Formulations comprising recombinant acid α-glucosidase

1 . A pharmaceutical formulation comprising:

(a) a population of recombinant human acid α-glucosidase (rhGAA) molecules, wherein the rhGAA molecules comprises at least 3.0% of the total N-glycans of the rhGAA are in the form of bis-M6P, and wherein the rhGAA molecules comprise from 2.0 mole to 8.0 mole sialic acid residues per mole rhGAA;

(b) at least one buffer selected from a citrate buffer, a phosphate buffer, and combinations thereof; and

(c) at least one excipient selected from the group consisting of mannitol, polysorbate 80, and combinations thereof, and

wherein the formulation has a pH of from 5.0 to 7.0.

2 . The pharmaceutical formulation of claim 1 , wherein the rhGAA molecules are present in a concentration of 5 mg/ml to 50 mg/mL.

3 . The pharmaceutical formulation of claim 2 , wherein the rhGAA molecules are present in a concentration of 15 mg/mL.

4 . The pharmaceutical formulation of claim 1 , wherein the formulation has a pH of from 5.5 to 7.0.

5 . The pharmaceutical formulation of claim 4 , wherein the formulation has a pH of 6.0.

6 . The pharmaceutical formulation of claim 1 , wherein the citrate buffer comprises a potassium, sodium, or ammonium salt.

7 . The pharmaceutical formulation of claim 1 , wherein the citrate buffer comprises sodium citrate.

8 . The pharmaceutical formulation of claim 1 , wherein the at least one buffer is present in a concentration of 10 mM to 100 mM.

9 . The pharmaceutical formulation of claim 8 , wherein the at least one buffer is present in a concentration of 25 mM.

10 . The pharmaceutical formulation of claim 1 , wherein trehalose, sucrose, glycine, or combinations thereof is excluded.

11 . The pharmaceutical formulation of claim 1 , wherein the at least one excipient is mannitol present in a concentration of 10 mg/ml to 50 mg/mL.

12 . The pharmaceutical formulation of claim 1 , wherein the at least one excipient is polysorbate 80 present in a concentration of 0.2 mg/ml to 0.5 mg/mL.

13 . The pharmaceutical formulation of claim 1 , wherein the at least one excipient comprises mannitol and polysorbate 80, and wherein the mannitol is present at a concentration of 20 mg/mL and the polysorbate 80 is present at a concentration of 0.5 mg/mL.

14 . The pharmaceutical formulation of claim 1 , further comprising:

(a) an alkalizing agent; and/or

(b) an acidifying agent,

wherein the alkalizing agent and/or acidifying agent are present in amounts to maintain the pharmaceutical formulation at a pH of from 5.0 to 6.0.

15 . The pharmaceutical formulation of claim 1 , wherein at least 75% of the rhGAA molecules comprise a glycan bearing bis-M6P at the first potential N-glycosylation site.

16 . The pharmaceutical formulation of claim 1 , wherein the rhGAA molecules comprise from 3.0 mole to 5.0 mole mannose-6-phosphate (M6P) residues per mole rhGAA.

17 . The pharmaceutical formulation of claim 1 , wherein the rhGAA molecules comprise from 4.0 mole to 7.0 mole sialic acid residues per mole rhGAA.

18 . The pharmaceutical formulation of claim 1 , wherein the rhGAA molecules comprise at least 3 mole M6P residues per mole of rhGAA, and at least 4.0 mole sialic acid residues per mole rhGAA.

19 . The pharmaceutical formulation of claim 1 , wherein 75% to 80% of the rhGAA molecules comprise a glycan bearing bis-M6P at the first potential N-glycosylation site, 40% to 60% of the rhGAA molecules comprise a glycan bearing mono-mannose-6-phosphate (mono-M6P) at the second potential N-glycosylation site, 40% to 60% of the rhGAA molecules comprise a glycan bearing bis-M6P at the fourth potential N-glycosylation site, and 25% to 40% of the rhGAA molecules comprise a glycan bearing mono-M6P at the fourth potential N-glycosylation site.

20 . The pharmaceutical formulation of claim 1 , wherein 20% of the rhGAA molecules comprise a sialic residue at the third potential N-glycosylation site, 70% of the rhGAA molecules comprise a sialic residue at the fifth potential N-glycosylation site, and 80% of the rhGAA molecules comprise a sialic acid residue at the sixth potential N-glycosylation site.

21 . The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation consists essentially of:

(a) the population of rhGAA molecules;

(b) sodium citrate;

(c) citric acid monohydrate;

(d) mannitol;

(e) polysorbate 80;

(f) water;

(g) optionally, an acidifying agent; and

(h) optionally, an alkalizing agent,

wherein the formulation has a pH of from 5.0 to 6.0.

22 . The pharmaceutical formulation of claim 1 , wherein the pharmaceutical formulation consists essentially of:

(a) the population of rhGAA molecules, present at a concentration of 15 mg/ml;

(b) sodium citrate buffer, present at a concentration of 25 mM;

(c) mannitol, present at a concentration of 20 mg/ml;

(d) polysorbate 80, present at a concentration of 0.5 mg/ml; and

(e) water;

(f) optionally, an acidifying agent; and

(g) optionally, an alkalizing agent,

wherein the formulation has a pH of from 5.0 to 6.0.

23 . The pharmaceutical formulation of claim 1 , wherein the rhGAA comprises on average at least 1 mole bis-M6P per mole of protein.

24 . The pharmaceutical formulation of claim 1 , wherein the rhGAA comprises on average 1.3 mole bis-M6P per mole of protein.

25 . The pharmaceutical formulation of claim 1 , wherein the rhGAA comprises at least 17% of the total N-glycans of the rhGAA are in the form of a bis-M6P N-glycan.

26 . The pharmaceutical formulation of claim 1 , wherein the rhGAA comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 2.

27 . A pharmaceutical composition comprising the formulation of claim 1 after lyophilization.

28 . A method of treating Pompe disease comprising:

(a) reconstituting the pharmaceutical composition of claim 27 ; and

(b) administering the reconstituted pharmaceutical composition to a patient in need thereof.