Human recombinant hyposialic acid-added erythropoietin, its purification method, and treatment applications.

Abstract

The present invention relates to the fields of biotechnology and medicine and describes pharmaceutical compositions of recombinant human erythropoietin characterized by a microheterogeneity of fucosylated N-glycans formed by di-, tri-, and tetra-antennary structures with mono- and bi-sialylated sialic acid residues representing 40-60% of total glycans, tri-sialylated sialic acid residues representing 40-43% of total glycans, and tetra-sialylated sialic acid residues representing 10-13% of total glycans. This glycosylation pattern confers properties that allow the composition to be used in disorders of the nervous system. Methods for obtaining the pharmaceutical compositions described herein are also described.

Description

Technical Field

[0001] Scope of the Technology The present invention relates to the fields of biotechnology and medicine, and more particularly to obtaining a pharmaceutical composition of recombinant human erythropoietin having a glycosylation pattern that confers properties enabling its use in disorders of the nervous system.

[0002] Background Erythropoietin (EPO) is a glycoprotein hormone formed by 166 amino acids with a molecular weight of 30.4 kDa (Lanfranco, F and Strasburger, C.J. (2016) Sports Endocrinology 47:115 - 27). EPO is naturally produced in perisinusoidal cells of the liver during fetal and perinatal periods and mainly in interstitial fibroblasts of the kidney in adulthood. This hormone stimulates the production of red blood cells in the bone marrow and plays an important role in the brain's response to neuronal injury (Siren L. et al. (2001) Proc Natl Acad Sci USA 98(7):4044 - 9).

[0003] EPO is a highly glycosylated molecule, and its carbohydrate moiety constitutes 40% of the molecular weight. This protein contains four complex chains of oligosaccharides linked to the polypeptide chain, three of which are by N - type bonds and one by an O - type bond, and their positions are well - described by different authors Elliott, S. et al. (2004) The Journal of Biological Chemistry, 279(16):16854 - 16862; Watson et al. (1994) Glycobiology 4(2):227 - 237. The oligosaccharides with N - type bonds may contain variable sialic acid terminal residues and are important for secretion, molecular stability, receptor binding, and in vivo activity (Egrie, J. and Browne, J. (2001) Br. J. Cancer, 84(l):3 - 10; Goldwasser et al. (1974) J. Biol. Chem 249:4202 - 4206).

[0004] From the 1990s to the present, a great deal of evidence has accumulated regarding the neuroprotective properties of recombinant human EPO (rhEPO). In 1998, Sakanara and colleagues demonstrated that in a gerbil model of total ischemia, supplying rhEPO through the lateral ventricle after occlusion of the common carotid artery resulted in reduced ischemic injury to hippocampal neurons in the CA1 region (Sakanara, M. et al. (1998) Proc. Natl. Acad. Sci. USA, 95:4635-4640).

[0005] The cytoprotective effects of EPO on the central nervous system were demonstrated by Maiese et al. in 2004 and subsequently by Viviani et al. in 2005 (Maiese, K. et al. (2004) Trends in Pharmacological Sciences 25(11):577-83; Viviani, D. et al. (2005) Journal of Neurochemistry 93(2):257-268). Despite all the information accumulated from non-clinical studies on hematopoietic EPO, the results obtained have not been replicated in clinical practice due to adverse events that occur in patients with long-term use.

[0006] In adults, EPO receptor expression in the nervous system is mainly found in neurons, astrocytes, and microglia, but astrocytes produce EPO, which is hyposialic acid-modified EPO (Nagai, A. et al. (2001) Journal of Neuropathology & Experimental Neurology, 60(4):317-319).

[0007] A considerable number of researchers are working on modifying rhEPO to obtain a drug that has the same neuroprotective properties but without the adverse events caused by hematopoietic effects. EPOs obtained by the total enzymatic desialication of rhEPO, called AsialoEPO (US2004 / 0122216), possess the desired properties described above. This type of EPO has high affinity for the rhEPO receptor, but its protective effect is limited due to its extremely short plasma half-life. Another example of erythropoietin modification is the conversion of lysine to homocitrulline by protein carbamylation, which results in a carbamylated EPO called CEPO (Leist, M., Ghezzi, P., Grasso, G. et al. (2004) 305(5681):239-242). Neither EPO showed hematopoietic effects, and clinical trials using CEPO did not demonstrate neuroprotective efficacy, even though no adverse effects were observed.

[0008] The patent application WO2007 / 009404 claims a different nasal formulation of EPO with a low sialic acid content, later referred to in the authors' publications as NeuroEPO (Garcia, JC and Sosa, I. (2009), The Scientific World Journal, 9:970-981). This rhEPO is obtained by a hollow fiber membrane fermentation process and ion exchange chromatography in purification to separate the most acidic isoform (from the lowest sialic acid content to the highest). NeuroEPO has 13 isoform profiles, 9 of which are shared with a hematopoietic rhEPO called EPOCIM®.

[0009] The inventors of the present invention describe for the first time a production process in a stirred tank (ST) combined with a purification step performed by chromatography using an integrated column as an anion exchanger having a quaternary ammonium ligand Q, which can increase the expression of rhEPO hyposialic acid-added isoforms without additional chemical and genetic modifications. These isoforms have an isoelectric point profile in the pH range of 4.25 to 5.85 and have a glycosylation-related tertiary structure different from NeuroEPO, resulting in higher efficacy in both in vitro and in vivo neuroprotective and neurorepair mechanisms.

[0010] Brief description of the invention In one embodiment, an object of the present invention is a pharmaceutical composition characterized by containing as an active ingredient rhEPO having an isotype profile with an isoelectric point in the range of 4.25 to 5.85. The rhEPO has minute heterogeneity of a fucosylated N-glycan formed by a 2, 3, and 4-branched structure having mono and bi-sialic acid-added sialic acid residues corresponding to 40-60% of the total glycan, trisialic acid-added sialic acid residues corresponding to 40-43%, and tetrasialic acid-added sialic acid residues corresponding to 10-13%, and a pharmaceutically acceptable excipient.

[0011] In particular, the O-glycosylation site of serine 126 has three sialylated forms, each containing 0 to 2 sialic acid residues. The monosialic acid addition structure is the most abundant, representing 78-82% of all glycans, while the asialylated structure accounts for 6-10% of all glycans.

[0012] The N-glycosylation site of asparagine 83 is - A fucosylated bibranched structure having 1 and 2 sialic acid residues, wherein this structure accounts for 8-12% of the total glycan, A fucosylated tribranched structure having 1, 2, and 3 sialic acid residues, wherein these structures account for 17-21% of the total glycan. - Fucosylated tetrabranched structures having 1 to 4 sialic acid residues, where these structures account for 27 to 31% of the total glycan, and - A fucosylated tetrabranched structure having N-acetyllactosamine type 1 and type 2 having 1 to 4 sialic acid residues, wherein these structures account for 38 to 42% of the total glycans. Includes.

[0013] Pharmaceutically acceptable excipients for the pharmaceutical compositions claimed in this invention include, but are not limited to, bioadhesive polymers such as hydroxypropyl methylcellulose, and protein stabilizers such as L-tryptophan, L-leucine, L-arginine hydrochloride, and L-histidine hydrochloride.

[0014] The above structure of the rhEPO isotype, which is part of the pharmaceutical composition targeted by the present invention, provides the composition with higher efficacy in both in vitro and in vivo neuroprotective and nerve repair mechanisms.

[0015] In another embodiment, an object of the present invention is a method for obtaining unmodified rhEPO, wherein the fermentation process is carried out in perfusion mode with ST using a protein-free culture medium with a pH range of 7.2–7.3 at a temperature range of 34±2°C, and the medium is supplemented with glutamine until a final concentration of 8–12 mmol / L is obtained. The method also includes a purification process having a chromatography step, in which an integrated column is used as an anion exchanger having a Q quaternary ammonium ligand, the equilibrium buffer is a 20 mmol / L Tris 10 mmol / L HCl solution with a pH range of 7.9–8.10 and a conductivity range of 1.35–1.65 mS / cm, and the purification process uses a 50 mmol / L sodium acetate elution buffer with a pH of 4.3–4.5 and a conductivity of 2–3.5 mS / cm.

[0016] In the method described herein, the pharmaceutical composition has an increased number of low-sialic acid-content isoforms having a tertiary structure related to glycosylation different from NeuroEPO, which results in higher efficacy in both in vitro and in vivo neuroprotective and nerve repair mechanisms.

[0017] The use of the pharmaceutical compositions described herein for treating dementia, stroke, Parkinson's disease, ataxia, craniocerebral injury, glaucoma, autism, neonatal hypoxia, multiple sclerosis, amyotrophic lateral sclerosis, and nerve damage induced by trauma, poisoning, or radiation is also an object of the present invention. In particular, a method for treating subjects requiring such treatment using such pharmaceutical compositions is described, wherein the composition is administered in a dose range of 0.1 mg to 4 mg in a volume of 1 mL, 1 to 3 times per week for a period of 6 to 12 months. [Modes for carrying out the invention]

[0018] Pharmaceutical composition The objective of the rhEPO of the present invention is to have an isoelectric point profile of 4.25 to 5.85 and glycosylation-unrelated secondary and tertiary protein structures similar to rhEPO that retain the same glycosylation-related tertiary structure having the O-glycosylation site of serine 126 and three N-glycosylation sites of asparagine 24, 38, and 83. The carbohydrate composition of the rhEPO described herein distinguishes it from other rhEPOs. The minute heterogeneity of the fucosylated N-glycan consists of 2, 3, and 4-branched structures having mono- and bi-sialic acid-added sialic acid residues in the range of 40 to 60%, preferably in the range of 43 to 50%, of the total carbohydrate, trisialic acid-added sialic acid residues in the range of 40 to 43%, and tetrasialic acid-added sialic acid residues in the range of 10 to 13% of the total carbohydrate.

[0019] In particular, the O-glycosylation site of serine 126 has three sialylated forms, each containing 0 to 2 sialic acid residues. The monosialic acid addition structure is the most abundant, accounting for 78-82% of all glycans, while the asialylated structure accounts for 6-10% of all glycans.

[0020] The N-glycosylation site of asparagine 83 is - A fucosylated bibranched structure having 1 and 2 sialic acid residues, wherein this structure accounts for 8-12% of the total glycan, - A fucosylated three-branched structure having 1, 2, and 3 sialic acid residues, where these structures account for 17 - 21% of the total glycans, the fucosylated three-branched structure, and - A fucosylated four-branched structure having 1 - 4 sialic acid residues, where these structures account for 27 - 31% of the total glycans, the fucosylated four-branched structure, and - A fucosylated four-branched structure having N - acetyl lactosamine types 1 and 2 with 1 - 4 sialic acid residues, where these structures are in the range of 38 - 42% of the total glycans, the fucosylated four-branched structure, and include.

[0021] The terms hypo - sialic acid - added rhEPO, EPO, HS, or basic isoform are used interchangeably in the present invention and refer to a pharmaceutical composition having the above - mentioned characteristics and is usually also referred to as NeuroEPO plus.

[0022] The pharmaceutical composition of the present invention has, as an active ingredient, a hypo - sialic acid - added isoform of rhEPO. These hypo - sialic acid - added isoforms are obtained by the process described as the present invention, which does not mean chemical modification and / or genetic modification of rhEPO to obtain the isoforms. The rhEPO isoforms, which are part of the active ingredient of the pharmaceutical composition, have a tertiary structure related to different glycosylation from NeuroEPO, which results in higher efficacy in both in vitro and in vivo neuroprotective and nerve - repair mechanisms.

[0023] The pharmaceutical composition of the present invention is administered by the nasal or ocular administration route, and its final dosage form is in the form of an aqueous solution which is a nasal drop, nasal spray, or eye drop. The pharmaceutical formulation contains hypo - sialic acid - added rhEPO as an active ingredient and optionally contains pharmaceutically suitable excipients and / or stabilizers.

[0024] Pharmacochemically acceptable excipients and / or stabilizers are nontoxic to the subjects to whom they are administered at the doses and concentrations used, and may include bioadhesive polymers such as hydroxymethylcellulose, hydroxypropylcellulose, and methylcellulose, and protein stabilizers such as L-tryptophan, L-leucine, L-arginine hydrochloride, and / or L-histidine hydrochloride, and their salts.

[0025] Treatment purpose and treatment method The present invention provides pharmaceutical compositions useful for treating neurological disorders such as cerebrovascular diseases, mental illnesses, and neurodegenerative diseases. In particular, these disorders may include dementia, stroke, Parkinson's disease, ataxia, craniocerebral injury, glaucoma, autism, neonatal hypoxia, multiple sclerosis, amyotrophic lateral sclerosis, and nerve damage induced by trauma, poisoning, or radiation.

[0026] The present invention further provides a method comprising administering hyposialic acid-modified rhEPO to subjects requiring such treatment 1 to 3 times per week over a period of 6 to 12 months. The administration is carried out gradually intranasally (IN) by dropping the drug into mucose. The dosage is in the range of 0.1 mg to 4 mg, preferably 0.5 mg to 1 mg. The maximum dose per application is 1 mL, and 0.5 mL per nostril for a total daily dose of 3 mL. The volume can be distributed in smaller volumes at time intervals of 5 to 15 minutes between each application, preferably every 15 minutes.

[0027] Method for obtaining the hyposialic acid-added isoform of rhEPO The method claimed in this invention comprises different steps and uses the cell lines described below.

[0028] cell line The cell lines that can be used to carry out the method objectives of the present invention are the same as those reported for rhEPO production. The most commonly used lines are CHO, COS, BHK, Namalwa, HeLa, Hep3B, and HepG2, and the CHO cell line is preferably used in the present invention.

[0029] Fermentation process The fermentation process of the present invention is carried out using ST technology. This process consists of several steps, the first of which includes thawing ampoules from a working cell bank until they reach room temperature (18-24°C). Subsequently, a proliferation step is carried out to adjust the cells to a cell concentration and cell viability that ensures proper seeding in the seed fermenter in order to increase biomass.

[0030] 1 x 10 6 Once a cell density of cells / mL or higher is reached, fermentation is initiated using a different operating mode. These modes can be batch culture, continuous culture, or biomass retention.

[0031] To obtain the hyposialic acid-added isoform, a temperature in the range of 34±2°C and a pH in the range of 6.8±0.4 should be maintained at this stage of fermentation.

[0032] Cells should be grown in protein-free culture medium until a final glutamine concentration in the range of 8–12 mmol / L is obtained.

[0033] Purification process The purification process for hyposialic acid-added rhEPO includes the following chromatographic steps.

[0034] First, pseudo-affinity chromatography is performed using a colored ligand. The purpose of this step is to capture rhEPO and partially remove major contaminants present in the supernatant (SN). Subsequently, gel filtration chromatography is performed to change the protein buffer to the buffer solution for the next chromatography step.

[0035] Next, pseudo-affinity chromatography using a metal chelate is performed. This step aims to capture rhEPO and completely remove any fractions of impurities that were not removed in the previous step. Gel filtration chromatography is performed again to change the buffer to the buffer solution used in the next chromatography step.

[0036] A key step in this manufacturing method is anion exchange chromatography using a Q quaternary ammonium ligand. An integrated column is used in this chromatography step. The objectives of this chromatography are separation of the hyposialic acid-added (biologically active) isoform from the acidic isoform, DNA retention, and product concentration. All of these ensure that the hyposialic acid-added isoform is obtained without contamination or mixing with the acidic isoform.

[0037] Finally, the buffer solution is changed, and gel filtration chromatography is performed again to elute the protein in the form of the active raw material. [Brief explanation of the drawing]

[0038] [Figure 1] Isotype Profile: A) Definition of isotype cleavage; B) Figure showing the SN generated in the culture under evaluated temperature and pH conditions. [Figure 2] This figure shows the proportion of hyposialic acid-added isotypes in the culture under the evaluated temperature and pH conditions. [Figure 3] This figure shows the proportion of hyposialic acid-added isotypes in each culture under pilot-scale evaluation conditions. [Figure 4] This figure shows the relative strength of the less acidic iso-type in SN. [Figure 5] This figure shows the effect of the pH and conductivity of the mobile phase on the static adsorption capacity of the rhEPO iso-type Q quaternary ammonium ligand: (A) Hyposialic acid addition (basic), (B) Acidic. [Figure 6] The diagram shows the breakthrough curves A) for the chromatography matrix "Q SFF" and B) for the integrated column "CIM QA". [Figure 7] This figure shows the effect of elution buffer pH on the performance of basic and acidic isoforms. [Figure 8]This figure shows the distribution of the hyposialic acid-added isoform of rhEPO at production scale. [Figure 9] This figure shows the study of N-glycans of hyposialic acid-added rhEPO using zwitterionic hydrophilic interaction liquid chromatography linked with mass spectrometry. [Figure 10] This figure shows the extracted ion electrophoresis (EIE) of the hyposialic acid-added rhEPO O126 glycopeptide glycoform observed due to trypsin and neuraminidase, and b) due to digestion by trypsin. [Figure 11] This figure shows the EIE of N83 glycopeptide, in which glycoforms resulting from trypsin digestion and neuraminidase digestion are observed. [Figure 12] a) This figure shows the EIE of hyposialic acid-added rhEPO N83 glycopeptide in which glycoforms are observed due to trypsin and neuraminidase, and b) due to trypsin-mediated digestion. [Figure 13] This figure shows the cell viability profiles of astrocyte cultures 24 and 48 hours after cell damage with 8% dimethyl sulfoxide (DMSO) and subsequent treatment with hyposialic acid-added rhEPO. [Figure 14] This is a Kaplan-Meier chart, a diagram showing the survival rate over a 7-day observation period. [Figure 15] This figure shows the neurological state of animals 24 hours after infarction. [Figure 16] This figure shows the effect of applying different rhEPO isotypes on the number of reticulocytes in a normocytic mouse model.

[0039] The present invention will be further described with reference to the following examples and drawings. However, these examples are not intended to be construed as limiting the scope of the invention.

[0040] example Example 1. Effects of pH and temperature on rhEPO isotype profiles in laboratory settings. A seed cell bank was obtained from CHO cell lines transfected with the human EPO gene, adapted for growth in a protein-free medium suspension. Complete adaptation to this culture medium was achieved after 37 generations, equivalent to 25 days of culture.

[0041] To evaluate the performance of cell lines under different pH and temperature conditions, experimental designs combining both variables were developed. The experimental conditions are shown in Table 1. The pH of the culture was controlled by adding 0.5 mol / L sodium hydroxide. [Table 1]

[0042] The isotype profiles of rhEPO corresponding to SN samples produced in cultures under different conditions were determined by isoelectric focusing. Mixtures of ampholines with pH ranges of 2–5 and 3–10 were used, with the internal EPOCIM® reference material used as a control. The intensity percentage of each isotype in the samples was analyzed by concentration measurement using the Gene Tools program. Isotypes with pH values ​​in the range of 2.80–4.25 were defined as acidic isotypes, and isotypes with pH values ​​in the range of 4.25–6.55 were defined as basic isotypes (Figure 1A).

[0043] Figure 1B shows that the isoform profile was strongly influenced by temperature. Regardless of pH, seven acid isoforms were observed when the corresponding SN samples were compared to the control at 37°C. On the other hand, a decrease in acid isoforms was observed at 35°C, which was more pronounced under pH 7.2 and 7.3 conditions.

[0044] Figure 2 shows that under conditions of pH 7.2 and 7.3 and a temperature of 35°C, the total (100%) of the obtained isoforms were basic isoforms.

[0045] Example 2. Effects of pH and temperature on the rhEPO isotype profile at a pilot scale. The effects of a more controlled and preferred environment for cell culture were evaluated against the best culture conditions obtained on a laboratory scale (temperature 35°C, pH 7.2 and 7.3). Four fermentation cycles were planned using a 3.5 L ST-type bioreactor (Infors-AGCH4103, Botmingen) from the seed cell bank described in Example 1. The operating conditions are shown in Table 2. Initial cell viability was over 90% under all evaluated conditions. [Table 2]

[0046] Samples were collected at the end of each fermentation cycle, and the isotype profiles of the SN samples produced in the cultures under the conditions evaluated in the bioreactor were determined by isoelectric focusing. Once these profiles were obtained, the Gene Tools program was used to determine the intensity of each band present in the gel, starting with the image corresponding to the isoelectric focusing gel, and to evaluate the proportion of hyposialic acid-added isotypes for each condition.

[0047] As shown in Figure 3 for culture conditions 2 and 3 (35°C, pH 7.2-7.3), the proportion of hyposialic acid-added isoforms was higher than in conditions 1 and 4. These results support the findings obtained on a laboratory scale, namely that by modifying the operating conditions, namely pH 7.2-7.3 and temperature 35°C, the isoform profile of rhEPO is also modified, and greater intensity can be obtained at pH values ​​of 4.25-5 and 85.

[0048] Example 3. Promotion of rhEPO basic isotype expression by increasing glutamine concentration in culture medium. To evaluate whether increasing the glutamine concentration in the culture medium affected the increase in hyposialic acid-added isoforms, the performance of rhEPO-producing CHO cell lines was assessed. Seed cell banks exposed to culture media at different glutamine concentrations were used, and acclimatization to the culture medium was performed for 15 days. The evaluation was performed using a rotating bottle held in an incubator at 37°C with a stirring speed of 600 rpm, measuring 0.5 × 10⁶ in a final volume of 300 mL of culture medium.6 The study began with an initial cell concentration of cells / mL. The final glutamine concentrations in the culture medium were 8, 12, and 16 mmol / L, with a culture medium containing 6 mmol / L of glutamine used as a control.

[0049] The relative intensity of the hyposialic acid-added isoform at the SN of the culture was evaluated by densitometry after treatment with different concentrations of glutamine for 7 days.

[0050] Figure 4 shows that in each of the three mutants evaluated, a higher proportion of hyposialic acid-added isoforms was obtained compared to the control. This supports the idea that increasing the glutamine concentration in the culture medium promotes the acquisition of SN hyposialic acid-added isoforms. The highest values ​​for these isoforms were observed at 8 mmol / L glutamine (87%).

[0051] Example 4. Effects of pH and conductivity on the adsorption of acidic and basic isoforms of Q-type quaternary ammonium anion exchangers in laboratory settings. To determine the optimal pH and conductivity conditions for the maximum adsorption of the acidic isoform of the Q-type strong quaternary ammonium anion exchanger, the following buffers were evaluated: 20 mmol / L sodium phosphate (anhydrous dibasic and monobasic dihydrate) and 20 mmol / L Tris 10 mmol / L HCl. The buffers were tested at different pH and conductivity values, with pH values ​​between 6 and 8 and conductivity values ​​between 1.5 and 5 mS / cm.

[0052] Maximum adsorption of the acidic iso-type exchanger was observed under conditions of pH 6 and conductivity 1.50 mS / cm. On the other hand, adsorption of the hyposialic acid-added (basic) iso-type was maximized at pH 8 and conductivity 1.50 mS / cm. The results are shown in Figure 5.

[0053] Example 5. Superior performance of a Q strong anion matrix using integrated column technology compared to conventional chromatographic gel technology in laboratory settings, regarding its ability to separate rhEPO isoforms. The dynamic adsorption capacity (Q) of each technology under study was calculated using two breakthrough curves, employing two of the linear flow rates recommended by the manufacturers of each technology: 100 cm / hour and 600 cm / hour for the chromatography gel technology (Q SFF), and 156 cm / hour and 624 cm / hour for the integrated column technology (CIM QA). The equilibrium solution used for the prior art experiments and the integrated column technology was Tris-HCl buffer with pH 8 and conductivity 1.5 mS / cm, as obtained in Example 4.

[0054] An rhEPO sample was applied to the column, and samples were collected at the column outlet at different times. The protein (C) concentration of each sample was determined by spectrophotometric analysis. Using the known C values ​​for each sample, the fraction of non-adsorbed protein C / C0 was calculated. The loading, which is the mass of protein applied to the column per unit volume of gel given in mg rhEPO / mL Q matrix, was calculated at each time point.

[0055] In Figure 6, the C / C0 versus dynamic adsorption capacity values ​​are graphically represented by a breakthrough curve that allows us to know the Q of the gel per fraction of non-adsorbed protein C / C0 = 0.1 at a specific flow rate. Figure 6A shows the breakthrough curve obtained with conventional techniques for chromatography gels, where the lowest flow rate had the highest dynamic capacity. Figure 6B shows that the integrated column can be operated at higher flow rates without experiencing fluctuations in the matrix's dynamic adsorption capacity, as it exhibits very similar dynamic adsorption capacities at the two linear flow rates tested.

[0056] Table 3 shows the Q values ​​obtained from different measurements. [Table 3]

[0057] Comparing the results of Q studies conducted with packed-bed strong anion exchangers and integrated column technologies, both technologies were observed to have similar Q values ​​at the lowest linear velocities studied. However, at the highest linear velocities, the integrated column exhibited a dynamic adsorption capacity 1.40 times higher than that of the conventional chromatography matrix evaluated. Therefore, it can be concluded that the integrated column enables increased process workflow without affecting the ability to process the rhEPO clumps being purified.

[0058] Example 6. Enhanced elution of hyposialic acid-added rhEPO isotypes in a laboratory-scale integrated column due to pH reduction. Experimental tests were performed using Q SFF and CIM QA columns. The equilibrium solution used for both techniques was Tris-HCl buffer with pH 8 and conductivity 1.5 mS / cm as described in Example 4. The working velocity for the Q SFF column was 600 cm / hour, and the working velocity for the integrated column was 624 cm / hour. The products used in the experiments were obtained from a Sephadex G-25 molecular exclusion chromatography column after changing the buffer in the equilibrium solution described above. For elution of the basic isoform, several runs were performed using a 0.01% integrated anion exchange column with 50 mmol / L Tween 20 sodium acetate buffer at pH values ​​of 4.41, 4.81, 5.06, and 5.20.

[0059] Figure 7 shows the recovery rates obtained from the elution of hyposialic acid-added (basic) isoforms and acidic isoforms in each operation. Analysis of these results confirmed that the performance of the elution buffer for the basic isoforms decreased as the pH increased; therefore, a 50 mmol / L sodium acetate buffer with a pH of 4.41 was selected for elution.

[0060] Example 7. Consistency regarding the separation and purity of isoforms in a process for obtaining hyposialic acid-added rhEPO on a production scale. When fermentation was performed using the seed cell bank described in Example 1, the initial cell viability was higher than 90%. The fermentation stage was carried out at a temperature of 35°C, and the pH of the culture medium was maintained at 7.2-7.3 in a protein-free culture medium supplemented to reach a final glutamine concentration of 8 mmol / L.

[0061] After obtaining four bacterial colonies, a purification step was performed, and in critical steps, an anion exchanger containing a Q quaternary ligand was used with an integrated column. A Tris-HCl solution with pH 8 and conductivity of 1.5 mS / cm was used as the equilibrium buffer, and a 50 mmol / L sodium acetate buffer with pH 4.41 was used for elution.

[0062] The isotype profiles of the four batches of active raw materials obtained after the purification process were determined by isoelectric focusing. A mixture of ampholines with pH ranges of 2–5 and 3–10 was used, with the internal EPOCIM® reference material used as a control. Figure 8 shows the consistency of isotype separation, and an isotype profile consisting of six major isotypes was observed, of which only two were shared with the control.

[0063] Furthermore, the sialic acid content of the purified isoform was determined according to the measurement procedure for this molecule and its purity by reverse-phase HPLC, as described in Europe 8.0 Pharmacopoeia (2014). The obtained sialic acid content and purity results are shown in Table 4. [Table 4]

[0064] The sialic acid content was less than 10 moles per protein molecule, and the purity was over 95% in four batches. From these findings, it can be concluded that the process for obtaining hyposialic acid-added rhEPO guarantees the acquisition of the isoform in the pH range of 4.25–5.85, which differs from that observed with NeuroEPO.

[0065] Example 8. Characteristic minute heterogeneity exhibited in the tertiary structure related to the glycosylation of hyposialic acid-added rhEPO. Glycan analysis To study its N-glycan profile, hyposialic acid-added rhEPO was subjected to denaturation and enzymatic digestion processes using peptide N-glycosidase F (PNGase F). After the N-glycans were released, they were purified by solid-phase extraction using a HyperSep Hypercab SPE cartridge (Mancera-Arteu, M. et al. (2016) Anal. Chim. Acta 940:92-103). Subsequently, derivatization was performed according to the procedure described by Gimenez et al. in 2015 (Gimenez, E et al. (2015) Anal. Chim. Acta, 866:59-68).

[0066] Zwitterion-hydrophilic interaction capillary liquid chromatography coupled with mass spectrometry was performed according to the methodology described by Mancera-Arteu, M. et al. (2016) Anal. Chim. Acta 940:92-103.

[0067] Figure 9 shows the mass spectra of the three glycans detected by hyposialic acid-added rhEPO. As shown above, glycan 3Ant2SiA1Fuc is the most abundant, while glycan 4Ant4SiA1Fuc is found in smaller quantities.

[0068] Table 5 shows the proportion of the relative area of ​​glycans based on their structure. [Table 5]

[0069] Table 6 shows the glycans detected by corresponding relative area, monoisotopic experimental molecular weight (Mexp), and mass error. As described above, there are a considerable number of glycans with few sialic acid addition structures. [Table 6]

[0070] As shown in Table 6, glycans with more sialic acid addition structures (four sialic acid molecules) are found at a low rate. Instead, structures with sialic acid molecules not found in other rhEPOs, such as 3Ant1SiA1Fuc, 4Ant1SiA1Fuc, 4Ant1LacNAc1SiA1Fuc, and 4Ant3LacNAc1SiA1Fuc, are detected. Glycan 4Ant3Sia1Fuc is abundant in hyposialic acid-added rhEPOs. Similarly, the proportion of the relative area due to branching relative to the total glycans detected differs from other rhEPOs, with structures having one or two sialic acid residues accounting for more than 50% of the relative area due to branching.

[0071] Glycopeptide analysis O 126 and N 83 To detect all glucoforms present in glycopeptides, rhEPO and rhEPO-CRS (pharmacopoeia reference product) were subjected to digestion with trypsin and neuraminidase (rhEPO HS-TN). All sialytes of each glucoform detected from trypsin digestion analysis (rhEPO HS-T) were studied. Samples were analyzed by mass spectrometry according to the procedure described in Gimenez, E. et al. (2011) Rapid Commun Mass Spectrom 25:2307~2316.

[0072] Figures 10A and 10B show the O detected in rhEPO HS-TN and HS-T digests. 126 The EIEs of the glycopeptide glycoforms are shown. In the first one, neuraminidase causes complete desialication of the glycopeptide, resulting in all sialic forms becoming a single glycoform, so O 126 A single peak corresponding to the / 0SiA glycoform is observed. In contrast, when digested with trypsin alone, three sialic acid forms with 0, 1, and 2 sialic acid molecules are observed.

[0073] Table 7 shows the O2 detected by corresponding relative area, monoisotopic experimental molecular weight (Mexp), and mass error. 126 It exhibits a sialomorphic form. [Table 7]

[0074] The results indicate that the most abundant sialylated form contains a single sialic acid molecule. The proportion of the relative area of ​​the non-sialylated isoform and the isoform with only one sialic acid molecule is higher compared to those described for other rhEPOs. Differences in the proportion of both sialylated forms are observed compared to CRS rhEPO.

[0075] In the case of N83 glycopeptide, analysis of the rhEPO HS-TN digest revealed six peaks corresponding to six different glycoforms that did not contain sialic acid. The obtained EIE is shown in Figure 11, and the detected glycoforms are shown in Table 8. The results obtained are N 83 This indicates that a higher proportion of complex desialic acid-added tetrabranched structures exist in that region. [Table 8]

[0076] Figure 12A shows no separation between the four different branched structures of the rhEPO HS-TN digest. In contrast, analysis of the sialic acid-added glycoforms of the rhEPO HS-T digest revealed clear separation between them, with those having three sialic acid residues exhibiting greater intensity (Figure 12B). These results are consistent with findings from glycan studies.

[0077] Table 9 shows all detected sialomorphisms and the corresponding errors in relative area, monoisotopic experimental molecular weight (Mexp), and mass. [Table 9]

[0078] The results support the finding that when proteins are digested solely by trypsin, hyposialic acid-added rhEPOs are characterized by fewer sialic acid-added structures (e.g., 2Ant1SiA1Fuc or 3Ant1SiA1Fuc). Examining the relative area ratio of mono- and bisialic acid-added structures, the results indicate that they account for approximately 60%.

[0079] The tetrabranched structure of N83 glycopeptide is found most frequently compared to other glycoforms, and the most common form has two sialic acid residues.

[0080] Example 9. The reversal effect of hyposialic acid-modified rhEPO on DMSO cytotoxicity in astrocytes. PG4 astrocytes were cultured in glucose-rich Dulbecco's modified Eagle medium (DMEM) supplemented with 3.7 g / L NaHCO3 and 10% fetal bovine serum (FBS). Cells were incubated at 37°C for 24 hours in a 5% CO2 / 95% air atmosphere. After the prescribed time, SN was extracted, and cells were subjected to damage induced with 8% DMSO and incubated again for 24 hours. Subsequently, SN was removed, and 2% DMEM culture medium containing different concentrations of hyposialic acid-added rhEPO (1.25, 2.5, 5, and 10 ng / mL) was added. Alamar Blue reagent was added at 24 and 48 hours, followed by incubation of cells for 6 hours and reading at 540 / 630 nm. All incubations were performed at 37°C in a 5% CO2 / 95% air atmosphere.

[0081] Figure 13 shows how cells were able to regain viability at different concentrations of hyposialic acid-modified rhEPO (1.25, 2.5, 5, and 10 ng / mL), with the best condition being 1.25 ng / mL of hyposialic acid-modified rhEPO. This demonstrates that astrocytes possess regenerative capacity.

[0082] Example 10. Hyposialate-added rhEPO exhibits superior neuroprotective activity compared to NeuroEPO. Following the methodology described by Kahn K. in 1972, gerbils from Mongolia were used to develop a model of persistent unilateral ischemia (Kahn K. (1972) Minneap 22:510-515). The animals were then randomly assigned to five experimental groups. Group 1: Vehicle 10 μL Group 2: Treated with 0.142 mg / kg of hyposialic acid-added rhEPO. Group 3: Treated with 0.0142 mg / kg of hyposialic acid-added rhEPO. Group 4: Treated with NeuroEPO at 0.142 mg / kg. Group 5: Treated with NeuroEPO at 0.0142 mg / kg.

[0083] Each treatment was administered intranasally three times a day for four days. The animals were evaluated during the first four days of treatment and the subsequent three-day recovery period.

[0084] The results showed that animals treated with hyposialic acid-added rhEPO at doses of 0.142 and 0.0142 mg / kg had significantly higher survival rates compared to the vehicle. However, NeuroEPO significantly reduced mortality only at a dose of 0.142 mg / kg, but not at 0.0142 mg / kg (Figure 14).

[0085] Neurological evaluation demonstrated the neuroprotective effect of hyposialic acid-added rhEPO (NeuroEPO plus), showing a significant reduction in neurological scale values ​​in animals treated with the used dose of NeuroEPO plus compared to the placebo group and the NeuroEPO-treated group. The same results obtained with hyposialic acid-added rhEPO could only be achieved at a dose of 0.142 mg / kg (Figure 15) (Duncan statistical test, p>0.05 for same letters, p<0.05 for different letters).

[0086] Example 11: Non-increasing effect of hyposialic acid-added rhEPO on reticulocyte count in a normocytic mouse model. Female B6D2F1 mice were randomly assigned to six experimental groups of six animals each, and each group received a single dose of treatment as follows. Group 1: 0.003 mg / mL of rhEPO working reference substance in a 200 μL volume via the subcutaneous route (SC). Group 2: 200 μL volume of 0.006 mg / mL hyposialic acid-added rhEPO via the SC pathway. Group 3: 200 μL volume of 0.5 mg / mL hyposialic acid-added rhEPO via the IN pathway. Group 4: 200 μL volume of rhEPO with 1 mg / mL hyposialic acid added via the IN pathway. Group 5: 200 μL volume of rhEPO with hyposialic acid added via the IN pathway. Group 6: Control, 200 μL of excipient via the SC pathway.

[0087] The results for reticulocyte count are shown in Figure 16. As described above, there was no significant difference between the control group and the group treated with hyposialic acid-added rhEPO using both SC and IN. The reticulocyte count in animals treated with rhEPO working reference was significantly higher than that of the control group and the group treated with hyposialic acid-added rhEPO using both pathways. (Duncan test, p>0.05 between identical letters; p<0.05 between different letters)

[0088] These results demonstrate that hyposialic acid-modified EPO does not increase reticulocyte counts, even at the highest dose established for this study. This indicates that hyposialic acid-modified EPO does not have hematopoietic effects.

Claims

1. A pharmaceutical composition characterized by containing recombinant human erythropoietin (rhEPO) having an isoelectric point profile between 4.25 and 5.85 as an active ingredient, The minute heterogeneity of fucosylated N-glycans is formed by 2,3 and 4-branched structures, wherein these branched structures have mono- and bi-sialic acid-added sialic acid residues in the range of 40-60% of the total glycans, tri-sialic acid-added sialic acid residues in the range of 40-43% of the total glycans, and tetra-sialic acid-added sialic acid residues in the range of 10-13% of the total glycans. Pharmaceutically acceptable excipients, A pharmaceutical composition characterized by the following: However, the rhEPO is obtained from the fermentation process of a CHO cell line transfected with the human EPO gene, which is carried out in a stirred tank at a temperature of 35°C and a pH range of 7.2 to 7.3, and A purification process using a chromatography step employing an integrated column as an anion exchanger containing a quaternary ammonium ligand. It is obtained by [method].

2. The pharmaceutical composition according to claim 1, characterized in that the O-glycosylation site of serine 126 has three sialylated forms having 0 to 2 sialic acid residues, the monosialic acid addition structure is the most abundant, accounting for 78 to 82% of the total glycan, and the non-sialic acid addition (asiallylated) structure accounts for 6 to 10% of the total glycan.

3. The pharmaceutical composition according to claim 1, characterized in that the N-glycosylation site of asparagine 83 includes the following: - A fucosylated bibranched structure having one and two sialic acid residues, wherein this structure accounts for 8-12% of the total glycan; - A fucosylated tribranched structure having 1, 2, and 3 sialic acid residues, wherein this structure accounts for 17-21% of the total glycan; - A fucosylated tetrabranched structure having 1 to 4 sialic acid residues, wherein this structure accounts for 27 to 31% of the total glycan; and - A fucosylated tetrabranched structure having N-acetyllactosamine type 1 and 2 having 1 to 4 sialic acid residues, wherein this structure accounts for 38 to 42% of the total glycans.

4. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutically acceptable excipients include a bioadhesive polymer and a protein stabilizer.

5. Bioadhesive polymers, - Hydroxymethylcellulose, - Hydroxypropylcellulose, - Methylcellulose, and The pharmaceutical composition according to claim 4, characterized by being selected from the group consisting of the following.

6. Protein stabilizers, - L-tryptophan, -L-leucine, -L-arginine hydrochloride, and -L-histidine hydrochloride, The pharmaceutical composition according to claim 4, characterized by being selected from the group consisting of the following.

7. The pharmaceutical composition according to claim 1, characterized in that the fermentation process for obtaining rhEPO is carried out in a protein-free culture medium supplemented with glutamine, giving a final concentration between 8 mmol / L and 12 mmol / L.

8. The pharmaceutical composition according to claim 1, characterized in that, in the purification process, a 20 mmol / L Tris 10 mmol / L HCl solution with a pH of 8 and a conductivity of 1.5 mS / cm is used as the equilibrium buffer, and a 50 mmol / L sodium acetate solution with a pH of 4.41 is used as the elution buffer.

9. A pharmaceutical composition according to any one of claims 1 to 6, for use in the treatment of dementia, stroke, Parkinson's disease, ataxia, craniocerebral injury, glaucoma, autism, neonatal hypoxia, multiple sclerosis, amyotrophic lateral sclerosis, and nerve damage induced by trauma, poisoning, or radiation.

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