Microparticle Formulations for Delivery to the Upper and Central Respiratory Tract and Methods of Manufacture

1 . A method of making a composition comprising microparticles comprising DAS 181, the method comprising:

a) providing a feedstock composition comprising DAS181, a counterion and an organic solvent; and

b) cooling the feedstock composition to below 25° C., whereby a composition comprising microparticle comprising DAS 181 is formed.

2 . The method of claim 1 wherein the DAS181 comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

3 . The method of claim 1 wherein the DAS 181 comprises a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

4 . The method of claim 1 wherein the DAS181 comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:1.

5 . The method of claim 1 wherein the DAS 181 comprises a polypeptide consisting of the amino acid sequence of SEQ ID NO:2.

6 . The method of claim 1 wherein the DAS 181 comprises a polypeptide consisting of amino acid sequence of SEQ ID NO:1 and a polypeptides consisting of the amino acid sequence of SEQ ID NO:2.

7 . The method of claim 1 wherein the feedstock composition comprises DAS 181 consisting of the amino acid sequence SEQ ID NO:1.

8 . The method of claim 1 wherein the feedstock composition comprises DAS 181 consisting of the amino acid sequence SEQ ID NO:2.

9 . The method of claim 1 wherein the feedstock composition comprises DAS 181 comprising or consisting of the amino acid sequence SEQ ID NO:1 and DAS 181 comprising or consisting of the amino acid sequence SEQ ID NO:2.

10 . The method of claim 1 where the feedstock composition comprises polypeptides consisting of the amino acid sequence SEQ ID NO:1 and polypeptides consisting of the amino acid sequence SEQ ID NO:2.

11 . The method of claim 1 wherein the step of cooling the feedstock composition includes cooling to a first temperature below 25° C. at a constant first rate and then cooling to a second temperate below the first temperature at a constant second rate that can be the same as or different from the first rate.

12 . The method of claim 1 wherein the step of cooling includes holding the feedstock composition at a first holding temperature below 25° C. for a first period of time.

13 . The method of claim 12 wherein the step of cooling includes holding the feedstock composition at a second holding temperature below the first holding temperature for a second period of time that can be the same as or different from the first period of time.

14 . The method of claim 1 wherein the step of providing a feedstock composition comprising DAS181, a counterion and an organic solvent comprises providing a solution comprising DAS 181 and a counterion in an aqueous solvent and adding an organic solvent to the solution comprising DAS 181 and a counterion in an aqueous solvent.

15 . The method of any of the forgoing claims further comprising isolating the microparticles from the composition comprising microparticles, whereby a dry powder formulation of microparticles is formed.

16 . The method of claim 15 , wherein the step of isolating the microparticles comprises heating the composition comprising microparticles to a temperature and for a time that reduces the amount of organic solvent the composition comprising microparticles.

17 . The method claim 1 , wherein the feedstock composition comprises two or more counterions.

18 . The method of claim 1 , wherein the counterion comprises citric acid.

19 . The method of claim 1 , wherein the counterion comprises magnesium sulfate.

20 . The method of claim 1 wherein the feedstock composition is 70-80% (wt/wt) DAS 181.

21 . The method of claim 1 wherein the feedstock composition is 8-12% (wt/wt) histidine and histidine HCl.

22 . The method of claim 1 wherein the feedstock composition is 8-10% (wt/wt) trehalose.

23 . The method of claim 1 wherein the feedstock composition is 5-9% (wt/wt) magnesium sulfate.

24 . The method of claim 1 wherein the feedstock composition is 1-4% (wt/wt) citrate.

25 . The method of claim 1 wherein the feedstock composition is 20-30% (wt/wt) isopropanol.

26 . The method of any of the forgoing claims, wherein the wt/wt amount of DAS181 in the resulting microparticles when substantially dry is about 60% to about 75% (wt/wt).

27 . The method of claim 26 wherein the resulting microparticles are 60% to 75% (wt/wt) a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:2.

28 . The method of claim 26 wherein the resulting microparticles are 60% to 75% (wt/wt) a polypeptide consisting of comprising the amino acid sequence SEQ ID NO:1 or SEQ ID NO:2.

29 . The method of claim 1 wherein the resulting microparticles comprise a polypeptide consisting of the amino acid sequence SEQ ID NO:1 and a polypeptide consisting of comprising the amino acid sequence SEQ ID NO:2 and wherein the polypeptide consisting of SEQ ID NO:1 and the polypeptide consisting of SEQ ID NO:2 together are 60% to 75% (wt/wt) of the microparticles.

30 . The method of any of the forgoing claims, wherein the predetermined temperature below about 25° C. is between about 4° C. to about −65° C.

31 . The method any of the forgoing claims, wherein the predetermined temperature below about 25° C. is between about −20° C. to about −65° C.

32 . The method of any of the forgoing claims, wherein the predetermined temperature below about 25° C. is between about −40° C. to about −65° C.

33 . The method of any of the forgoing claims, wherein the cooling rate is about −0.1° C./min to about −1° C./min.

34 . The method of any of the forgoing claims, wherein cooling rate is about −0.1° C./min to about −0.8° C./min.

35 . The method of any of the forgoing claims, wherein cooling rate is about −0.1° C./min to about −0.7° C./min, about −0.3° C./min to about −0.5° C./min, about −0.4° C./min.

36 . The method of any of the forgoing claims, wherein the microparticles have a mass median aerodynamic diameter of between about 9 microns to about 10 microns.

37 . The method of any of the forgoing claims, further comprising adding a scavenging agent to the feedstock composition.

38 . The method of claim 37 , wherein the scavenging agent is selected from among an amine, an antioxidant, a sugar, a chelator or a combination of two or more thereof.

39 . The method of claim 37 , wherein the scavenging agent is histidine.

40 . The method of claim 37 , wherein the scavenging agent further comprises a sugar.

41 . The method of claim 40 wherein the sugar is trehalose.

42 . The method of any of any of the forgoing claims, further comprising packaging the microparticle formulation in a container comprising between about 3 ppm to about 25 ppm aldehyde.

43 . The method of claim 42 wherein the aldehyde is formaldehyde.

44 . A composition comprising microparticles wherein the microparticles comprise about 60-70% wt/wt DAS181 (SEQ ID NO:17), 1-2% wt/wt histidine, 8-10% wt/wt histidine hydrochloride, 7-10% wt/wt trehalose, 4-8% wt/wt magnesium sulfate, 1-3% citrate, and 6-10% water.

45 . The composition of claim 44 wherein the microparticles are 60% to 75% (wt/wt) a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:2.

46 . The composition of claim 44 wherein the microparticles are 60% to 75% (wt/wt) a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 or the amino acid sequence of SEQ ID NO:2.

47 . The composition of claim 44 wherein the microparticles comprise a polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 and wherein the polypeptide consisting of the amino acid sequence of SEQ ID NO:1 and the polypeptide consisting of the amino acid sequence of SEQ ID NO:2 together are 60% to 75% (wt/wt) of the microparticles.

48 . A composition comprising the microparticle formulation of claim 44 wherein the microparticles have an MMAD of 8-15, 8-12 or 9-10 microns.

49 . The composition of claim 48 wherein the microparticles have a GSD of 1.3-1.9, 1.4-1.8 or 1.5-1.7.

50 . The composition of claim 48 wherein the weight percent of microparticles having a aerodynamic diameter below 5 microns is less than 35%, 30%, 25%, 20%, 15%, 10%, 8%, 7%, 6%, or 5%, on a volume basis.

51 . A capsule containing a composition comprising the microparticle formulation of claim 44 .

52 . A composition comprising microparticles, wherein the microparticles, when anhydrous, comprises about 65-75% wt/wt DAS181 (SEQ ID NO:1, SEQ ID NO:2 or a mixture thereof), 1-3% wt/wt histidine, 8-11% wt/wt histidine hydrochloride, 8-11% wt/wt trehalose, 5-8% wt/wt magnesium sulfate, and 1-3% citrate.

53 . The composition of claim 52 wherein the microparticles have an MMAD 8-15, 8-12 or 9-10 microns.

54 . The composition of claim 52 wherein the microparticles are 69% to 74% (wt/wt) a polypeptide comprising SEQ ID NO:1 or SEQ ID NO:2.

55 . The composition of claim 52 wherein the microparticles are 69% to 74% (wt/wt) a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:2.

56 . The composition of claim 52 wherein the microparticles comprise a polypeptide consisting of SEQ ID NO:1 and a polypeptide consisting of SEQ ID NO:2 and wherein the polypeptide consisting of SEQ ID NO:1 and the polypeptide consisting of SEQ ID NO:2 together are 66% to 74% (wt/wt) of the microparticles.

57 . The composition of claim 53 wherein the microparticles have a GSD of 1.3-1.9, 1.4-1.8 or 1.5-1.7.

58 . The composition of claim 57 wherein the weight percent of microparticles having a aerodynamic diameter below 5 microns is less than 35%, 30%, 25%, 20%, 15%, 10%, 8%, 7%, 6%, or 5%.

59 . A composition comprising microparticles wherein the microparticles comprise about 60-80% wt/wt DAS181, 4.5-6.5% wt/wt histidine, 3.5-5.5% wt/wt histidine hydrochloride, 7-11% wt/wt trehalose, 1-4% citrate, and 6-10% water.

60 . A composition of claim 59 wherein the microparticles have an MMAD of 8-15, 8-12 or 9-10 microns.

61 . The composition of claim 60 wherein the microparticles have a GSD of 1.3-1.9, 1.4-1.8 or 1.5-1.7.

62 . The composition of claim 59 wherein the microparticles are 60% to 80% (wt/wt) a polypeptide comprising SEQ ID NO:1 or SEQ ID NO:2.

63 . The composition of claim 59 wherein the microparticles are 60% to 80% (wt/wt) a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:2.

64 . The composition of claim 59 wherein the microparticles comprise a polypeptide consisting of SEQ ID NO:1 and a polypeptide consisting of SEQ ID NO:2 and wherein the polypeptide consisting of SEQ ID NO:1 and the polypeptide consisting of SEQ ID NO:2 together are 80% to 90% (wt/wt) of the microparticles.

65 . The composition of claim 64 wherein the weight percent of microparticles having an aerodynamic diameter below 5 microns is less than 35%, 30%, 25%, 20%, 15%, 10%, 8%, 7%, 6%, or 5%, on a volume basis.

66 . A composition comprising microparticles that, when anhydrous, comprise about 70-80% wt/wt DAS181, 5-7% wt/wt histidine, 4-6 wt/wt histidine hydrochloride, 9-11% wt/wt trehalose, and 1.5-3.5% citrate.

67 . The composition of claim 66 , wherein the microparticles are 70 to 80% (wt/wt) a polypeptide comprising SEQ ID NO:1 or SEQ ID NO:2.

68 . The composition of claim 66 , wherein the microparticles are 70% to 80% (wt/wt) a polypeptide consisting of SEQ ID NO:1 or SEQ ID NO:2.

69 . The composition of claim 66 , wherein the microparticles comprise a polypeptide consisting of SEQ ID NO:1 and a polypeptide consisting of SEQ ID NO:2 and wherein the polypeptide consisting of SEQ ID NO:1 and the polypeptide consisting of SEQ ID NO:2 together are 94% to 98% (wt/wt) of the microparticles.

70 . The composition of claim 66 wherein the microparticles have an MMAD of 8-15, 8-12 or 9-10 microns.

71 . The composition of claim 70 wherein the microparticles have GSD of 1.3-1.9, 1.4-1.8 or 1.5-1.7.

72 . The composition of claim 70 wherein the weight percent of microparticles having an aerodynamic diameter below 5 microns is less than 35%, 30%, 25%, 20%, 15%, 10%, 8%, 7%, 6%, or 5%, on a volume basis.

73 . A capsule containing the composition of any of claim 52 , 59 or 66 .

74 . The capsule of claim 73 wherein the capsule contains 9-14, 10-13, 11-12, 11 or 12 mg of DAS181.

75 . The capsule of claim 74 , when used in an inhaler, delivers a dose of 8-11, 9-10, 9 or 10 mg of DAS181.

76 . The capsule of claim 75 , wherein the capsule further contains lactose.