Method for cleaning chromatography matrix

Abstract

Provided herein are improved methods of cleaning protein A chromatography matrices that have previously been used to purify proteins comprising Fc. The methods generally involve washing a Protein A chromatography matrix with a series of three solutions, a first solution comprising an acid, a second solution comprising Tris, and a third solution comprising NaOH (e.g., 0.001-0.075 M NaOH).

Description

[0001] background

[0002] The preparation of recombinant Fc-containing proteins for therapeutic use typically involves expressing the proteins in mammalian cells and subsequently purifying them from host cell contaminants. Protein A affinity chromatography is commonly used as an integral part of the purification process for Fc-containing proteins because Protein A has the ability to selectively bind to the Fc region of Fc-containing proteins. To reduce the production costs associated with Protein A affinity chromatography, the Protein A chromatography matrix is ​​typically cleaned and reused multiple times. However, cleaning the Protein A matrix is ​​challenging because the chemicals that effectively clean the Protein A matrix can also damage the matrix, thereby shortening its useful life.

[0003] Therefore, there is a need for improved methods of cleaning Protein A chromatography matrices to extend the useful life of the matrices.

[0004] Overview

[0005] The present disclosure provides improved methods for cleaning Protein A chromatography matrices. These methods generally involve washing the Protein A chromatography matrix with a series of three solutions, the first solution comprising an acid, the second solution comprising Tris, and the third solution comprising NaOH (e.g., 0.001-0.075 M NaOH). A particularly advantageous aspect of the methods disclosed herein is that they can significantly extend the useful life of the Protein A chromatography matrix and thereby reduce the cost of purifying Fc-containing proteins.

[0006] In one aspect, provided herein is a method for cleaning a Protein A chromatography matrix previously used to purify dulaglutide, the method comprising washing a chromatography column comprising the Protein A chromatography matrix sequentially with: a) a first solution comprising one or more acids; b) a second solution comprising Tris; and c) a third solution comprising 0.001-0.075 M NaOH.

[0007] In one embodiment, the first solution comprises acetic acid. In one embodiment, the first solution comprises phosphoric acid. In one embodiment, the first solution comprises acetic acid and phosphoric acid. In one embodiment, the first solution comprises 0.5-5% acetic acid. In one embodiment, the first solution comprises 0.5-1.5% acetic acid. In one embodiment, the first solution comprises about 1% acetic acid. In one embodiment, the first solution comprises 0.5-5% phosphoric acid. In one embodiment, the first solution comprises 0.5-1.5% phosphoric acid. In one embodiment, the first solution comprises about 1% phosphoric acid. In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid.

[0008] In one embodiment, the second solution has a pH of 7-9. In one embodiment, the second solution has a pH of about 8. In one embodiment, the second solution comprises 10-100 mM Tris. In one embodiment, the second solution comprises 25-75 mM Tris. In one embodiment, the second solution comprises about 50 mM Tris.

[0009] In one embodiment, the third solution comprises 0.005-0.05 M NaOH. In one embodiment, the third solution comprises 0.009-0.015 M NaOH. In one embodiment, the third solution comprises about 0.01 M NaOH.

[0010] In one embodiment, the Protein A chromatography matrix is ​​washed with 1-10 column volumes of the first solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-3 column volumes of the first solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-10 column volumes of the second solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 1-2 column volumes of the second solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-10 column volumes of the third solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-3 column volumes of the third solution.

[0011] In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 270 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 270 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 270 cm / hr.

[0012] In one embodiment, the chromatographic column has a diameter of 75-150 cm. In one embodiment, the chromatographic column has a diameter of about 100 cm. In one embodiment, the chromatographic column has a diameter of about 140 cm.

[0013] 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.

[0014] In one embodiment, the Protein A chromatography matrix comprises a Protein A ligand having increased stability relative to wild-type Staphylococcus aureus Protein A under alkaline conditions.

[0015] In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid; the second solution comprises about 50 mM Tris, pH about 8; and the third solution comprises about 0.01 M NaOH.

[0016] In one embodiment, the Protein A chromatography matrix is ​​washed with: b) 2-3 column volumes of the first solution; b) 1-2 column volumes of the second solution; and c) 2-3 column volumes of the third solution. In one embodiment, the Protein A chromatography matrix is ​​washed with the first, second, and third solutions at a flow rate of about 270 cm / hr.

[0017] In one embodiment, the method further comprises washing the Protein A chromatography matrix with about 2 column volumes of the second solution after washing the Protein A chromatography matrix with the third solution. In one embodiment, the method further comprises washing the Protein A chromatography matrix with a fourth solution comprising about 0.1 M NaOH.

[0018] In one embodiment, the Protein A chromatography matrix is ​​washed with about 2 column volumes of the fourth solution. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-140 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of about 130 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-110 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution for about 25 minutes.

[0019] In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution, the second solution, and / or the third solution in an upstream or downstream direction.

[0020] In one embodiment, after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than 1% carryover from a previous application. In one embodiment, after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than 0.1% carryover from a previous application.

[0021] In one embodiment, the Protein A chromatography matrix is ​​used for 200-500 cycles of purification of dulaglutide. In one embodiment, the Protein A chromatography matrix is ​​used for 300-400 cycles of purification of dulaglutide. In one embodiment, the Protein A chromatography matrix is ​​used for about 304 cycles of purification of dulaglutide.

[0022] Additional embodiments of the present disclosure are as follows:

[0023] 1. A method for cleaning a protein A chromatography matrix, the method comprising washing a chromatography column containing the protein A chromatography matrix with the following substances in sequence:

[0024] a) a first solution comprising one or more acids;

[0025] b) a second solution comprising Tris; and

[0026] c) A third solution comprising 0.001-0.075 M NaOH.

[0027] 2. The method of embodiment 1, wherein the first solution comprises acetic acid.

[0028] 3. The method of embodiment 1 or 2, wherein the first solution comprises phosphoric acid.

[0029] 4. The method of any one of embodiments 1-3, wherein the first solution comprises acetic acid and phosphoric acid.

[0030] 5. The method of any one of embodiments 1 to 4, wherein the first solution comprises 0.5% to 5% acetic acid.

[0031] 6. The method of any one of embodiments 1 to 5, wherein the first solution comprises 0.5% to 1.5% acetic acid.

[0032] 7. The method of any one of embodiments 1-6, wherein the first solution comprises about 1% acetic acid.

[0033] 8. The method of any one of embodiments 1 to 7, wherein the first solution comprises 0.5% to 5% phosphoric acid.

[0034] 9. The method of any one of embodiments 1 to 8, wherein the first solution comprises 0.5% to 1.5% phosphoric acid.

[0035] 10. The method of any one of embodiments 1 to 9, wherein the first solution comprises about 1% phosphoric acid.

[0036] 11. The method of any one of embodiments 1 to 10, wherein the first solution comprises about 1% acetic acid and about 1% phosphoric acid.

[0037] 12. The method according to any one of embodiments 1 to 11, wherein the second solution has a pH of 7-9.

[0038] 13. The method of any one of embodiments 1 to 12, wherein the second solution has a pH of about 8.

[0039] 14. The method according to any one of embodiments 1 to 13, wherein the second solution comprises 10-100 mM Tris.

[0040] 15. The method according to any one of embodiments 1 to 14, wherein the second solution comprises 25-75 mM Tris.

[0041] 16. The method of any one of embodiments 1-15, wherein the second solution comprises about 50 mM Tris.

[0042] 17. The method of any one of embodiments 1 to 16, wherein the third solution comprises 0.005-0.05 M NaOH.

[0043] 18. The method of any one of embodiments 1 to 17, wherein the third solution comprises 0.009-0.015 M NaOH.

[0044] 19. The method of any one of embodiments 1-18, wherein the third solution comprises about 0.01 M NaOH.

[0045] 20. The method according to any one of embodiments 1 to 19, wherein the Protein A chromatography matrix is ​​washed with 1 to 10 column volumes of the first solution.

[0046] 21. The method according to any one of embodiments 1 to 20, wherein 2 to 3 column volumes of the first solution are used to wash the Protein A chromatography matrix.

[0047] 22. The method according to any one of embodiments 1 to 21, wherein the Protein A chromatography matrix is ​​washed with 2 to 10 column volumes of the second solution.

[0048] 23. The method according to any one of embodiments 1 to 22, wherein the Protein A chromatography matrix is ​​washed with 1 to 2 column volumes of the second solution.

[0049] 24. The method according to any one of embodiments 1 to 23, wherein the Protein A chromatography matrix is ​​washed with 2 to 10 column volumes of the third solution.

[0050] 25. The method according to any one of embodiments 1 to 24, wherein the Protein A chromatography matrix is ​​washed with 2 to 3 column volumes of the third solution.

[0051] 26. The method of any one of embodiments 1-25, wherein the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 270 cm / hr.

[0052] 27. The method of any one of embodiments 1-26, wherein the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 270 cm / hr.

[0053] 28. The method according to any one of embodiments 1 to 27, wherein the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 270 cm / hr.

[0054] 29. The method according to any one of embodiments 1 to 28, wherein the chromatography column has a diameter of 75 to 150 cm.

[0055] 30. The method according to any one of embodiments 1 to 29, wherein the chromatography column has a diameter of about 100 cm.

[0056] 31. The method according to any one of embodiments 1 to 29, wherein the chromatography column has a diameter of about 140 cm.

[0057] 32. The method according to any one of embodiments 1 to 31, wherein the Protein A chromatography matrix has an average particle size of 80-90 μm.

[0058] 33. The method of any one of embodiments 1-32, wherein the Protein A chromatography matrix has an average particle size of about 85 μm.

[0059] 34. The method of any one of embodiments 1 to 33, wherein the Protein A chromatography matrix comprises a Protein A ligand having increased stability under alkaline conditions relative to wild-type Staphylococcus aureus Protein A.

[0060] 35. The method of any one of embodiments 1-34, wherein

[0061] a) a first solution comprising about 1% acetic acid and about 1% phosphoric acid;

[0062] b) a second solution comprising about 50 mM Tris, pH about 8; and

[0063] c) The third solution contained about 0.01 M NaOH.

[0064] 36. The method of embodiment 35, wherein the Protein A chromatography matrix is ​​washed with:

[0065] a) 2-3 column volumes of the first solution;

[0066] b) 1-2 column volumes of a second solution; and

[0067] c) 2-3 column volumes of the third solution.

[0068] 37. The method of embodiment 35 or 36, wherein the Protein A chromatography matrix is ​​washed with the first, second and third solutions at a flow rate of about 270 cm / hr.

[0069] 38. The method of any one of embodiments 1-37, further comprising washing the Protein A chromatography matrix with about 2 column volumes of the second solution after washing the Protein A chromatography matrix with the third solution.

[0070] 39. The method of any one of embodiments 1-38, further comprising washing the Protein A chromatography matrix with a fourth solution comprising about 0.1 M NaOH.

[0071] 40. The method of embodiment 39, wherein the Protein A chromatography matrix is ​​washed with about 2 column volumes of the fourth solution.

[0072] 41. The method of embodiment 39 or 40, wherein the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-110 cm / hr.

[0073] 42. The method of embodiment 39 or 40, wherein the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of about 130 cm / hr.

[0074] 43. The method of any one of embodiments 39-42, wherein the Protein A chromatography matrix is ​​washed with the fourth solution for about 25 minutes.

[0075] 44. The method according to any one of embodiments 1 to 43, wherein the Protein A chromatography matrix is ​​washed with the first solution, the second solution and / or the third solution in an upflow or downflow direction.

[0076] 45. The method of any one of embodiments 1-44, wherein a Protein A chromatography matrix is ​​previously used to purify the Fc-containing protein.

[0077] 46. ​​The method of embodiment 45, wherein the Fc-containing protein is an antibody.

[0078] 47. The method of embodiment 45, wherein the Fc-containing protein is not an antibody.

[0079] 48. The method of embodiment 45, 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).

[0080] 49. The method of embodiment 45 or 48, wherein the Fc-containing protein comprises a GLP-1 analogue comprising the amino acid sequence of SEQ ID NO: 2.

[0081] 50. The method of any one of embodiments 45, 48 or 49, wherein the Fc-containing protein comprises a peptide linker.

[0082] 51. The method of embodiment 50, wherein the peptide linker comprises 1-10 G4S units (SEQ ID NO: 6).

[0083] 52. The method of any one of embodiments 44 or 47-51, wherein the Fc-containing protein comprises:

[0084] a) a GLP-1 analogue comprising the amino acid sequence of SEQ ID NO: 2;

[0085] b) a peptide linker comprising 1-10 G4S units (SEQ ID NO: 6); and

[0086] c) The Fc portion of an immunoglobulin.

[0087] 53. The method of embodiment 52, 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.

[0088] 54. The method of any one of embodiments 44 or 47-53, wherein the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 5.

[0089] 55. The method of any one of embodiments 44 or 47-54, 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: 5.

[0090] 56. The method of any one of embodiments 44 or 47-55, wherein the Fc-containing protein is dulaglutide.

[0091] 57. The method of any one of embodiments 44-56, wherein the Protein A chromatography matrix has less than 1% carryover from previous applications.

[0092] 58. The method of any one of embodiments 44-57, wherein the Protein A chromatography matrix has less than 0.1% carryover from previous applications.

[0093] 59. The method of any one of embodiments 1-58, wherein the Protein A chromatography matrix is ​​used for 200-500 cycles.

[0094] 60. The method of any one of embodiments 1-59, wherein a Protein A chromatography matrix is ​​used for purification of dulaglutide for greater than 250-400 cycles.

[0095] 61. The method according to any one of embodiments 1-60, wherein a Protein A chromatography matrix is ​​used for the purification of dulaglutide for greater than 300-400 cycles.

[0096] 62. The method of any one of embodiments 1-61, wherein the Protein A chromatography matrix is ​​used for about 304 cycles.

[0097] 63. The method of any of the above embodiments, wherein the method does not comprise static holding of the first solution.

[0098] 64. The method according to any of the above embodiments, wherein the method does not comprise static holding of the second solution.

[0099] 65. The method according to any of the previous embodiments, wherein the method does not comprise static holding of the third solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figures 1A-1BTo show the low pH viral inactivation (LpHVI) intermediate pH ( Figure 1A ) and conductivity ( Figure 1B ) picture.

[0102] Figure 2 Graph depicting the % yield and dynamic binding capacity (DBC) response for Protein A chromatography matrix throughout the 304 cycles of this study. The % yield for high loading (18 g / L) is shown in green, and the % yield for low loading (10 g / L) is shown in orange. The horizontal black dashed lines represent the minimum and maximum method validation acceptance criteria (PVAC) of 70% and 105%, respectively.

[0103] Figure 3 Figure 3 depicts the height equivalent to a theoretical plate (HETP) and asymmetry evaluation of a packed Protein A chromatography matrix bed, as a measure of column packing quality. The black dotted line indicates the minimum acceptable HETP of at least 1100 plates / m, and the red dotted lines indicate the minimum and maximum acceptable asymmetry values ​​of 0.7 and 1.8, respectively.

[0104] Figure 4 Graph depicting the percentage of carryover after simulated elution of the Protein A chromatography matrix throughout the 304 cycles of this study.

[0105] Figures 5A-5B is a graph depicting the assessment of product purity as determined by size exclusion chromatography. Figure 5A Figure 2 is a graph showing the percentage of dulaglutide monomer. The horizontal red dashed line represents the minimum acceptance standard of 91.2% monomer applied in the low pH viral inactivation unit operation. Figure 5B Yellow data points represent uncultured, low pH virus-inactivated samples.

[0106] Figure 6 Graph showing the assessment of product purity as determined by measuring residual host cell protein (rHCP) in neutralized low pH virus inactivation samples. The horizontal red dashed line represents the maximum acceptance standard of 482 ppm applied in the low pH virus inactivation unit operation.

[0107] Figure 7 Graph showing assessment of product purity as determined by measuring residual Protein A (rProA) leached from the column in neutralized low pH virus inactivation samples. The horizontal red dashed line represents the maximum acceptance standard of 523 ppm applied in the low pH virus inactivation unit operation.

[0108] Figure 8Figure 2 is a graph showing the assessment of product purity as determined by measuring residual DNA in neutralized low pH virus inactivated samples. The horizontal red dashed line represents the maximum acceptance standard of 500 ppb applied when the AEX unit was operated.

[0109] Figure 9 Graph showing assessment of product purity as determined by measuring residual Triton X-100 surfactant in neutralized low pH virus inactivated samples. The horizontal red dashed line represents the maximum acceptance standard of 518,000 ppm.

[0110] Figure 10 Graph showing comparison of percent yield from a previous Protein A chromatography matrix lifetime study compared to the current lifetime study. Outliers are represented by grey dots.

[0111] Detailed description

[0112] The present disclosure provides improved methods for cleaning Protein A chromatography matrices. These methods generally involve washing the Protein A chromatography matrix with a series of three solutions, the first solution comprising an acid, the second solution comprising Tris, and the third solution comprising NaOH (e.g., 0.001-0.075 M NaOH). A particularly advantageous aspect of the methods disclosed herein is that they can significantly extend the useful life of the Protein A chromatography matrix and thereby reduce the cost of purifying Fc-containing proteins.

[0113] I. Definition

[0114] As used herein, the term "cleaning" refers to the removal of residual substances (eg, proteins) bound to the chromatography matrix after the chromatography matrix is ​​used to purify an Fc-containing protein (eg, dulaglutide). In one embodiment, cleaning includes regeneration and disinfection steps.

[0115] As used herein, the term "upflow" refers to flowing a solution upward through a chromatography column.

[0116] As used herein, the term "downflow" refers to passing a solution or buffer downward through a chromatography column.

[0117] As used herein, the term "carryover" refers to proteins and other impurities that remain bound to the chromatography matrix after cleaning of the Protein A chromatography matrix, as measured by simulated elution. In one embodiment, carryover is calculated as a percentage of the total peak area of ​​the Fc-containing protein (e.g., dulaglutide) eluted in the previous cycle, based on an A280 path length of 2 mm, as follows: (Simulated elution peak area (ml*mAU) / previous cycle elution peak area (ml*mAU) x 100 = % carryover)

[0118] As used herein, the term "mock elution" refers to an elution procedure applied to a chromatography matrix that has not been loaded with protein after the last cleaning procedure.

[0119] 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 an antibody. In one embodiment, the Fc-containing protein is not an antibody.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] As used herein, the term "about" when referring to a numerical value or parameter herein includes a variability of ±5% of that numerical 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 (including the endpoints).

[0125] II. Methods for Cleaning Protein A Chromatography Matrix

[0126] Cleaning Protein A chromatography matrices is challenging because the chemicals that effectively clean Protein A matrices can also damage the matrices, thereby shortening their useful life. The methods disclosed herein significantly extend the useful life of Protein A chromatography matrices, such as those that have been used to purify Fc-containing proteins (e.g., dulaglutide), by minimizing carryover without damaging the matrix.

[0127] Typically, a purification cycle using a Protein A chromatography method comprises the following steps in sequence: preparing a loading composition comprising an Fc-containing protein, applying the loading composition to a chromatography column comprising a Protein A chromatography matrix, washing the Protein A chromatography matrix, eluting the Fc-containing protein, and cleaning the Protein A chromatography matrix. It will be appreciated by those skilled in the art that, depending on the desired purpose and results, a purification cycle using a Protein A chromatography method may include additional intermediate steps and / or additional steps before and / or after the Protein A purification process.

[0128] In one aspect, provided herein is a method for cleaning a Protein A chromatography matrix previously used to purify an Fc-containing protein, the method comprising washing a chromatography column comprising the Protein A chromatography matrix sequentially with: a) a first solution comprising one or more acids; b) a second solution comprising Tris; and c) a third solution comprising 0.001-0.075 M NaOH.

[0129] In one aspect, provided herein is a method for cleaning a Protein A chromatography matrix previously used to purify dulaglutide, the method comprising washing a chromatography column comprising the Protein A chromatography matrix sequentially with: a) a first solution comprising one or more acids; b) a second solution comprising Tris; and c) a third solution comprising 0.001-0.075 M NaOH.

[0130] The cleaning solutions and methods are described in detail below.

[0131] Cleaning solutions and methods

[0132] After the Protein A chromatography matrix is ​​used to purify an Fc-containing protein (e.g., dulaglutide), the Protein A chromatography matrix is ​​washed with a series of solutions to remove residual proteins and contaminants from the matrix without damaging the matrix. In one aspect, after purifying an Fc-containing protein using a chromatography column comprising the Protein A chromatography matrix, the Protein A chromatography matrix is ​​sequentially washed with the following: a) a first solution comprising one or more acids; b) a second solution comprising Tris; and c) a third solution comprising NaOH (0.001-0.075 M). A particularly advantageous aspect of the methods disclosed herein is that they can significantly extend the useful life of the Protein A chromatography column matrix and thereby reduce the cost of purifying Fc-containing proteins.

[0133] In one embodiment, the first solution comprises acetic acid. In one embodiment, the first solution comprises phosphoric acid. In one embodiment, the first solution comprises acetic acid and phosphoric acid.

[0134] In one embodiment, the first solution comprises 0.5-5% acetic acid. In one embodiment, the first solution comprises 0.5-1.5% acetic acid. In one embodiment, the first solution comprises about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about In one embodiment, the first solution comprises about 1% acetic acid.

[0135] In one embodiment, the first solution comprises 0.5-5% phosphoric acid. In one embodiment, the first solution comprises 0.5-1.5% phosphoric acid. In one embodiment, the first solution comprises about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about In one embodiment, the first solution comprises about 1% phosphoric acid.

[0136] In one embodiment, the first solution comprises 0.5-5% acetic acid and about 1% phosphoric acid. In one embodiment, the first solution comprises 0.5-1.5% acetic acid. In one embodiment, the first solution comprises about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, 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%, about 4. %, 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%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9% or about 5.0% acetic acid and about 1% phosphoric acid.

[0137] In one embodiment, the first solution comprises about 1% phosphoric acid and 0.5-5% phosphoric acid. In one embodiment, the first solution comprises about 1% phosphoric acid and 0.5-1.5% phosphoric acid. In one embodiment, the first solution comprises about 1% phosphoric acid and about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, 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%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9% or about 5.0% phosphoric acid.

[0138] In one embodiment, the first solution comprises 0.5-5% acetic acid and 0.5-5% phosphoric acid. In one embodiment, the first solution comprises 0.5-1.5% acetic acid and 0.5-1.5% phosphoric acid. In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid.

[0139] In one embodiment, the second solution has a pH of 7-9. In one embodiment, the second solution has a pH of about 7.0, 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 8.0, 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 9.0. In one embodiment, the second solution has a pH of about 8.

[0140] In one embodiment, the second solution comprises 10-100 mM Tris. In one embodiment, the second solution comprises 25-75 mM Tris. In one embodiment, the second solution comprises about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mM Tris. In one embodiment, the second solution comprises about 50 mM Tris.

[0141] In one embodiment, the second solution comprises 10-100 mM Tris and has a pH of about 8. In one embodiment, the second solution comprises 25-75 mM Tris and has a pH of about 8. In one embodiment, the second solution comprises about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 mM Tris and has a pH of about 8. In one embodiment, the second solution comprises about 50 mM Tris and has a pH of about 8.

[0142] In one embodiment, the third solution comprises 0.005-0.05M NaOH. In one embodiment, the third solution comprises 0.009-0.015M NaOH. In one embodiment, the third solution comprises about 0.005M, about 0.006M, about 0.007M, about 0.008M, about 0.009M, about 0.01M, about 0.011M, about 0.012M, about 0.013M, about 0.014M, about 0.015M, about 0.016M, about 0.017M, about 0.018M, about 0.019M, about 0.02M, about 0.021M, about 0.022M, about 0.023M, about 0.024M, about 0.025M, about 0.026M 0.48M, about 0.049M, or about 0.05M NaOH. In one embodiment, the third solution comprises about 0.01M NaOH.

[0143] In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid; the second solution comprises about 50 mM Tris, and the third solution comprises 0.005-0.05 M NaOH. In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid; the second solution comprises about 50 mM Tris, and the third solution comprises 0.009-0.015 M NaOH. In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid; the second solution comprises about 50 mM Tris, and the third solution comprises about 0.005 M, about 0.006 M, about 0.007 M, about 0.008 M, about 0.009 M, about 0.01 M, about 0.011 M, about 0.012 M, about 0.013 M, about 0.014 M, about 0.015 M, about 0.016 M, about 0.017 M, about 0.018 M, about 0.019 M, about 0.02 M, about 0.021 M, about 0.022 M, about 0.023 M, about 0.0 0.044M, about 0.045M, about 0.046M, about 0.047M, about 0.048M, about 0.049M, or about 0.05M NaOH.

[0144] In one embodiment, the first solution comprises about 1% acetic acid and about 1% phosphoric acid; the second solution comprises about 50 mM Tris, pH about 8; and the third solution comprises about 0.01 M NaOH.

[0145] In one embodiment, the method further comprises washing the Protein A chromatography matrix with the second solution after washing the Protein A chromatography matrix with the third solution.

[0146] In one embodiment, the method further comprises washing the Protein A chromatography matrix with a fourth solution comprising about 0.05-0.5 M NaOH. In one embodiment, the method further comprises washing the Protein A chromatography matrix with a fourth solution comprising about 0.05 M, about 0.06 M, about 0.07 M, about 0.08 M, about 0.09 M, about 0.1 M, about 0.11 M, about 0.12 M, about 0.13 M, about 0.14 M, about 0.15 M, about 0.16 M, about 0.17 M, about 0.18 M, about 0.19 M, about 0.2 M, about 0.25 M, about 0.3 M, about 0.35 M, about 0.4 M, about 0.45 M, or about 0.5 M NaOH. In one embodiment, the method further comprises washing the Protein A chromatography matrix with a fourth solution comprising about 0.1 M NaOH.

[0147] In one aspect, provided herein is a method for cleaning a Protein A chromatography matrix previously used to purify an Fc-containing protein, the method comprising: washing the Protein A chromatography matrix sequentially with: a) a first solution comprising about 1% acetic acid and about 1% phosphoric acid; b) a second solution comprising about 50 mM Tris, pH about 8; c) a third solution comprising about 0.01 M NaOH; d) the second solution; and e) a fourth solution comprising about 0.1 M NaOH.

[0148] In one aspect, provided herein is a method for cleaning a Protein A chromatography matrix previously used to purify dulaglutide, the method comprising: washing the Protein A chromatography matrix sequentially with: a) a first solution comprising about 1% acetic acid and about 1% phosphoric acid; b) a second solution comprising about 50 mM Tris, pH about 8; c) a third solution comprising about 0.01 M NaOH; d) the second solution; and e) a fourth solution comprising about 0.1 M NaOH.

[0149] In one embodiment, the Protein A chromatography matrix is ​​washed with 1-10 column volumes of the first solution. In one embodiment, the Protein A 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 the first solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-3 column volumes of the first solution. In one embodiment, the Protein A chromatography matrix is ​​washed with about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3 column volumes of the first solution.

[0150] In one embodiment, the Protein A chromatography matrix is ​​washed with 1-10 column volumes of the second solution. In one embodiment, the Protein A 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 the second solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 1-2 column volumes of the second solution. In one embodiment, the Protein A chromatography matrix is ​​washed with about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2 column volumes of the second solution.

[0151] In one embodiment, the Protein A chromatography matrix is ​​washed with 1-10 column volumes of the third solution. In one embodiment, the Protein A 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 the third solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-3 column volumes of the third solution. In one embodiment, the Protein A chromatography matrix is ​​washed with about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3 column volumes of the third solution.

[0152] In one embodiment, the method does not include static holding of the first solution. In one embodiment, the method does not include static holding of the second solution. In one embodiment, the method does not include static holding of the third solution. In one embodiment, the method does not include static holding of the first, second, and third solutions.

[0153] In one embodiment, the Protein A chromatography matrix is ​​washed with: 2-3 column volumes of the first solution; 1-2 column volumes of the second solution; and 2-3 column volumes of the third solution.

[0154] In one embodiment, the Protein A chromatography matrix is ​​washed with: about 3 column volumes of the first solution; about 1.1 column volumes of the second solution; and about 2.2 column volumes of the third solution.

[0155] In one embodiment, the method further comprises washing the Protein A chromatography matrix with the third solution followed by washing the Protein A chromatography matrix with about 2-10 column volumes of the second solution. In one embodiment, the method further comprises washing the Protein A chromatography matrix with the third solution followed by washing the Protein A chromatography matrix 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 the second solution. In one embodiment, the method further comprises washing the Protein A chromatography matrix with the third solution followed by washing the Protein A chromatography matrix with about 2 column volumes of the second solution.

[0156] In one embodiment, the Protein A chromatography matrix is ​​washed with 1-10 column volumes of the fourth solution. In one embodiment, the Protein A 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 the fourth solution. In one embodiment, the Protein A chromatography matrix is ​​washed with 2-3 column volumes of the fourth solution. In one embodiment, the Protein A chromatography matrix is ​​washed with about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3 column volumes of the fourth solution.

[0157] In one embodiment, the method further comprises storing the Protein A chromatography matrix in a solution comprising about 100 mM acetic acid, sodium acetate, at a pH of about 4.

[0158] In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 100-400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 100 cm / hr, 110 cm / hr, 120 cm / hr, 130 cm / hr, 140 cm / hr, about 150 cm / hr, 160 cm / hr, 170 cm / hr, 180 cm / hr, 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 270 cm / hr, about 300 cm / hr, about 310 cm / hr, about 320 cm / hr, about 330 cm / hr, about 340 cm / hr, about 350 cm / hr, about 360 cm / hr, about 370 cm / hr, about 380 cm / hr, about 390 cm / hr, or about 400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 270 cm / hr.

[0159] In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 100-400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 100 cm / hr, 110 cm / hr, 120 cm / hr, 130 cm / hr, 140 cm / hr, about 150 cm / hr, 160 cm / hr, 170 cm / hr, 180 cm / hr, 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about 260 cm / hr, about 270 cm / hr, about 280 cm / hr, about 290 cm / hr, about 300 cm / hr, about 310 cm / hr, about 320 cm / hr, about 330 cm / hr, about 340 cm / hr, about 350 cm / hr, about 360 cm / hr, about 370 cm / hr, about 380 cm / hr, about 390 cm / hr, about 400 cm / hr, about 410 cm / hr, about 420 cm / hr, about 430 cm / hr, about 440 cm / hr, about 450 cm / hr, about 460 cm / hr, about 470 cm / hr, about 480 cm / hr, about 490 cm / hr, about 500 cm / hr, about In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 270 cm / hr, about 300 cm / hr, about 310 cm / hr, about 320 cm / hr, about 330 cm / hr, about 340 cm / hr, about 350 cm / hr, about 360 cm / hr, about 370 cm / hr, about 380 cm / hr, about 390 cm / hr, or about 400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 270 cm / hr.

[0160] In one embodiment, the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 100-400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 100 cm / hr, 110 cm / hr, 120 cm / hr, 130 cm / hr, 140 cm / hr, about 150 cm / hr, 160 cm / hr, 170 cm / hr, 180 cm / hr, 190 cm / hr, about 200 cm / hr, about 210 cm / hr, about 220 cm / hr, about 230 cm / hr, about 240 cm / hr, about 250 cm / hr, about The Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 270 cm / hr, about 300 cm / hr, about 310 cm / hr, about 320 cm / hr, about 330 cm / hr, about 340 cm / hr, about 350 cm / hr, about 360 cm / hr, about 370 cm / hr, about 380 cm / hr, about 390 cm / hr, or about 400 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 270 cm / hr.

[0161] In one embodiment, the column is washed with the first solution at a flow rate of about 270 cm / hr, the second solution at a flow rate of about 270 cm / hr, and the third solution at a flow rate of about 270 cm / hr.

[0162] In one embodiment, the Protein A chromatography matrix is ​​washed with about 2 column volumes of the fourth solution. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-140 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of about 130 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-110 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of about 100 cm / hr, about 101 cm / hr, about 102 cm / hr, about 103 cm / hr, about 104 cm / hr, about 105 cm / hr, about 106 cm / hr, about 107 cm / hr, about 108 cm / hr, about 109 cm / hr, or about 110 cm / hr. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution for 10-30 minutes. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution for about 10, about 15, about 20, about 25 minutes, or about 30 minutes.

[0163] In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution, the second solution, and / or the third solution in an upstream or downstream direction. In one embodiment, the Protein A chromatography matrix is ​​washed with the first solution in an upstream direction. In one embodiment, the Protein A chromatography matrix is ​​washed with the second solution in an upstream direction. In one embodiment, the Protein A chromatography matrix is ​​washed with the third solution in an upstream direction. In one embodiment, the Protein A chromatography matrix is ​​washed with the fourth solution in an upstream direction.

[0164] In one aspect, any of the methods disclosed herein can reduce carryover from a previously used Protein A chromatography matrix. In one embodiment, after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than about 1% carryover from a prior application. In one embodiment, after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than about 0.1%, less than about 0.2%, less than about 0.3%, less than about 0.4%, less than about 0.5%, less than about 0.6%, less than about 0.7%, less than about 0.8%, less than about 0.9%, or less than about 1.0% carryover from a prior application. In one embodiment, after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than about 0.1% carryover from a prior application.

[0165] In one embodiment, any of the methods disclosed herein can remove one or more residual contaminants from a Protein A chromatography matrix. In one embodiment, the contaminant is Triton X-100, DNA, HCP, leached Protein A, or insulin.

[0166] In one embodiment, the Protein A chromatography matrix is ​​used for 200-500 cycles of purification of a protein comprising Fc. In one embodiment, the Protein A chromatography matrix is ​​used for 300-400 cycles of purification of a protein comprising Fc. In one embodiment, the Protein A chromatography matrix is ​​used for about 300, about 301, about 302, about 303, about 304, about 305, about 306, about 307, about 308, about 309, about 310, about 311, about 312, about 313, about 314, about 315, about 316, about 317, about 318, about 319, about 320, about 321, about 322, about 323, about 324, about 325, about 326, about 327, about 328, about 329, about 330, about 331, about 332, about 333, about 334, about 335, about 336, about 337, about 338, about 339, about 340, about 341, about 342, about 343, about 344, about 345, about 346, about 347, about 348, about 349, about 350, about 351, about 352, about 353, about 354, about 355, about 356, about 357, about 358, about 359, about 360, about 361, about 362, about 363, about 364, about 365, about 366, about 367, about 368, about 369, about 370, about 371, about 372, about 373, about 374, about 375, about 376 In one embodiment, the Protein A chromatography matrix is ​​used for about 304 cycles of purification of an Fc-containing protein.

[0167] In one embodiment, the Protein A chromatography matrix is ​​used for 200-500 cycles of purification of dulaglutide. In one embodiment, the Protein A chromatography matrix is ​​used for 300-400 cycles of purification of dulaglutide. In one embodiment, the Protein A chromatography matrix is ​​used for about 300, about 301, about 302, about 303, about 304, about 305, about 306, about 307, about 308, about 309, about 310, about 311, about 312, about 313, about 314, about 315, about 316, about 317, about 318, about 319, about 320, about 321, about 322, about 323, about 324, about 325, about 326, about 327, about 328, about 329, about 330, about 331, about 332, about 333, about 334, about 335, about 336, about 337, about 338, about 339, about 340, about 341, about 342, about 343, about 344, about 345, about 346, about 347, about 348, about 349 9, about 350, about 351, about 352, about 353, about 354, about 355, about 356, about 357, about 358, about 359, about 360, about 361, about 362, about 363, about 364, about 365, about 366, about 367, about 368, about 369, about 370, about 371, about 372, about 373, about 374, about 375, about In one embodiment, the Protein A chromatography matrix is ​​used for about 304 cycles of purification of dulaglutide.

[0168] Protein A chromatography

[0169] The methods provided herein generally comprise washing a chromatography column comprising a Protein A chromatography matrix.

[0170] In one embodiment, the chromatography column has a diameter of 10-150 cm. In one embodiment, the chromatography column has a diameter of 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.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] In one embodiment, the Protein A chromatography matrix comprises a Protein A ligand having increased stability under alkaline conditions relative to wild-type Staphylococcus aureus Protein A. 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.

[0175] In one embodiment, the Protein A chromatography matrix comprises a Protein A ligand having increased stability under alkaline conditions relative to wild-type Staphylococcus aureus Protein A and having an average particle size of about 85 μm.

[0176] 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.

[0177] 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 .

[0178] In one embodiment, provided herein is a method for cleaning MabSelect SuRe TM LX method, which has been used to purify Fc-containing proteins, makes MabSelect SuRe TM LX can be used for about 304 cycles of purifying Fc-containing proteins. In one embodiment, provided herein is a clean MabSelect SuRe TM The LX method, which has been used to purify dulaglutide, makes MabSelect SuRe TM LX can be used for approximately 304 cycles of purification of dulaglutide.

[0179] In one aspect, provided herein are methods of purifying an Fc-containing protein using a Protein A chromatography matrix that has been cleaned according to any of the methods disclosed herein.

[0180] In one aspect, provided herein are methods for purifying dulaglutide using a Protein A chromatography matrix that has been cleaned according to any of the methods disclosed herein.

[0181] III. Fc-containing proteins

[0182] The present disclosure provides methods for purifying an Fc-containing protein from a mixture of the Fc-containing protein and one or more contaminants.

[0183] 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, HEK cells, or murine myeloma cells.

[0184] In one embodiment, the Fc-containing protein comprises one or more of the amino acid sequences listed in Table 1 below.

[0185] 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).

[0186] In one embodiment, the Fc-containing protein comprises a GLP-1 analog comprising the amino acid sequence of SEQ ID NO:2.

[0187] 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 IgG4 Fc analog portion via a glycine-rich linker. In one embodiment, the peptide linker comprises 1-10 G4S units (SEQ ID NO: 6).

[0188] In one embodiment, the Fc-containing protein comprises: a GLP-1 analogue comprising the amino acid sequence of SEQ ID NO: 2; a peptide linker comprising 1-10 G4S units (SEQ ID NO: 6); 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 analogue, and the C-terminal residue of the peptide linker is fused directly to the N-terminal residue of the Fc portion.

[0189] In one embodiment, the Fc-containing protein comprises the amino acid sequence of SEQ ID NO: 5. 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: 5. The Fc-containing protein comprises a homodimer of the amino acid sequence of SEQ ID NO: 5.

[0190] In one embodiment, the Fc-containing protein is dulaglutide.

[0191] 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.

[0192] Dulaglutide is a human GLP-1 receptor agonist comprising a dimer of a GLP-1 analog fused at its C-terminus via a (G4S)3 peptide linker to the N-terminus of an immunoglobulin Fc portion analog, 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: 5.

[0193] 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 T2DM are described in more detail in U.S. Patent No. 7,452,966 and U.S. Patent Application Publication No. US20100196405. Dulaglutide agonizes 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.

[0194] 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:5, 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.

[0195] Table 1. Sequences contained in certain Fc-containing proteins

[0196]

[0197] In one embodiment, the Fc-containing protein is etanercept, alefacept, abatacept, rilonacept, romiplostim, belatacept, aflibercept, conbercept, emogram alfa, emogram alfa, afotacept alfa, or rotercept.

[0198] In one embodiment, the Fc-containing protein is an antibody. In one embodiment, the Fc-containing protein is not an antibody. Example

[0199] The following examples are offered by way of illustration only and not limitation.

[0200] Example 1: Study on the Lifespan of Protein A Chromatographic Matrix

[0201] During the Protein A purification step of the dulaglutide production process, the Protein A chromatography matrix was reused for up to 112 purification cycles using a cleaning protocol that included washing with an acid solution (1% acetic acid / 1% phosphoric acid) to regenerate the matrix and washing with a solution containing 50 mM NaOH and 1 M NaCl to sanitize the matrix. In this example, a novel method for cleaning the Protein A chromatography matrix was analyzed with the goal of extending the useful life of the Protein A chromatography matrix and maximizing the number of dulaglutide purification cycles using the Protein A chromatography matrix.

[0202] As a preliminary test to determine the importance of the first acid wash in the post-purification cleaning protocol for dulaglutide, a Protein A chromatography matrix (MabSelect SuRE TM LX) were cleaned in situ for four consecutive runs. The results of this test showed that washing with NaOH alone resulted in an increase in column pressure after each consecutive run. These preliminary results suggest that acid washes are necessary in the cleaning protocol for Protein A chromatography matrices after dulaglutide purification to control column pressure.

[0203] Furthermore, previous studies of the Protein A chromatography lifetime used for dulaglutide purification resulted in a continuous increase in the size of the pre-peak. To better control this increase, a caustic wash was introduced in the regeneration step of the cleaning protocol following the acid wash step, using 0.01 M NaOH for 2 column volumes.

[0204] The following study provides the MabSelect SuRe TM Results of a modified cleaning protocol for LX that included an additional wash with a caustic solution (0.01 M NaOH) between the acid wash (1% acetic acid / 1% phosphoric acid) and the sanitizing wash.

[0205] method

[0206] The Protein A chromatography matrix cleaning protocol used in this study included washing with an acid solution containing 1% acetic acid, 1% phosphoric acid without static holding, followed by washing with Tris equilibration buffer (50 mM Tris, pH 8), and then washing with a caustic solution (0.01 M NaOH). The Protein A chromatography matrix was then sanitized by washing the matrix again with Tris equilibration buffer, followed by washing with a solution containing 0.1 M NaOH. The general parameters for the Protein A chromatography matrix cleaning study are described in Table 4 below. MabSelect SuRe TM LX was used in this study and has a Protein A ligand engineered to have increased stability under alkaline conditions.

[0207] Table 4. Operating parameters for protein A dulaglutide purification lifespan study

[0208]

[0209]

[0210] 1 As the column size decreased, the flow rate for Run 1-88 was set at 107 cm / hr after repacking to 103 cm / hr.

[0211] 2 The target volume endpoint and flow rate differ from the current preparation protocol, but the target contact time of 25 minutes is equivalent.

[0212] 3 The elution flow rate used in the current preparation protocol was 210 cm / hr, and the flow rate of 270 cm / hr used in this study was within the parameter range of 200-330 cm / hr.

[0213] 4 Due to differences in elution characteristics across scales, the BS cut used in the current preparative protocol was set at 1.76CV, with a target of 1.5CV (within the range of 1.43–1.76CV) being more appropriate for the bench-scale model.

[0214] 5 The acid regeneration step in the current preparation protocol was set to 3CV of 1% acetic acid, 1% phosphoric acid.

[0215] Column packing and evaluation

[0216] Protein A chromatography matrix MabSelect SuRe LX (MSS LX) was loaded into a Millipore Vantage Pro 1.1 cm column using an AKTA Avant 25 liquid handling module (MST367). A second column was used to hold the matrix slurry. A ~50% slurry of MSS-LX in 20% EtOH 400 mM NaCl was introduced into the open column top; the top was then connected to consolidate the resin slurry at 2X the maximum method flow rate. Upon consolidation, the second column was removed. The column top adapter was placed on top of the resin bed and the flow was reapplied to the bed. The column was considered filled when no further decrease in bed height was observed. Finally, the column was conditioned after filling in packing buffer, which was passed through the column at the operating flow rate to achieve 5 column volumes (CV). According to the dulaglutide purification method, the column was filled to a bed height of 22.3 cm, ranging from 20 to 32 cm.

[0217] The suitability of the packed column is assessed by measuring the height of the theoretical plate (HETP) and asymmetry. Throughout this research, all column packing assessments (HETP and asymmetry methods) are completed under identical conditions and, when possible, are coupled with large-scale implementation. Use 100mM NaCl equilibrium column to reach conductivity baseline. The pulse test solution is 1M NaCl, and the injection volume is 2% of the column volume. With 100cm / hr equilibrium column, and injection and elution flow rate are 100cm / hr. If the number of theoretical plates is ≥1100 plates / m, and the asymmetry value is 0.7-1.8, then be suitable for using the column filling containing the original resin.

[0218] Column function assessment

[0219] After the column filling evaluation, the packed column was sterilized and stored. After storage, the column function evaluation was performed, consisting of simulated elution and dynamic binding capacity (DBC) evaluation. The operating parameters for simulated elution and DBC evaluation are shown in Tables 5 and 6 below, respectively.

[0220] Table 5. Simulated elution parameters for Protein A resin lifetime study

[0221]

[0222] *The flow rate for wash 1 in runs 65-80 was modified to reflect changes implemented in dulaglutide processing. These changes consisted of moving from 300 cm / hr for 5.0 CV to 150 cm / hr for the first 3.2 CVs, followed by 300 cm / hr for 2.2 CVs.

[0223] Table 6. Dynamic Binding Capacity (DBC) of Protein A Resin Lifetime Studies

[0224]

[0225] The mock elution simulated the conditions to which the Protein A chromatography matrix was exposed during the dulaglutide purification process, including column equilibration, washing, elution, cleaning, and post-use sterilization. The carryover from the blank peak was then calculated as a percentage of the total dulaglutide peak area eluted in the previous cycle, based on an A280 path length of 2 mm, using the following equation:

[0226]

[0227] The DBC evaluation provides a readout of the dynamic binding capacity of the resin (QB10). QB10 is determined using the following equation:

[0228] QB 10 =(V 10％ -V s -V 室 )xC / V c

[0229] In the above equation, V 10％ = volume at 10% breakthrough; VS = volume at the start of sample application; V 空 = Void volume of the AKTA flow path (equivalent to the column volume on the AKTA system); C = concentration of the sample solution, accurate to 3 digits; V C = column volume. (V 10％ -V S ) is determined by the Unicorn function.

[0230] Protein A affinity chromatography

[0231] Protein A affinity chromatography was performed using a column packed with MSSLX matrix. The matrix was filled with dulaglutide detergent viral inactivation (DVI) intermediate stored at least at -65°C. Before use, the charge aliquots were thawed and brought to room temperature, and four product cycles were performed using one 1 L aliquot per day. The final product cycles on the first, second, and fourth days of each block underwent low pH viral inactivation (LpHVI) incubation times (i.e., runs 4, 8, and 16), and the remainder was immediately neutralized. Throughout this study, the DVI batches used were cycled. Dulaglutide was loaded with the DVI intermediate within a validated acceptable range (PAR) of 10-18 g / L to evaluate parameter data throughout the resin life.

[0232] After thawing in a water bath set at 23°C and reaching room temperature, the material was 0.2 μm PES-filtered through a vacuum blood collection tube. Except for 2 runs from each block, all runs were loaded at 14.4 g / L, run 8 was loaded at 18 g / L (peak PAR loading), and run 16 was loaded at 10 g / L (lower PAR loading). Since the feed batch was originally formulated at a concentration of 1.62358 g / L, runs 145-148 were loaded at an incorrect loading. After the yield was low in this study, the batch was retested and a correction concentration of 1.439226 g / L was assigned. The recalculated loading applied based on the correction concentration of 1.439226 mg / L was calculated to be 12.8 g / L and was not considered to affect this study because it was still within the PAR range of loading (10-18 g / L).

[0233] The target resin life of the MSS-LX resin was 304 product runs. A total of 304 product runs were completed using the MSS LX resin, and analytical testing (rHCP, rProA, and SEC) was performed on the Protein A main stream in the first, fourth, eighth, and sixteenth cycles of blocks 1-19. Further testing (insulin, rDNA, and Triton X-100) was performed starting at the eighth cycle (peak PAR loading) in each second block of the lifespan.

[0234] Column packing evaluation

[0235] The criteria for height of a theoretical plate (HETP) and asymmetry (i.e., column asymmetry) are shown below in Table 7. These criteria are critical in determining acceptable performance of Protein A chromatography resins.

[0236] Table 7. Column Packing Evaluation Acceptance Criteria

[0237] property Acceptable Standards HETP (number of plates / m) ≥1100 Asymmetry 0.7-1.8

[0238] Column performance evaluation

[0239] Protein A / Low pH Viral Inactivation (LpHVI) % yield must be maintained within the 70-105% IPC range. During this study, the following parameters were evaluated to assess the consistent performance of the Protein A chromatography matrix: yield, mainstream pH, column packing quality, simulated elution carryover, dynamic binding, column outlet A280, pH, conductivity, and column delta pressure.

[0240] Critical Quality Attributes

[0241] During the dulaglutide manufacturing process, product purity is routinely tested during the low pH viral inactivation step, and therefore, the product validation acceptance criteria for low pH viral inactivation were also used in this study. For rDNA, this is typically tested after the AEX unit operation, and therefore the AEX acceptance criteria were used as representative limits in this study. The acceptance criteria applied throughout the study are shown in Table 8 below. The applicable low pH viral inactivation acceptance criteria and historical small-scale and / or large-scale experience were considered when evaluating the results. rHCP = residual host cell proteins; rDNA = residual deoxyribonucleic acid; rProA = residual protein A.

[0242] Table 8. Acceptance criteria for method validation of dulaglutide intermediates

[0243] test Unit Operation Method validation acceptance criteria rHCP (ppm) LpHVI ≤482 rDNA (ppb) AEX *≤500 rTriton X-100 (ppm) LpHVI ≤518000 SEC monomer % LpHVI ≥91.2 Residual insulin (μU / mL) LpHVI ≤20 rProA (ppm) LpHVI ≤523

[0244] *Acceptance criteria for low pH viral inactivation (LpHVI) after AEX unit operation are not defined

[0245] result

[0246] Column performance

[0247] The target column loading for this study was 14.4 g / L. This target was met for all runs except runs 145-148, where a DVI concentration error resulted in a reduced loading of 12.8 g / L. As described above, the loadings for all runs were within the acceptable range of 10-18 g / L and therefore had no impact on the conclusions of this study. The target for a run from each block was an upper limit of 18 g / L for the column loading for the Protein A unit operation, with a target of 10 g / L for the lower limit.

[0248] Elution peak morphology was consistent across the entire column loading range. In runs loaded at the lower column loading of 10 g / L, the pre-elution peak observed preceding the elution peak was absent, whereas at the higher column loading of 18 g / L, the pre-elution peak was larger. This pre-elution peak was observed to grow from 0.211 AU / cm to 0.468 AU / cm over the lifetime of this study at the upper limit of the demonstrated acceptable loading range, likely due to a combination of fouling and matrix ligand hydrolysis. Frontal cleavage was triggered at a UV signal of 4 AU / cm, indicating that there is no risk of triggering early elution at the upper limit of the demonstrated acceptable range for loading the pre-peak, as shown in previous studies, and is evidence of the effectiveness of the cleaning procedure for the Protein A chromatography matrix used in this study.

[0249] Throughout the life of the column, no peak broadening was observed, except for differences in loading dependence, while the pH shift and delta column pressure remained consistent. A wider peak was observed in the cycles run at a higher loading of 18 g / L, while conversely a narrower peak was observed at a lower loading of 10 g / L. These differences caused by loading also affected the percent yields of these cycles, with higher column loadings resulting in lower percent yields. This was a result of a lower percentage of mainstream collected from the wider peak due to backside cleavage occurring at the set volume (1.5 CV). The variability and tailing observed on the main elution peak occurred randomly throughout this study and were likely the result of batch variations in loading.

[0250] pH and conductivity

[0251] Offline pH and conductivity values ​​for charging and neutralization of low pH virus inactivation were monitored in all runs. Conductivity values ​​were read using an offline probe and temperature compensated to 25°C to normalize the values. The low pH virus inactivation and neutralization mainstream pH response observed in the lifespan study ranged from 8.21 to 8.48 (mean: pH 8.29 ± 0.04) ( Figure 1A The neutralized low pH virus inactivation conductivity was consistent throughout the life cycle study, with a response range of 7.14-7.85 μS / cm (average: 7.27 ± 0.06 μS / cm) ( Figure 1B ). Due to an error in the addition of neutralization buffer, high pH and conductivity values ​​were recorded at cycle 72.

[0252] Percent yield (% yield) and dynamic binding capacity (DBC)

[0253] The cleavage strategy for the purification of dulaglutide protein A was based on UV triggering and a set collection volume. The front-side cleavage occurred at a UV signal of 4 AU / cm at 280 nm, and the back-side cleavage in this study occurred 1.5 CV after the front-side cleavage. Figure 2 The % yield trend and decrease in dynamic binding capacity over the life of the Protein A resin are shown. The % yield was calculated based on the concentration of the neutralized low pH viral inactivation intermediate and the observed response range in the Protein A cycle life study was 84.4%-99.6%. The yield data showed that, with the exception of hardware pump issues (runs 221-236), this parameter remained consistent over the life of the resin ( Figure 2(Red box, pump failure resulted in reduced loading on the column but had no impact on the study conclusions). In runs targeting an upper bound of 18 g / L, a slight downward trend in % yield was observed toward the end of the lifetime, accounting for batch variation. This may be attributed to degradation of the MSS LX Protein A ligand, which exhibits a tendency to corrode and / or peak broadening at the demonstrated acceptable upper bound. However, % yield remained within the critical in-process control (CIPC) range of 70-105% throughout the lifetime of this study.

[0254] The observed DBC in the Protein A cycle life study ranged from 28.1 to 20.5 g / L. Based on these data, the Protein A DBC decreased by 0.025 g / L per column cycle, most likely due to a combination of binding site blockage caused by caustic sanitization exposure and low-level matrix contamination. Based on the slope equation of the DBC value over the resin life, the resin's DBC can be extrapolated to an upper acceptable capacity range of 18 g / L after 408 product cycles. Since the DBC value was 20.5 g / L at the end of this resin life study and the expected routine preparative capacity was 14.4 g / L, this study demonstrated sufficient resin capacity to bind dulaglutide at the target capacity ratio over 304 product cycles.

[0255] Column HETP and asymmetry

[0256] To detect changes in column packing quality, plates / meter and column asymmetry were regularly assessed throughout the column cycle life study. The HETP observed in the Protein A cycle life study was 1782-3180 plates / meter. This range was significantly broadened as column repacking after cycle 88 reduced column height and theoretical plate count, but only a slight decrease in plate count was observed subsequently, which is expected to be due to repeated cycling. During the column life study, the plate count remained above the post-pack acceptance standard of NLT 1100 plates / meter ( Figure 3 ).

[0257] The observed asymmetry ranged from 0.90 to 1.75. An asymmetry value of 0.97 was observed for the initial column packing using the original resin, which increased to 1.30 after the column was repacked after cycle 88. Over the next 216 product cycles, the asymmetry gradually increased, ultimately reaching 1.75 ( Figure 3 ), and this increase was not accompanied by any additional trend except for a broadening of the elution peak at the upper end of the demonstrated acceptable loading range, which could affect the yield at this loading.

[0258] Simulated elution carryover

[0259] To detect potential changes in elution carryover, mock elutions were performed at regular intervals throughout the study. The percentage of mock elution carryover observed in this Protein A cycle life study ranged from 0-0.01% ( Figure 4 ), remaining below the <1% guideline limit for the percentage of protein-borne residues.

[0260] Product purity was determined by size exclusion chromatography (SEC)

[0261] Figure 5A -B shows that the percentage of monomer and the percentage of total aggregates remained at acceptable levels throughout this study. During the preparation of dulaglutide, the neutralized low pH virus inactivation (nLpHVI) intermediate was analyzed by SEC with an acceptance criteria of ≥91.2%. The data trend shows that during the first column filling (cycles 1-88), the percentage of monomer loaded on all columns remained nearly constant. After the column was refilled, the data trend remained stable until cycle 200, after which a slight decrease in % monomer (~0.5%) was observed over the remaining lifetime ( Figure 5A As expected, the incubation time for low pH virus inactivation was associated with minimal aggregate growth, resulting in lower % monomer, but this shift was maintained throughout the lifetime and was not affected by column load or cycle number.

[0262] Residual host cell protein (rHCP)

[0263] Figure 6 The rHCP concentration (ppm) for low-pH viral inactivation, which showed neutralization, steadily decreased over the first 100 cycles, likely due to early cycling of the original matrix. At cycle 88, the column was repacked, with a slight decrease in bed height, after which rHCP levels remained consistent throughout the study, meeting the method validation acceptance criteria of ≤482 ppm for low-pH viral inactivation. This demonstrates the ability of the Protein A chromatography matrix to clear method-related impurities throughout the 304 cycles of the lifetime study.

[0264] Residual Protein A (rProA)

[0265] To evaluate the effect of increased caustic exposure on Protein A leaching, neutralization and low pH viral inactivation of rProA were assessed. Figure 7The results show that rProA levels in the main Protein A stream during the first column packing period were rising at the beginning of the study. After repacking the column, rProA levels stabilized and remained consistent for the remainder of the study, reaching a maximum of 126 ppm. Over the full 304 cycles of this study, all samples collected within the PAR loading range of 10-18 g / L met the method validation acceptance criteria of ≤523 ppm for low pH viral inactivation. This demonstrates the resin's ability to maintain acceptable levels of rProA over 304 product cycles.

[0266] Residual DNA (rDNA)

[0267] Figure 8 It is shown that after the initial column packing, the rDNA levels (ppb) in the Protein A mainstream steadily decreased. After repacking the column at cycle 88, the rDNA levels initially increased and then stabilized at ~250 ppb for the remainder of the life. All samples were collected at the upper PAR loading of 18 g / L, which represents a worst-case challenge, and met the method validation acceptance criteria of ≤500 ppb in AEX over the full 304 cycles of this study. This method validation acceptance criteria limit was used for representative purposes as there is no defined limit for rDNA for low pH viral inactivation. This confirms the ability of the resin to reduce rDNA to acceptable levels over 304 product cycles.

[0268] Residual Triton X-100 (rTX-100)

[0269] Figure 9 The concentration of rTX-100 (ppm) in the main stream of Protein A was shown to remain consistent throughout the first column filling period. After refilling the column, the level of rTX-100 increased approximately 3-fold but remained consistent throughout the remainder of the study, meeting the acceptance criteria of ≤518,000 ppm with values ​​between 371-572 ppm (after refilling). The levels of rTX-100 are displayed on a logarithmic scale in Figure 9 This was attributed to a shift between the acceptance criteria and the recorded values. This demonstrates the ability of the resin to reduce rTX-100 over 304 product cycles.

[0270] Residual insulin (rInsulin)

[0271] Throughout this study, the Protein A mainstream rInsulin concentration (μU / mL) remained consistent, meeting the acceptance criteria of ≤20 μU / mL (data not shown). This demonstrates the ability of the resin to reduce method-related impurities to acceptable levels over 304 product cycles.

[0272] Comparison of Protein A Lifespan Studies

[0273] This study confirmed the previous study of the chromatographic lifetime of dulaglutide MSS LX Protein A, which included a cleaning step of static storage with a solution containing 1% acetic acid, 1% phosphoric acid, followed by a sanitizing wash with 50 mM NaOH and 1 M NaCl. Figure 10 ,Notice Figure 10 The results demonstrate higher and more consistent yields compared to the previous study (grey points in the figure are outliers). The final yield achieved in this study (97.8% at 304 cycles) was significantly improved compared to the previous study of Protein A chromatography lifetime (88.9% at 112 cycles). Therefore, these results demonstrate an improved method for cleaning Protein A chromatography matrices that extends their useful lifetime. The inclusion of an additional 0.01 M NaOH caustic wash in the cleaning protocol described in this study eliminated the increase in pre-peaks observed in the previous lifetime study.

[0274] in conclusion

[0275] This study showed that yields remained consistently high over 304 runs while maintaining a dynamic binding capacity above the target loading until the end of the resin life, indicating that additional caustic exposure should not impact the CIPC yield of ≥70% over the resin life. No negative trends were observed for residual host cell proteins, residual DNA, residual insulin, residual protein A, and residual Triton X-100 tested over 304 dulaglutide purification cycles, with an additional step of washing the protein A chromatography matrix with a caustic solution containing 0.01 M NaOH having no effect on residual clearance. Quality attributes indicating stability, as measured by SEC, remained essentially unchanged over 304 product cycles.

[0276] This study demonstrates that by using an improved cleaning protocol including an additional caustic wash step (0.01 M NaOH), the Protein A chromatography matrix used for dulaglutide processing can purify dulaglutide for at least 304 purification cycles with acceptable product quality and acceptable unit operation performance. This number of cycles can even be extended beyond 304 cycles, as surprisingly, none of the evaluation criteria evaluated in the above study were negatively affected throughout the 304 cycles.

[0277] 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.

[0278] All references (e.g., publications or patents or patent applications) cited herein are hereby incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety. Other embodiments are within the scope of the following claims.

Claims

1. A method for cleaning a protein A chromatography matrix previously used for purifying dulaglutide, the method comprising washing a chromatography column comprising the protein A chromatography matrix with the following substances in sequence: a) a first solution comprising one or more acids; b) a second solution comprising Tris; and c) A third solution comprising 0.001-0.075 M NaOH.

2. The method of claim 1, wherein the first solution comprises acetic acid. The method according to claim 1 , wherein the first solution comprises phosphoric acid.

4. The method according to any one of claims 1 to 3, wherein the first solution comprises acetic acid and phosphoric acid.

5. The method according to any one of claims 1 to 4, wherein the first solution comprises 0.5 to 5% acetic acid.

6. The method according to any one of claims 1 to 5, wherein the first solution comprises 0.5 to 1.5% acetic acid.

7. The method of any one of claims 1 to 6, wherein the first solution comprises about 1% acetic acid.

8. The method according to any one of claims 1 to 7, wherein the first solution comprises 0.5 to 5% phosphoric acid.

9. The method according to any one of claims 1 to 8, wherein the first solution comprises 0.5 to 1.5% phosphoric acid.

10. The method of any one of claims 1 to 9, wherein the first solution comprises about 1% phosphoric acid.

11. The method of any one of claims 1 to 10, wherein the first solution comprises about 1% acetic acid and about 1% phosphoric acid.

12. The method according to any one of claims 1 to 11, wherein the second solution has a pH of 7 to 9.

13. The method of any one of claims 1 to 12, wherein the second solution has a pH of about 8.

14. The method according to any one of claims 1 to 13, wherein the second solution comprises 10-100 mM Tris.

15. The method according to any one of claims 1 to 14, wherein the second solution comprises 25-75 mM Tris.

16. The method of any one of claims 1-15, wherein the second solution comprises about 50 mM Tris.

17. The method according to any one of claims 1 to 16, wherein the third solution comprises 0.005-0.05 M NaOH.

18. The method of any one of claims 1 to 17, wherein the third solution comprises 0.009-0.015 M NaOH.

19. The method of any one of claims 1-18, wherein the third solution comprises about 0.01 M NaOH.

20. The method of any one of claims 1 to 19, wherein the Protein A chromatography matrix is ​​washed with 1 to 10 column volumes of the first solution.

21. The method according to any one of claims 1 to 20, wherein the Protein A chromatography matrix is ​​washed with 2 to 3 column volumes of the first solution.

22. The method of any one of claims 1 to 21, wherein the Protein A chromatography matrix is ​​washed with 2 to 10 column volumes of the second solution.

23. The method of any one of claims 1 to 22, wherein the Protein A chromatography matrix is ​​washed with 1 to 2 column volumes of the second solution.

24. The method according to any one of claims 1 to 23, wherein the Protein A chromatography matrix is ​​washed with 2 to 10 column volumes of the third solution.

25. The method according to any one of claims 1 to 24, wherein the Protein A chromatography matrix is ​​washed with 2 to 3 column volumes of the third solution.

26. The method of any one of claims 1 to 25, wherein the Protein A chromatography matrix is ​​washed with the first solution at a flow rate of about 270 cm / hr.

27. The method of any one of claims 1 to 26, wherein the Protein A chromatography matrix is ​​washed with the second solution at a flow rate of about 270 cm / hr.

28. The method of any one of claims 1 to 27, wherein the Protein A chromatography matrix is ​​washed with the third solution at a flow rate of about 270 cm / hr.

29. The method according to any one of claims 1 to 28, wherein the chromatography column has a diameter of 75 to 150 cm.

30. The method of any one of claims 1-29, wherein the chromatography column has a diameter of about 100 cm.

31. The method of any one of claims 1 to 30, wherein the Protein A chromatography matrix has an average particle size of 80-90 μm.

32. The method of any one of claims 1 to 31, wherein the Protein A chromatography matrix has an average particle size of about 85 μm.

33. The method of any one of claims 1 to 32, wherein the Protein A chromatography matrix comprises a Protein A ligand having increased stability under alkaline conditions relative to wild-type Staphylococcus aureus Protein A.

34. The method according to any one of claims 1 to 33, wherein a) a first solution comprising about 1% acetic acid and about 1% phosphoric acid; b) a second solution comprising about 50 mM Tris, pH about 8; and c) The third solution contained about 0.01 M NaOH.

35. The method of claim 34, wherein the Protein A chromatography matrix is ​​washed with: a) 2-3 column volumes of the first solution; b) 1-2 column volumes of a second solution; and c) 2-3 column volumes of the third solution.

36. The method of claim 34 or 35, wherein the Protein A chromatography matrix is ​​washed with the first, second and third solutions at a flow rate of about 270 cm / hr.

37. The method of any one of claims 1-36, further comprising washing the Protein A chromatography matrix with about 2 column volumes of the second solution after washing the Protein A chromatography matrix with the third solution.

38. The method of any one of claims 1-37, further comprising washing the Protein A chromatography matrix with a fourth solution comprising about 0.1 M NaOH.

39. The method of claim 38, wherein the Protein A chromatography matrix is ​​washed with about 2 column volumes of the fourth solution.

40. The method of claim 38 or 39, wherein the Protein A chromatography matrix is ​​washed with the fourth solution at a flow rate of 100-140 cm / hr.

41. The method of any one of claims 38-40, wherein the Protein A chromatography matrix is ​​washed with the fourth solution for about 25 minutes.

42. The method according to any one of claims 1 to 41, wherein the Protein A chromatography matrix is ​​washed with the first solution, the second solution and / or the third solution in an upstream or downstream direction.

43. The method of any one of claims 1 to 42, wherein after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than 1% carryover from a previous application.

44. The method of any one of claims 1 to 43, wherein after cleaning the Protein A chromatography matrix, the Protein A chromatography matrix has less than 0.1% carryover from a previous application.

45. The method according to any one of claims 1 to 44, wherein a Protein A chromatography matrix is ​​used for 200 to 500 cycles of purification of dulaglutide.

46. ​​The method according to any one of claims 1 to 45, wherein a Protein A chromatography matrix is ​​used for 300 to 400 cycles of purification of dulaglutide.

47. The method according to any one of claims 1 to 46, wherein a Protein A chromatography matrix is ​​used for about 304 cycles of purification of dulaglutide.

48. The method of any of the above claims, wherein the method does not involve static holding of the first solution.

49. The method of any of the above claims, wherein the method does not comprise static holding of the second solution.

50. The method of any of the above claims, wherein the method does not comprise static holding of the third solution.

51. Dulaglutide prepared by the method of any one of the preceding claims.

52. A composition comprising dulaglutide prepared by the method of any one of the preceding claims.

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