Process for concentration of a polypeptide
The present invention comprises a method of concentrating a composition comprising a polypeptide of interest and the use of such a concentrated composition for the treatment of diseases in mammals, in particular by subcutaneous injection.
1. A method of concentrating a solution, the method comprising:
(a) providing a solution that comprises α-mannosidase or an analogue thereof, wherein the analogue has (i) at least 95% sequence identity to the α-mannosidase of SEQ ID NO: 21 and (ii) at least 50% of the activity of the α-mannosidase;
(b) removing α-mannosidase aggregates from the solution by centrifugation or filtration; and then
(c) concentrating the solution, wherein the concentrated solution (i) is isotonic to mammalian tissue, (ii) comprises at least 25 mg/ml of the α-mannosidase or analogue, and (iii) comprises less than 5% w/w aggregated α-mannosidase or analogue based on the total α-mannosidase or analogue in the concentrated solution.
2. The method of claim 1 , wherein the α-mannosidase comprises amino acid sequence SEQ ID NO. 21.
3. The method of claim 1 , wherein the concentrating step is performed by freeze-drying or evaporation.
4. The method of claim 1 , wherein the concentrating step is performed by ultrafiltration.
5. The method of claim 1 , wherein the concentrating step is performed by tangential flow filtration.
6. The method of claim 1 , wherein the concentrating step is performed by a centrifugal device.
7. The method of claim 1 , wherein the solution further comprises one or more of glycine, L-serine, sucrose, or mannitol.
8. The method of claim 1 , wherein the solution further comprises one or more buffers selected from the group consisting of: TRIS-HCL, Na-citrate, and Na 2 HPO 4 .
9. The method of claim 1 , wherein the method further comprises sterilizing the concentrated solution.
10. The method of claim 1 , wherein the method further comprises freeze-drying the concentrated solution.
11. The method of claim 1 , wherein the centrifugation is performed at 1800-2500 g.
12. The method of claim 1 , wherein the filtration is with a filter that has a pore-size from about 0.20 to about 5.0 micrometer.
13. The method of claim 1 , wherein the α-mannosidase or analogue thereof is a recombinant protein.
14. The method of claim 1 , wherein said method further comprises one or more of the following steps prior to step a):
i) recombinantly expressing the α-mannosidase or the analogue thereof in a prokaryotic or vertebrate cell;
ii) purifying the α-mannosidase or the analogue thereof in a formulation buffer; and
iii) exchanging the formulation buffer.
15. The method according to claim 14 , wherein purifying comprises subjecting the α-mannosidase or the analogue thereof to one or more of affinity chromatography, ion exchange chromatography or hydroxyapatite chromatography.
16. The method according to claim 14 , wherein the purifying comprises subjecting the α-mannosidase or the analogue thereof to affinity chromatography and then subjecting the α-mannosidase or the analogue thereof to ion exchange chromatography.
17. The method according to claim 16 , wherein the purifying further comprises subjecting the α-mannosidase or the analogue thereof to a hydroxyapatite column after subjecting the α-mannosidase or the analogue thereof to the ion exchange chromatography.
18. The method according to claim 14 , wherein the recombinant expression is with a nucleic acid sequence comprising a sequence selected from the group consisting of (i) SEQ ID NO: 22 and (ii) a nucleic acid sequence which is at least 95% identical to SEQ ID NO: 22.
19. The method according to claims 16 , wherein said method further comprises a step of dilution or diafiltration of the α-mannosidase or the analogue thereof after the affinity chromatography but before the ion exchange chromatography.