Method for producing recombinant protein plasmin through improved feeding

Abstract

Production of recombinant Romanoplastin protein through an optimized feeding strategy, the method includes the construction of plasmid pET24a(+) containing the gene encoding the recombinant Romanoplastin protein; transfer of the plasmid containing the gene encoding the recombinant Romanoplastin protein to E. coli bacteria of the T7 Express strain; transfer of the Batch stage medium to the fermentor, addition of inoculum to the fermentor, initiation of the Fed Batch stage, initiation of the Induction stage, harvesting the culture medium in the fermentor; centrifugation of the culture medium harvested from the fermentor and purification of the recombinant Romanoplastin protein.

Description

Description of the invention Title of the invention Method for producing recombinant Romiplast protein through improved feeding Technical background of the relevant invention The technical field of the present invention is related to the field of biotechnology, in particular the field of production of recombinant proteins and more particularly the production of recombinant proteins as drugs. Technical problem and stating the objectives of the invention Romiplastim is a thrombopoietin analog peptidobody that controls platelet proliferation. The molecule consists of two main parts: the first part is a peptide sequence that is similar to the thrombopoietin sequence and can bind to thrombopoietin receptors, the second part contains the heavy chain of the IgG1 antibody (peptide + antibody = peptidebody). This drug is used to treat the disease immune thrombocytopenic purpura. Romiplastim is a fusion protein that increases platelet proliferation by activating the thrombopoietin receptor. This peptidobody has two identical single-chain subunits, each containing 269 amino acids. In each subunit, an IgG1 Fc sequence is covalently linked to the polypeptide sequence. The polypeptide sequence also includes two domains that bind to the thrombopoietin receptor c-Mpl, each domain consisting of 14 amino acids. The amino acid sequence of Romaplast is not identical to that of thrombopoietin and must be expressed in a host cell by recombinant technology. One of the steps in the production of this protein on an industrial scale is feeding it into a culture fermenter. One of the important factors in the feeding of the fermenter is Optical Density (OD). In products produced as air inclusions, higher ODs can help in higher yields. In the prior art, with the feedings presented in these documents, the maximum OD achieved is 100. However, cells containing the gene encoding this protein have a higher production capacity and, as a result, the ability to increase OD. Therefore, if the feeding environment in the fermenter is changed, this goal can be achieved. A description of the state of the prior art and the history of developments related to the claimed invention. Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disease with a complex and unknown mechanism in which antiplatelet autoantibodies lead to a decrease in the number of blood platelets. Although the role of CD4T cells in patients with ITP has been different in various studies, the role of this type of cell in the disease has been confirmed. According to studies, 1.6 to 2.25 people per 100,000 people are affected by this disease annually. The first step in the treatment of these patients is the use of corticosteroids or intravenous immunoglobulin (IVIg). Approximately 25 to 30 percent of patients who do not respond adequately to these drugs are candidates for splenectomy. Romiplostim, sold under the brand name Nplate, is a medication used to treat low blood platelet counts (thrombocytopenia) in people with immune thrombocytopenia (ITP). It works by mimicking thrombopoietin (TPO), a hormone that regulates platelet production. A method for the isolation and purification of Romiplastim has been presented in an invention published in 2022 with the application number WO / 2022 / 245259. This invention provides a method for the purification of recombinant Romiplastim obtained in a prokaryotic expression system, which includes the isolation and washing of inclusion bodies, protein refolding, and chromatographic purification, which is characterized by the fact that the initial removal of impurities contaminating cellular proteins, nucleic acids, lipids, and endotoxins in the isolation step of inclusion bodies is performed by successive washing of them, and the additional removal of impurities involves performing 2 successive stages of chromatography on the same cation exchange adsorbent, the first stage being separation and the second stage being polishing and concentration. The amino acid sequence of romiplastin is not identical to thrombopoietin and is expressed in a host bacterium by recombinant DNA technology. The World Health Organization has approved the use of romiplastin for adult patients with long-standing immune thrombocytopenic purpura who have not responded to other treatments such as corticosteroids, intravenous immunoglobulin (IVIG), Rho (D) immune globulin, and splenectomy. Romiplastin acts as a thrombopoietin receptor agonist and activates intracellular transcriptional pathways through c-Mpl, thereby increasing platelet proliferation. In addition, this drug can act similarly to thrombopoietin and control platelet proliferation in the bone marrow. Providing a solution to an existing technical problem along with an accurate, sufficient, and integrated description of the invention The present invention provides a solution for producing recombinant Romiplast protein through an improved feed-through strategy. In one embodiment of the present invention, said improved feed-through process for producing recombinant Romiplast protein includes a batch step, a fed-batch step, and an induction step. In one embodiment of the present invention, the batch phase medium is an aqueous solution comprising glucose at a weight percentage of between 30 and 40, dipotassium hydrogen phosphate at a weight percentage of between 40 and 45, monopotassium phosphate at a weight percentage of between 20 and 23, citric acid at a weight percentage of between 5 and 7, ammonium sulfate at a weight percentage of between 7 and 8, magnesium sulfate heptahydrate at a weight percentage of between 4 and 5, yeast extract at a weight percentage of between 2 and 4, trace elements at a weight percentage of between 2 and 3, and thiamine at a weight percentage of between 2 and 4. In one embodiment of the present invention, the fed-batch stage medium is an aqueous solution comprising glucose at a weight percentage between 400 and 500, yeast extract at a weight percentage between 55 and 65, peptone at a weight percentage between 7 and 9, ammonium sulfate at a weight percentage between 1 and 1.75, magnesium sulfate heptahydrate at a weight percentage between 14 and 15, and trace elements at a weight percentage between 3.5 and 5.5. In one embodiment of the present invention, the induction stage medium is an aqueous solution comprising glycerol at a weight percentage between 350 and 400, yeast extract at a weight percentage between 25 and 35, ammonium sulfate at a weight percentage between 0.5 and 1.5, magnesium sulfate heptahydrate at a weight percentage between 9 and 11, trace elements at a weight percentage between 2 and 3, thiamine at a weight percentage between 2 and 4. In one embodiment of the present invention, the recombinant Romiplast protein has an amino acid sequence according to Figure 1. Figure 1 provides the amino acid sequence of the recombinant Romiplast protein, according to one or more embodiments of the present invention. Figure 2 provides a flowchart for producing recombinant Romiplast protein via an improved feeding strategy, in accordance with one or more embodiments of the present invention. Referring to Figure 2, the production of recombinant Romiplast protein via an improved feeding strategy includes producing a recombinant cell expressing Romiplast (step 202); assaying expression of recombinant Romiplast protein in a host cell in a small-scale Erlenmeyer flask (step 204); producing recombinant Romiplast protein in a fermenter (step 206); harvesting the culture medium from the fermenter (step 208); centrifuging the culture medium harvested from the fermenter (step 210); and purifying recombinant Romiplast protein (step 212). Referring to further details of step 202, step 202 involves producing a recombinant cell expressing Romiplast. Details of step 202 are provided in Figure 3. Figure 3 provides a flowchart for producing a recombinant cell expressing Romiplast, in accordance with one or more embodiments of the present invention. Referring to Figure 3, producing a recombinant cell expressing Romiplast includes producing a plasmid containing a gene encoding a recombinant Romiplast protein (step 302); transferring the plasmid containing the gene encoding the recombinant Romiplast protein into a host bacterium (step 304), and selecting transgenic clones (step 306). Referring to step 302 in further detail, step 302 involves synthesizing a plasmid containing a gene encoding a recombinant Romiplast protein. In one embodiment of the present invention, the plasmid containing a gene encoding a recombinant Romiplast protein is plasmid pET24a(+). Referring to step 304 in further detail, step 304 involves transferring a plasmid containing a gene encoding a recombinant Romiplast protein into a host bacterium. In one embodiment of the present invention, the host bacterium is an E. coli bacterium of the T7 Express strain. Referring to step 204 in more detail, step 204 involves assaying the expression of the recombinant Romiplast protein in the host cell in a small Erlenmeyer flask. In one embodiment of the present invention, the purpose of assaying the expression in an Erlenmeyer flask is to ensure the expression of the recombinant Romiplast protein in the clone selected in step 306. In one embodiment of the present invention, to assay the expression of the recombinant protein, LB+0.5% Glucose culture medium, IPTG inducer level=0.5 mM, temperature 37 degrees Celsius, and induction time 24 hours were considered. In one embodiment of the present invention, Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to confirm the expression of the recombinant protein in the clone selected in step 306. Referring to step 206 in more detail, step 206 involves producing recombinant Romiplast protein in a fermenter. Figure 4 provides a flowchart for producing recombinant Romiplast protein in a fermenter, in accordance with one or more embodiments of the present invention. Referring to Figure 4, producing recombinant Romiplast protein in a fermenter includes transferring batch-stage medium to the fermenter (step 402), adding inoculum to the fermenter (step 404), starting the fed-batch process (step 406), starting the induction process (step 408), Referring to step 402 in more detail, step 402 includes transferring the batch stage medium to a fermenter. In one embodiment of the present invention, the fermenter includes an improved culture medium. In one embodiment of the present invention, the batch stage medium is an aqueous solution comprising glucose at a weight percent of between 30 and 40, dipotassium hydrogen phosphate at a weight percent of between 40 and 45, monopotassium phosphate at a weight percent of between 20 and 23, citric acid at a weight percent of between 5 and 7, ammonium sulfate at a weight percent of between 7 and 8, magnesium sulfate heptahydrate at a weight percent of between 4 and 5, yeast extract at a weight percent of between 2 and 4, trace elements at a weight percent of between 2 and 3, and thiamine at a weight percent of between 2 and 4 at a temperature of 35°C and a pH of 6.7±0.1. Referring to step 404 in more detail, step 404 involves adding inoculum to the fermenter. In one embodiment of the present invention, cells containing a gene encoding a recombinant protein consume nutrients in the batch medium until they are eventually starved of glucose. Referring to step 406 in more detail, step 406 includes initiating a fed-batch step. In one embodiment of the present invention, the fed-batch step medium is an aqueous solution comprising glucose at a weight percent between 400 and 500, yeast extract at a weight percent between 55 and 65, peptone at a weight percent between 7 and 9, ammonium sulfate at a weight percent between 1 and 1.75, magnesium sulfate heptahydrate at a weight percent between 14 and 15, trace elements at a weight percent between 3.5 and 5.5 at a temperature of 35°C and a pH of 6.7 ± 0.1. Referring to step 408 in more detail, step 408 includes initiating an induction step. In one embodiment of the present invention, the induction step medium is an aqueous solution comprising glycerol at a weight percent of between 350 and 400, yeast extract at a weight percent of between 25 and 35, ammonium sulfate at a weight percent of between 0.5 and 1.5, magnesium sulfate heptahydrate at a weight percent of between 9 and 11, trace elements at a weight percent of between 2 and 3, thiamine at a weight percent of between 2 and 4 at a temperature of 35°C and a pH of 6.7 ± 0.1. Because the cultivation conditions in Erlenmeyer flasks are different from those in fermentors, the parameters considered for optimization should be examined at the fermentor scale. In fermentor cultivation, the Fed-Batch cultivation process is performed. Fed-Batch cultivation consists of three stages: Batch, Fed-Batch, and Induction. During the Batch fermentation stage, the glucose in the initial culture medium is consumed and, with a sudden and sharp increase in the amount of oxygen due to the lack of a carbon source in the culture, it enters the Fed-Batch and feeding stage. In this stage, with continuous feeding, the number of cells increases and reaches the appropriate density for optimal protein expression. The OD parameter is used to monitor the cell population. After 12 hours of feeding with the first feed and reaching the appropriate OD (130±10), the Induction stage begins with the addition of Isopropyl ß-D-1-thiogalactopyranoside (IPTG) to the culture and feeding with the second feed. To confirm the expression of the recombinant protein during the fermentor, sampling is performed for SDS-PAGE at the beginning of the induction phase and also at different times during this phase.During the process, to confirm that the appropriate cell density has been achieved, sampling is performed to determine the dry weight of the cell mass. The presence of glucose in the first feed causes rapid cell proliferation and increases cell density. Glucose also suppresses the expression of proteins under the control of the lac operon at this stage. The presence of glycerol as a carbon source in the second feed instead of glucose causes protein expression. Referring to step 208 in more detail, step 208 involves harvesting the culture medium present in the fermenter. In one embodiment of the present invention, the culture medium is actually the bacterial cells propagated from the input clone in step 306 to the fermenter that have been propagated by the improved feeding strategy. Referring to step 210 in more detail, step 210 involves centrifuging the culture medium harvested from the fermenter. In one embodiment of the present invention, centrifugation is performed for 45 minutes at 4600 rpm. Referring to further details regarding step 212, step 212 involves purifying the recombinant Romiplast protein. . Figure 5 provides a flowchart for purifying the recombinant Romiplast protein, in accordance with one or more embodiments of the present invention. Referring to Figure 5, the purification of the recombinant Romiplast protein includes the steps of lysis and washing of the bacterial biomass (step 502), solubilization and refolding of the inclusion body (step 504); affinity chromatography using Mab select (step 506); IEX chromatography (step 508); ultrafiltration (step 510) and final filtration (step 512). Referring to step 502 in more detail, step 502 involves lysing and washing the bacterial biomass. Figure 6 provides a flowchart for lysing and washing the bacterial biomass, in accordance with one or more embodiments of the present invention. Referring to Figure 6, lysing and washing the bacterial biomass includes the steps of preparing a cell suspension from the isolated bacterial biomass in a lysis buffer (step 602); adding lysozyme and phenylmethylsulfonyl urea to the cell suspension (step 604); homogenizing the cell suspension (step 606); separating the air inclusion (step 608); washing the air inclusion with a first wash buffer (step 610); washing the air inclusion with a second wash buffer (step 612); and placing the final pellet in a freezer (step 614). Referring to step 602 in more detail, step 602 includes preparing a cell suspension of the isolated bacterial biomass in a lysis buffer. In one embodiment of the present invention, preparing a cell suspension of the isolated bacterial biomass includes dissolving the bacterial biomass in a lysis buffer. In one embodiment of the present invention, dissolving the bacterial biomass in a lysis buffer includes dissolving the bacterial biomass in a lysis buffer at a ratio of 1:10 (buffer:bacterial biomass). Referring to step 604 in more detail, step 604 includes adding lysozyme and phenylmethylsulfonyl urea to the cell suspension. In one embodiment of the present invention, adding lysozyme and phenylmethylsulfonyl urea to the cell suspension includes adding lysozyme at a concentration of 0.2 g / L while centrifuging on a stirrer at 700 rpm for 45 minutes. Referring to step 606 in further detail, step 606 involves homogenizing the cell suspension. In one embodiment of the present invention, homogenizing the cell suspension involves homogenizing the cell suspension in 3 consecutive cycles at a pressure of 700 bar. Referring to step 608 in more detail, step 608 involves separating the air inclusions. In one embodiment of the present invention, separating the air inclusions comprises centrifuging the homogenized cell suspension for 30 minutes at 9000 rpm (g) at 4 degrees Celsius and then removing the supernatant. Referring to step 610 in more detail, step 610 involves washing the air inclusion with a first wash buffer. In one embodiment of the present invention, washing the air inclusion with a first wash buffer involves washing the air inclusion with a first wash buffer in two steps, each for 20 minutes, at 4 degrees Celsius, at 9000 spin (g), and then removing the supernatant. In one embodiment of the present invention, air inclusion is washed with a first wash buffer at a weight:volume ratio of 16 grams of air inclusion per 40 ml of first wash buffer. Referring to step 612 in more detail, step 612 involves washing the air inclusion with a second wash buffer. In one embodiment of the present invention, washing the air inclusion with a second wash buffer involves washing the air inclusion with a second wash buffer in two steps, each for 20 minutes, at 4 degrees Celsius, at 9000 spin (g), and then removing the supernatant. In one embodiment of the present invention, air inclusion is washed with a second wash buffer at a weight:volume ratio of 16 grams of air inclusion per 40 ml of second wash buffer. Referring to step 614 in more detail, step 614 includes placing the final precipitate in a freezer. In one embodiment of the present invention, the final precipitate is placed in a freezer at a temperature of -20 degrees Celsius. In one embodiment of the present invention, the final precipitate can be stored in a freezer at a temperature of -20 degrees Celsius for up to 3 months. Referring to step 504 in more detail, step 504 involves solubilizing and refolding the inclusion body. Figure 7 provides a flowchart for solubilizing and refolding the inclusion body, in accordance with one or more embodiments of the present invention. Referring to Figure 7, solubilizing and refolding the inclusion body includes the steps of preparing a solution of the inclusion body (step 702); forming a solution soup containing the Romiplast protein (step 704); refolding the solution soup containing the Romiplast protein (step 706); and preparing a homogeneous solution containing the Romiplast protein (step 708). Referring to step 702 in more detail, step 702 includes the steps of preparing a solution of the air inclusion. In one embodiment of the present invention, preparing a solution of the air inclusion includes dissolving a certain amount of the air inclusion in a certain amount of a solvent buffer. In one embodiment of the present invention, dissolving a certain amount of the air inclusion in a certain amount of a solvent buffer includes dissolving a certain amount of the air inclusion in a certain amount of a buffer solvent with a weight / volume ratio of 1 g / 15 ml (air inclusion / solvent buffer) while stirring on a stirrer at 700 rpm for one hour at room temperature. Referring to step 704 in more detail, step 704 involves forming a broth solution containing the Romiplast protein. In one embodiment of the present invention, forming a broth solution containing the Romiplast protein comprises centrifuging the air inclusion solution at 9000 rpm (g) at 25 degrees Celsius for 45 minutes. Referring to step 706 in more detail, step 706 includes refolding the broth containing the Romiplast protein. In one embodiment of the present invention, refolding the broth containing the Romiplast protein comprises refolding the broth containing the Romiplast protein by reverse dilution. In one embodiment of the present invention, refolding the broth containing the Romiplast protein by reverse dilution comprises refolding the broth containing the Romiplast protein by a peristaltic pump at a temperature between 4 and 8 degrees Celsius and a dilution factor of 20 for 3 hours. Referring to step 708 in more detail, step 708 includes preparing a homogeneous solution containing the Romiplast protein. In one embodiment of the present invention, preparing a homogeneous solution containing the Romiplast protein includes stirring the solution containing the refolded protein with a stirrer at 4° C. for 24 hours. Referring to further details regarding step 508, step 508 involves affinity chromatography using Mab select. In one embodiment of the present invention, affinity chromatography using Mab select includes performing chromatography using Mab select resin. Referring to further details regarding step 508, step 508 comprises chromatography using Capto SP. In one embodiment of the present invention, chromatography using Capto SP comprises performing chromatography using Capto SP resin. Referring to step 510 in further detail, step 510 comprises ultrafiltration. In one embodiment of the present invention, the ultrafiltration is by an ultrafiltration system using a Hydrosart 10 kDa filter. Referring to step 510 in further detail, step 510 includes final filtration. In one embodiment of the present invention, final filtration is performed by a 0.22 µm Millipak filter at a maximum pressure of 1.5 bar and a maximum flow rate of 100 (ml / min). Example 1: Production of recombinant romaplast protein via improved feeding strategyFirst, a plasmid containing the gene encoding the recombinant protein Romiplast was produced. A suitable plasmid for this step is pET24a(+). Then, the said plasmid was introduced into the host cell of E. coli bacteria of the T7 Express strain. In order to create a recombinant cell, a susceptible cell was prepared from the desired host cell according to standard protocols, then transformation was performed and transgenic clones were selected. From the selected clones, an initial expression test was performed in an Erlenmeyer flask and the clone with the highest expression level was selected for preparation of a microbial bank. Then, the expression of the recombinant protein Romiplast was examined in a laboratory scale (Erlenmeyer flask). At this stage, LB+0.5% Glucose culture, IPTG inducer level=0.5 mM, was performed at 37°C and the induction time was 24 hours. The expression of the recombinant protein was confirmed using SDS-PAGE. Figure 8 shows an SDS-PAGE image regarding the confirmation of the expression of the recombinant protein in the host cell, according to one or more embodiments of the present invention. Referring to Figure 8 in more detail, the molecular weight of Romiplast is approximately 59 kDa. This image is a reduced gel that separates the Romiplast subunits.For this reason, the protein is observed in the range of 25 to 35 kDa. The desired band is visible in the Total and Pellet samples at 6 hours and 24 hours. This band is not present in the control sample, which was in the same conditions as the other samples, but without receiving IPTG. As it is clear, Romiplast is expressed in the insoluble form (IB) and is not present in the soluble sample. After the end of the process, the cells were lysed and the soluble proteins were separated from the insoluble ones. As can be seen in Figure 8, the band corresponding to the Romiplast protein is visible in the insoluble and Total samples. These results showed us that the target protein was expressed in an appropriate amount at 24 hours and is in the insoluble form. These results were sufficient for the development and scale-up of the culture. The next step is cultivation in the fermenter. For this purpose, the optimized feeding mentioned in the present invention is used. First, the batch stage medium is added to the fermenter. The batch stage medium is an aqueous solution and includes glucose by weight, dipotassium hydrogen phosphate by weight, monopotassium phosphate by weight, citric acid by weight, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, yeast extract by weight, trace elements by weight and thiamine by weight at a temperature of 35 ° C and a pH of 6.7 ± 0.1. In the next step, the cell clone containing the gene encoding the recombinant protein is added to the fermenter. In the next step, the fed-batch stage medium is added to the fermenter. The fed-batch medium consists of an aqueous solution containing glucose by weight, yeast extract by weight, peptone by concentration, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, trace elements by weight at a temperature of 35°C and a pH of 6.7 ± 0.1. In the final stage of this feeding, the induction medium is added to the fermenter.The induction phase medium consisted of an aqueous solution containing glycerol by weight, yeast extract by weight, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, trace elements by weight, thiamine by weight at a temperature of 35°C and a pH of 6.7 ± 0.1. In this phase, with continuous feeding, the number of cells increased and reached the appropriate density for optimal protein expression. The OD parameter was used to monitor the cell population. After 12 hours of feeding with the first feed and reaching the appropriate OD (130 ± 10), the induction phase began with the addition of IPTG to the culture and feeding with the second feed. The IPTG concentration at the time of induction was considered to be 0.1 mM. The induction phase continued with feeding for 12 hours. At the end of the process, the OD should be about 180 ± 10. The culture solution was harvested at the end of the process and centrifuged to separate the cells from the culture medium. The cell sediment is then stored in a -30 freezer for purification and purification processes.To confirm the expression of the recombinant protein during the fermentor, sampling is performed for SDS-PAGE at the beginning of the induction phase and also at different times of this phase. During the process, sampling is also performed to determine the dry weight of the cell mass to confirm that the appropriate cell density has been reached. Feeding in this culture process is by exponential feeding method and is maintained during the fed-batch and induction phases. In this feeding method, the feeding rate per unit time is calculated and applied according to the specific growth rate of the bacteria and the amount of biomass produced in exchange for the consumption of the carbon source. The presence of glucose in the first feed causes rapid cell proliferation and increases cell density. Glucose also suppresses the expression of the protein under the control of the lac operon at this stage. The presence of glycerol as a carbon source in the second feed instead of glucose causes protein expression. To investigate the production of Romiplast in the fermenter culture and the reproducibility of the process, 3 experiments were conducted under identical conditions. Figure 9 shows the optimal culture parameters of a 5-liter fermenter for an experiment to investigate the production of Romiplast in a fermenter according to one or more embodiments of the present invention. Figure 10 shows the results of the SDS-PAGE test in the first experiment according to one or more embodiments of the present invention. Referring to more details of Figure 10, sampling was performed at hours 0, 8, 6, 4 and 10 of the induction process. As with the expression process in the Erlenmeyer flask, the target protein band is visible in the Total and pellet samples in the fermentor. This confirms the correctness of the protein expression process and indicates that the protein is expressed in the IB form and in the appropriate amount. An increase in band intensity is also visible during the process. Figure 11 shows the results of the SDS-PAGE test in the second experiment according to one or more embodiments of the present invention. In this experiment, as in the previous experiment, the protein is expressed in the insoluble phase and during the induction step. Figure 12 shows the results of the SDS-PAGE test in the third experiment according to one or more embodiments of the present invention. In this experiment, the target protein is also expressed in the insoluble phase and during the induction step. In the cell lysis process, bacterial biomass is dissolved in lysis buffer at a 10-fold rate (1 g biomass in 10 ml buffer) and then 0.2 g / l lysozyme and PMSF at a concentration of 1 mM are added to the resulting suspension. The resulting cell suspension is homogenized on a stirrer at 700 rpm for 45 minutes and then homogenized for 3 cycles at 700 bar pressure. The resulting solution is centrifuged at 9000 rpm and 4 °C for 30 minutes and the supernatant is removed, and the resulting precipitate is collected. The air inclusion obtained from the lysis step is washed twice using the first wash buffer. For every 16 grams of initial biomass, 40 ml of washing buffer is used and centrifuged for 20 minutes at 4°C at 9000 (g) each time and the supernatant is removed. Then, the resulting precipitate is washed three times with the second washing buffer and centrifuged for 20 minutes at 4°C at 9000 (g) each time and the supernatant is removed. Finally, the remaining precipitate can be stored in a freezer at -20°C for up to 3 months.At this stage, SDS-PAGE was performed to confirm the presence of protein. Figure 13 shows SDS-PAGE after washing steps in accordance with one or more embodiments of the present invention. In the process of dissolving and refolding the Romiplast protein, 15 ml of solvent buffer is added per gram of air inclusion and it is stirred for 1 hour at room temperature at 700 rpm. Then, the resulting solution is centrifuged for 45 minutes at 25°C at 9000 rpm. The supernatant is separated and the precipitate is removed. The UV absorbance of the resulting soup is read at 280nm and the concentration of the dissolved protein is determined by taking into account the extinction coefficient of 1.58. The dissolved protein soup is refolded by Reverse Dilution with refolding buffer for 3 hours using a peristaltic pump at 4 to 8°C with a dilution factor of 20. The resulting solution is stirred at 4°C for 24 hours. Also, to confirm the presence of protein at this stage, SDS-PAGE is performed. Due to the nature of this fusion protein and the presence of the antibody heavy chain sequence therein, MabSelect resin was used for purification. Figure 14 shows the specifications for a MabSelect resin in accordance with one or more embodiments of the present invention. In this resin, protein A expressed in E. coli bacteria is linked to the agarose matrix of the resin via a disulfide bond. Figure 15 shows the chromatography parameters of MabSelect in accordance with one or more embodiments of the present invention. Capto SP resin was selected as one of the strong cationic resins for the purification of the Romiplast protein. The characteristics of the Capto SP resin are given in Figure 16. Figure 16 shows the characteristics of the Capto SP resin according to one or more embodiments of the present invention. This resin has a specific sulfonate group attached to the agarose matrix and has a high separation ability in protein purification due to the small particle size. Figure 17 shows the parameters approved for the packing of the CapTo SP column according to one or more embodiments of the present invention. Ion exchange chromatography was also used to purify Romiplast. The CapTo SP medium was packed in a chromatography column. The column packing method is evaluated by asymmetry and factor calculation. Acceptable ranges for asymmetry and h factor are 0.8-1.8 and less than 3, respectively. After packing, the column is washed with 1 CV of 1 M sodium hydroxide solution and then equilibrated with 2-3 column volumes of equilibration buffer. The column is connected to the apparatus and equilibration buffer equilibrates the column. This step is performed until the column conductivity unit reaches the range of 4.5±1 mS / cm. The chromatography steps are performed according to Figure 18. Figure 18 shows a summary of the steps for performing ion exchange chromatography using a CapTo Sp column in accordance with one or more embodiments of the present invention. After the last chromatography step, the elution obtained is concentrated by an ultrafiltration (TFF) system using a Hydrosart 10 kDa filter. Figure 19 summarizes the parameters of interest in TFF according to one or more embodiments of the present invention. The last step is the final filtration. This filtration is performed using a 0.22 µm Millipak filter. Before this filtration, Formulation 2 buffer is added to the solution from the previous step. The filtered solution is collected in sterile vials under a Grade A laminar flow hood and stored at 2-8°C. The filtration process is performed at a maximum pressure of 1.5 bar and a maximum velocity of about 100 mm / s. Explanation of shapes, maps and diagrams Figure 1 presents the amino acid sequence of a recombinant romaplast protein, according to one or more embodiments of the present invention; Figure 2 provides a flowchart for producing recombinant Romiplast protein via an improved feeding strategy, in accordance with one or more embodiments of the present invention; Figure 3 provides a flowchart for producing a recombinant cell expressing Romiplast, in accordance with one or more embodiments of the present invention; Figure 4 provides a flowchart for producing recombinant Romiplast protein in a fermenter, in accordance with one or more embodiments of the present invention; Figure 5 provides a flowchart for purifying recombinant Romiplastim protein, in accordance with one or more embodiments of the present invention; Figure 6 provides a flowchart for lysis and washing of bacterial biomass, in accordance with one or more embodiments of the present invention; Figure 7 provides a flowchart for solubilization and refolding of air inclusions, in accordance with one or more embodiments of the present invention; Figure 8 shows an SDS-PAGE image confirming expression of a recombinant protein in a host cell, in accordance with one or more embodiments of the present invention; Figure 9 shows the optimal culture parameters of a 5-liter fermenter for an experiment investigating the production of romaplast in a fermenter according to one or more embodiments of the present invention; Figure 10 shows the results of the SDS-PAGE test in the first experiment according to one or more embodiments of the present invention; Figure 11 shows the results of the SDS-PAGE test in the second experiment according to one or more embodiments of the present invention; Figure 12 shows the results of SDS-PAGE testing in the third experiment according to one or more embodiments of the present invention; Figure 13 shows SDS-PAGE after washing steps in accordance with one or more embodiments of the present invention; Figure 14 illustrates specifications for MabSelect resin in accordance with one or more embodiments of the present invention; Figure 15 illustrates MabSelect chromatography parameters in accordance with one or more embodiments of the present invention; Figure 16 illustrates specifications for Capto SP resin in accordance with one or more embodiments of the present invention; Figure 17 illustrates parameters approved for use in the CapTo SP column packing in accordance with one or more embodiments of the present invention; Figure 18 shows a summary of the steps for performing ion exchange chromatography using a CapTo Sp column in accordance with one or more embodiments of the present invention; and Figure 19 illustrates a summary of parameters of importance in TFF in accordance with one or more embodiments of the present invention. A clear and precise statement of the advantages of the claimed invention over prior inventions. One of the steps in the production of this protein on an industrial scale is feeding it into a culture fermenter. One of the important factors in the feeding of the fermenter is Optical Density (OD). In products produced as air inclusions, higher ODs can contribute to higher yields. In the prior art, with the feedings presented in these documents, the maximum OD achieved is 100. However, cells containing the gene encoding this protein have a higher production capacity and, as a result, the ability to increase OD. In this invention, with an improved feeding design, this factor has reached above 180. Description of at least one implementation method for implementing the invention First, a plasmid containing the gene encoding the recombinant protein Romiplast was constructed. The appropriate plasmid for this step is pET24a(+). Then, the plasmid was introduced into the host cell of E. coli from the T7 Express strain. To create the recombinant cell, a susceptible cell was prepared from the desired host cell according to standard protocols, then transformation was performed and transgenic clones were selected. From the selected clones, an initial expression test was performed in Erlenmeyer flask and the clone with the highest expression level was selected for preparation of the microbial bank. Then, the expression of the recombinant protein Romiplast was examined in a laboratory scale (Erlenmeyer flask). At this stage, LB+0.5% Glucose culture, IPTG inducer level= 0.5 mM, was performed at 37°C and the induction time was 24 hours. The expression of the recombinant protein was confirmed using SDS-PAGE. The next step is cultivation in the fermenter. For this purpose, the optimized feeding strategy mentioned in the present invention is used. First, the batch stage medium is added to the fermenter. The batch stage medium is an aqueous solution and includes glucose by weight, dipotassium hydrogen phosphate by weight, monopotassium phosphate by weight, citric acid by weight, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, yeast extract by weight, trace elements by weight and thiamine by weight at a temperature of 35 ° C and a pH of 6.7 ± 0.1. In the next step, the cell clone containing the gene encoding the recombinant protein is added to the fermenter. In the next step, the fed-batch stage begins. The fed-batch phase medium consists of an aqueous solution containing glucose by weight, yeast extract by weight, peptone by concentration, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, trace elements by weight at a temperature of 35 ° C and a pH of 6.7 ± 0.1. In the final phase of this feeding, the induction phase begins.The induction phase medium consisted of an aqueous solution containing glycerol by weight, yeast extract by weight, ammonium sulfate by weight, magnesium sulfate heptahydrate by weight, trace elements by weight, thiamine by weight at a temperature of 35°C and a pH of 6.7 ± 0.1. In this phase, with continuous feeding, the number of cells increased and reached the appropriate density for optimal protein expression. The OD parameter was used to monitor the cell population. After 12 hours of feeding with the first feed and reaching the appropriate OD (130 ± 10), the induction phase began with the addition of IPTG to the culture and feeding with the second feed. The IPTG concentration at the time of induction was considered to be 0.1 mM. The induction phase continued with feeding for 12 hours. At the end of the process, the OD should be about 180 ± 10. The culture solution was harvested at the end of the process and centrifuged to separate the cells from the culture medium. The cell sediment is then stored in a -30 freezer for purification and purification processes. In the cell lysis process, bacterial biomass is dissolved in lysis buffer at a 10-fold rate (1 g biomass in 10 ml buffer) and then 0.2 g / l lysozyme and PMSF at a concentration of 1 mM are added to the resulting suspension. The resulting cell suspension is homogenized on a stirrer at 700 rpm for 45 minutes and then homogenized for 3 cycles at 700 bar pressure. The resulting solution is centrifuged at 9000 rpm and 4 °C for 30 minutes and the supernatant is removed, and the resulting precipitate is collected. The air inclusion obtained from the lysis step is washed twice using the first wash buffer. For every 16 grams of initial biomass, 40 ml of washing buffer is used and centrifuged for 20 minutes at 4°C at 9000 (g) each time and the supernatant is removed. Then, the resulting precipitate is washed three times with the second washing buffer and centrifuged for 20 minutes at 4°C at 9000 (g) each time and the supernatant is removed. Finally, the remaining precipitate can be stored in a freezer at -20°C for up to 3 months. In the process of dissolving and refolding the Romiplast protein, 15 ml of solvent buffer is added per gram of air inclusion and it is stirred for 1 hour at room temperature at 700 rpm. Then the resulting solution is centrifuged for 45 minutes at 25°C at 9000 rpm. The supernatant is separated and the precipitate is removed. The UV absorbance of the resulting soup is read at 280nm and the concentration of the dissolved protein is determined by taking into account the extinction coefficient of 1.58. The dissolved protein soup is refolded by the Reverse Dilution method with refolding buffer for 3 hours using a peristaltic pump at 4 to 8°C with a dilution factor of 20. The resulting solution is stirred at 4°C for 24 hours. Due to the nature of this fusion protein and the presence of the antibody heavy chain sequence in it, MabSelect resin was used for purification. Ion exchange chromatography was also used for the purification of Romiplast. After the last chromatography step, the elution obtained was concentrated by ultrafiltration (TFF) system using a Hydrosart 10 kDa filter. The last step is the final filtration. This filtration is performed using a 0.22 µm Millipak filter. Before this filtration, Formulation 2 buffer is added to the solution from the previous step. The filtered solution is collected in sterile vials under a Grade A laminar flow hood and stored at 2-8°C. The filtration process is performed at a maximum pressure of 1.5 bar and a maximum velocity of about 100 mm / s. Explicit mention of the industrial application of the invention The amino acid sequence of romiplastin is not identical to thrombopoietin and is expressed in a host bacterium by recombinant DNA technology. The World Health Organization has approved the use of romiplastin for adult patients with long-standing immune thrombocytopenic purpura who have not responded to other treatments such as corticosteroids, intravenous immunoglobulin (IVIG), Rho (D) immune globulin, and splenectomy. Romiplastin acts as a thrombopoietin receptor agonist and activates intracellular transcriptional pathways through c-Mpl, thereby increasing platelet proliferation. In addition, this drug can act similarly to thrombopoietin and control platelet proliferation in the bone marrow.

Claims

Claims What is claimed: Claim 1) A method for producing recombinant Romiplast protein with an optical density (OD) of at least 180 through an optimized feeding strategy, said method comprising the following steps: -Constructing a pET24a(+) plasmid containing the gene encoding the recombinant Romiplast protein; -Transforming the plasmid containing the gene encoding the recombinant Romiplast protein into E. coli bacteria of the T7 Express strain; -Transferring the batch feed medium to the fermentor, at a temperature of 35°C and at a pH of 6.7±0.1, so that the batch feed medium is an aqueous solution containing glucose with a weight percentage between 30 and 40, dipotassium hydrogen phosphate with a weight percentage between 40 and 45, monopotassium phosphate with a weight percentage between 20 and 23, citric acid with a weight percentage between 5 and 7, ammonium sulfate with a weight percentage between 7 and 8, magnesium sulfate heptahydrate with a weight percentage between 4 and 5, yeast extract with a weight percentage between 2 and 4, trace elements with a weight percentage between 2 and 3, and thiamine with a weight percentage between 2 and 4; -Adding the inoculum to the fermentor; -Starting the Fed-Batch process, at a temperature of 35°C and at a pH of 6±0.1.7, where the Fed-Batch feeding medium is an aqueous solution containing glucose with a weight percentage between 400 and 500, yeast extract with a weight percentage between 55 and 65, peptone with a weight percentage between 7 and 9, ammonium sulfate with a weight percentage between 1 and 1.75, magnesium sulfate heptahydrate with a weight percentage between 14 and 15, and trace elements with a weight percentage between 3.5 and 5.5; -Starting the induction process, at a temperature of 35°C and at a pH of 6.7±0.1, so that the induction feeding medium is an aqueous solution containing glycerol with a weight percentage between 350 and 400, yeast extract with a weight percentage between 25 and 35, ammonium sulfate with a weight percentage between 0.5 and 1.5, magnesium sulfate heptahydrate with a weight percentage between 9 and 11, trace elements with a weight percentage between 2 and 3, thiamine with a weight percentage between 2 and 4; -Removing the culture medium from the fermentor; -Centrifuging the culture medium removed from the fermentor; and -Purifying the solution containing the recombinant Romiplast protein. Claim 2) A method for producing recombinant Romiplastim protein according to claim 1, wherein the batch feeding medium is an aqueous solution comprising glucose at 33% by weight, dipotassium hydrogen phosphate at 42% by weight, monopotassium phosphate at 21% by weight, citric acid at 6% by weight, ammonium sulfate at 7.5% by weight, magnesium sulfate heptahydrate at 4.5% by weight, yeast extract at 3% by weight, trace elements at 2.25% by weight, and thiamine at 3% by weight. Claim 3) The method of manufacturing the recombinant protein Romiplastim according to claim 1, such that the Fed-Batch feeding medium is an aqueous solution containing glucose with a weight percentage of 440, yeast extract with a weight percentage of 60, peptone with a weight percentage of 8, ammonium sulfate with a weight percentage of 1.20, magnesium sulfate heptahydrate with a weight percentage of 14.40, and trace elements with a weight percentage of 4. Claim 4) A method for producing recombinant Romiplastim protein according to claim 1, wherein the induction feeding medium is an aqueous solution comprising glycerol at 366% by weight, yeast extract at 30% by weight, ammonium sulfate at 1% by weight, magnesium sulfate heptahydrate at 10% by weight, trace elements at 2.5% by weight, and thiamine at 3% by weight.

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