CN114026108A - Seamless continuous flow chromatography method - Google Patents
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Abstract
The invention provides a seamless continuous flow chromatography method, which uses at least three chromatography devices, wherein at least two chromatography devices form a first-type chromatography device, at least one chromatography device forms a second-type chromatography device, the first-type chromatography device is used for realizing continuous flow chromatography, and the first-type chromatography device and the second-type chromatography device jointly carry out seamless connection chromatography. According to the seamless continuous flow chromatography method, the intermediate product does not need to be processed, the product after chromatography can be directly obtained, the process time is shortened, the production efficiency is improved, and the production cost is reduced.
Description
Reference to related applications
The present invention claims priority from an inventive patent application entitled "seamless continuous flow chromatography method" with application number 201910574121.X, filed in china on 28.6.2019, which is incorporated herein by reference in its entirety.
The invention relates to the technical field of separation and purification, in particular to the purification of biological agents, particularly relates to a chromatography method by using a chromatography device, and particularly relates to a seamless continuous flow chromatography method.
In the traditional chromatography process, an intermediate product generated after the previous step of chromatography is collected and uniformly mixed, and then the intermediate product is loaded for the next step of chromatography. Generally, the intermediate product needs to be treated by filtration, low-temperature storage, pH adjustment and the like, so that equipment resources are occupied; and a plurality of chromatographic processes need to be carried out one by one, so that the process time is long.
For example, referring to fig. 1, three working areas (P1, P2, P3) respectively perform the harvesting of cell fluid, chromatography and the obtaining of product Pr. Cell culture harvest from the first processing area P1 is collected for temporary storage in storage vessel T1. The harvest in the storage container T1 is then subjected to a first chromatography on column C1, and after elution on column C1, the captured intermediate product is stored temporarily in storage container T2. Then the intermediate product in the storage container T2 is subjected to a second chromatography through the chromatographic column C2, and after the chromatographic column C2 is eluted, the captured intermediate product is temporarily stored in the storage container T3. Then the intermediate product in the storage container T3 is subjected to a third chromatography through the chromatographic column C3, and after the chromatographic column C3 is eluted, the captured intermediate product is temporarily stored in the storage container T4. This requires not only the use of a plurality of buffer containers of large capacity, but also takes up additional buffer time.
Patent publication WO2014/180852a1 discloses a continuous flow chromatography method with which simultaneous operation of a plurality of chromatography processes can be achieved, but the use efficiency of chromatography packing is not improved, the overall process time is long (more than 30 hours), the reduction of production cost and the improvement of production efficiency are limited.
Disclosure of Invention
The present invention aims to overcome or at least alleviate the above-mentioned drawbacks of the prior art and to provide a seamless continuous flow chromatography method with seamless global connection of local continuous flow.
The present invention provides a seamless continuous flow chromatography process using at least three chromatography devices, wherein at least two chromatography devices constitute one type of chromatography device and at least one chromatography device constitutes a second type of chromatography device,
the chromatography device of the described type is used to carry out continuous flow chromatography,
the first-class chromatography device and the second-class chromatography device jointly realize seamless connection chromatography.
In at least one embodiment, no storage vessel is used between the one type of chromatography device and the second type of chromatography device.
In at least one embodiment, prior to the continuous flow chromatography, the preparation to be chromatographed is subjected to viral inactivation, preferably by an organic solvent detergent method.
In at least one embodiment, prior to the continuous flow chromatography, the preparation to be chromatographed is subjected to depth filtration.
In at least one embodiment, the start time and the end time of loading of each of the chromatographic devices of the one type are different; each of the chromatographic devices of one type can operate simultaneously; wherein the flow-through product of the sample from one of said chromatographic devices of one type can be captured by another of said chromatographic devices of one type; the chromatographic device of the type is continuously or discontinuously loaded and continuously or discontinuously collected.
In at least one embodiment, the type of chromatography device is used to capture a target product, and the purity of the target product in the capture harvest of the type of chromatography device is greater than 80%.
In at least one embodiment, the type two chromatography apparatus is used for fine purification, and the purity of the target product in the collection liquid of the type two chromatography apparatus is greater than 90%.
In at least one embodiment, the second type of chromatography device completes equilibration and waits for sample inflow before the first type of chromatography device collects product, and the chromatography process of the second type of chromatography device is initiated during the chromatography process performed by the first type of chromatography device.
In at least one embodiment, the chromatographic devices are chromatography columns, the packing of each of the one type of chromatographic device is the same, the packing of each of the two type of chromatographic devices is different, and the packing of each of the two type of chromatographic devices is different from the packing of the one type of chromatographic device; or
The chromatographic devices are membrane chromatographic devices, the chromatographic membranes of the first-class chromatographic devices are the same, the chromatographic membranes of the second-class chromatographic devices are different, and the chromatographic membranes of the second-class chromatographic devices are different from the chromatographic membranes of the first-class chromatographic devices.
In at least one embodiment, the one type of chromatography device is an affinity chromatography device and the ligand is protein a.
In at least one embodiment, the method is used to purify a biological agent, preferably an antibody.
In at least one embodiment, the method comprises:
s11, carrying out biological reaction to obtain cell culture harvest liquid as a reaction preparation;
s12, centrifuging the reaction preparation;
s13, precipitating or flocculating the reaction preparation;
s14, carrying out deep filtration on the reaction preparation;
s15, performing S/D virus inactivation on the reaction preparation;
s21, performing the continuous flow chromatography on the reaction preparation using the one type of chromatography device;
s22, performing the seamless connection chromatography on the reaction preparation by using the second type chromatography device;
s30, collecting the purified reaction preparation;
s31, performing virus removal and filtration on the reaction preparation;
s32, performing nanofiltration and ultrafiltration on the reaction preparation;
and S33, obtaining the product Pr.
In at least one embodiment, the type of chromatography device performs affinity chromatography, wherein the affinity chromatography is eluted at a pH of 3.2 to 4.0 and a conductivity of 30mS/cm or less.
In at least one embodiment, the second type of chromatography device performs cation chromatography, and the pH is controlled to be 5.0 to 8.0 when the cation chromatography is eluted.
According to the seamless continuous flow chromatography method, the intermediate product does not need to be processed, the product after chromatography can be directly obtained, and the process time is shortened. And the local continuous flow improves the use efficiency of the chromatographic packing, reduces the use cost of the packing and can indirectly reduce the equipment scale of the chromatographic column and the buffer storage tank. The method improves the production efficiency and reduces the production cost on the whole.
FIG. 1 is a schematic process flow diagram of a conventional multi-chromatography in the prior art.
FIG. 2 is a process flow diagram of a seamless continuous flow chromatography method according to a first embodiment of the invention.
Description of the reference numerals
P1, P2, P3 work area;
s11 biological reaction; s12 centrifuging; s13 precipitation or flocculation; s14 deep filtration; S15S/D virus inactivation; s31 virus removal and filtration; s32 nano-filtration and ultrafiltration;
c1, C2 and C3 chromatographic columns; a CC1 affinity continuous flow chromatography system; a C02 cation column; a C03 anion column;
t1, T2, T3, T4, T storage containers;
pr product.
Exemplary embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the detailed description is intended only to teach one skilled in the art how to practice the invention, and is not intended to be exhaustive or to limit the scope of the invention.
The seamless continuous flow chromatography method according to the invention uses at least three chromatography columns, wherein at least two chromatography columns (also called a type of chromatography device) are used for performing local continuous flow chromatography, the packing of the two chromatography columns being identical (e.g. for capturing the same first component); at least one further chromatography column (also called a second type of chromatography device, which is different from the packing of the first type of chromatography device, e.g. for capturing a second component or for fine purification) is connected in series with the local continuous flow chromatography system for achieving an integrated seamless connection chromatography.
In each type of chromatographic device of the local continuous flow chromatographic system, the time sequence of the chromatographic processes of at least two chromatographic devices is different, namely the starting time and the ending time (the starting time and the ending time) of each process are not completely the same; preferably, the start-stop time of loading is not exactly the same for each type of chromatographic device.
Each type of chromatography device of the local continuous flow chromatography system corresponds to a plurality of small chromatography columns which can perform chromatography operation without mutual influence, so that the purification amount of the local continuous flow chromatography completed in unit time is larger than the purification amount of a large chromatography column formed by the sum of the fillers of the plurality of small chromatography columns.
The chromatographic products of each first-class chromatographic device are conveyed to a downstream second-class chromatographic device, and a storage container is not needed between the first-class chromatographic device and the second-class chromatographic device for temporarily storing the preparation by reasonably arranging the chromatographic process time sequence of each first-class chromatographic device.
Since no intermediate storage vessel is required in the seamless continuous flow chromatography process, the process of organic solvent/detergent virus inactivation (S/D virus inactivation) needs to be advanced before the chromatography process. In addition, the harvesting process prior to the chromatography process may also include depth filtration. Preferably, the harvesting process further comprises centrifugation, flocculation or sedimentation.
The chromatography devices (first-type chromatography device and second-type chromatography device) may be chromatography columns or membrane chromatography devices.
When the chromatography device employs a chromatography column, the chromatography packing may be, for example, affinity packing, cationic packing, anionic packing, hydrophobic packing, or mixed mechanism packing.
When the chromatography device employs a membrane chromatography device, affinity chromatography, cation exchange chromatography, anion exchange chromatography, hydrophobic chromatography, or mixed mechanism chromatography, for example, may be carried out depending on the membrane medium.
(first embodiment)
In this embodiment, in the first working area P1, after the biological reaction in step S11, the obtained cell culture harvest is bevacizumab (bevacizumab) with an expression amount of 8 g/L.
Then the cell culture harvest liquid is subjected to centrifugation in the step S12, precipitation or flocculation (especially acid precipitation) in the step S13 and deep filtration in the step S14 to obtain cell supernatant.
Since the seamless continuous flow chromatography process is performed continuously, the cell supernatant needs to be subjected to step S15S/D virus inactivation before the chromatography process is started.
The virus-inactivated harvest was then subjected to 6-column seamless continuous flow chromatography in a second operating zone P2, wherein one type of chromatography device was 4 affinity columns and the second type of chromatography device was 1 cation column and 1 anion column. The affinity filler was prism A (product of GE Co., Ltd., product No. 17549803), the cation chromatography filler was Capto S Impact (product of GE Co., product No. 17371705), and the anion filler was POROS XQ (product of Thermo Co., product No. 4467818).
The 4 affinity columns constitute a local affinity continuous flow chromatography system CC1, and the preparation is subjected to continuous flow chromatography using the affinity continuous flow chromatography system CC1 at step S21. Followed by seamless connection chromatography using a cation column C02 and an anion column C03 in step S22. Wherein, the sample eluted by the affinity chromatography is loaded and combined to a cation column C02, and the cation column C02 is eluted by eluent with the pH value of 6.5-8.0; the eluent from the cation column C02 directly entered the anion column C03.
The flow-through of the anion column C03 enters the third operation zone P3 and the formulation is collected in the storage container T via step S30. And then performing virus removal filtration in the step S31, nanofiltration and ultrafiltration in the step S32, and performing the step S33 to obtain the product Pr.
Table 1 shows the sequence of chromatography processes of the CC1 continuous flow affinity chromatography system, wherein 4 affinity columns are assigned to columns No. 1 to No. 4, and the start and end times of each affinity column for each process during the two loading processes are shown.
As can be seen from the table, in the local affinity continuous flow chromatography, the starting time and the stopping time of the sample loading of each chromatographic column are different, so that the time for capturing the sample by each chromatographic column is staggered, and the downstream cation column C02 and anion column C03 can perform seamless connection chromatography.
TABLE 1 chromatographic Process timing sequence for the affinity continuous flow chromatography System CC1
Table 2(a) and table 2(b) show the process time required to purify one batch of monoclonal antibody of the present embodiment using a conventional process and using a seamless continuous flow chromatography process according to the present embodiment, respectively.
In the conventional process flow, about 8 hours are required for affinity chromatography, cation chromatography needs to be performed after the batch preparation is completely subjected to affinity chromatography, and anion chromatography needs to be performed after the batch preparation is completely subjected to cation chromatography, and the process time from S/D virus inactivation to completion of anion chromatography is three shifts and 22 hours.
Whereas with the seamless continuous flow chromatography process according to this embodiment, affinity continuous flow chromatography of the same batch of monoclonal antibody requires only about 5 hours, and cation chromatography and anion chromatography can be performed after a period of time has elapsed since the beginning of the affinity chromatography and the capture of the sample, without waiting for the batch of preparation to completely complete the affinity chromatography. It will be appreciated that affinity chromatography, cation chromatography and anion chromatography may even begin simultaneously, as the chromatography process involves a non-loading time such as equilibration.
With the seamless continuous flow chromatography process according to this embodiment, a 12 hour (two shift) operating time is saved compared to chromatography of conventional processes.
TABLE 2(a) Process time of the conventional Process flow
TABLE 2(b) seamless continuous flow chromatography Process time
(second embodiment)
In this embodiment, the cell culture harvest is bevacizumab (bevacizumab), and local continuous flow chromatography is performed using 2 affinity columns, followed by seamless connection chromatography using 1 cationic column and 1 hydrophobic column.
The affinity filler is Mabselect Sure (product of GE, cat # 17543803), and the cationic filler is fractogel EMD SO3 -(M) (product of Merck, cat. No. 1.16882.5000), and the hydrophobic filler is Toyopearl Phenyl-600M (product of TOSOH, cat. No. 0021890).
And centrifuging the cell culture harvest liquid, precipitating with acid, and performing deep filtration to obtain cell supernatant. S/D virus inactivation was performed before the start of chromatography, and the virus-inactivated harvest was then subjected to 4-column seamless continuous flow chromatography.
2 affinity columns as a continuous flow capture product, affinity chromatography elution sample loading was bound to the cation column. The cation column adopts saline eluent to carry out linear gradient elution, and the conductivity is less than or equal to 23mS/cm during elution. The collected liquid directly enters a hydrophobic chromatographic column, and the sample directly flows through and is collected in a storage container. Then the hydrophobic collection liquid is continuously subjected to nanofiltration, ultrafiltration and stock solution preparation to complete the whole process.
The process parameters during the affinity chromatography process include,
affinity equilibrium liquid: 20mmol/L Tris +150mmol/L NaCl, pH7.2 equilibrium 3CV, loading 50g/L, affinity wash solution: 100mmol/L citric acid-sodium citrate buffer, 3CV washed with pH5.5, affinity eluent: eluting with 100mmol/L citric acid-sodium citrate buffer solution with the pH value of 3.2-4.0, and carrying out affinity regeneration on the solution: regenerating 3CV by 100mmol/L citric acid solution and 0.1mmol/L NaOH CIP 3 CV.
The process parameters during the cation chromatography process include,
cation balance liquid: 20mmol/L citric acid-sodium citrate buffer solution, pH5.0 balance 5CV, loading capacity of 20-80 g/L, cation eluent B: 20mmol/L citric acid-sodium citrate buffer solution +0.5mol/L NaCl, pH5.0, 0-100B linear gradient elution 10CV, and conductivity less than or equal to 8 mS/cm.
The process parameters during the hydrophobic chromatography process include,
hydrophobic equilibrium liquid: 20mmol/L citric acid-sodium citrate buffer solution, pH5.0 balance 5 CV. The loading capacity is less than or equal to 150 g/L.
Table 3(a) and table 3(b) show the process time required to purify one batch of bevacizumab of this embodiment using a conventional process and using a seamless continuous flow chromatography process according to this embodiment, respectively.
With the seamless continuous flow chromatography process according to this embodiment, a 12 hour (two shift) operating time is saved compared to chromatography of conventional processes.
TABLE 3(a) Process time of the conventional Process flow
TABLE 3(b) seamless continuous flow chromatography Process time
Table 4 shows the comparison of the quality data of the seamless-junction chromatography product according to the present embodiment with the quality data of the chromatography product of the conventional single-column system, and it can be seen from the table that the sample prepared by the seamless-junction chromatography method according to the present embodiment has no significant difference in quality (e.g., purity and impurities) and process performance (e.g., yield) from the sample prepared by the conventional single-column chromatography process.
TABLE 4 comparison of the seamless-connection chromatography product quality data of the second embodiment with the conventional single-column system chromatography product quality data
(third embodiment)
In this embodiment, the cell culture harvest solution is recombinant anti-interleukin 23p19 subunit antibody injection, and the expression amount is 5 g/L. Local continuous flow chromatography was performed using 3 affinity columns followed by seamless connection chromatography using 1 cation column and 1 anion column.
And centrifuging the cell culture harvest liquid, precipitating with acid, and performing deep filtration to obtain cell supernatant. S/D virus inactivation was performed before the start of chromatography, and the virus-inactivated harvest was then subjected to 5-column seamless continuous flow chromatography.
The affinity filler was Mabselect SureLX (product of GE Co., Ltd., product No. 17547404), the cation chromatography filler was POROS HS (product of Thermo Co., product No. 1-3359-09), and the anion filler was POROS XQ (product of Thermo Co., product No. 4467818).
3 affinity columns as a continuous flow capture product, and affinity chromatography elution sample loading was bound to the cation column. The cation column is eluted by eluent with pH6.5-pH8.0. The eluent directly enters an anion chromatographic column, and a sample directly flows through the anion chromatographic column and is collected in a storage container. And then the whole process is completed after nanofiltration, ultrafiltration and stock solution preparation.
The process parameters during the affinity chromatography process include,
affinity equilibrium liquid: 20mmol/L Tris +150mmol/L NaCl, pH7.2 equilibrium 3CV, loading capacity 60g/L, affinity wash solution: 20mmol/L Tris-HCl +1mol/L sodium chloride, 3CV washed with pH7.2, affinity eluent: 50mmol/L citric acid-sodium citrate buffer solution, eluting with pH3.2-4.0, and affinity regeneration solution: 3CV regeneration with 3% acetic acid buffer, 0.2mmol/L NaOH CIP 3 CV.
The process parameters during the cation chromatography process include,
cation balance liquid: 20mmol/L citric acid-sodium citrate buffer solution, pH5.0 balance 5CV, loading capacity of 20-80 g/L, cation eluent: 20mmol/L Tris +50mmol/L NaCl, pH 6.5-8.0.
The process parameters during the anion chromatography process include,
anion balance liquid: 20mmol/L Tris-HCl, pH 7.5 to equilibrate 5 CV. The loading capacity is less than or equal to 150 g/L.
Table 5(a) and table 5(b) show the process time required to purify one batch of the recombinant anti-interleukin 23p19 subunit antibody injection solution of the present embodiment using the conventional process and using the seamless continuous flow chromatography process according to the present embodiment, respectively.
With the seamless continuous flow chromatography process according to this embodiment, a 12 hour (two shift) operating time is saved compared to chromatography of conventional processes.
TABLE 5(a) Process time of the conventional Process flow
TABLE 5(b) seamless continuous flow chromatography Process time
The invention has at least one of the following advantages:
(i) the invention realizes integral seamless connection chromatography by performing virus inactivation by a detergent method (SD method) before the chromatography step begins, does not need an intermediate storage container between each chromatography device, does not produce intermediate products, shortens the process time and reduces the equipment cost.
(ii) The invention integrates the local continuous flow chromatography technology on the basis of the integral seamless connection chromatography technology, so that the utilization rate of the packing of the chromatographic column is improved, the packing cost is directly reduced, and the scales of the chromatographic equipment and other storage tanks are indirectly reduced.
Of course, the present invention is not limited to the above-described embodiments, and those skilled in the art can make various modifications to the above-described embodiments of the present invention without departing from the scope of the present invention under the teaching of the present invention. For example:
the present invention is applicable to, but not limited to, the purification of biological agents, including, inter alia, antibodies, fusion proteins, and cytokines, and unless otherwise specified, the term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or antigen binding regions thereof that compete for specific binding with intact antibodies, including human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, polyclonal antibodies, and oligomers or antigen binding fragments thereof. Also included are proteins having antigen-binding fragments or regions, such as Fab, Fab ', F (ab')2, Fv, diabodies, Fd, dAb, single chain antibody molecules, Complementarity Determining Region (CDR) fragments, scFv, trifunctional antibodies, tetrafunctional antibodies, and polypeptides that contain at least a portion of an immunoglobulin sufficient to confer specific antigen-binding to a polypeptide of interest. Specific examples of known antibodies produced using the methods of the invention include, but are not limited to, certolizumab (sintilmab), adalimumab (adalimumab), bevacizumab (bevacizumab), albuterol (Aflibercept), infliximab (infliximab), nimotuzumab (nimotuzumab), pertuzumab (pertuzumab), ranibizumab (ranibizumab), rituximab (rituximab), and trastuzumab (trastuzumab).
Claims (12)
- A seamless continuous flow chromatography process using at least three chromatography devices, wherein at least two chromatography devices constitute one type of chromatography device and at least one chromatography device constitutes a second type of chromatography device,the chromatography device of the described type is used to carry out continuous flow chromatography,the first-class chromatography device and the second-class chromatography device jointly realize seamless connection chromatography.
- The seamless continuous flow chromatography method of claim 1, wherein no storage vessel is used between the one type of chromatography device and the second type of chromatography device.
- The seamless continuous-flow chromatography method according to claim 1, wherein prior to the continuous-flow chromatography, the preparation to be chromatographed is virus inactivated, preferably by an organic solvent detergent method.
- The seamless continuous-flow chromatography method according to claim 1, wherein the preparation to be chromatographed is subjected to depth filtration prior to the continuous-flow chromatography.
- The seamless continuous-flow chromatography method of claim 1, wherein the start time and the end time of loading of each of the chromatography devices of one type are different; each of the chromatographic devices of one type can operate simultaneously; wherein the flow-through product of the sample from one of said chromatographic devices of one type can be captured by another of said chromatographic devices of one type; the chromatographic device of the type is continuously or discontinuously loaded and continuously or discontinuously collected.
- The seamless continuous-flow chromatography method of claim 1, wherein the chromatography device of the one type is used to capture a target product, and the target product is greater than 80% pure in the capture collection of the chromatography device of the one type.
- The seamless continuous flow chromatography process of claim 6, wherein the chromatography device of the second type is used for fine purification, and the purity of the target product in the collection liquid of the chromatography device of the second type is greater than 90%.
- The seamless continuous flow chromatography method of claim 1, wherein the second type of chromatography device completes equilibration and waits for sample inflow before the first type of chromatography device collects product, and the chromatography process of the second type of chromatography device is started during the chromatography process performed by the first type of chromatography device.
- The seamless continuous-flow chromatography method of claim 1, wherein the chromatography devices are chromatography columns, the packing of each of the chromatography devices of one type is the same, the packing of each of the chromatography devices of the second type is different, and the packing of each of the chromatography devices of the second type is different from the packing of the chromatography device of one type; orThe chromatographic devices are membrane chromatographic devices, the chromatographic membranes of the first-class chromatographic devices are the same, the chromatographic membranes of the second-class chromatographic devices are different, and the chromatographic membranes of the second-class chromatographic devices are different from the chromatographic membranes of the first-class chromatographic devices.
- The seamless continuous-flow chromatography method of claim 1, wherein the one type of chromatography device is an affinity chromatography device and the ligand is protein a.
- The seamless continuous flow chromatography method according to claim 1, wherein the method is used for purifying a biological agent, preferably an antibody.
- The seamless continuous-flow chromatography method of claim 1, comprising:s11, carrying out biological reaction to obtain cell culture harvest liquid as a reaction preparation;s12, centrifuging the reaction preparation;s13, precipitating or flocculating the reaction preparation;s14, carrying out deep filtration on the reaction preparation;s15, performing S/D virus inactivation on the reaction preparation;s21, performing the continuous flow chromatography on the reaction preparation using the one type of chromatography device;s22, performing the seamless connection chromatography on the reaction preparation by using the second type chromatography device;s30, collecting the purified reaction preparation;s31, performing virus removal and filtration on the reaction preparation;s32, performing nanofiltration and ultrafiltration on the reaction preparation;and S33, obtaining the product Pr.
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| CN105358572A (en) * | 2013-05-06 | 2016-02-24 | 赛诺菲 | Continuous multistep process for purifying antibodies |
| CN109563124A (en) * | 2016-06-17 | 2019-04-02 | 豪夫迈·罗氏有限公司 | The purifying of multi-specificity antibody |
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