WO2019170145A1 - 一种生物大分子上游分阶段截留的生产方法、生产模块及在生产中的应用 - Google Patents
一种生物大分子上游分阶段截留的生产方法、生产模块及在生产中的应用 Download PDFInfo
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Definitions
- the invention belongs to the field of biological product production, and more specifically relates to industrial production of biological macromolecules, in particular, upstream process preparation of polypeptides, fusion proteins or immunoglobulins.
- Biomacromolecules especially fusion proteins and immunoglobulin molecules, are hotspots in the biopharmaceutical field today. Their production needs to be prepared by the upstream process of cell culture, and then obtained from the cell culture solution, purified, refined, and concentrated. And three major steps in the preparation of a preparation process for placing an antibody molecule in a formulation solution suitable for the human body.
- the expression system for the production of recombinant proteins in the upstream process is mainly established in mammalian cell culture systems, generally starting from the activation of cells in a single storage tube of the working cell bank, through a triangular flask, 2 liters, 10 liters, 50 liters, It is then produced in a 500 liter cell reactor.
- the medium involved in the cell culture process is as many as 70-80 kinds of substances, and in the cell culture process, it may accumulate secreted proteases, small cell fragments and host cell proteins, DNA, RNA molecules, etc. in the cell reaction. A variety of impurities. At the same time, it also includes defective antibody molecules or antibody molecules with different charge distribution due to redox.
- the downstream process it is necessary to remove cells by centrifugation and depth filtration, and then harvest the antibody by protein A affinity chromatography.
- the product yield of this step is more than 95%, but at this time, not only the vast majority of intact antibody molecules carrying the Fc fragment are harvested, but also the defective antibody molecules and the impurities related to the product properties are harvested together, resulting in Subsequent purification and refining steps are difficult to operate. Therefore, in such cases, in order to ensure the high quality of the product, or the similarity of different production batches, the purification and refining steps of the downstream process are often the result of sacrificing yield.
- the upstream cell culture stage of large-scale industrial production of existing biomacromolecules often adopts two forms of perfusion culture and fed-batch culture, and then combines different cell retention processes to achieve High-density cell culture while simultaneously filtering impurities in cell culture fluid.
- ATF technology Alternating Tangential Flow Filtration
- ATF technology is a state-of-the-art interception process, which is a filtration system that guarantees low shear to cells, large filtration area and strong anti-clogging ability.
- High density cell filtration culture can be achieved.
- due to the continuous growth of cells due to the continuous growth of cells, metabolites or product-related impurities of different properties besides expressed proteins are accumulated during large-scale cell culture, such as in the initial stage of cell culture.
- the invention provides a production method for phased retention in the upstream production process of biological macromolecules, a production module used in combination with the method, and the application of the method/module in industrial production of polypeptides, fusion proteins and immunoglobulin molecules,
- the method can effectively improve the efficiency, yield and purity of the upstream process of large-scale recombinant protein.
- a method of producing a phased retention of a biomacromolecule upstream comprising the use of a bioreactor in conjunction with a production system of a retention system, the method comprising at least two production stages, in the first production stage, Using a trapping system comprising a first filter module comprising a filter assembly having a filter aperture of from 0.05 ⁇ m to 1 ⁇ m; in a second production phase, using a trapping system comprising a second filter module, said The second filtration module comprises a filter element having a molecular weight cut-off of 1/15 to 2/3 of the molecular weight of the product.
- the first filtration module comprises a filter assembly having a pore size of from 0.1 ⁇ m to 0.5 ⁇ m, preferably from 0.2 ⁇ m to 0.3 ⁇ m.
- the second filtration module comprises a filter assembly having a cut-off molecular weight of the pores of from 2/15 to 7/15, preferably from 1/5 to 1/3, relative to the molecular weight of the product.
- the product has a molecular weight of from 50 to 300 KD; preferably, the product has a molecular weight of from 100 to 250 KD; more preferably, the product has a molecular weight of from 150 to 200 KD.
- the first stage of production is the initial stage of proliferation of cells, or may be referred to as the stage of product synthesis optimization. Due to the high culture temperature (37 ° C) in the initial environment, the cells are in a high-speed proliferation stage, and a large amount of cellular metabolic waste, lactic acid products, and a small amount of deaminated or degraded antibody impurities are easily accumulated.
- the second production stage is a product synthesis and collection stage, and the inventors have unexpectedly found that after the removal of metabolites and impurities generated in the first production stage, it is more advantageous for the synthesis and collection of high quality products in the second production stage. Therefore, a staged retention system was creatively introduced to improve the quality and yield of the later products.
- the intercepting system in the first production stage, mainly traps cells to remove metabolites and impurities; in the second production stage, the intercepting system mainly traps cells and enriches protein products, and removes small Molecular metabolites.
- the first stage of production in the upstream production of biomacromolecules, can be initiated after the cells are seeded into the bioreactor.
- the first production stage in the upstream production of the biomacromolecule, when the cell culture density reaches 6 ⁇ 1.5 ⁇ 10 6 cells/ml, the first production stage is initiated.
- the first production stage is initiated when the cell culture density reaches 10 ⁇ 5 x 10 6 cells/ml.
- the cell culture density in the second production stage is not less than 15 ⁇ 5 ⁇ 10 6 cells/ml; preferably, the cell density in the second production stage is not less than 35 ⁇ 5. ⁇ 10 6 cells/ml.
- the "culture density is not less than 15 ⁇ 5 ⁇ 10 6 cells / ml" is a culture density of 10 ⁇ 10 6 to 30 ⁇ 10 6 cells / ml.
- the "culture density is not less than 35 ⁇ 5 ⁇ 10 6 cells / ml" is a culture density of 30 ⁇ 10 6 to 50 ⁇ 10 6 cells / ml.
- the "culture density is not less than 15 ⁇ 5 ⁇ 10 6 cells / ml" is a culture density of 10 ⁇ 10 6 to 300 ⁇ 10 6 cells / ml until the culture to cell viability
- the cell fermentation broth is harvested to complete the second production stage; preferably, when the viability of the cultured cells is reduced to 70%, the cell fermentation broth is harvested; more preferably, when the viability of the cultured cells is reduced to 80%, the harvest is obtained.
- the cell fermentation broth optimally, when the viability of the cultured cells is reduced to 90%, the cell fermentation broth is harvested.
- the "culture density is not less than 35 ⁇ 5 ⁇ 10 6 cells / ml" is a culture density of 30 ⁇ 10 6 to 100 ⁇ 10 6 cells / ml.
- the cell culture density of the second production stage exhibits a relatively stable tendency, and the fluctuation range is not higher than 10 ⁇ 10 6 cells/ml.
- the cell culture density of the second stage of production is increasing, with a range of fluctuations greater than 10 x 106 cells per ml.
- the "fluctuation range is higher than 10 x 10 6 cells/ml" is a fluctuation range of 10 x 10 6 to 290 x 10 6 cells/ml.
- the "fluctuation range is higher than 10 x 10 6 cells/ml" is a fluctuation range of 10 x 10 6 to 90 x 10 6 cells/ml.
- the retention system includes, but is not limited to, an alternate tangential flow system, a tangential flow filtration system, a rotary filtration system, a depth filtration system, a centrifuge system, or a settling system.
- the bioreactor includes, but is not limited to, an airlift bioreactor, a mechanically agitated bioreactor, a bubble column bioreactor, a membrane bioreactor.
- the bioreactor includes, but is not limited to, a hatching shaker, a micro bioreactor, a benchtop bioreactor, a stainless steel bioreactor, a glass bioreactor, a disposable bioreactor, a rocking type Bioreactor or multiple parallel tanks.
- the cultured cells include, but are not limited to, mammalian cells, plant cells, insect cells, and microbial cells, and the like, which are suitable for industrialized fermentation culture; preferably, the mammalian cells include but not It is limited to CHO cells, NSO cells, Sp2/0 cells, HEK cells, BHK cells, and the like.
- the production process includes, but is not limited to, perfusion culture, concentrated perfusion culture, fed batch culture, and concentrated fed batch culture.
- the production process upstream of the cell includes the following steps:
- the intercepting system containing the first filtration module is combined with the bioreactor to perform cell culture production in the first production stage;
- the cut-off system containing the second filter module is replaced and the culture production is entered in the second production stage.
- the cut-off system containing the second filter module is replaced and the culture production is entered in the second production stage.
- the downstream production purification process is designed to rely on the characteristics of different biomolecule products
- the production of the biological analog of Herceptin is taken as an example to illustrate the production of the staged retention system in yield and The advantages of product quality.
- the production of the bio analog of Avastin is used as an example to demonstrate the advantages of the staged retention system in yield and product quality.
- a production module for upstream phased retention of a biomacromolecule the production module being capable of being incorporated into a retention system and used in conjunction with a bioreactor comprising at least two filtration modules, wherein A filter module comprises a filter assembly having a filter pore size ranging from 0.05 ⁇ m to 1 ⁇ m for removing metabolites and product impurities in the cell culture solution; the second filter module comprising a cut-off molecular weight of the filter pores relative to the molecular weight of the product 1 A filter assembly in the range of /15 to 2/3 for harvesting and enriching biomacromolecule products.
- the first filter module comprises a filter assembly having a filter pore of 0.1 ⁇ m to 0.5 ⁇ m, preferably 0.2 ⁇ m to 0.3 ⁇ m, and the second filter module comprises a molecular weight cutoff of the filter pore.
- the filter assembly is preferably from 1/5 to 7/15, preferably from 1/5 to 1/3, based on the molecular weight of the product.
- the impurity filtration module has a filtration pore size of 20-50 KD molecules; more preferably, the impurity filtration module has a filtration pore size of 50 KD molecules.
- the filter assembly includes, but is not limited to, a hollow fiber type, a tube type, a roll type, a flat type, and the like.
- the material of the filter assembly is cellulose ester or non-cellulose ester, and the cellulose ester material includes, but not limited to, cellulose acetate, nitrocellulose, ethyl cellulose, etc.;
- the esters include, but are not limited to, polysulfone (PS), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polyethersulfone (PES), and polyvinylidene fluoride (PVDF).
- the bioreactors include, but are not limited to, airlift bioreactors, mechanically agitated reactors, bubble column bioreactors, membrane bioreactors, and the like.
- the bioreactor includes, but is not limited to, a hatching shaker, a micro bioreactor, a benchtop bioreactor, a stainless steel bioreactor, a glass bioreactor, a disposable bioreactor, a rocking type Bioreactor or multiple parallel tanks.
- the retention system includes, but is not limited to, an alternate tangential flow system, a tangential flow filtration system, a rotary filtration system, a depth filtration system, a centrifuge system, or a settling system.
- the cultured cells include, but are not limited to, mammalian cells, plant cells, microbial cells, insect cells, and the like, which are suitable for large-scale fermentation culture; preferably, the mammalian cells include, but are not limited to, CHO cells, NSO cells. , Sp2/0 cells, HEK cells, BHK cells, and the like.
- a method for producing a production module/production method for phased filtration of a biomacromolecule upstream production wherein the biomacromolecule staged filtration production module/production method can be applied to industrialization of biomacromolecules in production.
- biomacromolecules include, but are not limited to, polypeptides or recombinant proteins.
- the recombinant protein is an immunoglobulin or a genetically engineered antibody, including but not limited to a human mouse chimeric antibody, a humanized antibody, a fully human antibody, a Fab fragment, F(ab') 2, Fv, ScFv, Single domain antibodies, dual/multispecific antibodies, and the like.
- the recombinant protein is a novel protein obtained by fusing a biologically active functional protein and an Fc segment.
- the application is suitable for microbial culture, culture, cloning or expression vector screening, small-scale protein supply, process development, optimization and characterization, seed expansion, production and other aspects.
- Figure 1 Schematic diagram of the first production stage of the upstream cell.
- Figure 2 Schematic diagram of the second production stage upstream of the cell.
- SEC-HPLC One of the methods for determining the purity of a product by molecular exclusion high performance liquid chromatography, which is calculated according to the area normalization method, and is divided into a monomer, a polymer (high molecular weight group) and a fragment (low molecular weight group). Among them, the greater the percentage of monomers, the higher the purity of the product.
- CEX-HPLC cation exchange high performance liquid chromatography, one of the methods for determining the charge purity of a product, used for the homogeneity of the reaction product, according to the charge of the product, is divided into an acid peak group, a main peak and a basic peak, wherein The higher the ratio of acid peaks or basic peaks, the higher the degree of charge heterogeneity of the product and the worse the product uniformity.
- rCE-SDS reduction capillary gel electrophoresis, using reduced capillary gel electrophoresis, driven by high-voltage electric field, after product reduction, according to the molecular weight of the size of the movement within the capillary is different, in which the heavy chain + light The higher the ratio of the sum of the chains, the higher the purity of the product.
- nrCE-SDS non-reducing capillary gel electrophoresis, using non-reducing capillary gel electrophoresis, using high-voltage electric field as the driving force, the product is separated according to the molecular weight and the moving speed in the capillary. The higher the proportion of monomer, The higher the purity of the representative product.
- Cell viability/cell viability The ratio of viable cells to total cell volume per unit volume.
- the antibody Since the antibody is susceptible to culture temperature, culture time, stirring rate, pH and the like during the fermentation process, various post-translational modification changes such as deamination, oxidation, saccharification, and N-terminal pyroglutamic acid cyclization occur. It affects the charge distribution of the antibody. These post-translational modifications determine whether they are key quality attributes depending on whether the location at which they occur is critical (such as whether the CDR region affects function, whether it occurs at the FcRn binding site affects PK, etc.). At the same time, charge distribution/charge purity is a very sensitive quality attribute, which can be used as a very good indicator for process consistency evaluation. For a long time, the control of charge heterogeneity is the focus and difficulty of antibody drug production.
- An advantage of the present invention is to maintain a higher charge purity of the product (reducing other post-translational forms or forms of degradation that may affect the charge) while further ensuring charge distribution similarity of the product in multi-batch mass production.
- substances produced during the cell expansion phase such as ammonium ions, proteases, other host proteins, DNA, etc., may have an effect on cell growth, may have an impact on product quality characteristics (such as some enzymes cause product degradation), and a large number Substituting impurities into the product harvesting liquid will greatly affect the downstream purification yield, and may not be completely removed, which has an impact on the yield and quality of the product.
- the system and method of the present invention can further solve the problem of product quality and uniformity existing in the existing fed culture system.
- the impurity control of the product in the fed-batch mode is not Stable, often switching to perfusion culture, resulting in increased process complexity.
- the invention can solve the problem of unstable and high content of product-related impurities in the fed-batch mode, and can obtain high-quality products without switching the process, and is a great innovation and progress in the field of large-scale antibody production.
- the present invention can ensure the product in the cell culture process by sequentially using a first filter component suitable for impurities and excretion of cellular metabolites and a second filter component suitable for product collection and enrichment in the cell culture process.
- a first filter component suitable for impurities and excretion of cellular metabolites and a second filter component suitable for product collection and enrichment in the cell culture process.
- the main technical advantages include:
- the present invention is advantageous for further reducing product-related impurities and ensuring high yield and high purity of the final product;
- Example 1 Cultivation of a 200 L bioreactor based on staged retention
- the antibody used in the production of the present invention is a biosimilar drug produced by the commercially available therapeutic antibody Herceptin.
- the expression cell line is Chinese hamster ovary cells (CHO), and the monoclonal antibody is of the IgG1/kappa type.
- Herceptin The same (INN: trastuzumab, IMGT number: 7637); its molecular weight is 145 kDa.
- the cell cryotube was taken out and directly immersed in a 37 ° C constant temperature water bath, and occasionally shaken to melt as soon as possible. Remove the cryotube from the 37 ° C water bath, disinfect the surface with 75% alcohol, unscrew the cryotube in the clean bench, aspirate the cell suspension with a sterile pipette, and place the CD with 1 mL pre-incubation. FortiCHO TM Medium basal medium in a 15 mL centrifuge tube, shake well; after 30-60 seconds, add 2 mL of pre-incubation basal medium and shake well; add another 4 mL of pre-incubation basal medium and shake for 30-60 seconds.
- CD OptiCHO TM medium manufactured by ThermoFisher
- the cell density is 2.5 ⁇ 4.5 ⁇ 10 6 cells/mL and the survival rate is ⁇ 90%.
- the specific control parameters are shown in Table 1:
- a 200L-alternating tangential flow system from Refine Techology is used.
- the hollow fiber column in the ATF device and the diaphragm at the top of the base are assembled, and then connected to the vacuum pump and the controller to intercept 0.2 um and 50 KD.
- the molecular weight vacuum fiber membrane was assembled into the ATF system, rinsed with 25% ethanol for 10 min, and then repeatedly rinsed with deionized water to remove residual ethanol, which was then sterilized in a high temperature damp heat sterilizer for 45 min.
- the ATF system containing the 0.2um filter module was inserted into a 200L bioreactor. After the cells were cultured in a 50L bioreactor for 3 days, they were inoculated into a 200L bioreactor with an initial cell density of 1x 10 6 cells. /mL specific control parameters are shown in Table 4:
- the ATF system When the cell density is grown to 10x 10 6 cells/ml, connect the ATF system, and turn on the controller and vacuum pump in the ATF device to calculate the volume of the medium exchange per day according to the amount of cell growth and the rate of cell growth. The rate at which the medium enters and the rate at which the culture waste is discharged.
- the ATF system replacing the 0.2 ⁇ m filter assembly is an ATF system containing a 50 KD cut-off molecular weight filter assembly, and the controller and vacuum pump in the ATF device are continuously activated to enter the second production stage. .
- the culture temperature of the bioreactor is adjusted to 34 ° C, the pH is adjusted to 6.9, and the dissolved oxygen is maintained at 50% before the cell growth density reaches 40 to 50 x 10 6 cells/ml.
- the culture speed was adjusted to 125 rpm (the results are presented in Table 6).
- the adjustment of the bioreactor temperature and culture conditions can be performed simultaneously with the replacement of an ATF system containing a 50 KD cut-off molecular weight filtration module. The supernatant was collected while growing to a cell growth density of approximately 100 x 10 6 cells/ml.
- Cell culture clarification is mostly performed by centrifugation or microfiltration combined with depth filtration to remove cells and cell debris from the culture.
- the cell suspension obtained from the 200 L bioreactor is centrifuged at 6000 ° C for 15 min at 4 ° C, the cells are removed, and the cell fermentation broth is retained; and then the deep filtration method currently used for cell culture clarification is used, which adopts The porous cellulose skeleton structure is loosened and filled with diatomaceous earth components, and the surface pore diameter of the injection end is funnel-shaped.
- the Millipore MX0HC10FS1 depth filter is used to connect the centrate to the peristaltic pump (the peristaltic pump can be selected as long as it meets the flow rate requirement), and the deep filtration membrane is washed with ultrapure water, and the flushing volume is 10 CV.
- the deep filtration membrane was then rinsed with a depth filtration buffer of 25 mM Tris-HCl + 100 mM NaCl, pH 7.4 ⁇ 0.2, with a flush volume of 1 CV and a flush flow rate of 100-300 LMH.
- the filter membrane was washed with a depth filtration buffer of 25 mM Tris-HCl + 100 mM NaCl, pH 7.4 ⁇ 0.2, and the filtrate was collected.
- the volume of the rinse was 2 CV, and the pressure before the membrane was controlled to not exceed 2 bar.
- Protein A affinity chromatography serves as a capture step in antibody purification and has a removal effect on host proteins, endotoxin, and DNA.
- the Protein A affinity chromatography material was Mabselect Sure (manufacturer: GE).
- the supernatant obtained by the depth filtration was loaded at a flow rate of 300 cm/h, and the column loading was guaranteed to be less than 35 mg/ml of the filler.
- the mixture was switched to a rinse solution of 25 mM Tris-HCl, 50 mM NaCl, pH 7.4, and the column was washed at a flow rate of 300 cm/h to 5 CV.
- the virus inactivation of this example uses an elution peak after incubation of the affinity chromatography at a low pH to inactivate the retrovirus and the enveloped virus.
- the eluate of the affinity chromatography was adjusted to pH 3.6-3.8 with 10% acetic acid and inactivated for 60-120 min.
- the virus inactivated samples were subjected to deep filtration using Millipore MX0HC10FS1 depth filter: 10 CV with ultrapure water, 50 mM acetic acid-sodium acetate, 50 liters of deep filtration membrane of PhC5.2, and rinsed after rinsing.
- the pipe was subjected to sample filtration at a flow rate of 100-300 LMH, and the filtration pressure was less than 2 bar.
- the 1 CV was washed with a buffer of 50 mM acetic acid-sodium acetate, Ph. 5.2, and the deep filtration membrane 2 CV was rinsed, and the rinse filtrate was recovered.
- this embodiment adopted three cycles of cation exchange chromatography in virus inactivation and depth filtration, and cation exchange chromatography selected Fractogel EMD COO-(M) (manufacturer) : Merck).
- the equilibration solution 50 mM acetic acid-sodium acetate, pH 5.2
- the equilibration solution 50 mM acetic acid-sodium acetate, pH 5.2
- switch to the balance solution 180cm / h flow rate wash column 5CV.
- the eluent is: 50 mM acetic acid-sodium acetate and 300 mM sodium chloride, pH 5.2, the equilibrium solution and the eluent 0-100% gradient elution 10 CV, the flow rate is 180 cm / h, during the elution process The eluted samples were collected.
- Anion exchange chromatography is used to effectively remove trace impurities such as various viruses, DNA, endotoxin, and the like in the sample. Due to the high isoelectric point of the monoclonal antibody, the anion exchange chromatography usually adopts a flow-through mode, that is, the impurity binds to the filler and the target protein does not bind to it through the column.
- the anion exchange chromatography of this example used Capto Q (manufacturer: GE), pre-equilibration solution (50Mm Tris-HCl+1M NaCl, Ph8.0) to equilibrate the column 2CV, and then use the balance solution (50mM Tris-HCl, Ph8.0) Balanced column 3CV, sample loading, flow rate 180cm / h, collected flow through the UV280 reading rose to 0.1AU began to collect the elution peak, the column loading to ensure less than 40mg / ml filler. After the sample is finished, switch to the balance solution 180cm/h flow rate to wash the column 3CV, and stop collecting the sample when the UV280 drops to 0.1AU.
- Capto Q manufactured by GE
- pre-equilibration solution 50Mm Tris-HCl+1M NaCl, Ph8.0
- balance solution 50mM Tris-HCl, Ph8.0
- Viral filtration can be used to remove non-enveloped viruses and is filtered using a 1.02 m 2 / batch of virus filtration membrane during the production process.
- the pressure tank was treated with 0.5 M NaOH, washed with water for injection and then compressed air for filtration.
- VPMG201NB1 manufactured by Merck Millipore
- rinse 10CV with ultrapure water, buffer 50 mM acetic acid-sodium acetate, and Ph.2 rinse the 2CV deep filtration membrane. After rinsing, connect the pipeline.
- the sample was filtered, the flow rate was 100-300 LMH, and the filtration pressure was 1.9-2.0 bar.
- the 1 CV was washed with a buffer of 50 mM acetic acid-sodium acetate, Ph5.2, and the deep filtration membrane 2CV was further washed, and the rinse filtrate was recovered.
- the ultrafiltration membrane of model P2C030C25 (manufacturer: Merck Millipore) having a pore size of 30 kD was cleaned and rinsed with 20 mM sodium phosphate pH 6.0 buffer to a pH of 6.0 at the back end of the membrane.
- the peristaltic pump was pumped into the ultrafiltration membrane, the flow rate at the inlet end was controlled to be 200 LMH, the transmembrane pressure was 0.35 bar, and the antibody product was harvested by using a 20 mM sodium phosphate pH 6.0 buffer.
- Example 1 The basic embodiment in Example 1, except that: When the cells were grown to a density 10x 10 6 cells / ml, the communication system ATF, ATF and open the device controller and a vacuum pump.
- the ATF system used was a vacuum fiber membrane module containing a molecular weight cut-off of 50 KD, and the fermentation step was completed until the cell growth density was about 100 x 10 6 cells/ml.
- the subsequent purification steps were the same as in Example 1.
- Example 1 After the Protein A affinity chromatography was completed, samples obtained in Example 1 and Comparative Example 1 were sampled for cation high performance liquid chromatography to examine the content of charge isomers, and the results of the distribution of acid-base peaks were obtained. As shown in Table 5:
- the antibody product obtained by the staged retention method has better charge purity than the antibody product of the single retention method, the content of the neutral peak is higher, and the content of the acid peak is lower.
- the staged retention method shows a significantly better biological activity than the single retention method, while the staged retention method yields a high yield of qualified end products; and the yield of qualified end products obtained by a single retention method The rate is significantly lower, as shown in Table 6.
- Example 2 2L bioreactor culture based on staged retention
- the antibody for production is a recombinant human anti-vascular endothelial growth factor (VEGF) humanized monoclonal antibody, which is obtained by fermentation and expression in mammalian cells in vitro, and the animal cell is Chinese hamster ovary cell (CHO), single
- VEGF human anti-vascular endothelial growth factor
- CHO Chinese hamster ovary cell
- the cloned antibody is of the IgG1/kappa type, and its amino acid sequence and the original drug Avastin The same (INN: bevacizumab, IMGT number: 8017, http://www.imgt.org/mAb-DB/); its molecular weight is 149 kDa.
- Cell culture technology using a conventional cell culture Seed / mL, seeded to meet the requirements of a living ⁇ 90%
- the cells were aseptically inoculated into the seed CD FortiCHO TM Medium containing basal medium Culture in a 2L bioreactor. After inoculation, the cell density was 1.0 ⁇ 10 6 cells/mL, and the viability was ⁇ 90%.
- the initial cell culture conditions were: culture temperature 37 ° C, stirring speed 95 rpm, pH: 7.20 ⁇ 0.05, dissolved oxygen: 50%.
- the hollow fiber column in the ATF device and the diaphragm at the top of the base are assembled first, and then connected to the vacuum pump and the controller.
- the top of the hollow fiber column with a pore size of 0.2 ⁇ m is first connected to the reactor.
- Pipe when the cells are cultured in a bioreactor to a cell density of 4.5 to 5.5 x 10 6 cells/mL, the controller and vacuum pump in the ATF device are turned on, and the daily medium is calculated according to the amount of cell growth and the rate of cell growth. The volume exchanged to adjust the rate at which fresh media enters and the rate at which the effluent is discharged.
- the daily perfusion volume reaches the desired setting, if the glucose is insufficient, an appropriate additional 30% glucose solution is added.
- the culture temperature is lowered to 32° C., and the pH is lowered to 7.00 ⁇ 0.05.
- the treatment will be treated.
- the sterile 50KD vacuum fiber membrane replaces the original 0.2 ⁇ m hollow fiber membrane and enters the second stage of production, maintaining the cell density in the range of 10 ⁇ 10 6 cells/mL until the cell viability begins to decrease to 90%. Culture the bioreactor and collect the cell culture fluid.
- the timing of collecting the cell culture fluid is controlled when the cell viability drops to 80%; in another embodiment, the timing of collecting the cell culture fluid is controlled when the cell viability drops to 70%; in another embodiment In this case, the timing of collecting the cell culture fluid is controlled when the cell viability drops to 50%.
- a deep filtration membrane (D0HC (No. MD0HC10FS1, manufacturer: Merck) is connected to the peristaltic pump (the peristaltic pump can be selected as long as it meets the flow rate requirement), and the deep filtration membrane is washed with ultrapure water, and the flushing volume is 10 CV.
- the deep filtration membrane was then rinsed with 25 mM Tris-HCl + 100 mM NaCl, pH 7.4 ⁇ 0.2, the flushing volume was 1 CV, and the rinsing flow rate was 100–300 LMH.
- the cell culture was filtered and the filtrate was collected immediately. The filtration flow rate was 100–300LMH.
- the filter membrane is washed with a depth filtration buffer of 25 mM Tris-HCl+100 mM NaCl, pH 7.4 ⁇ 0.2, and the filtrate is collected.
- the volume of the rinse is 2 CV, and the pressure before the membrane is controlled to not exceed 2 bar. .
- the Protein A affinity chromatography material was JWT 203 (manufacturer: JSR).
- JSR manufactured by the Engineering Chemical Society
- the supernatant obtained by the depth filtration was loaded at a flow rate of 300 cm/h, and the column loading was guaranteed to be less than 35 mg/ml of the filler.
- the mixture was switched to a rinse solution of 25 mM Tris-HCl, 50 mM NaCl, pH 7.4, and the column was washed at a flow rate of 300 cm/h to 5 CV.
- the eluent was 100 mM acetic acid-sodium acetate (pH 3.6 ⁇ 0.2, conductivity: 0.9 ⁇ 0.2 mS/cm), and the flow rate was 300 cm/h.
- the UV280 was tested, the UV280 reading was raised to 0.5 AU and the elution peak was collected, and the collection was stopped when it dropped to 0.5 AU again.
- the sample was adjusted to a pH of 3.5-3.7 at room temperature for 1-2 hours for a low pH virus inactivation procedure.
- the crude purified sample was subjected to preparative chromatography to prepare a stock solution.
- Anion exchange chromatography was performed on a Capto Q packing (manufacturer: GE healthcare).
- the pre-equilibration solution 50 mM Tris-HCl + 1 M NaCl, pH 8.0
- the pre-equilibration solution 50 mM Tris-HCl + 1 M NaCl, pH 8.0
- an equilibration solution 50 mM Tris-HCl, pH 8.0
- the collection flow through the UV280 reading rose to 0.1 AU and began to collect the elution peak.
- the column loading is guaranteed to be less than 40 mg/ml of filler.
- the sample was collected into the next cation exchange chromatography, and Fractogel EMD COO-(M) (manufacturer: Merck) was selected by cation exchange chromatography.
- the equilibration solution 50 mM acetic acid-sodium acetate, pH 5.2
- the equilibration solution 50 mM acetic acid-sodium acetate, pH 5.2
- the GB222 sample was loaded at a flow rate of 180 cm/h, and the column loading was guaranteed to be less than 45 mg/ml of the filler.
- the eluent is: 50 mM acetic acid-sodium acetate and 300 mM sodium chloride, pH 5.2, the equilibrium solution and the eluent 0-100% gradient elution 10 CV, the flow rate is 180 cm / h, during the elution process The eluted samples were collected.
- the purified sample is subjected to ultrafiltration and liquid exchange.
- the procedure is as follows: the ultrafiltration membrane of model P3C030C05 with a pore size of 30KD (manufacturer: Merck) is cleaned and rinsed with 20 mM sodium phosphate pH 6.0 buffer to the membrane back end.
- the pH is 6.0.
- the cation chromatographic sample was pumped into the ultrafiltration membrane by a peristaltic pump for ultrafiltration concentration, and the flow rate at the inlet end was controlled to be 300 LMH, the transmembrane pressure was 0.3-0.7 bar, and the initial concentration was about 20 mg/ml, followed by 20 mM sodium phosphate pH 6.0.
- the buffer is subjected to a 10-fold equal volume exchange, and after completion, the protein is recovered by evacuating the ultrafiltration membrane, which is a stock solution.
- Example 2 Cell culture medium The conditions are as in Example 2. The main difference is that when the cell density is grown to 4.5 to 7.5 x 10 6 cells/mL, the ATF system is connected, and the controller and vacuum pump in the ATF device are turned on.
- the ATF system used was a vacuum fiber membrane module containing a 50 KD cut-off molecular weight, which was cultured until the cell activity decreased to 90% to terminate the reaction.
- the subsequent purification steps were the same as in Example 2.
- the cell culture medium was taken for preliminary analysis, and the acid peak content was about 26%, while the acid peak on the 10th day of the staged retention method was only about 10%; when the subsequent purification step was completed, The acid peak content of the final product obtained by the stage cut-off process is also lower than the single cut-off method.
- the requirement for SEC-HPLC is that the purity of the antibody monomer is ⁇ 95%, while in the three single interception production batches, the value of the detected antibody monomer is only 93%, 92.8%. With 93%, it is much lower than the yield of the staged retention method used in Example 2.
- Example 3 Performing Staged Interception of Upstream Production at Different Cell Growth Densities
- the cell density of the staged retention process can be as low as 15 ⁇ 5 ⁇ 10 6 cells / ml, such as cell density reached 15 ⁇ 5 ⁇ 10 6 cells in low-density cell culture conditions / When it is in milliliters, it can enter the second production stage, and the effect is better when the cell density reaches 35 ⁇ 5 ⁇ 10 6 cells/ml. At the above cell density, entering the second production stage, the final product yield and product charge purity still show significantly better than the traditional single retention process. Taking the antibody of Example 2 as an example, as shown in Table 8, the cell density is 15 ⁇ 5 ⁇ 10 6 cells/ml, and the fluctuation range is 10 ⁇ 10 6 cells/ml to 20 ⁇ 10 6 cells/ml.
- the staged cut-off production process of the present invention is also applicable to the production of protein products of different molecular weights, taking recombinant genetically engineered antibodies as an example, the antibody Fab region, the average molecular weight is about 50KD; the double/multispecific antibody, the molecular weight can reach nearly 300KD. , can be well applied in the staged interception process.
- bis/multispecific antibodies have complex structures, often accompanied by specially designed mutations, and the possibility of product-related impurities is high. Therefore, the upstream production process using staged retention will be used in the first production stage.
- the relevant impurities are eliminated through larger pores, which is more conducive to later purification and development.
- the inventors have also explored the range of the filter pores of the filter assembly used in the two production stages, and the filter assembly in the range of 0.05 ⁇ m to 1 ⁇ m in the first production stage. It can satisfy the removal of small molecular metabolites and macromolecular product related impurities.
- using a filter assembly of 0.1 ⁇ m to 0.5 ⁇ m can more effectively improve the removal efficiency and product yield; in the second production stage,
- the present inventors take the molecular weight of the product as a reference, and when the cut-off pore diameter is set to a filter component in the range of 1/15 to 2/3 of the molecular weight of the product, a better product enrichment effect can be achieved, and when the cut-off pore diameter is set to be relative
- the yield is higher when the product has a molecular weight of 2/15 to 7/15, and the yield of the enriched antibody is highest when the cut-off pore diameter is set to be in the range of 1/5 to 1/3 of the molecular weight of the product.
- the inventors separately expressed and purified the Fab region ( ⁇ 50 KD) protein fragment of the Herceptin antibody sequence in Example 1 using a single interception method and a staged retention method, respectively, in a staged retention method.
- the first stage of culture was first installed with a hollow fiber column with a pore size of 0.1 ⁇ m.
- the treated sterile 10KD vacuum fiber was treated.
- the membrane replaces the original 0.1 ⁇ m hollow fiber membrane and enters the second stage of production.
- a single interception method installed a 10KD vacuum fiber membrane from the ATF system from the beginning.
- the two-stage retention method can effectively achieve a 4% increase in the Fab region yield of the Herceptin antibody sequence and a 2% increase in purity under the same culture conditions; the inventors A comparison of other protein fragments with a molecular weight of 50KD was also found.
- the staged retention method can effectively increase the yield by an average of 2-4% in the 50KD protein fragment preparation compared with the traditional single entrapment method; -2%.
- the inventors also performed the expression and purification of the bispecific antibody using a single interception method and a staged retention method, that is, the Fab region ( ⁇ 50 KD) of the Herceptin antibody sequence in Example 1.
- the terminal is directly ligated to the N-terminus of the variable region of the Avastin antibody of the original 2, and a bispecific antibody having a symmetrical structure and a molecular weight of about 250 KD is formed.
- the ATF-2L system of Refine Techology is still used.
- the first stage of culture is firstly installed with a hollow fiber column with a pore size of 0.5 ⁇ m. When the cell density reaches 30-40 ⁇ 10 6 , the treated one will be treated.
- the sterile 50KD vacuum fiber membrane replaces the original 0.5 ⁇ m hollow fiber membrane and enters the second stage of production.
- a single interception method installed a 50KD vacuum fiber membrane from the ATF system from the beginning. Both culture methods use the same culture conditions, compared to the single interception method, due to the complexity of the double-antibody molecule, the first production stage can be metabolites, multimers, and initial quality in the early stage of cell culture.
- the antibody molecule is removed, so the staged retention method can effectively achieve a 10% increase in the yield of the bispecific antibody sequence and a 5% increase in purity; the inventors also tried to compare other protein fragments having a molecular weight of 250 KD.
- the staged retention method can effectively increase the yield by an average of 5-10% in the preparation of a large molecular weight bispecific or trispecific antibody than the conventional single retention method; the purity is increased by an average of 3-5%.
- staged cut-off upstream production process of the present invention can be incorporated into the cultivation of different forms of bioreactors, or It can be used in various existing trapping systems, such as tangential flow filtration (TFF), rotary filtration systems, deep filtration systems, centrifuge systems or sedimentation systems. All changes and/or modifications are within the scope of the invention as defined by the appended claims.
Abstract
Description
摇床转速 | 温度 | 二氧化碳浓度 |
120rpm | 37℃ | 5% |
温度 | pH | 溶解氧 | 摇摆速度 | 摇摆角度 | 通气量 |
37℃ | 7 | 50% | 25rpm | 8° | 0.2L/分钟 |
温度 | pH | 溶解氧 | 转速 | 培养时间 |
37℃ | 7 | 50% | 90rpm | 3天 |
培养温度 | pH | 溶解氧 | 转速 |
37℃ | 7 | 50% | 90rpm |
Claims (18)
- 一种生物大分子上游分阶段截留的生产方法,包括使用生物反应器结合截留系统的生产方式,其特征在于,所述方法包含以下生产阶段:第一生产阶段,使用包含第一过滤模块的截留系统,所述的第一过滤模块包含有滤孔为0.05μm~1μm的过滤组件;第二生产阶段,使用包含有第二过滤模块的截留系统,所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量1/15~2/3的过滤组件。
- 如权利要求1所述的方法,其特征在于,所述的第一过滤模块包含有滤孔为0.1μm~0.5μm;和/或所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量的2/15~7/15。
- 如权利要求2所述的方法,其特征在于,所述的第一过滤模块包含有滤孔为0.2μm~0.3μm的过滤组件;和/或所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量的1/5~1/3的过滤组件。
- 如权利要求1~3任一所述的方法,其特征在于,所述产物的分子量为50~300KD。
- 如权利要求4所述的方法,其特征在于,所述产物的分子量为100~250KD或150~200KD。
- 如权利要求1所述的方法,其特征在于,所述的第二生产阶段的细胞培养密度不低于15±5×10 6细胞数/毫升。
- 如权利要求6所述的方法,其特征在于,所述的第二生产阶段的细胞密度不低于35±5×10 6细胞数/毫升。
- 如权利要求6所述的方法,其特征在于,所述的第二生产阶段的细胞培养密度的波动范围不高于10×10 6细胞数/毫升或所述的第二生产阶段的细胞培养密 度呈递增趋势,其波动范围高于10×10 6细胞数/毫升。
- 如权利要求1所述的方法,其特征在于,所述的截留系统包括交替式切向流系统、切向流过滤系统、旋转过滤系统、深层过滤系统、离心机系统或沉降系统;和/或所述的生物反应器包括气升式生物反应器、机械搅拌式生物反应器、鼓泡塔式生物反应器、膜生物反应器;和/或所述的培养细胞包括哺乳动物细胞、植物细胞、昆虫细胞和微生物细胞;较佳地为哺乳动物细胞。
- 如权利要求1所述的方法,其特征在于,生物大分子上游生产的生产工艺包括灌注培养、浓缩灌注培养、补料批次培养和浓缩补料批次培养。
- 如权利要求1所述的方法,其特征在于,所述的生物大分子包括多肽或重组蛋白。
- 如权利要求11所述的方法,其特征在于,所述的重组蛋白包括基因工程抗体、具有生物学活性的功能蛋白或Fc段融合而成的新型蛋白。
- 在如权利要求1所述的方法中所使用的生产模块,可安装在截留系统中,配合生物反应器使用,其特征在于,所述的生产模块包括第一过滤模块与第二过滤模块,所述的第一过滤模块包含有滤孔为0.05μm~1μm范围内的截留组件;所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量1/15~2/3范围内的过滤组件;所述的第一过滤模块与第二过滤模块可以分别置于两个截留系统中或通过替换的方法应用于同一个截留系统中。
- 如权利要求13所述的生产模块,其特征在于,所述的第一过滤模块包含有滤孔为0.2μm~0.3μm的过滤组件;和/或所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量的1/5~1/3的过滤组件。
- 如权利要求14所述的生产模块,其特征在于,所述的第一过滤模块包含 有滤孔为0.2μm~0.3μm的过滤组件;和/或所述的第二过滤模块包含有滤孔的截留分子量相对于产物分子量的1/5~1/3的过滤组件。
- 如权利要求1~12的任一方法或如权利要求13~15的任一生产模块的应用,其特征在于,所述方法或模块可应于生物大分子上游生产的过程中,其中所述的生物大分子为蛋白产物。
- 如权利要求16所述的应用,其特征在于,所述的生物大分子包括多肽或重组蛋白。
- 如权利要求17所述的应用,其特征在于,所述的重组蛋白包括基因工程抗体、具有生物学活性的功能蛋白或Fc段融合而成的新型蛋白。
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Also Published As
Publication number | Publication date |
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EP3722408A1 (en) | 2020-10-14 |
EP3722408A4 (en) | 2021-07-28 |
KR20200128108A (ko) | 2020-11-11 |
JP2021516971A (ja) | 2021-07-15 |
CN110241012A (zh) | 2019-09-17 |
US20200332248A1 (en) | 2020-10-22 |
RU2769767C1 (ru) | 2022-04-05 |
CN110241012B (zh) | 2022-11-01 |
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