METHOD FOR CONTROL, MONITORING AND/OR OPTIMIZATION OF A CHROMATOGRAPHIC PROCESS
20170241992 · 2017-08-24
Assignee
Inventors
- Thomas MULLER-SPATH (Zurichi, CH)
- Lars Aumann (Zurich, CH)
- Guido Strohlein (Zurich, CH)
- Michael Bavand (Lenzburg, CH)
- Nicole Ulmer (Schlieren, CH)
Cpc classification
B01D15/3809
PERFORMING OPERATIONS; TRANSPORTING
G01N30/468
PHYSICS
G01N33/52
PHYSICS
B01D15/1807
PERFORMING OPERATIONS; TRANSPORTING
International classification
G01N33/52
PHYSICS
Abstract
Provided is a method for control and/or monitoring and/or optimization of a chromatographic process, in which the method comprises at least 2 columns which are operated, alternatingly, wherein this operation can be carried out in that the at least 2 columns are operated in interconnected and disconnected states, wherein the columns switch positions after such a sequence of interconnected and disconnected state, and wherein downstream of at least one, or of each column, a detector is located capable of detecting the desired product and/or impurities when passing the detector.
Claims
1. A method for control and/or monitoring and/or optimization of a chromatographic process, in which the method comprises at least 2 columns which are operated, alternatingly, wherein this operation can be carried out in that the at least 2 columns are operated in interconnected and disconnected states, wherein the columns switch positions after such a sequence of interconnected and disconnected state, and wherein downstream of at least one, or of each column, a detector is located capable of detecting a desired product and/or impurities when passing the detector, wherein the method comprises the following steps: (a) measuring the disconnected areas (A.sub.Bi, and/or A.sub.peaki) corresponding to effluents of at least one, or of at least two, or of all columns in a product elution position in a disconnected state in at least one cycle or parts of one cycle using the detector(s) at the column(s) outlet(s); and/or (b) (i) measuring the area confined by the breakthrough curve and a horizontal baseline (A.sub.Bi) in a product load position in a disconnected state in at least one cycle or parts of one cycle using the detector(s) at the column(s) outlet(s), or (ii) measuring, in case of alternating operation in interconnected and disconnected state, the interconnected areas (A.sub.ICiU) corresponding to the effluents of at least one, or of all columns in an interconnected state upstream and/or downstream positions in at least one cycle or parts of one cycle using the detector(s) at the column(s) outlet(s); followed by at least one of the following steps: (c) using the measured values of the disconnected areas (A.sub.Bi,n, A.sub.peaki) in said step (a) and/or, in case of alternating operation in interconnected and disconnected state, the interconnected areas (A.sub.ICiU) in said step (b) of the at least one, or at least two detectors for control and/or monitoring and/or optimization of at least one process parameter; and (d) comparing the disconnected areas (A.sub.Bi,n and/or A.sub.peaki,n) of the at least one, or at least two detectors of the current cycle n with the disconnected area of at least one previous cycle (A.sub.Bi,n-1 and/or A.sub.peaki,n-1), optionally including a comparison by calculating the ratios between disconnected areas (A.sub.Bi,n/A.sub.Bi,n-1 and/or A.sub.peaki,n/A.sub.peaki,n-1) of the at least one detector; (e) comparing, in case of alternating operation in interconnected and disconnected state, the interconnected areas (A.sub.ICiU) of the current cycle n with the interconnected area of at least one previous cycle (A.sub.ICiU,n-1) of the at least one detector, or at least two detectors, optionally including a comparison by calculating the ratios between interconnected areas (A.sub.ICiU and A.sub.ICiU,n-1); and in case of either or both of said steps (d) or (e); and (f) using the comparison of the values of the disconnected areas (A.sub.Bi,n, A.sub.peaki) in said step (d) and/or, in case of alternating operation in interconnected and disconnected state, the interconnected areas (A.sub.ICiU) comparison in said step (e) of the at least one, or at least two detectors in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of at least one process parameter.
2. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 1, wherein the method, in case of a process without interconnected state, comprises the following steps: said step (a); said step (d); and said step (f), wherein in said step (f), the comparison of the values of the disconnected areas (A.sub.Bi,n, A.sub.peaki) in said step (d) is used in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of the at least one process parameter.
3. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 1, wherein the method, in case of a process without interconnected state, comprises the following steps: said step (a); and said step (c), wherein in said step (c), the measured values of the disconnected areas (A.sub.Bi,n, A.sub.peaki) in said step (a) of the at least one, or the at least two detectors for control and/or monitoring and/or optimization of the at least one process parameter are used.
4. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 1, wherein the method, in case of a process in which the at least 2 columns are operated in interconnected and disconnected state, wherein the columns switch positions after such a sequence of interconnected and disconnected state comprises the following steps: said step (b)(ii); said step (e); and said step (f), wherein in said step (f), the interconnected areas (A.sub.ICiU) comparison in said step (e) of the at least one, or the at least two detectors are used in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of the at least one process parameter.
5. The method for optimization of a chromatographic process according to claim 1, wherein in said step (a),in case of alternating operation in interconnected and disconnected state, loading the columns such that the initially determined disconnected area (A.sub.peaki) is smaller than 80% of the maximum disconnected area (A.sub.peaki,max) that is expected or known from previous chromatographic runs or cycles or parts thereof of the same chromatographic run, and wherein in said step (b) operating parameters are changed from cycle to cycle or multiples or parts thereof, such that the column load (L) is maximized while the ratio of the corresponding disconnected area (A.sub.peaki,n) and the load remains constant at the level defined by the column load and the disconnected area (A.sub.peaki) in said step (a).
6. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 1, in which the process comprises at least 2 columns which are operated alternatingly, in that the at least 2 columns are operated in alternating interconnected and disconnected states, wherein the columns switch positions after such a sequence of interconnected and disconnected state, and wherein downstream of at least one, or of each column, a detector is located capable of detecting the desired product and/or impurities when passing the detector, wherein the method comprises or consists of the following steps: said step (b)(ii); and said step (c), wherein in said step (c) the interconnected areas (A.sub.ICiU) in said step (b)(ii) of the at least one, or the at least two detectors for control and/or monitoring and/or optimization of the at least one process parameter are used.
7. A method for control and/or monitoring and/or optimization of a chromatographic process, in which the method comprises at least 2 columns which are operated, alternatingly, wherein this operation can be carried out in that the at least 2 columns are operated in interconnected and disconnected states, wherein the columns switch positions after such a sequence of interconnected and disconnected state, and wherein downstream of at least one, or of each column, a detector is located capable of detecting a desired product and/or impurities when passing the detector, wherein the method comprises the following steps: (1) measuring, in case of alternating operation in interconnected and disconnected state, the interconnected areas (A.sub.ICiU) corresponding to the effluents of at least one, or of two or all columns in an interconnected state upstream and/or downstream positions in at least one cycle or parts of one cycle using the detector(s) at the columns outlets; and (2) using the measured values of the interconnected areas (A.sub.ICiU) in said step (1) of the at least one, or at least two detectors for control and/or monitoring of at least one process parameter, including initiating a control action based on the measured values of the interconnected areas (A.sub.ICiU) reaching a specific target set point value, wherein the control action can be related to at least one of timing, flow rates, load, yield, including recovery, throughput, buffer consumption of the process, and wherein the control action can comprise stopping the interconnected loading phase upon the measured values of the interconnected areas (A.sub.ICiU) reaching a pre-defined or automatically calculated set point value and initiating the interconnected washing or the subsequent loading step.
8. A method for control and/or monitoring and/or optimization of a chromatographic process, in which the method comprises at least 2 columns which are operated, alternatingly, in interconnected and disconnected state, and in which the columns switch positions after such a sequence of interconnected and disconnected state, and wherein downstream of each column a detector is located capable of detecting a desired product and/or impurities when passing the detector, wherein the method comprises the following steps: (a) measuring the disconnected areas (A.sub.peaki) corresponding to the effluents of at least two, or of all columns in a product elution position in a disconnected state in at least one cycle or parts of one cycle using the detectors at the columns outlets; and/or (b) measuring the interconnected areas (A.sub.ICiU) corresponding to the effluents of at least one, or of all columns in an interconnected state upstream and/or downstream positions in at least one cycle or parts of one cycle using the detectors at the columns outlets; and (c) comparing the disconnected areas (A.sub.peaki) among the at least two detectors, by calculating the ratios between disconnected areas (A.sub.peaki) of the detectors; and/or (d) comparing the interconnected areas (A.sub.ICiU) among the at least two detectors, by calculating the ratios between interconnected areas (A.sub.ICiU) of the detectors; and (e) comparing the values of the disconnected areas (A.sub.peaki) comparison in said step (c) and/or the interconnected areas (A.sub.ICiU) comparison in said step (d) among at least two detectors in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of at least one process parameter.
9. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 8, wherein the method comprises the following steps: said step (a); said step (c);and said step (e), wherein in said step (e) the values of the disconnected areas (A.sub.peaki) comparison in said step (c) are compared in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of the at least one process parameter.
10. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 8, wherein the method comprises the following steps: said step (b); said step (d);and said step (e), wherein in said step (e), the values of the interconnected areas (A.sub.ICiU) comparison in said step (d) among at least one or at least two detector(s) are compared in order to quantify the degree of the signal magnitude difference and using this difference for control and/or monitoring and/or optimization of at least one process parameter.
11. The method for optimization of a chromatographic process according to claim 8, wherein the method comprises the following steps: said step (a), wherein in said step (a), the columns are loaded such that the initially determined disconnected area (A.sub.peaki) is smaller than 80% of the maximum disconnected area (A.sub.peaki,max) that is expected or known from previous chromatographic runs or cycles or parts thereof of the same chromatographic run, said step (b), wherein in said step (b), operating parameters are changed from cycle to cycle or parts thereof, such that the column load (L) is maximized while the ratio of the corresponding disconnected area (A.sub.peaki,n) and the load remains constant at the level defined by the column load and the disconnected area (A.sub.peaki) in said step (a).
12. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 8, wherein the method comprises the following steps: said step (b); said step (d), wherein in said step (d), the interconnected phase signals A.sub.ICiU among the at least two, preferably all detectors is compared by calculating the ratios between interconnected phase signals A.sub.ICiU of the different detectors and using this ratio for control and/or monitoring.
13. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 1, wherein the chromatographic process is a chromatographic purification method for the isolation of a desired product fraction from a mixture using 2 chromatographic columns (1,2), wherein the method further comprises, within one cycle to be carried out at least once, the following steps: a first batch step (B1), wherein during a batch timespan (t.sub.B) said columns are disconnected and a first column (1) is loaded with feed via its inlet using a first flow rate (Q.sub.feed,B) and its outlet is directed to waste, and from a second column (2) thereof desired product is recovered via its outlet and subsequently the second column (2) is regenerated; a first interconnected step (IC1), wherein the outlet of the first column (1) is connected to the inlet of the second column (2) during an interconnected timespan (t.sub.IC), the first column (1) is loaded beyond its dynamic breakthrough capacity with feed via its inlet using a second flow rate (Q.sub.feed,IC) which is the same or larger than the first flow rate (Q.sub.feed,B), and the outlet of the second column (2) is directed to waste, and wherein during a subsequent washing timespan (t.sub.wash,IC) which is larger or equal to 0 s the outlet of the first column (1) is connected to the inlet of the second column (2), the first column (1) is loaded with solvent and/or buffer which is free from feed material, and the outlet of the second column (2) is directed to waste, a second batch step (B2) analogous to the first batch step (B1) but with exchanged column positions, such that the first column (1) of the first batch step (B1) performs the tasks of the second column (2) of the first batch step (B1), and the second column (2) of the first batch step (B1) performs the tasks of the first column (1) of the first batch step (B1); a second interconnected step (IC2), analogous to the first interconnected step (IC1) but with exchanged column positions, such that the upstream column (1) of the first interconnected step (IC1) is the downstream column of the second interconnected step (IC2), and the downstream column (2) of the first interconnected step (B1) is the upstream column of the second interconnected step (IC2).
14. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein in the interconnected step (IC) the second flow rate (Q.sub.feed,IC) is adapted to be 1.0-10.0 times larger than the first flow rate (Q.sub.feed,B).
15. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein in the interconnected step (IC) the second flow rate (Q.sub.feed,IC) and/or the interconnected timespan (t.sub.IC) are adapted such that at the end of interconnected timespan (t.sub.IC) the feed concentration at the outlet of the upstream column is in the range of 30-90% of the feed concentration at the inlet of the upstream column, with the proviso that the values of the second flow rate (Q.sub.feed,IC) and/or the interconnected timespan (t.sub.IC) are adapted such that at the end of the interconnected timespan (t.sub.IC) at the outlet of the downstream column the feed concentration is below a breakthrough value of 0.25-5%, or of 1-2.5%, and that the elution volume corresponding to this breakthrough value can be multiplied with a safety factor in the range of 60-90%.
16. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein the batch timespan (t.sub.B) is chosen to be the accumulated time required for the recovery and regeneration of the respective column, wherein this accumulated time is given by the accumulated time required for the steps of: (i) washing with solvent and/or buffer under conditions that the product is not released from the stationary phase; (ii) elution with solvent and/or buffer under conditions that the product is released from the stationary phase; (iii) cleaning in place using a solvent and/or buffer to release everything from the stationary phase; and (iv) equilibration, by using the solvent and/or buffer under conditions similar or the same as in the subsequent process steps.
17. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein the columns are affinity chromatography material loaded columns, wherein further the chromatographic stationary phase can be in the form of particles, or in the form of beads and/or membranes and/or monoliths.
18. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein at the outlet of each column a detector for the analysis of the components at the outlet is located, both detectors being of the same type, wherein this is a detector selected from one or a combination of the following detectors: UV detector, visible light detector, IR detector, fluorescence detector, light scattering detector, refractive index detector, pH detector, conductivity detector, at-line HPLC analysis, Raman detector or mass spectrometry detector.
19. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein the first cycle is preceded by a startup step, in which the columns are interconnected and wherein a larger amount of feed solution is loaded into the upstream column in comparison with an interconnected step (IC) of a cycle of the method, and/or the last cycle is followed by a shutdown step, in which the two columns are disconnected, and both columns are subjected to product recovery and column regeneration.
20. The method for control and/or monitoring and/or optimization of a chromatographic process according to claim 13, wherein the desired product is one or a group of chemical reaction products, chemical separation products, biochemical reaction products, biological products, wherein preferably the reaction products are natural products, metals, antibodies, antibody fragments, fusion proteins, recombinant glycoproteins, and/or plasma proteins, or derivatives and/or combinations and/or mixtures thereof.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0303] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[0304] Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,
[0305]
[0306]
[0307]
[0308]
[0309]
[0310]
[0311]
[0312]
[0313]
[0314]
[0315]
DESCRIPTION OF PREFERRED EMBODIMENTS
EXAMPLE 1
Initial Determination of Operating Parameters for a Twin Column Countercurrent Sequential Loading Process
[0316] The initial operating parameters for a twin-column countercurrent sequential loading process for the purification of an IgG from clarified cell culture harvest using protein A affinity chromatography were determined based on the procedure outlined above. The breakthrough curves were recorded, fractionated and analyzed by offline Protein A analysis using a Poros A/20 column (Life technologies, USA) to determine the IgG concentrations. The concentration of IgG in the feed was 1.0 g/L. The columns were of 0.5 cm inner diameter and 5.0 cm length. A protocol for the elution and regeneration of a loaded column was developed including a wash step of 6 min, an elution step of 7 min, a cleaning step of 6 min, a first equilibration step of 3 min, and a second equilibration step of 3 min; all at a flow rate of 1 mL/min. Thus, the total duration of the elution and regeneration steps was t.sub.B=25 min.
[0317] A first breakthrough curve for a single column was recorded for a feed flow rate of 1 mL/min, a second breakthrough curve for a single column at a feed flow rate of 0.5 mL/min and a third breakthrough curve at a feed flow rate of 1 mL/min for two columns in series. With the load factor X=75%, and the safety factor Z=90%, the following elution volume values were obtained (see above descriptions and equations):
EV.sub.1H,1=19.9 mL, EV.sub.1L,1=23.4 mL, EV.sub.1H,75=48.4 mL and EV.sub.2=60.8 mL.
[0318] An overlay of the offline analysis results together with marks for the elution volume values is provided in
EXAMPLE 2
Operation of a Twin Column Countercurrent Sequential Loading Process
[0319] A twin column countercurrent sequential loading process was used for the capture of an IgG monoclonal antibody from clarified cell culture harvest using a Protein A affinity stationary phase packed into two columns of 0.5 cm inner diameter and 5.0 cm length. The process was operated with the following parameters on Contichrom Lab-10 equipment from ChromaCon AG, Switzerland. The UV detection wavelength was 305 nm. The process was run using the operating parameters determined in example 1, which are summarized in table 1.
TABLE-US-00001 TABLE 1 Operating parameters for twin-column countercurrent sequential loading process of example 2 including the startup phase. Q.sub.feed Phase step Q.sub.buffer buffer [mL/ t [—] [—] [mL/min] [—] min] [min] B wash 1.0 25 mM Phosphate, 0.5 6.0 pH 7.0 elute 1.0 25 mM Citrate, 0.5 6.0 pH 3.0 clean 1.0 0.1M NaOH 0.5 7.0 equilibrate 1.0 25 mM Citrate, 0.5 3.0 1 pH 3.0 equilibrate 1.0 25 mM Phosphate, 0.5 3.0 2 pH 7.0 IC feed 0 — 1.0 25.0 wash 1.0 25 mM Phosphate, 0.0 1.0 pH 7.0 startup feed 0 — 1.0 47.5 Q.sub.feed indicates the feed flow rate, Q.sub.buffer indicates the flow rate of all other steps; t is the duration of the substeps of the disconnected phase B and the interconnected phase IC, respectively. The parameters for the final elution correspond to the B parameters except for the feed flow rate, which is zero during the final elution step.
[0320] The operating parameters of the chromatographic process (such as feed flow rate and switch times) were not changed throughout the run. The feed concentration was artificially increased by approximately 0.01-0.02 g/L every cycle (see table 2) to simulate a subtle change in the upstream process.
[0321] After the initial startup phase the purification process was run continuously over five cycles before being eluted in the final elution phase. The effluents corresponding to the feed, the product fraction, the feed flow through fraction and the cleaning fraction were analyzed by offline protein A HPLC analysis in order to determine the IgG concentrations. From the volume of the fractions and the product concentrations, the product masses and performance parameters were computed.
[0322] The yield was calculated by comparing the product mass in the product fraction with the product mass in the feed fraction. The productivity was calculated by division of the product mass (obtained in one cycle) by the cycle duration and the total column volume (i.e. the volume of the two columns together). The buffer consumption was calculated by dividing the overall volume of buffers used within one cycle by the product mass in the product fraction of that cycle. The feed concentration and the performance parameters are summarized in table 2 and the evolution over time is shown in
TABLE-US-00002 TABLE 2 Feed concentration, IgG concentration, yield, productivity and buffer consumption of the sequential countercurrent loading process as average values for the five cycles of the process. buffer feed conc pool conc yield productivity consumption Cycle [g/L] [g/L] [%] [g/L/h] [L/g] 1 1.01 5.10 53.8 21.0 1.1 2 1.03 5.24 93.3 21.5 1.1 3 1.04 5.28 92.6 21.7 1.1 4 1.06 5.37 92.6 22.1 1.0 5 1.08 5.49 93.1 22.6 1.0
EXAMPLE 3
Detector Comparison, Low Impurity Signals
[0323] A twin column countercurrent sequential loading process was operated with the following parameters on Contichrom Lab-10 equipment from ChromaCon AG, Switzerland. Two detector cells giving different signals for the same sample were used for demonstration.
[0324] The operating parameters are summarized in Table 3.
TABLE-US-00003 TABLE 3 Operating parameters for twin-column countercurrent sequential loading process of example 3. Phase step Q.sub.buffer buffer Q.sub.feed t [—] [—] [mL/min] [—] [mL/min] [min] B wash 1.0 25 mM Phosphate, 0.2 6.0 pH 7.0 elute 1.0 25 mM Citrate, 0.2 6.0 pH 3.0 clean 1.0 0.1M NaOH 0.2 7.0 equilibrate 1.0 25 mM Citrate, 0.2 3.0 1 pH 3.0 equilibrate 1.0 25 mM Phosphate, 0.2 3.0 2 pH 7.0 IC feed 0 — 1.0 24.0 wash 1.0 25 mM Phosphate, 0.0 1.0 pH 7.0 startup feed 0 — 1.0 45.0 Q.sub.feed indicates the feed flow rate, Q.sub.buffer indicates the flow rate of all other steps; t is the duration of the substeps of the disconnected phase B and the interconnected phase IC, respectively. The parameters for the final elution correspond to the B parameters except for the feed flow rate, which is zero during the final elution step. The UV detection wavelength was 300 nm.
[0325] The chromatograms from the UV detectors UV1 and UV2 located at the outlet of each column, recorded during cycles 2, 3 and 4 are shown in
[0326] During the interconnected phases, the UV detectors record a signal that corresponds to the load flow-through of the upstream and the downstream column, respectively. The chromatograms of the process in cyclic steady state are explained referring to cycle 2 starting at 100 min.
[0327] In the following, the index n is used to describe the n-th cycle of the process. Thus, for instance, the area A.sub.peak2,3 corresponds to the elution peak area recorded for column 2 (by detector 2) in the 3.sup.rd cycle.
[0328] In the disconnected phase B the product was recovered from column 2. The disconnected phase typically includes at least one washing step prior to elution, and typically a strip and/or clean step followed by at least one re-equilibration step. The product peak is indicated by A.sub.peak2,2 (recorded by UV2). In parallel to the washing, elution, cleaning and re-equilibration of column 2, column 1 was loaded with feed at lower flow rate as the feed flow rate in the interconnected state. The signal UV1 corresponded to the non-adsorbing impurities that flowed through. The signal reached a constant plateau, indicating that no product was breaking through during the B phase.
[0329] In the subsequent interconnected phase IC, the columns were interconnected and column 1 was placed in the upstream position and continued to be loaded. The rising signal (UV1) recorded at the outlet of column 1 at the end of the interconnected phase indicated that product was breaking through (A.sub.ICiU,2). However, this product was not lost but re-adsorbed in the downstream column 2. While breakthrough was detected at column 1 (UV1), no breakthrough was detected at column 2 (UV2) indicating that the entire product was re-adsorbed. At the end of the interconnected phase B column 1 was washed with fresh buffer such that the product present in the liquid volume of the upstream column 1 was adsorbed in the downstream column 2.
[0330] In the following disconnected phase B the product was recovered from column 1 using the same protocol of washing, elution, strip and/or clean, re-equilibration steps that was used in the previous disconnected phase for column 2. The product peak is indicated by A.sub.peak1,2 (recorded by UV1). At the same time column 2 was loaded with feed at a lower flow rate as the feed flow rate in the interconnected state. The signal UV2 corresponded to the impurities that flowed through. Again, the signal reached a constant plateau, indicating that no product was breaking through.
[0331] In the subsequent interconnected phase IC, column 2 was continued to be loaded and the rising signal (UV2) at the end of the interconnected phase indicated that product was breaking through from column 2 (A.sub.IC2U,2). However, this product was not lost but re-adsorbed in the downstream column. While breakthrough was detected from column 2 (UV2), no breakthrough was detected at column 1 (UV1) indicating that the entire product was re-adsorbed. At the end of the interconnected phase B column 2 was washed with fresh buffer such that the product present in the liquid volume of column 2 was adsorbed in the downstream column 1.
[0332] After the interconnected phase had been completed, a new cycle was started.
[0333] In the process, before being washed and eluted in the disconnected phase, the respective columns are in the upstream positions in the interconnected phase and loaded such that product is breaking through into the downstream column where it is re-adsorbed (see example 2). The breakthrough in the interconnected phase is proportional to the areas designated with A.sub.IC2U,n (UV2, column 2) and A.sub.IC1U,n (for UV1, column 1) in
[0334] In
[0335] The determined areas are listed in Table 1.
[0336] In the ideal case of absolutely identical columns and detectors A.sub.IC1U,n (table 4, column 1) would be equal to A.sub.IC2U,n (table 4, column 3) and A.sub.peak1,n (table 4, column 2) would be equal to A.sub.peak2,n (table 3, column 4). In practice both columns and detectors are significantly different, thus in most cases the areas A.sub.IC1U and A.sub.IC2U are significantly different and the areas A.sub.peak1 and A.sub.peak2 are significantly different from each other.
[0337] This is confirmed by the ratio A.sub.IC2U/A.sub.IC1U (table 4, column 5) calculated for every single cycle. The ratio shows that A.sub.IC2U in cyclic steady state (cycles 3 and 4) is about 1.3 times larger (30%) than A.sub.IC1U. In the case of different areas it is very important to determine if the difference is due to a detector with different properties, e.g. amplification, or if it is due to a column with different, potentially deteriorated capacity. Making a judgment based on the different areas alone may lead to the erroneous replacement of a column that actually had an acceptable capacity.
[0338] By comparison of the ratios A.sub.peak2/A.sub.peak1 (table 4, column 6) it becomes clear that also A.sub.peak2 is about 1.3 times larger (30%) than A.sub.peak1. Also the ratio of the sums of the breakthrough and the peak areas (A.sub.IC2U,n+A.sub.peak2,n)/(A.sub.IC1U,n+A.sub.peak1,n) is about 1.3 (table 3, column 10).
[0339] Together, this information shows that the detector signal of UV2 is in general 1.3-fold larger than the detector signal of UV1.
[0340] In case the ratios A.sub.IC2U/A.sub.IC1U and A.sub.peak2/A.sub.peak1 would have been different, it would have indicated that the columns differ in capacity. In that case the ratio of the sums of the breakthrough and the peak areas (table 3, column 10) would have been used to determine the difference of the detector signals.
TABLE-US-00004 TABLE 4 Areas and ratios derived from FIG. 5, given in [mAU min]. The columns 5-10 marked with “ratio” represent ratios of values listed in other columns. For instance the ratio 3/1 (column 5) stands for a division of the values of column 3 by the values of column 1 (thus A.sub.peak2/A.sub.peak1) 10 1 2 3 4 5 6 7 8 9 ratio measured measured measured measured ratio ratio ratio ratio ratio (3 + 4)/ cycle A.sub.IC1U A.sub.peak1 A.sub.IC2U A.sub.peak2 3/1 4/2 2/1 4/3 7/8 (1 + 2) 2 128 1157 189 1500 1.48 1.30 9.0 7.9 1.14 1.31 3 111 1112 149 1419 1.34 1.28 10.0 9.5 1.05 1.28 4 113 1112 146 1419 1.29 1.28 9.8 9.7 1.01 1.28
EXAMPLE 4
Column Capacity Monitoring, Low Impurity Signals
[0341] The twin column countercurrent sequential loading process of example 3 was evaluated for column capacity changes. The feed was not changed during the run and the feed concentration was constant. Thus only the column capacity was potentially changing, for instance due to fouling or harsh cleaning. The explanations refer to the nomenclature used in
[0342] The fact that A.sub.ICiU,n-1=A.sub.ICiU,n-1 and/or A.sub.peaki,n=A.sub.peaki,n-1 for both columns (see example 2) indicates the column capacity has stayed constant from one cycle to the other.
[0343] If A.sub.ICiU,n>A.sub.ICiU,n-1 and/or A.sub.peaki,n<A.sub.peaki,n-1 would have been measured with significant difference it would have been indicative of a decreasing column capacity. Typically the accuracy of the area determination is 1%, thus an increase of A.sub.ICiU,n by 1% over A.sub.ICiU,n-1 or a decrease of A.sub.peaki,n by 1% over A.sub.peaki,n-1 cannot be attributed to a loss of capacity.
[0344] The method may be also used to evaluate the column performance of cycles that are not successive.
EXAMPLE 5
Column Capacity Monitoring, High Impurity Signals
[0345] A twin column countercurrent sequential loading process was operated for the capture of a product from feed material with a large impurity content. The feed material was the same throughout the entire run (constant product concentration in feed). The operating parameters are summarized in table 5.
TABLE-US-00005 TABLE 5 Operating parameters for twin-column countercurrent sequential loading process of example 5. Q.sub.feed Phase step Q.sub.buffer buffer [mL/ t [—] [—] [mL/min] [—] min] [min] B wash 1.0 25 mM Phosphate, 0.2 6.0 pH 7.0 elute 1.0 25 mM Citrate, 0.2 9.0 pH 3.0 clean 1.0 0.1M NaOH 0.2 7.0 equilibrate 1.0 25 mM Citrate, 0.2 2.0 1 pH 3.0 equilibrate 1.0 25 mM Phosphate, 0.2 4.0 2 pH 7.0 IC feed 0 — 1.0 24.5 wash 1.0 25 mM Phosphate, 0.0 1.0 pH 7.0 startup feed 0 — 1.0 45.0 Q.sub.feed indicates the feed flow rate, Q.sub.buffer indicates the flow rate of all other steps; t is the duration of the substeps of the disconnected phase B and the interconnected phase IC, respectively. The parameters for the final elution correspond to the B parameters except for the feed flow rate, which is zero during the final elution step. The UV detection wavelength was 305 nm.
[0346] Due to the large impurity content of the feed material the detectors were in the non-linear range of detection (over-saturated), as shown in
[0347] However, based only on the peak areas A.sub.peaki,n it was possible to determine if the column capacity had deteriorated from one cycle to the other. The product elution peak areas for the 2nd and the 3rd cycle of the process were determined to be A.sub.peak2,2=756 mAU min, A.sub.peak2,3=764 mAU min, A.sub.peak1,2=805 mAU min, A.sub.peak1,3=809 mAU min. By calculating the ratios A.sub.peak2,3/A.sub.peak2,3 and A.sub.peak1,3/A.sub.peak1,2, respectively, it becomes obvious that the areas are identical with a difference of 1% which corresponds to the accuracy of the area determination. This means that the column capacities are identical with a difference of maximum 1%. This is expected since typically the capacity does not change dramatically within two subsequent cycles. The method may be also used to evaluate the columns performance of cycles that are not successive.
[0348] It has to be noted that in the case of large impurity signals it is not possible to decide based on only A.sub.peaki data if the capacities of the two columns are different or if the detectors are give different signals for the same product concentrations. However, if A.sub.peaki measurements are carried out with a constant feed concentration it can be tested by changing the load L if the load is in the linear range of product adsorption. It is assumed that no dramatic column capacity decrease takes place within A.sub.peaki measurements of two cycles. If the load is in the linear range of product adsorption and the comparison is made between at least two peak areas A.sub.peaki from each detector the detectors signal difference can be determined by forming the average of the ratios of A.sub.peak2,n/A.sub.peak1,n. The detectors signal difference determination becomes more accurate if the comparison A.sub.peak2,n/A.sub.peak1,n is made for more cycles.
[0349] More information on the linear range of product adsorption is provided in example 10.
EXAMPLE 6
Feed Concentration and Column Capacity Monitoring, Low Impurity Signals
[0350] The twin column countercurrent sequential loading process of example 3 was evaluated for the effect of product concentration changes in the feed material and simultaneous column capacity changes.
[0351] The operating parameters of the chromatographic process (such as feed flow rate and switch times) were not changed over the cycles but the feed concentration is assumed to be variable and the column capacity is assumed to be constant or to decrease. The different scenarios for possible column degradation and potential feed concentration changes are summarized in table 5.
[0352] In example 3 the breakthrough areas are the same A.sub.ICiU,n=A.sub.ICiU,n-1 and the peak areas are the same A.sub.peaki,n=A.sub.peaki,n-1 for both columns in two consecutive cycles (3 and 4), indicating that neither the feed quality nor the column performance have significantly decreased from one cycle to the other.
[0353] In the following, more examples of relative behavior of A.sub.ICiU and A.sub.peaki and the possible causes with respect to feed concentration and column capacity are provided.
[0354] If A.sub.ICiU,n<A.sub.ICiU,n-1 and A.sub.peaki,n<A.sub.peaki,n-1 is detected the feed concentration must have decreased. The column capacity may have stayed the same from one cycle to the other but may have also decreased. An increasing ratio A.sub.peaki,n/A.sub.ICiU,n from one cycle to the other is indicative of the column quality staying constant or that the capacity is decreasing to a lesser extent than the column load. A decreasing ratio A.sub.peaki,n/A.sub.ICiU,n from one cycle to the other is indicative of the column quality staying constant or the capacity decreasing to a stronger extent than the column load.
[0355] If A.sub.ICiU,n>A.sub.ICiU,n-1 and A.sub.peaki,n≧A.sub.peaki,n-1 are measured the feed concentration must have increased from one cycle to the other. The column capacity may have stayed the same from one cycle to the other but may have also decreased. An increasing ratio A.sub.peaki,n/A.sub.ICiU,n from one cycle to the other is indicative of the column quality staying constant or that the capacity is decreasing to a lesser extent than the column load increases. A decreasing ratio A.sub.peaki,n/A.sub.ICiU,n from one cycle to the other is indicative of the column quality staying constant or the capacity decreasing to a stronger extent than the column load.
[0356] If A.sub.ICiU,n>A.sub.ICiU,n-1 and A.sub.peaki,n<A.sub.peaki,n-1 are measured the column capacity must have decreased. The feed concentration must have changed such that the load was increased to a stronger extent than the capacity of the columns decreased. Theoretically, the capacity of the columns may decrease to the same extent like the increase of the load such that the effects cancel out. In that case it is recommended to calculate the ratio of the areas corresponding to the total product that is eluting for two different cycles, (A.sub.peaki,n+A.sub.ICiU,n)/(A.sub.peaki,n-1+A.sub.ICiU,n-1), for the same detector. If the ratio is >1, the feed concentration must have increased from one cycle to the other.
TABLE-US-00006 TABLE 5 Changes in column capacity and feed concentration from cycle n-1 to n and the effects on the areas A.sub.ICiU and A.sub.peaki that are determined from the chromatograms. columns feed A.sub.ICiU A.sub.peaki = = A.sub.ICiU,n = A.sub.ICiU,.sub.n-1 A.sub.peaki,n = A.sub.peaki,n-1 =/↓ ↓ A.sub.ICiU,n < A.sub.ICiU,.sub.n-1 A.sub.peaki,n < A.sub.peaki,n-1 = ↑ A.sub.ICiU,n > A.sub.ICiU,.sub.n-1 A.sub.peaki,n ≧ A.sub.peaki,n-1 ↓ = A.sub.ICiU,n > A.sub.ICiU,.sub.n-1 A.sub.peaki,n < A.sub.peaki,n-1 ↓ ↑ A.sub.ICiU,n > A.sub.ICiU,.sub.n-1 effects may cancel out, check (A.sub.peaki,n + A.sub.ICiU,n)/(A.sub.peaki,n-1 + A.sub.ICiU,n-1) Column capacity changes include: capacity decrease “↓”, constant capacity “=”; Product concentration changes in the feed include the following: increase “↑”, decrease in “↓”, no change “=”.
EXAMPLE 7
Feed Concentration and Column Capacity Monitoring, High Impurity Signals
[0357] The twin column countercurrent sequential loading process of example 5 was evaluated for the effect of product concentration changes in the feed material and simultaneous column capacity changes. In the following, the product concentration in the feed will abbreviated with “feed concentration”
[0358] The operating parameters of the chromatographic process (such as feed flow rate and switch times) were not changed over the cycles but the feed concentration was assumed to be variable and the column capacity is assumed to be constant or to decrease.
[0359] Due to the large impurity content of the feed material the detectors were in the non-linear range of detection (over-saturated), as shown in
[0360] The fact that A.sub.peaki,n=A.sub.peaki,n-1 for both columns indicates that a.) neither the feed concentration nor the column performance have decreased significantly or b.) that the column capacity for both or one of the columns has decreased to the same extent as the feed concentration has increased from cycle n-1 to cycle n.
[0361] If A.sub.peaki,n>A.sub.peaki,n-1 is detected for both or one of the columns indicates that the feed concentration has increased from cycle n-1 to n and the column capacity has stayed equal or decreased to a lesser extent as the feed concentration has increased, for both or one of the columns.
[0362] If A.sub.peaki,n<A.sub.peaki,n-1 is detected for both or one of the columns, either the feed concentration has decreased from cycle n-1 to n or the column capacity has decreased or both the feed concentration and the column capacity have decreased. Possibly also the feed concentration has increased from cycle n-1 to n but the column capacity has decreased to a stronger extent.
EXAMPLE 8
Process Control, Low Impurity Signals
[0363] Two twin column countercurrent sequential loading processes for the capture of a product from feed material with a large impurity content were evaluated for the effect of product concentration changes in the feed material and simultaneous column capacity changes. In the following, the product concentration in the feed is abbreviated with “feed concentration”. The twin-column countercurrent sequential loading process conditions are listed in table 7.
TABLE-US-00007 TABLE 7 Operating parameters for the twin-column countercurrent sequential loading processes of example 8. Q.sub.feed Phase step Q.sub.buffer buffer [mL/ t [—] [—] [mL/min] [—] min] [min] B wash 1.0 25 mM Phosphate, 0.82/0.19 6.0 pH 7.0 elute 1.0 25 mM Citrate, 0.82/0.19 9.0 pH 3.0 clean 1.0 0.1M NaOH 0.82/0.19 7.0 equilibrate 1.0 25 mM Citrate, 0.82/0.19 2.0 1 pH 3.0 equilibrate 1.0 25 mM Phosphate, 0.82/0.19 4.0 2 pH 7.0 IC feed 0 — 1.0 24.5 wash 1.0 25 mM Phosphate, 0.0 1.0 pH 7.0 startup feed 0 — 1.0 45.0 Q.sub.feed indicates the feed flow rate, Q.sub.buffer indicates the flow rate of all other steps; t is the duration of the substeps of the disconnected phase B and the interconnected phase IC, respectively. Q.sub.feed was 0.82 mL/min and 0.19 mL/min, respectively. The parameters for the final elution correspond to the B parameters except for the feed flow rate, which is zero during the final elution step. The UV detection wavelength was 300 nm.
[0364] Based on the areas A.sub.ICiU, A.sub.Bi and A.sub.peaki, control actions can be derived to maintain certain levels of A.sub.peaki, which correspond to the concentration of the product of interest in the product pool.
[0365] As outlined in example 6, A.sub.ICiU and A.sub.peaki experience certain trends in response to feed concentration and column capacity changes. The aim of process control is to maintain A.sub.peaki within certain defined limits. The possible control actions include a change of the load and are listed in detail above.
[0366] The control actions based on the trends of the areas A.sub.ICiU and A.sub.peaki are summarized in the following: [0367] If the feed concentration remains constant and the column quality remains constant no control action is required. [0368] If the feed concentration decreases and the column capacity remains the same or decreases the load L may be increased to increase A.sub.peaki (see constraint reported below). [0369] In case the feed concentration is increasing and the column quality remains the same or decreases to a lesser extent, the load L must be reduced to maintain the desired level of A.sub.peaki. [0370] In case the feed concentration stays equal and the column capacity decreases the load L may be increased to increase A.sub.peaki (see constraint reported below). [0371] In case the feed concentration is increasing and the column capacity is decreasing to a larger extent, the load L may be increased to maintain the desired level of A.sub.peaki, taking into account the constraint explained below.
[0372] The above cases show that the effects of column capacity deterioration and feed concentration change in the periodic countercurrent loading process can be controlled by changing only the load. Summarizing the above control actions, if A.sub.peaki decreases beyond the specified limits the load should be increased (see constraint below) and if A.sub.peaki increases beyond the specified limit the load should be reduced.
[0373] The constraint for load increases is given by the dynamic binding capacity. The more the capacities of the downstream column in the interconnected state and of the column that is loaded in the disconnected state are exceeded, the more product is lost and the lower the final recovery of the process.
[0374] In order to avoid product losses, the breakthrough of product from the column that is loaded during the disconnected state and the breakthrough of product from the downstream column in the interconnected state must be minimized.
[0375] In process chromatograms these constraints imply that the signals from the columns that are loaded during the disconnected state and the signals from the downstream columns in the interconnected state must not exceed the plateau value that corresponds to the level of the impurities which are not adsorbed. This constraint is graphically illustrated in
[0376]
[0377] In the disconnected phase B2 the area in between the breakthrough curve and the impurity plateau baseline is zero for run II (area not indicated in
[0378] In the interconnected phase IC2 the areas in between the signal curve of the upstream column in the interconnected state and the impurity plateau baseline are designated A.sub.IC2U-I and A.sub.IC2U-II, respectively. In phase IC2 the breakthrough from the previously loaded column either starts (30 g/L load, UV2-II) or respectively continues (48 g/L load, UV2-I), however the column is in the upstream position and the product leaving the column is completely adsorbed in the downstream column. Therefore the product that corresponds to the areas A.sub.IC2U-I and A.sub.IC2U-II, recorded at the upstream column outlet in the interconnected phase, remains in the system and is not lost.
[0379] The area values, obtained by integration, that correspond to product losses are listed in Table 8.
[0380] In order to estimate if the product losses were significant in cyclic steady state the areas A.sub.ICiD and A.sub.Bi were compared with the respective elution peak areas A.sub.peaki by calculating the product loss ratios PLR1=(A.sub.B1+A.sub.IC1D)/(A.sub.peak1+A.sub.B1+A.sub.IC1D) for column 1 (signals not shown in
[0381] The yield values were confirmed by offline HPLC protein A analytics to be 90% (run I) and 100% (run II), which is in good agreement with the values determined by the online area evaluation. In the presented case the evaluation was carried out online by manual integration using the Contichrom evaluation software (ChromaCon AG, Zurich, Switzerland) but it can be fully automated using suitable integration algorithms and automatically trigger control actions for the subsequent cycles.
[0382] Consequently, based on the online analysis results, in run I the load would be lowered by means of a suitable control action in order to avoid product losses in future cycles. Possible control actions include but are not limited to a reduction of the feed flow rate in the disconnected state, a reduction of the feed flow rate in the interconnected state and a reduction of the interconnected state duration, as described further above.
[0383] It is worth noting that depending on the individual column capacity and detector calibration, the areas A.sub.B1 and A.sub.B2B may be significantly different. In order to reliably estimate if potential product losses are significant and require control actions, the areas A.sub.B1 and A.sub.B2 are put into perspective with the corresponding peak areas A.sub.peak1 and A.sub.peak2, respectively, and an average product loss ratio should be estimated based on both columns/detectors. The same applies for the areas A.sub.IC1D and A.sub.IC2DFurthermore it is worth noting that if A.sub.B1 and A.sub.B2 are zero, also A.sub.IC1D and A.sub.IC2D must be zero (see run II case).
TABLE-US-00008 TABLE 6 Data from the cyclic steady state from two operating points of twin-column sequential loading chromatography process. Run I: 48 g/L load, run II: 30 g/L load. The determined areas correspond to the areas indicated in FIG. 7 for UV2. The areas for UV1 were evaluated too but are not shown in FIG. 7 for clarity. Column 2 shows the feed flow rate in the disconnected state of the process. 4 7 2 3 UV1 5 6 UV2 1 Q.sub.feed,IC Load A.sub.peak1 A.sub.B1 A.sub.IC1D A.sub.peak2 Run [mL/min] [g/L] [mAU*min] [mAU*min] [mAU*min] [mAU*min] I 0.82 48 344 58.3 0 379 II 0.19 30 253 0.1 0 282 10 11 12 8 9 avg product Yield Yield 1 A.sub.B2 A.sub.IC2D loss ratio (online) (offline) Run [mAU*min] [mAU*min] [%] [%] [%] I 49 0 13.0 87.0 90.5 II 0 0 0.0 100.0 100.0
EXAMPLE 9
OPtimization of a Twin Column Countercurrent Sequential Loading Process, Low Impurity Signals
[0384] In order to optimize process performance it is desirable to maximize the load of the run in order to process more material in the same amount of time using the same columns.
[0385] In the previous examples suitable criteria for the identification of product losses have been explained in detail. The criteria can serve as optimization tool since they define the maximum load of the process. Based on the criteria, the load of the process may be increased just until the point where product losses start to occur.
[0386] A twin column countercurrent sequential loading process was run for the capture of an IgG monoclonal antibody from clarified cell culture harvest using a Protein A affinity stationary phase packed into two columns of 0.5 cm inner diameter and 5.0 cm length in order to demonstrate the optimization procedure.
[0387] The process was operated with the parameters listed in table 9 on Contichrom Lab-10 equipment from ChromaCon A. G., Switzerland.
TABLE-US-00009 TABLE 9 Operating parameters for the twin-column countercurrent sequential loading process of example 9. Phase step Q.sub.buffer buffer Q.sub.feed t [—] [—] [mL/min] [—] [mL/min] [min] B wash 1.0 25 mM Phosphate, pH 7.0 0.0/0.19/0.43/0.62/0.82 6.0 elute 1.0 25 mM Citrate, pH 3.0 0.0/0.19/0.43/0.62/0.82 9.0 clean 1.0 0.1M NaOH 0.0/0.19/0.43/0.62/0.82 7.0 equilibrate 1 1.0 25 mM Citrate, pH 3.0 0.0/0.19/0.43/0.62/0.82 2.0 equilibrate 2 1.0 25 mM Phosphate, pH 7.0 0.0/0.19/0.43/0.62/0.82 4.0 IC feed 0 — 1.0 24.5 wash 1.0 25 mM Phosphate, 0.0 1.0 pH 7.0 startup feed 0 — 1.0 45.0 Q.sub.feed indicates the feed flow rate, Q.sub.buffer indicates the flow rate of all other steps; t is the duration of the substeps of the disconnected phase B and the interconnected phase IC, respectively. Q.sub.feed was increased every two cycles. The parameters for the final elution correspond to the B parameters except for the feed flow rate, which is zero during the final elution step. The UV detection wavelength was 300 nm.
[0388] The same feed material was used throughout the run. The feed flow rate in the interconnected state was kept constant throughout the run at 1.0 mL/min.
[0389] The load flow rate in the disconnected phase was increased after every two cycles in steps from 0.0 mL/min to 0.82 mL/min (see Table 10, column 1). The flow rates corresponded to loads of 26 g/L to 48 g/L (see Table 10, column 2). For column 1, the areas A.sub.peak1, A.sub.B1, A.sub.IC1D were determined (see Table 10, columns 3-5) and for column 2, the areas A.sub.peak2, A.sub.B2 and A.sub.IC2D were determined (see Table 10, columns 6-8) as described in example 3 (Note: for 26 g/L load the areas of UV2 were not determined).
[0390] From the determined areas from UV1 and UV2, the average product loss ratios PLRavg and the yields were calculated (see Table 10, columns 9, 10). For comparison, the yield was determined by offline HPLC analysis (see Table 10, column 11). The productivity (see Table 10, column 12) was calculated as the amount of product produced within one cycle divided by the duration of the cycle and the total column volume based on the offline analysis values.
[0391] The buffer consumption (see Table 10, column 13) was calculated as the amount of buffer consumed (in liters) per gram of product purified based on the offline analysis values.
[0392] Table 10 shows that with increasing load the productivity increases significantly and the buffer consumption decreases. Increasing the load from 26 g/L to 43 g/L, which corresponds to an increase of more than 65%, still allows for product recovery with more than 97% yield. A further increase of the load to 48 g/L leads to significant product losses of at least 10%, resulting in a product yield of less than 90%. In the presented case, losses of more than 5% were considered inacceptable, so of the tested conditions 43 g/L was the optimal load that had a 65% larger productivity than the base case at 26 g/L and an over 65% reduced buffer consumption. Process yield, determined by online and offline analysis and the productivity are shown as a function of the load in
[0393] Summarizing, the presented example shows how the sequential loading process can be optimized by increasing the load successively and by monitoring and evaluating the areas under the signal curves corresponding to the positions of potential product loss.
[0394] The effect of a load increase control action becomes evident in the following cycle, potentially allowing for a new control action. In the presented example, the feed concentration was constant but the optimization method can also be applied if the feed concentration changes or the column capacity is different among the columns and/or the capacity of the columns is decreasing. The method can be fully automated using suitable integration algorithms and automatically trigger control actions for the subsequent cycles. Preferably the step size for load increase for process optimization is in the range of 5-10 g/L for an IgG capture using Protein A affinity chromatography. Through successive increase of the load the process the operating space corresponding to high yield is explored. As soon as an operating point with inacceptable yield is found, the process conditions should be reverted to a previous operating point that corresponded to 100% yield. The peak area of detector i corresponding to the highest possible load at 100% yield is called A.sub.peaki,max If desired, the process optimization can be continued by increasing the load again by smaller increments or a load safety margin can be left by not continuing the optimization.
[0395] Preferably the process optimization is started from a point that corresponds to an intermediate load. However, if only very little process knowledge is present an optimization starting point is defined by operating parameters corresponding to 0 g/L load in the disconnected phase.
TABLE-US-00010 TABLE 10 Optimization procedure data of the twin-column sequential loading chromatography process (example 9). Column 1 shows the feed flow rate in the disconnected state of the process; column 2 the load; columns 3-5 the areas A.sub.peak1, A.sub.B1, A.sub.IC1D, respectively, of UV1 determined in the 2nd cycle after the feed flow rate change; columns 6-8 the areas A.sub.peak2, A.sub.B2, A.sub.IC2D, respectively, of UV2 determined in the 2nd cycle after the feed flow rate change; column 9 the average product loss ration PLRavg, column 10 the yield determined online by the area evaluation method described in the example, column 11 the yield determined through HPLC offline analysis, column 12 the productivity and column 13 the buffer consumption based on the offline analyses. 3 4 5 6 7 1 2 UV1 UV1 UV1 UV2 UV2 Q Load A.sub.peak1 A.sub.B1 A.sub.IC1D A.sub.peak2 A.sub.B2 [mL/min] [g/L] [mAU*min] [mAU*min] [mAU*min] [mAU*min] [mAU*min] 0.00 26 219 0 0 n.a. n.a. 0.19 30 253 0.1 0 282 0 0.43 37 298 1.7 0 326 2 0.62 43 330 10.5 0 359 10 0.82 48 344 58.3 0 379 49 8 9 10 11 1 UV2 avg product Yield Yield 12 13 Q A.sub.IC2D loss ratio (online) (offline) Prod. B.C. [mL/min] [mAU*min] [%] [%] [%] [g/L/h] [L/g] 0.00 0 0.0 100.0 100.0 14.4 1.2 0.19 0 0.0 100.0 100.0 17.2 1.0 0.43 0 0.6 99.4 99.5 20.7 0.6 0.62 0 2.8 97.2 99.6 23.9 0.4 0.82 0 13.0 87.0 90.5 24.5 0.2
EXAMPLE 10
Process Control, High Impurity Signals
[0396] The example describes the control of the twin column countercurrent sequential loading process for the capture of a product from feed material with a large impurity signal (see
[0397] As shown in example 5, A.sub.B1 and A.sub.ICiD) as well and A.sub.B2 and A.sub.IC2D cannot be measured due to the large impurity signal.
[0398] However in the frequently observed case of an only slowly decreasing column capacity, and with implemented safety margins for the load it can be assumed that no product is lost during in the twin-column periodic countercurrent process. Safety margins can be determined by means of offline analysis for product concentration determination. Thus, with reference to example 9, A.sub.B1 and A.sub.IC1D) as well and A.sub.B2 and A.sub.IC2D can be assumed to be zero.
[0399] In the described case the area A.sub.peaki changes only in dependence of the load, for instance due to a change of the feed concentration.
[0400] Consequently, based on only the peak areas A.sub.peaki and the comparison of their development over at least two cycles, the process performance can be monitored and control actions can be derived to maintain certain levels of A.sub.peaki, which correspond to the desired concentration of the product of interest in the product pool.
[0401] In case the feed concentration remains constant the peak areas A.sub.peaki remain constant over the cycles (A.sub.peak1,n=A.sub.peak1,n-1 and A.sub.peak2,n=A.sub.peak2,n-1) and no control action is required. In case the feed concentration increases, the peak areas A.sub.peaki increase over the cycles (A.sub.peak1,n>A.sub.peak1,n-1 and A.sub.peak2,n>A.sub.peak2,n-1), and a decrease of the load is required if the peak areas have run out of specification.
[0402] In case the feed concentration decreases, the peak areas A.sub.peaki decrease over the cycles (A.sub.peak1,n<A.sub.peak1,n-1 and A.sub.peak2,n<A.sub.peak2,n-1), and an increase of the load is required if the peak areas have run out of specification.
[0403] The control actions may be also based on comparing areas of non-successive cycles or fractions of cycles.
EXAMPLE 11
Optimization of a Twin Column Countercurrent Sequential Loading Process, High Impurity Signals
[0404] The example describes the optimization of a twin column countercurrent sequential loading process for the capture of a product from feed material with a large impurity signal (see
[0405] However in order to optimize process performance it is desirable to maximize the load in order to process more material in the same amount of time using the same columns.
[0406] A successive increase of the load inevitably leads to product losses when the column capacity is exceeded. Due to the large impurity signal product losses cannot be directly identified by the areas A.sub.B1, A.sub.IC1D and A.sub.B2, A.sub.IC2D, respectively.
[0407] Nevertheless, process optimization based only on the peak areas A.sub.peaki is possible, when evaluating the peak areas of operating points with different loads.
[0408] The starting point of the process optimization corresponded to a load where no breakthrough was present, namely to a load during the interconnected state that corresponds to a value that is well below the breakthrough value of two sequential columns and with a feed flow rate of zero during the disconnected state. The starting load value corresponded to 26 g/L (see table 11, column 2). As described in example 9 the load was now increased every two cycles and the areas A.sub.peaki were determined by integrating the chromatograms (table 11, column 3 (UV1) and column 6 (UV2)). After having recorded the peak areas A.sub.peaki corresponding to three loads (26 g/L, 30 g/L and 37 g/L) and two loads (A.sub.peak2, 30 g/L, 37 g/L, the peak area from the first load was not measured), respectively, the area values were plotted as a function of the load and the data was fitted with two straight lines (see
[0409] It has to be noted that it is not possible to decide based on only A.sub.peaki data if the capacity of the two columns is different or if the detectors have a different signal magnitude. However, if A.sub.peaki measurements are carried out with a constant feed concentration in the linear region of the measurement range and the comparison is made between more than two peak areas A.sub.peaki from each detector, as outlined in this method, information on both detector calibration and column capacity decrease can be obtained.
[0410] Summarizing, the presented example shows how the sequential loading process can be optimized by changing the load and monitoring and evaluating only the peak areas without the need to evaluate areas under breakthrough curves. For this method information of successive cycles is required to determine if the column capacity has been exceeded. The advantage of the method is that it can also be applied in cases where the impurity signal is exceeding the measurement range of the detector. It is recommended to dynamically verify the performance data corresponding to historical operating points if the process is operated over time spans where significant column degradation is expected. For this purpose the load may be changed temporary.
[0411] Both presented optimization methods (example 9 and example 11) do not require detector calibration.
TABLE-US-00011 TABLE 11 Optimization procedure data of the twin-column sequential loading chromatography process (example 11). Column 1 shows the feed flow rate in the disconnected state of the process; column 2 the load; column 3 the area A.sub.peaki determined by integration of the chromatogram of UV1; column 4 the area A.sub.peaki as estimated from the linear correlation described in the example and in FIG. 9 for UV1; column 5 the difference between the two areas of column 4 and column 3 of UV1; column 6 the area A.sub.peaki determined by integration of the chromatogram of UV2; column 7 the area A.sub.peaki as estimated from the linear correlation described in the example and in FIG. 9 for UV2; column 8 the difference between the two areas of column 7 and column 6 of UV2; column 9 the average product loss ration PLRavg, column 10 the yield determined online by the area evaluation method described in the example, column lithe yield determined through HPLC offline analysis, column 12 the productivity and column 13 the buffer consumption based on the offline analyses. 3 4 6 7 1 2 UV1 UV1 expctd 5 UV2 UV2 expctd Q Load A.sub.peak1 A.sub.peak1 UV1 delta A.sub.peak2 A.sub.peak2 [mL/min] [g/L] [mAU*min] [mAU*min] [mAU*min] [mAU*min] [mAU*min] 0.00 26 219 219 1 n.a. 251 0.19 30 253 252 −1 282 282 0.43 37 298 299 0 326 326 0.62 43 330 338 8 359 362 0.82 48 344 377 33 379 399 9 10 11 1 8 avg product Yield Yield 12 13 Q UV2 delta loss ratio (online) (offline) Prod. B.C. [mL/min] [mAU*min] [%] [%] [%] [g/L/h] [L/g] 0.00 n.a. 0.3 99.7 100.0 14.4 1.2 0.19 0 −0.2 100.0 100.0 17.2 1.0 0.43 0 0.1 99.0 99.5 20.7 0.6 0.62 3 1.5 98.5 99.6 23.9 0.4 0.82 20 6.9 93.1 90.5 24.5 0.2
[0412] The twin column sequential process can be transformed into a somewhat simplified process by removing the interconnected phases, leaving only the batch phases (see
[0413] The tandem process generally speaking thus operates in a cyclic manner wherein one cycle comprises the following alternating phases: [0414] A first batch phase B1 wherein the columns are disconnected and the column that has been previously cleaned and re-equilibrated is loaded using a constant feed flow rate or a feed flow rate profile, and wherein the column that has been previously loaded performs the tasks of a typical chromatographic cycle that follow the feeding step (such as washing, elution, cleaning, re-equilibration); [0415] A second batch phase B2 where the functions are switched, i.e. wherein the columns are disconnected and the column that has been previously cleaned and re-equilibrated is loaded using a constant feed flow rate or a feed flow rate profile, and wherein the column that has been previously loaded performs the tasks of a typical chromatographic cycle that follow the feeding step (such as washing, elution, cleaning, re-equilibration).
[0416] What is also possible in view of the above is that the recovery of second column includes a preloading with feed. In the context of this application therefore the regeneration phase of the second column may also include, after at least one of the steps of cleaning, equilibration, a final step of loading with feed so as to preload the second column.
[0417] On the bottom of
[0418] The first cycle is usually preceded by a startup step, in which the two columns are disconnected and the column that performs the tasks of a typical chromatographic cycle that follow the feeding step (such as washing, elution, cleaning, re-equilibration) in the next phase B1 is loaded and the other column is idle or being prepared. The last cycle is followed by a shutdown step, in which the two columns are disconnected, and the column that has been loaded in the previous phase B2 performs the tasks of a typical chromatographic cycle that follow the feeding step (such as washing, elution, cleaning, re-equilibration) while the other column is idle, cleaned or is stored.
[0419] In order to maintain a high product yield in a tandem process it is usually the aim to minimize A.sub.Bi,n, which corresponds to product losses.
[0420] An analogous form of control and optimization as detailed above for the sequential loading process with alternating disconnected and interconnected phases can be applied to the tandem process as illustrated in
[0421] The determined areas are then compared among the detectors for the current and/or previous cycles and/or for the same detector for the current and previous cycles in the case of the process with alternating interconnected and disconnected phases (see
EXAMPLE 12
Control of a Twin Column Countercurrent Sequential Loading Process, Low Impurity Signals
[0422] The sequential loading process to be controlled, was used to purify an IgG.sub.1 monoclonal antibody from clarified cell culture harvest. The process was operated on a Contichrom CUBE Combined 30 system (ChromaCon A. G., Switzerland) with two columns of 5 mm inner diameter and 50 mm bed height (column volume CV=1 mL), packed with the protein A affinity material MAbSelect Sure. A UV detector was mounted behind each column and monitoring absorbance at 280 nm (A280).
[0423] The following buffers were used: 20 mM Phosphate pH 7.2 (buffer A), 10 mM Citrate pH 2.7 (buffer B), 0.1 M NaOH (cleaning buffer).
[0424] The process protocol included a washing step of 4 column volumes (CVs) of buffer A, an elution step of 8 CVs with buffer B, a cleaning step of 3 CVs with 0.1 M NaOH, a first re-equilibration step with 5 CVs with buffer B and a second re-equilibration step with 10 CVs of buffer A. An interconnected washing step of 3 CVs was used.
[0425] All steps were run at a flow rate of 2 mL/min.
[0426] The twin column sequential loading process was operated with two different set points A.sub.ICiU=1000 mAU min and A.sub.ICiU=2300 mAU min. In each case the interconnected loading phase was stopped after reaching the set point values and the interconnected wash step was started according to the method described in the preferred embodiments, which includes the following steps:
[0427] (a) measuring the interconnected phase signals A.sub.ICiU corresponding to the effluents of at least two, preferably all columns (i) in an interconnected state upstream and/or downstream positions in at least one cycle or parts of one cycle using the detectors at the columns outlets;
[0428] (b) initiating a control action based on A.sub.ICiU reaching a specific set point value, in this case A.sub.ICiU=1000 mAU min and A.sub.ICiU=2300 mAU min, respectively. As control action, the interconnected loading phase was stopped upon A.sub.ICiU reaching the pre-defined set point values and the interconnected washing step was initiated. The resulting chromatograms are shown in
[0429] The set point may also be determined automatically by a suitable computer software.
[0430] From a process performance optimization point of view it is desirable to achieve an early breakthrough of compound of interest from the upstream column when the columns are loaded in series. In the bottom part of
TABLE-US-00012 LIST OF REFERENCE SIGNS B Disconnected state or phase startup phase of the (batch state) of the sequential sequential loading process loading process Q.sub.feed,BT1L feed flow rate for single IC Interconnected state or phase column operation that is of the sequential loading significantly lower than process maximum desired feed flow t.sub.B duration of the disconnected rate phase of the sequential EV.sub.1H,1 elution volume corresponding loading process to low breakthrough based on t.sub.IC duration of the interconnected a single column breakthrough phase of the sequential curve recorded for Q.sub.feed,IC loading process EV.sub.1L,1 elution volume corresponding t.sub.startup duration of the startup phase to low breakthrough based on of the sequential loading a single column breakthrough process curve recorded for Q.sub.feed,BT1L t.sub.wash,IC duration of the wash phase of EV.sub.1H,X elution volume corresponding the sequential loading process to a high breakthrough based Q.sub.feed feed flow rate, general on a single column Q.sub.feed,IC second flow rate, feed flow breakthrough curve recorded rate during the interconnected for Q.sub.feed,IC phase of the sequential EV.sub.2 elution volume corresponding loading process to low breakthrough based on Q.sub.wash,IC wash flow rate during the a sequentially interconnected interconnected phase of the column breakthrough curve sequential loading process recorded for Q.sub.feed,IC, Q.sub.feed,B first flow rate, feed flow rate multiplied with the safety during the disconnected factor Z. phase of the sequential EV.sub.Y smaller of the two values loading process EV.sub.1H,X, EV.sub.2 Q.sub.feed,startup feed flow rate during the X magnitude of breakthrough, typically 30-90%. by other criteria such as W ratio between Q.sub.feed,BT1L and points of operating parameter Q.sub.feed,IC; Typically 50-90% changes Z safety factor for loading, i column index, detector index typically 60-90% n cycle number V.sub.dead dead volume, volume of A.sub.Bi area confined by interstitial liquid in one breakthrough curve and a column horizontal baseline V.sub.col empty column volume corresponding to plateau C.sub.feed feed concentration signal value of the non- DBC.sub.1H(EV) single column breakthrough adsorbing impurities curve, recorded at Q.sub.feed,IC, as measured at the outlet of a function of the elution column i during the volume disconnected phase B DBC.sub.2(EV) breakthrough curve of two A.sub.ICiU area confined by interconnected columns, breakthrough curve and a recorded at Q.sub.feed,IC as a horizontal baseline function of the elution corresponding to plateau volume signal value of the non- dEV elution volume increment for adsorbing impurities integration measured at the outlet of TL Target load value in the column i by detector i during disconnected phase of the the interconnected phase IC sequential loading process when column i is in the PL Preload value in the upstream position. interconnected phase of the A.sub.ICiD area confined by sequential loading process breakthrough curve and a A.sub.peaki area confined by the elution horizontal baseline peak curve of column i and a corresponding to plateau horizontal baseline signal value of the non- corresponding to the value of adsorbing impurities the equilibrated empty measured at the outlet of column or a baseline defined column i by detector i during the interconnected phase IC system during one cycle when column i is in the through the feed stream(s), downstream position. divided by the total bed A.sub.peaki,max Area A.sub.peaki that corresponds to volume of the the maximum possible load chromatographic columns of the process at 100% yield PLR product loss ratio L Load, corresponds to the amount of new product compound injected into the