicarbonate buffer, lysine protease is added at a protein:enzyme ratio of 20:1 (mass ratio), and the enzyme is digested at 37℃ for 15 h. After the enzyme digestion is completed, formic acid with a final concentration of 1% is added to terminate the enzyme digestion. The sample is centrifuged at 12000 rpm for 3 min, and the supernatant is transferred to a sample vial. The sample is analyzed by peptide mass fingerprint analysis using liquid chromatography-tandem mass spectrometry (LC-MS / MS). After obtaining the mass spectrometry data, the extractable ion chromatograms (XIC) of the truncated peptide segment and its corresponding full-length peptide segment with the strongest two charge forms are extracted, the peak areas of each segment are integrated, and R is calculated according to the following formula. 4 / S 5 Content of truncated variants (i.e., peptide bond breakage rate between Arg(4) and Ser(5):

[0076]

[0077] In specific implementations, sotexip is expressed in suitable host cells. Non-limiting examples of such host cells include, but are not limited to, CHO, DUK-B11, NSO, Sp2 / O, BHK, HEK 293, and BHK.

[0078] In the specific implementation plan, the CHO cells used are selected from CHO-S, CHOZN, CHO K1, CHO DG44, etc.

[0079] In some embodiments, cells capable of expressing sotexip are cultured in a bioreactor, the culture including a cell expansion phase and a protein production phase. Preferably, the culture temperature during the protein production phase is maintained substantially between about 29.5°C and 37°C.

[0080] In a specific implementation plan, the protein production stage refers to the time of the first feeding in fed-batch culture at ±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 from the start of perfusion culture to the end of the culture, or the time from the start of perfusion culture to the end of the culture 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 until the end of the culture.

[0081] The inventors of this application have discovered that the content of variants in sotsip samples is affected by the culture temperature during the protein production stage.

[0082] In a specific implementation, the culture temperature during the protein production stage is maintained at 29.5°C or higher and below or equal to 37°C (i.e., 29.5°C-37°C).

[0083] In specific implementations, the culture temperature is maintained substantially at, for example, 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 29.5°C and 37°C.

[0084] In a specific implementation plan, the protein production stage is maintained at 29.5°C-37°C for at least 50% to 100% of the time.

[0085] In a specific implementation plan, the culture temperature is maintained at 29.5°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.

[0086] In a specific implementation plan, the protein production stage is maintained at 29.5℃-37℃ for 95% of the time.

[0087] In the specific implementation plan, the culture temperature during the protein production stage is selected from values ​​such as 30℃, 32℃, 34℃, and 36.5℃.

[0088] In the specific implementation plan, as the culture temperature during the protein production stage gradually decreases, the content of truncated variants gradually decreases.

[0089] In some embodiments, the inventors of this application have found that the content of variants in Sotsip samples is affected by iron ions, such as ferric citrate, in the culture medium.

[0090] In the specific implementation plan, the average daily concentration of iron ions added to the culture medium during the cultivation period is 1.5 μmol / L / day to 125 μmol / L / day.

[0091] In the specific implementation plan, the above-mentioned culture period includes the entire culture period, including the cell expansion phase and the protein production phase.

[0092] In a specific implementation plan, if ferric citrate is used as the source of iron ions, then the exemplary formula for calculating the average daily addition concentration is as follows:

[0093]

[0094] In simple terms, the "total amount added" in the formula above refers to the cumulative amount of ferric citrate added to the bioreactor in any form throughout the entire culture process. Specifically, the total amount includes the amount of ferric citrate already present in the cell culture when it is inoculated into the bioreactor (which may also be zero), the amount of ferric citrate already added to the bioreactor beforehand (which may also be zero), the amount of ferric citrate in the feed medium throughout the entire culture process, and the amount of ferric citrate added in other forms.

[0095] In a specific implementation scheme, the average daily concentration of iron ions is 1.50 μmol / L / day to 5.00 μmol / L / day, 5.01 μmol / L / day to 15.00 μmol / L / day, 15.01 μmol / L / day to 30.00 μmol / L / day, 30.01 μmol / L / day to 45.00 μmol / L / day, 45.01 μmol / L / day to 60.00 μmol / L / day, 60.01 μmol / L / day to 75.00 μmol / L / day, 75.01 μmol / L / day to 90.00 μmol / L / day, 90.01 μmol / L / day to 115.00 μmol / L / day, or 115.01 μmol / L / day to 125.00 μmol / L / day, or any interval within any of the above ranges or any value within the intervals.

[0096] In a specific implementation plan, the average daily concentration of iron ions is selected from 1.77 μmol / L / day, 7.88 μmol / L / day, 62.85 μmol / L / day, 93.39 μmol / L / day, and 123.93 μmol / L / day.

[0097] In the specific implementation plan, after equilibrating the affinity chromatography column with a salt-containing buffer, the sample loading stage begins. After sample loading, the column is washed with a salt-containing buffer, then with a salt-free buffer, and finally eluted with a buffer at pH 3.0–4.0, collecting the eluted proteins.

[0098] In specific embodiments, the compositions disclosed herein containing sotexip and its variants contain variants in amounts of about 0.01% to about 10%, about 0.05% to about 10%, about 0.1% to about 10%, about 0.2% to about 10%, about 0.3% to about 10%, about 0.4% to about 10%, about 0.5% to about 10%, about 0.6% to about 10%, about 0.7% to about 10%, about 0.8% to about 10%, about 0.9% to about 10%, about 1% to about 10%, about 1.5% to about 10%, about 2 %-approximately 10%, approximately 2.5%-approximately 10%, approximately 3%-approximately 10%, approximately 3.5%-approximately 10%, approximately 4%-approximately 10%, approximately 4.5%-approximately 10%, approximately 5%-approximately 10%, approximately 5.5%-approximately 10%, approximately 6%-approximately 10%, approximately 6.5%-approximately 10%, approximately 7%-approximately 10%, approximately 7.5%-approximately 10%, approximately 8%-approximately 10%, approximately 8.5%-approximately 10%, approximately 9%-approximately 10%, approximately 9.5%-approximately 10%, or any interval within any of the above ranges or any value within the intervals.

[0099] In a more specific implementation, the content of the variant is 0.3%-9%, for example 0.33%-8.58%, or any interval within any of the above ranges or any value within the intervals.

[0100] This document also discloses pharmaceutical formulations for preparing the compositions disclosed herein, namely, mixing the compositions with optional pharmaceutically acceptable excipients and storing them as lyophilized formulations or aqueous solutions. Pharmaceutically acceptable excipients are non-toxic to recipients at the dosage and concentration used. Pharmaceutically acceptable excipients include pharmaceutically acceptable carriers and / or excipients.

[0101] In some implementations, pharmaceutically acceptable carriers and / or excipients include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, and / or disintegrants. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water; the binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, carboxymethylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropylcellulose, and hydroxypropyl methylcellulose; the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; the humectants include, but are not limited to, glycerol and starch; the adsorbents include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay; and the lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate. In some implementations, the pharmaceutical preparation does not contain pyrogens.

[0102] In some implementations, the pharmaceutical formulations disclosed in this application include gastrointestinal dosage forms and non-gastrointestinal dosage forms.

[0103] In specific implementation schemes, the gastrointestinal drug delivery dosage forms include solutions, drops, tablets, capsules, granules, films, gels, powders, emulsions, suspensions, pellets, suppositories, aerosols, sprays, powder sprays, patches, ointments, or creams.

[0104] In specific implementation schemes, the non-gastrointestinal dosage forms include injectable dosage forms, respiratory dosage forms, cavity dosage forms, mucosal dosage forms, and skin dosage forms.

[0105] In specific implementation schemes, the injectable dosage forms include, but are not limited to, various injectables such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections; the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder inhalers; the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and pills, for use in the rectum, vagina, urethra, nasal cavity, and ear canal; the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ointments, mouthwashes, sublingual tablets, adhesive tablets, and patches; and the skin dosage forms include, but are not limited to, topical solutions, lotions, liniments, ointments, plasters, pastes, and patches.

[0106] This document describes the preparation of sotexip using cell culture media. In some embodiments, the cell culture medium is a chemically defined culture medium (“CDM”).

[0107] In this specification and claims, the terms "culture medium" and "cell culture medium" refer to a nutrient source used for the growth or maintenance of cells. Those skilled in the art will understand that this nutrient source may contain components essential for cell growth and / or survival, or may contain components that aid cell growth and / or survival. Vitamins, essential or non-essential amino acids, and trace elements are components of the culture medium.

[0108] The term "chemically defined culture medium" indicates that it does not contain products of animal origin, such as animal serum and peptone. The term also covers culture media with a defined composition that do not contain undefined or partially defined components (such as animal serum, animal peptone, and plant peptone).

[0109] In this specification and claims, the terms “about” or “substantially” refer to a range of values ​​that a person skilled in the art would consider equivalent to the listed values ​​(e.g., having the same function or result), such as + / - 10% of the listed values.

[0110] In this specification and claims, the term "impurity" refers to a substance that is different from the target polypeptide product. Impurities include, but are not limited to: host cell material, such as host proteins; nucleic acids; variants, fragments, aggregates, or derivatives of the desired protein and polypeptide; other polypeptides; endotoxins; viral contaminants; cell culture medium components, etc.

[0111] In this specification and claims, the words “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0112] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0113] The foregoing disclosure generally describes this application, and the following embodiments further illustrate this application. These embodiments are described merely to illustrate the application and not to limit its scope. Although specific terms and values ​​are used herein, they are also understood to be exemplary and do not limit the scope of this application. Unless otherwise specified, the experimental methods and techniques in this specification are methods and techniques well known to those skilled in the art.

[0114] Example

[0115] Example 1: Preparation process of sotexip

[0116] (1) Construction of recombinant plasmids for monoclonal antibodies

[0117] The amino acid sequence of sotexip is shown in SEQ ID NO:2. The full sequence was synthesized by a gene synthesis company, and the gene sequence was ligated into the multiple cloning insertion site (MCS) of the corresponding vector. After verification by sequencing, the plasmid was extracted using a plasmid extraction kit (purchased from MN GmbH, Germany, catalog number 740422.50) and stored at -80℃ for later use.

[0118] (2) Host cell resuscitation and shake flask culture

[0119] The host cells used in this study were CHOZN ZFN-Modified GS. - / - The CHO cell line (hereinafter referred to as "CHOZN cells") was purchased from Merck. The CHOZN cells used in this application were removed from liquid nitrogen, rapidly thawed in a 37°C water bath, and then added to preheated EX-CELL CD CHO Fusion medium in a clean bench and mixed well before being cultured in an incubator.

[0120] (3) Cell transfection

[0121] On the day of transfection, after counting the cells, the cells to be transfected were mixed with plasmids and transfected using an exponential wave electroporator. Immediately after electroporation, the cells were mixed with preheated culture medium and transferred to a CO2 incubator for static culture.

[0122] (4) Cell pool screening

[0123] After electroporation and static culture for 24 hours, cell pools were selected using a mixed medium of 80% EX-CELL CHOCloning medium and 20% EX-CELL CD CHO Fusion medium, with an initial seeding density of 3000-10000 cells / well. After seeding, the 96-well plates were placed in a CO2 incubator for static culture. Once the cells had filled the wells, expression levels were assessed using OCTET, and cell pools with high expression levels were selected for expansion into 24-well plates. Cell growth was observed every 2-5 days. When cell density was high and cell condition was good, cell counting and yield assessment were performed. Based on the combined cell count and yield results, high-yielding cell pools were selected for monoclonal cell line seeding.

[0124] (5) Screening of monoclonal cell lines

[0125] Cell pools with high expression levels were sorted into single clones and cultured in 96-well plates. Single-clone cell imaging was performed daily. Once cells filled the wells, expression levels were assessed using OCTET. Cell pools with high expression levels were selected for expansion into 24-well plates. Cell growth was observed every 2-5 days. Once cell density was high and cell condition was good, cell counting and yield assessment were performed. Based on the combined cell count and yield results, high-expression single-clone cell lines were selected for shake-flask Fed-Batch culture and evaluation, assessing expression levels, product quality, and other indicators.

[0126] (6) Fed-batch culture experiment

[0127] Following typical cell culture processes for antibody drugs, sotecip was produced using a fed-batch culture process, involving cell resuscitation, stepwise expansion in shake flasks and bioreactors, and final production in a production tank. One cryopreserved cell tube was thawed in a 37°C water bath and cultured in shake flask culture medium. After continuous expansion in shake flasks and bioreactors, the culture was inoculated into a production tank for manufacturing. Based on typical cell culture processes for antibody drugs, appropriate parameters were set for the production stage, including culture temperature, pH, dissolved oxygen, stirring speed, and aeration. Appropriate feed medium and glucose solution were added as needed for cell growth, and antifoaming agent was added as required. After culture, the cell culture medium was subjected to deep filtration and sterile filtration, and the supernatant was collected for one-step ProA affinity chromatography purification.

[0128] Example 2: R in Sotsip 4 / S 5 Discovery of truncated variants

[0129] The structure of sotexip prepared in Example 1 is shown below. Figure 1 .

[0130] The samples prepared in Example 1 were subjected to complete molecular weight analysis. Since sotexip contains 6 N-glycosylation modification sites and 8 O-glycosylation modification sites, the heterogeneity caused by N-glycosylation and O-glycosylation is very severe, making determination impossible without cleaving the N-glycans and O-glycans. Therefore, before performing complete molecular weight analysis, N-glycan and O-glycan cleavage was performed using GENOVIS' OmniGLYZOR immobilized enzyme. The steps for complete molecular weight analysis after N-glycan and O-glycan cleavage are as follows:

[0131] Add 100 μg of protein to the column and seal the column with the top cap. Incubate the column upright (preventing resin from contacting the top cap) at 37°C and 300 rpm for 4 h in a shaker. After incubation, remove the bottom cap, place the column into a collection tube, loosen the top cap, and centrifuge at 1000×g for 1 min to recover the deglycosylated protein. To maximize sample collection, seal the column with the bottom cap, add 100 μl of reaction buffer, and seal the column to ensure complete suspension of the medium. Remove the bottom cap, place the column into a collection tube, loosen the top cap, and centrifuge at 1000×g for 1 min to recover the deglycosylated protein. Concentrate the recovered deglycosylated protein to approximately 50 μl using a centrifuge concentrator, and determine the protein content of the sample using Lunatic (extinction coefficient: 1.51).

[0132] The complete molecular weight analysis of the test samples was performed using liquid chromatography-mass spectrometry (LC-MS). The chromatographic conditions used are shown in Table 1, and the mass spectrometry conditions used are shown in Table 2.

[0133] Table 1 Chromatographic conditions

[0134]

[0135] Table 2 Mass Spectrometry Conditions

[0136]

[0137] Data processing was performed using Waters UNIFI 1.8 software. The complete molecular weight analysis total ion current chromatogram (TIC) and the deconvolution chromatograms of the major components are shown below. Figure 2 .

[0138] After the sample was treated with GENOVIS' OmniGLYZOR immobilized enzyme, both N-glycans and O-glycans on the protein were released. Deconvolution of the TIC spectrum revealed that the molecular weight of the main peak (peak B) was consistent with the molecular weight of the protein backbone (without N- and O-glycan modifications), with the error between the measured and theoretical molecular weight not exceeding 5.0 Da. The preceding peak (peak A) was approximately 439 Da shorter than the main peak, presumably related to protein truncation. Combined with sotexip sequence analysis, the theoretical molecular weight of the remaining component after truncation of one strand in the ActRIIA region (I1-R4) was 77190.2 Da, close to the measured molecular weight of peak A.

[0139] Example 3: R in Sotsip 4 / S 5 Identification of truncated variants

[0140] To confirm the breakpoint, peptide mass fingerprinting analysis was performed on the sample prepared in Example 1. The analytical procedure was as follows: approximately 100 μg of sample was added to 6 M urea and 10 mM TCEP, and incubated at 37°C for 60 min for denaturation and reduction; then, 20 mM IAM was added, and incubated at 37°C for 15 min for alkylation to block free thiol groups.

[0141] Urea was diluted to 1 M with 50 mM ammonium bicarbonate buffer. Lysyl endonuclease (Lys-C) was added at a protein:enzyme ratio of 20:1 (mass ratio), and digestion was carried out at 37℃ for 15 h. After digestion, formic acid was added to a final concentration of 1% to terminate the digestion. The sample was centrifuged at 12000 rpm for 3 min, and the supernatant was transferred to a sample vial. Peptide mass fingerprint analysis of the sample was performed using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The chromatographic conditions used are shown in Table 3, and the mass spectrometry conditions used are shown in Table 4.

[0142] Table 3 Chromatographic conditions used to identify the R4 / S5 truncated variant in sotexip

[0143]

[0144] Table 4. Mass spectrometry conditions used to identify the R4 / S5 truncated variant in Sotsip

[0145]

[0146] Data processing was performed using pFind software. The primary mass spectrometry (PMS) extractive ion chromatogram (XIC), primary and secondary mass spectra of the truncated peptide are shown below. Figure 3 As shown, Figure 3Image A shows the extraction ion chromatogram (I-I) of the sotexip variant, image B shows the I-I mass spectrum of the sotexip variant, and image C shows the secondary mass spectrum of the sotexip variant. The results indicate that a short peptide segment, ILGR, was found at a retention time of 44.3 min in the sample. 4 S 5 ETQECLFFNANWEK (where the strikethrough indicates the missing amino acid residue). The measured monoisotope peak m / z of this peptide is 951.9207, and the theoretical m / z is 951.9193, with an error of only 1.47 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. Sotexip is a homodimer formed by two identical peptide chains linked by disulfide bonds. Combined with the complete molecular weight analysis results, peak A is the R of one peptide chain. 4 The C-terminal peptide chain break at the site resulted in the deletion of the I1-R4 sequence in the ActRIIA region. The measured molecular weight of peak A was 77191.0 Da. The theoretical molecular weight of the remaining component after truncating I1-R4 at the N-terminus of one chain in the ActRIIA region was 77190.2 Da, further confirming that the break site was the R4 of one peptide chain. 4 Site.

[0147] Example 4: Content of truncated variants in Sotsip samples

[0148] Sotexip was prepared according to the preparation method in Example 1, and the obtained sample was identified according to the identification method in Example 3. R was calculated according to the following formula. 4 / S 5 Content of truncated variants:

[0149]

[0150] The sample pretreatment method and LC-MS / MS detection method are as shown in Example 2, and data processing is performed using Thermo's FreeStyle software. The R in batch 1 sample was calculated according to the formula. 4 / S 5 The content of the truncated variant was calculated to be 3.45% as follows:

[0151]

[0152] R was calculated in the same manner for multiple batches of samples. 4 / S 5 The content of truncated variants is shown in Table 5.

[0153] Table 5. Content of truncated variants in multiple batches of Sotticip samples

[0154]

[0155] Example 5: The effect of average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride, and the culture temperature during the protein production stage on R 4 / S 5 Effect of truncated variant content

[0156] The cell culture process for host cells containing a nucleic acid molecule or its vector encoding the amino acid sequence shown in SEQ ID NO:2 was performed according to the method described in Example 1. The effects of the average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride, as well as the culture temperature during the protein production stage, on the content of truncated variants were tested. After culture, the culture medium was collected once and subjected to one-step Protein A affinity chromatography. The obtained samples were identified according to the identification method in Example 3. The contents of truncated variants obtained under different average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride, and different culture temperatures during the protein production stage, are shown in Table 6.

[0157] Table 6. Content of truncated variants obtained under different average daily concentrations of riboflavin, ferric citrate, ammonium metavanadate, and lithium chloride, and at different culture temperatures during the protein production stage.

[0158]

[0159] Note: The temperature control accuracy is within ±0.5℃.

[0160] Data processing was performed using DOE experimental design software, and the summative results are as follows: Figure 4 As shown, the influencing trend is as follows Figure 5 As shown, the average daily concentration of ferric citrate and the culture temperature during the protein production stage had a significant impact on the content of truncated variants, while the average daily concentrations of riboflavin, ammonium metavanadate, and lithium chloride had no significant impact on the content of truncated variants.

[0161] As the culture temperature gradually decreased during protein production, the content of the sotracyp variant in the sotracyp samples gradually decreased. Furthermore, as the average daily concentration of ferric citrate gradually increased during culture, the content of the sotracyp variant in the sotracyp samples also gradually decreased.

[0162] During further analysis of Example 5, the applicant discovered that, without considering the average daily concentrations of riboflavin, ammonium metavanadate, and lithium chloride (which have no effect on the content of truncated variants), R under different culture temperatures and average daily concentrations of ferric citrate during the protein production stage... 4 / S 5Changes in the content of truncated variants, such as Figure 6 As shown.

[0163] The results showed that the culture temperature during protein production and the average daily concentration of ferric citrate during culture affected the content of soteccept variants. As the culture temperature gradually decreased and the average daily concentration of ferric citrate gradually increased during culture, the content of truncated variants gradually decreased. Furthermore, it is expected that the content of truncated variants can be further reduced after downstream purification processes.

[0164] Example 6: Effect of culture temperature on R during protein production stage 4 / S 5 Effect of truncated variant content

[0165] The cell culture process for host cells containing a nucleic acid molecule or its vector encoding the amino acid sequence shown in SEQ ID NO:2 was performed according to the method described in Example 1. The effect of culture temperature on the content of truncated variants during the protein production stage was further tested. After culture, the culture medium was collected once and subjected to a one-step Protein A affinity chromatography. The obtained samples were identified according to the identification method in Example 3. R under different culture temperature conditions during the protein production stage... 4 / S 5 Changes in the content of truncated variants, such as Figure 7 As shown in the figure, the culture temperature during protein production affects the content of sotexip variants. The content of truncated variants gradually decreases as the culture temperature during protein production decreases. Furthermore, a culture temperature between 29.5℃ and 37℃ during protein production can control the content of sotexip variants below 10%, and it is expected that the content of sotexip variants can be further significantly reduced after downstream purification processes.

[0166] Example 7: Effect of average daily iron ion concentration on R during cultivation 4 / S 5 Effect of truncated variant content

[0167] The cell culture process of host cells containing a nucleic acid molecule or its vector encoding the amino acid sequence shown in SEQ ID NO:2 was carried out in accordance with the method described in Example 1, and the effect of the average daily concentration of iron ions added during the culture process on the content of truncated variants was further tested.

[0168] In this embodiment and in Example 5, ferric citrate is used as the source of iron ions. The formula for calculating the average daily concentration of ferric citrate is as follows:

[0169]

[0170] After cultivation, the culture medium was collected once and subjected to one-step Protein A affinity chromatography. The obtained samples were identified according to the identification method in Example 3. R under different average daily ferric citrate concentrations... 4 / S 5 Changes in the content of truncated variants, such as Figure 8 As shown.

[0171] The results showed that the average daily concentration of iron ions added during cultivation affected the content of sotexip variants in the sotexip samples. As the average daily concentration of iron ions added gradually increased, the content of truncated variants gradually decreased. Furthermore, an average daily concentration of iron ions ranging from 1.77 to 123.93 μmol / L / day could control the content of sotexip variants below 10%, and it is expected that the content of sotexip variants can be further significantly reduced after downstream purification processes.

[0172] Example 8 Different contents of R 4 / S 5 Effect of truncated variants on Activin A affinity

[0173] The SPR technology (Biacore T200) was used to determine the concentrations of R at different levels. 4 / S 5 The affinity of the truncated variant of sotexip for recombinant human Activin A was investigated. A capture assay was used for analysis. Protein A was first immobilized on the reference and experimental channels of a CM5 chip via amino-coupling, with a coupling amount of approximately 10,000 RU. The test sample was captured onto the experimental channel at a flow rate of 30 μl / min. 2.5 nM recombinant human Activin A was serially diluted to 0.078125 nM with running buffer. Concentrations of recombinant human Activin A ranging from 2.5 nM to 0.078125 nM were sequentially passed through the reference and experimental channels, binding for 3 min and dissociating for 20 min, respectively. After each cycle, the protein was regenerated twice with pH 1.5 glycine hydrochloride for 30 s. The data were analyzed using Biacore T200 Evaluation software, and a 1:1 binding model was used for fitting.

[0174] Table 7. Table of different contents of R 4 / S 5 Affinity of truncated variant Sotsip samples to Activin A

[0175]

[0176] With different contents of R 4 / S 5The results of the affinity analysis of the truncated variant of Sotsyp for Activin A are shown in Table 7, combined with the dissociation curve fitting spectrum as follows: Figure 9 As shown, Figure 9 Image A shows the affinity binding-dissociation curve fitting of Activin A for a sotexip sample with a 0.33% R4 / S5 truncated variant; image B shows the affinity binding-dissociation curve fitting of Activin A for a sotexip sample with a 3.85% R4 / S5 truncated variant; and image C shows the affinity binding-dissociation curve fitting of Activin A for a sotexip sample with an 8.58% R4 / S5 truncated variant. The results show that within a 10% truncated variant content, different R4 / S5 truncated variant contents... 4 / S 5 The truncated variants of Sotsypium showed essentially the same affinity for Activin A, and their binding dissociation fit spectra were also essentially the same. Therefore, the R... 4 / S 5 The content of truncated variants less than or equal to about 10% does not affect the affinity and binding activity of the sotsip sample for Activin A.

[0177] Various changes and equivalent substitutions may be made to the embodiments disclosed in this application without departing from the spirit and scope of this disclosure. Unless the context otherwise requires, any feature, step, or embodiment of the embodiments disclosed herein may be used in combination with any other feature or embodiment.

Claims

1. A composition comprising sotexip and variants thereof, wherein the variant has a peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or two monomeric peptide chains of sotexip as shown in SEQ ID NO:2, and the percentage of the monomeric peptide chain with peptide bond cleavage between Arg(4)-Ser(5) is less than or equal to 10% of the total number of monomeric peptide chains in the composition, calculated based on peptide mass fingerprinting analysis, and wherein the amino acid sequence of one or two monomeric peptide chains of the variant is as shown in SEQ ID NO:

1.

2. The composition according to claim 1, wherein, Based on peptide quality fingerprint analysis, the percentage of monomeric peptide chains in the composition that have not undergone peptide bond cleavage between Arg(4) and Ser(5) is greater than or equal to 90% of the total number of monomeric peptide chains in the composition.

3. The composition according to claim 1 or 2, wherein the percentage of monomeric peptide chains in which peptide bonds are broken between Arg(4) and Ser(5) is greater than or equal to 0.1%, 0.5%, 1%, 1.5% or 2% of the total number of monomeric peptide chains in the composition.

4. The composition according to claim 1 or 2, wherein the percentage of monomeric peptide chains in which peptide bonds are broken between Arg(4) and Ser(5) is less than or equal to 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.9%, 0.8%, 0.7%, 0.6% or 0.5% of the total number of monomeric peptide chains in the composition.

5. The composition of claim 1 or 2, wherein the composition is prepared by a method comprising the steps of: culturing host cells containing a nucleic acid molecule or a vector thereof encoding an amino acid sequence as shown in SEQ ID NO:2 in a bioreactor, the culture comprising a cell expansion phase and a protein production phase, wherein the culture temperature of the protein production phase is maintained at 29.5°C-37°C.

6. The composition of claim 1 or 2, wherein the composition is prepared by a method comprising the steps of: culturing host cells containing a nucleic acid molecule or a vector thereof encoding an amino acid sequence as shown in SEQ ID NO:2 in a bioreactor, wherein the average daily concentration of iron ions added during culture is from 1.5 μmol / L / day to 125 μmol / L / day.

7. A method for preparing the composition according to any one of claims 1-4, comprising culturing host cells containing a nucleic acid molecule or a vector encoding an amino acid sequence as shown in SEQ ID NO:2 in a bioreactor, said culture comprising a cell expansion phase and a protein production phase, wherein the culture temperature during said protein production phase is maintained at 29.5°C-37°C; and / or using a culture medium supplemented with iron ions for culture.

8. The method according to claim 7, wherein the iron ions are derived from one or more of ferric citrate, ferric nitrate, ferrous sulfate, ferric ethylenediaminetetraacetate, and transferrin-bound iron.

9. The method according to claim 8, wherein the iron ions are derived from ferric citrate.

10. The method of claim 7, wherein the average daily concentration of iron ions added during the culture period is from 1.5 μmol / L / day to 125 μmol / L / day.

11. The method of claim 10, wherein the average daily concentration of iron ions is 1.50 μmol / L / day to 5.00 μmol / L / day, 5.01 μmol / L / day to 15.00 μmol / L / day, 15.01 μmol / L / day to 30.00 μmol / L / day, 30.01 μmol / L / day to 45.00 μmol / L / day, 45.01 μmol / L / day to 60.00 μmol / L / day, 60.01 μmol / L / day to 75.00 μmol / L / day, 75.01 μmol / L / day to 90.00 μmol / L / day, 90.01 μmol / L / day to 115.00 μmol / L / day, or 115.01 μmol / L / day to 125.00 μmol / L / day.

12. The method according to claim 11, wherein the average daily concentration of iron ions is selected from 1.77 μmol / L / day, 7.88 μmol / L / day, 62.85 μmol / L / day, 93.39 μmol / L / day, or 123.93 μmol / L / day.

13. The method of claim 7, wherein the culture temperature is maintained at at least 29.5°C, at least 30°C, at least 30.5°C, at least 31°C, at least 31.5°C, at least 32°C, at least 32.5°C, at least 33°C, at least 33.5°C, at least 34°C, at least 34.5°C, at least 35°C, at least 35.5°C, at least 36°C, at least 36.5°C, or at least 37°C.

14. The method of claim 7, wherein the culture temperature is maintained at 29.5°C-37°C for at least 50% to 100% of the time during the protein production phase.

15. The method of claim 14, wherein the culture temperature is maintained at 29.5°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.

16. The method of claim 15, wherein the culture temperature is maintained at 29.5°C-37°C for 95% of the time during the protein production stage.

17. The method according to any one of claims 7-16, wherein the host cell is a mammalian cell.

18. The method of claim 17, wherein the mammalian cell is derived from a mouse or a human.

19. The method of claim 18, wherein the human-derived cells comprise HEK 293 and PER.C6.

20. The method of claim 18, wherein the cells from the mouse comprise CHO, Hybridoma, YB2 / O, NSO, Sp2 / O, and BHK.

21. The method of claim 20, wherein the host cell is a CHO cell.

22. A pharmaceutical formulation comprising the composition of any one of claims 1-6 or the composition prepared by the method of any one of claims 7-21, and one or more pharmaceutically acceptable excipients.

23. The use of the composition according to any one of claims 1-6, the composition prepared by the method according to any one of claims 7-21, or the pharmaceutical preparation according to claim 22 in the preparation of a medicament for treating pulmonary hypertension.

24. The use of iron-containing culture media in reducing the content of sotexip variants in sotexip products, wherein the variants are characterized by peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or both monomeric peptide chains of sotexip as shown in SEQ ID NO:

2.

25. The application of claim 24, wherein the iron ions are derived from ferric citrate.

26. A method for detecting a variant of sotexip in a product containing sotexip, wherein the variant involves a peptide bond cleavage between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or both monomeric peptide chains of sotexip as shown in SEQ ID NO:2, the method comprising: The product was digested with lysyl endonuclease (Lys-C) to obtain the digested product; as well as, The enzyme digestion products were analyzed by peptide mass fingerprinting using LC-MS / MS, and the presence or absence of the variant was determined based on the primary and secondary mass spectra.

27. A method for quality inspection or quality control of a product containing sotexip, comprising detecting the content of a sotexip variant in the product containing sotexip, wherein the variant is characterized by a peptide bond break between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or both monomeric peptide chains of sotexip as shown in SEQ ID NO:

2.

28. The method of claim 27, wherein if the detected content of the sotexip variant is less than or equal to 10%, the sotexip product meets pharmaceutical requirements.

29. The application of sotexip variants in the quality inspection or quality control of sotexip-containing products, wherein the variants involve a peptide bond break between arginine at position 4 and serine at position 5 (Arg(4)-Ser(5)) of one or both monomeric peptide chains of sotexip as shown in SEQ ID NO:2.

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