Flow-through mode continuous refining method

Abstract

The invention provides a method for continuously refining a sample by adopting a flow-through mode, which comprises the following steps: (1) calculating the minimum number n of chromatographic columns used for purification, # imgabs0 #, determining the sample loading duration T1 and the post-treatment duration T2 by using the chromatographic columns in a batch chromatography manner, and representing an upward integer by a symbol # imgabs1 #; and (2) purifying by using at least n same chromatographic columns. The continuous refining process in the flow-through mode provided by the invention can improve the purification efficiency and reduce the use of consumables through an uninterrupted process, so that the production cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a continuous purification in a flow-through mode, which can be used for the continuous purification production of proteins such as antibodies and enzyme preparations, nucleic acids, blood products or vaccines to achieve low-cost, multi-cycle and automated purification. Background Art

[0002] At present, biopharmaceuticals are mainly produced in batches, that is, starting from the process starting point, after multiple intermediate steps with pauses, the final product is finally obtained. With the rapid development of biopharmaceuticals, the types of large molecule drugs are increasing, and the molecules are becoming more and more complex, which increases the difficulty of downstream purification; in addition, the upstream has been pursuing the increase of production capacity and the expansion of scale. Conventional batch production cannot meet the current needs, thus giving rise to continuous production processes. As a key technology of continuous production processes, continuous flow chromatography has gradually become a focus of attention.

[0003] In recent years, the application of continuous flow chromatography technology in antibody purification has been reported, but the current traditional continuous flow chromatography technology still has the following problems:

[0004] (1) Currently, most continuous flow chromatography processes are still at the continuous capture stage, while the subsequent purification steps are still mainly based on batch production processes. The continuous flow process and its development process for the purification steps in the flow-through mode have not yet been clarified.

[0005] (2) The current continuous affinity capture process uses multi-column operation to continuously load samples on two chromatographic columns in series, and achieves continuous sample loading and separation by sequentially switching the chromatographic columns (e.g. Figure 1 This operation is sometimes unnecessary for a polishing step in flow-through mode and increases the risk of accumulation of impurities that bind to the chromatography medium on the second column.

[0006] (3) During the continuous affinity capture stage, the sample loading is usually controlled by monitoring UV, such as the ΔUV control program of the AKTA PCC system and AutomAb control program of CUBE combined system. Unlike the affinity capture step, the main purpose of the polishing step is to remove trace impurities such as host cell proteins, DNA, antibody fragments, aggregates, heterogeneous bodies, etc., generally using ion exchange, hydrophobic interaction and mixed mode chromatography methods. Therefore, the loading capacity of the polishing step is determined according to the final product quality, and the traditional ΔUV or AutomAb program is no longer applicable.

[0007] (4) When using the traditional continuous chromatography method for double-column serial loading, it is impossible to monitor the pH and conductivity of the flow-through liquid, which is not applicable to the process of flow-through mode.

[0008] Therefore, a continuous purification method in flow-through mode is needed in the art. This method can continuously load a single column on a multi-column system at the highest filler loading condition while ensuring product quality, and simultaneously monitor the pH and conductivity of the flow-through liquid during the product flow-through process. Compared with batch production, the continuous purification process in flow-through mode can be faster, more stable, and more flexible through an uninterrupted process, reduce the use of consumables, and thus reduce production costs. It is the future trend of production process development in the pharmaceutical industry. Summary of the invention

[0009] In one aspect, the present invention provides a method for continuous purification of a sample using a flow-through mode, comprising:

[0010] (1) Calculate the minimum number of chromatography columns n used for purification, Wherein, the loading time T1 and the post-treatment time T2 are determined by using the chromatography column in a batch chromatography manner, and the symbol Indicates rounding up to an integer;

[0011] (2) Using at least n identical chromatography columns for purification.

[0012] In some embodiments, the loading is performed sequentially in all chromatography columns, and after the loading is completed, the column is immediately post-treated.

[0013] In some embodiments, all chromatography columns are equilibrated by adding an equilibration buffer before loading the sample.

[0014] In some embodiments, the loading is cycled through all chromatography columns sequentially, thereby enabling continuous processing of samples.

[0015] In some embodiments, while one of the chromatography columns is being loaded with a sample, all remaining chromatography columns are being post-processed.

[0016] In some embodiments, the switching between the loading state and the post-processing state is achieved by changing the flow system connected to the chromatography column.

[0017] In some embodiments, the continuous polishing method further comprises monitoring the pH or conductivity of the flow-through of the chromatography column on which the sample is loaded.

[0018] In some embodiments, the sample is previously captured by affinity chromatography purification or other chromatography steps.

[0019] In some embodiments, the chromatography column is selected from an ion chromatography column, a hydrophobic chromatography column, a mixed mode chromatography column, and a molecular sieve chromatography column.

[0020] In some embodiments, the chromatography column is an anion chromatography column.

[0021] In some embodiments, the chromatography column is a hydrophobic chromatography column.

[0022] In some embodiments, the height of the chromatography column is 5-25 cm.

[0023] In some embodiments, the sample loading time includes the time for sample loading and adding a wash buffer for elution.

[0024] In some embodiments, the post-treatment process includes, in sequence: adding a regeneration buffer, adding a disinfectant, pausing disinfection, adding a pre-equilibrium buffer, and adding an equilibrium buffer.

[0025] In some embodiments, the sample is a solution comprising a protein.

[0026] The continuous purification process in flow-through mode provided in this article can improve purification efficiency while reducing the use of consumables through an uninterrupted process, thereby reducing production costs.

[0027] The beneficial effects of the present invention include at least:

[0028] 1. The continuous purification method described in the present invention can realize continuous sample loading, reduce the interruption time in the purification process, shorten the time of process development of this part, and provide a new idea and method for downstream purification process.

[0029] 2. The continuous purification method described in the present invention uses a chromatographic device to achieve continuous purification in a flow-through mode for the first time. The method described in the present invention can simultaneously monitor the pH and conductivity of the flow-through liquid during the sample loading process.

[0030] 3. The continuous purification method described in the present invention can increase the purification efficiency by more than 64.8%, and the buffer consumption can be significantly reduced. In addition, the method described in the present invention can achieve continuous loading while using the least chromatography columns, thereby achieving high efficiency, low consumption and low cost.

[0031] 4. The continuous purification method of the present invention adopts a flow-through mode, with a high sample loading capacity and a high purity of the purified product. The product yield reaches 92% or more, the product purity reaches more than 95%, and the HCP (host cell protein) level of the product is extremely low, which improves the product stability and reduces the immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A typical sequential capture chromatography method - schematic diagram of dual-column serial loading (3-column system) is shown.

[0033] Figure 2 Schematic diagram showing single column loading in flow-through mode (2-column system).

[0034] Figure 3 Schematic diagram showing single column loading in flow-through mode (3-column system).

[0035] Figure 4 Schematic diagram showing single column loading in flow-through mode (4-column system).

[0036] Figure 5 Schematic diagram showing single column loading in flow-through mode (multi-column system).

[0037] Figure 6 Shows a continuous anion chromatogram in flowthrough mode.

[0038] Figure 7 Continuous anion chromatography flow-through product yields are shown.

[0039] Figure 8 Displays continuous anion chromatography flow-through product pH & conductivity.

[0040] Fig. 9 Shows the SEC-HPLC purity of the continuous anion chromatography flow-through product, where HMW is a high molecular weight impurity, LMW is a low molecular weight impurity, and the Main peak is the sample.

[0041] Fig.10 Showing the purity of the continuous anion chromatography flow-through product SDS-CE_NR.

[0042] Fig.11 Continuous anion chromatography showing HCP levels.

[0043] Fig.12 Shows the continuous hydrophobic chromatography profile in flowthrough mode.

[0044] Fig.13 Continuous hydrophobic chromatography flow-through product yield is shown.

[0045] Fig.14 Displays pH & conductivity of continuous hydrophobic chromatography flow-through product.

[0046] Fig.15 Shows the SEC-HPLC purity of the continuous hydrophobic chromatography flow-through product.

[0047] Fig.16 Continuous hydrophobic chromatography showing HCP levels. DETAILED DESCRIPTION

[0048] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0049] The term "or" refers to a single element of the listed optional elements, unless the context clearly indicates otherwise. The term "and / or" refers to any one, any two, any three, any more or all of the listed optional elements.

[0050] The term "comprising" or "including" means including the elements, integers or steps mentioned, but does not exclude any other elements, integers or steps. In the text, when the term "comprising" or "including" is used, unless otherwise specified, it also covers the situation consisting of the elements, integers or steps mentioned. For example, when referring to a method "comprising" a certain two steps, it is also intended to cover the method consisting of the two steps.

[0051] "Purification" in this article refers to the process of further purifying a sample (such as a crude product containing the target protein) that has been initially purified (roughly purified). In some cases, if the target product itself already has a fairly high purity in the sample, it is also possible to consider omitting the initial purification step. Rough purification and fine purification can use the same or different purification methods. For example, rough purification uses precipitation (such as using ammonium sulfate, organic solvents (such as acetone) or polyethylene glycol, etc.), dialysis or tangential flow filtration, and fine purification uses chromatography (or chromatography); or both rough purification and fine purification use chromatography. In some embodiments, the purification method described herein is anion exchange chromatography. In other embodiments, the purification method described herein is hydrophobic chromatography. In some embodiments, the chromatography column filler used in the purification method described herein is Poros XQ, Poros 50HQ or QFF. In other embodiments, the chromatography column filler used in the purification method described herein is Capto Phenyl (high sub), Phenyl Sepharose 6 Fast Flow (high sub), POROS Benzyl POROS Benzyl Ultra, Toyopearl Butyl-650S or Toyopearl Butyl-650M. In some embodiments, the same chromatography column described herein refers to a chromatography column with the same filler.

[0052] "Chromatography" in this article refers to a substance separation (or purification) technique that utilizes the differences in physicochemical properties between different components, thereby establishing differences in distribution coefficients between different phases. The chromatographic system used for chromatography usually includes two phases: a stationary phase, which is a solid substance or a component fixed on a solid substance; and a mobile phase, which is a substance that can flow, such as water and various solvents. When the mixture to be separated passes through the stationary phase with the solvent (mobile phase), due to the differences in the physicochemical properties of each component, the ability to interact with the two phases (adsorption, dissolution, binding, steric hindrance, etc.) is different, and the distribution in the two phases is different. Moreover, as the solvent moves forward, each component is constantly redistributed in the two phases. The weaker the interaction force with the stationary phase, the less retardation it encounters when moving with the mobile phase, and the faster it moves forward. Conversely, the stronger the interaction with the stationary phase, the slower it moves forward. By collecting the effluent in parts, each single component contained in the sample can be obtained, thereby achieving the purpose of separating each component. Examples of chromatographic techniques include affinity chromatography, ion exchange chromatography (including anion exchange chromatography and cation exchange chromatography), gel filtration chromatography (molecular sieve chromatography), hydrophobic chromatography or mixed mode chromatography, etc. Mixed mode chromatography uses more than one interaction mode, for example, the stationary phase used or its surface is combined with a hydrophobic ligand and an ion exchange ligand, and electrostatic and hydrophobic interactions can be used simultaneously to separate components. In some embodiments, the height of the chromatographic column described herein is 5-25 cm, for example, 5 cm, 6 cm, 7 cm, 8 cm, 9 cm, 10 cm, 11 cm, 12 cm, 13 cm, 14 cm, 15 cm, 16 cm, 17 cm, 18 cm, 19 cm, 20 cm, 21 cm, 22 cm, 23 cm, 24 cm or 25 cm. In some embodiments, the same chromatographic column described herein refers to a chromatographic column with the same height.

[0053] In some embodiments, the continuous polishing method described herein uses at least n identical chromatography columns for purification, wherein the Wherein, the loading time T1 and the post-treatment time T2 are determined by using the chromatography column in a batch chromatography manner, and the symbol Indicates rounding up to an integer. In some embodiments, "at least n identical chromatography columns" described herein refers to the minimum number of chromatography columns required for the purification method to achieve continuous loading. In some embodiments, the continuous purification method described herein uses 2 identical chromatography columns for purification. In some embodiments, the continuous purification method described herein uses 3 identical chromatography columns for purification. In some embodiments, the continuous purification method described herein uses 4 identical chromatography columns for purification. In some embodiments, the continuous purification method described herein uses 5, 6, 7, 8, 9 or 10 identical chromatography columns for purification.

[0054] "Affinity chromatography" refers to a chromatography technique that uses the specific affinity between the substance to be separated (such as the target product) and its specific ligand to separate it from other components. One of a pair of molecules with specific binding properties is connected to an insoluble carrier in the form of a covalent bond as a stationary phase adsorbent. When a sample containing mixed components passes through this stationary phase, only substances with specific affinity to the stationary phase molecules can be adsorbed and bound by the stationary phase, while irrelevant components flow out with the mobile phase. The bound substance to be separated can then be eluted by changing the mobile phase components. The carrier used in affinity chromatography is also called a medium, and the compound covalently linked to the medium is called a ligand. The main biological molecule pairs with specific affinity are: antigens and antibodies, DNA and complementary DNA or RNA, enzymes and substrates, hormones and receptors, vitamins and specific binding proteins, glycoproteins and plant lectins, etc. Affinity chromatography can be used to purify biological macromolecules, concentrate diluents, store unstable proteins, separate nucleic acids, etc.

[0055] "Ion exchange chromatography" refers to a method of separating ionic compounds by using a substance with ion exchange properties as a stationary phase and utilizing its property of reversibly exchanging ions with the mobile phase. This chromatography method can be used to separate amino acids, peptides and proteins, as well as nucleic acids, nucleotides and other charged biomolecules. The isoelectric point (pI) characteristics of different proteins result in different positive / negative net charges under different pH buffer conditions, and different ion exchange columns (anion or cation chromatography columns) can be selected to achieve separation.

[0056] "Hydrophobic chromatography" refers to a chromatography method that achieves separation based on the difference in hydrophobicity between biomolecules. Generally speaking, high ionic strength can enhance the hydrophobic interaction between the hydrophobic amino acids in the protein and the chromatography medium, weakening their electrostatic forces.

[0057] "Flow-through mode" in this article refers to a chromatography purification method in which the target product (such as a protein or antibody) is allowed to flow out of the chromatography column with the fluidity, while other components or impurities are adsorbed on the chromatography column. One advantage of using flow-through mode is that higher loadings can be used, and there are relatively few washing and elution steps.

[0058] "Continuous purification" and "continuous loading" refer to the samples described herein, starting from the loading of the purification process, and the purification is carried out uninterruptedly until the loading is completed. Correspondingly, "batch purification" refers to dividing a sample into multiple portions, and there is a time interval between the end of loading of one sample and the beginning of loading of the next sample. This time interval can be used, for example, for post-processing of a chromatography column. When a chromatography column is used for purification, continuous purification is also referred to as continuous chromatography, and batch purification is referred to as batch chromatography. In some embodiments, the loading / elution time, post-processing time, maximum loading capacity, and loading flow rate of the sample in the continuous purification method described herein are determined by batch chromatography. In some embodiments, the sample is a sample containing antibodies.

[0059] In some embodiments, the loading capacity is 100 mg / mL. In some embodiments, the loading flow rate is 0.4-1.0 mL / min; preferably, such as 0.5 mL / min, 0.6 mL / min, 0.7 mL / min; more preferably, such as 0.51 mL / min, 0.52 mL / min, 0.53 mL / min, 0.54 mL / min, 0.55 mL / min, 0.56 mL / min, 0.57 mL / min, 0.58 mL / min, 0.59 mL / min, 0.60 mL / min, 0.61 mL / min, 0.62 mL / min, 0.63 mL / min, 0.64 mL / min, 0.65 mL / min. In some embodiments, the loading capacity is not more than 60 mg / mL, preferably not more than 51 mg / mL. In some embodiments, the loading retention time is greater than 5 minutes, preferably greater than 6 minutes.

[0060] "Loading time", "loading / rinsing time" refers herein to the continuous loading time from the start of loading until the loading is completed when the maximum loading capacity of a single chromatography column is reached. The loading time can be determined by batch chromatography. When the purification process includes a rinsing step, the loading time also includes the rinsing time. In some embodiments, the loading time T1 described herein is determined by batch chromatography using the chromatography column. In some embodiments, the loading time is 80-100min. In some embodiments, the rinsing buffer used for rinsing is 25mM Na-Citrate. In some embodiments, the rinsing buffer is a mixture of 15mM Na-Citrate and 1M NaCl. In some embodiments, the volume of the rinsing buffer is at least 4 times the column volume, preferably, at least 5 times the column volume.

[0061] "Post-treatment duration" herein refers to, with respect to a single chromatography column, the total duration of the various steps of post-treatment thereof, which post-treatment steps include, for example, regeneration, disinfection, buffer equilibration, etc. In some embodiments, the post-treatment duration T2 described herein is determined by using the chromatography column in a batch chromatography manner.

[0062] When the purification method is an anion chromatography method: In some embodiments, the post-treatment process includes: adding 8 column volumes of regeneration buffer, adding 8 column volumes of disinfectant, disinfecting for 30 minutes, adding 8 column volumes of pre-equilibrium buffer, adding 10 column volumes of equilibrium buffer. In some embodiments, the regeneration buffer is 25mM Na-Citrate, 1M NaCl. In some embodiments, the disinfectant is 1M NaOH. In some embodiments, the pre-equilibrium buffer is 25mM Na-Citrate, 1M NaCl. In some embodiments, the equilibrium buffer is 25mM Na-Citrate. In some embodiments, the flow rate of each buffer and disinfectant in the post-treatment step is 1-2mL / min; preferably, such as 1.4mL / min, 1.5mL / min, 1.6mL / min, 1.7mL / min, 1.8mL / min; more preferably, such as 1.71mL / min.

[0063] When the purification method is a hydrophobic chromatography method: In some embodiments, the post-treatment process includes: adding 3 column volumes of regeneration buffer, adding 3 column volumes of disinfectant, pausing for 1-10 minutes, adding 3 column volumes of pre-equilibrium buffer, and adding 4 column volumes of equilibrium buffer. In some embodiments, the regeneration buffer is H 2 O. In some embodiments, the disinfectant is 1M NaOH. In some embodiments, the pre-equilibration buffer is 15mM Na-Citrate. In some embodiments, the equilibration buffer is 15mM Na-Citrate, 1M NaCl. In some embodiments, the flow rate of each buffer and disinfectant in the post-treatment process is 0.5-2.0mL / min; preferably, such as 0.6ml / min, 0.7mL / min, 0.8ml / min, 0.9ml / min, 1.0ml / min; more preferably, such as 1.0ml / min.

[0064] "Switching loading" refers to the method in this article that, due to the limited loading capacity of a single chromatography column, when the sample amount exceeds its processing capacity, the present invention switches the chromatography column so that the sample is replaced in turn to other column positions to achieve continuous loading (or continuous purification). The present invention can achieve large-scale continuous purification of target products, such as proteins or antibodies, by switching loading. Switching loading can be performed manually, but it is preferably performed by a chromatography instrument with a column switching function, and the chromatography column is switched to a loading state or a post-processing state by controlling the flow path of the access chromatography column. In some embodiments, the purification method described herein uses an AKTA instrument. In some embodiments, the purification method described herein uses an AKTA PCC instrument. In some embodiments, when the loading time T1 ≥ the post-processing time T2, two of the chromatography columns are used for switching loading and post-processing. In some embodiments, when the loading time T1 < the post-processing time T2, at least three of the chromatography columns are used for switching loading and post-processing. In some embodiments, when the polishing methods described herein are performed using chromatography equipment, real-time pH and conductivity monitoring of the convective stream can be achieved.

[0065] "Carry out sequentially" in this article means that the sample passes through all the chromatography columns sequentially, the chromatography columns that have not been loaded with samples are in the equilibrium solution waiting for loading, and the chromatography columns that have been loaded with samples start post-processing after the loading is completed. In some embodiments, such as a three-column system, when the first chromatography column is loaded with samples, the second and third chromatography columns are in a state of waiting for loading, and when the second chromatography column is loaded with samples, the first chromatography column is in a post-processing state, and the third chromatography column is in a state of waiting for loading.

[0066] "Perform a cycle sequentially" in the text means that after the sample passes through all the chromatography columns sequentially, it circulates to the first chromatography column to start loading, and all other remaining chromatography columns are in a post-processing state. In some embodiments, such as a three-column system, after the loading is sequentially performed in all the chromatography columns, the first chromatography column is switched again for loading, and the second and third chromatography columns are post-processed. When switching to the second chromatography column for loading, the first and third chromatography columns are post-processed.

[0067] In this article, the numerals, such as "first" and "second", are merely references to the temporal sequence for the convenience of description and do not indicate a specific relationship in space or position.

[0068] The technical scheme of the present invention is further described in detail below by examples and in conjunction with the accompanying drawings. Unless otherwise specified, the methods and materials of the embodiments described below are conventional products that can be purchased from the market. It will be understood by those skilled in the art that the present invention belongs to that the methods and materials described below are only exemplary and should not be considered as limiting the scope of the present invention.

[0069] The present invention provides a continuous flow chromatography process for a purification step in a flow-through mode and a development method thereof, which may include the following steps:

[0070] 1. Batch chromatography process development stage in flow-through mode: In this stage, it is necessary to first examine the individual factors that affect the continuous purification steps in batch mode, including filler screening, loading / elution condition screening, loading retention time optimization, loading capacity optimization, column height investigation, and loading / flow-through sample stability investigation.

[0071] (1) Filler screening: According to the charge of the protein and the process requirements, select suitable fillers for flow-through mode chromatography process development. Taking anion chromatography as an example, Poros XQ, Poros 50HQ, QFF and other fillers can generally be selected for continuous purification in flow-through mode. Considering that the continuous flow chromatography operation cycle is long and the fillers are recycled many times during the entire cycle, it is necessary to investigate the repeated service life of the fillers to be screened. The service life of the fillers can refer to the filler service life research provided by the filler manufacturer. Generally, the minimum number of uses cannot be less than the number of filler cycles in one production cycle.

[0072] After the filler to be screened is preliminarily selected, the process performance of the filler (such as loading capacity, recovery rate, column pressure performance, etc.) and the quality parameters of the flow-through product (such as SEC-HPLC purity, HCP, rDNA, etc.) can be investigated to screen the most suitable filler.

[0073] (2) Screening of loading / elution conditions: For the purification step, the impact of loading / elution conditions is generally more significant, such as loading / elution pH and loading / elution conductivity. Generally, several appropriate loading pH and conductivity conditions can be selected for screening based on the protein's own characteristics (such as isoelectric point, hydrophobicity, etc.) and the properties of the chromatographic filler. The comprehensive process performance (such as recovery rate, flowthrough peak shape, etc.) and the quality parameters of the flowthrough product (such as SEC-HPLC purity, HCP, rDNA, etc.) are examined to screen the optimal loading / elution conditions.

[0074] (3) Optimization of sample retention time: In order to better guide the design of continuous purification process, it is necessary to examine the performance of the purification process (such as recovery rate, column pressure performance, etc.) and product quality (such as SEC-HPLC purity, HCP, rDNA, etc.) at different retention times (such as 2, 4, 6 min) to determine the appropriate retention time.

[0075] (4) Sample loading capacity optimization: Unlike the affinity capture step, the sample loading capacity of the purification step is not determined based on the flow-through ratio of the sample, but on the process performance and the final product quality. For example, for anion chromatography in flow-through mode, the yield, flow-through peak shape and product quality of anion chromatography at different loading capacities such as 80 g / L, 100 g / L, 120 g / L and 140 g / L can be examined to finally determine the optimal sample loading capacity.

[0076] (5) Examination of the height of the chromatography column: The continuous purification process in the flow-through mode is based on the principle that the target protein first flows through the chromatography column, while impurities such as rDNA and HCP combine with the chromatography filler and do not flow through, ultimately achieving protein purification. Therefore, if the height of the chromatography column is too low, the impurity removal effect may be unsatisfactory; in addition, some purification fillers have small particle sizes and are subjected to greater pressure, and the continuous chromatography process may improve efficiency by reducing the sample retention time. If the chromatography column is too high, the column pressure may exceed the range during the chromatography process. Therefore, the column height of continuous purification needs to be optimized. It is generally recommended to examine 5-25cm, and confirm the acceptable column height range based on the pressure conditions, product yield and product quality parameters at different chromatography column heights.

[0077] (6) Stability investigation of loading / flow-through samples: In continuous purification experiments, in order to prevent program suspension and sample retention caused by certain abnormal events, it is still necessary to investigate the stability of loading / flow-through samples. In addition to paying attention to the quality parameters of samples placed under different conditions (such as SEC-HPLC purity, CE-SDS-NR purity, icIEF purity, etc.), it is also necessary to pay attention to whether there is an increase in column pressure during the loading process.

[0078] 2. Continuous purification confirmation phase of multiple systems in flow-through mode: Based on the results of the batch chromatography process development phase of flow-through mode, continuous purification of the multi-column system is achieved on the chromatography instrument to confirm its feasibility and stability in process performance (such as yield, pressure, etc.) and product quality (SEC-HPLC purity, CE-SDS-NR purity, HCP, etc.). The considerations at this stage mainly involve the following points:

[0079] (1) At this stage, the choice of a 2 / 3 / 4-column system or more can be determined based on the balance between the loading and elution time and the total column post-treatment time: if the loading / elution time T1 ≥ the total column post-treatment time T2, a 2-column system can be selected for switching loading; if the loading / elution time T1 < the total column post-treatment time T2, a 3-column system or more can be selected for switching loading. The principle of this stage is to keep the entire loading process (loading and elution steps) uninterrupted.

[0080] (2) In the specific implementation process, a chromatography device needs to be used to achieve single-column loading. This method can be combined with the single-column loading schematic diagram of different column systems ( Figure 2-5 ) for understanding. For multi-column systems, sample loading and post-processing operations are performed simultaneously. Simply put, cycle X performs single-column loading on column X, and the remaining columns perform post-processing separately. For example, in a 3-column system, cycle 1 performs single-column loading on column 1, and columns 2 and 3 perform post-processing separately; cycle 2 performs single-column loading on column 2, and columns 3 and 1 perform post-processing separately; cycle 3 performs single-column loading on column 3, and columns 1 and 2 perform post-processing separately; set cycles 1-3 as a loop, and when the method is running, the above operations can be repeated by setting the number of loops. Systems with 2 / 4 columns and more columns have the same logic as 3-column systems. During the loading process, the pH and conductivity of the flow-through can be monitored synchronously.

[0081] In some embodiments, the present invention provides a method for continuous purification in flow-through mode, which includes a process development stage and a confirmation stage, wherein the process development stage is used to develop suitable screening parameters, and the confirmation stage is used to achieve continuous purification of a multi-column system on a chromatographic instrument, and the loading and elution steps of the continuous purification process are uninterrupted.

[0082] In some embodiments, the multi-column system in the confirmation stage includes 2 column positions, 3 column positions, or more column positions.

[0083] In some embodiments, the method for selecting the column position system is: if the loading / elution time T1 ≥ the total column post-processing time T2, 2 column positions can be selected for switching loading; if the loading / elution time T1 < the total column post-processing time T2, 3 column positions or more can be selected for switching loading.

[0084] In some embodiments, during the confirmation phase, the pH and conductivity of the flow-through liquid can be monitored while achieving continuous sample loading on a single column in flow-through mode.

[0085] In some embodiments, the cyclic process of the multi-column position system is that the Xth column position performs single column loading at any Xth cycle, and the remaining column positions perform post-processing respectively.

[0086] In some embodiments, the screening parameters in the process development stage include filler material, loading / elution conditions, loading retention time, loading capacity, chromatography column height, and stability assessment of loading / flow-through samples.

[0087] In some embodiments, the chromatography type involved in the process development stage is one of flow-through mode ion chromatography, hydrophobic chromatography, mixed mode chromatography, and molecular sieve chromatography.

[0088] In some embodiments, the loading / rinsing conditions include a loading / rinsing pH of pH 5-9 and a loading / rinsing conductivity of 0.5-100 mS / cm. In some embodiments, the loading / rinsing conditions include a loading / rinsing pH of pH 5-9 and a loading / rinsing conductivity of 0.5-3.63 mS / cm. In some embodiments, the loading / rinsing conditions include a loading / rinsing pH of pH 5-9 and a loading / rinsing conductivity of 85-95 mS / cm. In some embodiments, the loading / rinsing conditions include a loading / rinsing pH of pH 5-9 and a loading / rinsing conductivity of less than or equal to 4.0 mS / cm.

[0089] In some embodiments, the chromatography column has a height of 5-25 cm.

[0090] The technical scheme of the present invention is further described in detail below by examples and in conjunction with the accompanying drawings. Unless otherwise specified, the methods and materials of the embodiments described below are conventional products that can be purchased from the market. It will be understood by those skilled in the art that the present invention belongs to that the methods and materials described below are only exemplary and should not be considered as limiting the scope of the present invention.

[0091] Example 1 Continuous Anion Chromatography in Flow-Through Mode

[0092] The following steps are involved:

[0093] 1. Batch chromatography process parameter development phase in flow-through mode

[0094] In this stage, the single factors that affect the continuous purification steps were first investigated in the batch purification process of monoclonal antibody molecule A, including filler screening, sample loading / elution condition screening, sample retention time optimization, sample loading capacity optimization, and sample loading / flow-through sample stability inspection. Other parameters are antibody platform processes in flow-through mode. Different projects use different processes, and the parameters are adjusted according to the required process. The specific operations are as follows:

[0095] (1) Filler screening: According to the charge of the protein and the process requirements, select appropriate fillers for flow-through mode chromatography process development. Considering that the continuous flow chromatography operation cycle is long and the fillers are recycled many times during the entire cycle, it is necessary to investigate the repeated service life of the fillers to be screened. The filler service life can refer to the filler service life research provided by the filler manufacturer. Generally, the minimum number of uses cannot be less than the number of filler cycles in one production cycle. In this experiment, the fillers Poros XQ, Poros 50HQ, and QFF were selected for continuous purification in flow-through mode.

[0096] The most suitable filler was selected based on the process performance of the above fillers (loading capacity, recovery rate, column pressure performance, etc.) and the quality parameters of the flow-through products (SEC-HPLC purity, HCP, rDNA, etc.).

[0097] (2) Screening of loading / elution conditions: For the purification step, the impact of loading / elution conditions is generally more significant, such as loading / elution pH and loading / elution conductivity. Generally, several appropriate loading pH and conductivity conditions can be selected for screening based on the protein's own characteristics (such as isoelectric point, hydrophobicity, etc.) and the properties of the chromatographic filler. The comprehensive process performance (recovery rate, flowthrough peak shape, etc.) and the quality parameters of the flowthrough product (SEC-HPLC purity, HCP, rDNA, etc.) are examined to screen the optimal loading / elution conditions.

[0098] (3) Optimization of sample retention time: In order to better guide the design of continuous purification process, it is necessary to examine the performance of the purification process (recovery rate, column pressure performance, etc.) and product quality (SEC-HPLC purity, HCP, rDNA, etc.) at different retention times (2, 4, 6 min) to determine the appropriate retention time.

[0099] (4) Optimization of sample loading capacity: Unlike the affinity capture step, the sample loading capacity of the purification step is not determined by the flow-through ratio of the sample, but by the process performance and the final product quality. This experiment investigated the yield, flow-through peak shape and product quality of anion chromatography at different loading capacities such as 80 g / L, 100 g / L, 120 g / L and 140 g / L, and finally determined the optimal sample loading capacity.

[0100] (5) Examination of the height of the chromatography column: The principle of the continuous purification process in the flow-through mode is that the target protein first flows through the chromatography column, while impurities such as rDNA and HCP combine with the chromatography filler and do not flow through, ultimately achieving protein purification. Therefore, if the height of the chromatography column is too low, the impurity removal effect may be unsatisfactory; in addition, some purification fillers have small particle sizes and large back pressures, and the continuous chromatography process may improve efficiency by reducing the sample retention time. If the chromatography column is too high, the column pressure may exceed the range during the chromatography process. Therefore, the column height of continuous purification needs to be optimized. It is generally recommended to examine 5-25cm, and confirm the acceptable column height range based on the pressure conditions, product yield and product quality parameters at different chromatography column heights.

[0101] (6) Stability investigation of loading / flow-through samples: In continuous purification experiments, in order to prevent program suspension and sample retention caused by certain abnormal events, it is still necessary to investigate the stability of loading / flow-through samples. In addition to paying attention to the quality parameters of samples placed under different conditions (SEC-HPLC purity, CE-SDS-NR purity, icIEF purity, etc.), it is also necessary to pay attention to whether the column pressure increases during the loading process.

[0102] According to the above steps, the final process parameters after single factor optimization are shown in Table 1.

[0103] Table 1 Anion chromatography process parameters after optimization

[0104]

[0105] *CV: Column volume, column volume

[0106] 2. Multi-system continuous purification confirmation stage in flow-through mode

[0107] Based on the results of the batch chromatography process parameter development phase in flow-through mode, continuous purification of the multi-column system was achieved on a continuous flow chromatography device to confirm its feasibility and stability in process performance (yield, pressure, etc.) and product quality (SEC-HPLC purity, CE-SDS-NR purity, HCP, etc.).

[0108] After adjusting the pH / conductivity of the affinity eluate, it was used as the loading sample for continuous anion chromatography. The sample concentration was 12.557 g / L, and the chromatography column size was selected to be 0.66 cm*9.4 cm&3.216 mL. The specific process parameters are shown in Table 2.

[0109] Table 2 Continuous anion chromatography process parameters in flow-through mode

[0110]

[0111]

[0112] The main considerations at this stage include:

[0113] (1) Selection of the number of chromatography columns: The number of chromatography columns is selected based on the balance between the total loading & elution time and the total column post-treatment time. Loading / elution time T1 = loading time + elution time = 25.61 mL / 0.54 mL / min + 20 mL / 0.54 mL / min = 84.5 min; total column post-treatment time T2 = regeneration time + post-use disinfection time + pre-equilibrium time + equilibrium time = (8 + 8 + 8 + 10) CV × 3.216 mL / 1.71 mL / min + 30 min = 93.9 min. Number of chromatography columns (The symbol According to the calculation, column position 3 was finally selected for switching and loading.

[0114] (2) In the specific operation process, column 1 is loaded with samples first. When the loading / elution is completed, the instrument with a chromatography column switching function automatically jumps to column 2 for loading / elution, and column 1 undergoes chromatography column post-processing such as regeneration, post-use disinfection, pre-equilibration, and equilibrium; when the loading / elution of column 2 is completed, the instrument automatically jumps to column 3 for loading / elution, and column 2 undergoes post-processing. During this process, column 1 completes the post-processing steps, and the flow rate returns to zero to wait for the next round of loading; when the loading / elution of column 3 is completed, the instrument automatically jumps to the column 1 with column equilibration for a new round of loading / elution, and column 3 undergoes post-processing. During this process, column 2 will complete the post-processing steps, and the flow rate returns to zero to wait for the next round of loading (refer to Figure 3 ). During the sample loading process, the pH and conductivity of the flow-through solution need to be monitored simultaneously.

[0115] According to the above process parameters, 40 loops (a total of 121 cycles) were successfully run, and no contamination occurred during the process. The results are as follows: Figure 6-11 As shown. Figure 6-8 In the continuous anion chromatography process of this flow-through mode, the flow-through peak performance is consistent, and the flow-through product yield is >96%, which is comparable to the batch mode. The product pH and conductivity of the entire process are also relatively stable. In addition, according to Figure 9-11 The product quality data, SEC-HPLC purity (Main peak: 92.11%-92.76%), SDS-CE_NR purity (Main peak: 99.21%-99.44%) and HCP level (43-53ppm) of the flow-through product were relatively stable throughout the production cycle, and the HCP level was low.

[0116] Considering the process performance and product quality throughout the entire production cycle, the continuous anion chromatography in this flow-through mode performed stably.

[0117] In addition, unlike the traditional batch process that uses only a single column for purification, the process of the present invention realizes continuous loading / elution during the purification process, and there is no need to wait for the column post-processing time. The same amount of sample is processed under the same process parameters (Table 2). The time and buffer dosage of the continuous process and the batch process are compared. The specific analysis data are shown in Tables 3 and 4.

[0118] Table 3 Comparison of time consumed by continuous process and batch process of anion chromatography

[0119]

[0120] Table 4 Comparison of buffer consumption in continuous and batch processes for anion chromatography

[0121]

[0122] Based on the data in Tables 3 and 4, the efficiency of the anion chromatography continuous process can be increased by 64.8% compared to the traditional batch process, and the buffer consumption is also significantly reduced, indicating that the continuous purification method can reduce material consumption while being more efficient.

[0123] Example 2 Continuous hydrophobic chromatography in flow-through mode

[0124] The following steps are involved:

[0125] 1. Batch chromatography process parameter development phase in flow-through mode

[0126] In this stage, the single factors affecting hydrophobic chromatography were first investigated in the batch purification process of fusion protein B, including filler screening, sample loading / washing (Wash) condition screening, sample retention time optimization, sample loading capacity optimization, and sample loading / flow-through sample stability investigation, etc. The investigation method can be carried out according to Example 1. Other parameters are antibody platform processes in flow-through mode. The processes used in different projects are different, and the process parameters are adjusted as needed. The process parameters after the final single factor optimization are shown in Table 5.

[0127] Table 5 Process parameters after hydrophobic chromatography optimization

[0128]

[0129] *This step is optional depending on the filler storage solution

[0130] 2. Multi-system continuous purification confirmation stage in flow-through mode

[0131] Based on the results of the batch chromatography process parameter development phase in flow-through mode, continuous hydrophobic polishing was achieved on a multi-column system on a continuous flow chromatography device to confirm its feasibility and stability in process performance (yield, pressure, etc.) and product quality (SEC_HPLC purity, CE-SDS-NR purity, HCP, etc.).

[0132] After adjusting the pH / conductivity of the affinity eluate, it was used as the loading sample for continuous hydrophobic chromatography. The sample concentration was 26.024 g / L, and the chromatography column size was selected to be 0.66*10.8 cm&3.695 mL. The specific process parameters are shown in Table 6.

[0133] Table 6 Continuous hydrophobic chromatography process parameters in flow-through mode

[0134]

[0135]

[0136] The main considerations at this stage include:

[0137] (1) Selection of the number of chromatography columns: The number of chromatography columns is selected based on the balance between the total loading / elution time and the total column post-treatment time. Loading / elution time T1 = loading time + elution time = 7.20 mL / 0.61 mL / min + 25 mL / 0.61 mL / min = 52.8 min; total column post-treatment time T2 = regeneration time + post-use disinfection time + pre-equilibrium time + equilibrium time = (3+3+3+4)CV×3.695 mL / 1.00 mL / min + 4 min = 52.0 min. Number of chromatography columns (The symbol Indicates rounding up). According to the calculation, column position 2 is finally selected for switching sample loading.

[0138] (2) During the specific operation, column 1 is loaded with sample first. When the loading / elution is completed, the instrument using the specific chromatography column switching function automatically jumps to column 2 for loading / elution, and column 1 undergoes chromatography column post-processing such as regeneration, disinfection, pre-equilibration, and equilibration. When the loading / elution of column 2 is completed, column 1 has also completed post-processing. The instrument automatically jumps to the well-equilibrated column 1 for the next round of loading / elution, and column 2 undergoes post-processing. After the processing steps are completed, the flow rate returns to zero and waits for the next round of loading (refer to Figure 2 ). During the sample loading process, the pH and conductivity of the flow-through solution need to be monitored simultaneously.

[0139] According to the above process parameters, 6 loops (a total of 19 cycles) were successfully run, which took 24 hours. No contamination occurred during the process. The results are as follows: Figure 12-16 .Depend on Figure 12-14 It can be seen that in the continuous hydrophobic chromatography process of this flow-through mode, the flow-through peak performance is consistent, and the flow-through product yield is above 92%, which is comparable to the batch mode. The product pH and conductivity of the entire process remain stable; in addition, Fig.14 and Fig.15 The product quality data in the data show that after this step of purification process, the HCP level of the flow-through product (10-12ppm) is significantly lower than that of the loaded sample, reducing the risks of product instability and high immunogenicity. It is relatively stable throughout the production cycle; in addition, the SEC-HPLC purity of the flow-through product (Main peak: 97.91%-97.84%) is also relatively stable throughout the production cycle.

[0140] Considering the process performance and product quality throughout the entire production cycle, the continuous hydrophobic chromatography in this flow-through mode performed stably.

[0141] In addition, this process also achieves continuous loading / elution during the purification process, without waiting for column post-processing time. The same amount of sample is processed under the same process parameters (Table 6). The time and buffer dosage of the continuous process and batch process are compared. The specific analysis data are shown in Tables 7 and 8.

[0142] Table 7 Comparison of time consumed by continuous process and batch process of hydrophobic chromatography

[0143] Protein processing amount (g) 4 Required number of cycles 19 Total time required for continuous hydrophobic chromatography (min) 1697 Total time required for traditional batch process (min) 5238 The efficiency of continuous hydrophobic process is improved by percentage compared with batch process (%) 67.6

[0144] Table 8 Comparison of buffer consumption in continuous and batch processes for hydrophobic chromatography

[0145]

[0146] Based on the data in Tables 7 and 8, the efficiency of the hydrophobic chromatography continuous process can be increased by 67.6% compared with the traditional batch process, and the consumption of various buffers is also significantly reduced, which once again verifies that the developed continuous purification method can reduce the consumption of reagents and consumables while being more efficient, thereby reducing production costs.

[0147] The implementation of the present invention is not limited to the above-mentioned embodiments. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and detail, and these are considered to fall within the protection scope of the present invention.

Claims

1. A method for continuous purification of a sample using a flow-through mode, comprising: (1) Calculate the minimum number of chromatography columns n used for purification, Wherein, the loading time T1 and the post-treatment time T2 are determined by using the chromatography column in a batch chromatography manner, and the symbol Indicates rounding up to an integer; (2) Using at least n identical chromatography columns for purification.

2. The continuous purification method according to claim 1, wherein the sample loading is performed sequentially in all chromatography columns, and after the sample loading is completed, the column is immediately post-treated.

3. The continuous purification method as claimed in claim 1, wherein an equilibration buffer is added to all chromatography columns for equilibration treatment before loading the sample.

4. The continuous polishing method according to claim 2, wherein the sample loading is circulated sequentially in all chromatography columns, thereby enabling continuous processing of samples.

5. The continuous purification method according to claim 4, wherein while one of the chromatography columns is being loaded with a sample, all the remaining chromatography columns are post-treated.

6. The continuous purification method according to claim 1, wherein the switching between the loading state and the post-processing state of the chromatography column is achieved by changing the flow path system connected to the chromatography column.

7. The continuous purification method according to any one of claims 1 to 6, further comprising monitoring the pH or conductivity of the flow-through liquid of the chromatography column on which the sample is loaded.

8. The continuous purification method according to claim 1, wherein the sample is previously captured by affinity chromatography purification or other chromatography steps.

9. The continuous polishing method according to claim 1, wherein the chromatography column is selected from the group consisting of an ion chromatography column, a hydrophobic chromatography column, a mixed mode chromatography column and a molecular sieve chromatography column.

10. The continuous polishing method according to claim 9, wherein the chromatography column is an anion chromatography column. The continuous polishing method according to claim 9 , wherein the chromatography column is a hydrophobic chromatography column.

12. The continuous polishing method according to claim 1, wherein the height of the chromatography column is 5-25 cm.

13. The continuous polishing method according to claim 1, wherein the sample loading time includes the time for sample loading and adding a washing buffer for elution.

14. The continuous purification method according to claim 1, wherein the post-treatment process comprises: Add regeneration buffer, add disinfectant, pause disinfection, add pre-equilibration buffer, add equilibration buffer.

15. The continuous polishing method according to claim 1, wherein the sample is a solution containing protein.

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