CN113880907A - Research method for development of Protein A affinity chromatography continuous flow process - Google Patents
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Abstract
The invention provides a research method for development of Protein A affinity chromatography continuous flow process, belonging to the technical field of biological pharmacy, comprising the following steps: development stage of batch affinity chromatography process parameters, confirmation stage of a penetration curve of a single-column affinity chromatography process, confirmation stage of sample loading capacity of a double-column affinity chromatography process, confirmation stage I of column position 3 or column position 4 continuous flow chromatography, and confirmation stage II of column position 3 or column position 4 continuous flow chromatography; the invention provides a certain guiding function for the development of other chromatography continuous flow processes such as affinity chromatography and the like through a simpler and faster test flow, shortens the development time of the partial processes and accelerates the application range of the continuous flow chromatography in the aspect of protein purification technology.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to a research method for developing a Protein A affinity chromatography continuous flow process.
Background
As therapeutic monoclonal antibody (mAb) drugs are developed throughout the drug development process, from development to clinical trials to regulatory agencies to market acceptance by marketable markets, every link of analysis, production and quality control (CMC) plays a critical role. Currently, almost all commercial mAb products are produced in batch mode, conventional column chromatography is run in batch mode, and buffer and media utilization is low. Secondly, the volume of the chromatographic column cannot be enlarged under the influence of column pressure, protein products are easy to inactivate, and the treatment efficiency and recovery rate are low. Such a production process is costly, long in manufacturing time, inefficient, and occupies a large area of equipment and facilities. Thus, biopharmaceutical companies are constantly seeking alternative strategies or new technologies to address these issues.
Biopharmaceuticals are currently produced in batches, starting from the beginning of the process, through a number of intermediate steps with pauses, and finally obtaining the final product. Continuous production processes, i.e., continuous production, have been used for many years in food, petrochemical and chemical production, and, unlike batch production, continuous production in which raw materials are continuously added at the beginning of the process and the final product is continuously harvested at the end, theoretically without a step of residence in between, are called continuous production. In the process, the quality of the medicine can be well controlled in the production process, and the quality is not released only by the standard reaching of the final product. The advantage of continuous processing is evident from an economic standpoint compared to batch processing, which can increase productivity in shorter processing times while reducing overall cost, reducing equipment footprint and ensuring product quality.
One strategy for protein drug manufacturing is to employ Continuous Biological Processes (CBPs), a technique that has been successfully used to produce amino acids, vitamins and antibiotics for decades. CBP has demonstrated a number of advantages, including high efficiency, high productivity, and high consistency. CBP also takes advantage of real-time monitoring, control and automation, small footprint of hardware and other facilities (e.g., chromatography columns, chromatography resins, reactors). In order to overcome the defects of the traditional chromatography process, adapt to the continuous expansion of upstream cell culture scale and the improvement of protein expression, a process development expert develops a continuous purification technology. Continuous multi-column chromatography techniques such as continuous loop chromatography (CAC), Radial Flow Chromatography (RFC), expanded bed adsorption chromatography (EBA), multi-layer countercurrent solvent gradient chromatography (MSCGP), periodic countercurrent flow chromatography (PCC), Continuous Countercurrent Tangential Chromatography (CCTC), and Simulated Moving Bed (SMB) have been developed.
Currently, continuous flow chromatography is gradually applied in the biopharmaceutical industry, and protein affinity chromatography is often used as a first step process of downstream purification in antibody production, wherein the cost of affinity filler is high, and the price of the filler per liter is 5-10 ten thousand RMB. In the continuous flow chromatography, 3 column positions or a plurality of column positions can be adopted for carrying out repeated chromatography for a plurality of times, and different chromatographic columns are relatively independent, so that the production efficiency is improved, and the use amount of the filler is reduced. The process development procedures for affinity chromatography and other continuous flow chromatography have not been well defined and have been adapted in different ways by different researchers.
Disclosure of Invention
In view of the above, the present invention provides a research method for development of Protein a affinity chromatography continuous flow process, so as to solve the following problems in the conventional development process: the development process of the continuous flow chromatography process is complex, and the process method with strong robustness and applicability can be developed only by carrying out reasonable design and experimental verification on the basis of fully researching batch process parameters. The continuous flow process is developed mainly by adopting a simple and quick test flow.
In order to solve the technical problems, the invention provides a research method for developing a Protein A affinity chromatography continuous flow process, which comprises the following steps:
(1) development stage of batch affinity chromatography process parameters: the method comprises the steps of loading, washing, eluting, washing and storing in the chromatography process, optimizing buffer solution components, pH, conductivity, retention time and washing volume process parameters required by each stage, developing recommended process parameters and platform process technical conditions based on chromatography packing, and considering the stability characteristics of a target protein sample in a project;
(2) confirming the penetration curve of the single-column affinity chromatography process: on the basis of the determination of the affinity technological parameters, the dynamic loading capacity of the affinity chromatography sample loading is investigated. And (3) inspecting the dynamic loading capacity of the samples under different loading retention times, determining the proper loading retention time and the maximum loading capacity under the retention time when the maximum combined loading capacity of the samples under different retention times changes to a certain extent. The difference between different projects is large, and experimental tests need to be carried out according to different project characteristics and process characteristics;
(3) and (3) confirming the loading capacity of the double-column affinity chromatography process: on the basis of the determination of the single-column affinity chromatography penetration curve, the sample adsorption distribution proportion of the column position 1 and the column position 2 in the double-column affinity chromatography process is determined. Inspecting the mass of the eluted sample at the column position 1 and the mass proportion of the eluted sample at the column position 2 under different sample loading capacity, determining the mass ratio of the combined samples of the column 1 and the column 2, determining the optimal sample loading capacity of the double columns, and ensuring that no sample penetrates through the 2 nd chromatographic column in the 2 nd column position series sample loading;
(4) 3-column or 4-column continuous flow chromatography confirmation stage I: on the basis of determining the loading capacity result of the double-column series process, continuous chromatography is carried out on 3 column positions or 4 column positions, and the feasibility of the process in the aspect of yield process attributes is confirmed. This procedure was performed using a 3PCC continuous chromatography system, setting the initial 1 st tandem loading volume V1 and the subsequent tandem loading volume V2, the loading volume V1: V2 ratio being as m 1: the proportion of m2 is ensured to be consistent, and the sample accumulation effect is not generated in the continuous circulation process;
(5) 3-column or 4-column continuous flow chromatography confirmation stage II: mainly on the basis of an affinity continuous chromatography confirmation stage I, the multi-cycle continuous chromatography, the service life of a chromatographic column filler and the system stability are determined.
Further, the retention time of each process in the step 1 is more than or equal to 5 min.
Further, the retention time of each process in the steps 2-5 is more than or equal to 2.5 min.
The technical scheme of the invention has the following beneficial effects:
the invention provides a certain guiding function for the development of other chromatography continuous flow processes such as affinity chromatography and the like through a simpler and faster test flow, shortens the development time of the partial processes and accelerates the application range of the continuous flow chromatography in the aspect of protein purification technology.
Drawings
FIG. 1 is a schematic representation of the binding mode of continuous flow chromatography, chromatographic process (Cytiva 3PCC mode) of the present invention;
FIG. 2 is a logic diagram of the continuous flow tomography programming of the present invention (Cytiva 3PCC mode);
FIG. 3 is a graph of the breakthrough event at step 2 of the present invention;
FIG. 4 is a graph of the loading capacity at step 3 of the present invention;
FIG. 5 is a graph of data for a sample loading ratio of 60% at step 4 of the present invention;
FIG. 6 is a graph of data for a sample loading ratio of 70% at step 4 of the present invention;
FIG. 7 is a graph of data for a sample loading ratio of 80% at step 4 of the present invention;
FIG. 8 is a graph of data for the loading ratio of 100% at step 4 of the present invention.
Detailed Description
The following examples are intended to illustrate the present invention in detail and should not be construed as limiting the scope of the present invention in any way. The instruments and devices referred to in the following examples are conventional instruments and devices unless otherwise specified; the related reagent raw materials are all conventional reagent raw materials which are sold on the market if not specifically mentioned; the methods involved are conventional methods unless otherwise specified.
Example 1
As shown in fig. 1-7: a research method for developing a Protein A affinity chromatography continuous flow process comprises the following steps:
the method comprises the following steps of (I) a batch affinity chromatography process parameter development stage: the method mainly comprises the steps of loading, washing, eluting, washing and the like in the chromatography process, optimizing process parameters such as buffer solution components, pH, conductivity, retention time, washing volume and the like required by each stage, developing recommended process parameters and platform process technical conditions based on chromatography packing, and considering characteristics such as stability of a target protein sample in a project;
the Protein A affinity chromatography process parameters used in the patent are antibody affinity chromatography platform process technology, the specific processes adopted are shown in the following table, the processes adopted in different projects are different, and the process parameters are adjusted according to requirements.
| Serial number | Step (ii) of | Buffer or sample | Retention time | Volume (CV) |
| 1 | Washing with water | WFI | ≥5min | ≥ |
| 2 | CIP | 0.1M NaOH | ≥5min | ≥ |
| 3 | Balancing | 50mM Tris-HCl,150mM NaCl,pH 7.0 | ≥5min | ≥3CV |
| 4 | Sample loading | Supernatant fluid | ≥5min | The loading capacity is less than or equal to 35g/L |
| 5 | Leaching 1 | 50mM Tris-HCl,150mM NaCl,pH 7.0 | ≥5min | ≥2CV |
| 6 | |
50mM Tris,1.5M NaCl,pH 8.0 | ≥5min | ≥3CV |
| 7 | |
20mM NaAc/HAc,pH 5.0 | ≥5min | ≥ |
| 8 | Elution is carried out | 20mM HAc,pH 3.0 | ≥5min | ≥4CV |
| 9 | CIP | 0.1M NaOH | ≥5min | ≥3CV |
| 10 | Washing with water | WFI | ≥5min | ≥2CV |
| 11 | Preservation of | 20％EtOH | ≥5min | ≥2CV |
In the development of the subsequent continuous chromatography process, the improvement of the sample loading capacity is mainly focused, and meanwhile, the retention time in the sample loading process is ensured to be within a reasonable range. Based on the technological parameters of batch purification, the retention time of the process is 5min, and in continuous chromatography, because double columns are connected in series for sampling, the retention time of a single chromatographic column is 2.5min, which is equal to the retention time of the process in batch chromatography. Therefore, in the subsequent development of the continuous flow process, the retention time of the single-root chromatography was set to be ≧ 2.5 min.
(II) a single-column affinity chromatography process penetration curve confirmation stage: the method is mainly used for investigating the dynamic loading capacity of the sample loading of affinity chromatography on the basis of determining the parameters of the affinity process. And (3) inspecting the dynamic loading capacity of the samples under different loading retention times, determining the proper loading retention time and the maximum loading capacity under the retention time when the maximum combined loading capacity of the samples under different retention times changes to a certain extent. The difference between different projects is large, and experimental tests need to be carried out according to different project characteristics and process characteristics;
in this experiment, the sample breakthrough data during affinity chromatography was analyzed for 3min retention time as described above. In the stage, a chromatographic column with the diameter of 1cm, the height of the column of 10cm and the volume of 7.85ml is adopted, a sample is a clarified liquid obtained after fermentation of a certain project, and the protein expression C0 is 0.43 mg/ml. The C1 concentration is the concentration value of the breakthrough sample during chromatography.
According to penetration curve analysis, when the retention time is 3min and the loading capacity reaches 104g/L, the sample is completely penetrated, the chromatographic column cannot capture the sample, and the chromatographic column reaches the maximum binding capacity. From the analysis of the yield of the eluted sample, the maximum binding capacity of the filler is less than or equal to 57 g/L. In the subsequent continuous flow chromatography, the sample loading should be in the range of 57-114g/L under the condition of two columns in series. The loading capacity is less than or equal to 57g/L, and a second chromatographic column can be combined without a first penetrated sample, so that the utilization rate of the packing capacity of the chromatographic column is reduced. The loading capacity of the sample is more than or equal to 114g/L, the second chromatographic column cannot be combined with the first penetrated sample, the sample has larger loss, and the yield is reduced.
(III) a sample loading capacity confirmation stage of the double-column affinity chromatography process: mainly on the basis of the determination of a single-column affinity chromatography penetration curve, the sample adsorption distribution proportion of a column position 1 and a column position 2 in the double-column affinity chromatography process is determined. Inspecting the mass of the eluted sample at the column position 1 and the mass proportion of the eluted sample at the column position 2 under different sample loading capacity, determining the mass ratio of the combined samples of the column 1 and the column 2, determining the optimal sample loading capacity of the double columns, and ensuring that no sample penetrates through the 2 nd chromatographic column in the 2 nd column position series sample loading;
in this experiment, as described above, the sample loading capacity of the sample during the double-column tandem affinity chromatography was confirmed and analyzed under the condition of 3min retention time of a single column. The diameter of a chromatographic column adopted in the stage is 1.6cm, the height of the column is 9cm, the volume of the column is 18ml, a sample is a clarified solution after fermentation of a certain project, and the protein expression amount is 0.516 mg/ml.
According to the analysis of the sample yield results of the column 1 and the column 2 in the double-column series affinity chromatography process, when the retention time is 3min and the loading capacity reaches 70g/L, the sample does not penetrate through the two chromatographic columns, part of the sample is washed away in the process of removing a small amount of impurities, the two chromatographic columns almost completely capture the loaded sample, the sample yield of the two chromatographic columns is 95.6%, the ratio of the sample binding mass m1 of the column 1 to the sample binding mass m2 of the column 2 is 53:13, and the ratio is about 4.3: 1. therefore, in the subsequent continuous chromatography process, on the basis of meeting the design of the process, the loading capacity can be set to 70g/L, and in the circulation process, the volume of the sample loaded in the second round of chromatography is set to be 80% of the volume of the sample loaded in the first round of chromatography, and the calculation formula is m2/(m1+ m2), so that the situation that the number of unbound samples of the front chromatographic column is gradually increased in an accumulative manner in the circulation chromatography process, and the sample cannot be completely recovered is avoided.
(IV) 3-column or 4-column continuous flow chromatography confirmation stage I: the method is mainly characterized in that on the basis of determining the loading capacity result of the double-column series process, continuous chromatography is carried out on 3 or 4 column positions, and the feasibility of the process in the aspect of yield process attributes is confirmed. The process was carried out using primarily the Cytiva corporation 3PCC continuous chromatography system, setting the initial 1 st tandem loading volume V1 and the subsequent tandem loading volume V2, with the loading volume V1: V2 ratios as m 1: the proportion of m2 is ensured to be consistent, and the sample accumulation effect is not generated in the continuous circulation process;
according to the loading capacity of 70g/L, a continuous flow chromatography experiment is carried out, 3 chromatographic columns with the diameter of 1.6cm, the height of the packed column of 9cm and the volume of 18ml are adopted in the stage, and the sample loading volume proportion in the circulating sample loading process is respectively researched, wherein the setting proportion is respectively 60%, 70%, 80% and 100%. The sample is a clarified liquid after fermentation of a certain item, and the protein expression amount is 0.516 mg/ml.
Table 1 (loading ratio 60%):
table 2 (loading ratio 70%):
table 3 (loading ratio 80%):
table 4 (loading proportion 100%):
carrying out chromatography under different sample loading proportion values, and analyzing the overall sample recovery rate, wherein the sample recovery rates are all more than or equal to 90% under the condition that the sample loading times are 3. When the sample loading proportion is 80%, the overall recovery rate of the sample is highest, and the average value of the mass of the total elution samples of AC-E-1, AC-E-2 and AC-E-3 is as follows: the AC-E-4 eluted sample mass was 5.5:1, which is greater than 4.3: 1. During the multi-cycle loading process, the sample does not produce accumulation effect.
And (V) 3-column or 4-column continuous flow chromatography confirmation stage II: mainly on the basis of an affinity continuous chromatography confirmation stage I, the multi-cycle continuous chromatography, the service life of a chromatographic column filler and the system stability are determined.
According to the sample loading capacity of 70g/L, carrying out continuous flow chromatography experiments, adopting 3 chromatographic columns with the diameter of 1.6cm, the height of the packed column of 11cm and the volume of 22ml at the stage, setting the sample loading proportion to be 80% in the continuous sample loading process and the sample loading retention time to be 3min, and carrying out 6 sample loading rounds in total. And result analysis shows that the total recovery rate of the sample is 89.6%, and the requirement required by process design is met.
While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (3)
1. A research method for development of Protein A affinity chromatography continuous flow process is characterized in that: the method comprises the following steps:
(1) development stage of batch affinity chromatography process parameters: the method comprises the steps of loading, washing, eluting, washing and storing in the chromatography process, optimizing buffer solution components, pH, conductivity, retention time and washing volume process parameters required by each stage, developing recommended process parameters and platform process technical conditions based on chromatography packing, and considering the stability characteristics of a target protein sample in a project;
(2) confirming the penetration curve of the single-column affinity chromatography process: on the basis of the determination of the affinity technological parameters, the dynamic loading capacity of the affinity chromatography loading is investigated, the dynamic loading capacity of the sample is investigated at different loading retention times, the maximum combined loading capacity of the sample at different retention times is changed to a certain extent, the proper loading retention time and the maximum loading capacity at the retention time are determined, the difference between different projects is large, and experimental tests are required to be carried out according to different project characteristics and technological characteristics;
(3) and (3) confirming the loading capacity of the double-column affinity chromatography process: on the basis of determining a single-column affinity chromatography penetration curve, confirming the adsorption distribution proportion of the samples at the column positions 1 and 2 in the double-column affinity chromatography process, inspecting the mass of the eluted samples at the column positions 1 and the mass proportion of the eluted samples at the column positions 2 under different loading capacity, determining the mass ratio of the combined samples of the column positions 1 and 2, determining the optimal loading capacity of the double-column, and ensuring that no sample penetrates through the 2 nd chromatographic column in the 2 nd column position series loading;
(4) 3-column or 4-column continuous flow chromatography confirmation stage I: on the basis of determining the loading capacity result of the double-column tandem process, continuous chromatography is carried out on 3 or 4 column positions, and the feasibility of the process in the aspect of yield process attributes is confirmed, the process is carried out by using a 3PCC continuous chromatography system, the initial 1 st tandem loading volume V1 and the subsequent tandem loading volume V2 are set, and the loading volume V1 to V2 ratio is as m 1: the proportion of m2 is ensured to be consistent, and the sample accumulation effect is not generated in the continuous circulation process;
(5) 3-column or 4-column continuous flow chromatography confirmation stage II: mainly on the basis of an affinity continuous chromatography confirmation stage I, the multi-cycle continuous chromatography, the service life of a chromatographic column filler and the system stability are determined.
2. The method of claim 1, wherein the method is used for developing a Protein A affinity chromatography continuous flow process, and comprises the following steps: the retention time of each process in the step 1 is more than or equal to 5 min.
3. The method of claim 1, wherein the method is used for developing a Protein A affinity chromatography continuous flow process, and comprises the following steps: the retention time of each process in the steps 2-5 is more than or equal to 2.5 min.
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