Etanercept variants and related processes

Abstract

The present disclosure relates to etanercept variants and related processes, wherein the variants are specific truncated variants of etanercept. The present application also provides pharmaceutical formulations comprising the variants described above. The invention further provides a method for reducing the variant, the content of truncated variant fragments can be effectively optimized through the method, and the method is simple in process and easy to operate.

Description

Technical Field

[0001] This disclosure relates to the pharmaceutical field, specifically to an etanercept variant and related processes. Background Technology

[0002] Tumor necrosis factor (TNF) is a key pathogenic factor in rheumatoid arthritis. Etanercept, a recombinant human type II tumor necrosis factor receptor-protein, consists of the extracellular ligand-binding moiety of the type II tumor necrosis factor receptor and the Fc fragment of human IgG1. It can bind to free TNF with high affinity and acts as a natural TNF antagonist by preventing the binding of TNF molecules to cellular receptors. By inhibiting the biological activity of TNF, it blocks TNF-mediated cellular responses, fundamentally halting disease progression. It also participates in regulating biological responses controlled by other downstream molecules induced or regulated by TNF (such as cytokines, adhesion molecules, or proteases). It not only relieves common symptoms such as joint swelling, pain, stiffness, and morning stiffness, but also blocks joint destruction and improves joint function. It possesses biological activity in neutralizing TNF-α and TNF-β.

[0003] With the continuous development and progress of biotechnology and engineering, and with the maturity and continuous improvement of upstream cell culture technology, protein expression levels are constantly increasing, and the content of related substances of target proteins, such as variants and impurities, is also becoming more obvious. Further research is needed on the analysis of variants or impurities of etanercept and the process of reducing the content of variants or impurities. Summary of the Invention This disclosure relates to specific variants of etanercept and related processes for reducing the content of these variants.

[0004] On one hand, this disclosure provides a composition comprising etanercept and truncated variants thereof, said truncated variants comprising one or more of the following: (1) A truncated variant resulting from a breakage of at least one chain in etanercept between positions G234 and D235. (2) A truncated variant resulting from a breakage of at least one chain in etanercept between positions D235 and E236. (3) A truncated variant resulting from a breakage of at least one chain in etanercept between T233 and G234. (4) A truncated variant resulting from a breakage of at least one chain in etanercept between positions S239 and C240. (5) A truncated variant resulting from a breakage of at least one chain in etanercept between positions K238 and S239. (6) A truncated variant resulting from a break in at least one chain in etanercept between C240 ​​and D241.

[0005] The above site numbering is based on SEQ ID NO: 1.

[0006] Etanercept is formed by two identical peptide chains linked by disulfide bonds. In some embodiments, the sequence of each of the two protein chains of etanercept is shown in SEQ ID NO: 1.

[0007] In a preferred embodiment, the truncated variant includes one or more of the following: (1) A truncated variant resulting from a break between G234 and D235 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (2) A truncated variant resulting from a break in one chain of etanercept between D235 and E236 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (3) A truncated variant resulting from a break between T233 and G234 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (4) A truncated variant resulting from a break between S239 and C240 ​​in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (5) A truncated variant resulting from a break in one chain of etanercept between positions K238 and S239 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (6) A truncated variant resulting from a break between C240 ​​and D241 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; The above site numbering is based on SEQ ID NO: 1.

[0008] In some embodiments, the total content of the truncated variant in the composition is less than or equal to about 5%, as determined by SEC-HPLC.

[0009] In a preferred embodiment, the total content of the truncated variant in the composition is less than or equal to about 4.5%, less than or equal to about 4%, less than or equal to about 3.5%, 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 1.7%, and less than or equal to about 1.5%.

[0010] In a preferred embodiment, the total content of the truncated body 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%, or greater than or equal to about 1%.

[0011] In some embodiments, the composition is prepared by a method comprising the steps of purifying cell culture harvest containing etanercept protein molecules using protein A affinity chromatography, wherein sodium sulfate is added to the protein A affinity chromatography elution buffer used in the purification step.

[0012] On the other hand, this disclosure provides pharmaceutical formulations comprising the compositions described herein, and one or more pharmaceutically acceptable carriers.

[0013] On the other hand, this disclosure provides a method for purifying etanercept, wherein a composition containing etanercept protein molecules is purified by protein A affinity chromatography, wherein sodium sulfate is added to the protein A affinity chromatography elution buffer used in the purification step.

[0014] In some embodiments, the composition is a cell culture harvest medium.

[0015] On the other hand, this disclosure provides a method for reducing the content of truncated variants of etanercept in an etanercept composition, wherein the composition containing etanercept protein molecules, such as cell culture harvest broth, is purified by protein A affinity chromatography, wherein sodium sulfate is added to the protein A affinity chromatography elution buffer used in the purification step, and the truncated variants include one or more of the following: (1) A truncated variant resulting from a breakage of at least one chain in etanercept between positions G234 and D235. (2) A truncated variant resulting from a breakage of at least one chain in etanercept between positions D235 and E236. (3) A truncated variant resulting from a breakage of at least one chain in etanercept between T233 and G234. (4) A truncated variant resulting from a breakage of at least one chain in etanercept between positions S239 and C240. (5) A truncated variant resulting from a breakage of at least one chain in etanercept between positions K238 and S239. (6) A truncated variant resulting from a breakage of at least one chain in etanercept between positions C240 ​​and D241. The above site numbering is based on SEQ ID NO: 1.

[0016] In some implementations, the sequence of each of the two protein chains of etanercept is shown in SEQ ID NO: 1.

[0017] In some preferred embodiments, the truncated variant includes one or more of the following: (1) A truncated variant resulting from a break between G234 and D235 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (2) A truncated variant resulting from a break in one chain of etanercept between D235 and E236 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (3) A truncated variant resulting from a break between T233 and G234 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (4) A truncated variant resulting from a break between S239 and C240 ​​in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (5) A truncated variant resulting from a break in one chain of etanercept between positions K238 and S239 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (6) A truncated variant resulting from a break between C240 ​​and D241 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO: 1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; The above site numbering is based on SEQ ID NO: 1.

[0018] This disclosure has one or more of the following beneficial effects: This disclosure identifies a series of truncated variants of etanercept, and their specific sequence structures are determined through analysis. Further optimization of the purification method revealed that adding sodium sulfate to the elution buffer of protein A affinity chromatography significantly reduces the content of these truncated variants. The purification process described in this disclosure is rapid, simple, easily scalable, and suitable for large-scale production.

[0019] the term All publications, patents and patent applications mentioned in this specification are incorporated herein by reference as if specifically and individually indicated that each individual publication, patent or patent application is incorporated herein by reference.

[0020] Before this disclosure is described in detail below, it should be understood that this disclosure is not limited to the specific methodologies, procedures, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0021] Some embodiments disclosed herein include numerical ranges, and certain aspects of this disclosure may be described using ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions using ranges are for purposes of brevity and convenience only and should not be considered as a strict limitation of the scope of this disclosure. Therefore, descriptions using ranges should be considered as specifically disclosing all possible subranges and all possible specific numerical points within those ranges, as these subranges and numerical points have been explicitly stated herein. The above principles apply equally regardless of the breadth of the numerical values ​​described. When a range description is used, the range includes the endpoints of the range.

[0022] The term "protein" refers to a product obtained by fusing antibody fragments with other bioactive proteins using genetic engineering techniques. Due to the differences in fusion proteins, these proteins possess a variety of biological functions, and the expressed recombinant protein does not affect the antigen-binding ability of the single-chain antibody, nor does it affect the biological properties of the protein it is fused with.

[0023] The term "fragment" includes at least a portion of a complete protein. As used herein, a "fragment" of a protein molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that specifically binds to or reacts with a selected antigen or its immunogenicity-determining moiety, or a fusion protein product further derived from such fragment, such as a single-chain antibody. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the molecular structure of etanercept.

[0025] Figure 2 Analysis of the non-reduced complete molecular weights of etanercept LMWS (Low molecule weights) fractions, including N-glycan, sialic acid, and O-glycan cleavage.

[0026] Figure 3 Analysis of the complete molecular weight of etanercept LMWS (Low molecule weights) fractions after cleavage of N-glycan, sialic acid, and O-glycan.

[0027] Figure 4 This is a schematic diagram of the etanercept fracture site.

[0028] Figure 5 The images show the SEC-HPLC chromatograms before and after etanercept affinity chromatography optimization. Detailed Implementation

[0029] Etanercept is a fusion protein, a homodimer formed by two identical peptide chains linked by disulfide bonds. Specifically, it consists of the extracellular ligand-binding moiety of type II tumor necrosis factor receptor and the Fc fragment of human IgG1, as shown in the figure. Figure 1 As shown in the diagram. In a specific implementation, the sequence of each of the two identical peptide chains of etanercept is shown in SEQ ID NO:1.

[0030] In some implementations, the truncated variants of etanercept disclosed herein include one or more of the following: (1) A truncated variant resulting from a breakage of at least one chain in etanercept between positions G234 and D235. (2) A truncated variant resulting from a breakage of at least one chain in etanercept between positions D235 and E236. (3) A truncated variant resulting from a breakage of at least one chain in etanercept between T233 and G234. (4) A truncated variant resulting from a breakage of at least one chain in etanercept between positions S239 and C240. (5) A truncated variant resulting from a breakage of at least one chain in etanercept between positions K238 and S239. (6) A truncated variant resulting from a breakage of at least one chain in etanercept between positions C240 ​​and D241. The above site numbers are all based on SEQ ID NO: 1.

[0031] In some specific implementations, the truncated variants of etanercept disclosed herein mainly include truncated variants resulting from a break between positions G234 and D235, and truncated variants resulting from a break between positions S239 and C240.

[0032] In some specific implementations, the truncated variants of etanercept disclosed herein also include truncated variants resulting from fractures at positions D235 / E236, T233 / G234, K238 / S239, or C240 / D241.

[0033] In some specific implementations, cleavage occurs at the cleavage sites G234 / D235, D235 / E236, and T233 / G234, resulting in the loss of the receptor-binding regions (the TNFR domain sequence that binds to the TNF antigen is L1-D235) of both heavy chains of etanercept, and the resulting truncated variants have significantly reduced biological activity.

[0034] In some specific implementations, cleavage at the cleavage sites S239 / C240, K238 / S239, and C240 / D241 can result in the loss of the receptor-binding region of one of the heavy chains, and this truncated variant also reduces activity.

[0035] In some specific implementations, the truncated variant of etanercept is generated by the breakage of both chains of etanercept between the G234 and D235 positions, and the resulting truncated variant can be represented as HC:D235-G466+HC:D235-G466.

[0036] In some specific implementations, the truncated variant of etanercept is generated by breaking one chain of etanercept between positions S239 and C240 ​​while the other chain remains unchanged, and the resulting truncated variant can be represented as HC+HC:C240-G466.

[0037] In some specific implementations, a truncated variant of etanercept, in which one chain breaks between positions D235 and E236 and the other chain breaks between positions G234 and D235, can be represented as HC:E236-G466+HC:D235-G466.

[0038] In some specific implementations, a truncated variant of etanercept, in which one chain breaks between T233 and G234 and the other chain breaks between G234 and D235, can be represented as HC:G234-G466+HC:D235-G466.

[0039] In some specific implementations, a truncated variant of etanercept, in which one chain breaks between positions K238 and S239 while the other chain remains unchanged, can be represented as HC+HC:S239-G466.

[0040] In some specific implementations, a truncated variant of etanercept, in which one chain breaks between positions C240 ​​and D241 while the other chain remains unchanged, can be represented as HC+HC:D241-G466.

[0041] The present disclosure is further described in detail through the following embodiments. Specific embodiments are listed below to illustrate the present disclosure; however, it should be understood that these embodiments are provided merely for illustrative purposes and not for limiting the scope of the present disclosure.

[0042] All chromatography media described in this disclosure are commercially available. The cross-linked agarose affinity chromatography media MabSelect and MabSelect Sure were purchased from Cytiva Sweden AB.

[0043] Example 1: Preparation process of etanercept Energizer structure such as Figure 1 As shown. The sequence of each of the two identical peptide chains of etanercept is shown in SEQ ID NO:1.

[0044] Upstream cell culture process The host cell line used in this embodiment is Chinese hamster ovary (CHO) CHO-K1 cells from the China Center for Type Culture Collection (CCC). The expression vector used is an expression plasmid independently constructed by Qilu Pharmaceutical Co., Ltd., which can achieve efficient expression of exogenous recombinant proteins in mammalian cells. The production culture medium contains the following main components: glucose, sodium chloride, potassium chloride, manganese sulfate, ethanolamine, ferric citrate, zinc sulfate, copper sulfate, glutathione, magnesium chloride, sodium dihydrogen phosphate, sodium bicarbonate, alanine, asparagine, arginine, aspartic acid, cysteine, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, biotin, choline chloride, nicotinamide, vitamin B6, vitamin B2, vitamin B1, vitamin B12, and linoleic acid.

[0045] Referring to the typical cell culture process for antibody drugs, etanercept stock solution was produced following the process of cell resuscitation, stepwise expansion in shake flasks and bioreactors, and batch feeding in production tanks. During the production stage, appropriate process parameters such as culture temperature, pH, dissolved oxygen, stirring speed, and aeration were set. Appropriate feeding medium and glucose solution were added according to the needs of cell growth, and antifoaming solution was added as needed. After the culture was completed, the cell culture medium was centrifuged, deep filtered, and sterilized filtered, and the supernatant was collected for purification.

[0046] Capture of etanercept protein using affinity chromatography The MabSelect Sure affinity chromatography column (11×250 mm) was equilibrated with at least 5 column volumes of a buffer solution containing 0.02 mol / L Tris-HCl, 0.15 mol / L NaCl, and pH 7.2.

[0047] Cell culture harvest medium containing etanercept protein molecules was loaded onto the chromatography medium to adsorb the antibody molecules. After loading, the sample was washed with at least 3 column volumes of equilibration buffer (0.02 mol / L Tris-HCl, 0.15 mol / L NaCl, pH 7.2), followed by at least 5 column volumes of Wash1 buffer (0.02 mol / L Tris-HCl, 1 mol / L sodium chloride, pH 7.2). Then, the sample was washed with at least 3 column volumes of Wash2 buffer (0.02 mol / L acetate-sodium acetate, pH 5.5). Finally, elution was performed with 0.05 mol / L acetic acid, pH 3.6 ± 0.2. The collection interval was defined as the peak value dropping to 200 mAU after reaching the initial 200 mAU, at which point collection ceased. This yielded the etanercept affinity harvest medium. The obtained sample was analyzed using SEC-HPLC. SEC-LMWS (Low The molecule weights were 2.1%, indicating a high fragment content.

[0048] Example 2: Characterization of etanercept protein fragments using mass spectrometry The SEC-LMWS components of the affinity chromatography samples collected in Example 1 were separated and collected using SEC-HPLC. The separated LMWS samples were then subjected to N-glycan, sialic acid, and O-glycan cleavage, followed by non-reduced and reduced intact molecular weight analysis. The results of the non-reduced intact molecular weight analysis are as follows: Figure 2 As shown, the results of the analysis to restore the complete molecular weight are as follows: Figure 3 As shown (attached) Figure 2 Appendix Figure 3 The midpoint numbering is based on SEQ ID NO: 1. The LMWS component types are mainly broken at the G234 / D235 and S239 / C240 positions, producing HC:D235-G466+HC:D235-G466 and HC:L1-G234 components, and HC+HC:C240-G466 and HC:L1-S239 components, respectively. Additionally, minor fractures were found at positions D235 / E236, T233 / G234, K238 / S239, and C240 / D241, producing components HC:E236-G466+HC:D235-G466 and HC:L1-D235, HC:G234-G466+HC:D235-G466 and HC:L1-T233, HC+HC:S239-G466 and HC:L1-K238, and HC+HC:D241-G466 and HC:L1-C240, respectively.

[0049] The fracture sites of all LMWS components occurred above the hinge region, and the fracture sites were as follows: Figure 4 As shown, six breakpoints were found in the LMWS fraction. Three of these breakpoints—G234 / D235, D235 / E236, and T233 / G234—result in the loss of the receptor-binding domains (the TNFR domain sequence binding to the TNF antigen is L1-D235) of both heavy chains, leading to inactivation of this type of LMWS fraction. The other three breakpoints—S239 / C240, K238 / S239, and C240 / D241—result in the loss of the receptor-binding domain of one heavy chain, also reducing the activity of this truncated LMWS fraction. Due to the loss of receptor-binding domains in these truncated etanercept variants, their biological activity is significantly affected. To improve the product's efficacy, further process optimization is needed to reduce the content of these variants in the etanercept product.

[0050] Example 3: Effect of sodium sulfate on etanercept protein fragment content in affinity chromatography Sample source A cell harvesting solution containing etanercept protein was obtained according to Example 1. The cell harvesting solution was subjected to affinity chromatography, and the fragments were optimized using an elution buffer containing a certain concentration of sodium sulfate.

[0051] Affinity Chromatography The MabSelect Sure affinity chromatography column (11×250 mm) was equilibrated with at least 5 column volumes of a buffer solution containing 0.02 mol / L Tris-HCl, 0.15 mol / L NaCl, and pH 7.2.

[0052] Cell culture harvest medium containing etanercept protein molecules was loaded onto the chromatography medium to adsorb the antibody molecules. After loading, the medium was further irradiated with 0.02 mol / L Tris-HCl and 0.15 mol / L Tris-HCl. Wash with at least 3 column volumes of NaCl equilibration buffer (pH 7.2), followed by at least 5 column volumes of Wash1 buffer (0.02 mol / L Tris-HCl, 1 mol / L sodium chloride, pH 7.2). Then wash with at least 3 column volumes of Wash2 buffer (0.02 mol / L acetate-sodium acetate, pH 5.5). Finally, elute with 0.05 mol / L acetic acid and different concentrations of sodium sulfate (5 / 15 / 30 / 45 / 60 mmol / L), pH 3.6 ± 0.2. The collection interval is defined as follows: collection stops when the peak value drops below 200 mAU after the initial collection point of 200 mAU. This yields the optimized etanercept affinity harvest. The obtained samples were analyzed using SEC-HPLC. The results are shown in Table 1. The SEC-LMWS (Low molecule weights) was reduced to 1.5–1.7%, a reduction of 19–29% compared to the control process in Example 1.

[0053]

[0054] From the appendix Figure 5 The SEC-HPLC chromatograms show the changes in SEC-LMWS (Low molecule weights) content in the samples before and after affinity chromatography optimization.

[0055] Example 4: Effect of sodium chloride in affinity chromatography on etanercept protein fragment content Sample source A cell harvesting solution containing nacip protein was obtained according to Example 1. The harvesting solution was subjected to affinity chromatography, and the fragments were optimized using an elution buffer containing a certain concentration of sodium chloride.

[0056] Affinity Chromatography The MabSelect Sure affinity chromatography column (11×250 mm) was equilibrated with at least 5 column volumes of a buffer solution containing 0.02 mol / L Tris-HCl, 0.15 mol / L NaCl, and pH 7.2.

[0057] Cell culture harvest medium containing etanercept protein molecules was loaded onto the chromatography medium to adsorb the antibody molecules. After loading, the medium was further irradiated with 0.02 mol / L Tris-HCl and 0.15 mol / L Tris-HCl. Wash with at least 3 column volumes of NaCl equilibration buffer (pH 7.2), followed by at least 5 column volumes of Wash1 buffer (0.02 mol / L Tris-HCl, 1 mol / L sodium chloride, pH 7.2). Then wash with at least 3 column volumes of Wash2 buffer (0.02 mol / L acetate-sodium acetate, pH 5.5). Finally, elute with 0.05 mol / L acetic acid, different concentrations of sodium chloride (15 / 60 mmol / L), pH 3.6 ± 0.2. The collection interval is defined as the peak value decreasing from 200 mAU to 200 mAU, at which point the optimized etanercept affinity harvest is obtained. The obtained samples were analyzed using SEC-HPLC. The results are shown in Table 2. The SEC-LMWS (Low molecule weights) was reduced to a maximum of 1.9%, a reduction of up to 10% compared to the control process in Example 1.

[0058]

[0059] Surprisingly, the experimental results of Examples 3-4 above showed that adding Na2SO4 to the elution buffer during affinity chromatography significantly reduced the content of a series of truncated variants, while using other salts such as NaCl did not yield such a significant effect.

Claims

1. A composition, characterized in that, This includes etanercept and its truncated variants, said truncated variants comprising one or more of the following: (1) A truncated variant resulting from a breakage of at least one chain in etanercept between positions G234 and D235. (2) A truncated variant resulting from a breakage of at least one chain in etanercept between positions D235 and E236. (3) A truncated variant resulting from a breakage of at least one chain in etanercept between T233 and G234. (4) A truncated variant resulting from a breakage of at least one chain in etanercept between positions S239 and C240. (5) A truncated variant resulting from a breakage of at least one chain in etanercept between positions K238 and S239. (6) A truncated variant resulting from a breakage of at least one chain in etanercept between positions C240 ​​and D241. The above site numbering is based on SEQ ID NO:

1.

2. The composition according to claim 1, characterized in that, The truncated variants include one or more of the following: (1) A truncated variant resulting from a break between G234 and D235 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (2) A truncated variant resulting from a break in one chain of etanercept between D235 and E236 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (3) A truncated variant resulting from a break between T233 and G234 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (4) A truncated variant resulting from a break between S239 and C240 ​​in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (5) A truncated variant resulting from a break in one chain of etanercept between positions K238 and S239 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; or (6) A truncated variant resulting from a break between C240 ​​and D241 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; The above site numbering is based on SEQ ID NO:

1.

3. The composition according to any one of claims 1-2, characterized in that, The total content of the truncated variant in the composition was less than or equal to about 5%, as determined by SEC-HPLC.

4. The composition according to claim 3, characterized in that, The total content of the truncated variant in the composition is less than or equal to about 4.5%, less than or equal to about 4%, less than or equal to about 3.5%, 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 1.7%, and less than or equal to about 1.5%.

5. The composition according to any one of claims 1-4, characterized in that, The total 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%, and greater than or equal to about 1%.

6. The composition according to any one of claims 1-5, characterized in that, The composition is prepared by a method comprising the steps of purifying cell culture harvest containing etanercept protein molecules by protein A affinity chromatography, wherein sodium sulfate is added to the protein A affinity chromatography elution buffer used in the purification step.

7. A pharmaceutical preparation comprising the composition of any one of claims 1 to 6, and one or more pharmaceutically acceptable carriers.

8. A method for purifying etanercept, characterized in that, The composition containing etanercept protein molecules was purified by protein A affinity chromatography, wherein sodium sulfate was added to the protein A affinity chromatography elution buffer used in the purification step.

9. The method of claim 8, wherein the composition is a cell culture harvest medium containing etanercept protein molecules.

10. A method for reducing the content of a truncated variant of etanercept in an etanercept composition, characterized in that, The composition containing etanercept protein molecules was purified by protein A affinity chromatography, wherein sodium sulfate was added to the protein A affinity chromatography elution buffer used in the purification step, and the truncated variant included one or more of the following: (1) A truncated variant resulting from a breakage of at least one chain in etanercept between positions G234 and D235. (2) A truncated variant resulting from a breakage of at least one chain in etanercept between positions D235 and E236. (3) A truncated variant resulting from a breakage of at least one chain in etanercept between T233 and G234. (4) A truncated variant resulting from a breakage of at least one chain in etanercept between positions S239 and C240. (5) A truncated variant resulting from a breakage of at least one chain in etanercept between positions K238 and S239. (6) A truncated variant resulting from a breakage of at least one chain in etanercept between positions C240 ​​and D241. The above site numbering is based on SEQ ID NO:

1.

11. The method of claim 10, wherein the composition is a cell culture harvest medium containing etanercept protein molecules.

12. The method according to any one of claims 10-11, characterized in that, The truncated variants include one or more of the following: (1) A truncated variant resulting from a break between G234 and D235 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (2) A truncated variant is produced by a break in one chain of etanercept between positions D235 and E236, and the other chain is selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (3) A truncated variant resulting from a break between T233 and G234 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (4) A truncated variant resulting from a break between S239 and C240 ​​in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; (5) A truncated variant resulting from a break in one chain of etanercept between positions K238 and S239 and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; or (6) A truncated variant resulting from a break between C240 ​​and D241 in one chain of etanercept and another chain selected from one of the following a~g: a. No breakage occurred; the sequence is as shown in SEQ ID NO:

1. b. A fracture occurred between positions G234 and D235; c. A break occurred between positions D235 and E236; d. A fracture occurred between positions T233 and G234; e. A fracture occurred between positions S239 and C240; f. A break occurred between positions K238 and S239; g. A break occurred between positions C240 ​​and D241; The above site numbering is based on SEQ ID NO: 1.

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