Method for chromatography resin slurry determination

Abstract

The present invention relates to a method for determining slurry concentration of a chromatography resin slurry during column packing. The present invention provides a more accurate and consistent method for determining slurry concentration for packing a chromatography column. The method of the present invention utilizes an automated pump to provide a controlled flow rate to consolidate a resin sample. Based on the consolidated resin, a slurry concentration can be determined. The determined slurry concentration can be used to pack the resin in a chromatography column with a highly accurate bed height.

Description

Technical Field

[0001] This invention relates to a method for determining the resin slurry of a chromatographic resin slurry used during column packing. More specifically, the invention provides a more accurate and consistent method for determining the slurry concentration used to pack a chromatographic column, for example, by means of a mode of axial compression. Background of the Invention

[0003] Resin slurry determination is a common method used to determine the amount of chromatographic media present in an aqueous slurry. The resin slurry percentage, or slurry concentration, is used in calculations during column packing operations to determine how much aqueous slurry should be placed in the column to ensure proper operation and target bed height. The slurry concentration must be determined and entered into automated chromatographic packed columns (e.g., [missing information]). The main variables in the column are consistently filled to the correct height and fill factor. In fact, such as... Automated chromatographic packed columns provide a packing operation that is highly automated but relies on accurate slurry concentration for reproducible execution.

[0004] Current methods for determining resin slurry composition include gravity sedimentation, centrifugation, and flow consolidation. These methods obtain representative samples from the resin slurry bulk and subject the samples to forces that induce resin sedimentation, which in turn allows analysis of the ratio of sedimented resin to the supernatant (slurry concentration). However, these methods have several drawbacks. For example, in current methods, discrepancies are frequently observed between the slurry concentration results and the achieved filling factor (defined as the ratio of consolidated bed height to final packed bed height) and column height. Additionally, if various waiting times are employed after centrifugation, high variability in centrifugation results has been shown between different resin types and proportions within the same standard. Gravity sedimentation is considered more consistent, but it can be time-consuming and may require empirically determined and applied correction factors, increasing the uncertainty of the method.

[0005] Recently, GE Healthcare has specifically designed automated filling columns. A recommended consolidation method is provided. This method involves obtaining a representative sample from the resin bulk, loading the sample into a 10 mm (diameter) × 100 mm Tricorn column, and manually operating a syringe to generate consolidation force on the sample. After final consolidation is achieved, the resin slurry is determined based on the resin-to-supernatant ratio. However, this method is largely inconsistent due to variations in the force with which the plunger on the syringe is pressed down.

[0006] Therefore, a more accurate and consistent method is still needed to determine the slurry concentration used for packing chromatographic columns, such as automated column packing. Summary of the Invention

[0007] A method is provided for determining the resin slurry percentage or slurry concentration of a chromatographic column. The method of the present invention provides a highly accurate slurry concentration, which in turn provides a precise target bed height when the resin is packed into a chromatographic column, such as an automated packed column.

[0008] In one embodiment, the present invention provides a method for determining a slurry concentration, the method comprising: adding a slurry comprising a resin to a first chromatographic column; pumping a volume of liquid (e.g., deionized water, distilled water, purified water, salt solution, or organic solvent) through the chromatographic column at a constant flow rate to form a solidified resin, wherein the volume of liquid is pumped through the chromatographic column by an automated pumping system; measuring the bed height of the solidified resin after the pumping step is completed; and determining the slurry concentration based on the measured bed height. In some embodiments, the method may further include the step of adding water to the chromatographic column prior to pumping and allowing the slurry to settle for approximately five minutes to approximately one hour. In other embodiments, the method may further include filling a second chromatographic column with the determined slurry concentration. In this aspect, the second chromatographic column includes a solidification rate, and the flow rate is equal to the solidification rate. In another embodiment, the automated pumping system may include a syringe operatively coupled to the pump. In some embodiments, the pumping system is programmed to cycle on and off over a predetermined time period or multiple time periods.

[0009] In another embodiment, the present invention provides a method for determining the slurry concentration of a chromatographic resin slurry during column packing, the method comprising: adding a slurry comprising resin to a chromatographic column; setting an automatic pump to operate at a constant flow rate for a period of time, e.g., from pump start-up to resin settling; pumping a volume of liquid through the chromatographic column at a constant flow rate to form solidified resin, wherein the volume of liquid is pumped through the chromatographic column by the automatic pump; measuring the bed height of the solidified resin after the pumping step is completed; and determining the slurry concentration based on the measured bed height. In some embodiments, the method may include a step of providing a flow rate based on the type of resin used in the slurry. The flow rate may be in the range of about 20 cm / hr to about 1500 cm / hr. In other embodiments, the method may include a step of mixing the slurry before adding the slurry to the chromatographic column. In yet another embodiment, the method may include a step of packing resin into a second chromatographic column using the determined slurry concentration, wherein the second chromatographic column includes a solidification rate and the flow rate is equal to the solidification rate. In another embodiment, the liquid may be deionized water, distilled water, purified water, salt solution, or organic solvent.

[0010] In another embodiment, the invention includes a method for determining the slurry concentration of a chromatographic resin slurry during column packing, the method comprising: providing a slurry comprising resin; mixing the slurry to uniformly distribute the resin; adding the slurry to a chromatographic column; providing an automated injection pump system including a pump having a controller and an injector operatively coupled to the pump, wherein the automated injection pump system is configured to pump a volume of water through the chromatographic column, and the controller is configured to control the flow rate of the volume of water such that the flow rate is constant over a period of time; pumping a volume of water through the chromatographic column at a constant flow rate to form a solidified resin; measuring the bed height of the solidified resin after the pumping step is completed; determining the slurry concentration based on the measured bed height; inputting the determined slurry concentration into a second chromatographic column; and packing the resin into the second chromatographic column using the determined slurry concentration. In some embodiments, the flow rate is in the range of about 30 cm / hr to about 60 cm / hr. In another embodiment, the second chromatographic column includes a solidification rate, and the flow rate is equal to the solidification rate. In other embodiments, the second column includes a consolidation rate, and the flow rate is different from the consolidation rate. In other embodiments, the packed second column has a bed height within 1 cm of the target bed height. Attached Figure Description

[0011] Other features and advantages of the invention can be identified from the following detailed description provided in conjunction with the accompanying drawings:

[0012] Figure 1 This is a flowchart of a method for determining resin slurry according to the present invention.

[0013] Figure 2A and Figure 2B It is based on the invention and Figure 1 A schematic diagram of the associated system for determining resin slurry; and

[0014] Figure 3 This is a bar chart showing the average sedimentation percentage of the resin after centrifugation.

[0015] Figure 4 It is a bar chart, thus showing the percentage error for determining the resin slurry by centrifugation (shaded bars) and the current claimed process for various types of resins (filled bars).

[0016] Figure 5This is a bar graph showing the relationship between the achieved bed height and the ideal bed height obtained using centrifugation, the GE slurry kit, or the method according to the invention. In each group of three columns, the first column represents centrifugation, the middle column represents the GE slurry kit, and the last of the three columns represents the results using the disclosed method for various types of resins. The horizontal solid line represents the target bed height for individual resin filling. The horizontal dashed line represents the bed height limit (±1 cm) before filling beyond the acceptable bed height range. The X-axis represents the type of resin slurry, and the Y-axis represents the bed height (cm).

[0017] Figure 6 This is a bar graph showing the theoretical bed heights achieved by different operators using the GE kit for various resin types. In each group of three columns, the first column represents centrifugation, the middle column represents the GE slurry kit, and the last column represents the results using the disclosed methods for various resin types. In each group of three columns, the first column represents the initial theoretical final bed height, the middle column represents the theoretical final bed height including pauses, and the last column represents the theoretical final bed height with variable forces. The horizontal solid line represents the ideal bed height for individual resin filling. The horizontal dashed line represents the bed height limit (±1 cm) before filling beyond the acceptable bed height range. The X-axis represents the type of resin slurry, and the Y-axis represents the bed height (cm).

[0018] Figure 7 This is a bar chart showing the theoretical bed height of MabSelect SuRe resin at different flow rates that can be experienced using the uncontrolled flow GE kit. In each group of two columns, the solid line represents the theoretical bed height after the first flow, and the white line represents the theoretical bed height after the second flow. The X-axis represents the flow rate (ml / min), and the Y-axis represents the theoretical bed height (cm). The horizontal solid line represents the ideal bed height for individual resin filling. The horizontal dashed line represents the bed height limit (±1cm) before filling beyond the acceptable bed height range. Detailed Implementation

[0019] This invention relates to a method for determining the resin slurry percentage or slurry concentration of a chromatographic column slurry. More specifically, the invention utilizes an automated pump system to provide a controlled flow rate to solidify the resin sample. Based on the solidified resin sample, the slurry concentration can be determined and used to fill a second chromatographic column with the same resin. It has been found that by controlling the delivery flow rate to match the solidification rate used on the column to be filled with resin, a highly reproducible slurry concentration corresponding to the slurry concentration required for accurate filling of the second chromatographic column without correction factors can be achieved.

[0020] This invention offers numerous advantages over conventional slurry determination methods, including centrifugation, gravity settling, and manual injection. Compared to any of the conventional slurry determination methods, the methods and systems of this invention can be assembled, executed, and disassembled in a shorter timeframe. Furthermore, the methods of this invention have been shown to produce more consistent and reproducible slurry concentration results than conventional slurry determination methods, with lower standard deviations between repeated measurements.

[0021] Figure 1 A flowchart of a method for determining resin slurry according to one embodiment of the present invention is shown. In step 101, the assembly of the column and pump is performed. Figure 2A and Figure 2B Various apparatuses for determining the percentage of resin slurry according to the present invention are shown. For example... Figure 2A As shown, system 200 includes a column 201, an automatic pump system 204 including a pump 203 with a controller 206, and a flow-through collection container 205. Pump 203 provides a liquid flow to column 201. In this respect, column 201 is vertically oriented, and pump 203 is operatively connected to the top portion of column 201 such that liquid flows from pump 203 into column 201. The liquid flow is provided using automatic pump 203. Pump 203 includes controller 206, which is operable to start and stop pump 203 and define the flow rate of liquid leaving pump 203. The system of the present invention also includes flow-through collection container 205, which is operatively connected to the bottom portion of column 201. Flow-through collection container 205 is designed to collect liquid flowing through column 201.

[0022] In another implementation, such as Figure 2B As shown, the automatic pump system 204 may further include a syringe 202 operatively coupled to the pump 203. The syringe 202 can supply a flow of liquid to the column 201 using the automatic pump 203. In one embodiment, a controller 206 of the pump 203 is operable to define the flow rate of liquid exiting the syringe 202. In this respect, the column 201 is vertically oriented, and the syringe 202 is operatively connected to the top portion of the column 201 such that liquid flows from the syringe 202 into the column 201.

[0023] Column 201 can be operatively connected to pump 203 or syringe 202 and flow-through collection container 205 via any connecting member, which allows liquid to flow from pump 203 or syringe 202 into column 201 and outward from column 201 into flow-through collection container 205. For example, column 201 can be operatively connected to pump 203 or syringe 202 and flow-through collection container 205 via tubing. In this respect, a single tubing can connect pump 203 or syringe 202 to the top of column 201, and a separate tubing can connect the bottom of column 201 to flow-through collection container 205.

[0024] The type and size of column 201 can vary. In one embodiment, column 201 is a chromatographic column. In another embodiment, column 201 is an analytical-scale chromatographic column. For example, column 201 could be a 1 cm × 25 cm column. Analytical-scale chromatographic columns are advantageous for use with this invention because such columns are small, easy to transport, and provide sufficient vertical resolution to perform accurate slurry ratio readings.

[0025] The type and size of the pump system 204 can vary as long as the pump 203 can provide a constant liquid flow rate to the column 201. In one embodiment, the pump 203 may include any automatic or mechanical pump that delivers a precise flow starting from 0.2 mL / min. For example, the pump 203 may include any type of automatic injector with a fully automatic system. In another embodiment, as described above, the pump 203 may include a syringe 202 that provides a liquid flow to the column 201. In this respect, the syringe 202 should have a volume of at least 20 mL. In another embodiment, the syringe 202 may have a volume of at least 30 mL. In another embodiment, the syringe 202 may have a volume of 20 mL to 60 mL. The flow-through collection container 205 may be any container operable to receive the flow from the column 201. Suitable flow-through collection containers 205 include, but are not limited to, beakers, flasks, graduated cylinders, test tubes, bottles, and wide-mouth bottles.

[0026] After assembling system 200, a slurry comprising resin is added to column 201 (step 102). The present invention contemplates any resin suitable for chromatographic columns. In some embodiments, the present invention contemplates the use of resins suitable for protein purification. Examples of resins contemplated by the present invention include, but are not limited to: rProtein A Fast Flow, MabSelect TM Xtra, MabSelect SuRe TM Capto TM Phenyl, Q Fast Flow, Phenyl HP 50HS EMD Hicap SE, Capto TM Q and 200HR.

[0027] The resin should be thoroughly mixed in the slurry to ensure uniform distribution before being added to column 201. Any technique known in the art can be used to add the mixed resin slurry to column 201. In one embodiment, the resin slurry can be pipetted into column 201, for example, using a pipette controller. The amount of resin slurry added will depend on the desired sedimentation resin bed volume. In one embodiment, the slurry can be added to column 201 until the meniscus is at a height of approximately 23 cm to approximately 25 cm. For example, the slurry can be added to column 201 until the meniscus is at a height of approximately 25 cm. In this respect, increased accuracy is achieved when utilizing the large amount of available space within the column. After adding the slurry to the column, the final height of the slurry should be determined and recorded for later use. After adding the resin slurry to column 201 and determining the height of the resin slurry, purified water can be added to column 201 until the water approaches the top of column 201. This allows for the installation of a top adapter or piston on column 201 and the venting of air, which is important for laminar flow. The resin can then be allowed to settle for a period of time. In one embodiment, the slurry can be allowed to settle for about five minutes to about one hour. In another embodiment, the slurry can be allowed to settle for about 20 minutes to about 45 minutes. For example, the slurry can be allowed to settle for about 30 minutes.

[0028] In step 103, pump system 204 is configured to provide a liquid flow at a constant rate. In this regard, a controller 206 for pump 203 can be configured to define the flow rate of the liquid exiting pump 203. The liquid flow rate is selected based on the specific resin used in the slurry (e.g., the physical properties of the resin, such as bead size, bead matrix, and matrix density) and the consolidation rate employed by a second column (e.g., an autopacked column), which will be filled with the resin used for the chromatographic operation. In one embodiment, the liquid flow rate can be selected such that the flow rate is equal to the consolidation rate of the second column (e.g., an autopacked column). In practice, by controlling the liquid flow rate to match the consolidation rate employed by the column to be resin-filled, a highly reproducible slurry concentration corresponding to the slurry concentration required for precise filling of the second chromatographic column without correction factors can be achieved. However, a highly reproducible slurry concentration can still be achieved when the liquid flow rate differs from the consolidation rate. Therefore, in some embodiments, the liquid flow rate may not be equal to the consolidation rate of the second column.

[0029] The flow rate of the liquid can vary depending on the column, resin, and filling technology used. In one embodiment, the flow rate can range from about 20 cm / hr to about 1500 cm / hr. In another embodiment, the flow rate can range from about 30 cm / hr to about 1000 cm / hr. In another embodiment, the flow rate can range from about 50 cm / hr to about 600 cm / hr. In yet another embodiment, the flow rate can range from about 60 cm / hr to about 200 cm / hr. In some embodiments, the flow rate can be about 30 cm / hr. In other embodiments, the flow rate can be about 60 cm / hr.

[0030] In step 104, pump system 204 is activated so that a liquid flow is pumped from pump 203 to and through column 201. In one embodiment, the liquid pumped through column 201 is water. For example, water may be distilled, deionized, and / or purified. In another embodiment, the liquid pumped through column 201 may be a salt solution. In yet another embodiment, the liquid pumped through column 201 may be any organic solvent. For example, the liquid may be an alcohol. Suitable alcohols include, but are not limited to, ethanol, propanol, butanol, pentanol, hexanol, heptanol, and octanol. In this respect, to provide more general and consistent results, the liquid pumped through column 201 should be the same liquid used during the filling operation of the selected resin.

[0031] The controller 206 of pump 203 can be used to initiate automatic liquid flow from pump 203 to column 201. As discussed above, a liquid flow is pumped from pump 203 to and through column 201 at a selected flow rate, which remains constant throughout step 104. Pumping of the liquid flow should continue until the resin stops settling. In one embodiment, the liquid can be pumped through column 201 for a period of about 10 minutes to about 50 minutes. In another embodiment, the liquid can be pumped through column 201 for a period of about 15 minutes to about 40 minutes. In yet another embodiment, the liquid can be pumped through column 201 for a period of about 15 minutes to about 30 minutes. For example, in some embodiments, the liquid can be pumped through column 201 for about 30 minutes when the liquid flow rate is about 30 cm / hr. In other embodiments, the liquid can be pumped through column 201 for about 15 minutes when the liquid flow rate is about 60 cm / hr. In some implementations, the flow rate is stopped or paused for one or more time periods. For example, the flow rate can be paused for 1 to 5 minutes, 5 to 10 minutes, 10 to 20 minutes, 20 to 30 minutes, 40 to 50 minutes, or for one or several hours. The flow rate can be restarted after each pause. In some implementations, stopping the liquid flow is unnecessary to achieve accurate readings. For example, the system can be in a continuous solidified flow state for more than 1 hour. This is an additional benefit of automated flow and is not possible when using GE reagent kits. Controller 206 can be used to stop the automated flow of liquid from pump 203 to column 201.

[0032] In one embodiment, the method includes programming an automatic pump to stop the flow of liquid through the column and allow resin settling, and then restarting the pump to allow liquid flow through the column before the pumping step is complete. The pumping pause can be for a period of time longer than one time period. In one embodiment, the pumping pause is for a period of 1 minute to 60 minutes. In some embodiments, the pump is programmed to pump intermittently. In one embodiment, for all consolidation rate resins of 60 cm / hr, the cycle is 15 minutes flow > 5 minutes pause > 5 minutes additional flow. Consolidation at 30 cm / hr will require twice the flow duration. In some embodiments, result readings are obtained immediately before or after the final process operation ends.

[0033] After the flow of the liquid is stopped, the resin bed can be allowed to stabilize for a period of time without flow. In one embodiment, after the flow of the liquid is stopped, the resin bed can be allowed to stabilize for about 10 minutes to about 45 minutes. For example, the resin bed can be allowed to stabilize for about 30 minutes. In another embodiment, after the flow of the liquid is paused, the resin bed can be allowed to stabilize for 5 minutes to 10 minutes, and then the injection pump system 204 can be restarted at the same flow rate for an additional 5 minutes. In this respect, performing sequential flow can minimize variability. In step 105, the height of the settling / consolidating resin bed can be measured. Any measuring device that allows for accurate reading of the bed height can be used to perform the measurement step. In some embodiments, column 201 may include a scale vertically positioned on the outer surface. After measuring the height of the consolidated resin bed, the slurry concentration can be determined based on the measured bed height (step 106). The measured slurry concentration (expressed as a percentage) is given directly from the bed height (in centimeters).

[0034] After determining the slurry concentration of the resin to be used, this slurry concentration can be used to pack the second column. The second column can be any chromatographic column, including an automatically packed column. For example, an automatically packed column could be... Column. In this respect, the slurry concentration can be input into the automatically filled column. In some embodiments, the operator can manually input the determined slurry concentration into the filled column. In other embodiments, the determined slurry concentration can be automatically input into the filled column using a network or hardware circuit that connects system 200 to the automatically filled column. Regardless of the type of column used for filling the resin, the determined slurry concentration is highly reproducible and corresponds to the slurry concentration required for accurately filling the second column.

[0035] The method of the present invention provides a highly accurate slurry concentration, which in turn provides a precise target bed height when the resin is packed into a chromatographic column. In one embodiment, column packing with the slurry concentration determined according to the present invention achieves a bed height within 2 cm of the target bed height. In another embodiment, column packing with the slurry concentration determined according to the present invention achieves a bed height within 1 cm of the target bed height. In another embodiment, column packing with the slurry concentration determined according to the present invention achieves a bed height within 0.75 cm of the target bed height. In another embodiment, column packing with the slurry concentration determined according to the present invention achieves a bed height within 0.50 cm of the target bed height. For example, column packing with the slurry concentration determined according to the present invention achieves a bed height within 0.10 cm of the target bed height.

[0036] Furthermore, when the resin is packed into the chromatographic column, a highly accurate packing factor is achieved using column packing with a slurry concentration determined according to the present invention. For example, a packing factor within 5% of the ideal packing factor is achieved using column packing with a slurry concentration determined according to the present invention. In another embodiment, a packing factor within 3% of the ideal packing factor is achieved using column packing with a slurry concentration determined according to the present invention.

[0037] Example

[0038] The following non-limiting examples illustrate a method for determining slurry concentration according to the present invention. These examples are merely illustrative of preferred embodiments of the invention and should not be construed as limiting the scope of the invention, which is defined by the appended claims.

[0039] Invention Example 1

[0040] A method for determining slurry concentration was performed according to the present invention. The method was performed using a 1 cm × 25 cm chromatographic SNAP column. A KD Scientific syringe pump was used to provide a controlled flow rate to solidify the resin sample. The following resin was tested: MabSelect SuRe. TM , 50HQ and Capto TM Phenyl.

[0041] Table 1 shows the results of the method performed according to the present invention.

[0042] Table 1: Results of the Inventive Method

[0043]

[0044]

[0045] As can be seen from the data in Table 1, the method of the present invention demonstrates consistent and reproducible results, with low standard deviation between repeated measurements. The slurry concentration obtained according to the present invention results in an average bed height that differs from the target bed height by 0.4 cm or less. The low standard deviation indicates that the results are highly reproducible.

[0046] Comparison Example 2

[0047] Centrifugal separation

[0048] It utilizes centrifuge tubes with 13mL capacity. A 6KR centrifuge was used. The slurry was adjusted to a 50% ratio by removing the supernatant before centrifugation. Centrifugation of 10 mL slurry samples was performed at 1000 rpm for 5 minutes and at 3500 rpm for 15 minutes. Table 2 below shows the resins used.

[0049] Table 2: Resins tested

[0050]

[0051] The resin failed to solidify properly.

[0052] Figure 3 The results of the centrifugation method were demonstrated. More specifically, Figure 3 The average sedimentation percentage is shown for centrifugation at 1000 rpm for 5 minutes and at 3500 rpm for 15 minutes. (As shown from...) Figure 3 It can be seen that the results of centrifugation are variable among resins and consistently overestimate the known 50% resin volume. This indicates that centrifugation is not a sufficient sedimentation method.

[0053] fluid solids

[0054] The AKTA Avant chromatography system (with a 2.5cm × 50cm SNAP column) was used for simulation. The consolidation effect of the technology. All resins were consolidated at 60 cm / hr. The resins shown in Table 2 were also used for the fluidization test. The resins were allowed to settle by gravity overnight. Before fluidization, the slurry was adjusted to a 50% ratio by removing the supernatant.

[0055] Table 3 below shows the average fluid-solid results between round 1 and round 2.

[0056] Table 3: Average fluid-solid results

[0057]

[0058] As can be seen from Table 3, the consolidation results are variable between resins, and there is no consolidation that returns to the original 50% ratio. This indicates that gravity is not a sufficient settling method.

[0059] Comparison of results from centrifugation and consolidation methods

[0060] Table 4 below compares the results of each of the centrifugal separation method and the fluid-solid method.

[0061] Table 4: Comparison of centrifugation results and consolidation results

[0062]

[0063] As can be seen from the table above, there is a significant difference between the consolidation and centrifugation results. Six out of the eight resins exhibited a difference greater than 5% between the consolidation reading and the initial centrifugation reading. This difference remained even after a given extended duration of 2 hours. In fact, after 2 hours, three out of the eight resins still showed a difference greater than 5% between the consolidation and sedimentation centrifugation readings. This indicates that due to the variability of the test, the long sedimentation time, and the variations between resins, consolidation and centrifugation are insufficient to determine the slurry concentration.

[0064] In addition, several readings did not meet the target fill factor. To calculate the target fill factor, 5% of 20cm (target bed height) equals 1cm, which is the maximum permissible bed height range. A difference greater than 5% indicates that the reading did not meet the target fill factor. As can be seen from the data above, several readings did not meet the target fill factor.

[0065] Comparison Example 3

[0066] Table 5 below illustrates a comparison of centrifugation slurry percentage and final bed height between the centrifugation method and the GE kit.

[0067] Table 5: Comparison of centrifugation results and GE reagent kit results

[0068]

[0069] As can be seen from Table 5, in several of the tested resin types, neither the centrifugation method nor the GE reagent kit reached the target bed height, indicating inconsistency in the methods.

[0070] Compare the results of centrifugation, GE reagent kit and the method according to this disclosure.

[0071] Figure 4The percentage error of slurry concentration was compared between the centrifugal separation method and the method according to this disclosure. Several different types of resins were tested, including MabSelect SuRe, POROS50HQ, Capto Phenyl, Q Sepharose FF, and MabSelect SuRe PCC. For all resin types, the method according to this disclosure showed a significant reduction in error compared to the centrifugal separation method. Additionally, for several of the tested resin types, the centrifugal separation method had an error percentage reaching the 5% error limit, indicating that the filling compromised the ideal fill factor to achieve the minimum permissible height requirement. A compromised fill factor can lead to column degradation and reduced column reuse. In some embodiments, the column must meet final desired metrics, including desired height and fill factor. As the column consolidates, the height decreases and the fill factor increases. In one embodiment, the column achieves a height of 19 cm to 21 cm and is targeted for compression.

[0072] Figure 5 A direct comparison of bed heights achieved using centrifugation, a GE reagent kit, and the method according to this disclosure is shown. Figure 5 As demonstrated in the study, the method according to this disclosure is superior to other methods tested. The bed height achieved by the method according to this disclosure is closer to the ideal bed height compared to other methods tested. Furthermore, the bed heights obtained by centrifugation and the GE kit reach or exceed error limits, which indicate that the bed filling exceeds the acceptable bed height range and will require refilling.

[0073] GE Reagent Kit Operator Variability Testing and Limit Assessment

[0074] To enable operator variability in accessing currently available GE reagent kits, operators are provided with incremental guidance and experience on how to use the system, and measurements are performed from the batch solution after each increment. Figure 6 The results of the operator variability test are shown. The data indicate that the GE kit experiences operator variability, variability due to familiarity and experience, and variability due to the material being tested. This also demonstrates that the GE kit lacks sufficient control to produce consistent results in a dynamic manufacturing environment.

[0075] To assess the impact of uncontrolled flow rates used in currently commercially available GE kits, an experiment was conducted based on potential operator-mandated variations in flow rates during sample processing. Figure 7 The experimental results are shown, in which water flows through the column for the first time and the bed height measurement is recorded. Water is then allowed to flow through the column a second time, and the second bed height measurement is recorded. Figure 7The study shows that flow rates exceeding the recommended range can lead to potentially unacceptable changes in bed height.

[0076] While the numerical ranges and parameters illustrating the broad scope of the invention are approximate, the values ​​described in specific examples are reported as precisely as possible. However, any numerical value inherently contains some error, which must be caused by the standard deviation found in their corresponding test measurements. Furthermore, when numerical ranges of varying ranges are described herein, it is contemplated that any combination of these values, including those listed, can be used. In fact, all ranges disclosed herein are inclusive and composable. For example, all ranges include the endpoints of the range and all intermediate values.

[0077] The scope of the invention described and claimed herein is not limited to the specific embodiments disclosed herein, as these embodiments are intended to illustrate several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. In fact, various modifications to the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. All patents and patent applications cited above are expressly and integrally incorporated herein by reference.

Claims

1. A method for determining slurry concentration, the method comprising: A slurry containing resin is added to the first chromatographic column; A certain volume of liquid is pumped through the first chromatographic column at a constant flow rate; The flow rate is controlled to match the consolidation rate used by the second column to form a consolidated resin in the first column, wherein the volume of liquid is pumped through the first column by an automatic pump system, wherein the automatic pump system is programmed to cycle on and off within a predetermined time period or multiple time periods. After the pumping step is completed, the bed height of the solidified resin in the first chromatographic column is measured. The slurry concentration is determined in the first chromatographic column based on the measured bed height; and the second chromatographic column is filled with the slurry concentration determined in the first chromatographic column, wherein the filled second chromatographic column has a fill factor within 5% of the ideal fill factor.

2. The method of claim 1, wherein the liquid is deionized water, distilled water, purified water, salt solution or organic solvent.

3. The method of claim 1, wherein the step of adding the slurry further comprises the following steps: Water is added to the first chromatographic column before pumping and the slurry is allowed to settle for five minutes to one hour.

4. The method of claim 1, wherein the second chromatographic column includes a consolidation rate, and wherein the flow rate of the first chromatographic column is equal to the consolidation rate of the second chromatographic column.

5. The method of claim 1, wherein the automatic pump system includes an injector operatively coupled to the pump.

6. The method of claim 1, wherein the packed second chromatographic column has a bed height within 1 cm of the target bed height.

7. A method for determining the slurry concentration of a chromatographic resin slurry during column packing, the method comprising: A slurry containing resin is added to the first chromatographic column; Set the automatic pump to run at a constant flow rate for a period of time; A volume of liquid is pumped through the first column at a constant flow rate matching the consolidation rate used in the second column to form a consolidated resin in the first column, wherein the volume of liquid is pumped through the first column by the automatic pump, wherein the automatic pump is programmed to cycle on and off within a predetermined time period or multiple time periods. After the pumping step is completed, the bed height of the consolidated resin in the first chromatographic column is measured. The slurry concentration is determined based on the bed height measured in the first chromatographic column; and The second chromatographic column is filled with the slurry concentration determined in the first chromatographic column, wherein the filled second chromatographic column has a fill factor within 5% of the ideal fill factor.

8. The method of claim 7, further comprising pausing the automatic pump to stop the liquid flow through the first chromatographic column and allow the resin to settle, and restarting the pump to allow liquid flow through the first chromatographic column before completing the pumping step.

9. The method of claim 8, wherein the pumping is paused for more than one time period.

10. The method of claim 8 or 9, wherein the pumping is paused for a period of 1 minute to 60 minutes.

11. The method of claim 7, wherein the step of setting the automatic pump further comprises providing a flow rate based on the type of resin used in the slurry and the consolidation rate employed by the second chromatographic column.

12. The method of claim 11, wherein the flow rate is in the range of 20 cm / hr to 1500 cm / hr.

13. The method of claim 7, further comprising mixing the slurry before adding it to the first chromatographic column.

14. The method of claim 7, wherein the liquid is deionized water, distilled water, purified water, salt solution or organic solvent.

15. The method of claim 7, wherein the packed second chromatographic column has a bed height within 1 cm of the target bed height.

16. A method for determining the slurry concentration of a chromatographic resin during column packing, the method comprising: Provide slurries including resin; The slurry is mixed to ensure that the resin is evenly distributed; The slurry is added to the first chromatographic column; An automated injection pump system is provided, the automated injection pump system including a pump having a controller and a syringe operably coupled to the pump, wherein the automated injection pump system is configured to pump a volume of water through a first chromatographic column, and the controller is configured to control the flow rate of the volume of water to match the consolidation rate employed by a second chromatographic column, wherein the flow of the volume of water will fill the resin in the first chromatographic column. The volume of water is pumped through the first chromatographic column at a constant flow rate to form a solidified resin, wherein the automatic injection pump system is programmed to cyclically turn on and off within a predetermined time period or multiple time periods. After the pumping step is completed, the bed height of the consolidated resin in the first chromatographic column is measured. The slurry concentration is determined based on the measured bed height of the first chromatographic column; The determined slurry concentration is input into the automated injection pump system; and the resin is filled into the second chromatographic column using the determined slurry concentration in the first chromatographic column, wherein the second chromatographic column has a fill factor within 5% of the ideal fill factor.

17. The method of claim 16, wherein the flow rate is in the range of 30 cm / hr to 60 cm / hr.

18. The method of claim 16, wherein the second chromatographic column includes a consolidation rate, and the flow rate is equal to the consolidation rate.

19. The method of claim 16, wherein the packed second column includes a consolidation rate, and the flow rate of the first column is the same as the consolidation rate of the second column.

20. The method of claim 16, wherein the packed second chromatographic column has a bed height within 1 cm of the target bed height.

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