Downstream Processing of an Alkaline Phosphatase

1 . A method for isolating an alkaline phosphatase (AP) comprising:

(a) providing a solid phase comprising a ligand of formula (I):

wherein R is a spacer molecule which links the ligand to the solid phase,

(b) contacting said ligand with a composition comprising an AP that comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1 and a host cell protein (HCP);

(c) performing two or more wash steps, wherein at least one wash step is performed using a wash buffer comprising between 20-100 mM arginine (Arg), between 0.5-2 M Urea, or between 5-15% ethylene glycol, or any combination thereof; and

(d) eluting said AP from the ligand with an elution buffer;

wherein said eluate comprises less than 100 ppm HCP.

2 . The method according to claim 1 , wherein the wash buffer comprises less than 1 mM NaCl.

3 . The method according to claim 1 , wherein the elution buffer comprises less than 1 mM NaCl.

4 . The method according to claim 1 , wherein the washing buffer comprises about 40 mM Arg.

5 . The method according to claim 1 , the method further comprising:

(i) providing a second solid phase comprising a second ligand of formula (II):

(ii) contacting said second ligand with a composition comprising said AP and a HCP, and

(iii) performing two or more wash steps, wherein at least one wash step is performed using a second washing buffer having a pH between 7.5-8.5 and comprising between 0.05-0.2 M NaCl and between 0.1-0.5 M L-Arg.

6 . The method according to claim 5 , wherein said second washing buffer comprises between 1-20% glycerol.

7 . The method according to claim 5 , wherein (i)-(ii) precedes (a)-(d).

8 . The method according to claim 1 , further comprising at least one further purification step selected from the group consisting of anion exchange chromatography, ultrafiltration/diafiltration, viral filtration, and hydrophobic interaction chromatography, and any combination thereof.

9 . A method for producing a composition comprising an isolated alkaline phosphatase (AP), the method comprising

dissolving or diluting said AP in a buffer, which results in a composition having a pH of between 6.5 and 7.5 and comprising between 200-300 mM sorbitol, and/or between 10-40% glycerol, and/or between 5-40 mM histidine, and/or between 10-40 mM citrate, or any combination thereof;

wherein there is no visible particle formation in said composition, using a stability test at 2-8° C. for 2 months.

10 . The method according to claim 9 , wherein the composition comprises less than 100 ppm host cell protein (HCP).

11 . A composition comprising an isolated alkaline phosphatase, wherein the composition comprises less than 100 ppm of a host cell protein (HCP) and wherein the composition does not show visible particle formation during stability testing at 2-8° C. for 2 months.

12 . The composition according to claim 11 , wherein the composition has a pH of between 6.5-7.5, and comprises between 10-40 mM citrate or between 5-40 mM histidine.

13 . The composition according to claim 12 , wherein the composition comprises between 200-300 mM sorbitol and/or between 10-40% glycerol.

14 . The method according to claim 1 , wherein the alkaline phosphatase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

15 . The method according to claim 1 , wherein the HCP is a cathepsin-like protein and/or wherein said alkaline phosphatase is obtained from a cell-based expression system.

16 . The method for treating a disease or condition that can be improved by the administration of alkaline phosphatase, the method comprising administering to an individual in need thereof the composition according to claim 11 .

17 . The method according to claim 10 , wherein the composition comprises AP obtained by a method comprising:

(a) providing a solid phase comprising a ligand of formula (I):

wherein R is a spacer molecule which links the ligand to the solid phase,

(b) contacting said ligand with a composition comprising an AP that comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1 and a host cell protein (HCP);

(c) performing two or more wash steps, wherein at least one wash step is performed using a wash buffer comprising between 20-100 mM arginine (Arg), between 0.5-2 M Urea, or between 5-15% ethylene glycol, or any combination thereof; and

(d) eluting said AP from the ligand with an elution buffer;

wherein said eluate comprises less than 100 ppm HCP.

18 . The composition according to claim 11 , wherein the alkaline phosphatase comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.

19 . The composition according to claim 11 , wherein the HCP is a cathepsin-like protein and/or wherein said alkaline phosphatase is obtained from a cell-based expression system.