JPWO2021099529A5

JPWO2021099529A5 -

Info

Publication number: JPWO2021099529A5
Application number: JP2022529291A
Authority: JP
Publication date: 2023-11-27

Claims

A method for extracting a target protein from a precipitate, the method comprising:
a) mixing a precipitate containing a protein of interest with a liquid in a first tank to form a first suspension having a first dilution factor;
b) a first filtration of the first suspension comprising a dynamic filter element adapted to produce a first retentate depleted in the protein of interest and a first permeate enriched in the protein of interest; supplying the unit;
c) diluting the first suspension in the first tank to a second dilution factor by adding a liquid;
d) collecting the first permeate enriched with the protein of interest in a second tank;
e) precipitating one or more impurities in the first permeate enriched with the protein of interest to produce a second suspension;
f) removing precipitated impurities from the second suspension to produce a solution containing the protein of interest.

2. The method of claim 1, wherein step c) comprises diluting the first suspension in the first tank by flowing the first residual liquid into the first tank.

3. The method of claim 1 or 2, comprising concentrating the first permeate enriched in the protein of interest prior to the step of precipitating one or more impurities in the first permeate.

Removing precipitated impurities from the second suspension involves:
i. The second suspension is filtered into a second suspension containing a dynamic filter element adapted to produce a second retentate containing one or more precipitated impurities and a second permeate enriched in the protein of interest. supplying a filtration unit of 2;
ii. 4. A method according to any one of claims 1 to 3, comprising collecting the second permeate enriched in the protein of interest in a further tank.

5. The method of claim 4, further comprising step ia) flowing the second residual liquid into a tank containing the second suspension.

Removing precipitated impurities from the second suspension involves:
i. The second permeate in a further tank was subjected to a continuous concentration process in a third filtration unit containing a cross-flow filter element, thereby depleting a third retentate enriched in the protein of interest and the protein of interest. producing a third permeate;
ii. Claim 4 or 5, further comprising either returning the third retentate enriched in the protein of interest to a third tank and/or collecting the third retentate enriched in the protein of interest. the method of.

Additionally, step ia) dilutes the suspension in the tank containing the second suspension by flowing the third permeate into the tank containing the second suspension, thereby diluting the suspension in the tank containing the second suspension; 7. The method of claim 6, comprising diluting to a third dilution factor.

A method for extracting a target protein from a precipitate, the method comprising:
a) mixing the precipitate with a liquid in a first tank to form a first suspension having a first dilution factor;
b) a first filtration of the first suspension comprising a dynamic filter element adapted to produce a first retentate depleted in the protein of interest and a first permeate enriched in the protein of interest; supplying the unit;
c) diluting the first suspension in the first tank to a second dilution factor by adding liquid, optionally by flowing the first residual liquid into the first tank;
d) collecting the first permeate enriched with the protein of interest in a second tank;
d1) including a cross-flow filter element in the first permeate in the second tank adapted to produce a second retentate enriched in the protein of interest and a second permeate depleted in the protein of interest; applying a continuous concentration process in a second filtration unit;
d2) either returning the second retentate enriched in the protein of interest to the second tank and/or collecting the second retentate enriched in the protein of interest;
e) precipitating one or more impurities from the second retentate enriched with the protein of interest to produce a second suspension;
f) removing precipitated impurities from the second suspension to produce a solution containing the protein of interest.

Further, step d2a) diluting the first suspension in the first tank by flowing a second permeate into the first tank, thereby diluting said suspension to a second dilution factor. 9. The method of claim 8, comprising:

Removing precipitated impurities from the second suspension involves:
i. The second suspension is filtered into a second suspension containing a dynamic filter element adapted to produce a third retentate containing one or more precipitated impurities and a third permeate enriched in the protein of interest. supplying the filtration unit of 3;
ii. 10. A method according to claim 8 or 9, comprising collecting a third permeate rich in the protein of interest in a further tank.

11. The method of claim 10, further comprising step ia) flowing the third residual liquid to a tank containing the second suspension to a third dilution factor.

Removing precipitated impurities from the second suspension involves:
i. The third permeate in a further tank was subjected to a continuous concentration process in a fourth filtration unit containing a cross-flow filter element, thereby depleting a fourth retentate enriched in the protein of interest and the protein of interest. producing a fourth permeate;
ii. 12. The method of claim 10 or 11, further comprising either returning the fourth retentate enriched in the protein of interest to a further tank and/or collecting the fourth retentate enriched in the protein of interest. .

Additionally, step ia) dilutes the suspension in the tank containing the second suspension by flowing the fourth permeate into the tank containing the second suspension, thereby diluting the suspension in the tank containing the second suspension; 13. The method of claim 12, comprising diluting to a third dilution factor.

An industrial-scale method for the extraction of proteins of interest from precipitates with high yields, comprising:
a) mixing the precipitate with a liquid in a first tank to form a suspension having a first dilution factor;
b) feeding the first suspension to a first filtration unit comprising a rotating cross-flow filter element comprising a filter disc with a ceramic membrane having an average pore size between 5 nm and 5000 nm, the filter element comprising: producing a first retentate that is depleted in the protein of interest and a first permeate that is enriched in the protein of interest;
c) diluting the first suspension in the first tank to a second dilution factor by adding liquid, in part by flowing the first residual liquid into the first tank; ;
d) collecting the first permeate enriched with the protein of interest in a second tank;
e) subjecting the first permeate in the second tank to a continuous concentration process in a second filtration unit containing a cross-flow filter element, thereby forming a second retentate rich in the protein of interest and a second retentate rich in the protein of interest; producing a second permeate depleted in protein;
f) either returning the second retentate enriched in the protein of interest to the second tank and/or collecting the second retentate enriched in the protein of interest;
g) precipitating one or more impurities in the second retentate enriched with the protein of interest to produce a second suspension;
h) removing precipitated impurities from a second suspension to produce a solution containing the protein of interest. .

Furthermore, step e1) dilutes the first suspension in the first tank by continuously flowing the second permeate into the first tank, thereby diluting the suspension to a second dilution factor. 15. The method of claim 14, comprising diluting to .

The second suspension is filtered into a second suspension containing a dynamic filter element adapted to produce a third retentate containing one or more precipitated impurities and a third permeate enriched in the protein of interest. supplying the filtration unit of 3;
16. A method according to claim 14 or 15, wherein precipitated impurities are removed from the second suspension by collecting a third filtrate rich in the protein of interest in a further tank.

The second suspension is filtered into a second suspension containing a dynamic filter element adapted to produce a third retentate containing one or more precipitated impurities and a third permeate enriched in the protein of interest. 17. The method of claim 16, wherein after feeding the third filtration unit, the third retentate is passed to a tank containing the second suspension.

Removing precipitated impurities from the second suspension comprises:
The third permeate in a further tank was subjected to a continuous concentration process in a fourth filtration unit containing a cross-flow filter element, thereby depleting a fourth retentate enriched in the protein of interest and the protein of interest. producing a fourth permeate;
18. The method of claim 16 or 17, further comprising either returning the fourth retentate enriched in the protein of interest to a further tank and/or collecting the fourth retentate enriched in the protein of interest. .

After producing a fourth retentate enriched in the protein of interest and a fourth permeate depleted in the protein of interest, further flowing the fourth permeate into a tank containing the second suspension; 19. The method of claim 18, comprising diluting a suspension in a tank containing a second suspension, thereby diluting the suspension to a third dilution factor.

The first retentate and the second permeate are sequentially flowed into the first tank to dilute the suspension to a second dilution factor, and/or the second suspension is diluted to a third dilution factor. 20. According to any one of claims 14 to 19, wherein the third retentate and the fourth permeate are successively introduced into a tank containing the second suspension for dilution to a dilution factor. Method.

A filtration unit adapted to produce a permeate enriched with a protein of interest includes two or more hollow shafts adapted to collect permeate, each shaft having at least one filter disk containing a ceramic membrane. 21. The method according to any one of claims 14 to 20, wherein the method is conjugated.

22. A method according to any one of claims 14 to 21, wherein the filtration unit for carrying out the concentration process comprises a dynamic cross-flow filter element.

23. A method according to any one of claims 1 to 22, wherein precipitating one or more impurities from the permeate or retentate rich in the protein of interest is by changing the solvation capacity of the solvent.

24. Altering the solvation capacity of the solvent comprises reducing the solubility of one or more impurities by addition of reagents and/or changing the pH or conductivity of the solution. Method.

25. The method of claim 24, wherein the reagent that precipitates one or more impurities is an ionized organic additive.

26. The method of claim 25, wherein the ionizable organic additive is a fatty acid or a salt or ester thereof.

27. The method of claim 26, wherein the fatty acid has the general structure CH ₃ (CH ₂ ) _n COOH, and preferably the fatty acid is a C7 to C10 carboxylic acid or a salt or ester thereof.

The fatty acid is selected from the group consisting of enanthic acid (heptanoic acid), caprylic acid (octanoic acid), octenoic acid, pelargonic acid (nonanoic acid), nonenoic acid or capric acid (decanoic acid), or salts or esters thereof. 28. The method of claim 27.

The fatty acid is caprylic acid (octanoic acid), about 0.1 g/g total protein, about 0.25 g/g total protein, about 0.5 g/g total protein, about 0.75 g/g total protein, about 1 .0g/1g total protein, about 1.5g/1g total protein, about 2.0g/1g total protein, about 2.5g/1g total protein, about 3.0g/1g total protein, about 3.5g/1g total protein 1g of total protein, or about 4.0g/g of total protein, preferably the amount of fatty acid, preferably caprylic acid (octanoic acid), is about 0.275g/g of total protein, about 0.280g/g of total protein. 29. The method of claim 28, wherein the amount is 1 g protein, about 0.285 g/g total protein, about 0.290 g/g total protein, about 0.300 g/g total protein, or about 0.325 g/g total protein.

Caprylic acid is added to the permeate or retentate rich in the protein of interest at a temperature of between about 22°C and about 40°C, preferably between about 25°C and about 38°C, preferably between about 27°C and about 37°C. 32. The method according to claim 29, wherein the co-incubations are carried out at a temperature between about 32<0>C, more preferably about 32[deg.]C.

26. A method according to any one of claims 1 to 25, wherein the reagent for precipitating one or more impurities from the permeate or retentate rich in the protein of interest is a non-ionic organic polymer.

32. The method of claim 31, wherein the nonionic organic polymer is selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, polyvinylpyrrolidone, dextran, cellulose, and other polymers.

The method according to any one of claims 1 to 32, wherein the precipitate is an insoluble solid containing the protein of interest.

34. The method of claim 33, wherein the insoluble solid is in the form of pellets or paste.

35. A method according to any one of claims 1 to 34, wherein the precipitate containing the protein of interest is an intermediate product of an alcohol fractionation process.

36. The method of claim 35, wherein the alcohol fraction is human plasma.

37. The method according to any one of claims 1 to 36, wherein the precipitate is in the form of a lyophilizate and/or a crystallized solid containing the protein of interest.

38. A method according to any one of claims 1 to 37, wherein the protein of interest is an immunoglobulin, preferably human immunoglobulin G (IgG).

39. Any one of claims 1 to 38, wherein the first suspension is produced by mixing a protein-containing precipitate with a liquid, thereby providing a starting composition having a first dilution factor. The method described in.

The first suspension is continuously fed to the first filtration unit, preferably until the first suspension is diluted to at least a second dilution factor. 40. The method according to any one of claims 1 to 39, wherein one filtration unit is continuously fed.

The second suspension is continuously fed to the further filtration unit, preferably the second suspension is fed to the further filtration unit until the second suspension is diluted to at least a third dilution factor. 41. The method according to any one of claims 1 to 40, wherein the method is continuously fed to.

42. A filtration unit comprising a dynamic filter element adapted to produce a first retentate and a first permeate enriched with the protein of interest comprises a rotating cross-flow filter element. The method described in Section 1.

The dynamic filter element is a dynamic cross-flow filter element, preferably a rotating cross-flow filter element, preferably the dynamic filter element in at least the first filter unit is a rotating cross-flow filter element. A method according to any one of claims 1 to 42.

44. A method according to any preceding claim, wherein the one or more filter elements comprise a ceramic membrane.

45. A method according to any preceding claim, wherein the dynamic filter element comprises a filter membrane having an average pore size in the range from 5 nm to 2 μm.

46. A method according to any one of claims 1 to 45, wherein the one or more filtration units comprises a pressure vessel, preferably at least the first filtration unit comprises a pressure vessel.

47. A method according to any preceding claim, wherein the temperature of the one or more filtration units is controlled at a temperature between 2°C and 25°C.

48. A method according to any one of claims 1 to 47, wherein the intermembrane filtration pressure of the one or more filtration units is between 0.1 and 2.5 bar.

the flow rate and/or residence time of the suspension or solution into the filter unit and/or the flow rate of the retentate/raffinate containing impurities/precipitants and/or the flow rate of the first permeate/filtrate enriched with the protein of interest. 49. A method according to any one of claims 1 to 48, comprising modulating.

50. A method according to any one of claims 1 to 49, wherein the flow of liquid through the filtration unit is reversed at regular intervals and for defined times, including periodic backflow periods.

51. The method of claim 50, wherein the time between backflow periods increases as the turbidity and protein concentration of the liquid in the filtration unit decreases.

The duration of the reflux period is less than the duration of the filtration period, preferably the duration of the reflux period is at most one quarter, one eighth, one tenth or 16 minutes of the duration of the filtration period. 52. The method according to claim 50 or 51, wherein one or less of:

53 of claims 1 to 52, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or typically at least 98% of the protein of interest in the protein-containing precipitate is recovered. The method described in any one of the above.

54. A method according to any one of claims 1 to 53, wherein the filter element for carrying out the concentration process comprises a filtration membrane with a mean pore size suitable for ultrafiltration and/or diafiltration.

55. A method according to any one of claims 1 to 54, wherein the filter element for carrying out the concentration process is a rotating cross-flow filter element adapted to carry out a continuous concentration process.

The filter element for carrying out the concentration process comprises a membrane with a molecular weight cutoff that is less than the molecular weight of the protein of interest, and preferably the membrane cutoff is lower than the molecular weight of the protein of interest so that the protein of interest is retained in the retentate. 56. A method according to any one of claims 1 to 55, wherein the molecular weight of the protein is at most one-third that of the protein.

57. A method according to any one of claims 1 to 56, wherein the filter element for carrying out the concentration process comprises an ultrafiltration device comprising a membrane in the form of a polymeric membrane such as polyethersulfone or regenerated cellulose.

The product of the method is subjected to one or more further treatments, such as a chromatography step, a virus inactivation step, a concentration and formulation step, so that the final product is suitable for administration to a subject. , the method according to any one of claims 1 to 57.