Methods for enhanced impurity removal during protein a chromatography
阅读说明：本技术 用于在蛋白a色谱法期间增强杂质去除的方法 (Methods for enhanced impurity removal during protein a chromatography ) 是由 C·A·贝吉 于 2018-12-20 设计创作，主要内容包括：本文提供涉及经由蛋白A色谱法纯化包含Fc区的多肽(例如,抗体)的方法；涉及在蛋白A色谱法期间使用包含苯甲酸盐和/或苯甲醇的洗涤溶液的方法；以及在蛋白A色谱法之前使用苯甲酸钠调节收获物的方法。(Provided herein are methods involving purification of polypeptides (e.g., antibodies) comprising an Fc region via protein a chromatography; methods involving the use of wash solutions comprising benzoate and/or benzyl alcohol during protein a chromatography; and a method of conditioning the harvest using sodium benzoate prior to protein a chromatography.)
1. A method of purifying a polypeptide comprising an Fc region, the method comprising the steps of:
(a) contacting a protein a chromatography matrix with a sample comprising (i) the polypeptide comprising an Fc region and (ii) one or more impurities under conditions wherein the polypeptide comprising an Fc region binds to protein a; and
(b) washing the substrate with a wash solution, wherein the wash solution comprises one or both of (i) benzoate salt at a concentration of about 0.1M to about 1.0M and (ii) benzyl alcohol at a concentration of about 0.5% to about 4% volume/volume (v/v), and wherein the wash solution has a pH of about 4.0 to about 10.0.
2. The method of claim 1, wherein the wash solution comprises: 1) a benzoate salt; 2) benzyl alcohol; or 3) benzoate and benzyl alcohol.
3. The process according to claim 1 or claim 2, wherein the concentration of benzoate salt is from about 0.1M to about 0.5M.
4. The process according to any one of claims 1-3, wherein the benzoate salt is an alkali metal benzoate salt.
5. The process according to any one of claims 1-4, wherein the benzoate salt is sodium benzoate.
6. The method of claim 5, wherein the concentration of sodium benzoate is from about 0.1M to about 0.3M.
7. The method of claim 5, wherein the concentration of sodium benzoate is about 0.3M.
8. The method of claim 5, wherein the concentration of sodium benzoate is about 0.5M.
9. The method of any one of claims 1-8, wherein the benzyl alcohol is at a concentration of about 1% to about 4% (v/v).
10. The method of any one of claims 1-8, wherein the benzyl alcohol is at a concentration of about 1% to about 2% (v/v).
11. The method of any one of claims 1-8, wherein the concentration of benzyl alcohol is from about 2% (v/v) to about 4% (v/v).
12. The method of any one of claims 1-11, wherein the wash solution further comprises a buffer.
13. The method of claim 11, wherein the buffer is selected from the group consisting of: phosphate, Tris, arginine, acetate and citrate.
14. The method of claim 11 or claim 13, wherein the buffer is at a concentration of about 10mM to about 500 mM.
15. The method of claim 11 or claim 13, wherein the buffer is at a concentration of about 50mM or 500 mM.
16. The method of any one of claims 1-15, wherein the wash solution has a pH of about 5.0 to about 10.0.
17. The method of any one of claims 1-16, wherein the wash solution has a pH of about 5.0 to about 9.0.
18. The method of any one of claims 1-16, wherein the wash solution has a pH of about 5.0, about 6.0, about 7.0, about 9.0, or about 10.0.
19. The method of any one of claims 1-18, wherein the wash solution further comprises sodium benzenesulfonate.
20. The method of claim 19, wherein the concentration of sodium benzenesulfonate is from about 0.1M to about 0.5M.
21. The method of any one of claims 1-20, wherein the wash solution further comprises caprylic acid.
22. The method of claim 21, wherein the concentration of caprylic acid is about 10mM to about 50 mM.
23. The method of any one of claims 1-22, wherein the wash solution further comprises hexylene glycol.
24. The method of claim 23, wherein the concentration of hexylene glycol is from about 1% to about 10% (v/v).
25. The method of any one of claims 1-24, wherein the wash solution further comprises creatine.
26. The method of claim 25, wherein the creatine is at a concentration of about 10mM to about 100 mM.
27. The method of any one of claims 1-26, wherein the wash solution further comprises arginine.
28. The method of claim 27, wherein the arginine is at a concentration of about 0.1M to about 1.0M.
29. The method of claim 27, wherein the arginine is at a concentration of about 0.5M.
30. The method of any one of claims 27-29, wherein the arginine is arginine-HCl.
31. The method of any one of claims 27-30, wherein the wash solution comprising arginine has a pH of about 4.0 to about 6.0.
32. The method of any one of claims 27-30, wherein the wash solution comprising arginine has a pH of about 8.0 to about 10.0.
33. The method of any one of claims 1-32, wherein the wash solution further comprises one or more non-buffered salts.
34. The method of claim 33, wherein the one or more non-buffered salts are selected from the group consisting of: sodium chloride, sodium bromide, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, and any combination thereof.
35. The method of claim 33, wherein the one or more non-buffered salts are sodium chloride and/or potassium chloride.
36. The method of any one of claims 33-35, wherein the concentration of the one or more non-buffering salts is from about 0.1M to about 1.0M.
37. The method of any one of claims 1-36, wherein the wash solution is a solution selected from the group consisting of:
(i) a solution having a pH of about 10.0 comprising sodium benzoate at a concentration of about 0.5M and sodium bicarbonate at a concentration of about 50 mM;
(ii) a solution having a pH of about 9.0 comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2%, arginine at a concentration of 0.5M, and sodium phosphate at a concentration of 50 mM;
(iii) a solution having a pH of about 7.0 comprising sodium benzoate at a concentration of about 0.5M and benzyl alcohol at a concentration of about 2% (v/v);
(iv) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and sodium chloride at a concentration of 0.5M, having a pH of about 7.0;
(v) a solution comprising hexylene glycol at a concentration of about 10% (v/v), sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v), having a pH of about 7.0;
(vi) a solution having a pH of about 7.0 comprising a benzenesulfonate salt at a concentration of about 0.5M, sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v);
(vii) a solution having a pH of about 7.0 comprising caprylic acid at a concentration of about 50mM, sodium benzoate at a concentration of 0.5M, arginine at a concentration of about 0.5M, and sodium chloride at a concentration of about 0.5M;
(viii) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M, having a pH of about 6.0;
(ix) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M, having a pH of about 5.0; and
(x) A solution comprising benzyl alcohol at a concentration of about 2% (v/v) and arginine at a concentration of about 0.5M, having a pH of about 5.0.
38. The method of any one of claims 1-37, further comprising the step of washing the substrate with a first solution prior to washing the substrate with the wash solution of step (b) in claim 1.
39. The method of claim 38, wherein the first solution comprises a buffer selected from the group consisting of: phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof.
40. The method of claim 39, wherein the first solution comprises a buffer at a concentration of about 10mM to about 100 mM.
41. The method of any one of claims 38-40, wherein the first solution is a phosphate buffer.
42. The method of any one of claims 1-41, further comprising the step of washing the substrate with a second solution after washing the substrate with the wash solution of step (b) in claim 1.
43. The method of claim 42, wherein the second solution comprises a buffer selected from the group consisting of: phosphate buffer, Tris buffer, acetate buffer, carbonate buffer, citrate buffer, and any combination thereof.
44. The method of claim 43, wherein the second solution comprises a buffer at a concentration of about 10mM to about 100 mM.
45. The method of any one of claims 42-44, wherein the second solution has a pH of about 5.0 to about 7.0.
46. The method of any one of claims 42-45, wherein the second solution comprises substantially low or no salt.
47. The method of any one of claims 1-46, further comprising the step of contacting the protein A chromatography matrix with an elution solution after one or more washing steps.
48. The method of claim 47, further comprising the step of collecting an eluate comprising the polypeptide comprising an Fc region.
49. The method of claim 48, further comprising the step of filtering the eluate via depth filtration.
50. The method of claim 48 or claim 49, wherein the eluate comprises less than about 500 parts per million (ppm) of the one or more impurities.
51. The method of any one of claims 1-50, wherein the method results in the polypeptide comprising an Fc region being purified from one or more impurities to a greater extent than a corresponding method lacking the step of washing the substrate with the wash solution.
52. The method of any one of claims 1-51, wherein the one or more impurities is a Host Cell Protein (HCP).
53. The method of claim 52, wherein the one or more HCPs is selected from the group consisting of: phospholipase, clusterin, serine protease, elongation factor, and any combination thereof.
54. The method of claim 52 or claim 53, wherein the HCP is putative phospholipase B-like 2(PLBL 2).
55. The method of claim 52 or claim 53, wherein the host cell is a mammalian host cell.
56. The method of any one of claims 52-55, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.
57. The method of any one of claims 1-56, wherein the Fc region is a human Fc region.
58. The method of claim 57, wherein the human Fc region comprises a human IgG1, IgG2, or IgG4Fc region.
59. The method of any one of claims 1-56, wherein the Fc region is a mouse Fc region.
60. The method of claim 59, wherein the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region.
61. The method of any one of claims 1-60, wherein the polypeptide comprising an Fc region is an antibody.
62. The method of claim 61, wherein the antibody is a human, humanized, or chimeric antibody.
63. The method of claim 61 or claim 62, wherein the antibody is a monoclonal antibody.
64. The method of claim 61 or claim 62, wherein the antibody is a bispecific antibody or a trispecific antibody.
65. The method of any one of claims 1-64, further comprising, prior to step (a), adjusting the harvest of the polypeptide comprising the Fc region to achieve a final concentration of benzoate salt of 0.1M to about 0.5M and a pH of about 7.0 to about 9.0 to produce a sample comprising (i) the polypeptide comprising the Fc region and (ii) one or more impurities.
66. A method of purifying a polypeptide comprising an Fc region, the method comprising the steps of:
(A) conditioning the harvest comprising the Fc region-comprising polypeptide to achieve a final concentration of benzoate salt of about 0.1M to about 0.5M and a pH of about 7.0 to about 9.0 to produce a sample comprising (i) the Fc region-comprising polypeptide and (ii) one or more impurities; and
(B) contacting the sample with at least one chromatography matrix.
67. The method according to claim 65 or 66, wherein the benzoate salt is an alkali metal benzoate salt.
68. The method according to claim 67, wherein said benzoate salt is sodium benzoate.
69. The method of any one of claims 65-68, wherein the final concentration of benzoate in the harvest is from about 0.4M to about 0.5M.
70. The method of any one of claims 65-69, wherein the pH of the harvest is from about 7.0 to about 8.0.
71. The method of any one of claims 65-69, wherein the pH of the harvest is from about 8.0 to about 9.0.
72. The method of any one of claims 65-67, wherein the harvest is produced from a culture comprising a host cell engineered to express the polypeptide.
73. The method of claim 72, wherein the host cell is a eukaryotic host cell.
74. The method of claim 73, wherein the eukaryotic host cell is a Chinese Hamster Ovary (CHO) cell.
75. The method of any one of claims 65-74, wherein said harvest is clarified prior to said adjusting.
76. The method of any one of claims 65-74, wherein said harvest is clarified after said adjusting.
77. The method of any one of claims 66-76, wherein the at least one chromatography matrix comprises an affinity chromatography matrix.
78. The method of claim 77, wherein the affinity chromatography matrix is a protein A chromatography matrix or a protein G chromatography matrix.
79. The method of any one of claims 65-78, further comprising the step of contacting the at least one chromatography matrix with at least one wash solution.
80. The method of any one of claims 65-79, further comprising the step of contacting the at least one chromatography matrix with an elution solution.
81. The method of claim 80, further comprising the step of collecting an eluate comprising the polypeptide comprising an Fc region.
82. The method of claim 81, further comprising the step of filtering the eluate via depth filtration.
83. The method of claim 81 or claim 82, wherein the eluate comprises less than about 500 parts per million (ppm) of the one or more impurities.
84. The method of any one of claims 66-83, wherein the method results in the polypeptide comprising an Fc region being purified from the one or more impurities to a greater extent to produce the sample as compared to a corresponding method lacking the step of modulating the harvest of the polypeptide comprising an Fc region.
85. The method of any one of claims 66-84, wherein the one or more impurities is a Host Cell Protein (HCP).
86. The method of claim 85, wherein the one or more HCPs are selected from the group consisting of: phospholipase, clusterin, serine protease, elongation factor, and any combination thereof.
87. The method of claim 85 or claim 86, wherein the HCP is putative phospholipase B-like 2(PLBL 2).
88. The method of any one of claims 66-87, wherein the Fc region is a human Fc region.
89. The method of claim 82, wherein the human Fc region comprises a human IgG1, IgG2, or IgG4Fc region.
90. The method of any one of claims 66-89, wherein the Fc region is a mouse Fc region.
91. The method of claim 90, wherein the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region.
92. The method of any one of claims 66-91, wherein the polypeptide comprising an Fc region is an antibody.
93. The method of claim 92, wherein the antibody is a human, humanized, or chimeric antibody.
94. The method of claim 92 or claim 93, wherein the antibody is a monoclonal antibody.
95. The method of claim 92 or claim 93, wherein the antibody is a bispecific antibody or a trispecific antibody.
Technical Field
The present disclosure relates to methods of purifying polypeptides (e.g., antibodies) comprising an Fc region via protein a chromatography.
Background
Antibodies and other Fc region-containing proteins (e.g., immunoadhesins) have found widespread use in drug/therapeutic applications. The use of these molecules (e.g., in human patients) requires careful purification to remove any contaminants/impurities that may be generated during protein production. Purification of therapeutic proteins is often achieved using one or more chromatographic purification steps; one particularly useful type of chromatographic purification of proteins (e.g., antibodies) containing an immunoglobulin Fc region is protein a chromatography. However, it has been shown that Host Cell Proteins (HCPs) co-elute with antibodies during conventional capture mode protein chromatography, including protein a chromatography, which can create problems for downstream applications of these antibodies. Typically, one or more washing steps are employed after the product (e.g., a protein containing an immunoglobulin Fc region) is bound to the chromatography resin prior to elution. Unfortunately, current wash formulations consisting of salts and buffer substances may not be sufficient to disrupt the interaction of HCPs and other impurities with various monoclonal antibody (mAb) products. Accordingly, there is a need for improved purification methods (e.g., implementing new wash formulations) that reduce the concentration/number of impurities (e.g., HCPs) that co-purify with antibodies (e.g., during protein a affinity chromatography).
All references, including patent applications, patent publications, non-patent documents, and UniProtKB/Swiss-Prot accession numbers, cited herein are hereby incorporated by reference in their entirety as if each individual reference were specifically and individually indicated to be incorporated by reference.
Summary of The Invention
To meet the above and other needs, disclosed herein are improved methods for purifying a polypeptide comprising an Fc region from one or more impurities. These methods comprise contacting a protein a chromatography matrix with a sample (e.g., a cell lysate) comprising (i) a polypeptide comprising an Fc region and (ii) one or more impurities, and washing the matrix with a wash solution having a pH of about 4.0-10.0 and comprising benzoate and/or benzyl alcohol. The present disclosure is based, at least in part, on the following surprising findings: the use of benzoate (e.g., sodium benzoate) and/or benzyl alcohol in wash solutions at pH of about 4.0-10.0 during protein a chromatography provides superior impurity (e.g., host cell impurities) clearance over currently utilized wash formulations (see figure 1, example 1). The present disclosure is also based, at least in part, on the following findings: the inclusion of one or more additional components selected from benzene sulfonate (e.g., sodium benzene sulfonate), caprylic acid, hexylene glycol, and/or arginine may further improve the removal of impurities when included in the wash solution (see fig. 2 and 3, example 1).
Accordingly, in one aspect, provided herein is a method of purifying a polypeptide comprising an Fc region, the method comprising the steps of: contacting (a) a protein a chromatography matrix with a sample comprising (i) a polypeptide comprising an Fc region and (ii) one or more impurities under conditions in which the polypeptide comprising the Fc region binds to protein a; and (b) washing the substrate with a wash solution, wherein the wash solution comprises one or both of (i) benzoate salt in a concentration of about 0.1M to about 1.0M and (ii) benzyl alcohol in a concentration of about 0.5% to about 4% volume/volume (v/v), and wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprises: (1) a benzoate salt; (2) benzyl alcohol; or (3) benzoate and benzyl alcohol. In some embodiments, the concentration of benzoate salt is from about 0.1M to about 0.5M. In some embodiments that may be combined with any of the preceding embodiments, the benzoate salt is an alkali metal benzoate (benzoate alkali salt). In some embodiments that may be combined with any of the preceding embodiments, the benzoate salt is sodium benzoate. In some embodiments, the concentration of sodium benzoate is from about 0.1M to about 0.3M. In some embodiments, the concentration of sodium benzoate is about 0.3M. In some embodiments, the concentration of sodium benzoate is about 0.5M. In some embodiments that may be combined with any of the preceding embodiments, the concentration of benzyl alcohol is from about 1% to about 4% (v/v). In some embodiments that may be combined with any of the preceding embodiments, the concentration of benzyl alcohol is from about 1% to about 2% (v/v). In some embodiments that may be combined with any of the preceding embodiments, the concentration of benzyl alcohol is about 2% (v/v). In some embodiments that may be combined with any of the preceding embodiments, the concentration of benzyl alcohol is about 4% (v/v).
In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises a buffering agent. In some embodiments, the buffer is selected from the group consisting of phosphate, Tris, arginine, acetate, and citrate. In some embodiments, the buffer is at a concentration of about 10mM to about 50mM or about 10mM to about 500 mM. In some embodiments, the concentration of the buffer is about 50 mM. In some embodiments, the concentration of the buffer is about 500 mM. In some embodiments, the wash solution has a pH of about 5.0 to about 10.0. In some embodiments, the wash solution has a pH of about 5.0 to about 9.0. In some embodiments, the wash solution has a pH of about 5.0, about 6.0, about 7.0, about 8.0, about 9.0, or about 10.0.
In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises sodium benzenesulfonate. In some embodiments, the concentration of sodium benzenesulfonate is from about 0.1M to about 0.5M. In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises caprylic acid. In some embodiments, the concentration of caprylic acid is about 10mM to about 50 mM. In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises hexylene glycol. In some embodiments, the concentration of hexylene glycol is from about 1% to about 10% (v/v). In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises creatine. In some embodiments, the concentration of creatine is from about 10mM to about 100 mM. In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises arginine. In some embodiments, the concentration of arginine is from about 0.1M to about 1.0M. In some embodiments, the concentration of arginine is about 0.5M. In some embodiments, the arginine is arginine-HCl. In some embodiments, the wash solution comprising arginine has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution comprising arginine has a pH of about 8.0 to about 10.0. In some embodiments that may be combined with any of the preceding embodiments, the wash solution further comprises one or more non-buffered salts. In some embodiments, the one or more non-buffering salts are selected from the group consisting of sodium chloride, sodium bromide, potassium chloride, potassium bromide, magnesium chloride, magnesium bromide, calcium chloride, calcium bromide, and any combination thereof. In some embodiments, the one or more non-buffering salts are sodium chloride and/or potassium chloride. In some embodiments, the concentration of the one or more non-buffered salts is from about 0.1M to about 1.0M.
In some embodiments that may be combined with any of the preceding embodiments, the wash solution is a solution selected from the group consisting of: (i) a solution comprising sodium benzoate at a concentration of about 0.5M and sodium bicarbonate at a concentration of about 50mM, pH about 10.0; (ii) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2%, arginine at a concentration of about 0.5M, and sodium phosphate at a concentration of about 50mM, pH about 9.0; (iii) a solution comprising sodium benzoate at a concentration of about 0.5M and benzyl alcohol at a concentration of about 2% (v/v), pH about 7.0; (iv) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and sodium chloride at a concentration of about 0.5M, at a pH of about 7.0; (v) a solution comprising hexylene glycol at a concentration of 10% (v/v), sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v), pH about 7.0; (vi) a solution comprising a benzenesulfonate salt at a concentration of about 0.5M, sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v), at a pH of about 7.0; (vii) a solution comprising caprylic acid at a concentration of about 50mM, sodium benzoate at a concentration of about 0.5M, arginine at a concentration of about 0.5M, and sodium chloride at a concentration of about 0.5M, at a pH of about 7.0; (viii) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M, at a pH of about 6.0; (ix) a solution comprising sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M, at a pH of about 5.0; (x) A solution comprising benzyl alcohol at a concentration of about 4% (v/v) at a pH of about 5.0 to about 10; (xi) A solution comprising benzyl alcohol at a concentration of about 4% (v/v) and having a pH of about 9.0; and (xii) a solution comprising benzyl alcohol at a concentration of about 2% (v/v) and arginine at a concentration of about 0.5M at a pH of about 5.0.
In some embodiments that may be combined with any of the preceding embodiments, the method further comprises the step of washing the substrate with a first solution prior to washing the substrate with a wash solution as described above. In some embodiments, the first solution comprises a buffer selected from the group consisting of a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, a citrate buffer, and any combination thereof. In some embodiments, the first solution comprises a buffer at a concentration of about 10mM to about 100mM or about 10mM to about 500 mM. In some embodiments, the first solution is a phosphate buffer.
In some embodiments that may be combined with any of the preceding embodiments, the method further comprises the step of washing the substrate with a second solution after washing the substrate with the wash solution as described above. In some embodiments, the second solution comprises a buffer selected from the group consisting of a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, a citrate buffer, and any combination thereof. In some embodiments, the second solution comprises a buffer at a concentration of about 10mM to about 100mM or about 10mM to about 500 mM. In some embodiments, the second solution has a pH of about 5.0 to about 7.0. In some embodiments, the second solution comprises a substantially low salt or no salt.
In some embodiments that may be combined with any of the preceding embodiments, the method further comprises the step of contacting the protein a chromatography matrix with an elution solution after the one or more washing steps. In some embodiments, the method further comprises the step of collecting the eluate comprising the polypeptide comprising the Fc region. In some embodiments, the method further comprises the step of filtering the eluate via depth filtration. In some embodiments, the eluate comprises less than about 500 parts per million (ppm) of the one or more impurities.
In some embodiments that may be combined with any of the preceding embodiments, application of the methods described herein results in a polypeptide comprising the Fc region being purified from one or more impurities to a greater extent than a corresponding method lacking a step of washing the substrate with a wash solution. In some embodiments that may be combined with any of the preceding embodiments, the one or more impurities are Host Cell Proteins (HCPs). In some embodiments, the one or more HCPs are selected from phospholipases (e.g., putative phospholipase B-like 2), clusterin, serine proteases, elongation factors, and any combination thereof. In some embodiments, the host cell is a mammalian host cell. In some embodiments, the host cell is a Chinese Hamster Ovary (CHO) cell.
In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human IgG1, IgG2, or IgG4Fc region. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments that may be combined with any of the preceding embodiments, the polypeptide comprising an Fc region is an antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.
In some embodiments, any of the above methods, prior to contacting the protein a chromatography matrix with a sample comprising (i) a polypeptide comprising an Fc region and (ii) one or more impurities, the harvest of the polypeptide comprising the Fc region is adjusted to achieve a final concentration of benzoate salt of about 0.1M to about 0.5M, pH of about 7.0 to about 9.0, e.g., to produce a sample comprising (i) the polypeptide comprising an Fc region, and (ii) one or more impurities. In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the final concentration of benzoate in the harvest is from about 0.4M to about 0.5M. In some embodiments, the pH of the adjusted harvest is from about 7.0 to about 8.0. In some embodiments, the pH of the adjusted harvest is from about 8.0 to about 9.0. In some embodiments, the harvest is produced from a culture comprising a host cell engineered to express the polypeptide. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the harvest is clarified prior to conditioning. In some embodiments, the harvest is clarified after conditioning.
In a related aspect, there is provided a method of purifying a polypeptide comprising an Fc region, the method comprising the steps of: (A) conditioning the harvest comprising the Fc region-containing polypeptide to achieve a final concentration of benzoate salt of about 0.1M to about 0.5M, pH of about 7.0 to about 9.0, e.g., to produce a sample comprising (i) the Fc region-containing polypeptide and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, the at least one chromatography matrix comprises an affinity chromatography matrix. In some embodiments, the affinity chromatography matrix is a protein a chromatography matrix or a protein G chromatography matrix. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with an eluate solution. In some embodiments, the method further comprises the step of collecting the eluate comprising the polypeptide comprising the Fc region. In some embodiments, the method further comprises the step of filtering the eluate via depth filtration. In some embodiments, the eluate comprises less than about 500 parts per million (ppm) of the one or more impurities.
In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the final concentration of benzoate in the harvest is from about 0.4M to about 0.5M. In some embodiments, the pH of the adjusted harvest is from about 7.0 to about 8.0. In some embodiments, the pH of the adjusted harvest is from about 8.0 to about 9.0. In some embodiments, the harvest is produced from a culture comprising a host cell engineered to express the polypeptide. In some embodiments, the host cell is a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a Chinese Hamster Ovary (CHO) cell. In some embodiments, the harvest is clarified prior to conditioning. In some embodiments, the harvest is clarified after conditioning. In some embodiments, the method results in the polypeptide comprising an Fc region being purified from the one or more impurities to a greater extent than a corresponding method lacking the step of modulating a harvest comprising the polypeptide comprising an Fc region to produce a sample. In some embodiments, the one or more impurities are Host Cell Proteins (HCPs). In some embodiments, the one or more HCPs are selected from the group consisting of: phospholipase, clusterin, serine protease, elongation factor, and any combination thereof. In some embodiments, the HCP is putative phospholipase B-like 2(PLBL 2). In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human IgG1, IgG2, or IgG4Fc region. In some embodiments, the Fc region is a mouse Fc region. In some embodiments, the mouse Fc region comprises a mouse IgG1, IgG2, or IgG3 Fc region. In some embodiments, the polypeptide comprising an Fc region is an antibody. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.
It will be appreciated that features of one, some or all of the various embodiments described above and herein may be combined to form further embodiments of the disclosure. These and other aspects of the disclosure will become apparent to those skilled in the art. These and other embodiments of the present disclosure are further described by the following detailed description.
Brief Description of Drawings
Figure 1 shows the concentration of Chinese Hamster Ovary (CHO) Host Cell Protein (HCP) impurities in antibody samples eluted from a protein a column after washing with the indicated control or test wash solutions.
FIGS. 2A-B show the concentration of specific HCP (HCP-A) in antibody samples eluted from ｃA protein A column. Figure 2A shows the concentration of HCP- ｃA in antibody samples eluted from the protein ｃA column after washing with 2% benzyl alcohol ± 0.5M sodium benzoate and/or 0.5M arginine acid, as assessed by elisｃA, compared to control washes. Figure 2B shows the concentration of HCP- ｃA in antibody samples eluted from the protein ｃA column after washing with various wash solutions at pH9.0 or 10.0, as assessed by elsｃA, compared to control washes.
Figure 3 shows the concentration of HCP- ｃA in antibody samples eluted from the protein ｃA column after washing with the indicated wash solution containing additional test compound, as assessed by elisｃA.
Fig. 4A-B show the concentration of general HCP and PLBL2 in antibody samples eluted from the protein a column. Figure 4A shows the concentration of general HCP in antibody samples eluted from the protein a column after washing with 0.5M arginine, 0.5M sodium benzoate, or 4% benzyl alcohol, as assessed by ELISA, compared to a process control wash. Figure 4B shows the general concentration of PLBL2 in antibody samples eluted from the protein a column after washing with 0.5M arginine, 0.5M sodium benzoate, or 4% benzyl alcohol, as assessed by ELISA, compared to a process control wash.
Figure 5 shows the improvement in visual clarity of antibody samples eluted from a protein a column washed with an intermediate wash comprising 2% benzyl alcohol and 0.5M sodium benzoate.
FIGS. 6A-B show the decrease in off-column yield (off-column yield) and removal of PLBL2 when protein A column loading exceeded 40 g/L. FIG. 6A shows that as the loading density of the protein A column was increased from 40g/L to 60g/L, the percentage of the off-column yield decreased linearly from 93.1% to 78.1%. FIG. 6B shows that the level of PLBL2 eluted from the protein A column decreased from 32.1ppm to 17ppm as the loading density of the protein A column increased from 40g/L to 60 g/L.
Figure 7 shows that adjusting the harvest to 0.5M sodium benzoate and pH 7.2 or to 0.5M sodium benzoate and pH9 prior to protein a purification results in improved removal of PLBL2 and HCP impurities. 0.5M sodium benzoate, which adjusted the harvest to pH9.0, showed the lowest level of PLBL2 and HCP impurities, and demonstrated a greater log of PLBL2 clearance relative to pH adjustment alone.
Fig. 8 shows that the relationship between PLBL2 content and sodium benzoate concentration is approximately sigmoidal. For concentrations above 0.4M sodium benzoate, a decrease in the increase in clearance of PLBL2 was observed.
Detailed Description
Described herein are methods for reducing the amount of co-purified impurities (e.g., host cell protein impurities) during protein a-based isolation of proteins containing an Fc region. The methods of the present disclosure employ an intermediate washing step using a novel wash solution containing benzoate and/or benzyl alcohol that has been shown to significantly reduce the levels of host cell protein impurities in the eluate collected during protein a affinity chromatography (see examples 1 and 2). Inclusion of one or more additives (e.g., benzenesulfonic acid (salts), octanoic acid, hexylene glycol, creatine, and/or arginine) in this novel wash solution further improves the removal of impurities from the protein eluate comprising the Fc region-containing protein after capture and elution from the protein a matrix.
I. Definition of
Before the present disclosure is described in detail, it is to be understood that this disclosure is not limited to particular compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "molecule" optionally includes a combination of two or more such molecules, and the like.
As used herein, the term "about" refers to the usual error range for individual values as would be readily known to one skilled in the art. References herein to "about" a value or parameter include (and describe) embodiments that refer to the value or parameter itself.
It is to be understood that the aspects and embodiments of the present disclosure described herein include, consist of, and "consist essentially of the aspects and embodiments.
The term "and/or" as used herein as the phrase "a and/or B" is intended to include both a and B; a or B; (only) A; and (only) B. Also, as used herein, the term "and/or" as the phrase "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; (only) A; (only) B; and (only) C.
The terms "polypeptide" or "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation to a labeling component or toxin. Also included within this definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
The term "antibody" is used herein in the broadest sense and specifically includes monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), antibody fragments, or synthetic polypeptides that carry one or more CDRs or CDR-derived sequences so long as the polypeptide exhibits the desired activity. Antibodies (abs) and immunoglobulins (igs) are glycoproteins with the same structural features. Generally, antibodies are considered igs with a defined or recognized specificity. Thus, while antibodies exhibit binding specificity to a particular target, immunoglobulins include antibodies and other antibody-like molecules that lack target specificity. Antibodies of the present disclosure can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, etc.) or subclass (e.g., IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.). "type" and "class" and "subtype" and "subclass" are used interchangeably herein. Natural or wild-type (obtained from a non-human manipulated member of the population) antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The antibodies described herein can be human antibodies, humanized antibodies, non-human animal (e.g., mouse, rat, hamster, rabbit, camelid, etc.) antibodies, or chimeric antibodies.
In the context of the variable domains of antibodies, the term "variable" may refer to certain portions of the molecule of interest that differ widely in sequence between two antibodies and between more antibodies, and that are used for specific recognition and binding of a particular antibody to its particular target. However, the variability is not evenly distributed throughout the variable domain of the antibody. Variability is concentrated in three segments in both the light and heavy chain variable domains, called Complementarity Determining Regions (CDRs), also called hypervariable regions. The more highly conserved portions of the variable domains are referred to as Framework (FR) regions or sequences. The variable domains of native heavy and light chains each comprise four FR regions, primarily in a β -sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β -sheet structure. The CDRs in each chain are typically held together adjacent to the FR regions and, together with CDR2 from the other chain, contribute to the formation of the target (epitope or determinant) binding site of the antibody (see Kabat et al Sequences of proteins of Immunological Interest, nature Institute of Health, Bethesda, MD (1987)). As used herein, unless otherwise indicated, the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al. One CDR may have the ability to specifically bind to a homologous epitope.
The term "hinge" or "hinge region" as used herein may refer to a flexible polypeptide comprising amino acids between a first constant domain and a second constant domain of an antibody.
The term "bispecific antibody" can refer to a molecule that combines the antigen binding sites of two antibodies within a single molecule. Thus, bispecific antibodies are capable of binding two different antigens simultaneously.
The term "monoclonal antibody" as used herein may refer to an antibody obtained from a population of substantially homologous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they retain the desired activity.
As used herein, the term "multivalent antibody" or "multivalent antibody" may refer to an antibody that comprises two or more antigen binding sites, and thus is capable of simultaneously binding two or more antigens, which may have the same or different structures. The term "bivalent" means that the antibody comprises two antigen binding sites. The term "tetravalent" means that the antibody comprises four antigen binding sites.
As used herein, the term "antigen binding site" may refer to a portion of an antibody that comprises a region that specifically binds to and is complementary to a portion or all of an antigen. When the antigen is large, the antibody may bind only to a specific portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by one or more antibody variable domains and may be constituted by the association of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
"humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof that contain sequences derived from non-human immunoglobulins as compared to human antibodies. Typically, a humanized antibody will comprise substantially all of one variable domain, typically two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin template sequence. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (typically that of a selected human immunoglobulin template). Generally, the goal is to obtain antibody molecules with minimal immunogenicity in humans. Thus, it is possible to also vary one or more amino acids in one or more CDRs to less immunogenic amino acids for a human host without substantially minimizing the specific binding function of the one or more CDRs to the target. Alternatively, the FRs may be non-human, but those with the most immunogenic properties are replaced by less immunogenic amino acids. Nonetheless, CDR grafting (as described above) is not the only method to obtain humanized antibodies. For example, modifying only the CDR regions may be insufficient because it is not uncommon for framework residues to play a role in determining the three-dimensional structure of the CDR loops and the overall affinity of an antibody for its ligand. Thus, any means can be practiced such that the non-human parent antibody molecule is modified to be a less immunogenic molecule to humans, and global sequence identity to human antibodies is not always necessary.
The term "impurity" may refer to any foreign or undesired molecules present in solution (e.g., a sample comprising a polypeptide comprising an Fc region). The impurity may be a biological (molecule) (e.g., a macromolecule), such as a protein, DNA, or RNA, that is also present in the sample containing the protein of interest. Impurities may include undesired protein variants (e.g., aggregated proteins, misfolded proteins, disulfide-insufficiently bound (undersized-bound) proteins, fragments, etc.), other proteins from the host cell, components from the cell culture medium, molecules that are part of the absorbent for affinity chromatography (e.g., protein a), endotoxins, nucleic acids, viruses, etc.
Method for isolating and/or purifying a polypeptide comprising an Fc region
SUMMARY
Certain aspects of the present disclosure relate to methods of purifying polypeptides (e.g., antibodies) comprising an Fc region via protein a chromatography. In some embodiments, the method comprises the steps of: contacting a protein a chromatography matrix or resin with a sample comprising: (1) a polypeptide comprising an Fc region (e.g., an antibody) and (2) one or more impurities (e.g., host cell impurities); and washing the substrate with a washing solution comprising benzoate and/or benzyl alcohol. In some embodiments, the wash solution comprises benzoate salt at a concentration of from about 0.1M to about 1.0M. In some embodiments, the wash solution comprises benzyl alcohol at a concentration of about 0.5% to about 4% volume/volume (v/v). In some embodiments, the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprises one or more additives (e.g., one or more of benzenesulfonic acid (salt), octanoic acid, hexylene glycol, non-buffering salts (such as sodium chloride), creatine, and/or arginine). In some embodiments, the wash solution further comprises a buffering agent. In some embodiments, the harvest comprising the Fc region-containing polypeptide is adjusted to achieve a final concentration of benzoate salt of about 0.1M to 0.5M and a pH of about 7 to about 9 to produce a sample comprising: (1) a polypeptide (e.g., an antibody) comprising an Fc region and (2) one or more impurities (e.g., host cell impurities).
Contacting the sample with a protein A matrix or resin
Certain aspects of the present disclosure relate to methods of purifying polypeptides (e.g., antibodies) comprising an Fc region via protein a chromatography. In some embodiments, the method comprises the steps of: contacting a protein a chromatography matrix or resin with a sample comprising: (1) a polypeptide comprising an Fc region (e.g., an antibody) and (2) one or more impurities (e.g., host cell impurities).
In some embodiments, the disclosure relates to methods of purifying a polypeptide (e.g., an antibody, an immunoadhesin, a fusion protein, etc.) comprising an Fc region from a sample (e.g., a cell lysate sample, a cell culture supernatant sample, etc.). In some embodiments, the sample is a cell culture supernatant (e.g., a supernatant from a cell (e.g., a CHO cell) that is engineered to produce and secrete the polypeptide), or is derived from a cell culture supernatant (e.g., a partially purified cell culture supernatant sample). In some embodiments, the polypeptide comprising an Fc region is a secreted antibody. In some embodiments, the Fc region is a C-terminal region of an immunoglobulin heavy chain, and may include a native sequence Fc region and a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is typically defined as extending from an amino acid residue at position Cys226 or Pro230 to its carboxy terminus (the numbering of the residues in the Fc region is that of the EU index as in Kabat. the Fc region of an immunoglobulin typically comprises two constant domains, CH2 and CH3, and optionally comprises a CH4 domain. in some embodiments, the Fc region is an Fc region obtained from any suitable immunoglobulin, such as an IgG1, IgG2, IgG3, or IgG4 subtype, IgA, IgE, IgD, or igm An IgG2 or IgG3 Fc region. In some embodiments, the Fc region is a human Fc region. In some embodiments, the human Fc region comprises a human IgG1, IgG2, and/or IgG4Fc region.
In some embodiments, the polypeptide comprising an Fc region is an antibody. In some embodiments, "antibody" is used herein in the broadest sense and specifically encompasses monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multivalent antibodies (e.g., bivalent, trivalent, tetravalent, etc.), and multispecific antibodies (e.g., bispecific, trispecific, etc.). The antibody can be from any source, including, for example, human, non-human primate, rodent (e.g., mouse, rat, hamster, etc.), rabbit, camelid, shark, and/or recombinantly produced. In some embodiments, the antibody is a human, humanized, or chimeric antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a multispecific antibody and/or a multivalent antibody. In some embodiments, the antibody is a bispecific antibody or a trispecific antibody.
In some embodiments, the sample comprising the polypeptide comprising an Fc region (e.g., a cell lysate sample, a cell culture supernatant sample, etc.) further comprises one or more impurities. In some embodiments, the one or more impurities are present in the sample as a result of a method for producing a polypeptide comprising an Fc region (e.g., a process for producing a secreted antibody). In some embodiments, the one or more impurities are one or more impurities derived from the host cell (e.g., one or more host cell proteins, one or more host cell nucleic acids, one or more host cell lipids, etc.). The host cell may be any host cell known in the art suitable for producing a polypeptide comprising an Fc region, including, for example, prokaryotic cells (e.g., e.coli cells, aspergillus niger (a. niger) cells, etc.), eukaryotic cells (e.g., yeast cells, plant cells, insect cells (e.g., S1 cells), and/or mammalian (mouse, rat, hamster, rabbit, human, non-human primate, etc.) cells (e.g., hybridomas, CHO cells, 293T cells, per.c6 cells, NS0 cells, etc.) One or more (e.g., one or more, two or more, three or more, four or more, etc.) Host Cell Proteins (HCPs) of the elongation factor and/or any combination thereof. In some embodiments, the host cell is a CHO cell. In some embodiments, the one or more impurities are one or more CHO cell HCPs. In some embodiments, the one or more CHO cell HCPs are one or more of phospholipases, clusterin, serine proteases, elongation factors, and any combination thereof.
In some embodiments, the disclosure relates to methods of purifying a polypeptide comprising an Fc region from one or more impurities in a sample via protein a chromatography. In some embodiments, the sample is contacted with a protein a matrix or resin. In some embodiments, the sample is contacted with a protein a matrix or resin under conditions suitable for binding of a polypeptide comprising an Fc region in the sample to protein a. Methods and suitable conditions for contacting and binding the Fc region-containing polypeptide to the protein a matrix or resin are readily understood by one of ordinary skill in the art (e.g., as described in the manufacturer's protocol for commercially available protein a matrices or resins). Any suitable protein a matrix or resin known in the art may be used in the methods of the present disclosure, including, for example: MabSelect, MabSelect Xtra, MabSelect Sure LX protein A, MabSelectpcc, MabSelect prism A, rProtein A Sepharose CL-4B, and nProtein A Sepharose 4FF (GEHealthcare); EshMunoa, ProSep A, ProSep-vA High Capacity, ProSep-vA Ultra and ProSep-vA Ultra plus (Millipore); poros A and Mabcapture A (Poros); IPA-300, IPA-400, and IPA-500(RepliGen Corp.); affigel protein A and Affiprep protein A (Bio-Rad); mabsorbet A1PP and mabsorbet A2P (Affinity Chromatography Ltd.); protein a Ceramic Hyper D F (PallCorp.); ultralink immobilized protein a and sepharose protein a (pierce); protein a
Washing protein A substrates or resins with washing solutions
Certain aspects of the present disclosure relate to methods of purifying polypeptides comprising an Fc region via protein a chromatography by washing a protein a matrix or resin bound to the polypeptide (e.g., antibody) comprising the Fc region with a wash solution comprising benzoate and/or benzyl alcohol. In some embodiments, the method comprises the steps of: contacting a protein a chromatography matrix or resin with a sample comprising (1) a polypeptide comprising an Fc region (e.g., an antibody) and (2) one or more impurities (e.g., host cell impurities) under conditions wherein the polypeptide comprising an Fc region is contacted with protein a; and washing the substrate or resin with a wash solution comprising benzoate salt at a concentration of about 0.1M to about 1.0M and/or benzyl alcohol at a concentration of about 0.5% to about 4% volume/volume (v/v), wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprises benzoate salt. In some embodiments, the wash solution comprises benzyl alcohol. In some embodiments, the wash solution comprises benzoate and benzyl alcohol.
In some embodiments, the present disclosure relates to wash solutions comprising benzoate and/or benzoic acid (e.g., pH adjusted). Any suitable source or form of benzoate (e.g., alkali metal salt) and/or benzoic acid known in the art may be used in the wash solutions of the present disclosure, including, for example, sodium benzoate, potassium benzoate, lithium benzoate, calcium benzoate, magnesium benzoate, beryllium benzoate, barium benzoate, strontium benzoate, rubidium benzoate, cesium benzoate, and/or any combination thereof. In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate or potassium benzoate. In some embodiments, the benzoate salt is sodium benzoate.
In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution in a concentration of from about 0.1M to about 1.0M. For example, benzoate (e.g., sodium benzoate) and/or benzoic acid may be present in the wash solution at the following concentrations: about 0.1M to about 1.0M, about 0.1M to about 0.9M, about 0.1M to about 0.8M, about 0.1M to about 0.7M, about 0.1M to about 0.6M, about 0.1M to about 0.5M, about 0.1M to about 0.4M, about 0.1M to about 0.3M, about 0.1M to about 0.2M, about 0.2M to about 1.0M, about 0.2M to about 0.9M, about 0.2M to about 0.8M, about 0.2M to about 0.7M, about 0.2M to about 0.6M, about 0.2M to about 0.5M, about 0.2M to about 0.4M, about 0.2M to about 0.3M, about 0.3M to about 1.0M, about 0.3M to about 0.0.0, about 0.0, about 0.3M to about 0.0.0, about 0.0, about 0.3M to about 0.0, about 0.0.0, about 0, about 0.0, about 0.0.0.0, about 0, about 0.0, about 0.0.0, about 0.0, about 0, about 0.0, about 0.0.0, about 0.0, about 0.0.0.0, about 0.0, about 0, about 0.0.0, about 0, about 0., About 0.5M to about 0.7M, about 0.5M to about 0.6M, about 0.6M to about 1.0M, about 0.6M to about 0.9M, about 0.6M to about 0.8M, about 0.6M to about 0.7M, about 0.7M to about 1.0M, about 0.7M to about 0.9M, about 0.7M to about 0.8M, about 0.8M to about 1.0M, about 0.8M to about 0.9M, or about 0.9M to about 1.0M. In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution in a concentration of from about 0.1M to about 0.5M. In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution in a concentration of from about 0.1M to about 0.3M.
In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of any of about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M. In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of about 0.5M. In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution at a concentration of about 0.1M or less than about 0.1M, about 0.3M or less than about 0.3M, about 0.5M or less than about 0.5M, about 0.75M or less than about 0.75M or about 1.0M or less than about 1.0M. In some embodiments, the benzoate (e.g., sodium benzoate) and/or benzoic acid is present in the wash solution in a concentration of about 0.5M or less than about 0.5M.
In some embodiments, the present disclosure relates to a wash solution comprising benzyl alcohol. Any suitable source or form of benzyl alcohol known in the art can be used in the wash solutions of the present disclosure.
In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 0.5% to about 4.0% volume/volume (v/v). For example, benzyl alcohol can be about 0.5% to about 4%, about 1% to about 4%, about 1.5% to about 4%, about 2% to about 4%, 2.5% to about 4%, about 3% to about 4%, about 3.5% to about 4%, about 0.5% to about 3.5%, about 1% to about 3.5%, about 1.5% to about 3.5%, about 2% to about 3.5%, about 2.5% to about 3.5%, about 3% to about 3.5%, about 0.5% to about 3%, the concentration of about 1% to about 3%, about 1.5% to about 3%, about 2% to about 3%, about 2.5% to about 3%, about 0.5% to about 2.5%, about 1% to about 2.5%, about 1.5% to about 2.5%, about 2% to about 2.5%, about 0.5% to about 2%, about 1% to about 2%, about 1.5% to about 2%, about 0.5% to about 1.5%, about 1% to about 1.5%, or about 0.5% to about 1% (v/v) is present in the wash solution. In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 1% to about 4% volume/volume (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 1% to about 2% volume/volume (v/v).
In some embodiments, the benzyl alcohol is present in the wash solution at any concentration of about 0.5%, 0.75%, 1%, 1.25%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, or about 4% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 2% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 1% or less than about 1%, about 2% or less than about 2%, about 3% or less than about 3% or about 4% or less than about 4%. In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 4% or less than about 4.0% (v/v). In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 4% (v/v) or less than about 2% (v/v).
Additive agent
In some embodiments, the wash solutions of the present disclosure further comprise one or more (e.g., one or more, two or more, three or more, four or more, or all five) of the following additives: benzenesulfonic acid (salt), octanoic acid, hexylene glycol, nonbuffering salts, and/or creatine at any of the concentrations described herein. In some embodiments, the wash solution comprising one or more additives has a pH of about 4.0 to about 10.0. In some embodiments, the inclusion of one or more additives in the wash solution further improves the purification of the polypeptide comprising an Fc region from one or more impurities (e.g., host cell impurities) by the methods described herein.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol and one of benzene sulfonic acid (salt), octanoic acid, hexanediol, non-buffered salt, and/or creatine at a pH of about 4.0 to about 10.0. For example, the wash solution may comprise: benzoate and/or benzyl alcohol and benzene sulfonic acid (salt); benzoate and/or benzyl alcohol and caprylic acid; benzoate and/or benzyl alcohol and hexylene glycol; benzoate and/or benzyl alcohol and non-buffering salts; or benzoate and/or benzyl alcohol and creatine, at a pH of from about 4.0 to about 10.0.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol and two of benzene sulfonic acid (salt), octanoic acid, hexylene glycol, non-buffered salt, and/or creatine at a pH of about 4.0 to about 10.0. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, benzenesulfonate and octanoic acid; benzoate and/or benzyl alcohol, benzenesulfonate and hexylene glycol; benzoate and/or benzyl alcohol, benzenesulfonate and nonbuffering salts; benzoate and/or benzyl alcohol, benzenesulfonate and creatine; benzoate and/or benzyl alcohol, octanoic acid and hexylene glycol; benzoate and/or benzyl alcohol, caprylic acid and nonbuffering salts; benzoate and/or benzyl alcohol, caprylic acid and creatine; benzoate and/or benzyl alcohol, hexylene glycol and nonbuffering salts; benzoate and/or benzyl alcohol, hexylene glycol and creatine; or benzoate and/or benzyl alcohol, a nonbuffering salt, and creatine, at a pH of about 4.0 to about 10.0.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol and three of benzene sulfonic acid (salt), octanoic acid, hexylene glycol, non-buffered salt, and/or creatine at a pH of about 4.0 to about 10.0. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, benzene sulfonic acid (salts), octanoic acid and hexanediol; benzoate and/or benzyl alcohol, benzene sulfonic acid (salts), octanoic acid and non-buffer salts; benzoate and/or benzyl alcohol, benzene sulfonic acid (salts), octanoic acid and creatine; benzoate and/or benzyl alcohol, benzene sulfonic acid (salt), hexylene glycol and non-buffer salts; benzoate and/or benzyl alcohol, benzene sulfonic acid (salts), hexylene glycol and creatine; benzoate and/or benzyl alcohol, benzenesulfonic acid (salts), nonbuffering salts and creatine; benzoate and/or benzyl alcohol, caprylic acid, hexylene glycol and non-buffer salts; benzoate and/or benzyl alcohol, octanoic acid, hexanediol and creatine; or benzoate and/or benzyl alcohol, caprylic acid, nonbuffering salts and creatine; benzoate and/or benzyl alcohol, hexylene glycol, nonbuffering salts, and creatine; the pH is from about 4.0 to about 10.0.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol and four of benzene sulfonic acid (salt), octanoic acid, hexanediol, nonbuffering salts, and/or creatine at a pH of about 4.0 to about 10.0. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, benzene sulphonic acid (salt), octanoic acid, hexylene glycol and non-buffer salts; benzoate and/or benzyl alcohol, benzene sulfonic acid (salt), octanoic acid, hexanediol and creatine; benzoate and/or benzyl alcohol, benzene sulfonic acid (salt), octanoic acid, nonbuffering salts and creatine; benzoate and/or benzyl alcohol, benzene sulfonic acid (salt), hexylene glycol, nonbuffering salt and creatine; or benzoate and/or benzyl alcohol, caprylic acid, hexylene glycol, nonbuffering salts, and creatine, at a pH of about 4.0 to about 10.0.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol and all five of benzene sulfonic acid (salt), octanoic acid, hexylene glycol, nonbuffering salts, and creatine, at a pH of about 4.0 to about 10.0.
In some embodiments, the present disclosure relates to a wash solution comprising benzenesulfonic acid (salt). Any suitable form or source of benzenesulfonic acid (salt) known in the art may be used in the wash solutions of the present disclosure, including, for example, benzenesulfonates (e.g., alkali metal salts) such as sodium or potassium benzenesulfonates, benzenesulfonic acid, and/or any combination thereof. In some embodiments, the benzene sulfonic acid (salt) is sodium benzene sulfonate.
In some embodiments, a benzenesulfonate salt (e.g., sodium benzenesulfonate) is present in a wash solution in a concentration of from about 0.1M to about 0.5M. For example, the sodium benzenesulfonate may be present in the wash solution in a concentration of from about 0.1M to about 0.5M, from about 0.1M to about 0.4M, from about 0.1M to about 0.3M, from about 0.1M to about 0.2M, from about 0.2M to about 0.5M, from about 0.2M to about 0.4M, from about 0.2M to about 0.3M, from about 0.3M to about 0.5M, from about 0.3M to about 0.4M, or from about 0.4M to about 0.5M. In some embodiments, the sodium benzenesulfonate is present in the wash solution in a concentration of from about 0.1M to about 0.3M.
In some embodiments, the sodium benzenesulfonate is present in the wash solution at a concentration of any one of about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, or 0.5M. In some embodiments, the sodium benzenesulfonate is present in a wash solution at a concentration of about 0.5M. In some embodiments, the sodium benzenesulfonate is present in the wash solution in a concentration of about 0.1M or less than about 0.1M, about 0.3M or about or less than about 0.3M or about 0.5M or less than about 0.5M. In some embodiments, the sodium benzenesulfonate is present in the wash solution at a concentration of about 0.5M or less than about 0.5M.
In some embodiments, the present disclosure relates to a wash solution comprising caprylic acid. Any suitable form or source of octanoic acid known in the art may be used in the wash solutions of the present disclosure.
In some embodiments, the caprylic acid is present in the wash solution at a concentration of about 1mM to about 50 mM. For example, the caprylic acid may be present in a wash solution at a concentration of about 1mM to about 50mM, about 10mM to about 50mM, about 20mM to about 50mM, about 30mM to about 50mM, about 40mM to about 50mM, about 1mM to about 40mM, about 10mM to about 40mM, about 20mM to about 40mM, about 30mM to about 40mM, about 1mM to about 30mM, about 10mM to about 30mM, about 20mM to about 30mM, about 1mM to about 20mM, about 10mM to about 20mM, or about 1mM to about 10 mM. In some embodiments, the caprylic acid is present in the wash solution at a concentration of about 10mM to about 50 mM.
In some embodiments, the caprylic acid is present in the wash solution at a concentration of any one of about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, or 50 mM. In some embodiments, the caprylic acid is present in the wash solution at a concentration of about 50 mM. In some embodiments, the caprylic acid is present in the wash solution at a concentration of about 10mM or less than about 10mM, about 30mM or less than about 30mM, or about 50mM or less than about 50 mM. In some embodiments, the caprylic acid is present in the wash solution at a concentration of about 50mM or less than about 50 mM.
In some embodiments, the present disclosure relates to a wash solution comprising hexylene glycol. Any suitable form or source of hexylene glycol known in the art may be used in the wash solutions of the present disclosure.
In some embodiments, the hexylene glycol is present in the wash solution at a concentration of about 0.5% to about 10% (v/v). For example, the hexylene glycol may be present in a wash solution at a concentration of about 0.5% to about 10%, about 1% to about 10%, about 2% to about 10%, about 4% to about 10%, about 6% to about 10%, about 8% to about 10%, about 9% to about 10%, 0.5% to about 9%, about 1% to about 9%, about 2% to about 9%, about 4% to about 9%, about 6% to about 9%, about 8% to about 9%, about 0.5% to about 8%, about 1% to about 8%, about 2% to about 8%, about 4% to about 8%, about 6% to about 8%, about 0.5% to about 6%, about 1% to about 6%, about 4% to about 6%, about 0.5% to about 4%, about 1% to about 4%, about 2% to about 4%, about 0.5% to about 2%, about 1% to about 2%, or about 0.5% to about 1% v% (v/v). In some embodiments, the hexylene glycol is present in the wash solution at a concentration of about 1% to about 10% (v/v).
In some embodiments, the benzyl alcohol is present in the wash solution at a concentration of about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% (v/v). In some embodiments, the hexylene glycol is present in the wash solution at a concentration of about 10% (v/v). In some embodiments, the hexylene glycol is present in the wash solution at a concentration of about 1% or less than about 1%, about 2% or less than about 2%, about 4% or less than about 4%, about 6% or less than about 6%, about 8% or less than about 8%, or about 10% or less than about 10%. In some embodiments, the hexylene glycol is present in the wash solution at a concentration of about 10% or less than 10% (v/v).
In some embodiments, the present disclosure relates to wash solutions comprising one or more (e.g., one or two or more, three or more, etc.) non-buffered salts. Any suitable form or source of non-buffered salts known in the art may be used in the wash solutions of the present disclosure. The non-buffering salt may comprise a halogen salt (e.g. a salt comprising Cl or Br), in particular a halogen salt comprising an alkali metal (e.g. Na or K) or an alkaline earth metal (e.g. Ca or Mg). In some embodiments, the non-buffering salt is sodium chloride and/or potassium chloride. In some embodiments, the non-buffered salt is sodium chloride.
In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.1M to about 1.0M. For example, the non-buffering salt (e.g., sodium chloride) may be present in the wash solution at a concentration of about 0.1M to about 1.0M, about 0.1M to about 0.8M, about 0.1M to about 0.5M, about 0.1M to about 0.4M, about 0.1M to about 0.2M, about 0.2M to about 1.0M, about 0.2M to about 0.8M, about 0.2M to about 0.6M, about 0.2M to about 0.5M, about 0.2M to about 0.4M, about 0.4M to about 1.0M, about 0.4M to about 0.8M, about 0.4M to about 0.6M, about 0.4M to about 0.5M, about 0.5M to about 1.0M, about 0.5M to about 0.8M, about 0.6M, about 0.4M to about 0.5M to about 0.8M, about 0.6M, about 0M to about 0.8M, about 0M, or about 0M to about 0.8M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.1M to about 0.5M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5M to about 1.0M.
In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of any of about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 1.0M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.1M or less than about 0.1M, about 0.2M or less than about 0.2M, about 0.4M or less than about 0.4M, about 0.5M or less than about 0.5M, about 0.6M or less than about 0.6M, about 0.8M or less than about 0.8M, or about 1.0M or less than about 1.0M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 0.5M or less than about 0.5M. In some embodiments, the non-buffering salt (e.g., sodium chloride) is present in the wash solution at a concentration of about 1.0M or less than about 1.0M.
In some embodiments, the present disclosure relates to a wash solution comprising creatine. Any suitable form or source of creatine known in the art may be used in the wash solutions of the present disclosure, including, for example, creatine-HCl, creatine esters, creatine pyruvate, creatine phosphate, creatine alpha-ketoglutarate, creatine citrate, and/or any combination thereof. In some embodiments, the creatine is creatine-HCl.
In some embodiments, the creatine is present in the wash solution at a concentration of about 1mM to about 100 mM. For example, the creatine may be present in a wash solution at a concentration of about 1mM to about 100mM, about 10mM to about 100mM, about 25mM to about 100mM, about 50mM to about 100mM, about 75mM to about 100mM, about 1mM to about 75mM, about 10mM to about 75mM, about 25mM to about 75mM, about 50mM to about 75mM, about 1mM to about 50mM, about 10mM to about 50mM, about 25mM to about 50mM, about 1mM to about 25mM, about 10mM to about 25mM, or about 1mM to about 10 mM. In some embodiments, the creatine is present in the wash solution at a concentration of about 10mM to about 100 mM. In some embodiments, the creatine is present in the wash solution at a concentration of about 10mM to about 50 mM. In some embodiments, the creatine is present in the wash solution at a concentration of any one of about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100 mM. In some embodiments, the creatine is present in the wash solution at a concentration of about 50 mM.
Arginine
In some embodiments, the present disclosure relates to a wash solution further comprising arginine and/or an arginine derivative. In some embodiments, the inclusion of arginine and/or arginine derivative in the wash solution further improves the purification of a polypeptide comprising an Fc region from one or more impurities (e.g., host cell impurities) by the methods described herein. Any suitable form or source of arginine and/or arginine derivatives known in the art including, for example, arginine-HCl, acetyl arginine, agmatine, arginine (arginic acid), N- α -butyryl-L-arginine, N- α -valeryl-arginine, and/or any combination thereof, may be used in the wash solutions of the present disclosure. The arginine and/or arginine derivative may be L-arginine and/or D-arginine, and derivatives thereof. In some embodiments, the arginine and/or arginine derivative is arginine-HCl.
In some embodiments, the present disclosure relates to the use of arginine and/or arginine derivatives (e.g., arginine-HCl) in wash solutions comprising benzoate and/or benzyl alcohol. In some embodiments, the wash solution comprises benzoate and arginine and/or arginine derivatives (e.g., arginine-HCl). In some embodiments, the wash solution comprises benzyl alcohol and arginine and/or an arginine derivative (e.g., arginine-HCl). In some embodiments, the wash solution comprises benzoate salt, benzyl alcohol, and arginine and/or arginine derivatives (e.g., arginine-HCl). In some embodiments, the wash solution further comprises one or more (e.g., one or more, two or more, three or more, four or more, or all five) of a benzenesulfonate, octanoic acid, hexanediol, a non-buffer salt, and/or creatine at any concentration described herein. In some embodiments, the wash solution comprising arginine and/or arginine derivatives has a pH of about 4.0 to about 10.0. In some embodiments, the wash solution comprising arginine and/or arginine derivatives has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution comprising arginine and/or arginine derivatives has a pH of about 4.0 to about 5.0. In some embodiments, the wash solution comprising arginine and/or arginine derivatives has a pH of about 8.0 to about 10.0. In some embodiments, the wash solution comprising arginine and/or arginine derivatives has a pH of about 8.0 to about 9.0.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol, arginine and/or an arginine derivative (e.g., arginine-HCl), and one of benzenesulfonic acid (salt), octanoic acid, hexylene glycol, non-buffered salt, and/or creatine. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, arginine and benzenesulfonic acid (salts); benzoate and/or benzyl alcohol, arginine and caprylic acid; benzoate and/or benzyl alcohol, arginine and hexylene glycol; benzoate and/or benzyl alcohol, arginine and nonbuffering salts; or benzoate and/or benzyl alcohol, arginine and creatine.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol, arginine and/or arginine derivatives (e.g., arginine-HCl), and two of benzene sulfonic acid (salt), caprylic acid, hexylene glycol, non-buffered salts, and/or creatine. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt) and octanoic acid; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt) and hexylene glycol; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts) and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt), and creatine; benzoate and/or benzyl alcohol, arginine, caprylic acid and hexylene glycol; benzoate and/or benzyl alcohol, arginine, caprylic acid and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, caprylic acid and creatine; benzoate and/or benzyl alcohol, arginine, hexylene glycol and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, hexylene glycol and creatine; or benzoate and/or benzyl alcohol, arginine, nonbuffering salts and creatine.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol, arginine and/or arginine derivatives (e.g., arginine-HCl), and three of benzenesulfonic acid (salt), octanoic acid, hexylene glycol, non-buffered salts, and/or creatine. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), octanoic acid and hexanediol; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), octanoic acid and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), octanoic acid and creatine; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), hexylene glycol and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), hexylene glycol and creatine; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salts), nonbuffering salts and creatine; benzoate and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol, and non-buffer salts; benzoate and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol and creatine; benzoate and/or benzyl alcohol, arginine, caprylic acid, nonbuffering salts, and creatine; or benzoate and/or benzyl alcohol, arginine, hexylene glycol, nonbuffering salts and creatine.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol, arginine and/or arginine derivatives (e.g., arginine-HCl), and four of benzene sulfonic acid (salt), caprylic acid, hexylene glycol, non-buffered salts, and/or creatine. For example, the wash solution may comprise: benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt), octanoic acid, hexylene glycol and nonbuffering salts; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt), octanoic acid, hexanediol and creatine; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt), octanoic acid, nonbuffering salts, and creatine; benzoate and/or benzyl alcohol, arginine, benzenesulfonic acid (salt), hexylene glycol, nonbuffering salts, and creatine; benzoate and/or benzyl alcohol, arginine, caprylic acid, hexylene glycol, nonbuffering salts, and creatine.
In some embodiments, the wash solution comprises benzoate and/or benzyl alcohol, arginine and/or arginine derivatives (e.g., arginine-HCl), and all five of benzenesulfonic acid (salt), octanoic acid, hexylene glycol, non-buffered salts, and/or creatine.
In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1M to about 1.0M. For example, the arginine and/or arginine derivative (e.g., arginine-HCl) may be present in an amount of about 0.1M to about 1.0M, about 0.1M to about 0.9M, about 0.1M to about 0.8M, about 0.1M to about 0.7M, about 0.1M to about 0.6M, about 0.1M to about 0.5M, about 0.1M to about 0.4M, about 0.1M to about 0.3M, about 0.1M to about 0.2M, about 0.2M to about 1.0M, about 0.2M to about 0.9M, about 0.2M to about 0.8M, about 0.2M to about 0.7M, about 0.2M to about 0.6M, about 0.2M to about 0.5M, about 0.2M to about 0.4M, about 0.2M to about 0.7M, about 0.2M to about 0.0.0M, about 0M to about 0.3M, about 0.0M to about 0.0.0M, about 0M to about 0.3M to about 0.0, about 0M to about 0.0M, about 0M to about 0.0.0, about 0M to about 0.0, about 0M to about 0.0, about 0M to about 0.0, about 0M to about 0.0, about 0 to about 0M, about 0.0 to about, The concentration of about 0.4M to about 0.6M, about 0.4M to about 0.5M, about 0.5M to about 1.0M, about 0.5M to about 0.9M, about 0.5M to about 0.8M, about 0.5M to about 0.7M, about 0.5M to about 0.6M, about 0.6M to about 1.0M, about 0.6M to about 0.9M, about 0.6M to about 0.8M, about 0.6M to about 0.7M, about 0.7M to about 1.0M, about 0.7M to about 0.9M, about 0.7M to about 0.8M, about 0.8M to about 1.0M, about 0.8M to about 0.9M, or about 0.9M to about 1.0M is present in the wash solution. In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1M to about 0.5M. In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1M to about 0.3M.
In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of any one of about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, or 1.0M. In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.5M. In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.1M or less than about 0.1M, about 0.2M or less than about 0.2M, about 0.3M or less than about 0.3M, about 0.4M or less than about 0.4M, about 0.5M or less than about 0.5M, about 0.75M or less than about 0.75M, or about 1.0M or less than about 1.0M. In some embodiments, the arginine and/or arginine derivative (e.g., arginine-HCl) is present in the wash solution at a concentration of about 0.5M or less than about 0.5M.
pH
In some embodiments, the present disclosure relates to wash solutions having a pH of about 4.0 to about 10.0. For example, the wash solution may have a pH of about 4.0 to about 10.0, about 5.0 to about 10.0, about 6.5 to about 10.0, about 7.0 to about 10.0, about 7.5 to about 10.0, about 8.0 to about 10.0, about 9.0 to about 10.0, 4.0 to about 9.0, about 5.0 to about 9.0, about 6.0 to about 9.0, about 6.5 to about 9.0, about 7.0 to about 9.0, about 7.5 to about 9.0, about 8.0 to about 9.0, 4.0 to about 8.0, about 5.0 to about 8.0, about 6.0 to about 8.0, about 6.5 to about 8.0, about 7.0 to about 8.0, about 7.5 to about 7.0, about 6.0 to about 5.0, about 6.0 to about 7.0, about 6.0 to about 5.0, about 6.0 to about 7.0, about 6.0 to about 6.0. In some embodiments, the wash solution has a pH of about 5.0 to about 9.0. In some embodiments, the wash solution has a pH of about 4.0 to about 6.0. In some embodiments, the wash solution has a pH of about 4.0 to about 5.0. In some embodiments, the wash solution has a pH of about 8.0 to about 10.0. In some embodiments, the wash solution has a pH of about 8.0 to about 9.0.
In some embodiments, the wash solution has a pH of about any one of 4.0, 4.25, 4.5, 4.75, 5.0, 5.25, 5.5, 5.75, 6.0, 6.25, 6.5, 6.75, 7.0, 7.25, 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, or 10.0. In some embodiments, the wash solution has a pH of about 4.0. In some embodiments, the wash solution has a pH of about 5.0. In some embodiments, the wash solution has a pH of about 6.0. In some embodiments, the wash solution has a pH of about 6.5. In some embodiments, the wash solution has a pH of about 7.0. In some embodiments, the wash solution has a pH of about 7.5. In some embodiments, the wash solution has a pH of about 9.0. In some embodiments, the wash solution has a pH of about 10.0.
Buffering agent
In some embodiments, the wash solutions of the present disclosure further comprise one or more (e.g., one or more, two or more, three or more, four or more, five or more, etc.) buffers. Any suitable buffer known in the art may be used in the wash solutions of the present disclosure, including, for example, phosphate, Tris (hydroxymethyl) methylamine), Bis-Tris propane, arginine, histidine, triethanolamine, diethanolamine, formate, acetate, carbonate MES (2- (N-morpholino) ethanesulfonic acid), citrate, HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid), TAPS (3- { [ Tris (hydroxymethyl) methyl ] amino } propanesulfonic acid), Bicine (N, N-Bis (2-hydroxyethyl) glycine), Tricine (N-Tris (hydroxymethyl) methylglycine), TES (2- { [ Tris (hydroxymethyl) methyl ] amino } ethanesulfonic acid, PIPES (piperazine-N, n' -bis (2-ethanesulfonic acid), cacodyae (dimethylarsinic acid), SSC (sodium citrate brine), and/or any combination thereof. In some embodiments, the buffer is one or more of phosphate, Tris, arginine, acetate, and citrate.
In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 1mM to about 100mM or about 1mM to about 500 mM. For example, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) may be present in an amount of about 1mM to about 500mM, 10mM to about 500mM, 50mM to 500mM, 100mM to about 500mM, 150mM to about 500mM, 200mM to about 500mM, 250mM to about 500mM, 300mM to about 500mM, 350mM to about 500mM, 400mM to about 500mM, 450mM to about 500mM, 1mM to about 450mM, 1mM to about 400mM, 1mM to about 350mM, 1mM to about 300mM, 1mM to about 250mM, 1mM to about 200mM, 1mM to about 150mM, 1mM to about 100mM, about 10mM to about 100mM, about 25mM to about 100mM, about 40mM to about 100mM, about 50mM to about 100mM, about 60mM to about 100mM, about 75mM to about 100mM, about 1mM to about 75mM, about 10mM to about 40mM, about 75mM to about 75mM, about 50mM to about 75mM, about 75mM to about 50mM, about 75mM, about 500, About 1mM to about 60mM, about 10mM to about 60mM, about 25mM to about 60mM, about 40mM to about 60mM, about 50mM to about 60mM, about 1mM to about 50mM, about 10mM to about 50mM, about 25mM to about 50mM, about 40mM to about 50mM, about 1mM to about 40mM, about 10mM to about 40mM, about 25mM to about 40mM, about 1mM to about 25mM, about 10mM to about 25mM, or about 1mM to about 10mM is present in the wash solution. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 10mM to about 50mM, or about 10mM to about 500 mM.
In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at any concentration of about 1mM, 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at any one of a concentration of about 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 500 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 50 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 10mM or less than about 10mM, about 25mM or less than about 25mM, about 50mM or less than about 50mM, about 75mM or less than about 75mM, or about 100mM or less than about 100mM, or about 500mM or less than about 500 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 50mM or less than about 50 mM. In some embodiments, the buffer (e.g., phosphate, Tris, arginine, acetate, and/or citrate) is present in the wash solution at a concentration of about 500mM or less than about 500 mM.
Exemplary washing solutions
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate and/or benzyl alcohol, and have a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M and/or benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M and benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).
In some embodiments, a wash solution of the present disclosure comprises sodium benzoate, benzyl alcohol, and/or sodium chloride, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and/or sodium chloride at a concentration of 0.5M, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and sodium chloride at a concentration of 0.5M, and has a pH of about 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).
In some embodiments, a wash solution of the present disclosure comprises sodium benzoate, benzyl alcohol, arginine, and/or sodium chloride, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), arginine at a concentration of 0.5M, and/or sodium chloride at a concentration of 0.5M, and has a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), arginine at a concentration of 0.5M, and sodium chloride at a concentration of 0.5M, and has a pH of about 7.0. In some embodiments, the wash solution further comprises a phosphate buffer (e.g., at a concentration of about 50 mM).
In some embodiments, a wash solution of the present disclosure comprises sodium benzoate, benzyl alcohol, a phosphate buffer, and/or arginine, and has a pH of about 9.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), phosphate buffer at a concentration of 50mM, and/or arginine at a concentration of 0.5M, and has a pH of about 9.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), phosphate buffer at a concentration of about 50mM, and arginine at a concentration of 0.5M, and has a pH of about 9.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate, benzyl alcohol, and/or arginine, and have a pH of about 6.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and/or arginine at a concentration of 0.5M, and has a pH of about 6.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and sodium chloride at a concentration of 0.5M, and has a pH of about 6.0.
In some embodiments, the wash solutions of the present disclosure comprise hexylene glycol, sodium benzoate, and/or benzyl alcohol, and have a pH of about 7.0. In some embodiments, the wash solution comprises hexylene glycol at a concentration of about 10% (v/v), sodium benzoate at a concentration of about 0.5M, and/or benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0. In some embodiments, the wash solution comprises hexylene glycol at a concentration of about 10% (v/v), sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0.
In some embodiments, a wash solution of the present disclosure comprises benzenesulfonic acid (salt), sodium benzoate, and/or benzyl alcohol, and has a pH of about 7.0. In some embodiments, the wash solution comprises benzenesulfonic acid (salt) at a concentration of about 0.5M, sodium benzoate at a concentration of about 0.5M, and/or benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0. In some embodiments, the wash solution comprises benzenesulfonic acid (salt) at a concentration of about 0.5M, sodium benzoate at a concentration of about 0.5M, and benzyl alcohol at a concentration of about 2% (v/v), and has a pH of about 7.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate, benzyl alcohol, and/or arginine (e.g., arginine-HCl), and have a pH of about 5.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and/or arginine at a concentration of about 0.5M (e.g., arginine-HCl), having a pH of about 5.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M (e.g., arginine-HCl), having a pH of about 5.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate, benzyl alcohol, and/or arginine (e.g., arginine-HCl), and have a pH of about 6.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and/or arginine at a concentration of about 0.5M (e.g., arginine-HCl), having a pH of about 6.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, benzyl alcohol at a concentration of about 2% (v/v), and arginine at a concentration of about 0.5M (e.g., arginine-HCl), having a pH of about 6.0.
In some embodiments, the wash solutions of the present disclosure comprise benzyl alcohol and/or arginine (e.g., arginine-HCl), and have a pH of about 5.0. In some embodiments, the wash solution comprises benzyl alcohol at a concentration of about 2% (v/v) and/or arginine (e.g., arginine-HCl) at a concentration of about 0.5M, and has a pH of about 5.0. In some embodiments, the wash solution comprises benzyl alcohol at a concentration of about 2% (v/v) and arginine (e.g., arginine-HCl) at a concentration of about 0.5M, and has a pH of about 5.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate, arginine (e.g., arginine-HCl), caprylic acid, and/or sodium chloride, and have a pH of about 9.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, arginine (e.g., arginine-HCl) at a concentration of about 0.5M, caprylic acid at a concentration of about 50mM, and/or sodium chloride at a concentration of 0.5M, at a pH of about 9.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, arginine (e.g., arginine-HCl) at a concentration of about 0.5M, caprylic acid at a concentration of about 50mM, and sodium chloride at a concentration of 0.5M, having a pH of about 9.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate, arginine (e.g., arginine-HCl), caprylic acid, and/or sodium chloride, and have a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, arginine (e.g., arginine-HCl) at a concentration of about 0.5M, caprylic acid at a concentration of about 50mM, and/or sodium chloride at a concentration of 0.5M, having a pH of about 7.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M, arginine (e.g., arginine-HCl) at a concentration of about 0.5M, caprylic acid at a concentration of about 50mM, and sodium chloride at a concentration of 0.5M, having a pH of about 7.0.
In some embodiments, the wash solutions of the present disclosure comprise sodium benzoate and/or sodium bicarbonate, and have a pH of about 10.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M and/or sodium bicarbonate at a concentration of about 50mM, and has a pH of about 10.0. In some embodiments, the wash solution comprises sodium benzoate at a concentration of about 0.5M and sodium bicarbonate at a concentration of about 50mM, and has a pH of about 10.0.
In some embodiments, the wash solutions of the present disclosure comprise benzyl alcohol at a concentration of about 4% (v/v), and have a pH of about 5.0 to about 10. In some embodiments, the wash solution comprises benzyl alcohol at a concentration of about 4% (v/v), and has a pH of about 9.0.
Conditioning harvest comprising a polypeptide comprising an Fc region prior to chromatography
In one aspect, there is provided a method of purifying a polypeptide comprising an Fc region, the method comprising the steps of: (A) conditioning a harvest comprising a polypeptide comprising an Fc region to achieve a final concentration of benzoate salt from about 0.1M to about 0.5M, pH of about 7.0 to about 9.0 to produce a sample comprising (i) the polypeptide comprising the Fc region and (ii) one or more impurities; and (B) contacting the sample with at least one chromatography matrix. In some embodiments, the at least one chromatography matrix is an affinity chromatography matrix, e.g., a protein a chromatography matrix or a protein G chromatography matrix. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with an elution solution. In some embodiments, the method further comprises the step of collecting the eluate comprising the Fc region-containing polypeptide.
In some embodiments, the term "harvest" refers to the fluid present at the end of or after a cell culture, such as a cell lysate sample or a cell culture supernatant sample (e.g., supernatant from cells, such as CHO cells, that are engineered to produce and secrete a polypeptide). In some embodiments, the harvest comprises intact host cells and/or cell debris. In some embodiments, the harvest does not comprise intact host cells and/or cell debris. For example, in some embodiments, the fluid present at the end of or after the cell culture is subjected to one or more centrifugation and/or filtration steps prior to conditioning to obtain a final concentration of benzoate salt of about 0.1M to about 0.5M and a pH of about 7.0 to about 9.0. In some embodiments, the harvest is derived from a fluid present at the end of the cell culture or after the cell culture. For example, in some embodiments, the fluid present at the end of or after the cell culture is subjected to one or more pretreatment steps to optimize cell isolation and/or purification of the polypeptide comprising the Fc region.
In related aspects, any of the methods of purifying a polypeptide comprising an Fc region described herein further comprises the step of adjusting the harvest comprising the polypeptide comprising an Fc region to obtain a final concentration of benzoate salt of 0.1M to about 0.5M, and a pH of about 7.0 to about 9.0, to produce a sample comprising (i) the polypeptide comprising an Fc region and (ii) one or more impurities.
In some embodiments, the benzoate salt is an alkali metal benzoate salt. In some embodiments, the benzoate salt is sodium benzoate. In some embodiments, the harvest is adjusted to obtain a final concentration of benzoate (e.g., sodium benzoate) of any one of (including any range between) 0.025M, 0.05M, 0.075M, 0.1M, 0.125M, 0.15M, 0.175M, 0.2M, 0.225M, 0.25M, 0.275M, 0.3M, 0.325M, 0.35M, 0.375M, 0.4M, 0.425M, 0.45M, 0.475M, 0.5M, 0.525M, 0.55M, 0.575M, 0.6M, 0.625M, 0.65M, 0.675M, 0.7M, 0.725M, 0.75M, 0.775M, or 0.8M. In some embodiments, the pH of the harvest is adjusted to about any one of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0, including any range between these values. In some embodiments, the harvest is clarified prior to conditioning (e.g., addition of sodium benzoate and adjustment of pH). In some embodiments, the harvest is clarified after conditioning (e.g., addition of sodium benzoate and adjustment of pH).
In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.5M and a pH of about 7. In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.1M and a pH of about 9. In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.2M, pH9. In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.3M and a pH of about 9. In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.4M at a pH of 9. In some embodiments, the method comprises adjusting the harvest to achieve a final sodium benzoate concentration of about 0.5M, pH9.
In some embodiments, modulating the harvest (e.g., adding sodium benzoate and adjusting the pH) results in the polypeptide comprising the Fc region being purified from one or more impurities to a greater extent to produce the sample, as compared to a corresponding method lacking the step of modulating the harvest comprising the polypeptide comprising the Fc region. In some embodiments, the one or more impurities are Host Cell Proteins (HCPs), such as phospholipases, clusterin, serine proteases, elongation factors, and any combination thereof. In some embodiments, the HCP is putative phospholipase B-like 2(PLBL 2).
In some embodiments, the conditioned harvest (e.g., to which sodium benzoate has been added to achieve the final concentrations described herein and whose pH has been adjusted as described herein) or is a sample comprising (i) an Fc-containing polypeptide and (ii) one or more impurities. In some embodiments, the sample is contacted with (e.g., the sample is subjected to at least one chromatography step) at least one chromatography matrix. In some embodiments, the at least one chromatography matrix comprises any one or more of: affinity chromatography matrices, mixed mode chromatography matrices (e.g., multimodal chromatography matrices), Hydrophobic Interaction (HIC) chromatography matrices, anion exchange chromatography matrices, cation exchange chromatography matrices, size exclusion chromatography matrices, Ceramic Hydroxyapatite (CHT) chromatography matrices, and/or hydrophilic interaction liquid chromatography (HILIC) matrices, and the like, in any order. In some embodiments, the sample is contacted with an affinity chromatography matrix, such as a protein a matrix or a protein G matrix. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with at least one wash solution. In some embodiments, the method further comprises the step of contacting the at least one chromatography matrix with an elution solution. In some embodiments, the method further comprises the step of collecting the eluate comprising the Fc region-containing polypeptide.
Impurity removal
Certain aspects of the present disclosure relate to methods of purifying a polypeptide comprising an Fc region (e.g., an antibody) by washing a protein a matrix bound to the polypeptide with a wash solution comprising benzoate and/or benzyl alcohol via protein a chromatography to improve purification of the polypeptide from one or more impurities. In some embodiments, the method comprises the steps of: contacting a protein a chromatography matrix with a sample comprising: (1) a polypeptide comprising an Fc region (e.g., an antibody) and (2) one or more impurities (e.g., host cell impurities); and washing the substrate with a wash solution comprising benzoate salt at a concentration of about 0.1M to about 1.0M and/or benzyl alcohol at a concentration of about 0.5% to about 4% volume/volume (v/v), wherein the wash solution has a pH of about 4.0 to about 10.0. In some embodiments, washing the protein a matrix with the wash solution results in the polypeptide comprising the Fc region being purified from the one or more impurities to a greater extent than a corresponding method (as described above) lacking a step of washing the protein a matrix with the wash solution.
Standard protein a procedures typically result in a product purity of about 95% without the use of washing steps as described herein. The largest proportion of impurities in the product is due to High Molecular Weight (HMW) aggregates and/or Low Molecular Weight (LMW) fragments of the product. These product variants are considered impurities because they have the ability to separate from the product based on various parameters (e.g., charge, hydrophobicity, size differences, etc.). These HMW and LMW impurities comprise about 4-5% of the protein A pool. In addition, the absence of standard protein a procedures using washing steps as described herein also typically results in Host Cell Protein (HCP) impurities on the order of 1000ppm or 0.1% of the product pool (order). However, due to the specifications set for injectable mAb products (see, e.g., FDA guidelines), it is important to reduce and/or completely remove this 0.1% HCP impurity. The step comprising applying the wash solution described herein can reduce the amount of HCP present in the cell to 100-10ppm (a HCP reduction of 1/10 to 1/100 relative to the same protein a process lacking the wash step described herein), representing a 90-99% relative improvement. Methods of measuring protein purity and/or impurity levels of a sample (e.g., by ELISA assay) are generally known to those of ordinary skill in the art. Exemplary purifications of monoclonal antibodies (mabs) purified from one or more host cell proteins using standard methods relative to any of the methods described herein are shown in table a below.
Table a: exemplary purification Process
In some embodiments, the methods described herein produce a protein pool containing polypeptides comprising an Fc region (e.g., monoclonal antibodies) after protein a elution, the protein pool comprising less than about 500ppm (parts per million) HCP (e.g., one or more HCP from CHO cells). For example, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein can contain less than about 500ppm, less than about 450ppm, less than about 400ppm, less than about 350ppm, less than about 300ppm, less than about 250ppm, less than about 200ppm, less than about 150ppm, less than about 100ppm, less than about 75ppm, less than about 50ppm, less than about 25ppm, less than about 10ppm, or less than about 1ppm HCP (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein comprises less than about 100ppm HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool comprising a polypeptide comprising an Fc region produced by the methods described herein contains less than about 10ppm HCPs (e.g., one or more HCPs from CHO cells).
In some embodiments, the methods described herein produce a protein pool containing polypeptides (e.g., monoclonal antibodies) comprising an Fc region after protein a elution, the protein pool comprising less than about 0.1% HCPs (e.g., one or more HCPs from CHO cells). For example, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein can contain less than about 0.1%, less than about 0.09%, less than about 0.08%, less than about 0.07%, less than about 0.06%, less than about 0.05%, less than about 0.04%, less than about 0.03%, less than about 0.02%, or less than about 0.01% HCP (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein contains less than about 0.05% HCPs (e.g., one or more HCPs from CHO cells). In some embodiments, a protein pool containing a polypeptide comprising an Fc region produced by the methods described herein contains less than about 0.01% HCPs (e.g., one or more HCPs from CHO cells).
In some embodiments, the methods described herein reduce the amount and/or concentration (e.g., parts per million) of one or more impurities co-purified with a polypeptide comprising an Fc region by at least about 10% relative to the amount of the one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with the polypeptide comprising an Fc region by a corresponding method lacking a step of washing the protein a matrix with a wash solution. For example, the methods described herein reduce the amount and/or concentration (e.g., parts per million) of one or more impurities co-purified with a polypeptide comprising an Fc region by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% relative to the amount of the one or more impurities co-purified with the polypeptide comprising an Fc region (e.g., one or more HCPs, such as one or more HCPs from CHO cells) purified by a corresponding method that lacks a step of washing the protein a matrix with a wash solution. In some embodiments, the methods described herein reduce the amount and/or concentration (e.g., parts per million) of the one or more impurities co-purified with the polypeptide comprising an Fc region to at most about 1/1.5 relative to the amount of the one or more impurities co-purified with the polypeptide comprising an Fc region purified by a corresponding method lacking a step of washing the protein a substrate with a wash solution. For example, the methods described herein reduce the amount and/or concentration (e.g., parts per million) of one or more impurities (e.g., one or more HCPs, such as one or more HCPs from CHO cells) co-purified with a polypeptide comprising an Fc region to at most about 1/1.5, at most about 1/2, at most about 1/2.5, at most about 1/3, at most about 1/3.5, at most about 1/4, at most about 1/4.5, at most about 1/5, at most about 1/5.5, at most about 1/6, at most about 1/6.5, at most about 1/7, at most about 1/7.5, at most about 1/8, at most about 1/8.5, at most about, At most about 1/9, at most about 1/9.5, at most about 1/10, at most about 1/50, or at most about 1/100.
Additional step
In some embodiments, the methods described herein further comprise one or more additional washing steps. In some embodiments, the methods described herein further comprise one or more elution steps. In some embodiments, the methods described herein further comprise one or more washing steps and one or more elution steps.
In some embodiments, the present disclosure relates to washing a protein a substrate with a first solution prior to washing the substrate with a wash solution. In some embodiments, the substrate is washed with the first solution one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) prior to washing the substrate with the wash solution. In some embodiments, the substrate is washed once with the first solution prior to washing the substrate with the wash solution. In some embodiments, the first solution comprises a buffer. Any suitable buffer known in the art may be used in the first solution, including, for example, phosphate, Tris (hydroxymethyl) methylamine), acetate, carbonate, citrate, Bis-Tris propane, arginine, histidine, triethanolamine, diethanolamine, formate, MES (2- (N-morpholino) ethanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid), TAPS (3- { [ Tris (hydroxymethyl) methyl ] amino } propanesulfonic acid), Bicine (N, N-Bis (2-hydroxyethyl) glycine), Tricine (N-Tris (hydroxymethyl) methylglycine), TES (2- { [ Tris (hydroxymethyl) methyl ] amino } ethanesulfonic acid, PIPES (piperazine-N, n' -bis (2-ethanesulfonic acid), cacodyae (dimethylarsinic acid), SSC (sodium citrate brine), and/or any combination thereof. In some embodiments, the first solution comprises a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, and/or a citrate buffer. In some embodiments, the first solution comprises a phosphate buffer. In some embodiments, the first solution comprises one or more additional components (e.g., benzoate salt, benzyl alcohol, one or more additives described herein, etc.). In some embodiments, the first solution has a pH of about 5.0 to about 10.0 (e.g., about 6.0 to about 10.0, about 6.0 to about 9.0, about 7.0 to about 10.0, about 7.0 to about 9.0, about 8.0 to about 10.0, about 8.0 to about 9.0, about 9.0 to about 10.0, about 5.0 to about 8.0, about 6.0 to about 8.0, about 7.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 7.0, or about 5.0 to about 6.0). In some embodiments, the first wash solution has a pH of about 7.0.
In some embodiments, the first solution comprises a buffer at a concentration of about 10mM to about 100mM or about 10mM to about 500 mM. For example, the first solution may comprise a concentration of about 10mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 250mM to about 500mM, about 300mM to about 500mM, about 350mM to about 500mM, about 400mM to about 500mM, about 450mM to about 500mM, about 10mM to about 450mM, about 10mM to about 400mM, about 10mM to about 350mM, about 10mM to about 300mM, about 10mM to about 250mM, about 10mM to about 200mM, about 10mM to about 150mM, about 10mM to about 100mM, about 25mM to about 100mM, about 40mM to about 100mM, about 50mM to about 100mM, about 60mM to about 100mM, about 75mM to about 100mM, about 10mM to about 75mM, about 25mM to about 75mM, about 40mM to about 75mM, about 50mM to about 60mM, about 60mM to about 60mM, about 50mM to about 60mM, about 50mM, about 60mM, about 500mM, about, About 10mM to about 50mM, about 25mM to about 50mM, about 40mM to about 50mM, about 10mM to about 40mM, about 25mM to about 40mM, or about 10mM to about 25 mM. In some embodiments, the first solution comprises a buffer at a concentration of about 10mM to about 50mM or about 10mM to about 500 mM.
In some embodiments, the first solution comprises a buffer at a concentration of any one of about 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100 mM. Alternatively, the first solution comprises a buffer at a concentration of any one of about 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM. In some embodiments, the first solution comprises a buffer at a concentration of about 500 mM. In some embodiments, the first solution comprises a buffer at a concentration of about 50 mM. In some embodiments, the first solution comprises a phosphate buffer at a concentration of about 500 mM. In some embodiments, the first solution comprises a phosphate buffer at a concentration of about 50 mM.
In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) and sodium chloride. In some embodiments, the first solution comprises a phosphate buffer solution (e.g., sodium phosphate) and sodium chloride, and has a pH of about 7.0. In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50mM and sodium chloride at a concentration of about 0.5M. In some embodiments, the first solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50mM and sodium chloride at a concentration of about 0.5M, and has a pH of about 7.0.
In some embodiments, the present disclosure relates to washing a protein a substrate with a second solution after washing the substrate with a wash solution. In some embodiments, the substrate is washed with the second solution one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.) prior to washing the substrate with the wash solution. In some embodiments, after washing the substrate with the wash solution, the substrate is washed once with the second solution. In some embodiments, the second solution comprises a buffer. Any suitable buffer known in the art may be used in the second solution including, for example, phosphate, Tris (hydroxymethyl) methylamine), acetate, carbonate, citrate, Bis-Tris propane, arginine, histidine, triethanolamine, diethanolamine, formate, MES (2- (N-morpholino) ethanesulfonic acid), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), MOPS (3- (N-morpholino) propanesulfonic acid), TAPS (3- { [ Tris (hydroxymethyl) methyl ] amino } propanesulfonic acid), Bicine (N, N-Bis (2-hydroxyethyl) glycine), Tricine (N-Tris (hydroxymethyl) methylglycine), TES (2- { [ Tris (hydroxymethyl) methyl ] amino } ethanesulfonic acid, PIPES (piperazine-N, n' -bis (2-ethanesulfonic acid), cacodyae (dimethylarsinic acid), SSC (sodium citrate brine), and/or any combination thereof. In some embodiments, the second solution comprises a phosphate buffer, a Tris buffer, an acetate buffer, a carbonate buffer, and/or a citrate buffer. In some embodiments, the second solution comprises a phosphate buffer. In some embodiments, the second solution comprises a substantially low salt or no salt. In some embodiments, the second solution has a pH of about 4.0 to about 8.0 (e.g., about 5.0 to about 8.0, about 6.0 to about 8.0, about 7.0 to about 8.0, about 4.0 to about 7.0, about 5.0 to about 7.0, about 6.0 to about 7.0, about 4.0 to about 6.0, about 5.0 to about 6.0, or about 4.0 to about 5.0). In some embodiments, the second solution has a pH of about 5.0 to about 7.0. In some embodiments, the pH of the second solution is about 7.0. In some embodiments, the second solution comprises a substantially low salt. In some embodiments, the second solution does not comprise a salt.
In some embodiments, the second solution comprises a buffer at a concentration of about 10mM to about 100mM or about 10mM to about 500 mM. For example, the second solution may comprise a concentration of about 10mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 250mM to about 500mM, about 300mM to about 500mM, about 350mM to about 500mM, about 400mM to about 500mM, about 450mM to about 500mM, about 10mM to about 450mM, about 10mM to about 400mM, about 10mM to about 350mM, about 10mM to about 300mM, about 10mM to about 250mM, about 10mM to about 200mM, about 10mM to about 150mM, about 10mM to about 100mM, about 25mM to about 100mM, about 40mM to about 100mM, about 50mM to about 100mM, about 60mM to about 100mM, about 75mM to about 100mM, about 10mM to about 75mM, about 25mM to about 75mM, about 40mM to about 75mM, about 50mM to about 60mM, about 60mM to about 60mM, about 50mM to about 60mM, about 50mM, about 60mM, about 500mM, about, About 10mM to about 50mM, about 25mM to about 50mM, about 40mM to about 50mM, about 10mM to about 40mM, about 25mM to about 40mM, or about 10mM to about 25 mM. In some embodiments, the second solution comprises a buffer at a concentration of about 10mM to about 50mM or about 10mM to about 500 mM.
In some embodiments, the second solution comprises a buffer at a concentration of any one of about 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, or 100 mM. Alternatively, the second solution comprises a buffer at a concentration of any one of about 150mM, 200mM, 250mM, 300mM, 350mM, 400mM, 450mM, or 500 mM. In some embodiments, the second solution comprises a buffer at a concentration of about 500 mM. In some embodiments, the second solution comprises a buffer at a concentration of about 50 mM.
In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate). In some embodiments, the second solution comprises a phosphate buffer solution (e.g., sodium phosphate) and has a pH of about 7.0. In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50 mM. In some embodiments, the second solution comprises a phosphate buffer (e.g., sodium phosphate) at a concentration of about 50mM and has a pH of about 7.0.
In some embodiments, the methods of the present disclosure involve washing a protein a substrate with a wash solution, and do not include a step of washing the substrate with a first solution (prior to the wash solution) or a second solution (after the wash solution). In some embodiments, the methods of the present disclosure involve washing the protein a substrate with a first solution followed by washing the substrate with a wash solution, and do not include the step of washing the substrate with a second solution (after the wash solution). In some embodiments, the methods of the present disclosure involve washing the protein a substrate with a wash solution followed by washing the substrate with a second solution, and do not include the step of washing the substrate with the first solution (prior to the wash solution). In some embodiments, the methods of the present disclosure involve washing the protein a substrate with a first solution, then washing the substrate with a wash solution, and then washing the substrate with a second solution.
In some embodiments, after one or more washing steps, the protein a matrix is contacted with the elution solution one or more times (e.g., one or more times, two or more times, three or more times, four or more times, five or more times, etc.). In some embodiments, the matrix is contacted with the elution solution once. Any solution known in the art suitable for eluting polypeptides bound to a protein a matrix can be used as an elution solution in the methods of the present disclosure (e.g., an elution solution comprising 40mM sodium acetate having a pH of about 3.1). In some embodiments, the elution solution further comprises one or more additional components (e.g., arginine at any concentration described herein). In some embodiments, the eluate comprising the Fc region-containing polypeptide is collected after contacting the matrix with the eluate. In some embodiments, two or more elutions comprising the Fc region-containing polypeptide are collected after contacting the matrix with the elution solution two or more times. In some embodiments, the two or more elutes are combined after elution. In some embodiments, the one or more elutes are filtered. Any suitable method of filtering the eluate known in the art may be used, including, for example, via depth filtration. In some embodiments, the one or more elutions are filtered via depth filtration.
In some embodiments, the eluate from a protein a matrix as described herein can be further processed and/or purified (e.g., using additional chromatography and/or filtration steps (e.g., by using one or more of ion exchange chromatography, mixed mode chromatography, affinity chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, size exclusion chromatography, diafiltration, ultrafiltration, and/or virus removal filtration)), and/or formulated (e.g., prepared for administration to a subject in need thereof (e.g., a human subject)).
The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the disclosure. The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Indeed, various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.
Examples
- 上一篇：一种医用注射器针头装配设备
- 下一篇：澳瑞他汀E衍生物的白蛋白结合产物