Method for continuous purification in flow-through mode

Abstract

A method for using a flow-through mode to continuously purify a sample, comprising: (1) calculating the minimum number n of chromatography columns used for purification, formula (I), wherein a sample loading duration T1 and a post-treatment duration T2 are determined by means of batch chromatography by using the chromatography columns, and symbol (II) representing ceiling; and (2) using at least n same chromatography columns to perform purification. By means of an uninterrupted flow, the continuous purification process in the flow-through mode can improve purification efficiency while reducing the use of consumables, thereby reducing production costs.

Description

A method for continuous purification in flow-through mode

[0001] This application claims priority to the Chinese patent application No. 202311083893.6 filed with the China Patent Office on August 25, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a flow-through mode continuous purification method, which can be used for the continuous purification production of proteins such as antibodies and enzyme preparations, nucleic acids, blood products or vaccines, so as to achieve low-cost, multi-cycle and automated purification. Background Art

[0003] Currently, biopharmaceuticals are primarily produced in batches, starting with the process's starting point and proceeding through multiple intermediate steps with pauses to ultimately obtain the final product. However, with the rapid development of biopharmaceuticals, the variety of large-molecule drugs has increased, and the molecules are becoming increasingly complex, increasing the difficulty of downstream purification. Furthermore, upstream manufacturers are constantly pursuing increased production capacity and scale expansion, making conventional batch production unable to meet current demands, thus giving rise to continuous production processes. As a key technology in continuous production processes, continuous flow chromatography has gradually become a focus of attention.

[0004] 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:

[0005] (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.

[0006] (2) The current continuous affinity capture process uses a multi-column operation, where samples are continuously loaded onto two chromatographic columns connected in series. Continuous sample loading and separation are achieved by sequentially switching the columns (as shown in Figure 1). This operation is sometimes unnecessary for the polishing step in flow-through mode and increases the risk of accumulation of impurities that are easily bound to the chromatographic medium on the second column.

[0007] (3) During the continuous affinity capture phase, the sample loading capacity is generally controlled by monitoring UV, such as the ΔUV control program of the AKTAPCC system and The AutomAb control program for the CUBE combined system. Unlike the affinity capture step, the polishing step primarily aims to remove trace impurities such as host cell proteins, DNA, antibody fragments, aggregates, and heterogeneous species. Chromatographic methods such as ion exchange, hydrophobic interaction, and mixed-mode chromatography are typically used. Therefore, the loading capacity of the polishing step is determined by the final product quality, making traditional ΔUV or AutomAb procedures inapplicable.

[0008] (4) When using the traditional continuous chromatography method for double-column serial loading, the pH and conductivity of the flow-through liquid cannot be monitored, which is not applicable to the process of flow-through mode.

[0009] Therefore, a continuous purification method using flow-through mode is needed in this field. This method allows for continuous single-column loading on a multi-column system at maximum filler loading capacity, while ensuring product quality, and simultaneously monitors the pH and conductivity of the flow-through solution during product flow-through. Compared to batch production, continuous purification processes using flow-through mode offer faster, more stable, and more flexible processes through an uninterrupted process, reducing consumables and, consequently, production costs. This represents a future trend in pharmaceutical production process development.

[0010] Summary of the Invention

[0011] In one aspect, the present invention provides a method for continuous purification of a sample using flow-through mode, comprising:

[0012] (1) Calculate the minimum number of chromatography columns n used for purification, The loading time T1 and the post-processing time T2 are determined by batch chromatography using the chromatography column, and the symbol Indicates rounding up to an integer;

[0013] (2) Use at least n identical chromatography columns for purification.

[0014] In some embodiments, the sample loading is performed sequentially on all chromatography columns, and after the sample loading is completed, the column is immediately post-treated.

[0015] In some embodiments, all chromatography columns are equilibrated by adding an equilibration buffer before loading the sample.

[0016] In some embodiments, the loading is cycled through all chromatography columns sequentially, thereby enabling continuous processing of samples.

[0017] In some embodiments, while one of the chromatography columns is being loaded with a sample, all remaining chromatography columns are being post-processed.

[0018] 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.

[0019] In some embodiments, the continuous polishing method further comprises monitoring the pH or conductivity of the flow-through of the chromatography column undergoing the sample loading operation.

[0020] In some embodiments, the sample is previously captured by affinity chromatography purification or other chromatography steps.

[0021] 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.

[0022] In some embodiments, the chromatography column is an anion chromatography column.

[0023] In some embodiments, the chromatography column is a hydrophobic chromatography column.

[0024] In some embodiments, the height of the chromatography column is 5-25 cm.

[0025] In some embodiments, the sample loading time includes the time for sample loading and adding wash buffer for elution.

[0026] In some embodiments, the post-treatment process includes, in sequence: adding a regeneration buffer, adding a disinfectant, pausing disinfection, adding a pre-equilibration buffer, and adding an equilibration buffer.

[0027] In some embodiments, the sample is a solution comprising protein.

[0028] On the other hand, this document also provides a non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that when the program is executed by a processor, the steps of the method described in the first aspect of this document are implemented.

[0029] In another aspect, the present invention provides a protein purification device comprising:

[0030] A memory and a processor, wherein the memory stores a computer program that, when executed by the processor, implements the steps of the method described in the first aspect of the present invention. The continuous purification process in flow-through mode provided herein can improve purification efficiency while reducing consumables through an uninterrupted process, thereby reducing production costs.

[0031] The beneficial effects of the present invention include at least:

[0032] 1. The continuous purification method of the present invention can achieve 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.

[0033] 2. The continuous purification method described in the present invention uses chromatography equipment to achieve continuous purification in 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.

[0034] 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 sample loading while using a minimum of chromatography columns, thereby achieving high efficiency, low consumption and low cost.

[0035] 4. The continuous purification method described in this invention uses a flow-through mode, which allows for high sample loading capacity and high-purity purified products. The yield reaches 92% or higher, the purity reaches over 95%, and the HCP (host cell protein) level in the product is extremely low, improving product stability and reducing immunogenicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 shows a typical continuous capture chromatography method - a schematic diagram of dual-column serial loading (3-column system).

[0037] Figure 2 shows a schematic diagram of single-column loading in flow-through mode (2-column system).

[0038] Figure 3 shows a schematic diagram of single-column sample loading in flow-through mode (3-column system).

[0039] Figure 4 shows a schematic diagram of single-column sample loading in flow-through mode (4-column system).

[0040] Figure 5 shows a schematic diagram of single-column sample loading in flow-through mode (multi-column system).

[0041] Figure 6 shows the continuous anion chromatogram in flow-through mode.

[0042] Figure 7 shows the product yield of continuous anion chromatography flow-through.

[0043] Figure 8 shows the pH and conductivity of the continuous anion chromatography flow-through product.

[0044] Figure 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.

[0045] Figure 10 shows the purity of the continuous anion chromatography flow-through product SDS-CE_NR.

[0046] Figure 11 shows continuous anion chromatography HCP levels.

[0047] Figure 12 shows the continuous hydrophobic chromatography profile in flow-through mode.

[0048] Figure 13 shows the continuous hydrophobic chromatography flow-through product yield.

[0049] Figure 14 shows the pH and conductivity of the continuous hydrophobic chromatography flow-through product.

[0050] FIG15 shows the SEC-HPLC purity of the continuous hydrophobic chromatography flow-through product.

[0051] Figure 16 shows sequential hydrophobic chromatography HCP levels. DETAILED DESCRIPTION

[0052] 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.

[0053] The term "or" refers to a single element of the listed alternative 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 alternative elements.

[0054] The terms "comprising" or "including" refer to the inclusion of the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. As used herein, when the terms "comprising" or "including" are used, unless otherwise indicated, the context also encompasses the context consisting of the stated elements, integers, or steps. For example, when reference is made to a method "comprising" two steps, the context also encompasses a method consisting of the two steps.

[0055] "Purification" in this article refers to the process of further purifying a sample (such as a crude product containing the target protein) that has undergone preliminary purification (crude purification). 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 preliminary purification step. Crude purification and refined purification can use the same or different purification methods. For example, crude purification uses precipitation (such as using ammonium sulfate, organic solvents (such as acetone) or polyethylene glycol, etc.), dialysis or tangential flow filtration, and refined purification uses chromatography (or chromatography); or both crude purification and refined purification use chromatography. In some embodiments, the refined purification method described herein is anion exchange chromatography. In other embodiments, the refined purification method described herein is hydrophobic chromatography. In some embodiments, the chromatography column filler used in the refined 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.

[0056] "Chromatography," as used herein, refers to a technique for separating (or purifying) substances by exploiting differences in the physicochemical properties of different components, resulting in differences in their distribution coefficients between different phases. Chromatographic systems typically consist of two phases: a stationary phase, which is a solid substance or a component fixed to a solid substance; and a mobile phase, which is a mobile substance such as water or various solvents. As the mixture to be separated passes through the stationary phase along with the solvent (mobile phase), the components' differing physicochemical properties and abilities to interact (adsorb, dissolve, bind, sterically hinder, etc.) with the two phases lead to different distributions between them. Furthermore, as the solvent moves forward, the components are continuously redistributed between the two phases. Components with weaker interactions with the stationary phase experience less retardation when moving with the mobile phase and move faster. Conversely, components with stronger interactions with the stationary phase move slower. By collecting the effluent fractions, the individual components contained in the sample can be obtained, thus achieving the goal of separating the components. 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 to separate components at the same time. In some embodiments, the height of the chromatography 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 chromatography column described herein refers to a chromatography column with the same height.

[0057] In some embodiments, the continuous polishing method described herein uses at least n identical chromatography columns for purification, wherein the The loading time T1 and the post-processing time T2 are determined by batch chromatography using the chromatography column, and the symbol In some embodiments, the "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.

[0058] "Affinity chromatography" refers to a chromatographic technique that exploits the specific affinity between the target substance (e.g., the product of interest) and its specific ligand to separate it from other components. One of a pair of molecules with specific binding properties is covalently linked to an insoluble carrier as a stationary phase adsorbent. When a sample containing mixed components passes through this stationary phase, only substances with specific affinity for the stationary phase molecules are adsorbed and bound by the stationary phase, while unrelated components are discharged with the mobile phase. The bound target substance can then be eluted by changing the mobile phase composition. The carrier used in affinity chromatography is also called the medium, and the compound covalently linked to the medium is called the ligand. Examples of biomolecule pairs with specific affinities include: antigen and antibody, DNA and complementary DNA or RNA, enzyme and substrate, hormone and receptor, vitamin and specific binding protein, glycoprotein and plant lectin, etc. Affinity chromatography can be used to purify biomacromolecules, concentrate dilutions, store unstable proteins, and isolate nucleic acids.

[0059] "Ion exchange chromatography" refers to a method for separating ionic compounds using a stationary phase with ion exchange properties, exploiting its ability to reversibly exchange ions with the mobile phase. This chromatographic 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) of different proteins results in varying net positive and negative charges under different pH buffer conditions, necessitating the selection of different ion exchange columns (anionic or cationic) for separation.

[0060] Hydrophobic chromatography refers to a chromatographic method that separates biomolecules based on their hydrophobicity. Generally speaking, high ionic strength enhances the hydrophobic interactions between the hydrophobic amino acids in proteins and the chromatography medium, weakening their electrostatic forces.

[0061] In this context, "flow-through mode" refers to a chromatography purification method in which the target product (e.g., protein or antibody) is allowed to flow through the column, while other components or impurities are adsorbed on the column. One advantage of using flow-through mode is that higher loadings can be used with relatively few wash and elution steps.

[0062] "Continuous purification" and "continuous loading" herein refer to, for the samples described herein, the uninterrupted purification process from the moment the sample is loaded until loading is complete. Correspondingly, "batch purification" refers to the process of dividing a sample into multiple portions, with a time interval between the end of loading one sample and the beginning of loading the next. This time interval can be used, for example, for post-processing of a chromatography column. When purification is performed using a chromatography column, continuous purification is also referred to herein as continuous chromatography, and batch purification is referred to herein as batch chromatography. In some embodiments, the sample loading / washing time, post-processing time, maximum loading capacity, and loading flow rate in the continuous purification methods described herein are determined by batch chromatography. In some embodiments, the sample comprises an antibody.

[0063] In some embodiments, the sample loading capacity is 100 mg / mL. In some embodiments, the sample 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 sample loading capacity is not greater than 60 mg / mL, preferably not greater than 51 mg / mL. In some embodiments, the sample retention time is greater than 5 minutes, preferably greater than 6 minutes.

[0064] "Loading time" and "loading / rinsing time" refer 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 80-100 min. In some embodiments, the rinsing buffer used for rinsing is 25 mM Na-Citrate. In some embodiments, the rinsing buffer is a mixture of 15 mM Na-Citrate and 1 M 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.

[0065] "Post-treatment duration" herein refers to, with respect to a single chromatography column, the total duration of the various steps of post-treatment thereof, including, 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.

[0066] When the polishing method is an anion chromatography method: In some embodiments, the post-treatment process includes, in sequence: adding 8 column volumes of regeneration buffer, adding 8 column volumes of disinfectant, disinfecting for 30 minutes, adding 8 column volumes of pre-equilibration buffer, and adding 10 column volumes of equilibration buffer. In some embodiments, the regeneration buffer is 25 mM Na-Citrate, 1 M NaCl. In some embodiments, the disinfectant is 1 M NaOH. In some embodiments, the pre-equilibration buffer is 25 mM Na-Citrate, 1 M NaCl. In some embodiments, the equilibration buffer is 25 mM Na-Citrate. In some embodiments, the flow rate of each buffer and disinfectant in the post-treatment steps is 1-2 mL / min; preferably, such as 1.4 mL / min, 1.5 mL / min, 1.6 mL / min, 1.7 mL / min, or 1.8 mL / min; more preferably, such as 1.71 mL / min.

[0067] When the polishing method is hydrophobic chromatography: In some embodiments, the post-treatment process includes, in sequence: adding 3 column volumes of regeneration buffer, adding 3 column volumes of disinfectant, pausing for 1-10 minutes, adding 3 column volumes of pre-equilibration buffer, and adding 4 column volumes of equilibration buffer. In some embodiments, the regeneration buffer is HO. 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 during the post-treatment process is 0.5-2.0 mL / min; preferably, such as 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min, or 1.0 mL / min; more preferably, such as 1.0 mL / min.

[0068] "Switching loading" in this article means 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 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 connected to the 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 instrumentation, real-time pH and conductivity monitoring of the convective stream can be achieved.

[0069] "Sequentially" as used herein means that the sample passes through all chromatography columns sequentially, with unloaded chromatography columns remaining in the equilibration solution awaiting sample loading, and loaded chromatography columns beginning post-processing upon completion of sample loading. In some embodiments, such as a three-column system, while the first chromatography column is being loaded, the second and third chromatography columns are in a state awaiting sample loading. While the second chromatography column is being loaded, the first chromatography column is in a post-processing state, while the third chromatography column is in a state awaiting sample loading.

[0070] As used herein, "cycle sequentially" means that after the sample has passed through all columns, it cycles to the first column for loading, while all remaining columns are in post-processing mode. In some embodiments, such as a three-column system, after loading has occurred sequentially through all columns, the sample is then transferred back to the first column for loading, while the second and third columns undergo post-processing. When loading is transferred to the second column, the first and third columns undergo post-processing.

[0071] 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.

[0072] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0073] 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:

[0074] 1. Batch chromatography process development stage in flow-through mode: In this stage, individual factors that affect the continuous purification steps need to be examined 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.

[0075] (1) Filler screening: Based on the charge of the protein and the process requirements, select appropriate fillers for flow-through mode chromatography process development. Taking anion chromatography as an example, fillers such as Poros XQ, Poros 50HQ, and QFF 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 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.

[0076] 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 examined to screen the most suitable filler.

[0077] (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, flow-through peak shape, etc.) and the quality parameters of the flow-through product (such as SEC-HPLC purity, HCP, rDNA, etc.) are examined to screen the optimal loading / elution conditions.

[0078] (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.

[0079] (4) Sample loading capacity optimization: Unlike the affinity capture step, the sample loading capacity of the polishing step is not determined by the flow-through ratio of the sample, but by 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 ultimately determine the optimal sample loading capacity.

[0080] (5) Examination of column height: The principle of the continuous purification process in the flow-through mode is that the target protein first flows through the column, while impurities such as rDNA and HCP bind to the chromatographic filler and do not flow through, ultimately achieving protein purification. Therefore, if the column height 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 column height 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 5-25 cm, and the acceptable column height range should be determined based on the pressure conditions, product yield and product quality parameters at different column heights.

[0081] (6) Stability investigation of sample loading / flow-through: 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 sample loading / flow-through. 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.

[0082] 2. Continuous purification confirmation phase for multiple systems in flow-through mode: Based on the results of the batch chromatography process development phase in flow-through mode, continuous purification of the multi-column system is performed on the chromatography instrument to confirm its feasibility and stability in terms of process performance (e.g., yield, pressure, etc.) and product quality (SEC-HPLC purity, CE-SDS-NR purity, HCP, etc.). The main considerations in this phase include the following:

[0083] (1) At this stage, the choice of a 2 / 3 / 4 or more column position system can be determined based on the balance between the loading and elution time and the total column post-processing time: if the loading / elution time T1 ≥ the total column post-processing time T2, a 2-column position system can be selected for switching loading; if the loading / elution time T1 < the total column post-processing time T2, a 3-column position 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.

[0084] (2) In the specific implementation process, a chromatographic device is required to realize single-column loading. This method can be understood in conjunction with the single-column flow loading diagram of different column systems (Figures 2-5). For a multi-column system, 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 loading on column 2, and columns 3 and 1 perform post-processing separately; cycle 3 performs 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 the 3-column system. During the loading process, the pH and conductivity of the flow-through can be monitored simultaneously.

[0085] In some embodiments, the present invention provides a continuous purification method in flow-through mode, which includes a process development stage and a confirmation stage. 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. The loading and elution steps of the continuous purification process are uninterrupted.

[0086] In some embodiments, the multi-column system in the confirmation stage includes 2 columns, 3 columns, or more columns.

[0087] 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 or more column positions can be selected for switching loading.

[0088] 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.

[0089] In some embodiments, the cyclic process of the multi-column system is that the Xth column position performs single column loading at any Xth cycle, and the remaining column positions perform post-processing respectively.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the loading / washing conditions include a loading / washing pH of pH 5-9 and a loading / washing conductivity of 0.5-100 mS / cm. In some embodiments, the loading / washing conditions include a loading / washing pH of pH 5-9 and a loading / washing conductivity of 0.5-3.63 mS / cm. In some embodiments, the loading / washing conditions include a loading / washing pH of pH 5-9 and a loading / washing conductivity of 85-95 mS / cm. In some embodiments, the loading / washing conditions include a loading / washing pH of pH 5-9 and a loading / washing conductivity of less than or equal to 4.0 mS / cm.

[0093] In some embodiments, the chromatography column has a height of 5-25 cm.

[0094] In some embodiments, the "non-transitory computer-readable storage medium" described herein may be, for example, an optical disk or a hard disk.

[0095] The technical solutions of the present invention will be further described in detail below by way of examples and in conjunction with the accompanying drawings. Unless otherwise stated, the methods and materials of the embodiments described below are all conventional products that can be purchased on the market. Those skilled in the art will appreciate that the methods and materials described below are merely exemplary and should not be construed as limiting the scope of the present invention.

[0096] Example 1 Continuous Anion Chromatography in Flow-Through Mode

[0097] The following steps are involved:

[0098] 1. Batch chromatography process parameter development phase in flow-through mode

[0099] In this phase, we first investigated the individual factors that affect the continuous purification steps 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. Other parameters refer to the antibody platform process in flow-through mode. The processes used in different projects vary, and the parameters are adjusted according to the required process. The specific operations are as follows:

[0100] (1) Filler screening: Based on the protein charge and process requirements, select appropriate fillers for flow-through 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 the flow-through mode.

[0101] The optimal 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 product (SEC-HPLC purity, HCP, rDNA, etc.).

[0102] (2) Screening of loading / elution conditions: For the purification step, the influence 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, flow-through peak shape, etc.) and the quality parameters of the flow-through product (SEC-HPLC purity, HCP, rDNA, etc.) are examined to screen the optimal loading / elution conditions.

[0103] (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.

[0104] (4) Sample loading capacity optimization: Unlike the affinity capture step, the sample loading capacity of the polishing 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 of 80 g / L, 100 g / L, 120 g / L, and 140 g / L, and ultimately determined the optimal sample loading capacity.

[0105] (5) Examination of column height: The principle of the continuous purification process in flow-through mode is that the target protein first flows through the column, while impurities such as rDNA and HCP combine with the chromatographic filler and do not flow through, ultimately achieving protein purification. Therefore, if the column height 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 column height 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 5-25 cm, and the acceptable column height range should be determined based on the pressure conditions, product yield and product quality parameters at different column heights.

[0106] (6) Stability investigation of loaded / 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 loaded / 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 there is an increase in column pressure during the loading process.

[0107] According to the above steps, the final process parameters after single factor optimization are shown in Table 1.

[0108] Table 1 Anion chromatography process parameters after optimization *CV: Column volume, column volume

[0109] 2. Multi-system continuous purification confirmation stage in flow-through mode

[0110] 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.).

[0111] The affinity eluate was adjusted for pH and conductivity and used as the loading sample for continuous anion chromatography. The sample concentration was 12.557 g / L, and the column dimensions were 0.66 cm*9.4 cm and 3.216 mL. The specific process parameters are shown in Table 2.

[0112] Table 2 Continuous anion chromatography process parameters in flow-through mode

[0113] The main considerations at this stage include the following:

[0114] (1) Selection of the number of chromatography columns: The number of chromatography columns is selected based on the balance between the total loading and 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-equilibration time + equilibrium time = (8 + 8 + 8 + 10) CV × 3.216 mL / 1.71 mL / min + 30 min = 93.9 min. Number of chromatography columns (where the symbol According to the calculation, column position 3 was finally selected for switching and loading.

[0115] (2) During the specific operation, column 1 is loaded first. When the loading / rinsing is completed, the instrument with a chromatography column switching function automatically jumps to column 2 for loading / rinsing. Column 1 undergoes chromatography column post-processing such as regeneration, post-use disinfection, pre-equilibration, and equilibration. When the loading / rinsing of column 2 is completed, the instrument automatically jumps to column 3 for loading / rinsing. Column 2 undergoes post-processing. During this process, column 1 completes the processing steps and the flow rate returns to zero to wait for the next round of loading. When the loading / rinsing of column 3 is completed, the instrument automatically jumps to the balanced column 1 for a new round of loading / rinsing. Column 3 undergoes post-processing. During this process, column 2 completes the post-processing steps and the flow rate returns to zero to wait for the next round of loading (see Figure 3). During the loading process, the pH and conductivity of the flow-through need to be monitored simultaneously.

[0116] Based on the above process parameters, 40 loops (a total of 121 cycles) were successfully run without any contamination, as shown in Figures 6-11. As shown in Figures 6-8, during the continuous anion chromatography process in this flowthrough mode, the flowthrough peak performance was consistent, and the flowthrough product yield was >96%, comparable to that in batch mode. The product pH and conductivity were also relatively stable throughout the process. Furthermore, according to the product quality data in Figures 9-11, the 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 flowthrough product were relatively stable throughout the production cycle, with low HCP levels.

[0117] Considering the process performance and product quality throughout the entire production cycle, the continuous anion chromatography in this flow-through mode performed stably.

[0118] In addition, unlike traditional batch processes that use only a single column for purification, the process of the present invention achieves continuous loading / elution during the purification process, eliminating the need to wait for column post-processing. The same amount of sample was processed under consistent process parameters (Table 2). The time and buffer usage of the continuous process and batch process were compared. Specific analytical data are shown in Tables 3 and 4.

[0119] Table 3 Comparison of time consumed by continuous process and batch process for anion chromatography

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

[0121] Based on the data in Tables 3 and 4, the efficiency of the continuous anion chromatography process can be increased by 64.8% compared to the traditional batch process, and the buffer consumption is also significantly reduced, showing that this continuous purification method is more efficient and can also reduce material consumption.

[0122] Example 2 Continuous hydrophobic chromatography in flow-through mode

[0123] The following steps are involved:

[0124] 1. Batch chromatography process parameter development phase in flow-through mode

[0125] In this phase, individual factors influencing hydrophobic chromatography were first investigated in the batch purification process of fusion protein B, including filler screening, sample loading / wash condition screening, sample retention time optimization, sample loading capacity optimization, and sample loading / flowthrough sample stability testing. The investigation method can be carried out according to Example 1. Other parameters refer to the antibody platform process in flowthrough mode. The process used in different projects varies, and process parameters are adjusted as required. The final process parameters after single-factor optimization are shown in Table 5.

[0126] Table 5 Process parameters after hydrophobic chromatography optimization *This step is optional depending on the filler storage solution

[0127] 2. Multi-system continuous purification confirmation stage in flow-through mode

[0128] 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.).

[0129] The affinity eluate was adjusted for pH and conductivity and used as the loading sample for continuous hydrophobic chromatography. The sample concentration was 26.024 g / L, and the column dimensions were 0.66 x 10.8 cm x 3.695 mL. The specific process parameters are shown in Table 6.

[0130] Table 6 Continuous hydrophobic chromatography process parameters in flow-through mode

[0131] The main considerations at this stage include the following:

[0132] (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-equilibration time + equilibrium time = (3 + 3 + 3 + 4) CV × 3.695 mL / 1.00 mL / min + 4 min = 52.0 min. Number of chromatography columns (where the symbol According to the calculation, column position 2 was finally selected for switching and loading.

[0133] (2) During the specific operation, column 1 is loaded first. When the loading / rinsing is completed, the instrument automatically switches to column 2 using the specific column switching function for loading / rinsing. Column 1 undergoes post-processing such as regeneration, disinfection, pre-equilibration, and equilibration. When the loading / rinsing of column 2 is completed, column 1 has also completed post-processing. The instrument automatically switches to the equilibrated column 1 for the next round of loading / rinsing. Column 2 undergoes post-processing. After the post-processing steps are completed, the flow rate returns to zero and waits for the next round of loading (see Figure 2). During the loading process, the pH and conductivity of the flow-through need to be monitored simultaneously.

[0134] Based on the above process parameters, six loops (a total of 19 cycles) were successfully run over a 24-hour period, with no contamination observed. The results are shown in Figures 12-16. As shown in Figures 12-14, during the continuous hydrophobic chromatography process in this flow-through mode, the flow-through peaks exhibited consistent performance, with flow-through product yields exceeding 92%, comparable to batch mode. The product pH and conductivity remained stable throughout the entire process. Furthermore, the product quality data in Figures 14 and 15 show that after this polishing step, the HCP level (10-12 ppm) of the flow-through product was significantly reduced compared to the loaded sample, reducing the risks of product instability and high immunogenicity. Furthermore, the product remained relatively stable throughout the production cycle. Furthermore, the SEC-HPLC purity of the flow-through product (Main peak: 97.91%-97.84%) was also relatively stable throughout the production cycle.

[0135] Considering the process performance and product quality throughout the entire production cycle, the continuous hydrophobic chromatography in this flow-through mode performed stably.

[0136] In addition, this process also achieves continuous loading / elution during the purification process, eliminating the need to wait for column post-processing. The same amount of sample was processed under consistent process parameters (Table 6). The time and buffer usage of the continuous process and batch process were compared. Specific analytical data are shown in Tables 7 and 8.

[0137] Table 7 Comparison of time consumed by continuous process and batch process of hydrophobic chromatography

[0138] Table 8 Comparison of buffer consumption in continuous and batch processes for hydrophobic chromatography

[0139] Based on the data in Tables 7 and 8, the efficiency of the hydrophobic chromatography continuous process can be increased by 67.6% compared to the traditional batch process, and the consumption of various buffers is also significantly reduced. This once again verifies that the developed continuous purification method is more efficient while also reducing the consumption of reagents and consumables, thereby reducing production costs.

[0140] The embodiments of the present invention are not limited to the above 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 details, and these are all considered to fall within the scope of protection 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.

16. A non-transitory computer-readable storage medium having a computer program stored thereon, characterized in that: When the program is executed by a processor, the steps of the method according to any one of claims 1 to 15 are implemented.

17. A device for protein purification, comprising: A memory and a processor, wherein the memory stores a computer program executable on the processor, and when the computer program is executed by the processor, the steps of the method according to any one of claims 1 to 15 are implemented.

Images