阅读说明：本技术 分析托酚酮的方法 (Method for analyzing tropolone ) 是由 B·A·维尔克斯 于 2018-12-04 设计创作，主要内容包括：本文公开了用于检测和/或定量样品中托酚酮的方法,例如在产物(例如,重组蛋白,如抗体)的最终制剂中或其生产期间产生的托酚酮。通过添加部分或完全氟化的烷基或芳基(例如五氟苯基丙基),其(共价)结合托酚酮样的化合物,将托酚酮及其衍生物(环庚三烯酮)从混合物分离。然后通过紫外或串联质谱法对其进行分析。还公开了托酚酮样的化合物和氟化烷基或芳基的反应混合物。(Disclosed herein are methods for detecting and/or quantifying tropolone in a sample, such as tropolone produced in a final formulation of a product (e.g., a recombinant protein, such as an antibody) or during production thereof. Tropolone and its derivatives (tropone) are separated from the mixture by addition of a partially or fully fluorinated alkyl or aryl group (e.g. pentafluorophenylpropyl) which (covalently) binds to the tropolone-like compound. It is then analyzed by uv or tandem mass spectrometry. Also disclosed are reaction mixtures of tropolone-like compounds and fluorinated alkyl or aryl groups.)

1. A method of separating a compound of formula I, such as tropolone, from another component in a sample, the method comprising:

contacting the sample with a partially or fully fluorinated alkyl or aryl moiety, e.g., a fluorophenyl group, such as pentafluorophenylpropyl group, under conditions wherein a compound of formula I, e.g., tropolone, is associated with the moiety to a greater extent than with the component, e.g., is bound to or retained by the moiety,

thereby, a compound of formula I, such as tropolone, is separated from the component, wherein formula I is:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a) C (O) R5; or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and is

n is 0, 1,2, 4 or 5.

2. The method of claim 1, wherein the moiety comprises pentafluorophenylpropyl.

3. The method of claim 1 or 2, wherein the pentafluorophenylpropyl group is associated with, e.g., bound, e.g., covalently bound, to the matrix.

4. The method of claim 3, wherein the matrix comprises an insoluble matrix, such as a chromatography matrix, e.g., a silica gel.

5. The method of any one of claims 1-4, further comprising: in cases where the compound preferentially elutes, the moiety is contacted with one or more mobile phases (e.g., one or two mobile phases).

6. The method of any one of claims 1-5, wherein the method comprises: the sample is subjected to Liquid Chromatography (LC) separation.

7.A method of assessing the presence, e.g., level, of a compound of formula I, e.g., tropolone, in a sample comprising a product, the method comprising:

a) i) providing an aliquot of the sample, e.g. a depleted phase, e.g. a mobile phase, of a compound of formula I (e.g. tropolone), wherein the compound of formula I, e.g. tropolone, has been separated from another component of the sample, or

ii) subjecting the sample to conditions wherein a compound of formula I, e.g., tropolone, separates from another component of the sample, e.g., to form an enriched phase or aliquot of the compound of formula I, e.g., tropolone, and a depleted phase or aliquot of the compound of formula I, e.g., tropolone; and

b) assessing the presence, e.g. the level, of a compound of formula I, e.g. tropolone, e.g. determining a value for the level of a compound of formula I, e.g. tropolone, in a sample:

i) using tandem Mass Spectrometry (MS)²) Or is or

ii) using Ultraviolet (UV) absorption (e.g., UV absorption of about 242nm or about 238 nm);

whereby the sample is analyzed in a manner such that,

wherein formula I is:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a) C (O) R5; or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halo, oxocyano; and is

n is 0, 1,2, 4 or 5.

8. The method of claim 7, wherein a) comprises: an aliquot of the sample, e.g., a depleted phase, e.g., a mobile phase, of a compound of formula I, e.g., tropolone, is provided, wherein the compound of formula I, e.g., tropolone, has been separated from another component of the sample.

9. The method of claim 7, wherein a) comprises: the sample is subjected to conditions in which a compound of formula I, e.g., tropolone, separates from another component of the sample, e.g., to form an enriched phase or aliquot of the compound of formula I, e.g., tropolone, and a depleted phase or aliquot of the compound of formula I, e.g., tropolone.

10. The method of any one of claims 7-9, wherein a) comprises: the sample is subjected to Liquid Chromatography (LC) separation.

11. The method of any one of claims 7-10, wherein a) comprises: the sample is contacted with a partially or fully fluorinated alkyl or aryl moiety, e.g., a fluorophenyl group, such as pentafluorophenylpropyl group, under conditions wherein a compound of formula I, e.g., tropolone, is associated with the moiety to a greater extent than with the component, e.g., bound to or retained by the moiety.

12. The method of claim 11, wherein the moiety comprises pentafluorophenylpropyl.

13. The method of any one of claims 7-12, wherein b) comprises: assessing the level or presence of a compound of formula I, e.g. tropolone, e.g. using tandem Mass Spectrometry (MS)²) A value for the level of a compound of formula I, e.g., tropolone, in a sample is determined.

14. The method of any one of claims 7-12, wherein b) comprises: the level or presence of a compound of formula I, e.g., tropolone, is assessed, e.g., using Ultraviolet (UV) absorption, e.g., ultraviolet absorption at about 242nm or about 238nm, to determine a value for the level of a compound of formula I, e.g., tropolone, in a sample.

15. The method of any one of claims 7, 11, or 12, further comprising: a) i) and b) i).

16. The method of any one of claims 7, 11, or 12, further comprising: a) i) and b) ii).

17. The method of any one of claims 7, 11, or 12, further comprising: a) ii) and b) i).

18. The method of any one of claims 7, 11, or 12, further comprising: a) ii) and b) ii).

19. The method of any one of claims 7-18, wherein the method has a linear range of about 0.1-10000 μ g/ml, 0.2-8000 μ g/ml, 0.3-7000 μ g/ml, 0.4-6000 μ g/ml, 0.5-5000 μ g/ml, 0.5-4000 μ g/ml, 0.5-3000 μ g/ml, 0.5-2000 μ g/ml, or 0.5-1000 μ g/ml, such as 0.5-1000 μ g/ml, with respect to the value determining the level of a compound of formula I, such as tropolone, present in a sample.

20. The method of any one of claims 7-19, wherein the lower limit of the linear range of the method with respect to the value determining the level of a compound of formula I, e.g., tropolone, present in the sample is about 0.01 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.35 μ g/ml, 0.4 μ g/ml, 0.45 μ g/ml, 0.5 μ g/ml, 0.6 μ g/ml, 0.7 μ g/ml, 0.8 μ g/ml, 0.9 μ g/ml, or 1 μ g/ml, e.g., 0.5 μ g/ml.

21. The method of any one of claims 7 to 20, wherein the upper limit of the linear range of the method with respect to the value for determining the level of a compound of formula I, e.g. tropolone, present in a sample is about 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml, 1000 μ g/ml, 1200 μ g/ml, 1400 μ g/ml, 1600 μ g/ml, 1800 μ g/ml, 2000 μ g/ml, 3000 μ g/ml, 4000 μ g/ml, 5000 μ g/ml, 6000 μ g/ml, 7000 μ g/ml, 8000 μ g/ml, 9000 μ g/ml, or 10,000 μ g/ml, e.g. 1000 μ g/ml.

22. The method of any one of claims 7-21, wherein the accuracy of the method (e.g., as represented by the standard deviation between replicate samples) can be less than or equal to about 0%, 40%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, e.g., 17%, 16.5%, or 16%, with respect to the value determining the level of a compound of formula I, e.g., tropolone, present in a sample.

23. The method of any one of claims 7-22, wherein the accuracy of the method (e.g., as represented by the average single point addition recovery in three different samples) can be greater than or equal to about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, e.g., 91%, with respect to the value determining the level of a compound of formula I, e.g., tropolone, present in a sample.

24. The method of any one of claims 7-23, wherein the lower limit of detection for the method is about 1 μ g/ml, 1.5 μ g/ml, 2 μ g/ml, 2.5 μ g/ml, 3 μ g/ml, 3.5 μ g/ml, 4 μ g/ml, 4.5 μ g/ml, 5 μ g/ml, 5.5 μ g/ml, 6 μ g/ml, 6.5 μ g/ml, 7 μ g/ml, 7.5 μ g/ml, 8 μ g/ml, 8.5 μ g/ml, 9 μ g/ml, 9.5 μ g/ml, or 10 μ g/ml, e.g. 5 μ g/ml, with respect to the value determining the level of a compound of formula I, e.g. tropolone, present in the sample.

25. The method of any one of claims 6 or 10, wherein LC is reverse phase chromatography.

26. The method of any one of claims 6 or 10, wherein the LC is not reverse phase chromatography.

27. A method according to any of claims 6 or 10, wherein LC comprises using a stationary phase comprising a partially or fully fluorinated alkyl or aryl group, for example a fluorophenyl group, such as pentafluorophenylpropyl.

28. The method of claim 27, wherein the LC comprises a stationary phase using a fluorophenyl-containing group.

29. The method of claim 27, wherein LC comprises using a stationary phase comprising pentafluorophenylpropyl.

30. The method of any one of claims 6, 10, or 25-29, wherein LC comprises using a first mobile phase and a second mobile phase.

31. The method of claim 30, wherein the first mobile phase comprises formic acid in water, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid in water.

32. The method of claim 31, wherein the first mobile phase comprises about 0.1% formic acid in water.

33. The method of claim 30, wherein the second mobile phase comprises formic acid in acetonitrile, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid in acetonitrile.

34. The method of claim 33, wherein the second mobile phase comprises about 0.1% formic acid in acetonitrile.

35. The method of any one of claims 33 or 34, wherein the second mobile phase comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% acetonitrile, e.g., about 100% acetonitrile.

36. The method of any one of claims 6, 10, or 25-35, wherein LC comprises: using a stationary phase comprising pentafluorophenylpropyl and using a first mobile phase and a second mobile phase, wherein the first mobile phase comprises about 0.1% formic acid in water and the second mobile phase comprises about 0.1% formic acid in acetonitrile.

37. The method of any one of claims 6, 10, or 25-36, wherein LC comprises: a Discovery HSF5-3 column was used.

38. The method of any one of claims 7-13, 15, 17 and 19-37, wherein MS is used²Including Selective Reaction Monitoring (SRM).

39. The method of any one of claims 7-13, 15, 17 and 19-37, wherein MS is used²Including Multiple Reaction Monitoring (MRM), such as Parallel Reaction Monitoring (PRM).

40. The method of any one of claims 38 or 39, wherein SRM or MRM (e.g. PRM) is used to monitor one or more transitions selected from transitions i, ii, iii, iv, v and vi from Table 1.

41. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transition i.

42. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transition ii.

43. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transition iii.

44. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transition iv.

45. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transition v.

46. The method of claim 40, wherein SRM or MRM (e.g., PRM) is used to monitor transitions vi.

47. A reaction mixture comprising a partially or fully fluorinated alkyl or aryl moiety, such as a fluorophenyl group, e.g. pentafluorophenylpropyl group, wherein formula I is given by the formula:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a) C (O) R5; or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently isC₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halo, oxocyano; and is

n is 0, 1,2, 4 or 5.

48. A method of manufacturing a product, such as a recombinant polypeptide, comprising: providing a sample comprising the product and optionally a compound of formula I, such as tropolone, wherein:

the sample is analyzed by the method of any one of claims 7-43, 45 or 46, or

Separating a compound of formula I, such as tropolone, from another component of the sample by the method of any one of claims 1 to 6,

wherein formula I is given by:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a) C (O) R5; or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halo, oxocyano; and is

n is 0, 1,2, 4 or 5.

49. The method of claim 48, wherein the method of manufacturing comprises: expressed and secreted by a plurality of cells, e.g., a plurality of CHO cells, e.g., a plurality of GS-CHO cells.

50. A method or reaction mixture according to any one of claims 1 to 49, wherein the sample comprises a culture supernatant.

51. The method or reaction mixture of any of claims 1-49, wherein the sample comprises a cell lysate.

52. The method or reaction mixture of any of claims 1-51, wherein the sample comprises culture supernatant and cell lysate.

53. The method or reaction mixture of any of claims 1-52, wherein the sample is produced by a method of manufacture of a product, such as a recombinant polypeptide.

54. The method or reaction mixture of any of claims 1-53, wherein the sample comprises a final product, e.g., a final product formulated for delivery, e.g., administration, to a patient.

55. A process or reaction mixture according to any one of claims 1 to 54, wherein the product or recombinant polypeptide is a homo-or heteropolymeric polypeptide, such as a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or antibody fragment, such as a human or humanized antibody or fragment thereof, such as a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep or cow antibody, typically a humanized antibody or fragment thereof derived from rabbit origin.

56. The method or reaction mixture of any of claims 1-55, wherein the product or recombinant polypeptide is a therapeutic polypeptide.

57. The method or reaction mixture of any of claims 1-56, wherein the product or recombinant polypeptide is disclosed in Table 1, Table 2, Table 3, or Table 4.

58. The method or reaction mixture of any of claims 1-57, wherein the product or recombinant polypeptide is an antibody.

59. The method or reaction mixture of claim 58, wherein the antibody is a monoclonal antibody.

60. The method or reaction mixture of any of claims 58 or 59, wherein the monoclonal antibody is a therapeutic antibody.

61. A method or reaction mixture according to any one of claims 49 to 60, wherein the cells are mammalian cells.

62. The method or reaction mixture of claim 61, wherein the cell is a mouse, rat, chinese hamster, syrian hamster, monkey, ape, dog, horse, ferret, or cat.

63. The method or reaction mixture of claim 61, wherein the cells are Chinese Hamster Ovary (CHO) cells.

64. The method or reaction mixture of claim 63, wherein the CHO cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell.

65. The method or reaction mixture of claim 61, wherein the cell is HeIa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER. C6, BHK (baby hamster kidney cells), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, or any cell derived therefrom.

Technical Field

The present disclosure relates to methods for detecting and/or quantifying tropolone during production of a product (e.g., a recombinant protein, such as an antibody).

Background

Tropolone (2-hydroxy-2, 4, 6-cycloheptatrien-1-one) is a small molecule used in cell culture media to promote uptake of metal ions, which is essential for the growth of cells, such as those used in biological manufacturing. Because tololone is a synthetic chemical added to cell cultures during product production, regulatory agencies governing biological products often require evidence of the clearance of tololone.

Accordingly, there is a need for methods for isolating, detecting and quantifying tololone in a variety of biopharmaceutical products in a simple, rapid, and efficient manner.

Disclosure of Invention

The methods and compositions described herein provide for rapid and easy isolation of compounds of formula I (e.g., tropolone) from other sample components and testing for compounds of formula I (e.g., tropolone), levels, and clearance. This allows the purity of the product to be assessed. The methods and compositions described herein can minimize regulatory delays (regulatory delays) as well as time and resource consumption testing of compounds of formula I (e.g., tropolone).

Accordingly, in one aspect the present invention relates to a method of isolating a compound of formula I (e.g. tropolone) from another component of a sample, the method comprising:

in cases where a compound of formula I (e.g., tropolone) is associated with (e.g., bound to or retained by) a moiety to a greater extent than the component, the sample is contacted with a partially or fully fluorinated alkyl or aryl (e.g., fluorophenyl, such as pentafluorophenylpropyl) moiety,

thereby, a compound of formula I, for example tropolone, is separated from the component, wherein formula I is:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

in another aspect, the invention relates to a method of assessing the presence (e.g., level) of a compound of formula I (e.g., tropolone) in a sample comprising a product, the method comprising:

a) i) providing an aliquot of the sample, e.g. a depleted phase (e.g. mobile phase) of a compound of formula I (e.g. tropolone), wherein the compound of formula I (e.g. tropolone) has been separated from another component of the sample, or

ii) subjecting the sample to conditions wherein a compound of formula I, e.g. tropolone, separates from another component of the sample, e.g. to form a compound of formula I (e.g. tropolone) enriched phase or aliquot and a compound of formula I (e.g. tropolone) depleted phase or aliquot; and

b) assessing the presence (e.g., level) of a compound of formula I (e.g., tropolone), e.g., determining a value for the level of a compound of formula I (e.g., tropolone) in a sample:

i) using tandem Mass Spectrometry (MS)²) Or is or

ii) using Ultraviolet (UV) absorption (e.g., UV absorption of about 242nm or about 238 nm);

thereby analyzing the sample.

In another aspect, the invention relates to a reaction mixture comprising a partially or fully fluorinated alkyl or aryl (e.g., fluorophenyl, such as pentafluorophenylpropyl) moiety and a sample comprising a compound of formula I (e.g., tropolone), another component, and optionally a product.

In another aspect, the invention relates to a method of producing a product (e.g., a recombinant polypeptide), the method comprising: providing a sample comprising the product and optionally a compound of formula I (e.g., tropolone), wherein:

analyzing the sample by the methods described herein, or

The compound of formula I (e.g., tropolone) is separated from another component of the sample by the methods described herein.

Drawings

FIG. 1 shows two views of a chromatogram for detecting tropolone isolated by RP-HPLC using UV; the bottom view is an enlarged view of the top view.

FIG. 2 shows the use of Luna-NH₂Total Ion Current (TIC) profile of SRM transition in table 1 after column separation.

FIG. 3 shows TIC traces of SRM transitions (transitions) in Table 1 after separation using a Discovery HS F5-3(Supelco) chromatography column.

FIG. 4 shows a graph showing a calibration curve of tropolone standards in water and determining the linear range.

FIG. 5 shows TIC traces of samples from three treatments, each showing no tropolone peak.

Fig. 6 shows TIC traces of samples in three treatments, three of which were either doped with tropolone (top three) or undoped with tropolone (bottom three).

Figure 7 shows two chromatograms using uv absorbance at 242nm (top) and 238nm (bottom) to detect tropolone in a tropolone standard treated by the chromatographic method identified in example 2.

Fig. 8A and 8B show detailed parameters of an exemplary LC method of the present disclosure.

Detailed Description

In order for a recombinant biopharmaceutical protein to be acceptable for administration to a human patient, it is important to remove residual contaminants resulting from the manufacturing and purification processes from the final bioproduct (e.g., a recombinant polypeptide). These process contaminants include compounds added to the culture medium during the culturing of cells and the purification of biological products.

U.S. and foreign regulations typically require the removal of such contaminants. For example, the U.S. Food and Drug Administration (FDA) requires that biopharmaceuticals intended for in vivo use in humans be as free as possible of exogenous immunoglobulin and non-immunoglobulin impurities, and requires tests to be performed to detect and quantify potential impurities. Also, the international harmonization conference (ICH) provides guidelines on testing procedures and acceptance criteria for biotechnology/bioproducts.

Tropolone (2-hydroxy-2, 4, 6-cycloheptatrien-1-one) is a seven-membered aromatic ring. It has a variety of uses, including use as an antioxidant in cosmetic and topical pharmaceutical formulations, as an ultraviolet absorber in sunscreens, and as a catechol-O-methyltransferase (COMT) inhibitor. Tropolone may be added to the cell culture medium to facilitate uptake of metal ions into the cultured cells. In some embodiments, the tropolone is added to the cell culture medium at a concentration of less than or equal to 0.1mg/ml, 0.5mg/ml, 1mg/ml, 1.25mg/ml, 1.5mg/ml, 1.75mg/ml, 2mg/ml, 2.25mg/ml, 2.5mg/ml, 2.75mg/ml, 3mg/ml, 3.5mg/ml, 4mg/ml, 4.5mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, or 10 mg/ml.

In some embodiments, a compound of formula I (e.g., tropolone) may be added to the cell culture medium to facilitate uptake of metal ions into the cultured cells. In some embodiments, a compound of formula I (e.g., tropolone) is added to the cell culture medium at a concentration of less than or equal to 0.1mg/ml, 0.5mg/ml, 1mg/ml, 1.25mg/ml, 1.5mg/ml, 2mg/ml, 2.25mg/ml, 2.5mg/ml, 2.75mg/ml, 3mg/ml, 3.5mg/ml, 4mg/ml, 4.5mg/ml, 5mg/ml, 6mg/ml, 7mg/ml, 8mg/ml, 9mg/ml, or 10 mg/ml.

As a synthetic chemical added to cell cultures used for the production of biologicals, many regulatory agencies require: for example, the removal of a compound of formula I (e.g., tropolone) from a biological product is demonstrated before it can be declared safe for use in humans. Many methods of making or producing a biological product involve an affinity chromatography step, for example, using a chromatography column containing a resin that selectively retains the desired biological product, and it is desired that a compound of formula I (e.g., tropolone) pass through the affinity column before the desired biological product is eluted. Any compound of formula I (e.g., tropolone) remaining in a sample of a biological product can be determined using the following procedure: (i) suitable chromatography steps to separate potentially residual compounds of formula I (e.g., tropolone) from other components of the biological product, and (ii) suitable detection and/or quantification steps to determine the presence and abundance of compounds of formula I (e.g., tropolone). Suitable chromatographic steps and detection methods are described herein.

The present disclosure describes, inter alia, methods of analyzing a sample comprising a product and optionally a compound of formula I (e.g., tropolone) to determine a value for the level of the compound of formula I (e.g., tropolone) present in the sample, wherein the methods of the present disclosure have advantages over previously available methods (e.g., RP-HPLC and UV/fluorescence detection) in one or more of linear range, accuracy, precision, and detection limits. In some embodiments, the methods of the present disclosure are not affected or significantly detrimentally affected (e.g., substantially unaffected) in one or more of the linear range, accuracy, precision, and detection limits of the various products and/or product formulations, as compared to previously available methods (e.g., RP-HPLC and UV/fluorescence detection). For example, the methods of the present disclosure can determine a value for the level of a compound of formula I (e.g., tololone) in a sample containing multiple buffer components without a significant decrease in accuracy, while previously available methods can determine a value for the level of a compound of formula I (e.g., tololone) in a sample containing one buffer component, but with decreased accuracy when determining a value for the level of a compound of formula I (e.g., tololone) in a sample containing another buffer component.

In some embodiments, with respect to determining the value of the level of a compound of formula I (e.g., tropolone) present in a sample, the disclosed methods have a linear range of about 0.1 μ g/ml to 10000 μ g/ml, 0.2 μ g/ml to 8000 μ g/ml, 0.3 μ g/ml to 7000 μ g/ml, 0.4 μ g/ml to 6000 μ g/ml, 0.5 μ g/ml to 5000 μ g/ml, 0.5 μ g/ml to 4000 μ g/ml, 0.5 μ g/ml to 3000 μ g/ml, 0.5 μ g/ml to 2000 μ g/ml, or 0.5 μ g/ml to 1000 μ g/ml (e.g., 0.5 μ g/ml to 1000 μ g/ml). In some embodiments, with respect to the value determining the level of a compound of formula I (e.g., tropolone) present in a sample, the lower limit of the linear range of the disclosed methods is about 0.01 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.35 μ g/ml, 0.4 μ g/ml, 0.45 μ g/ml, 0.5 μ g/ml, 0.6 μ g/ml, 0.7 μ g/ml, 0.8 μ g/ml, 0.9 μ g/ml, or 1 μ g/ml (e.g., 0.5 μ g/ml). In some embodiments, the upper limit of the linear range for the methods of the present disclosure is about 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml, 1000 μ g/ml, 1200 μ g/ml, 1400 μ g/ml, 1600 μ g/ml, 1800 μ g/ml, 2000 μ g/ml, 3000 μ g/ml, 4000 μ g/ml, 5000 μ g/ml, 6000 μ g/ml, 7000 μ g/ml, 8000 μ g/ml, 9000 μ g/ml, or 10,000 μ g/ml (e.g., 1000 μ g/ml) for determining the level of a compound of formula I (e.g., tropolone) present in a sample.

In some embodiments, the disclosed methods have an accuracy as expressed by the standard deviation between replicate samples with respect to determining the value of the level of a compound of formula I (e.g., tropolone) present in the sample. In the same embodiment, the precision may be less than or equal to about 50%, 40%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, e.g., 17%, 16.5%, or 16%.

In some embodiments, the disclosed methods have an accuracy as shown by the average single point addition recovery (spike recovery) in three different samples with respect to the value determining the level of a compound of formula I (e.g., tropolone) present in the sample. In the same embodiment, the accuracy can be greater than or equal to about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, e.g., 91%.

In some embodiments, the methods of the present disclosure have a lower limit of detection with respect to the value that determines the level of a compound of formula I (e.g., tropolone) present in a sample. In the same embodiment, the lower limit of detection can be about 1. mu.g/ml, 1.5. mu.g/ml, 2. mu.g/ml, 2.5. mu.g/ml, 3. mu.g/ml, 3.5. mu.g/ml, 4. mu.g/ml, 4.5. mu.g/ml, 5. mu.g/ml, 5.5. mu.g/ml, 6. mu.g/ml, 6.5. mu.g/ml, 7. mu.g/ml, 7.5. mu.g/ml, 8. mu.g/ml, 8.5. mu.g/ml, 9. mu.g/ml, 9.5. mu.g/ml, or 10. mu.g/ml.

The present disclosure also describes, inter alia, methods of making products (e.g., recombinant polypeptides), wherein a sample of the product is analyzed for the presence or level of a compound of formula I (e.g., tololone) by the sample analysis methods described herein.

In some embodiments, the sample is a sample of a cosmetic formulation, for example including a product for a cosmetic formulation.

In some embodiments, the sample is a sample of a topical pharmaceutical formulation, e.g., including a product for a pharmaceutical formulation.

In some embodiments, the sample is a sample of a sunscreen, for example including a product for a sunscreen, such as a compound of formula I (e.g., tropolone), and/or another product for a sunscreen, for example, another UV blocker.

In some embodiments, the sample is a sample of a COMT inhibitor, e.g., it includes a product that acts as a COMT inhibitor, such as a compound of formula I (e.g., tropolone), and/or another product that acts as a COMT inhibitor. In some embodiments, the sample is a sample comprising L-DOPA (e.g., levodopa or L-3, 4-dihydroxyphenylalanine) and/or an aromatic L-amino acid decarboxylase inhibitor (e.g., a DOPA decarboxylase inhibitor, DDCI, or AAADI).

The present disclosure further describes, inter alia, a reaction mixture comprising a fluorophenyl moiety (e.g., a pentafluorophenylpropyl group) and a sample, wherein the sample comprises a compound of formula I (e.g., tropolone), another component, and optionally a product. In one embodiment, the reaction mixture may be used to isolate a compound of formula I (e.g., tropolone) from the components and/or products, and in other embodiments, subsequently used to detect the presence or determine the level of a compound of formula I (e.g., tropolone). A portion of the reaction mixture may be associated with, e.g., bound or covalently bound to, a matrix, wherein the matrix comprises an insoluble matrix, e.g., a chromatography matrix, a resin, a gel, or a bead, e.g., a silica, agarose, cellulose, dextran, polyacrylamide, or latex matrix, a resin, a gel, or a bead.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice and/or testing of the present invention, the preferred methods and materials are described below. In describing and claiming the present invention, the following terminology, which provides a definition, will be used in accordance with the definitions set forth below.

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.

The articles "a" and "an" are used herein to mean one or more than one (i.e., at least one) of the grammatical object of the article. For example, "a cell" may mean one cell or more than one cell.

As used herein, "about" and "approximately" shall generally refer to an acceptable degree of error in the measured quantity given the nature or accuracy of the measurement. Exemplary degrees of error are within 20%, typically within 10%, and more typically within 5% of a given value range or value.

As used herein, the term "semi-quantitative" refers to the comparative evaluation of different chemical substances by mass spectrometry without reference to a particular standard for each individual substance.

The term "endogenous", as used herein, refers to any substance that is naturally produced from or within an organism, cell, tissue or system.

The term "exogenous" as used herein refers to any substance introduced into or produced outside of an organism, cell, tissue or system. Thus, "exogenous nucleic acid" refers to a nucleic acid that is introduced into or produced outside of an organism, cell, tissue, or system. In one embodiment, the sequence of the exogenous nucleic acid is not naturally occurring or naturally occurring within the organism, cell, tissue or system into which the exogenous nucleic acid is introduced. In one embodiment, the sequence of the exogenous nucleic acid is a non-naturally occurring sequence, or encodes a non-naturally occurring product.

As used herein, the term "heterologous" refers to any substance from one species being introduced into an organism, cell, tissue or system of a different species.

As used herein, the terms "nucleic acid," "polynucleotide," or "nucleic acid molecule" are used interchangeably to refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a combination of DNA or RNA thereof, as well as polymers thereof in single-stranded or double-stranded form. The term "nucleic acid" includes, but is not limited to, a gene, cDNA or mRNA. In one embodiment, the nucleic acid molecule is synthetic (e.g., chemically synthesized or artificial) or recombinant. Unless specifically limited, the term encompasses molecules comprising analogs or derivatives of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally or non-naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, nucleic acid Res.19:5081 (1991); Ohtsuka et al, J.biol. chem.260:2605-2608 (1985); and Rossolini et al, mol.cell. probes 8:91-98 (1994)).

As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by or in addition to peptide bonds. The protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that constitute the protein or peptide sequence. In one embodiment, the protein may comprise more than one, e.g., two, three, four, five or more than five polypeptides, wherein each polypeptide is bound to each other by covalent or non-covalent bonds/interactions. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds or by means other than peptide bonds. As used herein, the term refers to two short chains, which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and to longer chains, which are commonly referred to in the art as proteins, including many types. "polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like.

As used herein, "product" refers to a molecule, nucleic acid, polypeptide, or any hybrid thereof that is produced (e.g., expressed) by a cell that is modified or engineered to produce the product. In one embodiment, the product is a naturally occurring product or a non-naturally occurring product, such as a synthetic product. In one embodiment, a portion of the products are naturally occurring and another portion of the products are non-naturally occurring. In one embodiment, the product is a polypeptide, e.g., a recombinant polypeptide. In one embodiment, the product is suitable for diagnostic or preclinical use. In another embodiment, the product is suitable for therapeutic use, for example for the treatment of a disease. In one embodiment, the product is selected from table 1, table 2, table 3 or table 4. In one embodiment, the modified or engineered cell comprises an exogenous nucleic acid that controls the expression of the product or encodes a therapeutic polypeptide. In other embodiments, the modified or engineered cell comprises other molecules, such as non-nucleic acid molecules, which control the expression or construction of the product in the cell.

In one embodiment, the modification of the cell comprises introducing an exogenous nucleic acid comprising a nucleic acid sequence that controls or alters (e.g., increases) the expression of an endogenous nucleic acid sequence (e.g., an endogenous gene). In this embodiment, the modified cell produces an endogenous polypeptide product that is naturally or endogenously expressed by the cell, but the modification increases the production and/or improves the quality of the product as compared to an unmodified cell (e.g., as compared to the endogenous production or quality of the polypeptide).

In another embodiment, the modification of the cell comprises introducing an exogenous nucleic acid encoding a recombinant polypeptide described herein. In this embodiment, the modified cell produces a recombinant polypeptide product that may be naturally-occurring or non-naturally-occurring. In this embodiment, the modified cell produces a recombinant polypeptide product that may also be expressed endogenously or non-endogenously by the cell. In embodiments where the recombinant polypeptide product is also expressed endogenously by a cell, the modification increases production and/or improves quality of the product as compared to an unmodified cell (e.g., as compared to the endogenous production or quality of the polypeptide).

As used herein, "recombinant polypeptide" or "recombinant protein" refers to a polypeptide that can be produced by a cell as described herein. The recombinant polypeptide is a polypeptide that: at least one nucleotide of the sequence encoding the polypeptide or at least one nucleotide of the sequence controlling the expression of the polypeptide is formed by genetic engineering (cell or precursor cell). For example, at least one nucleotide is altered, e.g., it is introduced into a cell, or it is the product of a genetically engineered rearrangement. In one embodiment, the sequence of the recombinant polypeptide is not distinguishable from the naturally occurring isoform of the polypeptide or protein. In one embodiment, the amino acid sequence of the recombinant polypeptide is different from the sequence of a naturally occurring isoform of the polypeptide or protein. In one embodiment, the recombinant polypeptide and the cell are of the same species. In one embodiment, the recombinant polypeptide is endogenous to the cell, in other words, the cell is from a first species and the recombinant polypeptide is native to the first species. In one embodiment, the amino acid sequence of the recombinant polypeptide is the same as or substantially the same as, or differs by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% from the polypeptide encoded by the endogenous genome of the cell. In one embodiment, the recombinant polypeptide and the cell are from different species, e.g., the recombinant polypeptide is a human polypeptide, and the cell is a non-human (e.g., rodent, such as CHO) cell or an insect cell. In one embodiment, the recombinant polypeptide is exogenous with respect to the cell, in other words, the cell is from a first species and the recombinant polypeptide is from a second species. In one embodiment, the polypeptide is a synthetic polypeptide. In one embodiment, the polypeptide is derived from a non-naturally occurring source. In one embodiment, the recombinant polypeptide is a human polypeptide or protein that differs from the amino acid sequence from the naturally-occurring isoform of the human polypeptide or protein. In one embodiment, the recombinant polypeptide differs from the naturally occurring isoform of the human polypeptide or protein by no more than 1,2,3,4, 5, 10, 15, or 20 amino acid residues. In one embodiment, the recombinant polypeptide differs from the naturally-occurring isoform of the human polypeptide by no more than 1,2,3,4, 5,6, 7,8, 9, 10, or 15% of the amino acid residues.

The term "obtaining" or "obtaining" as used herein refers to the process of obtaining a physical entity or value (e.g., a numerical value) by either "directly obtaining" or "indirectly obtaining" the physical entity or value. By "directly obtaining" is meant performing a process (e.g., performing a synthetic or analytical method) to obtain a physical entity or value. "indirectly obtaining" refers to receiving a physical entity or value from another party or source (e.g., a third party laboratory that directly obtains the physical entity or value). Directly acquiring the physical entity includes: a process is performed that includes a physical change in a physical substance (e.g., a raw material). Exemplary variations include: physical entities from two or more starting materials, shearing or fragmenting a substance, isolating or purifying a substance, combining two or more separate entities into a mixture, performing a chemical reaction comprising breaking or forming covalent or non-covalent bonds. Directly obtaining a value includes: performing a process that includes a physical change in a sample or another substance, e.g., performing an analytical process that includes a physical change in a substance (e.g., a sample, analyte, or reagent (sometimes referred to herein as a "physical assay"), performing an analytical method, e.g., including one or more of: isolating or purifying a substance, such as an analyte, or a fragment or other derivative thereof, from another substance; combining the analyte or fragment or other derivative thereof with another substance (e.g., a buffer, solvent, or reactant); or altering the structure of the analyte or fragment or other derivative thereof, for example by breaking or forming a covalent or non-covalent bond between the first and second atoms of the analyte; or by altering the structure of the agent or fragment or other derivative thereof (e.g., by disrupting or forming a covalent or non-covalent bond) between the first and second atoms of the agent.

As used herein, "method of manufacture" and "method of production" are used interchangeably and are one or more of a series of operations and/or conditions that produce a sample comprising a product (e.g., a recombinant polypeptide or a therapeutic product).

As used herein, MS¹Represents a mass spectrum.

As used herein, MS²Represents a tandem mass spectrum.

The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to particular aspects, it is apparent that other aspects and variations of the present invention may be devised by others skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the following claims be interpreted to embrace all such aspects and equivalent variations.

Sample preparation

Samples for use in the methods of the present disclosure can be generated by a number of steps of the methods of making and producing the product (e.g., a recombinant polypeptide). In some embodiments, the sample comprises one or more of a culture supernatant, a cell lysate, a product purification intermediate (e.g., a product partially purified from cellular proteins or other contaminants), a purified product, and a final formulated product (e.g., formulated for in vivo use in humans). The product contained in the sample or produced by the manufacturing and production methods may be any product described herein or known in the art.

Chromatography

Suitable chromatographs for use in the methods described herein are known to those skilled in the art, and include: for example, affinity chromatography, gel filtration chromatography, ion exchange chromatography, reverse phase chromatography, hydrophobic interaction chromatography. In some embodiments, the chromatographic method is HPLC reverse phase chromatography. The chromatogram comprises: high Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), capillary electrophoresis, ion migration. See also, for example, Process Scale Purification of Antibodies (Process Scale Purification of Antibodies), UweGottschalk 2011 John Wiley father (John Wiley & Sons) ISBN: 1118210743; antibody volume 1: production and Purification (Antibodies Vol 1Production and Purification), G.Subramanian 2013, Springer Science & Business Media, Germany; basic methods for Antibody Production and Characterization (basic methods in Antibody Production and Characterization), Gary C.Howard 2000CRC Press (CRC Press).

Other exemplary chromatographic methods include, but are not limited to: strong anion exchange chromatography (SAX), Liquid Chromatography (LC), High Performance Liquid Chromatography (HPLC), ultra high performance liquid chromatography (UPLC), Thin Layer Chromatography (TLC), amide column chromatography, and combinations thereof.

In some embodiments, the methods of the present disclosure employ LC comprising one or more (e.g., one, two, or more) mobile phases and a stationary phase. In some embodiments, LC comprises the use of a mobile phase. In some embodiments, LC includes the use of two mobile phases (e.g., a first mobile phase and a second mobile phase). In some embodiments, the mobile phase (e.g., the first and/or second mobile phase) comprises formic acid in water, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19% in water,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid. In some embodiments, the mobile phase (e.g., the first and/or second mobile phase) comprises formic acid in acetonitrile, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid in acetonitrile, e.g., 0.1% formic acid in acetonitrile. In some embodiments, "in acetonitrile" refers to a solution, e.g., a mobile phase, wherein at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (e.g., solvent) of the solution is acetonitrile, e.g., about 100% of the solvent is acetonitrile. In some embodiments, the stationary phase comprises a partially or fully fluorinated alkyl or aryl group, for example a fluorophenyl group, such as pentafluorophenylpropyl. In some embodiments, the stationary phase comprises silica gel particles attached to a partially or fully fluorinated alkyl or aryl group (e.g., a fluorophenyl group, such as pentafluorophenylpropyl group). In some embodiments, the stationary phase pore size is about Or(e.g. in). In some embodiments, the LC comprises use of a Discovery HS F5 stationary phase, e.g., a Discovery HS F5 column.

Without wishing to be bound by theory, it is believed that the partially or fully fluorinated alkyl or aryl (e.g., fluorophenyl, such as pentafluorophenyl) coating of the column resin alters the manner in which tropolone remains on the chromatography column. While more traditional reverse phase chromatography columns do not adequately separate tololone from interfering components, a partially or fully fluorinated alkyl or aryl (e.g., fluorophenyl, such as pentafluorophenyl) resin coating is believed to more readily retain hydrophobic groups and, therefore, more readily elute hydrophilic groups.

Mass spectrometry

Mass spectrometry methods suitable for use in the methods described herein are known to those skilled in the art and include, for example: electrospray ionization MS, matrix-assisted laser desorption/ionization MS (MALDI-MS), time-of-flight MS, Fourier transform ion cyclotron resonance MS, quadrupole time-of-flight MS, linear quadrupole, quadrupole ion trap MS, orbital trap, cylindrical ion trap, three-dimensional ion trap, quadruple mass filter (quadruple mass filter), tandem mass spectrometry, LC-MS/MS, Fourier Transform Mass Spectrometry (FTMS), ion mobility separation mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), and combinations thereof. In some embodiments, the mass spectrum is a tandem Mass Spectrum (MS)²). See also, e.g., Protein Mass Spectrometry (Protein Mass Spectrometry), Julian Whitelegge, 2008, Elsevier; protein Sequencing and identification Using Tandem Mass Spectrometry (Protein Sequencing and Sequencing Using Tandem Mass Spectrometry), Michael Kinter, 2005, John Wiley father, Inc. (John Wiley)&Sons); protein Therapeutics (chromatography of protein Therapeutics using Mass Spectrometry), Guodong Chen, 2014, spagling, germany, were characterized using Mass Spectrometry.

In some embodiments, mass spectrometry suitable for use in the methods described herein includes Selective Reaction Monitoring (SRM), e.g., monitoring selected pairs of precursor and product ions, e.g., transitions. In some embodiments, mass spectrometry suitable for use in the methods described herein includes Multiplex Reaction Monitoring (MRM), e.g., monitoring a plurality of product ions derived from one or more precursor ions, e.g., a plurality of transitions. In some embodiments, mass spectrometry suitable for use in the methods described herein includes Parallel Reaction Monitoring (PRM), e.g., monitoring multiple transformations in a single analytical step, e.g., using a high resolution mass spectrometer. In some embodiments, mass spectrometry suitable for use in the methods described herein comprises monitoring transitions as described in table 1, e.g., under the conditions described in table 1.

Compound and tropolone useful for biological production

In some embodiments, compounds may be added to the cell culture medium to enhance cell growth. For example, compounds can be used to promote uptake of metal ions in cultured cells. In some embodiments, the compound added to the cell culture medium is a compound of formula (I), a pharmaceutically acceptable salt, stereoisomer, racemate, or solvate thereof:

wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently of each other is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently of each other is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

in some embodiments, X is O. In some embodiments, R¹Is OR³(e.g., OH). In some embodiments, n is 0. In some embodiments, the compound of formula (I) is tropolone (i.e., 2-hydroxy-2, 4, 6-cycloheptatrien-1-one). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, X is O. In some embodiments, R¹Is OR³(e.g., OH). In some embodiments, R²Is OR³OR C (O) OR³(e.g., OH or C (O) OH). In some embodiments, n is 3. In some embodiments, n is 3, and R is²Are OH, OH and C (O) OH. In some embodiments, the compound of formula (I) is penicillanic acid (i.e., 4,5, 6-trihydroxy-3-oxocyclohepta-1, 4, 6-triene-1-carboxylic acid). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, X is O. In some embodiments, R¹Is hydrogen. In some embodiments, R²Is OR³OR C (O) OR³(e.g., OH or C (O) OH). In some embodiments, n is 3. In some embodiments, n is 3, 2R²Is OH and 1R²Is C (O) OH. In some embodiments, the compound of formula (I) is picromyceric acid (i.e., 5, 6-dihydroxy-3-oxocyclohepta-1, 4, 6-triene-1-carboxylic acid). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, X is O. In some embodiments, R¹Is OR³(e.g., OH). In some embodiments, R²Is OR³、C(O)R⁵OR C (O) OR³(e.g., OH or C (O) OH). In some embodiments, n is 3. In some embodiments, n is 3, and 1R²Is OH. In some embodiments, 2R²Joined to form a heterocyclyl ring (e.g., a five-membered heterocyclyl ring, such as maleic anhydride). In some embodiments, the compound of formula (I) is penicillic acid (stipitatoninic acid) (i.e., 4, 7-dihydroxy-1H-cyclohepta [ c ]]Furan-1, 3, 6-trione). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, X is O. In some embodiments, R¹Is OR³(e.g., OH). In some embodiments, R²Is OR³、C(O)R⁵OR C (O) OR³(e.g., OH or C (O) OH). In some embodiments, n is 3. In some embodiments, n is 4, and 2R²Is OH. In some embodiments, 2R²Joined to form a heterocyclyl ring (e.g., a five-membered heterocyclyl ring, such as succinic anhydride). In some embodiments, the compound of formula (I) is penicillium lanuginosa dianhydride (pubulinocaicid) (i.e., 6,7, 8-trihydroxy-1H-cyclohepta [ c ]]Furan-1, 3, 5-trione). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, X is O. In some embodiments, R¹Is OR³(e.g., OH). In some embodiments, R²Is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR OR³(e.g., OH). In some embodiments, n is 3. In some embodiments, n is 3, and 1R²Is OH. In some embodiments, 2R²Are linked to form a group consisting of one or more R⁶An optionally substituted heterocyclyl ring (e.g., a six membered heterocyclyl ring, such as a pyran ring). In some embodiments, R⁶Is OR³(e.g. OH) or C₁-C₆Alkyl (e.g. CH)₃). In some embodiments, the compound of formula (I) is xanthomonas (i.e., 3,7, 9-trihydroxy-3-methyl-3, 4-dihydrocyclohepta [ c)]Pyran-1-one). In some embodiments, the compound of formula (I) isOr a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound disclosed in U.S. patent No. 3,135,768, which is incorporated herein by reference in its entirety.

Selected chemical definition

The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the periodic table of elements (CAS version Handbook of Chemistry and Physics, 75 th edition, inner page), and specific functional groups are generally defined as described herein. In addition, the general principles of Organic Chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry (Organic Chemistry), University Science Books (University Science Books), Sausalit, 1999; smith and March, Advanced Organic Chemistry by March (March's Advanced Organic Chemistry), 5 th edition, john wilford, new york, 2001; larock, Integrated Organic Transformations (Comprehensive Organic Transformations), VCH publishing Co., New York, 1989; and Carruther, Some Modern Methods of organic Synthesis (Some Modern Methods of organic Synthesis), third edition, Cambridge university Press, Cambridge, 1987.

Unless otherwise indicated, structures described herein also include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S conformations of the asymmetric centers, the Z and E double bonds are differentA isomer, and Z and E conformers. Thus, individual stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the compounds of the present invention are within the scope of the invention. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention. In addition, unless otherwise indicated, structures described herein are also meant to include such compounds: the compounds differ by the presence of one or more isotopically enriched atoms. For example, compounds having the structure of the invention (including replacement of hydrogen by deuterium or tritium, or replacement of carbon by deuterium or tritium)¹³C or¹⁴C enriched carbon substitution) is within the scope of the present disclosure. According to the invention, the compounds are useful, for example, as analytical tools, as probes in biological assays or as therapeutic agents.

Where a particular enantiomer is preferred, it may be provided in some embodiments that it is substantially free of the corresponding enantiomer, and may also be referred to as "optically enriched". The term "optically enriched" as used herein means that the compound is composed of a significantly larger proportion of one enantiomer. In certain embodiments, the compounds consist of at least about 90% by weight of the preferred enantiomer. In other embodiments, the compound consists of at least about 95%, 98%, or 99% by weight of the preferred enantiomer. Preferred enantiomers may be separated from racemic mixtures by any method known to those skilled in the art, including chiral High Pressure Liquid Chromatography (HPLC), and formation and crystallization of chiral salts or prepared by chiral synthesis. See, e.g., Jacques et al, Enantiomers, Racemates and Resolution (enertiomers, racemes and Resolution) (willy Interscience, new york, 1981); wilen et al, Tetrahedron (Tetrahedron) 33: 2725 (1977); and the Stereochemistry of Eliel, e.l. Carbon Compounds (Stereochemistry of Carbon Compounds) (McGraw-Hill, new york, 1962); wilen, s.h., Resolving Agents and optical resolution Tables (Tables of Resolving Agents and optical resolutions), page 268 (e.l. eliel, university of saint press (univ.of note dam Pres), indiana, 1972).

The term "alkyl" as used herein refers to a monovalent saturated straight or branched chain hydrocarbon, e.g., a straight or branched chain group of 1-12, 1-10, or 1-6 carbon atoms, referred to herein as C, respectively₁-C₁₂Alkyl radical, C₁-C₁₀Alkyl, and C₁-C₆An alkyl group. Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, sec-pentyl, isopentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl and the like.

The term "heterocyclyl" refers to monocyclic or fused, spiro-fused and/or bridged bicyclic and polycyclic ring systems in which at least one ring is saturated or partially unsaturated (but not aromatic) and contains heteroatoms. The heterocyclic group may be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure, and any ring atom may be optionally substituted. Representative heterocyclyl groups include ring systems wherein (i) each ring is non-aromatic and at least one ring contains a heteroatom, e.g., tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidinonyl, piperidinyl, pyrrolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, oxazapinyl, diazepinyl, oxazapinyl, thiazapinyl, morpholinyl, and quinuclidinyl; (ii) at least one ring is non-aromatic and contains heteroatoms and at least another ring is an aromatic carbocyclic ring, e.g. 1,2,3, 4-tetrahydroquinolinyl, 1,2,3, 4-tetrahydroisoquinolinyl; (iii) at least one ring is non-aromatic and contains heteroatoms and at least one other ring is aromatic and contains heteroatoms, such as 3, 4-dihydro-1H-pyrano [4,3-c ] pyridine and 1,2,3, 4-tetrahydro-2, 6-naphthyridine.

As described herein, the compounds of the present invention may comprise an "optionally substituted" moiety. The term "substituted", whether before or after the term "optionally", generally means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise specified, an "optionally substituted" group may have suitable substituents at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituents at each position may be the same or different. The combinations of substituents envisaged in the present invention are preferably those which result in the formation of stable or chemically feasible compounds. The term "stable" as used herein refers to a compound that is not substantially altered when subjected to conditions to allow its production, detection, and in certain embodiments its recovery, purification, and use for one or more of the purposes disclosed herein.

The term "pharmaceutically acceptable salt" as used herein refers to salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without excessive toxicity, irritation, allergic response, and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts are described in detail, for example, by Berge et al in j.pharmaceutical Sciences,1977,66, 1-19, which is incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of the present invention include those of suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable salts are salts of amino groups formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid), or organic acids (such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid), or by using other methods known in the art, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, citrates, cyclopentanepropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, dodecylsulfates, malates, maleates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, oxalates, palmitates, dihydronaphthalenesulfonates, pectinates, pernates, persulfonates, and tartratesSulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propionates, stearates, succinates, sulfates, tartrates, thiocyanates, p-toluenesulfonates, undecanoates, valerates, and the like. Salts derived from suitable bases include alkali metal salts, alkaline earth metal salts, ammonium salts and N⁺(C_1-4Alkyl radical)₄ ^-And (3) salt. Representative alkali or alkaline earth salts include sodium, lithium, potassium, calcium, magnesium, and the like. Other pharmaceutically acceptable salts include: non-toxic ammonium salts, quaternary ammonium salts, and salts formed using a counter ion (e.g., halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate) with an amine cation.

The term "solvate" refers to a form of a compound that is associated with a solvent, typically by a solvolysis reaction. The physical association includes hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds of formula (I) may be prepared, for example, in crystalline form or may be solvated. Suitable solvates include pharmaceutically acceptable solvates, and also includes stoichiometric and non-stoichiometric solvates. In some cases, the solvate will be capable of isolation, for example when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid. "solvate" encompasses both solution phase and isolatable solvates. Representative solvates include hydrates, ethanolates, and methanolates.

It is also understood that compounds having the same molecular formula but differing in the nature or order of bonding of their atoms or the arrangement of their atoms in space are referred to as "isomers". Isomers in which the arrangement of atoms in space is different are referred to as "stereoisomers". Stereoisomers that are not mirror images of each other are referred to as "diastereomers", and stereoisomers that are not superimposable mirror images of each other are referred to as "enantiomers". For example, when the center of a compound is asymmetric, it is bonded to four different groups, and then a pair of enantiomers is possible. Enantiomers can be characterized by the absolute configuration of their asymmetric centers and described by the R and S sequencing rules of Cahn and Prelog, or by the way the molecules are rotated about the plane of polarized light and called dextrorotatory or levorotatory (i.e., the (+) or (-) isomers, respectively). The chiral compounds may exist as individual enantiomers or as mixtures thereof. Mixtures containing equal proportions of enantiomers are referred to as "racemic mixtures".

Production parameters

The methods described herein can be used to analyze samples produced by methods of making and producing products (e.g., recombinant polypeptides). The method of manufacture and production may be characterized by various production parameters.

A production parameter as used herein is a parameter or element in the production process. Production parameters that may be selected include: for example, cells or cell lines, culture media, culture processes or bioreactor variables (e.g., batch, fed-batch or perfusion), preparations of glycoprotein preparation and purification processes for the production of glycoproteins.

The main production parameters include: 1) the type of host; 2) the genetics of the host; 3) medium type, 4) fermentation platform; 5) a purification step; 6) and (4) preparing the preparation. A secondary production parameter as used herein is a production parameter that is adjustable or variable within each primary production parameter. Examples include: selecting a host subclone according to the desired properties of the polysaccharide; modulating constitutive or inducible host gene levels; introducing a new gene or promoter element; media additives (e.g., part of the list in table IV); physiochemical growth properties; growth vessel type (e.g., bioreactor type, T-flask); cell density; the cell cycle; enrichment of products with the desired polysaccharide type (e.g., by lectin or antibody-mediated enrichment, ion exchange chromatography, CE, or similar methods); or similar secondary production parameters, will be apparent to those skilled in the art.

Culture medium

The manufacturing and production methods described herein can include determining and/or selecting media components and/or media component concentrations that have a positive correlation with a desired property or properties of one or more polysaccharides. The medium components may be added or administered during the production of the glycoprotein or upon a change in the medium, depending on the culture conditions. The medium components include components added directly to the culture as well as components that are byproducts of cell culture.

The culture medium comprises the following components: for example, buffer, amino acid content, vitamin content, salt content, mineral content, serum content, carbon source content, lipid content, nucleic acid content, hormone content, trace element content, ammonia content, cofactor content, indicator content, small molecule content, hydrolysate content, and enzyme modulator content.

Examples of the various media components are provided below:

exemplary buffers include: tris, N- (Tris (hydroxymethyl) methyl) glycine (Tricine), HEPES, MOPS, PIPES, TAPS, N-bis (2-hydroxyethyl) glycine (bicine), BES, TES, diarsenate, MES, acetate, MKP, ADA, ACES, glycinamide, and acetamidoglycine (acetamidoglycine). The culture medium may be serum-free, or may include animal-derived products (e.g., Fetal Bovine Serum (FBS), calf serum (FCS), Horse Serum (HS), human serum), animal-derived serum substitutes (e.g., Ultroser G, SF, and HY; skim milk powder; bovine EX-CYTE), fetuin, Bovine Serum Albumin (BSA), serum albumin, and transferrin. When serum-free medium is selected, lipids may be included, such as palmitic acid and/or stearic acid.

Lipid components include oils, saturated fatty acids, unsaturated fatty acids, glycerides, steroids, phospholipids, sphingolipids and lipoproteins. Exemplary amino acids that can be included in or eliminated from the culture medium include: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, proline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Examples of vitamins that can be present in or eliminated from the medium include vitamin a (retinoid), vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D, vitamin E, and vitamin K.

Minerals that may be present in or eliminated from the culture medium include: bismuth, boron, calcium, chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium, manganese, molybdenum, nickel, phosphorus, potassium, rubidium, selenium, silicon, sodium, strontium, sulfur, tellurium, titanium, tungsten, vanadium, and zinc. Exemplary salts and minerals include: CaCl2 (anhydrous), CuSO 45H 2O, Fe (NO3). sub.9H 2O, KCl, KNO3, KH2PO4, MgSO4 (anhydrous), NaCl, NaH2PO4H2O, NaHCO3, Na2SE3 (anhydrous), ZnSO4.7H2O; linoleic acid, lipoic acid, D-glucose, hypoxanthine 2Na, phenol red, putrescine 2HCl, sodium pyruvate, thymidine, pyruvic acid, sodium succinate, succinic acid, sodium sulfate hexahydrate, glutathione (reduced), p-aminobenzoic acid (PABA), methyl linoleate, bacto peptone G (bacto peptone G), adenosine, cytidine, guanosine, 2' -deoxyadenosine HCl, 2' -deoxycytidine HCl, 2' -deoxyguanosine, and uridine. When the desired polysaccharide characteristic is reduced fucosylation, the production parameters may comprise culturing the cell, e.g., a CHO cell, such as a dhfr-deficient CHO cell, in the presence of manganese (e.g., present at a concentration of about 0.1 μ Μ to 50 μ Μ). Reduced fucosylation can also be obtained, for example, by culturing cells (e.g., CHO cells, e.g., dhfr-deficient CHO cells) at an osmotic pressure of about 350 to 500 mOsm. The osmotic pressure can be adjusted by adding salt to the medium or by producing salt as a by-product due to evaporation during production.

Hormones include, for example, somatostatin, growth hormone releasing factor (GRF), insulin, prolactin, human growth hormone (hGH), growth hormone (somatotropin), estradiol, and progesterone. The growth factors include: for example, Bone Morphogenic Proteins (BMP), Epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), Nerve Growth Factor (NGF), Bone Derived Growth Factor (BDGF), transforming growth factor β 1(TGF- β 1), [ growth factor from U.S. patent No. 6,838,284B2 ], hemin (hemin), and NAD. Examples of surfactants that may be present in the medium or eliminated from the medium include Tween-80 and Pluronic F-68. Small molecules may include, for example, butyrate, ammonia, unnatural sugars, unnatural amino acids, chloroquine, and betaines.

Physiological chemical parameter

The production parameters may also include physiochemical parameters. The conditions may include: temperature, pH, osmotic pressure, shear or agitation rate, oxidation, gushing rate (surging rate), growth vessel, tangential flow, DO, CO₂Nitrogen, batch feed, redox, cell density and feed strategy. Examples of physiochemical parameters that may be selected may include, for example: pH, osmotic pressure, shear or agitation rate, oxidation, gush rate, growth vessel, tangential flow, batch dissolved O₂、CO₂Nitrogen, fed batch, redox, cell density, perfusion culture, feeding strategy, temperature and culture time.

Other Production parameters are known to those skilled in the art, see, e.g., antibody expression and Production (2011), Mohamed Al-Rubeai eds; the press publishing house (springer publishing).

Products and nucleic acids encoding same

Provided herein are methods of analyzing samples, such as samples produced by, for example, methods of making and producing recombinant polypeptides. The method of manufacture and production may comprise: cells or cell lines capable of producing a product (e.g., cells and products described herein) are identified, selected, or prepared. Products encompassed by the present disclosure include, but are not limited to: molecules, nucleic acids, polypeptides (e.g., recombinant polypeptides, such as antibodies, bispecific antibodies, multispecific antibodies), or hybrids thereof, which can result, for example, from expression in a cell. In some embodiments, the cell is engineered or modified to produce a product. Such modifications include the introduction of molecules that control or result in the production of the product. For example, a cell is modified by introducing an exogenous nucleic acid encoding a polypeptide, e.g., a recombinant polypeptide, and the cell is cultured under conditions suitable for, e.g., production (e.g., expression and secretion) of the polypeptide, e.g., the recombinant polypeptide.

In some embodiments, the cultured cells are used to produce proteins (e.g., antibodies, such as monoclonal antibodies) and/or recombinant proteins for therapeutic use. In some embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in some embodiments, molecules having a molecular weight of about 4000 daltons (dalton) to greater than about 140,000 daltons may be produced. In some embodiments, these molecules may have a range of complexities, and may include post-translational modifications, including glycosylation.

In some embodiments, the polypeptide is: for example, botulinum toxin (BOTOX), botulinum toxin (Myobloc), botulinum toxin preparations (Neuroloc), lisu (Dysport) (or other botulinum neurotoxins of serotypes), alfurosidase alpha (aldosidase alpha), daptomycin (daptomycin), YH-16, chorionic gonadotropin alpha (chlorogonotropic alpha), filgrastim (filgrastim), cetrorelix (cetrorelix), interleukin-2 (interleukin-2), aldesleukin (aldesleukin), teskin (tecellulin), dinierein (diferutox), interferon alpha-n 3(interferon alpha-n3) (injections), interferon alpha-nl (interferon alpha-nl), DL-8234, interferon (interferon), mulberrin (interferon), interferon gamma (interferon) (interferon gamma-1), interferon gamma-interferon gamma (interferon alpha-n3) (injections), interferon alpha-nl (interferon alpha-nl), DL-8234, interferon (interferon gamma-interferon (interferon), interferon gamma-interferon (interferon alpha-gamma-interferon alpha (interferon alpha-gamma-1, interferon gamma-interferon alpha (interferon alpha-gamma-1, interferon alpha (interferon alpha, gamma-interferon alpha-alpha, gamma-interferon alpha (interferon alpha, gamma-alpha, interferon alpha-alpha, interferon (interferon alpha-alpha, gamma-alpha, interferon alpha, gamma-alpha-, croFab, nesiritide (nesiritide), abamectin (abatacept), alefacept (alefacept), Ribis (Rebif), alfa eltoprim (epcotinalfa), teriparatide (teriparatide), calcitonin (calcupin), etanercept (etanercept), hemoglobin glutamate 250(bovine) (hemoglobin), dutrex alpha (droxygen alpha), collagenase (collagenase), carnipeptide (carperite), recombinant human epidermal growth Factor (recombinant human epidermal growth Factor), DWP401, darbepotein alpha (darbepoetin), recombinant human synetin omega (epoetinometeomer alpha), recombinant human synemet beta (epoetin alpha), recombinant human synephrine alpha (vw alpha), recombinant human erythropoietin (mangiolein alpha + erythropoietin (mangiolein alpha), recombinant human erythropoietin (vw alpha + erythropoietin (mangiolein alpha), recombinant human erythropoietin (mangiolein alpha (vw + mangiolein alpha), recombinant human erythropoietin (mangiolein alpha (mangiol), recombinant human erythropoietin (mangiolein alpha), recombinant human erythropoietin (mangiol alpha (mangiol + vw), recombinant human erythropoietin (mangiolein alpha (mangiol), recombinant human erythropoietin (mangiol), human erythropoietin (mangiol alpha), human erythropoietin (mangiolin + mangiol alpha), human erythropoietin (mangiol), recombinant human erythropoietin (mangolexin alpha), human erythropoietin (, Recombinant (Recombinate), recombinant Factor VIII (Recombinant Factor VIII), Factor VIII (recombinant), alfamet (Alphnmate), ortex alpha (octocog alpha), Factor VIII, palifermin (palifermin), addinine (Indikinase), tenecteplase (tenecteplase), alteplase (alteplase), plectase (pamiteplase), reteplase (reteplase), nateplase (nateplase), monteplase (monteplase), follitropin alpha (follitropin alpha), rPFSH, HypFSH, micafungin (micafungin), pefilstim (pegfilstim), letograstin (letograstin), natrofemin (nartomodulograstin), sertralin (seromycin), netriline (netriline), netrilysin (netrilysin), netrilysin (lutein), netrilysin (luteolin (netrilysin), netrilysin (luteolin) (L), netrilysin (luteolin) (L), netrilysin (netrilysin) (L), netrilysin (netrilysin), netrilysin) (L (D), D (D) (L (D), D (D), D (D), D (D, Histrelin (histrelin), nafarelin (nafarelin), leuprorelin (leuprolide) (ATRIGEL), leuprorelin (DUROS), goserelin (goserelin), europine (Eutropin), growth hormone (somatropin), mecamylamine (mecastin), enfavirtide (enfavirtide), Org-33408, insulin glargine (insulin glargine), insulin glufosinate (insulin glargine), insulin (inhalation), insulin lispro (insulin lispro), insulin detemir (insulin deterronir), insulin (rapidmist), mecamylamine (mevalon farnate), anakinra (amacrine), western interleukin (uklein), 99 mTc-western-peptide (mtisopeptide (99 mtp), interferon alpha (interferon), interferon alpha-interferon (interferon alpha-interferon), interferon alpha-interferon (interferon beta), interferon alpha-interferon (interferon alpha-interferon), interferon alpha-gamma-interferon (interferon, interferon alpha-interferon, interferon alpha-gamma-interferon (interferon alpha-interferon, interferon alpha-gamma-interferon, interferon alpha-gamma-interferon, gamma-interferon, gamma-, Doublet (bile), insulin (recombinant), recombinant human insulin (recombinant human insulin), insulin aspart (insulin aspart), meccanonin (mecasenin), roscovitine-A (Roferon-A), interferon-alpha 2(interferon-alpha 2), lucernone (Alfaferone), consensus interferon-1 (interferon alfacon-1), interferon alpha (interferon), atherebic recombinant human luteinizing hormone (avonexex' recombinant human luteinizing hormone), streptokinase alpha (dornase alpha), troffemine (transfermin), ziconotide (zipotide), taltirelin (taltirelin), abeditetramine (bibominalfa), atosiban (bazineb), kalilemin (bocavir), balamin (balancer), balancer (balancer), SHAzan peptide (zeatin), zeatin (eclipt-B), zeatin (zeatin), zeatin-beta-fetidide (zeatin), zeatin (zeatin), zeatin (zeatin), zeatin (zea, Galactosidase beta (agalisase beta), galactosidase alpha (agalisase alpha), laronidase (laronidase), cupreotide acetate (prezatide copper acetate), labyrinse (rasburicase), ranibizumab (ranibizumab), alemtinimide (actimmume), PEG-Intron (PEG-Intron), certinin (Tricomin), recombinant human parathyroid hormone (PTH)1-84, recombinant human erythropoietin (epoetin delta), transgenic antithrombin III (transgenic antithrombin III), granditerol, hyaluronidase (Vitrase), recombinant insulin (albumin), interferon-alpha (interferon-alpha), GEM-21S, valosin (vapreotide), trypsin (trypsin alpha), trypsin inhibitor (bovine serum albumin (85bovine serum albumin), bovine serum albumin (85monoclonal antibody), bovine serum albumin (85monoclonal), bovine serum albumin (bovine serum albumin), bovine serum albumin (85bovine serum albumin (bovine serum albumin), bovine serum albumin (85bovine serum albumin), bovine serum albumin (bovine serum albumin), bovine serum albumin (interferon delta), interferon-alpha (interferon-alpha), human interferon-21S, bovine serum albumin (bovine serum albumin), bovine serum albumin (bovine serum albumin) 1), bovine serum albumin (interferon-alpha), bovine serum albumin (interferon beta), bovine serum albumin (interferon-interferon, Lanoteplase (lanoteplase), recombinant human growth hormone (recombinant human growth hormone hormomone), enfuvirtide (enfuvirtide), VGV-1, interferon (alpha) (interferon (alpha)), lucetinate (lucinantant), empadil (aviptadil), icatin (icatibant), icariside (ecalanide), omiganan (omiganan), aurobium (aurora), pexiganan acetate (pexiganacetate), ADI-PEG-20, LDI-200, degarelix (degarelix), pseudomonas exotoxin (cinquedotoxin), Favld, MDX-1379, ISISISX-247, liraglutide (liraglutide), terticoidin (tericotinase), lipotropin (proximate enzyme), lipotropin (4504), lipocalin (450123), amatoxin (4504), amatoxin (amatoxin), amaurosporine (4504), amaurosporine (4500), lipocalin (lipocalin), lipocalin (4504), lipocalin (lipocalin), lipocalin (4504), lipocalin (lipocalin) and (lipocalin) in (lipocalin) as shown in (lipocalin) in (lipocalin) as shown in (e) and (e) as shown in (e), and (lipocalin) in (e) as shown in, e, teduglutide (teduglutide), Dammard (Diamyd), DWP-412, growth hormone, recombinant G-CSF, insulin (insulin), insulin (Technosphere), insulin (AERx), RGN-303, DiaPep277, interferon beta (interferon beta), interferon alpha-n 3(interferon alpha-n3), belicep (belitacept), transdermal insulin patch (transdermal insulin patches), AMG-531, MBP-8298, Xerecept, Opbaman (ebopacan), AIDSVAX, GV-1001, Linfoscarnan (LymphosScan), ranpirnase (ranpirnase), Lipoxysase (lupulin), MP52, Sickle-T (Sipulivichel-T), thrombin-37, thrombin (thrombin-37), thrombin (thrombin), thrombin (thrombin-37), thrombin (thrombin ), thrombin (thrombin-IV), thrombin (thrombin-thrombin, thrombin-37), thrombin (thrombin, thrombin-IV), thrombin (thrombin-37), thrombin (thrombin, thrombin-beta-E, thrombin (E), thrombin (thrombin, thrombin, EUR-1008M, recombinant FGF-I, BDM-E, rotigotine (rotigotide), ETC-216, P-113, MBI-594AN, duramycin (duramycin), SCV-07, OPI-45, Endostatin (Endostatin), Angiostatin (Angiostatin), ABT-510, Bowman birk inhibitor (Bowman birk inhibitor), XMP-629, 99 mTc-Hynic-annexin V, Kahalalide F (kahalalide F), CTCE-9908, teverelix (teverelix), azariix (orezalix), romidepsin (roridisin), AY-504798, interleukin4 (interlein uk 4), PRX-321, peptide scan (Pepsic), interleukin (irkin), interleukin (rhbutein), interferon-alpha (ATY-35015), interferon-albumin (Albizin-161), interferon-albumin (Albizine-2), interferon-albumin (Albizine-161), interferon-2-albumin (Albizine-161), interferon-2-Albizine (Albizine-I-X-1, interferon-2, interferon-Albizine (Albizine-I), interferon-X-D, interferon-I, interferon-2, interferon-I, interferon-D, interferon-I, interferon-, Omega interferon (omega interferon), PCK-3145, CAP-232, pasireotide (pasireotide), huN901-DMI, SB-249553, Oncovax-CL, Oncovax-P, BLP-25, Cervax-16, MART-1, gp100, neuraminidase (tyrosinase), nano-non-peptide (nemifitide), rAAT, CGRP, pernaprox (pegsunecrcept), thymosin beta4 (thymosina beta4), plebeidulipicin (plipidepsin), GTP-200, ramoplanin (ramoplanin), GRASPA, OBI-1, AC-100, salmon calcitonin (salmon calcitonin) Elegan (eligen), elselin (examoraxelin), capromorelin (procarythrin), carmine (decahydramin (nape), napestrin-131), napestrin (KK-52), recombinant peptide (PEG 2), caprolactamine (caprolactamin), caprolactamine (napestrin), capromorelin (napestrin), napestrin-52), caprolactamin (napestrin), capromorelin-200, napestrin (napestrin), napestrian (napestrian-2) and (napestrin), napestrin (napestrin), nap, bFGF, PEGylated recombinant staphylokinaseviatant (PEGylated recombinant staphylokinase), V-10153, prourokinase with ultrasonication (SonoLysis Prolyse), neuroVax, CZEN-002, rGLP-1, BIM-51077, LY-548806, exenatide (medisarb), AVE-0010, GA-GCB, Avorelin (avorelin), ACM-9604, linaclotide acetate (linaclotid eactate), CETi-1, Helmossban (Hemospan), VAL, rapid acting insulin (injectable) (Viadel), intranasal insulin (Ellisgen), recombinant methionyl human leptin (immunogenic human leptin), Picraka (cortikinra), interleukin (Mukine), interleukin (RG-33, Irelargol-11, RG-3632, ORT-3632, RG-368, ORZygien-3660231, RG-3632, ORE-368, ORE-60232, ORE-E-D-E3, and E-D, Recombinant human insulin (recombinant human insulin), RPI-78M, Omepleren (orelvekin), CYT-99007CTLA4-Ig, DTY-001, Valerast (valategart), interferon alpha-n 3(interferon alpha-n3), IRX-3, RDP-58, tafferon (Tauferon), bile salt-stimulated lipase (bile salt stimulated lipase), meripase (meripase), alanine phosphatase (alanine phosphatase), EP-2104R, merinopentan-II (melaanotan-II), brimonide (bremelanotide), ATL-104, recombinant human microfibrillar lysozyme (dynamic human fibroblast), SEMAX-200, SEMAX, ACV-1, CJXen-4, CJ-1008, BCR-03, BCA-03, ALKANORMB-L-104, recombinant human microplasmin lyase (AEI-L-728-1, AEOMB-728-1, GAMMA-728, AEOMB-14, GAMMA-D-IV-1, GAEMITA-1, GAL-1008, GAL-L-D, ALOMB-D-L-D-L-104, recombinant human microplasmin, GAL-D, GAL-L-D, GAL-, Vacc-5q, Vacc-4x, Tat-toxoid (TatToxoid), YSPSL, CHS-13340, PTH (1-34) (Novasome), osthol-C (Ostabolin-C), PTH analogue ((PTH analg), MBRI-93.02, MTB72F, MVA-Ag85A, FARA04, BA-210, recombinant pestis FIV (recombinant bindant flag), AG-702, OxSODrol, rBetV1, Der-P1/Der-P2/Der-P7, PR1 peptide antigen (PR1 peptide antigen), mutant ras vaccine (mutan ras vaccine), HPV-16E7 lipopeptide (HPV-16E 6 lipopeptide vaccine), rabeprint-5 toxin (Bytein), intein 1, triptolide-D (WT-11), Pentox (CDT-9808), Pentox-D (CDT-9808), Oryza-D, Var-P3528/Der-P493-12, recombinant pestis vaccine (Va-P) and Pyth-P11, wherein, Kriginose (reticulose), rGRF, HA, alpha-galactosidase A (alpha-galactosidase A), ACE-011, ALTU-140, CGX-1160, angiotensin (angiotensin), D-4F, ETC-642, APP-018, rhMBL, SCV-07, DRF-7295, ABT-828, ErbB2-specific immunotoxin (ErbB2-specific immunotoxin), DT3SSIL-3, TST-10088, PRO-1642, Combotox, cholecystokinin-B/gastrin receptor binding peptides (cholecystokinin-B/gastrin-receptor binding peptides), 111In-hEGF, AE-37, trastuzumab (trastuzumab) -DM, antagonist G (antangg), IL-12, PM-BP 34, IGF-321, IGF-19, BLOL19-IRA-8819, BLOCK-8819, IGF-IRF-8819, and BLT-887, Re-188-P-2045, AMG-386, DC/1540/KLH, VX-001, AVE-9633, AC-9301, NY-ESO-1 (polypeptide), NA17.A2 (polypeptide), (CBP-501), recombinant human lactoferrin (recombinant human lactoferrin), FX-06, AP-214, WAP-8294A, ACP-HIP, SUN-11031, polypeptide YY [3-36], FGLL, asecept (atacicept), BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34, F-18-CCR1, AT-1100, JPD-003, PTH (7-34) (Novasome), duramycin (duramycin), CAB-2, CTCE-0214, GlycoPEGylated erythropoietin (GlycoPEGylated erythropoietin), EPO-1540, EPO-114, AMG-528, and EPO-6, JR-013, factor XIII, aminocandin (aminocandin), PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, teverelix (teverelix), EP-51216, hGH, OGP-I, sifuvirtide (sifuvirtide), TV4710, ALG-889, Org-41259, rhCC10, F-991, thymopentin (thymopentin), r (m) CRP, liver-selective insulin (hepatective insulin), sutilin (subalin), L19-IL-2fusion protein (L19-IL-2fusion protein), elastin ((elafin), NMK-150, ALTU-139, EN-122004, rhTPO, platelet receptor agonist (thrombopoietin-receptor agonist), neuroagonist-108, neuroagonist-208, growth factor-105, growth factor-X-1007, and interferon, Teriparatide (Elliparate), GEM-OS1, AC-162352, PRX-302, LFn-p24fusion (LFn-p24fusion), EP-1043, gpE1, gpE2, MF-59, hPTH (1-34), 768974, SYN-101, PGN-0052, avicumnine (avicumnine), BIM-23190, multi-epitope tyrosinase peptide (multi-epitope tyrosinase peptide), Enkastit (enkastim), APC-8024, GI-5005, ACC-001, TTS-CD3, vascular targeting TNF (vascular-targeted TNF), desmopressin (deperssin), onacept (onerpt), and TP-9201.

In some embodiments, the polypeptide is adalimumab (HUMIRA), infliximab (REMICADE)^TM) Rituximab (rituximab) (RITUXAN)^TM/MABTHERA^TM) Etanercept (ENBREL)^TM) Bevacizumab (avacetizumab) (AVASTIN)^TM) Trastuzumab (trastuzumab) (HERCEPTIN)^TM) Pegrilagram (NEULASTA)^TM) Or any other suitable polypeptide, including biosimilar (biovaillar) and biomutate (biobeter).

Other suitable polypeptides are listed in the following table and in table 1 of US 2016/0097074:

TABLE 1

In some embodiments, the polypeptide is a hormone, clotting factor/blood clotting factor, cytokine/growth factor, antibody molecule, fusion protein, protein vaccine, or peptide, as shown in table 2.

Table 2: exemplary product

In some embodiments, the protein is a multispecific protein, such as a bispecific antibody shown in table 3.

Table 3: bispecific formats

TABLE 4

TABLE 4

TABLE 4

TABLE 4

TABLE 4

TABLE 4

In some embodiments, the polypeptide is an antigen expressed by a cancer cell. In some embodiments, the recombinant or therapeutic polypeptide is a tumor-associated antigen or a tumor-specific antigen. In some embodiments, the recombinant or therapeutic polypeptide is selected from the group consisting of: HER2, CD20, 9-O-acetyl-GD 3, beta hCG, A33 antigen, CA19-9 marker, CA-125 marker, calreticulin, carbonic anhydrase IX (MN/CAIX), CCR5, CCR8, CD19, CD22, CD25, CD44v 25, CD25, CC123, CD138, carcinoembryonic antigen (CEA; CD66 25), desmoglein 4, E-cadherin neo-epitope, endosialin, ephrin A25 (EphAh 25), Epidermal Growth Factor Receptor (EGFR), epithelial cell adhesion molecule (EpCAM), Erb3672, fetal acetylcholine receptor, fibroblast activation antigen (LIP), fucosyl (fucosyl) GM 25, GM 25, HER-II-G72, HER-II-G25, HER-II-G-II receptor, GLOB3672, HER-25, GLE-like receptor, LIP-25, LIP-G-II, and its receptor, Melanoma-specific chondroitin sulfate proteoglycan (MCSCP), mesothelin, MUC1, MUC2, MUC3, MUC4, and MUC2,

MUC5_AC、MUC5_BMUC7, MUC16, Muller inhibitor type II (MIS) receptor type II), plasma cell antigen, poly SA, PSCA, PSMA, sonic hedgehog (SHH), SAS, STEAP, sTn antigen, TNF- α precursor, and combinations thereof.

In some embodiments, the polypeptide is an activating receptor and is selected from the group consisting of 2B4(CD244), α₄β₁Integrins, β₂Integrins, CD2, CD16, CD27, CD38, CD96, CD100, CD160, CD137, CEACAM1(CD66), CRTAM, CS1(CD319), DNAM-1(CD226), GITR (TNFRSF18), activated forms of KIR, NKG2C, NKG2D, NKG2E, one or more natural cytotoxic receptors, NTB-A, PEN-5, and combinations thereof, optionally wherein β₂The integrin comprises CD11a-CD 18, CD11b-CD 18, or CD11c-CD 18, optionally wherein the activated form of KIR comprises KIR2DS1, KIR2DS4, or KIR-S, and optionally wherein the natural cytotoxic receptor comprises NKp30, NKp44, NKp46, or NKp 80.

In some embodiments, the polypeptide is an inhibitory receptor and is selected from the group consisting of: KIR, ILT2/LIR-L/CD85j, an inhibitory form of KIR, KLRG1, LAIR-1, NKG2A, NKR-P1A, Siglec-3, Siglec-7, Siglec-9, and combinations thereof, optionally wherein the inhibitory form of KIR comprises KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, KIR3DL2, or KIR-L.

In some embodiments, the polypeptide is an activating receptor and is selected from the group consisting of: CD3, CD2(LFA2, OX34), CD5, CD27(TNFRSF7), CD28, CD30(TNFRSF8), CD40L, CD84(SLAMF5), CD137(4-1BB), CD226, CD229(Ly9, SLAMF3), CD244(2B4, SLAMF4), CD319(CRACC, BLAME), CD352(Ly 08, NTBA, SLAMF6), CRTAM (CD355), DR3(TNFRSF25), GITR (CD357), HVEM (CD270), ICOS, LIGHT, LT β R (TNFRSF3), OX40(CD134), NKG2D, SLAM (CD150, SLAMF1), TCR α, TCR β, TCR γ, TIM1 (cr, KIM1), and combinations thereof.

In some embodiments, the polypeptide is an inhibitory receptor and is selected from the group consisting of: PD-1(CD279), 2B4(CD244, SLAMF4), B71(CD80), B7H1(CD274, PD-L1), BTLA (CD272), CD160(BY55, NK28), CD352(Ly108, NTBA, SLAMF6), CD358(DR6), CTLA-4(CD152), LAG3, LAIR1, PD-1H (VISTA), TIGIT (VSIG9, VSTM3), TIM2(TIMD2), TIM3(HAVCR 68692, KIM3), and combinations thereof.

Other exemplary proteins include, but are not limited to: leader et al "protein therapeutics: summary and pharmacological classification "(Protein therapeutics: a summary and pharmacological classification), nature reviews Drug Discovery, 2008, 7: 21-39 (incorporated herein by reference) of any of the proteins described in tables 1-10; or any conjugate, variant, analog, or functional fragment of a recombinant polypeptide described herein.

Other recombinant protein products include non-antibody scaffolds or alternative protein scaffolds such as, but not limited to: darpins, affibodies and mimobody proteins (adnectins). The non-antibody scaffold or alternative protein scaffold can be engineered to identify or bind to one or two or more (e.g., 1,2,3,4, or 5 or more) different targets or antigens.

Also provided herein are nucleic acids, e.g., exogenous nucleic acids encoding a product (e.g., a polypeptide, e.g., a recombinant polypeptide described herein). The nucleic acid sequence encoding the desired recombinant polypeptide can be obtained using recombinant methods known in the art, for example, by screening libraries, e.g., genes, from cells expressing the desired nucleic acid sequence, by obtaining the nucleic acid sequence from vectors known to contain the same sequence using standard techniques, or by isolating it directly from cells and tissues containing it. Alternatively, nucleic acids encoding recombinant polypeptides may be produced synthetically, rather than cloned. Recombinant DNA techniques and processes are very advanced and mature in the art. Thus, a nucleic acid sequence encoding a recombinant polypeptide can be readily envisioned or produced by one of ordinary skill in the art familiar with the amino acid sequences of recombinant polypeptides described herein.

In some embodiments, the exogenous nucleic acid controls the expression of a product endogenously expressed by the host cell. In this embodiment, the exogenous nucleic acid comprises one or more nucleic acid sequences that increase expression of the endogenous product (also referred to herein as "endogenous product transcription activation sequences"). For example, a nucleic acid sequence that increases expression of an endogenous product comprises a constitutively active promoter or a stronger promoter, e.g., increases transcription at a desired site, e.g., increases expression of a desired endogenous gene product. Following introduction of an exogenous nucleic acid comprising an endogenous product transcriptional activation sequence, the exogenous nucleic acid is integrated into the chromosomal genome of the cell, e.g., at a preselected location near the genomic sequence encoding the endogenous product, such that the endogenous product transcriptional activation sequence increases transcriptional activation or expression of the desired endogenous product. Other methods of modifying cells, such as the introduction of exogenous nucleic acids to increase expression of endogenous products are described, for example, in U.S. Pat. nos. 5,272,071; hereby incorporated by reference in its entirety.

Expression of the products described herein is typically achieved by operably linking a nucleic acid encoding the recombinant polypeptide or portion thereof to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into a eukaryotic or prokaryotic cell. Typical cloning vectors contain other regulatory elements such as transcription and translation terminators, initiation sequences, and promoters which may be used to regulate the expression of the desired nucleic acid sequence.

The nucleic acid sequences described herein may be cloned into various types of vectors, which encode a product (e.g., a recombinant polypeptide) or comprise a nucleic acid sequence that can control the expression of an endogenous product. For example, the nucleic acid may be cloned into a vector, including but not limited to: plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include: expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In some embodiments, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al, 2012, molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual), volumes 1-4, Cold spring harbor Press, New York; and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to: retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and lentiviruses. Generally, suitable carriers comprise: an origin of replication, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers that function in at least one organism (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193). Viral-derived vectors are suitable tools for achieving long-term gene transfer, as they allow long-term, stable integration of transgenes and their propagation in daughter cells.

The vector may further comprise: for example, signal sequences to promote secretion, polyadenylation signals and transcription terminators (e.g., from the Bovine Growth Hormone (BGH) gene), elements that allow prokaryotes to replicate and episome gene replication (e.g., the SV40 origin and ColE1 or others known in the art), and/or elements that allow selection, such as selectable markers or reporter genes.

In one embodiment, a vector comprising a nucleic acid sequence encoding a polypeptide (e.g., a recombinant polypeptide) further comprises: a promoter sequence responsible for recruiting a polymerase to enable initiation of transcription to express a polypeptide, such as a recombinant polypeptide. In one embodiment, a promoter sequence suitable for use in the methods described herein is typically associated with an enhancer to drive mass transcription and thus mass replication of the target exogenous mRNA. In one embodiment, the promoters include the Cytomegalovirus (CMV) major immediate early promoter (Xia, Bringmann et al, 2006) and the SV40 promoter (Chernajovsky, Mory et al, 1984), both of which are derived from the same name virus or promoters derived therefrom. Several other less common viral promoters have been successfully used to drive transcription after inclusion in expression vectors including the Rous Sarcoma virus long terminal repeat (RSV-LTR) and the Moloney murine leukemia virus (Moloney murine leukemia virus) LTR (Papadaikis, Nicklin et al, 2004). In another embodiment, specific endogenous mammalian promoters may be used to drive constitutive transcription of a gene of interest (Pontiller, Gross et al, 2008). The CHO specific Chinese hamster elongation factor 1-alpha (CHEF1 alpha) promoter provides a high-yielding alternative to virus-based sequences (De, Allison 2004). In addition to a promoter, the vectors described herein comprise an enhancer region as described above; specific nucleotide motif regions near the core promoter that can recruit transcription factors to upregulate transcription rates (Riethoven 2010). Like the promoter sequences, these regions are typically derived from viruses and are contained in promoter sequences, such as hCMV and SV40 enhancer sequences, or may be additionally contained in, for example, adenovirus-derived sequences (gailet, Gilbert et al, 2007).

In one embodiment, the vector comprises a nucleic acid sequence encoding a product (e.g., a polypeptide, e.g., a recombinant polypeptide) as described herein, and the vector further comprises a nucleic acid sequence encoding a selectable marker. In one embodiment, the selectable marker comprises Glutamine Synthetase (GS); and dihydrofolate reductase (DHFR), such as enzymes that confer resistance to Methotrexate (MTX); or antibiotic markers, such as enzymes that confer antibiotic resistance, e.g., hygromycin, neomycin (G418), bleomycin, puromycin, or blasticidin. In another embodiment, the selection marker comprises a Selexis selection system (e.g., SUREMENT Platform available from Selexis SA, Inc.)^TMAnd Selexis Genetic Elements^TM) Or a catalan selection system or compatible therewith.

In one embodiment, a vector comprising a nucleic acid sequence encoding a recombinant product described herein comprises a selectable marker that can be used to identify one or more cells comprising a nucleic acid encoding a recombinant product described herein. In one embodiment, a selectable marker can be used to identify one or more cells that comprise integration of a nucleic acid sequence encoding a recombinant product into the genome, as described herein. The identification of one or more cells that have integrated a nucleic acid sequence encoding a recombinant protein can be used to select and engineer a cell or cell line that stably expresses the product.

Suitable vectors for use are commercially available, including the GS Expression System available from Longza Biologics, Inc^TM、GS Xceed^TMGene expression systems orVectors relevant to the CHOK1SV technology, for example, are described in, e.g., Fan et al, pharm. bioprocess (2013); 487-502, which is incorporated herein by reference in its entirety. The GS expression vector comprises a GS gene or functional fragment thereof (e.g., a GS minigene), and one or more (e.g., 1,2, or 3 or more) high efficiency transcription cassettes for expressing a gene of interest, e.g., a nucleic acid encoding a recombinant polypeptide described herein. The GS minigene comprises, e.g., consists of intron 6 of the genomic CHO GS gene. In one embodiment, the GS vector comprises a GS gene and one or two polyA signals operably linked to the SV40L promoter. In another embodiment, the GS vector comprises a GS gene, SV40 splicing, and polyadenylation signal operably linked to the SV40L promoter. In this embodiment, for example, a transcription cassette for expressing a gene or recombinant polypeptide of interest described herein includes: the hCMV-MIE promoter and the 5' untranslated sequences from the hCMV-MIE gene, including the first intron. Other vectors may be constructed based on the GS expression vector, for example, where other selection markers replace the GS gene in the expression vectors described herein.

Vectors suitable for use in the methods described herein include, but are not limited to, other commercially available vectors such as pcDNA3.1/Zeo, pcDNA3.1/CAT, pcDNA3.3TOPO (Sermer Feishel, Inc. (Thermo Fisher), formerly Invitrogen); pTarget, HaloTag (Promega Corp.); pUC57(GenScript Co.); pFLAG-CMV (Sigma-Aldrich Co.; pCMV6(Origene Co.); pEE12 or pEE14 (Longsa Biopreparation Co., Ltd.), or pBK-CMV/pCMV-3Tag-7/pCMV-Tag2B (Stratagene Co., Ltd.).

Cells and cell culture

In one embodiment, the cell is a mammalian cell. In one embodiment, the cell is a cell other than a mammalian cell. In one embodiment, the cell is a mouse, rat, chinese hamster, syrian hamster, monkey, ape, dog, horse, ferret, or cat. In some embodiments, the cell is a mammalian cell, e.g., a human cell or a rodent cell, e.g., a hamster cell, a mouse cell, or a rat cell. In some embodiments, the cell is from a duck, parrot, fish, insect, plant, fungus, or yeast. In one embodiment, the cell is an Archaebacteria (Archaebacteria). In one embodiment, the cell is an actinomycete species, such as Mycobacterium tuberculosis (Mycobacterium tuberculosis).

In one embodiment, the cell is a Chinese Hamster Ovary (CHO) cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1SV GS knockout cell (Longsa Biopreparation). The CHO FUT8 knock-out cell is, for example, CHOK1SV (biosimica Longsa).

In another embodiment, the cell is HeIa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, per.c6, BHK (baby hamster kidney cells), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, CHOK1, CHOK1SV, Potelligent CHOK1SV, CHO GS knockout, CHOK1 SV-KO, CHOs, CHO 44, CHO DXB11 and CHOZN, or any cell derived therefrom. In one embodiment, the cell is a stem cell. In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

In one embodiment, the cell is any cell described herein comprising an exogenous nucleic acid encoding a recombinant polypeptide, e.g., expressing a recombinant polypeptide, e.g., a recombinant polypeptide selected from table 1 or table 2.

Mass production

The methods described herein can be used to analyze samples, e.g., samples produced by devices, facilities, and methods of manufacture and production. The devices, facilities and methods of manufacture and production described herein are suitable for culturing any desired cell line, including prokaryotic and/or eukaryotic cell lines. Furthermore, in some embodiments, the devices, facilities, and methods of manufacture and production are suitable for culturing suspension cells or anchorage-dependent (adherent) cells, and for configuring production operations for the production of pharmaceuticals and biopharmaceutical products (e.g., polypeptide products, nucleic acid products (e.g., DNA or RNA), or cells and/or viruses, such as those used for cell and/or viral therapy).

In some embodiments, the cell expresses or produces a product, such as a recombinant therapeutic or diagnostic product. Examples of products produced by cells include, but are not limited to: antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), antibody mimetics (polypeptide molecules that specifically bind to an antigen but are not structurally related to an antibody, e.g., DARPins, affibodies, mimobody proteins, or ignars), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), viral therapeutic agents (e.g., anti-cancer oncolytic viruses, viral vectors for gene therapy, and viral immunotherapy), cellular therapeutic agents (e.g., pluripotent stem cells, mesenchymal stem cells, and adult stem cells), vaccines or lipid-encapsulated particles (e.g., exosomes, virus-like particles), RNA (e.g., siRNA) or DNA (e.g., plasmid DNA), antibiotics, or amino acids. In some embodiments, the devices, facilities, and methods can be used to produce biosimilar drugs.

As described above, in some embodiments, the methods described herein can be used to analyze samples, e.g., samples produced by devices, facilities, and methods of manufacture and production. The devices, facilities and methods of manufacture and production allow the production of eukaryotic cells, such as mammalian cells, or lower eukaryotic cells (lower eukaryotic cells), such as yeast cells or filamentous fungal cells, or prokaryotic cells, such as gram-positive or gram-negative cells, and/or products of eukaryotic or prokaryotic cells synthesized on a large scale by eukaryotic cells, such as proteins, peptides, antibiotics, amino acids, nucleic acids (e.g., DNA or RNA). Unless otherwise indicated herein, the apparatus, facilities, and methods may include any desired volume or production yield, including but not limited to laboratory scale yields, semi-industrial scale yields, and full production scale yields.

In some embodiments, the devices, facilities and methods of manufacture and production allow for the production of cells and products of cells, particularly proteins, peptides (as discussed in detail above), antibodies or amino acids synthesized by cells (e.g., mammalian cells) in large scale.

A wide variety of flasks, bottles, reactors, and controllers allow for the production and expansion of cell culture systems. The system can be selected based at least in part on its correlation to one or more desired glycan properties. The cells may be grown, for example, in batch, fed-batch, perfusion, or continuous culture. Production parameters that may be selected include: for example, the addition or removal of media, including when (early, medium or late during the culture time) and the frequency of harvest of the media; increasing or decreasing the speed of agitation of the cell culture; increasing or decreasing the temperature of the cultured cells; adding or removing culture medium to adjust culture density; selecting a time to start or stop cell culture; and selecting a time for altering the cell culture parameter. The parameters may be chosen for any of batch, fed-batch, perfusion or continuous culture conditions.

In some embodiments, the cultured cells for large scale production are eukaryotic cells, e.g., animal cells, e.g., mammalian cells. The mammalian cell may be, for example, a human cell line, a mouse myeloma (NSO) cell line, a Chinese Hamster Ovary (CHO) cell line, or a hybridoma cell line. Preferably the mammalian cell is a CHO cell line.

In some embodiments, cells in culture for large-scale production are used to produce antibodies (e.g., monoclonal antibodies) and/or recombinant proteins discussed in detail above for therapeutic use. In some embodiments, the cell produces a peptide, amino acid, fatty acid, or other useful biochemical intermediate or metabolite.

In some embodiments, the cells used for large scale production are eukaryotic cells, biochemical markers, recombinant peptides or nucleotide sequences of interest, proteins, yeast, insect cells, stable or virus-infected avian or mammalian cells, e.g., CHO cells, monkey cells, lysates for medical, research or commercial purposes, and the like.

In some embodiments, the cells used for large scale production are prokaryotic cells, gram positive cell strains, such as bacillus and streptomyces. In some embodiments, the host cell is a Firmicutes (phylum Firmicutes), e.g., the host cell is a bacillus. Bacillus that can be used are, for example, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto, bacillus megaterium, and the like. In some embodiments, the host cell is bacillus subtilis, such as bacillus subtilis 3NA and bacillus subtilis 168. The bacillus may be obtained from: such as Bacillus genetic stock center, bioscience 556, West 12 street 484, Columbus OH 43210-1214.

In some embodiments, the prokaryotic cells used for large-scale production are gram-negative cells, such as Salmonella spp or e.coli (e.coli), such as the commercially available strains TG1, W3110, DH1, XL1-Blue and Origami. .

Suitable host cells are commercially available, for example from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Brenrek, Germany).

In one embodiment, the cell culture is performed as a batch culture, a fed-batch culture, a perfusion culture or a continuous culture. In one embodiment, the cell culture is a suspension culture. In one embodiment, the cell or cell culture is placed in vivo to express the recombinant polypeptide, e.g., in a model organism or a human subject.

In one embodiment, the medium is serum free. Serum-free and protein-free media are commercially available, for example, from lomsa bio-preparations.

Suitable media and culture methods for mammalian cell lines are well known in the art, as described in U.S. Pat. No.5,816,038. Examples of standard cell culture media used in laboratory flasks or low density cell culture and meeting the needs of specific cell types are, for example: los visv Park Memorial Institute (RPMI) 1640 medium (Morre, g., Journal of the American Medical Association), 199, p.519, 1967), L-15 medium (Leibovitz, a. et al, am. j. of hygene, 78,1p.173ff,1963), dubek's modified Eagle medium (Dulbecco's modified Eagle's medium, DMEM), Eagle's minimal essential medium (Eagle's minimal essential medium, MEM), hamm's F12 medium (Ham's F12 medium) (Ham, r. et al, proc. nature. acad. sc.53, p.1965) or isco's modified transferrin, isco's medium, isco's modified, p.928, DMEM. et al, lacking phospholipids. For example, Hamm's F10 or F12 medium was specifically designed for CHO cell culture. Other media particularly suitable for CHO cell culture are described in EP-481791. This medium is known to be supplemented with fetal bovine serum (FBS, also known as fetal calf serum FCS), which provides a natural source of large amounts of hormones and growth factors. Cell culture of mammalian cells is today a routine procedure described in detail in scientific textbooks and manuals, which include, for example, in detail: for example, R.IanFresnel, "Culture of Animal cells (Culture of Animal cells), handbook, 4 th edition, Wiley-Liss/N.Y., 2000.

Other suitable culturing methods are known to the skilled artisan and may depend on the recombinant polypeptide product and the host cell utilized. Determining or optimizing conditions suitable for expression and production of recombinant polypeptides expressed by the cell is within the skill of the ordinarily skilled artisan.

In one aspect, the cell or cell line used for large scale production comprises an exogenous nucleic acid encoding a product (e.g., a recombinant polypeptide). In one embodiment, the cell or cell line expresses a product, such as a therapeutic or diagnostic product. Methods for genetically modifying or engineering cells to express a desired polypeptide or protein are well known in the art and include, for example, transfection, transduction (e.g., viral transduction), or electroporation.

Physical methods for introducing nucleic acids (e.g., exogenous nucleic acids or vectors described herein) into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al, 2012, "molecular cloning: a LABORATORY Manual (Molecular CLONING: A Laboratory Manual), Vol.1-4, Cold spring harbor Press, N.Y..

Chemical methods for introducing nucleic acids (e.g., exogenous nucleic acids or vectors described herein) into host cells include colloidally dispersed systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles). Other methods of targeted delivery of nucleic acids of the prior art are available, such as polynucleotide delivery with targeted nanoparticles or other suitable delivery systems of submicron size.

In some embodiments, it is desirable to integrate the exogenous nucleic acid into the nucleic acid of the host cell, e.g., the genomic or chromosomal nucleic acid of the host cell. Methods for determining whether an exogenous nucleic acid has been integrated into the genome of a host cell may include the GS/MSX selection method. The GS/MSX selection method uses complementation of glutamine auxotrophs by recombinant GS genes to select for high level expression of proteins from cells. Briefly, the GS/MSX selection method comprises including a nucleic acid encoding glutamine synthetase on a vector comprising an exogenous nucleic acid encoding a recombinant polypeptide product. Administration of Methionine Sulfoximine (MSX) cells were selected in which the exogenous nucleic acid encoding the recombinant polypeptide and GS had been stably integrated into the genome. Since GS can be endogenously expressed by certain host cells, such as CHO cells, the concentration and duration of selection with MSX can be optimized to identify high-yield cells in which the exogenous nucleic acid encoding the recombinant polypeptide product is stably integrated into the host genome. GS selection and its system are further described in the following documents: fan et al, pharm. bioprocess, (2013); 487-502, which is incorporated herein by reference in its entirety.

Other methods for identifying and selecting cells in which the exogenous nucleic acid has been stably integrated into the host cell genome may include, but are not limited to: including a reporter gene on the exogenous nucleic acid and assessing the cells for the presence of the reporter gene, and performing PCR analysis and detection of the exogenous nucleic acid.

In one embodiment, a cell selected, identified, or produced using the methods described herein is capable of producing a higher yield of a protein product as compared to a cell selected using only the selection method for stable expression (e.g., integration of an exogenous nucleic acid encoding a recombinant polypeptide). In one embodiment, a cell selected, identified, or produced using the methods described herein produces 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold or more of a product, e.g., a recombinant polypeptide, as compared to a cell that has not been contacted with a protein degradation inhibitor or that has been selected for stable expression only (e.g., incorporating an exogenous nucleic acid encoding the recombinant polypeptide).

Cell lines and methods of recombinant polypeptide production

Methods for recovering and purifying products (e.g., recombinant polypeptides) are well established in the art. To recover the recombinant polypeptide product, physical, or chemical, or physicochemical methods are used. The physical or chemical or physicochemical method may be a filtration method, a centrifugation method, an ultracentrifugation method, an extraction method, a lyophilization method, a precipitation method, a crystallization method, a chromatography method, or a combination of two or more methods thereof. In one embodiment, the chromatographic method comprises one or more of size exclusion chromatography (or gel filtration), ion exchange chromatography, e.g., anion or cation exchange chromatography, affinity chromatography, hydrophobic interaction chromatography and/or multimodal chromatography.

The methods described herein are suitable for analysis of samples produced by production and preparation methods for culturing any desired cells, including prokaryotic and/or eukaryotic cells. The methods of manufacture and production may be carried out, for example, in a reactor (e.g., a bioreactor). Furthermore, in some embodiments, samples and products can be produced using apparatus, facilities, and methods suitable for the manufacture and production of cultured suspension cells or anchorage-dependent (adherent) cells, and suitable for configuring production operations for the production of molecular products (e.g., polypeptide products) or cells and/or viruses, such as those used for cell and/or virus therapy.

In some embodiments, the cell expresses or produces a product, such as a recombinant therapeutic or diagnostic product. As described in more detail below, examples of products produced by the cells include, but are not limited to: antibody molecules (e.g., monoclonal antibodies, bispecific antibodies), fusion proteins (e.g., Fc fusion proteins, chimeric cytokines), other recombinant proteins (e.g., glycosylated proteins, enzymes, hormones), or lipid-encapsulated particles (e.g., exosomes, virus-like particles). In some embodiments, the devices, facilities, and methods can be used to produce biosimilar drugs.

In some embodiments, the devices, facilities, and production methods allow for the production of eukaryotic cells (e.g., mammalian cells) and/or products of eukaryotic cells, e.g., proteins, peptides, antibodies, or amino acids synthesized by eukaryotic cells, in large scale. Unless otherwise indicated herein, the apparatus, facilities, and methods may include any desired volume or production yield, including but not limited to laboratory scale yields, semi-industrial scale yields, and full production scale yields.

Further, unless otherwise specified herein, the apparatus, facilities, and methods may include any suitable reactor, including but not limited to: stirred tank, air lift (airlife), fiber, microfiber, hollow fiber, ceramic matrix, fluidized bed, fixed bed, spouted bed, and/or stirred tank bioreactor. For example, in some aspects, an exemplary bioreactor unit may perform one or more or all of the following: feeding of nutrients and/or carbon sources, injection of suitable gases (e.g., oxygen), flow of fermentation or cell culture media, separation of gas and liquid phases, maintenance of temperature, maintenance of pH, agitation (e.g., stirring), and/or cleaning/disinfecting. Exemplary reactor units (e.g., fermentation units) can comprise 1,2,3,4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more bioreactors. In various embodiments, the bioreactor may be adapted for use with batch, fed-batch, perfusion, or continuous fermentation processes. Any suitable reactor diameter may be used. In some embodiments, the volume of the bioreactor may be from about 100mL to about 50,000L. Non-limiting examples include: a volume of 100mL, 250mL, 500mL, 750mL, 1 liter, 2 liters, 3 liters, 4 liters, 5 liters, 6 liters, 7 liters, 8 liters, 9 liters, 10 liters, 15 liters, 20 liters, 25 liters, 30 liters, 40 liters, 50 liters, 60 liters, 70 liters, 80 liters, 90 liters, 100 liters, 150 liters, 200 liters, 250 liters, 300 liters, 350 liters, 400 liters, 450 liters, 500 liters, 550 liters, 600 liters, 650 liters, 700 liters, 750 liters, 800 liters, 850 liters, 900 liters, 950 liters, 1000 liters, 1500 liters, 2000 liters, 2500 liters, 3000 liters, 3500 liters, 4000 liters, 4500 liters, 5000 liters, 6000 liters, 7000 liters, 8000 liters, 9000 liters, 10,000 liters, 15,000 liters, 20,000 liters, and/or 50,000 liters. Additionally, suitable reactors may be multi-purpose, single-purpose, disposable, or non-disposable, and may be formed of any suitable material, including metal alloys, such as stainless steel (e.g., 316L or any other suitable stainless steel), and Inconel (Inconel), plastic, and/or glass. In some embodiments, suitable reactors may be circular, e.g., cylindrical. In some embodiments, a suitable reactor may be square, e.g., rectangular. In one case, a square reactor can provide advantages over a round reactor, such as ease of use (e.g., loading and setup by a technician), greater mixing and homogeneity of the reactor contents, and a lower footprint.

In some embodiments, unless otherwise specified herein, the apparatuses, facilities, and production methods described herein may further include any suitable unit operations and/or equipment not otherwise mentioned, such as operations and/or equipment for the separation, purification, and isolation of the product. Any suitable facility and environment may be used, such as a conventional stick-build facility, a modular facility, or any other suitable configuration, facility, and/or layout. For example, in some embodiments, a modular cleanroom may be used. In addition, unless otherwise specified, the devices, systems, and methods described herein may be housed and/or performed in a single location or facility, or may alternatively be housed and/or performed in separate or multiple locations and/or facilities.

By way of non-limiting example, and not limitation, U.S. publication nos. 2013/0280797; U.S. publication No. 2012/0077429; U.S. publication No. 2011/0280797; U.S. publication No. 2009/0305626; and U.S. patent No. 8,298,054; U.S. patent nos. 7,629,167; and U.S. patent No.5,656,491, which is incorporated herein by reference in its entirety, describe exemplary facilities, devices, and/or systems that may be suitable.

In some embodiments, the cell is a eukaryotic cell, such as a mammalian cell. The mammalian cell may be, for example, a human or rodent or bovine cell line or cell line. Examples of such cells, cell lines or cell strains are: for example, a mouse myeloma (NS0) cell line, a Chinese Hamster Ovary (CHO) cell line, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2/0, YB2/0, Y0, C127, L cells, COS, such as COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, Hela, EB1, EB2, EB3, an oncolytic or hybridoma cell line. Preferably the mammalian cell is a CHO cell line. In one embodiment, the cell is a CHO cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1SV GS knockout cell. CHO FUT8 knock-out cells are, for exampleCHOK1SV (biosimica lonza). The eukaryotic cell may also be an avian cell, cell line or cell line, e.g.Cells, EB14, EB24, EB26, EB66 or EBv 13.

In one embodiment, the eukaryotic cell is a stem cell. The stem cells may be: for example, pluripotent stem cells, including Embryonic Stem Cells (ESCs), adult stem cells, induced pluripotent stem cells (ipscs), tissue-specific stem cells (e.g., hematopoietic stem cells) and Mesenchymal Stem Cells (MSCs).

In some embodiments, the cultured cells are eukaryotic cells, such as mammalian cells. The mammalian cell may be, for example, a human cell line, a mouse myeloma (NSO) cell line, a Chinese Hamster Ovary (CHO) cell line, or a hybridoma cell line. Preferably the mammalian cell is a CHO cell line. In one embodiment, the cell is a CHO cell. In one embodiment, the cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1SV GS knockout cell. CHO FUT8 knock-out cells are, for exampleSV (biospecification of Longsa).

In some embodiments, the cell is a yeast cell (e.g., saccharomyces cerevisiae (s. cerevisae), trichoderma reesei (t. reesei)), an insect cell (e.g., Sf9), an algal cell (e.g., cyanobacteria), or a plant cell (e.g., tobacco (tobaco), alfalfa (alfa), Physcomitrella patens (physiocornitella patents)). In one embodiment, the cell is a rodent cell. In another embodiment, the cell is HeLa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER. C6, BHK (baby hamster kidney cells), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, CHO-K1.

In some embodiments, the cell is a stem cell. In one embodiment, the cell is a differentiated form of any of the cells described herein. In one embodiment, the cell is a cell derived from any primary cell in culture.

In some embodiments, the thin filmThe cell is a hepatocyte, such as a human hepatocyte, an animal hepatocyte or an nonparenchymal cell. For example, the cell can be a human hepatocyte that is eligible for platelet metabolism, a human hepatocyte that is eligible for platelet induction, a platelet quick Transporter Certified^TMHuman hepatocytes, suspension-qualified human hepatocytes (including 10 donors and 20 donor pooled hepatocytes), human hepatokupffer cells (human hepatotic kupffer cells), human hepatic stellate cells, dog hepatocytes (including single and pooled Beagle canine hepatocytes (Beagle hepatocytes)), mouse hepatocytes (including CD-1 and C57BI/6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus monkey hepatocytes), cat hepatocytes (including brachiocephalus brevis cat hepatocytes) and rabbit hepatocytes (including new zealand white rabbit hepatocytes). Exemplary hepatocytes are available from Triangle research labs, LLC, Davis Drive research Triangle science park 6, north carona, usa 27709.

In one embodiment, the eukaryotic cell is a lower eukaryotic cell, such as a yeast cell (e.g., Pichia (Pichia) such as Pichia pastoris (Pichia pastoris), Pichia methanolica (Pichia pastoris), Pichia kluyveri (Pichia kluyveri) and Pichia angusta), a Kanga (Komagataella genus) such as Pasteur congtahariella (Komagataella torula), Saccharomyces (Saccharomyces) such as Candida quasii (Candida), Saccharomyces cerevisiae (Saccharomyces cerevisiae), e.g., Saccharomyces cerevisiae (Candida), Candida utilis (Candida), Saccharomyces cerevisiae (Saccharomyces cerevisiae) (e.g., Candida), Saccharomyces cerevisiae (Candida utilis), candida cacao (Candida cacao), Candida boidinii (Candida boidinii)), Geotrichum (Geotrichum genus) (e.g., Geotrichum fermentum), Hansenula polymorpha (Hansenula polymorpha), Yarrowia lipolytica (Yarrowia lipolytica), or Schizosaccharomyces pombe (Schizosaccharomyces pombe). Preferred are the species Pichia pastoris. Examples of pichia strains are X33, GS115, KM71, KM 71H; and CBS 7435.

In one embodiment, the eukaryotic cell is a fungal cell (e.g., Aspergillus (Aspergillus) (e.g., Aspergillus niger (a. niger), Aspergillus fumigatus (a. fumigus), Aspergillus oryzae (a. oryzae), Aspergillus rubrus (a. nidulans)), Acremonium (e.g., Acremonium thermophilum), Chaetomium (e.g., Chaetomium thermophilum), Chrysosporium (e.g., Chrysosporium thermophilum), Cordyceps (Cordyceps) (e.g., Cordyceps militaris (c. milaritis)), corynebacterium (corynespora), Myceliophthora (e.g., Myceliophthora thermophila), Fusarium (e) (e.g., Fusarium oxysporum (f. chrysosporum)), plasmopara (gloeophyta) (e.g., Myceliophthora (Myceliophthora)), Myceliophthora (e.g., Myceliophthora cinerea). Chaetomium (necatria) (e.g. red coccobacillus (n. hematomatococcus)), Neurospora (Neurospora) (e.g. Neurospora crassa (n. crassa), Penicillium (Penicillium), Sporotrichum (sportomium) (e.g. thermothermophile), Neurospora (Thielavia) (e.g. Thielavia terrestris (t. terrestris), Sporotrichum (t. heterotheca), Trichoderma (Trichoderma) (e.g. Trichoderma reesei (t. reesei)) or Verticillium (verticillicium) (e.g. Verticillium dahlia)).

In one embodiment, the eukaryotic cell is an insect cell (e.g., Sf9, Mimic)^TMSf9，Sf21，HighFive^TM(BT1-TN-5B1-4) or BT1-Ea88 cells), algal cells (e.g.of the genus Geotrichum, class Diatomae (Bacillariophyceae), genus Dunaliella, genus Chlorella, genus Chlamydomonas, genus Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina or Zornia (Ochromonas)), or plant cells (e.g.from monocotyledonous plants (e.g.maize, rice, wheat or green bristlegrass) or from dicotyledonous plants (e.g.cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella or Dictyocaulus (E.g.Arabidopsis thaliana).

In one embodiment, the cell is a bacterium or a prokaryotic cell.

In some embodiments, the prokaryotic cell is a gram-positive cell, such as a bacillus, streptomyces, streptococcus, staphylococcus, or lactobacillus. Bacillus that can be used are, for example, bacillus subtilis, bacillus amyloliquefaciens, bacillus licheniformis, bacillus natto, or bacillus megaterium. In some embodiments, the cell is bacillus subtilis, e.g., bacillus subtilis 3NA and bacillus subtilis 168. Bacillus may be obtained, for example, from the Bacillus genetic stock center, bioscience 556, West 12 street 484, Columbus OH 43210-.

In one embodiment, the prokaryotic cell is a gram-negative cell, such as Salmonella (Salmonella pp.) or Escherichia coli (Escherichia coli), such as TG1, TG2, W3110, DH1, DHB4, DH5a, HMS174, HMS174(DE3), NM533, C600, HB101, JM109, MC4100, XL1-Blue and Origami, as well as those derived from Escherichia coli B strains, such as BL-21 or BL21(DE3), all of which are commercially available.

Suitable host cells are commercially available, for example from culture collections such as DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Brenrek, Germany) or the American Type Culture Collection (ATCC).

In some embodiments, the cultured cells are used to produce proteins (e.g., antibodies, such as monoclonal antibodies) and/or recombinant proteins for therapeutic use. In some embodiments, the cultured cells produce peptides, amino acids, fatty acids, or other useful biochemical intermediates or metabolites. For example, in some embodiments, molecules having a molecular weight of about 4000 daltons (dalton) to greater than about 140,000 daltons may be produced. In some embodiments, these molecules may have a range of complexities, and may include post-translational modifications, including glycosylation.

Description of several embodiments

1. A method of separating a compound of formula I, such as tropolone, from another component in a sample, the method comprising:

in cases where a compound of formula I (e.g., tropolone) is associated with (e.g., bound to or retained by) a moiety to a greater extent than the component, the sample is contacted with a partially or fully fluorinated alkyl or aryl (e.g., fluorophenyl, such as pentafluorophenylpropyl) moiety,

thereby, a compound of formula I, for example tropolone, is separated from the component, wherein formula I is:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

2. the method of paragraph 1 wherein the moiety comprises pentafluorophenylpropyl.

3. The method of paragraph 1 or 2, wherein the pentafluorophenylpropyl group is associated with, e.g., bound, e.g., covalently bound, to the matrix.

4. The method of paragraph 3 wherein the matrix comprises an insoluble matrix, such as a chromatographic matrix, e.g., a silica gel.

5. The method of any of paragraphs 1-4, further comprising: in cases where the compound preferentially elutes, the moiety is contacted with one or more mobile phases (e.g., one or two mobile phases).

6. The method of any of paragraphs 1-5, wherein the method comprises: the sample is subjected to Liquid Chromatography (LC) separation.

7.A method of assessing the presence, e.g., level, of a compound of formula I, e.g., tropolone, in a sample comprising a product, the method comprising:

a) i) providing an aliquot of the sample, e.g. a depleted phase (e.g. mobile phase) of a compound of formula I (e.g. tropolone), wherein the compound of formula I (e.g. tropolone) has been separated from another component of the sample, or

ii) subjecting the sample to conditions wherein a compound of formula I, e.g., tropolone, separates from another component of the sample, e.g., to form a compound of formula I (e.g., tropolone) enriched phase or aliquot and a compound of formula I (e.g., tropolone) depleted phase or aliquot; and

b) assessing the presence (e.g., level) of a compound of formula I (e.g., tropolone), e.g., determining a value for the level of a compound of formula I (e.g., tropolone) in a sample:

i) using tandem Mass Spectrometry (MS)²) Or is or

ii) using Ultraviolet (UV) absorption (e.g., UV absorption of about 242nm or about 238 nm);

whereby the sample is analyzed in a manner such that,

wherein formula I is:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

8. the method of paragraph 7 wherein a) comprises: an aliquot of the sample, e.g., a depleted phase, e.g., a mobile phase, of a compound of formula I, e.g., tropolone, is provided, wherein the compound of formula I, e.g., tropolone, has been separated from another component of the sample.

9. The method of paragraph 7 wherein a) comprises: the sample is subjected to conditions in which a compound of formula I, e.g., tropolone, separates from another component of the sample, e.g., to form an enriched phase or aliquot of the compound of formula I, e.g., tropolone, and a depleted phase or aliquot of the compound of formula I, e.g., tropolone.

10. The method of any of paragraphs 7-9, wherein a) comprises: the sample is subjected to Liquid Chromatography (LC) separation.

11. The method of any of paragraphs 7-10, wherein a) comprises: the sample is contacted with a partially or fully fluorinated alkyl or aryl moiety, for example a fluorophenyl group such as pentafluorophenylpropyl group, under conditions in which a compound of formula I, for example tropolone, is associated with (e.g., bound to or retained by) the moiety to a greater extent than the component.

12. The method of paragraph 11 wherein the moiety comprises pentafluorophenylpropyl.

13. The method of any of paragraphs 7-12, wherein b) comprises: assessing the level or presence of a compound of formula I, e.g. tropolone, e.g. using tandem Mass Spectrometry (MS)²) A value for the level of a compound of formula I, e.g., tropolone, in a sample is determined.

14. The method of any of paragraphs 7-12, wherein b) comprises: the level or presence of a compound of formula I, e.g., tropolone, is assessed, e.g., using Ultraviolet (UV) absorption, e.g., ultraviolet absorption at about 242nm or about 238nm, to determine a value for the level of a compound of formula I, e.g., tropolone, in a sample.

15. A method as claimed in any of paragraphs 7, 11 or 12, comprising a) i) and b) i).

16. The method of any of paragraphs 7, 11 or 12, comprising a) i) and b) ii).

17. The method of any of paragraphs 7, 11 or 12, comprising a) ii) and b) i).

18. The method of any of paragraphs 7, 11 or 12, comprising a) ii) and b) ii).

19. The method of any of paragraphs 7 to 18, wherein the method has a linear range of about 0.1 to 10000 μ g/ml, 0.2 to 8000 μ g/ml, 0.3 to 7000 μ g/ml, 0.4 to 6000 μ g/ml, 0.5 to 5000 μ g/ml, 0.5 to 4000 μ g/ml, 0.5 to 3000 μ g/ml, 0.5 to 2000 μ g/ml, or 0.5 to 1000 μ g/ml, for example 0.5 to 1000 μ g/ml, with respect to the value for determining the level of a compound of formula I, e.g. tropolone, present in a sample.

20. The method of any of paragraphs 7 to 19, wherein the lower limit of the linear range for the method with respect to the value for determining the level of a compound of formula I, e.g. tropolone, present in the sample is about 0.01 μ g/ml, 0.05 μ g/ml, 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.35 μ g/ml, 0.4 μ g/ml, 0.45 μ g/ml, 0.5 μ g/ml, 0.6 μ g/ml, 0.7 μ g/ml, 0.8 μ g/ml, 0.9 μ g/ml, or 1 μ g/ml, e.g. 0.5 μ g/ml.

21. The method of any of paragraphs 7 to 20, wherein the upper limit of the linear range for the method with respect to the value for determining the level of a compound of formula I, e.g. tropolone, present in a sample is about 500 μ g/ml, 600 μ g/ml, 700 μ g/ml, 800 μ g/ml, 900 μ g/ml, 1000 μ g/ml, 1200 μ g/ml, 1400 μ g/ml, 1600 μ g/ml, 1800 μ g/ml, 2000 μ g/ml, 3000 μ g/ml, 4000 μ g/ml, 5000 μ g/ml, 6000 μ g/ml, 7000 μ g/ml, 8000 μ g/ml, 9000 μ g/ml, or 10,000 μ g/ml, e.g. 1000 μ g/ml.

22. The method of any of paragraphs 7-21, wherein the accuracy of the method (e.g., as expressed by the standard deviation between replicate samples) can be less than or equal to about 0%, 40%, 30%, 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, e.g., 17%, 16.5%, or 16%, with respect to determining the value of the level of a compound of formula I, e.g., tropolone, present in a sample.

23. The method of any of paragraphs 7-22, wherein the accuracy of the method (e.g., as expressed by the average single point addition recovery in three different samples) can be greater than or equal to about 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, e.g., 91%, with respect to the value determining the level of a compound of formula I, e.g., tropolone, present in the sample.

24. The method of any of paragraphs 7-23, wherein the lower limit of detection for the method is about 1 μ g/ml, 1.5 μ g/ml, 2 μ g/ml, 2.5 μ g/ml, 3 μ g/ml, 3.5 μ g/ml, 4 μ g/ml, 4.5 μ g/ml, 5 μ g/ml, 5.5 μ g/ml, 6 μ g/ml, 6.5 μ g/ml, 7 μ g/ml, 7.5 μ g/ml, 8 μ g/ml, 8.5 μ g/ml, 9 μ g/ml, 9.5 μ g/ml, or 10 μ g/ml, e.g. 5 μ g/ml, with respect to the value determining the level of a compound of formula I, e.g. tropolone, present in the sample.

25. The method of any of paragraphs 6 or 10 wherein the LC is reverse phase chromatography.

26. The method of any of paragraphs 6 or 10 wherein the LC is not reverse phase chromatography.

27. A method as claimed in any of paragraphs 6 or 10 wherein LC comprises the use of a stationary phase comprising a partially or fully fluorinated alkyl or aryl group, for example a fluorophenyl group such as pentafluorophenylpropyl group.

28. The method of paragraph 27 wherein the LC comprises a stationary phase using a fluorophenyl-containing group.

29. The method of paragraph 27 wherein the LC comprises using a stationary phase comprising pentafluorophenylpropyl.

30. The method of any of paragraphs 6, 10 or 25-29, wherein LC comprises using a first mobile phase and a second mobile phase.

31. The method of paragraph 30, wherein the first mobile phase comprises formic acid in water, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid in water.

32. The method of paragraph 31 wherein the first mobile phase comprises about 0.1% formic acid in water.

33. The method of paragraph 30, wherein the second mobile phase comprises formic acid in acetonitrile, e.g., about 0.01%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% formic acid in acetonitrile.

34. The method of paragraph 33 wherein the second mobile phase comprises about 0.1% formic acid in acetonitrile.

35. The method of any of paragraphs 33 or 34, wherein the second mobile phase comprises at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% acetonitrile, e.g., about 100% acetonitrile.

36. The method of any of paragraphs 6, 10 or 25-35, wherein the LC comprises: using a stationary phase comprising pentafluorophenylpropyl and using a first mobile phase and a second mobile phase, wherein the first mobile phase comprises about 0.1% formic acid in water and the second mobile phase comprises about 0.1% formic acid in acetonitrile.

37. The method of any of paragraphs 6, 10 or 25-36, wherein the LC comprises: a Discovery HSF5-3 column was used.

38. The method of any of paragraphs 7-13, 15, 17 and 19-37, wherein MS is used²Including Selective Reaction Monitoring (SRM).

39. The method of any of paragraphs 7-13, 15, 17 and 19-37, wherein MS is used²Including Multiple Reaction Monitoring (MRM), such as Parallel Reaction Monitoring (PRM).

40. A method as described in any of paragraphs 38 or 39, wherein an SRM or MRM (e.g., a PRM) is used to monitor one or more transitions selected from transitions i, ii, iii, iv, v, and vi from Table 1.

41. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition i.

42. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition ii.

43. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition iii.

44. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition iv.

45. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition v.

46. A method as paragraph 40 recites, wherein an SRM or MRM (e.g., PRM) is used to monitor the transition vi.

47. A reaction mixture comprising a partially or fully fluorinated alkyl or aryl moiety, such as a fluorophenyl group, e.g. pentafluorophenylpropyl group, wherein formula I is given by the formula:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl or C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

48. a method of manufacturing a product, such as a recombinant polypeptide, comprising: providing a sample comprising the product and optionally a compound of formula I, such as tropolone, wherein:

the sample is analyzed by the method of any one of paragraphs 7-43, 45 or 46, or

Separating a compound of formula I, such as tropolone, from another component of the sample by the method of any one of paragraphs 1-6,

wherein formula I is given by:

and wherein:

x is O or S;

R¹is hydrogen, C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, OR³、C(O)R⁵、C(O)OR³、N(R^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵；

Each R²Independently is C₁-C₆Alkyl radical, C₁-C₆Heteroalkyl, N (R)^4a)(R^4b)、C(O)N(R^4a)(R^4b) Or N (R)^4a)C(O)R⁵(ii) a Or

Two R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring; or R¹And R²Linked to form a linkage optionally substituted with one or more R⁶A substituted heterocyclyl ring;

R³is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R^4aand R^4bIndependently of each other is hydrogen, C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

R⁵is C₁-C₆Alkyl or C₁-C₆A heteroalkyl group;

each R⁶Independently is C₁-C₆Alkyl or C₁-C₆Heteroalkyl, halogen, oxo, or cyano; and

n is 0, 1,2, 4 or 5;

49. the method of paragraph 48 wherein the method of manufacturing includes: expressed and secreted by a plurality of cells (e.g., a plurality of CHO cells, e.g., a plurality of GS-CHO cells).

50. The method or reaction mixture of any of paragraphs 1-49, wherein the sample comprises a culture supernatant.

51. The method or reaction mixture of any of paragraphs 1-49, wherein the sample comprises a cell lysate.

52. The method or reaction mixture of any of paragraphs 1-51, wherein the sample comprises a culture supernatant and a cell lysate.

53. The method or reaction mixture of any of paragraphs 1-52, wherein the sample is produced by a method of manufacture of a product, such as a recombinant polypeptide.

54. The method or reaction mixture of any of paragraphs 1-53, wherein the sample comprises a final product, e.g., a final product formulated for delivery (e.g., administration to a patient).

55. The method or reaction mixture of any of paragraphs 1-54, wherein the product or recombinant polypeptide is a homo-or heteropolymeric polypeptide, such as a hormone, growth factor, receptor, antibody, cytokine, receptor ligand, transcription factor or enzyme, preferably an antibody or antibody fragment, such as a human or humanized antibody or fragment thereof, such as a humanized antibody or fragment thereof derived from a mouse, rat, rabbit, goat, sheep or cow antibody, typically a humanized antibody or fragment thereof derived from a rabbit source.

56. The method or reaction mixture of any of paragraphs 1-55, wherein the product or recombinant polypeptide is a therapeutic polypeptide.

57. The method or reaction mixture of any of paragraphs 1-56, wherein the product or recombinant polypeptide is disclosed in table 1, table 2, table 3, or table 4.

58. The method or reaction mixture of any of paragraphs 1-57, wherein the product or recombinant polypeptide is an antibody.

59. The method or reaction mixture of paragraph 58 wherein the antibody is a monoclonal antibody.

60. The method or reaction mixture of any of paragraphs 58 or 59, wherein the monoclonal antibody is a therapeutic antibody.

61. The method or reaction mixture of any of paragraphs 49-60, wherein the cells are mammalian cells.

62. The method or reaction mixture of paragraph 61, wherein the cell is a mouse, rat, Chinese hamster, Syrian hamster, monkey, ape, dog, horse, ferret, or cat.

63. The method or reaction mixture of paragraph 61, wherein the cells are Chinese Hamster Ovary (CHO) cells.

64. The method or reaction mixture of paragraph 63, wherein the CHO cell is a CHO-K1 cell, a CHO-K1SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell.

65. The method or reaction mixture of paragraph 61, wherein the cell is HeIa, HEK293, HT1080, H9, HepG2, MCF7, Jurkat, NIH3T3, PC12, PER. C6, BHK (baby hamster kidney cells), VERO, SP2/0, NS0, YB2/0, Y0, EB66, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, or any cell derived therefrom.