| CPC C07K 16/00 (2013.01) [B01D 15/1864 (2013.01); B01D 15/1885 (2013.01); B01D 15/20 (2013.01); B01D 15/3804 (2013.01); B01D 15/424 (2013.01); C07K 1/36 (2013.01); C12M 33/00 (2013.01); G01N 30/468 (2013.01); G01N 30/88 (2013.01); C07K 2317/55 (2013.01); C07K 2317/622 (2013.01); G01N 2030/8831 (2013.01)] | 26 Claims |
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1. A method for producing a biopolymer using a chromatography system (20), wherein the chromatography system comprises:
an eluting system (10) and a capturing system (11), wherein the capturing system (11) comprises a feed container (1) for holding a medium comprising the biopolymer and waste products, wherein the feed container (1) has an outlet (16),
a first chromatography unit (2) and a second chromatography unit (3) both comprising material having affinity for the biopolymer, wherein the first chromatography unit (2) has an inlet (12) and an outlet (13) and the second chromatography unit (3) has an inlet (14) and an outlet (15),
wherein the outlet (16) of the feed container (1) is in fluid connection with the inlet (12) of the first chromatography unit (2) and in fluid connection with the inlet (14) of the second chromatography unit (3), wherein a first valve means (31) is located between the outlet (16) and the inlet (12), and a second valve means (32) is located between the outlet (16) and the inlet (14),
wherein the outlet (13) of the first chromatography unit (2) is in fluid connection with the inlet (14) of the second chromatography unit (3), and a third valve means (33) is located between the outlet (13) of the first chromatography unit (2) and the inlet (14) of the second chromatography unit (3), and the outlet (15) of the second chromatography unit (3) is in fluid connection with the inlet (12) of the first chromatography unit (2), and a fourth valve means (34) is located between the outlet (15) of the second chromatography unit (3) and the inlet (12) of the first chromatography unit (2);
wherein the eluting system (10) comprises a wash buffer container (4), having an outlet (21),an elution buffer container (5), having an outlet (22), optionally a cleaning buffer container (6), having an outlet (23), optionally an equilibration buffer container (7), having an outlet (24), a water supply (8), having an outlet (25),
an inline buffer dilution system (9) having an inlet (26a) and an outlet (27), wherein the outlet (21) of the wash buffer container (4) is in fluid connection with the inlet (26a) of the inline buffer dilution system (9) and wherein a fifth valve means (35) is located between the outlet (21) of the wash buffer container (4) and the inlet (26a) of the inline buffer dilution system (9), wherein the outlet (22) of the elution buffer container (5) is in fluid connection with the inlet (26a) of the inline buffer dilution system (9) and wherein a sixth valve means (36) is located between the outlet (22) of the elution buffer container (5) and the inlet (26a) of the inline buffer dilution system (9),
optionally, wherein the outlet (23) of the cleaning buffer container (6) is in fluid connection with the inlet (26a) of the inline buffer dilution system (9) and wherein a seventh valve means (37) is located between the outlet (23) of the cleaning buffer container (6) and the inlet (26a) of the inline buffer dilution system (9),
optionally, wherein the outlet (24) of the equilibration buffer container (7) is in fluid connection with the inlet (26a) of the inline buffer dilution system (9) and wherein an eighth valve means (38) is located between the outlet (24) of the equilibration buffer container (7) and the inlet (26a) of the inline buffer dilution system (9),
wherein the outlet (25) of the water supply (8) is in fluid connection with the inlet (26a) of the inline buffer dilution system (9) or is in fluid connection with a separate inlet (26b) of the inline buffer dilution system (9) and wherein a ninth valve means (39) is located between the outlet (25) of the water supply (8) and the inlet (26a) or the separate inlet (26b) of the inline buffer dilution system (9); and
wherein the outlet (27) of the inline buffer dilution system (9) is in fluid connection with the inlet (12) of the first chromatography unit (2) and in fluid connection with the inlet (14) of the second chromatography unit (3), wherein a tenth valve means (40) is located between the outlet (27) of the inline buffer dilution system (9) and the inlet (12) of the first chromatography unit (2), and an eleventh valve means (41) is located between the outlet (27) of the inline buffer dilution system (9) and inlet (14) of the second chromatography unit (3),
the method comprising:
(a) leading a medium comprising the biopolymer and waste products through the fluid connection from the outlet (16) of the feed container (1) to the inlet (12) of the chromatography unit (2) wherein the biopolymer is captured on the chromatography unit (2), and medium and waste products continues through the outlet (13) of the chromatography unit (2) until a first pre-determined level of binding capacity is reached in the chromatography unit (2), wherein the fluid connection from the outlet (16) of the feed container (1) to the inlet (14) of the second chromatography unit (3) is closed by the second valve means (32) and wherein the fluid connection from the outlet (13) of the first chromatography unit (2) to the inlet (14) of the second chromatography unit (3) is closed by the third valve means (33),
(b) when the first pre-determined level of binding capacity is reached, leading the medium comprising the biopolymer and waste products through the fluid connection from the outlet (13) of the first chromatography unit (2) to the inlet (14) of the second chromatography unit (3) and through the second chromatography unit (3) for a specified setting for the biopolymer un-captured by the first chromatography unit (2) to be captured on the second chromatography unit (3), and medium and waste products continues through the outlet (15) of the chromatography unit (3), until a second pre-determined level of binding capacity is reached in the chromatography unit (2) and/or (3), wherein the fluid connection from the outlet (16) of the feed container (1) to the inlet (14) of the second chromatography unit (3) is closed by the second valve means (32),
(c) when the second pre-determined level of binding capacity is reached, leading the medium comprising the biopolymer and waste products through the fluid connection from the outlet (16) of the feed container (1) to the inlet (14) of the chromatography unit (3) wherein the biopolymer is captured on the chromatography unit (3), and medium and waste products continues through the outlet (15) of the chromatography unit (3), until a third pre-determined level of binding capacity is reached in the chromatography unit (2) and/or (3), wherein the fluid connection from the outlet (16) of the feed container (1) to the inlet (12) of the first chromatography unit (2) is closed by the first valve means (31), and wherein the fluid connection from the outlet (15) of the second chromatography unit (3) to the inlet (12) of the first chromatography unit (2) is closed by the fourth valve means (34),
wherein during or after step c)
(i) washing the first chromatography unit (2) with a specified concentration of water and buffer by leading concentrated wash buffer through the fluid connection from the outlet (21) of the wash buffer container (4) to the inlet (26a) of the inline buffer dilution system (9) and by leading the water from the supply (8) through the fluid connection from the outlet (25) of the water from the supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) wherein the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9) is closed by the sixth valve means (36), and leading the diluted wash buffer through the outlet (27) of the inline buffer dilution system (9) to the inlet (12) of the first chromatography unit (2), through the first chromatography unit (2), and through the outlet (13) of the first chromatography unit (2), until a first pre-determined level of washing is reached, wherein the fluid connection from the outlet (27) of the inline buffer dilution system (9) to the inlet (14) of the second chromatography unit (3) is closed by the eleventh valve means (41), (ii) when the first pre-determined level of washing is reached, eluting the biopolymer from the chromatography unit (2) by leading:
(a1) concentrated elution buffer through the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9) and by leading the water from the supply (8) through the fluid connection from the outlet (25) of the water from the supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) or alternatively by leading:
(a2) a working solution ready elution buffer through the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9), wherein the fluid connection from the outlet (21) of the wash buffer container (4) to the inlet (26a) of the inline buffer dilution system (9) is closed by the fifth valve means (35) and, wherein when the working solution ready elution buffer is used the fluid connection from the outlet (25) of the water supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) is closed by the ninth valve means (39) and leading the working solution ready elution buffer the outlet (27) of the inline buffer dilution system (9) to the inlet (12) of the first chromatography unit (2), through the first chromatography unit (2), and through the outlet (13) of the first chromatography unit (2), until a first pre-determined level of eluting is reached, and collecting the eluate, wherein the fluid connection from the outlet (27) of the inline buffer dilution system (9) to the inlet (14) of the second chromatography unit (3) is closed by the eleventh valve means (41),
(d) when the third pre-determined level of binding capacity is reached, leading the medium through the fluid connection from the outlet (15) of the second chromatography unit (3) to the inlet (12) of the first chromatography unit (2) and through the first chromatography unit (2) for a specified setting for the biopolymer un-captured by the second chromatography unit (3) to be captured on the first chromatography unit (2), and medium and waste products continues through the outlet (13) of the chromatography unit (2) until a fourth pre-determined level of binding capacity is reached in the chromatography unit (2) and/or (3), wherein the fluid connection from the outlet (16) of the feed container (1) to the inlet (12) of the first chromatography unit (2) is closed by the first valve means (31),
(e) when the fourth pre-determined level of binding capacity is reached, leading the medium comprising the biopolymer and waste products through the fluid connection from the outlet (16) of the feed container (1) to the inlet (12) of the chromatography unit (2) wherein the biopolymer is captured on the chromatography unit (2), and medium and waste products continues through the outlet (13) of the chromatography unit (2), until a fifth pre-determined level of binding capacity is reached in the chromatography unit (2) and/or (3), wherein the fluid connection from the outlet (16) of the feed container (1) to the inlet (14) of the second chromatography unit (3) is closed by the second valve means (32), wherein during or after step e)
(iii) washing the chromatography unit (3) with a specified concentration of water and buffer by leading concentrated wash buffer through the fluid connection from the outlet (21) of the wash buffer container (4) to the inlet (26a) of the inline buffer dilution system (9) and by leading the water from the supply (8) through the fluid connection from the outlet (25) of the water from the supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) wherein the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9) is closed by the sixth valve means (36), and leading the diluted wash buffer through the outlet (27) of the inline buffer dilution system (9) to the inlet (14) of the second chromatography unit (3), through the second chromatography unit (3), and through the outlet (15) of the second chromatography unit (3), until a second pre-determined level of washing is reached, wherein the fluid connection from the outlet (27) of the inline buffer dilution system (9) to the inlet (12) of the first chromatography unit (2) is closed by the tenth valve means (40), (iv) when the second pre-determined level of washing is reached, eluting the biopolymer from the second chromatography unit (3) by leading:
(b1) concentrated elution buffer through the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9) and by leading the water from the supply (8) through the fluid connection from the outlet (25) of the water from the supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) or alternatively by leading:
(b2) a working solution ready elution buffer through the fluid connection from the outlet (22) of the elution buffer container (5) to the inlet (26a) of the inline buffer dilution system (9), wherein the fluid connection from the outlet (21) of the wash buffer container (4) to the inlet (26a) of the inline buffer dilution system (9) is closed by the fifth valve means (35) and, wherein when the working solution ready elution buffer is used the fluid connection from the outlet (25) of the water supply (8) to the inlet (26a) or the inlet (26b) of the inline buffer dilution system (9) is closed by the ninth valve means (39) and leading the diluted elution buffer through the outlet (27) of the inline buffer dilution system (9) to the inlet (14) of the second chromatography unit (3), through the second chromatography unit (3), and through the outlet (15) of the second chromatography unit (3), until a second pre-determined level of eluting is reached, and collecting the eluate, wherein the fluid connection from the outlet (27) of the inline buffer dilution system (9) to the inlet (12) of the first chromatography unit (2) is closed by the tenth valve means (40),
(f) optionally, repeating step (a) to (e), and
(g) optionally, purifying the biopolymer from the collected eluate(s).
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