Method for reducing contaminants in protein purification

Abstract

Provided herein are improved methods of purifying an Fc-containing protein from a mixture comprising an Fc-containing protein and one or more host cell proteins (HCPs). The methods generally involve applying the mixture to a chromatography column comprising a protein A chromatography matrix, and washing the chromatography matrix with a sodium caprylate wash buffer.

Description

[0001] background

[0002] The production of recombinant Fc-containing proteins for therapeutic use typically involves expressing the proteins in mammalian cells, followed by purification of these proteins from host cell contaminants. Protein A affinity chromatography is often used as part of the Fc-containing protein purification process because Protein A is able to selectively bind to the Fc region of Fc-containing proteins. Although Protein A affinity chromatography removes a large number of host cell contaminants, a large number of host cell proteins (HCPs), including proteases such as cathepsin D, may still be present after such chromatography. These proteases can cleave or fragment the Fc-containing protein and result in a reduced yield of intact protein.

[0003] Therefore, there is a need for improved methods of purifying Fc-containing proteins using Protein A affinity chromatography to remove HCPs, such as proteases (eg, cathepsin D).

[0004] Overview

[0005] The present disclosure provides an improved method for purifying an Fc-containing protein (e.g., dulaglutide) from a mixture of the Fc-containing protein and one or more host cell proteins (HCPs). The method generally involves applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions in which dulaglutide is bound to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer (e.g., a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate). A particularly advantageous aspect of the methods disclosed herein is that they significantly reduce the level of cathepsin D in the protein A chromatography column eluate compared to other washing methods known in the art and significantly increase the amount of intact Fc-containing protein recovered during the purification process.

[0006] In one aspect, provided herein is a method for purifying dulaglutide from a mixture comprising dulaglutide and one or more host cell proteins (HCPs), the method comprising the steps of: (a) applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the dulaglutide binds to the chromatography matrix; and (b) washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0007] In one embodiment, the sodium octanoate wash buffer comprises 150-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises 200-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises approximately 300 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises approximately 50 mM Tris. In one embodiment, the sodium octanoate wash buffer comprises 0.5-1.5 M NaCl. In one embodiment, the sodium octanoate wash buffer comprises approximately 1 M NaCl. In one embodiment, the sodium octanoate wash buffer has a pH of 7-9. In one embodiment, the sodium octanoate wash buffer has a pH of approximately 8.

[0008] In one embodiment, the method further comprises washing the chromatography matrix with a NaCl wash buffer comprising 0.5-1.5 M NaCl. In one embodiment, the NaCl wash buffer comprises about 1 M NaCl. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris. In one embodiment, the NaCl wash buffer has a pH of 7-9. In one embodiment, the NaCl wash buffer has a pH of about 8.

[0009] In one embodiment, the chromatography column is washed with a NaCl wash buffer prior to washing with the sodium octanoate wash buffer. In one embodiment, the chromatography column is washed with a sodium octanoate wash buffer prior to washing with the NaCl wash buffer.

[0010] In one embodiment, the chromatography matrix is ​​equilibrated with a Tris buffer having a pH of 7-9 between steps (a) and (b). In one embodiment, the Tris buffer comprises 10-100 mM Tris. In one embodiment, the Tris buffer comprises about 50 mM Tris. In one embodiment, the Tris buffer has a pH of about 8.0.

[0011] In one embodiment, the chromatography matrix is ​​washed with 2-10 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 2 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with 2-10 volumes of NaCl wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 5 volumes of NaCl wash buffer.

[0012] In one embodiment, the chromatography matrix is ​​washed sequentially with about 5 column volumes of a NaCl wash buffer comprising about 50 mM Tris and about 1 M NaCl, pH about 8; and about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate, pH about 8.

[0013] In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of 300 ± 30 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a sodium octanoate wash buffer at a flow rate of 300 ± 30 cm / hr.

[0014] In one embodiment, the method further comprises washing the chromatography matrix with a Tris buffer comprising 10-100 mM Tris, optionally comprising about 50 mM Tris, pH 8.0.

[0015] In one embodiment, the method further comprises contacting the chromatography matrix with an elution buffer to prepare an eluate comprising dulaglutide. In one embodiment, the elution buffer has a pH of 2.5-4. In one embodiment, the elution buffer has a pH of about 3. In one embodiment, the elution buffer comprises sodium citrate. In one embodiment, the elution buffer comprises 5-25 mM sodium citrate. In one embodiment, the elution buffer comprises about 10 mM sodium citrate.

[0016] In one embodiment, the eluate is monitored by absorbance using a spectrophotometer. In one embodiment, the eluate comprises at least about 70% of the dulaglutide present in the mixture prior to performing the method.

[0017] In one embodiment, the chromatography column has a diameter of 50-150 cm. In one embodiment, the chromatography column has a diameter of 75-150 cm. In one embodiment, the chromatography column has a diameter of about 100 cm. In one embodiment, the Protein A chromatography matrix has an average particle size of 80-90 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of about 85 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of 50-70 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of about 60 μm.

[0018] In one embodiment, the Protein A chromatography matrix is ​​stable in base.

[0019] In one embodiment, the one or more HCPs are proteases selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, metalloproteases, and aminopeptidases, or a combination thereof. In one embodiment, the one or more HCPs are cathepsin D.

[0020] In one embodiment, the eluate comprises a reduced amount of one or more HCPs compared to the mixture. In one embodiment, the eluate comprises no more than 100 ng / mg of HCPs. In one embodiment, the amount of one or more HCPs is determined by mass spectrometry or ELISA.

[0021] In one embodiment, the eluate comprises a reduced amount of cathepsin D compared to the mixture. In one embodiment, the eluate comprises no more than 100 ng / mg of cathepsin D. In one embodiment, the amount of cathepsin D is determined by mass spectrometry or ELISA. In one embodiment, the amount of cathepsin D is determined by an enzymatic assay. In one embodiment, the amount of cathepsin D is determined by an enzymatic assay in which a fluorogenic substrate for cathepsin D is added to the sample and its cleavage products are determined by HPLC with fluorescence detection.

[0022] In one embodiment, the mass spectrometry is LC-MS.

[0023] In one embodiment, the eluate comprises less than 300 μU / mL of cathepsin D activity.

[0024] In one aspect, provided herein is dulaglutide produced by any of the methods disclosed herein.

[0025] Other embodiments of the present disclosure are described below:

[0026] 1. A method for purifying an Fc-containing protein from a mixture comprising the Fc-containing protein and one or more host cell proteins (HCPs), the method comprising the following steps:

[0027] (a) applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the Fc-containing protein binds to the chromatography matrix; and

[0028] (b) Wash the chromatography matrix with sodium octanoate wash buffer containing 150-500 mM sodium octanoate.

[0029] 2. The method of embodiment 1, wherein the sodium octanoate wash buffer comprises 200-500 mM or 150-350 mM sodium octanoate.

[0030] 3. The method of embodiment 1 or 2, wherein the sodium octanoate wash buffer comprises 200-350 mM sodium octanoate.

[0031] 4. The method of any one of embodiments 1-3, wherein the sodium octanoate wash buffer comprises about 300 mM sodium octanoate.

[0032] 5. The method of any one of embodiments 1-4, wherein the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate.

[0033] 6. The method of any one of embodiments 1-5, wherein the sodium octanoate wash buffer comprises 0.5-1.5 M NaCl.

[0034] 7. The method of any one of embodiments 1-6, wherein the sodium octanoate wash buffer comprises about 1 M NaCl.

[0035] 8. The method of any one of embodiments 1-7, wherein the sodium caprylate wash buffer has a pH of 7-9.

[0036] 9. The method of any one of embodiments 1-8, wherein the sodium caprylate wash buffer has a pH of about 8.

[0037] 10. The method of embodiment 1, further comprising washing the chromatography matrix with a NaCl wash buffer comprising 0.5-1.5 M NaCl.

[0038] 11. The method of embodiment 10, wherein the NaCl wash buffer comprises about 1 M NaCl.

[0039] 12. The method of embodiment 10 or 11, wherein the NaCl wash buffer comprises about 50 mM Tris and about 1 M NaCl.

[0040] 13. The method according to any one of embodiments 10-12, wherein the NaCl wash buffer has a pH of 7-9.

[0041] 14. The method according to any one of embodiments 10-13, wherein the NaCl wash buffer has a pH of about 8.

[0042] 15. The method according to any one of embodiments 10-14, wherein the chromatography column is washed with NaCl wash buffer before washing with sodium octanoate wash buffer.

[0043] 16. The method according to any one of embodiments 10-14, wherein the chromatography column is washed with a sodium octanoate wash buffer before washing with the NaCl wash buffer.

[0044] 17. The method according to any one of embodiments 1 to 16, wherein the chromatography matrix is ​​equilibrated with Tris buffer at pH 7-9 between step (a) and step (b).

[0045] 18. The method of embodiment 17, wherein the Tris buffer comprises 10-100 mM Tris.

[0046] 19. The method of embodiment 17 or 18, wherein the Tris buffer comprises about 50 mM Tris.

[0047] 20. The method according to any one of embodiments 17-19, wherein the Tris buffer has a pH of about 8.0.

[0048] 21. The method according to any one of embodiments 1 to 20, wherein the chromatography matrix is ​​washed with 2 to 10 column volumes of sodium octanoate wash buffer.

[0049] 22. The method according to any one of embodiments 1 to 21, wherein the chromatography matrix is ​​washed with about 2 column volumes of sodium octanoate wash buffer.

[0050] 23. The method according to any one of embodiments 10-22, wherein the chromatography matrix is ​​washed with 2-10 volumes of NaCl wash buffer.

[0051] 24. The method according to any one of embodiments 10-23, wherein the chromatography matrix is ​​washed with about 2 or about 5 volumes of NaCl wash buffer.

[0052] 25. The method of embodiment 1, wherein the chromatography matrix is ​​washed sequentially with:

[0053] (a) about 2 column volumes of NaCl wash buffer comprising about 50 mM Tris and about 1 M NaCl, pH about 8; and

[0054] (b) About 2 column volumes of sodium octanoate wash buffer, which contains about 50 mM Tris and about 300 mM sodium octanoate, pH about 8.

[0055] 26. The method according to any one of embodiments 10-25, wherein the chromatography matrix is ​​washed with NaCl wash buffer at a flow rate of 300±30 cm / hr.

[0056] 27. The method according to any one of embodiments 1 to 26, wherein the chromatography matrix is ​​washed with sodium octanoate wash buffer at a flow rate of 300±30 cm / hr.

[0057] 28. The method according to any one of embodiments 1-27, further comprising washing the chromatography matrix with a Tris buffer comprising 10-100 mM Tris, optionally wherein the Tris buffer comprises about 50 mM Tris, pH about 8.0.

[0058] 29. The method according to any one of embodiments 1 to 28, further comprising contacting the chromatography matrix with an elution buffer to produce an eluate comprising the Fc-containing protein.

[0059] 30. The method according to embodiment 29, wherein the elution buffer has a pH of 2.5-4.

[0060] 31. The method of embodiment 29 or 30, wherein the elution buffer has a pH of about 3.

[0061] 32. The method according to any one of embodiments 29-31, wherein the elution buffer comprises sodium citrate.

[0062] 33. The method according to any one of embodiments 29-32, wherein the elution buffer comprises 5-25 mM sodium citrate.

[0063] 34. The method according to any one of embodiments 29-33, wherein the elution buffer comprises about 10 mM sodium citrate.

[0064] 35. The method according to any one of embodiments 29 to 34, wherein the eluate is monitored by absorbance using a spectrophotometer.

[0065] 36. The method of any one of embodiments 29-35, wherein the eluate comprises at least 70% of the Fc-containing proteins present in the mixture.

[0066] 37. The method according to any one of embodiments 1-36, wherein the chromatography column has a diameter of 75-150 cm.

[0067] 38. The method according to any one of embodiments 1-37, wherein the chromatography column has a diameter of about 100 cm.

[0068] 39. The method according to any one of embodiments 1-38, wherein the Protein A chromatography matrix has an average particle size of 80-90 μm.

[0069] 40. The method of any one of embodiments 1-39, wherein the Protein A chromatography matrix has an average particle size of about 85 μm.

[0070] 41. The method according to any one of embodiments 1 to 40, wherein the Protein A chromatography matrix is ​​stable in base.

[0071] 42. The method according to any one of embodiments 1 to 41, wherein the one or more HCPs is a protease selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, metalloproteases and aminopeptidases, or a combination thereof.

[0072] 43. The method of embodiments 1-42, wherein the one or more HCPs is cathepsin D.

[0073] 44. The method of any one of embodiments 29-43, wherein the eluate comprises a reduced amount of one or more HCPs compared to the mixture.

[0074] 45. The method of any one of embodiments 29-44, wherein the eluate comprises no more than 100 ng / mg of HCP.

[0075] 46. ​​The method of embodiment 43 or 44, wherein the one or more HCPs are determined by mass spectrometry or ELISA.

[0076] 47. The method according to any one of embodiments 29-46, wherein the eluate comprises a reduced amount of cathepsin D compared to the mixture.

[0077] 48. The method according to any one of embodiments 29-47, wherein the eluate comprises no more than 100 ng / mg cathepsin D.

[0078] 49. The method according to embodiment 47 or 48, wherein the amount of cathepsin D is determined by mass spectrometry or ELISA.

[0079] 50. The method of embodiment 46 or 49, wherein the mass spectrometry is LC-MS.

[0080] 51. The method of any one of embodiments 29-50, wherein the eluate comprises less than 300 μU / mL of cathepsin D activity.

[0081] 52. The method according to any one of embodiments 1-51, wherein the Fc-containing protein is not an antibody.

[0082] 53. The method of any one of embodiments 1-52, wherein the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog comprising one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0083] 54. The method of any one of embodiments 1-53, wherein the Fc-containing protein comprises a GLP-1 analogue comprising the amino acid sequence of SEQ ID NO: 2.

[0084] 55. The method of any one of embodiments 1-54, wherein the Fc-containing protein comprises a peptide linker.

[0085] 56. The method according to embodiment 55, wherein the peptide linker comprises 1-10 G4S units (SEQ ID NO: 3).

[0086] 57. The method of any one of embodiments 1-56, wherein the Fc-containing protein comprises:

[0087] (a) a GLP-1 analogue comprising the amino acid sequence of SEQ ID NO: 2;

[0088] (b) a peptide linker comprising the amino acid sequence of SEQ ID NO: 3; and

[0089] c) Fc portion of an immunoglobulin.

[0090] 58. The method of embodiment 57, wherein the N-terminal residue of the peptide linker is directly fused to the C-terminal residue of the GLP-1 analogue, and the C-terminal residue of the peptide linker is directly fused to the N-terminal residue of the Fc portion.

[0091] 59. The method of any one of embodiments 1-58, wherein the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 4.

[0092] 60. The method according to any one of embodiments 1-59, wherein the Fc-containing protein is a homodimer comprising two identical amino acid chains, each amino acid chain comprising the amino acid sequence of SEQ ID NO: 4.

[0093] 61. The method according to any one of embodiments 1-60, wherein the Fc-containing protein is dulaglutide.

[0094] 62. An Fc-containing protein prepared by the method of any of the above claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] Figure 1 UV chromatograms of the flow-through from washes on a Protein A column using the indicated buffer solutions. Prior to the wash step, the Protein A column was preloaded with a mixture containing dulaglutide and host cell proteins.

[0097] Figure 2 Graph showing HCP content (ng / mg) as assessed by ELISA, HCP content per unit dulaglutide (ppm) as assessed by LC-MS, and cathepsin D activity (µU / mL) in the dulaglutide eluate after the dulaglutide mixture was preloaded onto a Protein A column and washed with 50 mM Tris, pH 8.0 buffer containing the indicated concentrations of sodium octanoate. Wash conditions were: 5 CV 50 mM Tris, pH 8.0, then 5 CV 50 mM Tris, 1 M NaCl, pH 8, then 5 CV 50 mM Tris, X mM (300, 200, 100, 50 mM, as indicated in the graph) sodium octanoate, pH 8, followed by 5 CV 50 mM Tris, pH 8.0.

[0098] Figure 3Graph showing HCP content (ng / mg) as assessed by ELISA, HCP content per unit dulaglutide (ppm) as assessed by LC-MS, and cathepsin D activity (µU / mL) in the dulaglutide eluate after the dulaglutide mixture was preloaded onto a Protein A column and washed with the indicated combinations or a series of washes with NaCl wash buffer and sodium octanoate wash buffer. The first wash was identical for all conditions tested and consisted of 5 column volumes of 50 mM Tris, pH 8.0.

[0099] Figure 4 Graph showing HCP content (ng / mg) as assessed by ELISA, HCP content per unit dulaglutide (ppm) as assessed by LC-MS, and cathepsin D activity (µU / mL) in the dulaglutide eluate after preloading the dulaglutide mixture onto a Protein A column followed by two washes: the first wash was with 5 column volumes (CV) of 50 mM Tris, pH 8.0, and the second wash was with 5 CV of wash buffer containing 1 M NaCl (as a control) or the indicated CVs of wash buffer containing 300 mM sodium octanoate. Control conditions were 5 CV 50 mM Tris, pH 8.0, followed by 5 CV 50 mM Tris, 1 M NaCl, pH 8, then 5 CV 50 mM Tris, pH 8.0; 5 CV wash, 3 CV wash, and 2 CV wash conditions were 5 CV 50 mM Tris, pH 8.0, then X CV (5, 3, 2 CV as indicated in the figure) 50 mM Tris, 1 M NaCl, pH 8, then X CV 50 mM Tris, 300 mM sodium octanoate, pH 8, then X CV 50 mM Tris, pH 8.0.

[0100] Detailed description

[0101] Provided herein are methods for purifying an Fc-containing protein (e.g., dulaglutide) from a mixture of an Fc-containing protein and one or more host cell proteins (e.g., HCP). The method generally involves applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions in which dulaglutide is bound to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer (e.g., a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate). A particularly advantageous aspect of the methods disclosed herein is that they significantly reduce the level of cathepsin D in the protein A chromatography column eluate compared to other washing methods known in the art and significantly increase the amount of intact Fc-containing protein recovered during the purification process.

[0102] I. Definitions

[0103] As used herein, the term "Fc-containing protein" refers to a protein that comprises an Fc region. In one embodiment, the Fc-containing protein comprises a variable Fc region that comprises one or more amino acid substitutions, additions, and / or deletions relative to a naturally occurring Fc region. In one embodiment, the Fc-containing protein is not an antibody.

[0104] As used herein, the term "contaminant" refers to any material, particularly biomacromolecules such as DNA, RNA, or protein, other than the recombinantly produced Fc-containing protein present in the mixture. Contaminants include, but are not limited to, cellular and viral proteins or nucleic acids produced during the production of the Fc-containing protein, or their byproducts. Contaminants also include any host cell protein (HCP), host cell nucleic acid, or host cell fragment produced at any stage of the Fc-containing protein production process.

[0105] As used herein, the terms "host cell protein" and "HCP" refer to any undesired protein derived from the cells (eg, mammalian cells) used to produce the Fc-containing protein.

[0106] As used herein, the term "purification" refers to reducing the amount of contaminants (e.g., HCPs) in a composition comprising an Fc-containing protein. Purification may or may not result in the complete removal of contaminants from the composition. In certain embodiments, purification refers to a reduction in contaminants of at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold.

[0107] As used herein, the term "eluate" refers to a solution containing an Fc-containing protein (e.g., dulaglutide) that has been purified according to the methods disclosed herein. In one embodiment, the eluate may further contain one or more HCPs. In one embodiment, the eluate contains a lower amount of HCPs than the mixture.

[0108] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above.

[0109] As used herein, the term "about" when referring to a value or parameter herein includes a variability of ±5% of that value or parameter. For example, when referring to a pH value, "about" means a range that includes values ​​5% below the referenced value and values ​​5% above the referenced value. Thus, a pH of about 10 refers to a pH that includes pH 9.5 to pH 10.5 (inclusive).

[0110] II. Protein A Chromatography Method

[0111] One challenge in downstream processing of Fc-containing proteins, such as dulaglutide, is effectively separating the Fc-containing protein from contaminants and impurities, such as host cell proteins (HCPs). In particular, during the preparation of dulaglutide, residual cathepsin D can trap the dulaglutide product and result in a reduced yield of intact dulaglutide. The methods disclosed herein significantly reduce the levels of HCPs, including cathepsin D, in the eluate from a Protein A chromatography column and significantly increase the amount of intact Fc-containing protein recovered during purification.

[0112] In one aspect, provided herein is a method for purifying an Fc-containing protein from a mixture comprising the Fc-containing protein and one or more host cell proteins (HCPs), the method comprising the steps of: applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the Fc-containing protein binds to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0113] Typically, a Protein A chromatography method comprises the following steps, in sequence: preparing a loading composition comprising a mixture of an Fc-containing protein and one or more HCPs, applying the loading composition to a chromatography column comprising a Protein A chromatography matrix, washing the Protein A chromatography matrix, and eluting the Fc-containing protein. It will be appreciated by those skilled in the art that the Protein A chromatography method may include additional intermediate steps and / or additional steps before or after the Protein A purification process, depending on the desired purpose and results.

[0114] In one aspect, provided herein is a method for purifying an Fc-containing protein from a mixture of the Fc-containing protein and one or more contaminants, the method comprising the steps of: applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the Fc-containing protein binds to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0115] In one embodiment, the method further comprises washing the Protein A chromatography matrix with a NaCl wash buffer comprising 0.5-1.5 M NaCl.

[0116] In one embodiment, the chromatography column is washed with a NaCl wash buffer before or after washing the chromatography matrix with a sodium octanoate wash buffer. In one embodiment, NaCl and sodium octanoate wash buffers are combined. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer followed by a sodium octanoate wash buffer.

[0117] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with a NaCl buffer comprising 0.5-1.5 M NaCl, and then washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0118] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 1 M NaCl, followed by washing the chromatography matrix with about 2 column volumes of a sodium octanoate wash buffer comprising about 300 mM sodium octanoate.

[0119] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl, followed by washing the chromatography matrix with about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate.

[0120] In one embodiment, the method further comprises equilibrating the chromatography matrix with a Tris buffer having a pH of 7-9 after applying the mixture to the chromatography column. In one embodiment, the Tris buffer comprises 10-100 mM Tris. In one embodiment, the Tris buffer comprises about 50 mM Tris. In one embodiment, the Tris buffer has a pH of about 8.0.

[0121] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; equilibrating the chromatography matrix with a Tris buffer comprising about 50 mM Tris, pH about 8.0; and washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl, followed by washing the chromatography matrix with about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate.

[0122] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of: applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; equilibrating the chromatography matrix with a Tris buffer comprising about 50 mM Tris, pH about 8.0; washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl, followed by about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate; and contacting the chromatography matrix with an elution buffer comprising about 10 mM sodium citrate, pH about 3.0, to prepare an eluate comprising dulaglutide.

[0123] In one aspect, provided herein is a method for purifying an Fc-containing protein from a mixture of the Fc-containing protein and one or more HCPs, the method comprising the steps of: applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions in which the Fc-containing protein is bound to the chromatography matrix; equilibrating the chromatography matrix with a Tris buffer comprising about 50 mM Tris, pH about 8.0; washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl, followed by about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate; and contacting the chromatography matrix with an elution buffer comprising about 10 mM sodium citrate, pH about 3.0, to prepare an eluate comprising the Fc-containing protein.

[0124] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of: applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; equilibrating the chromatography matrix with a Tris buffer comprising about 50 mM Tris, pH about 8.0; washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl, pH about 8.0, followed by about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate, pH about 8.0; and contacting the chromatography matrix with an elution buffer comprising about 10 mM sodium citrate, pH about 3.0, to prepare an eluate comprising dulaglutide.

[0125] Protein A chromatography

[0126] The methods provided herein generally comprise applying a mixture of an Fc-containing protein and one or more contaminants to a chromatography column comprising a Protein A chromatography matrix under conditions that allow the Fc-containing protein to bind to the Protein A chromatography matrix, and washing the Protein A chromatography matrix with a sodium octanoate wash buffer (e.g., a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate). The methods disclosed herein purify an Fc-containing protein (e.g., dulaglutide) from one or more contaminants (e.g., HCPs).

[0127] Contaminants can be any material present at any stage of the methods disclosed herein other than the desired Fc-containing protein. Contaminants include, but are not limited to, viral and cellular proteins or nucleic acids produced during the preparation of the Fc-containing protein, or their byproducts. Contaminants also include any undesirable byproducts of the Fc-containing protein (e.g., fragments of the Fc-containing protein).

[0128] In one aspect, the methods of the present disclosure provide improved methods for purifying a desired Fc-containing protein from one or more HCPs, resulting in an eluate containing lower amounts of HCPs compared to the mixture applied to a Protein A chromatography matrix.

[0129] In one aspect, the methods of the present disclosure provide improved methods for purifying dulaglutide from one or more HCPs, resulting in an eluate containing lower amounts of HCPs compared to the mixture applied to a Protein A chromatography matrix.

[0130] In one aspect, the methods disclosed herein utilize a Protein A chromatography matrix.

[0131] In one embodiment, the chromatography column has a diameter of 10-150 cm. In one embodiment, the chromatography column has a diameter of about 1 cm, about 2 cm, about 3 cm, about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 15 cm, about 20 cm, about 25 cm, about 30 cm, about 35 cm, about 40 cm, about 45 cm, about 50 cm, about 55 cm, about 60 cm, about 65 cm, about 70 cm, about 75 cm, about 80 cm, about 85 cm, about 90 cm, about 95 cm, about 100 cm, about 110 cm, about 120 cm, about 130 cm, about 140 cm, or about 150 cm.

[0132] In one embodiment, the chromatography column has a bed height of 10-40 cm. In one embodiment, the chromatography column has a bed height of about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm, about 19 cm, about 20 cm, about 21 cm, about 22 cm, about 23 cm, about 24 cm, about 25 cm, about 26 cm, about 27 cm, about 28 cm, about 29 cm, about 30 cm, about 30.5 cm, about 31 cm, about 31.5 cm, about 32 cm, about 32.5 cm, about 33 cm, about 33.5 cm, about 34 cm, about 34.5 cm, about 35 cm, about 35.5 cm, about 36 cm, about 36.5 cm, about 37 cm, about 37.5 cm, about 38 cm, about 38.5 cm, about 39 cm, about 39.5 cm, or about 40 cm.

[0133] In one embodiment, the chromatography column is loaded at a temperature of about 10-40°C. In one embodiment, the chromatography column is loaded at a temperature of about 15-35°C. In one embodiment, the chromatography column is loaded at a temperature of about 15-30°C.

[0134] In one embodiment, the Protein A chromatography matrix has an average particle size of 80-90 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of about 80 μm, about 81 μm, about 82 μm, about 83 μm, about 84 μm, about 85 μm, about 86 μm, about 87 μm, about 88 μm, about 89 μm, or about 90 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of about 85 μm.

[0135] In one embodiment, the Protein A chromatography matrix has an average particle size of 50-70 μm. In one embodiment, the Protein A chromatography matrix has an average particle size of about 60 μm.

[0136] In one embodiment, the Protein A chromatography matrix is ​​stable in alkali. In one embodiment, the Protein A chromatography matrix comprises an engineered Protein A variant that is more stable in alkali than wild-type Protein A. In one embodiment, the Protein A chromatography matrix comprises an engineered variant of Protein A that has been modified to replace specific amino acids that are sensitive to alkali with amino acids that are more stable in alkali.

[0137] A variety of Protein A chromatography matrices suitable for use with the methods disclosed herein can be utilized. The Protein A chromatography matrix can have a variety of backbone compositions, including, for example, glass or silica-based matrices, agarose-based matrices, and organic polymer-based matrices. In one embodiment, the Protein A chromatography matrix comprises a modified variant of Protein A. In one embodiment, the Protein A amino acid sequence comprises a C-terminal cysteine ​​for cross-linking to the matrix. In one embodiment, the Protein A chromatography matrix is ​​an agarose matrix. In one embodiment, the Protein A chromatography matrix comprises a Protein A tetramer cross-linked to the agarose matrix via a C-terminal cysteine ​​on the Protein A. In one embodiment, the Protein A chromatography matrix comprises a Protein A tetramer cross-linked to the agarose matrix via an epoxy bond.

[0138] In one embodiment, the Protein A chromatography matrix is ​​MabSelect from Cytiva (Marlborough, MA). TM Protein A chromatography matrix. In one embodiment, MabSelect TM Protein A chromatography matrix is ​​MabSelectSuRe TM 、MabSelect SuRe TM LX, MabSelect SuRe TM pcc or MabSelect PrismA TM .

[0139] In one embodiment, the present disclosure provides a composition comprising a Protein A chromatography matrix and a mixture comprising an Fc-containing protein and one or more contaminants. In one embodiment, the present disclosure provides a composition comprising a Protein A chromatography matrix and a mixture comprising dulaglutide and one or more HCPs. Various components of the mixture are further described herein.

[0140] mixture

[0141] The methods of the present disclosure comprise contacting a mixture of an Fc-containing protein and at least one contaminant with a Protein A chromatography matrix under conditions such that the Fc-containing protein binds to the Protein A chromatography matrix.

[0142] In one aspect, the method of the present disclosure comprises contacting a mixture of dulaglutide and at least one contaminant with a Protein A chromatography matrix under conditions such that an Fc-containing protein binds to the Protein A chromatography matrix.

[0143] In one embodiment, the contaminant is one or more HCPs.

[0144] In one embodiment, the contaminant is leached Protein A, host cell nucleic acid, a fragment of an Fc-containing protein, an aggregate of an Fc-containing protein, or a derivative of an Fc-containing protein, endotoxin, a viral contaminant, or a cell culture medium component.

[0145] The methods of the present disclosure comprise contacting a mixture of an Fc-containing protein and one or more HCPs with a Protein A chromatography matrix under conditions that allow the Fc-containing protein to bind to the Protein A chromatography matrix.

[0146] In one aspect, the method of the present disclosure comprises contacting a mixture of dulaglutide and one or more HCPs with a Protein A chromatography matrix under conditions such that an Fc-containing protein binds to the Protein A chromatography matrix.

[0147] In one embodiment, the one or more HCPs are proteases selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, metalloproteases, and aminopeptidases, or combinations thereof.

[0148] In one embodiment, the HCP is selected from protein S100-A6, lysosomal acid lipase / cholesteryl ester hydrolase, CC motif chemokine 2, phospholipid transfer protein isoform X2, sulfhydryl oxidase 1 isoform X1, farnesyl pyrophosphate synthase isoform X1, retinoid-inducible serine carboxypeptidase isoform X1, T-complex protein 1 subunit δ, 60S ribosomal protein L18 isoform X1, cytoplasmic dynein 1 heavy chain 1 isoform X1, clathrin heavy chain 1 isoform X1, metalloproteinase inhibitor 1, pigment epithelium-derived factor isoform X1, acid ceramidase isoform X1, coat subunit β isoform X1, 60S ribosomal protein L10a isoform X1, dynamin-related NEDD8-dissociation protein 1, α-L-iduronidase isoform X1, and anti-torsion protein-1B-like isoform X1. In one embodiment, the contaminant HCP is cathepsin D.

[0149] In one embodiment, the host cell protein is from a mammalian host cell. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK) cell, a murine hybridoma cell, a HEK cell, or a murine myeloma cell.

[0150] Wash buffers and methods

[0151] After applying the mixture to a chromatography column comprising a Protein A chromatography matrix, the Protein A chromatography matrix is ​​washed with one or more wash buffers to remove one or more contaminants. In one aspect, after applying the mixture comprising dulaglutide and one or more HCPs to a chromatography column comprising a Protein A chromatography matrix, the Protein A chromatography matrix is ​​washed with one or more wash buffers to remove one or more HCPs. In one embodiment, the Protein A chromatography matrix is ​​washed with a sodium octanoate wash buffer (e.g., a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate).

[0152] Sodium caprylate (also known as sodium octanoate) is an eight-carbon saturated fatty acid (chemical formula: C8H 15 NaO2).

[0153] In one aspect, provided herein is a method for purifying an Fc-containing protein from a mixture of the Fc-containing protein and one or more contaminants, the method comprising the steps of: applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the Fc-containing protein binds to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0154] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more contaminants, the method comprising the steps of applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate.

[0155] In one embodiment, the sodium octanoate wash buffer comprises about 150-500 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 200-500 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 150-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 200-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 250-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 275-325 mM sodium octanoate.

[0156] In one embodiment, the sodium octanoate wash buffer comprises about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM, about 480 mM, about In one embodiment, the sodium octanoate wash buffer comprises about 300 mM sodium octanoate.

[0157] In one embodiment, the sodium octanoate wash buffer comprises about 10-100 mM Tris. In one embodiment, the sodium octanoate wash buffer comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM Tris. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris.

[0158] In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 150-500 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 200-500 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 150-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 200-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 250-350 mM sodium octanoate. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 275-325 mM sodium octanoate.

[0159] In one embodiment, a sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM, about 300 mM, about 310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about 360 mM, about 370 mM, about 380 mM, about 390 mM, about 400 mM, about 410 mM, about 420 mM, about 430 mM, about 440 mM, about 450 mM, about 460 mM, about 470 mM In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate.

[0160] In one embodiment, the pH of the sodium caprylate wash buffer is from about 7 to about 9. In one embodiment, the pH of the sodium caprylate wash buffer is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In one embodiment, the pH of the sodium caprylate wash buffer is about 8.

[0161] In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM sodium octanoate and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 200 mM sodium octanoate and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 250 mM sodium octanoate and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 350 mM sodium octanoate and has a pH of about 8.

[0162] In one embodiment, the sodium octanoate wash buffer comprises about 0.1-2 M NaCl. In one embodiment, the sodium octanoate wash buffer comprises about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M, or about 2.0 M NaCl. In one embodiment, the sodium octanoate wash buffer comprises about 1 M NaCl.

[0163] In one embodiment, the Protein A chromatography matrix is ​​washed with a NaCl wash buffer comprising 0.5-1.5 M NaCl and a sodium octanoate wash buffer comprising 150-500 mM sodium octanoate. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer before or after washing the chromatography matrix with the sodium octanoate wash buffer.

[0164] In one embodiment, the NaCl wash buffer comprises about 0.1-2 M NaCl. In one embodiment, the NaCl wash buffer comprises about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M or about 2.0 M NaCl. In one embodiment, the NaCl wash buffer comprises about 1 M NaCl.

[0165] In one embodiment, the NaCl wash buffer comprises about 10-100 mM Tris. In one embodiment, the NaCl wash buffer comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 mM Tris. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris.

[0166] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.1-2 M NaCl. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.1 M, about 0.2 M, about 0.3 M, about 0.4 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.8 M, about 0.9 M, about 1.0 M, about 1.1 M, about 1.2 M, about 1.3 M, about 1.4 M, about 1.5 M, about 1.6 M, about 1.7 M, about 1.8 M, about 1.9 M or about 2.0 M NaCl. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1 M NaCl.

[0167] In one embodiment, the pH of the NaCl wash buffer is about 7 to about 9. In one embodiment, the pH of the NaCl wash buffer is about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, or about 9. In one embodiment, the pH of the NaCl wash buffer is about 8.

[0168] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8. In one embodiment, the sodium octanoate wash buffer comprises about 50 mM Tris and about 1.1 M NaCl and has a pH of about 8.

[0169] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.1 M NaCl, and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 150 mM sodium octanoate, and has a pH of about 8.

[0170] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 200 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 200 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 200 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.1 M NaCl, and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 200 mM sodium octanoate, and has a pH of about 8.

[0171] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 250 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 250 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 250 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.1 M NaCl, and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 250 mM sodium octanoate, and has a pH of about 8.

[0172] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.1 M NaCl, and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 300 mM sodium octanoate, and has a pH of about 8.

[0173] In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.8 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 350 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 0.9 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 350 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.0 M NaCl and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 350 mM sodium octanoate and has a pH of about 8. In one embodiment, the NaCl wash buffer comprises about 50 mM Tris and about 1.1 M NaCl, and has a pH of about 8; and the sodium octanoate wash buffer comprises about 50 mM Tris and about 350 mM sodium octanoate, and has a pH of about 8.

[0174] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer.

[0175] In one embodiment, the chromatography matrix is ​​equilibrated with Tris buffer having a pH of about 7-9 after applying the mixture to the chromatography column and before washing the chromatography matrix.

[0176] In one embodiment, the Tris buffer comprises about 10-100 mM Tris. In one embodiment, the Tris buffer comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM or about 100 mM Tris. In one embodiment, the Tris buffer comprises about 50 mM Tris.

[0177] In one embodiment, the Tris buffer comprises about 10-100 mM Tris and has a pH of about 8. In one embodiment, the Tris buffer comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM Tris and has a pH of about 8. In one embodiment, the Tris buffer comprises about 50 mM Tris and has a pH of about 8.

[0178] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of sodium octanoate wash buffer.

[0179] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 1 column volume of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 1 column volume of sodium octanoate wash buffer.

[0180] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 2 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 2 column volumes of sodium octanoate wash buffer.

[0181] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 3 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 3 column volumes of sodium octanoate wash buffer.

[0182] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 4 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 4 column volumes of sodium octanoate wash buffer.

[0183] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 5 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 5 column volumes of sodium octanoate wash buffer.

[0184] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 6 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 6 column volumes of sodium octanoate wash buffer.

[0185] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 7 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 7 column volumes of sodium octanoate wash buffer.

[0186] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 8 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 8 column volumes of sodium octanoate wash buffer.

[0187] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 9 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 9 column volumes of sodium octanoate wash buffer.

[0188] In one embodiment, the chromatography matrix is ​​washed with about 1-10 column volumes of NaCl wash buffer followed by about 10 column volumes of sodium octanoate wash buffer. In one embodiment, the chromatography matrix is ​​washed with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 column volumes of NaCl wash buffer followed by about 10 column volumes of sodium octanoate wash buffer.

[0189] In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of about 100-400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of about 100 cm / hr, about 150 cm / hr, about 200 cm / hr, about 250 cm / hr, about 300 cm / hr, about 350 cm / hr, or about 400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of 300±30 cm / hr.

[0190] In one embodiment, the chromatography matrix is ​​washed with a sodium octanoate wash buffer at a flow rate of about 100-400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a sodium octanoate wash buffer at a flow rate of about 100 cm / hr, about 150 cm / hr, about 200 cm / hr, about 250 cm / hr, about 300 cm / hr, about 350 cm / hr, or about 400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a sodium octanoate wash buffer at a flow rate of 300±30 cm / hr.

[0191] In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of 300 ± 30 cm / hr and a sodium octanoate wash buffer at a flow rate of about 100-400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of 300 ± 30 cm / hr and a sodium octanoate wash buffer at a flow rate of about 100 cm / hr, about 150 cm / hr, about 200 cm / hr, about 250 cm / hr, about 300 cm / hr, about 350 cm / hr, or about 400 cm / hr. In one embodiment, the chromatography matrix is ​​washed with a NaCl wash buffer at a flow rate of 300 ± 30 cm / hr and a sodium octanoate wash buffer at a flow rate of 300 ± 30 cm / hr.

[0192] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that the dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 1 M NaCl, followed by about 2 column volumes of a sodium octanoate wash buffer comprising about 300 mM sodium octanoate, thereby purifying dulaglutide from the one or more HCPs in the mixture.

[0193] In one aspect, provided herein is a method for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs, the method comprising the steps of applying the mixture to a chromatography column comprising a protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; and washing the chromatography matrix with about 2 column volumes of a NaCl buffer comprising about 50 mM Tris and about 1 M NaCl at a flow rate of 300±30 cm / hr, followed by about 2 column volumes of a sodium octanoate wash buffer comprising about 50 mM Tris and about 300 mM sodium octanoate at a flow rate of 300±30 cm / hr, thereby purifying dulaglutide from the one or more HCPs in the mixture.

[0194] In any of the methods disclosed herein, the method further comprises washing the chromatography matrix with a Tris buffer comprising 10-100 mM Tris after washing with the sodium octanoate buffer. In one embodiment, the Tris buffer comprises about 10-100 mM Tris and has a pH of about 8. In one embodiment, the Tris buffer comprises about 10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM Tris and has a pH of about 8. In one embodiment, the Tris buffer comprises about 50 mM Tris and has a pH of about 8.

[0195] Elution from Protein A

[0196] The method of the present disclosure further comprises eluting the Fc-containing protein from the Protein A chromatography matrix. The Fc-containing protein bound to the Protein A chromatography matrix can be eluted using an acid or a combination of acids (eg, a weak acid and a strong acid).

[0197] The Fc-containing protein (eg, dulaglutide) bound to the Protein A chromatography matrix is ​​eluted by contacting the Protein A chromatography matrix with an elution buffer to prepare an eluate.

[0198] In one embodiment, the Protein A chromatography matrix is ​​washed with a Tris buffer comprising about 50 mM Tris, pH about 8.0, prior to the elution step.

[0199] In one embodiment, the elution buffer comprises sodium citrate. In one embodiment, the elution buffer comprises about 5-25 mM sodium citrate. In one embodiment, the elution buffer comprises about 10 mM sodium citrate. In one embodiment, the elution buffer comprises about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM or about 25 mM sodium citrate.

[0200] In one embodiment, the elution buffer has a pH of about 2.5 to 4. In one embodiment, the elution buffer has a pH of about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0.

[0201] In one embodiment, the elution buffer comprises about 10 mM sodium citrate and has a pH between 2.5 and 4. In one embodiment, the elution buffer comprises about 10 mM sodium citrate and has a pH of about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, or about 4.0.

[0202] Those skilled in the art will appreciate that the elution buffer may contain additional components that aid in the elution of the Fc-containing protein from the Protein A chromatography matrix. These components may include additional buffers and additives that aid, for example, dissociation and solubilization.

[0203] In one embodiment, the eluate is monitored by absorbance using a spectrophotometer.

[0204] In one embodiment, the elution buffer comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of the Fc-containing protein in the mixture. For example, the percent recovery can be determined by calculating the percentage of Fc-containing protein in the eluate relative to the amount in the mixture applied to the chromatography column.

[0205] In one embodiment, the eluate comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% of the dulaglutide in the mixture. For example, the percent recovery can be determined by calculating the percentage of dulaglutide in the eluate relative to the amount in the mixture applied to the chromatography column.

[0206] In one embodiment, the eluate is applied to another chromatography matrix to further purify dulaglutide from any remaining contaminants (e.g., HCPs). In one embodiment, the eluate is applied to an anion exchange (AEX) chromatography matrix, size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography.

[0207] Host cell protein detection

[0208] The methods provided herein result in a significant reduction in host cell proteins (HCPs) following Protein A purification of Fc-containing proteins (eg, dulaglutide) compared to other methods known in the art.

[0209] In one embodiment, the one or more HCPs include a protease selected from the group consisting of a serine protease, an aspartic protease, a cysteine ​​protease, a metalloprotease, and an aminopeptidase, or a combination thereof.

[0210] In one embodiment, the HCP is selected from protein S100-A6, lysosomal acid lipase / cholesteryl ester hydrolase, CC motif chemokine 2, phospholipid transfer protein isoform X2, sulfhydryl oxidase 1 isoform X1, farnesyl pyrophosphate synthase isoform X1, retinoid-inducible serine carboxypeptidase isoform X1, T-complex protein 1 subunit δ, 60S ribosomal protein L18 isoform X1, cytoplasmic dynein 1 heavy chain 1 isoform X1, clathrin heavy chain 1 isoform X1, inhibitor of metalloproteinase 1, pigment epithelium-derived factor isoform X1, acid ceramidase isoform X1, coat subunit β isoform X1, 60S ribosomal protein L10a isoform X1, dynamin-associated NEDD8-dissociating protein 1, α-L-iduronidase isoform X1, antitorsion protein-1B-like isoform X1 and cathepsin D. In one embodiment, the HCP is cathepsin D.

[0211] Methods for determining the concentration of host cell proteins (HCPs) are known in the art. In one embodiment, an immunoassay is used to detect the amount of HCPs in the eluate. In one embodiment, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In an HCP ELISA, primary antibodies are specific for HCPs produced in specific host cells, such as CHO cells, that are used to generate Fc-containing proteins. In one embodiment, the ELISA is a Gyrolab assay. ® CHO-HCP Kit 1 (Cygnus Technologies, Warren, NJ) ELISA assay.

[0212] In one embodiment, by the amount of HCP in mass spectrometry eluent.In one embodiment, mass spectral analysis is LC-MS.In LC-MS measures, by peptide mapping / LC-MS / MS HCP collection of illustrative plates, by the ultra high performance liquid chromatography (UPLC) analysis sample of for example coupling ThermoScientific mass spectrometer.In this analysis, make sample experience trypsin digestion, use dithiothreitol (DTT) reduction / precipitation, transfer subsequently and the supernatant in acidifying HPLC bottle carries out LC-MS / MS analysis.LC-MS / MS data can be analyzed by Proteome Discoverer for CHO-K1 protein database.HCP content is reported as the total parts per million (ppm) of HCP of each sample in HCP total content (such as ng of HCP / mg product).

[0213] Methods for determining the level of cathepsin D activity in the eluate are known in the art. Generally, any assay that can reliably detect cathepsin D activity can be used.

[0214] In one embodiment, the eluate comprises a reduced amount of one or more HCPs compared to the mixture. In one embodiment, the eluate comprises a reduced amount of cathepsin D compared to the mixture. In one embodiment, the eluate comprises reduced cathepsin D activity compared to the mixture.

[0215] In one embodiment, the eluate comprises less than about 100 ng / mg of HCP. In one embodiment, the eluate comprises less than about 90 ng / mg, less than about 80 ng / mg, less than about 70 ng / mg, less than about 60 ng / mg, less than about 50 ng / mg, or less than about 40 ng / mg.

[0216] In one embodiment, the eluent comprises less than about 100 ppm, less than about 90 ppm, less than about 80 ppm, less than about 70 ppm, less than about 60 ppm, less than about 50 ppm, less than about 40 ppm, less than about 30 ppm, or less than about 20 ppm of HCP.

[0217] In one embodiment, the eluate comprises less than about 90 ng / mg, less than about 80 ng / mg, less than about 70 ng / mg, less than about 60 ng / mg, less than about 50 ng / mg, less than about 40 ng / mg, less than about 30 ng / mg, less than about 20 ng / mg, less than about 10 ng / mg, less than about 9 ng / mg, less than about 8 ng / mg, less than about 7 ng / mg, less than about 6 ng / mg, less than about 5 ng / mg, less than about 4 ng / mg, less than about 3 ng / mg, less than about 2 ng / mg, or less than about 1 ng / mg of cathepsin D.

[0218] In one embodiment, the eluate comprises less than about 100 ppm, less than about 90 ppm, less than about 80 ppm, less than about 70 ppm, less than about 60 ppm, less than about 50 ppm, less than about 40 ppm, less than about 30 ppm, or less than about 20 ppm of cathepsin D.

[0219] In one embodiment, the amount of HCP is determined by mass spectrometry or ELISA. In one embodiment, the amount of cathepsin D is determined by mass spectrometry or ELISA. In one embodiment, the mass spectrometry is LC-MS.

[0220] In one embodiment, the eluate comprises less than 300 μU / mL of cathepsin D activity. In one embodiment, the eluate comprises less than 350 μU / mL, less than 325 μU / mL, less than 300 μU / mL, less than 275 μU / mL, less than 250 μU / mL, less than 225 μU / mL, less than 200 μU / mL, less than 175 μU / mL, less than 150 μU / mL, less than 125 μU / mL, less than 100 μU / mL of cathepsin D activity, or less than 50 μU / mL of cathepsin D activity.

[0221] III. Fc-containing proteins

[0222] The present disclosure provides methods for purifying an Fc-containing protein from a mixture of the Fc-containing protein and one or more contaminants.

[0223] In one embodiment, the Fc-containing protein is produced in a mammalian host cell. In one embodiment, the Fc-containing protein is produced in Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, murine hybridoma cells, or murine myeloma cells.

[0224] In one embodiment, the Fc-containing protein comprises one or more of the amino acid sequences listed in Table 1 below.

[0225] In one embodiment, the Fc-containing protein comprises a glucagon-like peptide 1 (GLP-1) analog comprising one or more modifications compared to the wild-type GLP-1 amino acid sequence (SEQ ID NO: 1).

[0226] In one embodiment, the Fc-containing protein comprises a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2.

[0227] In one embodiment, the protein comprising Fc comprises a peptide linker. In one embodiment, the C-terminal amino acid of the GLP-1 analog portion of the protein comprising Fc is fused to the N-terminus of the Fc portion of the immunoglobulin via a peptide linker. In one embodiment, the peptide linker comprises 1-10 G4S units (SEQ ID NO: 3).

[0228] In one embodiment, the Fc-containing protein comprises: a GLP-1 analog comprising the amino acid sequence of SEQ ID NO: 2; a peptide linker comprising the amino acid sequence of SEQ ID NO: 3; and an Fc portion of an immunoglobulin. In one embodiment, the N-terminal residue of the peptide linker is fused directly to the C-terminal residue of the GLP-1 analog, and the C-terminal residue of the peptide linker is fused directly to the N-terminal residue of the Fc portion.

[0229] In one embodiment, the Fc-containing protein is a homodimer comprising two identical amino acid chains, each amino acid chain comprising the amino acid sequence of SEQ ID NO: 4.

[0230] In one embodiment, the Fc-containing protein is dulaglutide.

[0231] In one aspect, provided herein are methods for purifying dulaglutide from a mixture of dulaglutide and one or more HCPs. In one embodiment, dulaglutide is produced in CHO cells.

[0232] Dulaglutide is a human GLP-1 receptor agonist comprising a dimer of a GLP-1 analog fused at its C-terminus via a peptide linker to the N-terminus of an analog of the Fc portion of an immunoglobulin, and is identified by CAS Registry Number 923950-08-7, which provides the following chemical names: 7-37-Glucagon-Like Peptide I [8-Glycine, 22-Glutamic Acid, 36-Glycine] (Synthetic Human) Fusion Protein and Peptide (Synthetic 16-Amino Acid Linker) Fusion Protein and Immunoglobulin G4 (Synthetic Human Fc Fragment), Dimer. Each dulaglutide monomer has the amino acid sequence set forth in SEQ ID NO: 4.

[0233] The two monomers are linked to form a dimer via a disulfide bond between cysteine ​​residues 55 and 58 of SEQ ID NO: 4. The structure, function, preparation, and use of dulaglutide for treating type 2 diabetes mellitus (T2DM) are described in more detail in U.S. Patent No. 7,452,966 and U.S. Patent Application Publication No. US20100196405. Dulaglutide stimulates the GLP-1 receptor, leading to stimulation of insulin synthesis and secretion, and has been shown to provide improved glycemic control in patients with T2DM.

[0234] As used herein, the term "dulaglutide" refers to any GLP-1 receptor agonist protein dimer of two monomers having the amino acid sequence of SEQ ID NO: 4, including any protein that is the subject of a regulatory submission seeking approval of a GLP-1 receptor agonist product that relies in whole or in part on data relating to dulaglutide submitted to regulatory agencies by Eli Lilly and Company, regardless of whether the party seeking approval of the protein actually identifies the protein as dulaglutide or uses some other terminology.

[0235] Table 1. Sequence of dulaglutide

[0236]

[0237] In one embodiment, the Fc-containing protein is etanercept, alefacept, abatacept, rilonacept, romiplostim, belatacept, aflibercept, conbercept, emotagmin alfa, emotagmin alfa, afotacept alfa, or rotercept.

[0238] In one aspect, provided herein is an Fc-containing protein produced by any of the methods disclosed herein.

[0239] In one aspect, provided herein is dulaglutide prepared by any of the methods disclosed herein.

[0240] Example

[0241] The following examples are offered by way of illustration only and not limitation.

[0242] Example 1: Analysis of wash buffer additives for removal of host cell proteins (HCPs)

[0243] Protein A purification is used as a step in the preparation of dulaglutide to reduce process-related impurities, such as medium components, Triton X-100, HCPs, and DNA, and to reduce potential viral contaminants. Dulaglutide is bound to a protein A chromatography matrix, and contaminants or impurities are removed by washing the matrix with a series of wash buffers. However, after protein A purification, residual host cell proteins (HCPs) may be present in the eluate, which may affect product quality and patient safety by causing, for example, aggregation, product fragmentation due to catalytic activity, and / or immunogenicity considerations. In this example, a variety of washing conditions were tested to reduce the level of HCPs in the protein A eluate.

[0244] CHO cells expressing dulaglutide were harvested and purified by methods known in the art. The dulaglutide-purified cell broth was then loaded onto a chromatography column containing MabSelect SuReLX™ Protein A matrix using the loading conditions in the wash study column shown in Table 4 below. The column was equilibrated with 50 mM Tris, pH 8.0.

[0245] To analyze optimal wash conditions, various buffers with buffer additives designed to disrupt non-covalent interactions were compared as Protein A wash buffers. The primary goal of this study was to improve the overall clearance of host cell proteins (HCPs) during dulaglutide purification. The flow-through of these wash buffers was monitored spectrophotometrically to detect peaks that would indicate potential removal of undesirable species such as HCPs. Figure 1 The results in show that washing the Protein A chromatography matrix with 50 mM Tris, pH 8.0 buffer containing 300 mM sodium octanoate as an additive produces a rather large UV peak at the beginning of the wash step, suggesting possible removal of undesirable HCPs. Other additives including 1 M sodium chloride, 1 M lysine, and 1 M arginine initially produced much smaller peaks. Wash buffers with sodium benzoate produced broad peaks throughout the wash process, which may mask any protein breakthrough.

[0246] To analyze the effect of wash buffer additives on HCP clearance, the protein A chromatography matrix was washed once with a wash buffer containing 1 M sodium benzoate, 1 M NaCl, 1 M lysine, 1 M arginine, or 300 mM sodium octanoate. Dulaglutide was then eluted from protein A with elution buffer (10 mM sodium citrate, pH 3.0), and the HCP levels in the eluate were analyzed by ELISA and LC-MS.

[0247] For the host cell protein (HCP) measurement by LC-MS, by peptide mapping / LC-MS / MS HCP analysis of illustrative plates sample, for example, by ultra-high performance liquid chromatography (UPLC) coupled with a Thermo Scientific mass spectrometer. In this analysis, sample undergoes trypsin digestion, is reduced / precipitated with dithiothreitol (DTT), then shifted and acidified supernatant in HPLC vials for LC-MS / MS analysis. By Proteome Discoverer, the CHO-K1 protein database to which a control protein sequence has been added is analyzed LC-MS / MS data. HCP content is reported as parts per million (ppm) HCP of each sample HCP total content.

[0248] For HCP ELISA, use Gyrolab ®ELISA assays were performed using CHO-HCP Kit 1 (Cygnus Technologies, according to the manufacturer's instructions). HCP content was reported as ng of HCP / mg of product.

[0249] As shown in Table 2 below, 300 mM sodium octanoate as a single additive resulted in significantly lower levels of the most immunogenic HCPs (i.e., HCPs with the highest EpiMatrix scores) compared to the other additives tested. The yield of dulaglutide was similar in all wash buffers tested. Furthermore, as shown in Table 3, wash buffers containing combinations of additives were tested, and the wash buffers containing the combinations containing sodium octanoate consistently had the lowest levels of the most immunogenic HCPs.

[0250] These results demonstrate that the addition of sodium octanoate to the wash buffer used for Protein A purification of dulaglutide can effectively reduce the levels of HCPs in the Protein A eluate, including those that are most immunogenic and therefore most potentially harmful to patient safety.

[0251] Table 2. Host Cell Protein (HCP) Analysis of Protein A Dulaglutide Wash Buffer Supplement

[0252]

[0253] *EpiMatrix score represents the concentration of T cell epitopes in a protein sequence.

[0254] Table 3. Host Cell Protein (HCP) Analysis of Protein A Dulaglutide Wash Buffer Additive Combinations

[0255]

[0256]

[0257] *EpiMatrix score represents the concentration of T cell epitopes in a protein sequence.

[0258] Example 2: Analysis of Sodium Caprylate Protein A Wash for HCP Removal

[0259] To further analyze Protein A wash conditions, wash buffers containing NaCl or sodium octanoate were compared with Protein A wash buffer to improve overall host cell protein (HCP) clearance during dulaglutide purification. In particular, the removal of cathepsin D from the dulaglutide eluate is important because residual cathepsin D proteolytically cleaves dulaglutide, generating the undesirable des(1-22) / des(1-25) sheared form of dulaglutide. The Protein A purification parameters used for this wash study, as well as the parameters of the dulaglutide production process, are described in Table 4 below.

[0260] Table 4. Purification parameters of dulaglutide protein A

[0261]

[0262] a The flow rate was adjusted to a 6 minute residence time (based on a target preparative bed height of 31.5 cm).

[0263] b The flow rate was adjusted to a residence time of 6.3 minutes (based on a target preparative bed height of 31.5 cm).

[0264] c The flow rate was adjusted to a residence time of 12.6 minutes (based on a target preparative bed height of 31.5 cm).

[0265] Several dulaglutide Protein A purification runs were designed to test various wash conditions, such as continuous washes and different wash volumes, to find the optimal wash conditions for sodium octanoate wash buffer in this dulaglutide purification step, see Table 5 below.

[0266] CHO cells expressing dulaglutide were harvested and purified by methods known in the art. The dulaglutide-purified cell broth was then loaded onto a chromatography column containing MabSelect SuRe LX Protein A matrix using the loading conditions in the wash study column described in Table 4 above. The column was equilibrated with 50 mM Tris, pH 8.0.

[0267] In the first histone A purification run, wash buffers with a series of sodium octanoate concentrations were tested to determine the optimal strength (volume molarity range) of sodium octanoate wash buffer required for washing, while minimizing the amount of octanoate added to the process, which may then need to be removed (see Runs 3-6, Table 5). After washing, dulaglutide was eluted according to the conditions listed in Table 4. The level of HCP in the eluate was determined by HCP ELISA and LC-MS assays (as described in Example 1). The level of cathepsin D in the eluate was assessed by cathepsin D activity assay.

[0268] The results of this analysis showed that a wash buffer containing 300 mM sodium octanoate significantly reduced cathepsin D activity in the eluate from Protein A purification, while also minimizing the levels of HCPs compared to wash buffers with 1 M NaCl or wash buffers with lower concentrations of sodium octanoate (see Figure 2 ).

[0269] In a second set of Protein A purification runs, washing the Protein A chromatography matrix with a wash buffer containing a NaCl / sodium octanoate combination was compared to serial washes with wash buffers containing either NaCl or sodium octanoate to determine the orthogonality of the washes, or the benefit of serial washes compared to combined washes (see Runs 7-9, Table 5). Figure 3 As shown in , in the series, washing with sodium chloride followed by washing with sodium octanoate resulted in the greatest reduction in cathepsin D activity and HCP levels in the eluate, as assessed by ELISA, cathepsin D activity assay, and LC-MS.

[0270] In a third set of Protein A purification runs, the effect of reducing the number of column volumes (CVs) of wash buffer containing 300 mM sodium octanoate in the second wash step was determined. The Protein A chromatography matrix was first equilibrated by washing with equilibration buffer containing 50 mM Tris, pH 8.0, followed by washes using the following control conditions: 5 CV 50 mM Tris, pH 8.0, then 5 CV 50 mM Tris, 1 M NaCl, pH 8, then 5 CV 50 mM Tris, pH 8.0; or a 5 CV wash, a 3 CV wash, or a 2 CV wash condition: 5 CV 50 mM Tris, pH 8.0, then X CV (5, 3, 2 CV) 50 mM Tris, 1 M NaCl, pH 8, then X CV 50 mM Tris, 300 mM sodium octanoate, pH 8, then X CV 50 mM Tris, pH 8.0. Dulaglutide was eluted according to the conditions in Table 4 and analyzed for HCP and cathepsin D as described above.

[0271] like Figure 4 As shown in , the eluate from each run using sodium octanoate wash buffer significantly reduced HCP and cathepsin D activity compared to the control wash buffer containing NaCl. In addition, although 2 CV resulted in a slight increase in cathepsin D activity compared to larger wash volumes (3 or 5 CV), the HCP levels were comparable. In addition, 5 CV of wash buffer containing 300 mM sodium octanoate resulted in a 0.6% increase in dulaglutide aggregation compared to the control, while 2 or 3 CV had no significant effect on aggregation (data not shown). Therefore, using 2 CV of sodium octanoate in the second wash step significantly reduced HCP levels and cathepsin D activity compared to the control condition, while also reducing the total wash volume required and minimizing aggregation during dulaglutide purification.

[0272] Tables 5-7 below disclose the wash conditions tested in this study (Table 5), the % yield of dulaglutide after Protein A purification under each wash condition (Table 6), and the HCP levels and cathepsin D activity in the eluate under each wash condition (Table 7).

[0273] These results of the tested dulaglutide Protein A purification conditions demonstrate that optimal wash conditions for the Protein A purification step during dulaglutide production reduce the amount of residual HCPs. Specifically, these results indicate that a wash buffer containing 300 mM sodium octanoate used in the wash series for Protein A purification can effectively reduce HCP levels, and in particular, cathepsin D activity levels, in the dulaglutide eluate while also reducing the total wash volume required without increasing aggregation.

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described, will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0282] Other implementations are within the scope of the following claims.

Claims

1. A method for purifying dulaglutide from a mixture comprising dulaglutide and one or more host cell proteins (HCP), the method comprising the following steps: (a) applying the mixture to a chromatography column comprising a Protein A chromatography matrix under conditions such that dulaglutide binds to the chromatography matrix; and (b) Wash the chromatography matrix with sodium octanoate wash buffer containing 150-500 mM sodium octanoate.

2. The method of claim 1, wherein the sodium octanoate wash buffer comprises 150-350 mM sodium octanoate.

3. The method of claim 1, wherein the sodium octanoate wash buffer comprises 200-350 mM sodium octanoate.

4. The method of claim 1, wherein the sodium octanoate wash buffer comprises about 300 mM sodium octanoate.

5. The method of any one of the preceding claims, wherein the sodium caprylate wash buffer comprises about 50 mM Tris.

6. The method according to any one of the preceding claims, wherein the sodium caprylate wash buffer comprises 0.5-1.5 M NaCl.

7. The method of any one of the preceding claims, wherein the sodium octanoate wash buffer comprises about 1 M NaCl.

8. The method according to any one of the preceding claims, wherein the sodium caprylate wash buffer has a pH of 7-9.

9. The method of any one of the preceding claims, wherein the sodium caprylate wash buffer has a pH of about 8.

10. The method according to any one of the preceding claims, further comprising washing the chromatography matrix with a NaCl wash buffer comprising 0.5-1.5 M NaCl. The method of claim 10 , wherein the NaCl wash buffer comprises about 1 M NaCl.

12. The method of claim 10 or 11, wherein the NaCl wash buffer comprises about 50 mM Tris.

13. The method according to any one of claims 10-12, wherein the NaCl wash buffer has a pH of 7-9.

14. The method of any one of claims 10-13, wherein the NaCl wash buffer has a pH of about 8.

15. The method according to any one of claims 10 to 14, wherein the chromatography column is washed with a NaCl wash buffer before washing with a sodium octanoate wash buffer.

16. The method according to any one of claims 10 to 14, wherein the chromatography column is washed with a sodium octanoate wash buffer before washing with a NaCl wash buffer.

17. The method according to any one of the preceding claims, wherein the chromatography matrix is ​​equilibrated with Tris buffer at pH 7-9 between steps (a) and (b). The method according to claim 17 , wherein the Tris buffer comprises 10-100 mM Tris.

19. The method of claim 17 or 18, wherein the Tris buffer comprises about 50 mM Tris.

20. The method of any one of claims 17-19, wherein the Tris buffer has a pH of about 8.

0.

21. A method according to any one of the preceding claims, wherein the chromatography matrix is ​​washed with 2-10 column volumes of sodium octanoate wash buffer.

22. The method of claim 21, wherein the chromatography matrix is ​​washed with about 2 column volumes of sodium octanoate wash buffer.

23. The method according to any one of claims 10 to 22, wherein the chromatography matrix is ​​washed with 2 to 10 column volumes of NaCl wash buffer.

24. The method of claim 23, wherein the chromatography matrix is ​​washed with about 2 or about 5 column volumes of NaCl wash buffer.

25. The method of claim 1, wherein the chromatography matrix is ​​washed sequentially with: (a) about 2 column volumes of NaCl wash buffer comprising about 50 mM Tris and about 1 M NaCl, pH about 8; and (b) About 2 column volumes of sodium octanoate wash buffer, which contains about 50 mM Tris and about 300 mM sodium octanoate, pH about 8.

26. The method according to any one of claims 10 to 25, wherein the chromatography matrix is ​​washed with NaCl wash buffer at a flow rate of 300 ± 30 cm / hr.

27. The method according to any one of the preceding claims, wherein the chromatography matrix is ​​washed with sodium octanoate wash buffer at a flow rate of 300 ± 30 cm / hr.

28. The method of any of the above claims, further comprising washing the chromatography matrix with a Tris buffer comprising 10-100 mM Tris, optionally wherein the Tris buffer comprises about 50 mM Tris, pH about 8.

0.

29. The method according to any one of the preceding claims, further comprising contacting the chromatography matrix with an elution buffer to produce an eluate comprising dulaglutide.

30. The method of claim 29, wherein the elution buffer has a pH of 2.5-4.

31. The method of claim 29 or 30, wherein the elution buffer has a pH of about 3.

32. The method of any one of claims 29-31, wherein the elution buffer comprises sodium citrate.

33. The method of any one of claims 29-32, wherein the elution buffer comprises 5-25 mM sodium citrate.

34. The method of any one of claims 29-33, wherein the elution buffer comprises about 10 mM sodium citrate.

35. The method according to any one of claims 29 to 34, wherein the eluate is monitored by absorbance using a spectrophotometer.

36. The method of any one of claims 29-35, wherein the eluate comprises at least 70% of the dulaglutide present in the mixture.

37. A method according to any one of the preceding claims, wherein the chromatography column has a diameter of 75-150 cm.

38. The method of any preceding claim, wherein the chromatography column has a diameter of about 100 cm.

39. The method according to any one of the preceding claims, wherein the Protein A chromatography matrix has an average particle size of 80-90 μm.

40. The method of any one of the above claims, wherein the Protein A chromatography matrix has an average particle size of about 85 μm.

41. The method of any one of claims 1-38, wherein the Protein A chromatography matrix has an average particle size of about 60 μm.

42. A method according to any one of the preceding claims, wherein the Protein A chromatography matrix is ​​alkali-stabilised.

43. The method of any of the above claims, wherein the one or more HCPs is a protease selected from the group consisting of serine proteases, aspartic proteases, cysteine ​​proteases, metalloproteases, and aminopeptidases, or a combination thereof.

44. The method of any of the above claims, wherein the one or more HCPs is cathepsin D.

45. The method of any one of claims 29-44, wherein the eluate comprises a reduced amount of one or more HCPs compared to the mixture.

46. ​​The method of any one of claims 29-45, wherein the eluate contains no more than 100 ng / mg of HCP.

47. The method of claim 45 or 46, wherein the amount of HCP is determined by mass spectrometry or ELISA.

48. The method of any one of claims 29-47, wherein the eluate comprises a reduced amount of cathepsin D compared to the mixture.

49. The method of any one of claims 29-48, wherein the eluate comprises no more than 100 ng / mg of cathepsin D.

50. The method of claim 48 or 49, wherein the amount of cathepsin D is determined by mass spectrometry or ELISA.

51. The method of claim 48 or 49, wherein the amount of cathepsin D is determined by an enzymatic assay.

52. The method of claim 47 or 50, wherein the mass spectrometry is LC-MS.

53. The method of any one of claims 29-52, wherein the eluate comprises less than 300 μU / mL of cathepsin D activity.

54. Dulaglutide prepared by the method according to any one of the preceding claims.

55. A composition comprising dulaglutide prepared by the method according to any one of the preceding claims.

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