Methods for producing glycoprotein compositions

Abstract

The present invention relates to a method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein, the method comprising culturing recombinant host cells expressing the glycoprotein in a culture medium in a bioreactor having a pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium, wherein (a) the culturing is performed under sterile conditions, (b) the pH value measured by the pH measuring device differs from the pH value of the culture medium by 0.05 units or less, and (c) the relative content of at least one glycosylated variant in the glycoprotein composition has reduced batch-to-batch variability compared to a method in which the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units, thereby producing the glycoprotein composition. The present invention further relates to the use of a method for carbon dioxide-based pH calibration to improve batch-to-batch variability.

Description

[Technical Field]

[0001] The present invention relates to a method for recombinantly producing a glycoprotein composition with reduced batch-to-batch variability. The method disclosed herein comprises a pH measurement step in which the measured pH value differs from the pH value of the culture medium by no more than 0.05 units, leading to reduced batch-to-batch variability of one or more glycosylated variants in the glycoprotein composition compared to a method in which the pH value measured by the pH measurement device differs from the pH value in the culture medium by no more than 0.05 units. [Background technology]

[0002] Recombinant glycoproteins are commonly produced in eukaryotic expression systems because eukaryotic cells possess the necessary glycosylation machinery, i.e., the enzymes required to attach sugar moieties to proteins. Variation in the glycan pattern on a protein has a profound effect on protein function. For example, the structure of N-linked glycans on a protein affects various characteristics, including protease susceptibility, intracellular transport, secretion, tissue targeting, biological half-life, and antigenicity of the protein in a cell or organism. Alterations in one or more of these characteristics can significantly affect the efficacy of a glycoprotein in its natural context and, consequently, its effectiveness as a therapeutic agent. The glycosylation state of a glycoprotein is tightly regulated, and even small variations in glycosylation can have profound effects.

[0003] In glycoproteins, sugars are attached either to the amide nitrogen atom of the side chain of asparagine (N-linked) or to the oxygen atom of the side chain of serine or threonine (O-linked). Glycosylation begins with the formation of N-linkages in the endoplasmic reticulum, so-called "core glycosylation." After this, the polypeptide is transported to the Golgi apparatus, where O-linked sugars are added and the N-linked glycans can be modified in many different ways, for example, by removal of mannose residues, addition of N-acetylglucosamine, galactose and / or fucose and / or sialic acid residues.

[0004] Erythropoiesis-stimulating glycoproteins contain several N-glycosylation sites. Erythropoietin (EPO) is a glycoprotein with three N-glycosylation sites and one O-glycosylation site. Erythropoietin has been biosynthetically produced using recombinant DNA technology (Egrie, JC, Strickland, TW, Lane, J et al. (1986) Immunobiol. 72:213-224). It is the product of a cloned human EPO gene inserted into and expressed in Chinese hamster ovary (CHO) cells. The primary structure of the major, fully processed form of human erythropoietin (hEPO) is shown in Figure 1. Two disulfide bridges exist between Cys7-Cys161 and Cys29-Cys33. The molecular weight of the polypeptide chain of erythropoietin, excluding the sugar moiety, is 18,236 Da. In the intact erythropoietin glycoprotein, approximately 40% of the molecular weight is accounted for by carbohydrate groups that glycosylate the protein (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262:12059 (Non-Patent Document 2)).

[0005] Typical N-glycans of erythropoietin include biantennary, triantennary, and tetraantennary structures with one or two N-acetyllactosamine repeats (see, for example, Postnikov et al. 2016 Russ. Chem. Rev. 85-99 (Non-Patent Document 3)). It is known that a high number of terminal sialic acid moieties on the N-glycan of erythropoietin increases the specific activity of erythropoietin compared to less sialylated erythropoietin glycoproteins (see, for example, Imai et al., Eur. J. Biochem. 194 (1990), pp. 457-462 (Non-Patent Document 4)). Desialylation of erythropoietin reduces the half-life of erythropoietin in circulation (Ridley et al. J Natl Med Assoc. 1994 Feb;86(2):129-35 (Non-Patent Document 5)). It has been reported that an increase in the number of poly-N-acetyllactosamine repeats or an increase in the number of N-glycan branches of erythropoietin are both associated with higher specific bioactivity of erythropoietin (see, for example, International Publication No. 99 / 28346). Glycosylated variants of erythropoietin are important influencers not only on the biological activity, in vivo bioactivity, and pharmacokinetics of erythropoietin, but also alter the solubility and lifetime of the glycoprotein in blood.

[0006] In ICH guidelines, a critical quality attribute (CQA) is defined as a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, Pharmaceutical Development, Q8(R2)). Due to its described potential impact on biological activity, N-glycosylation of recombinantly produced erythropoietin is considered a critical quality attribute (CQA) and is carefully monitored during production to ensure consistent product quality. The relative content of glycosylated variants is highly dependent on several factors, including cell culture conditions. Due to the significant impact of glycosylated variants on bioavailability, the ability to consistently produce a narrow range of glycosylation variants in a pharmaceutical erythropoietin product also leads to lower batch-to-batch variability with respect to erythropoietin specific activity.

[0007] Yoon et al. (Biotechnol Bioeng, 89(3):345-356, 2005) (Non-Patent Document 6) discloses that optimization of culture temperature and pH in serum-free suspension culture of CHO cells can result in increased maximum erythropoietin concentration and volumetric productivity.

[0008] WO 2017 / 072340 (Patent Document 2) discloses a system for monitoring deviations of the state of a cell culture in a bioreactor from a reference state of the cell culture in a reference bioreactor, the bioreactor containing the same medium as the reference bioreactor.

[0009] WO 2017 / 072346 (Patent Document 3) discloses a system and method for recalibrating a pH meter using CO2 concentrations measured in two or more different tanks to determine whether the pH measurement device is affected by a pH measurement problem and to identify pH measurement deviations in pH measurement devices operably connected to the different tanks.

[0010] Therefore, there is ongoing interest in methods for producing erythropoietin reliably and with high quality. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 99 / 28346 [Patent Document 2] International Publication No. 2017 / 072340 [Patent Document 3] International Publication No. 2017 / 072346 [Non-patent literature]

[0012] [Non-Patent Document 1] Egrie, JC, Strickland, TW, Lane, J et al. (1986) Immunobiol.72:213-224 [Non-patent document 2] Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262:12059 [Non-patent document 3] Postnikov et al.2016 Russ.Chem.Rev.85-99 [Non-patent document 4] Imai et al.,Eur.J.Biochem.194(1990),p.457-462 [Non-Patent Document 5] Ridley et al.J Natl Med Assoc.1994 Feb;86(2):129-35 [Non-patent document 6] Yoon at al.Biotechnol Bioeng,89(3):345-356,2005 Summary of the Invention

[0013] The present invention is based on the discovery that tight control of the pH of the culture medium in the production bioreactor during fermentation significantly improves the variability of glycoprotein products, particularly the variability of the N-glycosylation profile of erythropoietin, particularly with respect to the presence of sialylated isoforms of erythropoietin and tetraantennary +1 repeat structures.

[0014] The inventors have found that by adjusting the bioreactor pH probe signal to a pH reference that accurately reflects the pH in the culture medium, without the influence of any offset that may be introduced by sampling and offline measurements, and optionally also by subsequently controlling the pH in the culture medium using such a pH probe, it is possible to significantly reduce variability with respect to N-glycosylation and sialylation during the production of glycoprotein compositions in recombinant mammalian host cells.

[0015] 1. A method for calibrating an in-line pH measurement device after sterilizing a bioreactor and filling it with culture medium, comprising: a) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor; b) calculating the pH value of the culture medium in the bioreactor using a medium-specific correlation; c) adjusting the pH measuring device to said pH value By using a method including A method, in particular a method that relies on offline measurement of pH to (re)calibrate an in-line pH measuring device, in which the pH value measured by the pH measuring device in the bioreactor differs from the pH of the culture medium by more than 0.05 units, preferably by more than 0.03 units. It has been found that the standard deviation of the relative content of one or more glycosylation variants in a glycoprotein composition produced in such a bioreactor is reduced compared to the standard deviation of the relative content of the same glycosylation variant in a glycoprotein composition produced using a fermentation method, whereby the standard deviation of the relative content of one or more glycosylation variants is calculated for the glycoprotein composition from at least two fermentation batches.

[0016] Thus, one aspect of the present invention is a method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein, comprising the steps of: Culturing recombinant host cells that express a glycoprotein in a culture medium in a bioreactor having a pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium, (a) The culture is carried out under sterile conditions; (b) the pH value measured by the pH measuring device differs from the pH value of the culture medium by less than 0.05 units; and (c) the relative content of at least one glycosylated variant in the glycoprotein composition is wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. has reduced batch-to-batch variability compared to a process characterized by: Thereby producing a glycoprotein composition.

[0017] In certain embodiments of all aspects and embodiments, wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. is a method comprising removing a sample of culture medium from a bioreactor, particularly under sterile conditions, measuring the pH value of the sample using a pH measuring device placed outside the bioreactor ("offline pH measurement"), and adjusting the pH measuring device within the bioreactor to the measured pH value.

[0018] Another aspect is a method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein in a recombinant host cell that expresses the glycoprotein, comprising: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells; and (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing the glycoprotein. and the calibrating step of step (d) comprises: (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust gas of the bioreactor; (iii) calculating the pH of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH calculated in step (iii); The method includes:

[0019] In particular embodiments of the aspect, the pH measured using the pH measuring device after calibration step (d) differs from the pH value of the culture medium by no more than 0.05 units, preferably no more than 0.03 units.

[0020] In certain embodiments of all aspects and embodiments, the glycoprotein composition comprises at least one glycosylated variant of the glycoprotein, and the standard deviation of the relative content of the glycosylated variant calculated for the glycoprotein composition from at least two fermentation batches is: wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using the method is reduced.

[0021] In certain embodiments, the difference between the pH value measured by the pH measuring device in the bioreactor and the pH in the culture medium is the only parameter that is altered in the production of a fermentation batch when comparing the standard deviation of a glycosylated variant of a glycoprotein composition produced according to the present invention with the standard deviation of another glycoprotein composition.

[0022] In certain embodiments of all aspects and embodiments, the standard deviation is calculated for glycoprotein compositions from at least three, preferably at least five, and more preferably at least ten fermentation batches.

[0023] In particular embodiments of all aspects and embodiments, the recombinant host cell is a mammalian cell, in particular a CHO cell.

[0024] In certain embodiments of all aspects and embodiments, the culture medium comprises a carbonate buffer.

[0025] In certain embodiments of all aspects and embodiments, the medium-specific correlation is determined by collecting at least one medium-specific dataset for a culture medium, the dataset comprising: (a) charging a culture medium into a bioreactor, the bioreactor having at least one pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium; (b) introducing a gas mixture comprising carbon dioxide into the bioreactor; (c) measuring the pH in the culture medium using a pH measuring device, wherein the pH measuring device is calibrated (under non-sterile conditions) to measure the pH in the culture medium; (d) Any of the following: (i) varying the pH value in the culture medium and measuring the carbon dioxide concentration in the headspace and / or exhaust air of the bioreactor for at least two different pH values; or (ii) varying the carbon dioxide concentration in the gas mixture and measuring the pH value for at least two different carbon dioxide concentrations in the gas mixture; and (e) Obtaining medium-specific correlations by mathematically fitting at least two pairs of headspace / exhaust carbon dioxide concentrations and corresponding pH values ​​of the culture medium. is collected using

[0026] In certain embodiments of all aspects and embodiments, the recombinant host cells have been cultured in a pre-culture medium in at least one pre-culture step prior to inoculation, and the bioreactor used for the pre-culture has at least one pH measurement device disposed within the bioreactor and configured to be in physical contact with the pre-culture medium, and the pH measurement device in the bioreactor used for the pre-culture is (i) introducing a gas mixture containing carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust gas; (iii) calculating the pH value of the pre-culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). It is calibrated according to

[0027] In certain embodiments of all aspects and embodiments, the glycoprotein composition is an erythropoiesis composition. In certain embodiments of all aspects and embodiments, the glycoprotein is an erythropoiesis-stimulating glycoprotein. In certain embodiments of all aspects and embodiments, the glycoprotein is erythropoietin.

[0028] One aspect of the present invention is a method for reducing the variability in the relative content of at least one glycosylated variant between batches of a recombinant glycoprotein composition, comprising the steps of: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells expressing the glycoprotein; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein, Here, the calibrating step of step (d) is (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust gas; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH value calculated in step iii); Including, Steps (a)-(g) result in a first batch of a recombinant glycoprotein composition having a defined relative content of glycosylated variants; process, (h) repeating steps (a)-(g) to produce at least one subsequent batch of glycoprotein composition, wherein the relative content of the at least one glycosylated variant of the first batch and the at least one subsequent batch has reduced batch-to-batch variability.

[0029] In particular embodiments of all aspects and embodiments, the standard deviation of the calculated relative glycoprotein content for said first and at least one subsequent batch is 1% or less, particularly 0.8% or less, and most particularly 0.5% or less of the median pH value.

[0030] One aspect of the present invention is A method for measuring pH, wherein the measured pH value differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. and a method for producing a glycoprotein composition using a carbon dioxide-based method for calibrating a pH measuring device configured to be disposed within a bioreactor and to be in physical contact with the culture medium in the bioreactor, to reduce the standard deviation of the relative content of one or more glycosylated variants in a glycoprotein composition calculated between at least two batches compared to a glycoprotein composition produced using a carbon dioxide-based method for calibrating a pH measuring device configured to be disposed within a bioreactor and to be in physical contact with the culture medium in the bioreactor.

[0031] A further aspect of the present invention is an erythropoiesis-stimulating composition comprising at least one erythropoiesis-stimulating glycoprotein, wherein: (a) between about 3.3 area% and about 3.8 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have biantennary structures; (b) between about 8.6 area% and about 9.5 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have triantennary structures; (c) between about 5.6 area% and about 5.9 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have triantennary + 1 repeat structures; (d) between about 42.2 area% and about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have tetraantennary structures; (e) between about 27.4 area% and about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have tetraantennary + 1 repeat structures; and (f) between about 10.7 area% and about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein. (g) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; (h) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; (i) about 23.5 area% to about 24.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 4; (j) about 17.1 area% to about 18.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 5; (k) about 9.4 area% to about 11.8 area% of the erythropoiesis-stimulating glycoprotein is isoform 6; (l) about 3.7 area% to about 5.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 7; and / or (m) about 0.9 area% to about 1.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 8. In certain embodiments of all aspects and embodiments, the area percent of the N-glycans of the erythropoiesis-stimulating glycoprotein is determined by anion exchange chromatography after enzymatic release and (enzymatic) desialylation of N-glycoside-linked oligosaccharides from the glycoprotein. In certain embodiments, the anion exchange chromatography is high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). In certain embodiments of all aspects and embodiments, the area percent of the sialylated isoforms of the erythropoiesis-stimulating glycoprotein in the erythropoiesis composition is determined by capillary zone electrophoresis.

[0032] The present invention provides a method for producing glycoproteins, in particular for erythropoiesis-stimulating glycoproteins, such as erythropoietin, with reduced batch-to-batch variability with regard to glycosylation, in particular with regard to N-glycans having tetraantennary, tetraantennary +1 repeat, tetraantennary +2 repeat and tetraantennary +3 repeat structures, as well as with regard to sialylated isoforms, in particular isoforms 2 and 3, which may ensure a specific product quality and / or improve the biological function, such as the specific biological activity, of the (erythropoiesis-stimulating) glycoprotein. Amino acid sequence description TIFF2025536587000001.tif52170 [Brief explanation of the drawings]

[0033] [Figure 1] Structure of erythropoietin and its glycosylation sites (revised from Postnikov et al 2016 Russ.Chem.Rev.85-99). [Figure 2] Typical glycosylation patterns of erythropoietin. (Figure 2A) N-glycan with two branches. (Figure 2B) N-glycan with three branches. (Figure 2C) N-glycan with four branches. (Figure 2D) N-glycan with four branches and one poly-N-acetyllactosamine repeat. (Figure 2E) N-glycan with four branches and three poly-N-acetyllactosamine repeats. [Figure 3] Correlation between bioreactor pH and the corresponding exhaust carbon dioxide concentration ("ACO") produced independently at three different pressures: 20 mbar (top), 50 mbar (middle), and 135 mbar (bottom). Lines represent quadratic fitting. [Figure 4] Improved range and standard deviation of glycosylated variants of isoform 2 erythropoietin. [Figure 5] Improved range and standard deviation of glycosylated variants of erythropoietin with tetraantennary +1 repeat N-glycan structures. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description 1.Definition In order that the present invention may be more readily understood, certain terms are first defined. Before the present invention is further described, it is to be understood that the present invention is not limited to the particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings generally given to them in the art.However, the meaning and scope of the terms should be clear, and in the event of potential ambiguity, the definitions provided herein shall prevail over dictionary or external definitions.Although any methods and materials similar or equivalent to those described herein can also be used to implement or test the present invention, preferred methods and materials are described herein.The publications mentioned herein are incorporated by reference to disclose and describe the relevant methods and / or materials to which the publications are cited.

[0036] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0037] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Accordingly, this reference is intended to serve as a predicate for use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of a "negative" limitation.

[0038] The term "about" means within a range of + / - 20% of the numerical value that follows. In certain embodiments, the term "about" means within a range of + / - 10% of the numerical value that follows. In certain embodiments, the term "about" means within a range of + / - 5% of the numerical value that follows.

[0039] The term "comprising" also encompasses the term "consisting of."

[0040] It is understood that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed. Furthermore, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0041] As used herein, the term "accuracy" refers to the closeness of a reported or estimated value from the true value. An inaccurate measurement, observation, or estimate will differ from the true value. An accurate measurement, observation, or estimate will not differ from the true value.

[0042] The terms "aerate," "aeration," or "gassing" are used interchangeably herein and refer to the dispersion of a gaseous material into a flowable fluid, such as a culture medium, to provide a diffusion surface for introducing molecules or compounds from the gas to the flowable surface. The term is not limited to the dispersion of air itself, but rather refers to the introduction of any gas, such as oxygen, carbon dioxide, nitrogen, and mixtures thereof, into a culture medium. In certain aspects and embodiments, the cell culture is aerated with a defined gas mixture, including, for example, process air and carbon dioxide. For normal cell growth, a specific concentration of dissolved oxygen must be maintained. Additionally, controlled introduction of carbon dioxide is used to maintain pH at a desired level. In certain embodiments, the gas mixture introduced into the bioreactor during calibration of a pH measurement device and / or during cultivation comprises 93% process air and 7% CO2. Aeration can be achieved by headspace aeration, but for large bioreactors, the gas exchange achieved by headspace aeration may be too slow. The most efficient method of introducing gas into a bioreactor fluid is submerged gassing or sparging (these two terms are used interchangeably herein), which involves forming small gas bubbles in the fluid. Aeration of the culture medium by sparging is preferred because it allows molecules and / or compounds to diffuse into the culture medium through the fluid-gas bubble interface after expelling the gas bubbles into the culture medium. In one embodiment, aeration is achieved by sparging the culture medium with a gas at a defined carbon dioxide concentration. In a specific embodiment, the culture medium is sparged with a gas mixture containing 93% process air and 7% CO2. In some embodiments, sparging is provided by a porous solid material (such as titanium) or a metal sparging ring with small pre-drilled holes associated with the bioreactor.

[0043] As used herein, the term "area percent" or "area %" refers to the percentage of the integrated area under the peak (hereinafter "AUP") of a particular species (e.g., a specific glycosylated variant of a glycoprotein) measured by a detector relative to the total integrated peak area of ​​the entire chromatogram, e.g., a chromatogram generated by high-performance anion exchange chromatography or capillary zone electrophoresis using pulsed amperometric detection. The AUP can be determined by using an appropriate integrator. Each peak in the chromatogram corresponds to a different component in the mixture loaded onto the chromatography column, and the ratio of the AUP of each detectable component to the total AUP of all sample components yields the area percentage. Area percent can be expressed mathematically as follows: area i -%=100×(AUP i ) / (Σ of all AUPs)

[0044] As used herein, the term "bioreactor" refers to any biocompatible tank or vessel, such as a large fermentation chamber, used to grow mammalian cell cultures. Typically, a bioreactor is at least 0.25 L and may be 1, 10, 100, 250, 500, 1000, 2500, 5000, 8000, 10,000, 12,000 L or more, or any volume in between. In one embodiment of all aspects and embodiments herein, the bioreactor is a large-scale bioreactor, i.e., the bioreactor is at least 10 L, preferably at least 50 L, more preferably at least 500 L, and particularly preferably at least 5000 L, and / or is used for commercial recombinant glycoprotein production. Typically, the internal conditions of the bioreactor, including but not limited to pH, dissolved oxygen, agitation, temperature, and / or pH, can be monitored, adjusted, and controlled during cultivation. When the bioreactor is filled with culture medium, the internal volume of the bioreactor is typically not completely filled but is divided into a working volume and a headspace volume. The working volume is the portion of the total volume occupied by the culture medium, and the remaining volume above the culture medium is referred to herein as the "headspace" or "headspace volume." Typically, the working volume is approximately 70% to 80% of the total volume of the bioreactor. The bioreactor contains at least one inlet and one outlet, allowing the introduction and removal of gas streams, respectively. These may be equipped with an air inlet filter to provide a sterile gas mixture for aseptic conditions within the bioreactor. The gas stream exiting the bioreactor through the outlet is also referred to herein as "exhaust." The outlet may be equipped with a sensor that allows the determination of the carbon dioxide concentration in the exhaust. Such a sensor may be, for example, an infrared-based off-gas analyzer, mass spectrometry, a Raman sensor, or an optical device. The bioreactor is also typically in fluid communication with other containers, such as a culture medium tank or a harvest tank, to allow the introduction or removal of liquid medium into or from the bioreactor.It will be apparent to those skilled in the art that one or more additional access ports may be provided for greater access to the interior of the bioreactor, including access under aseptic / sterile conditions, for example, to remove samples that may be used for offline measurements. The bioreactor may further include additional equipment, such as impellers, baffles, spargers, and / or ports, specifically to allow for the culture and growth of mammalian cells. The bioreactor may be constructed of any material suitable for holding a mammalian cell culture suspended in a medium under the culture conditions of the present invention, including glass, plastic, or metal, or a combination thereof. The bioreactor may be multiple-use or single-use, reusable, disposable, or recyclable.

[0045] As used herein, the term "buffer" refers to a substance whose presence in a solution increases the amount of acid or alkali that must be added to produce a unit change in pH. A buffer solution resists changes in pH due to the action of its acid-base conjugate components. Buffer solutions for use with biological reagents generally maintain a constant concentration of hydrogen ions so that the pH of the solution is within a desired range, often close to physiological pH. Traditional buffer components include, but are not limited to, organic and inorganic salts, acids, and bases. Well-known buffer substances include, for example, phosphate buffer solutions consisting of phosphoric acid and / or its salts, acetate buffer solutions consisting of acetic acid and its salts, carbonate and / or bicarbonate buffer solutions, citrate buffer solutions consisting of citric acid and / or its salts, morpholine buffer solutions, 2-(N-morpholino)ethanesulfone buffer solutions, histidine buffer solutions, glycine buffer solutions, and tris(hydroxymethyl)aminomethane (TRIS) buffer solutions.

[0046] The terms "carbon dioxide concentration" and "percent carbon dioxide (gas)" are used interchangeably herein and refer to the relative amount of carbon dioxide gas present in a gas mixture. Carbon dioxide concentration is expressed herein as [%].

[0047] The term "cell culture" or "cell culture" refers to a cell population suspended in a culture medium under conditions suitable for the survival and / or growth of the cell population. The term also applies to the combination of the culture medium and the cell population suspended therein. The term includes cell culture processes, also referred to as "fermentation" or "fermentation processes," at all scales (e.g., from Ambr® systems to large-scale industrial bioreactors, i.e., milliliter scale to >10,000 L scale), all different process modes (e.g., batch, fed-batch, perfusion, continuous culture), all process control modes (e.g., uncontrolled, fully automated, and controlled systems controlling pH, temperature, and oxygen content), and all types of cell culture systems (e.g., single-use systems, stainless steel systems, glassware systems). In general, the following parameters are often determined daily and include viable cell concentration, product concentration, and several metabolic products such as glucose or lactate, pH, osmolality, osmolality, and ammonium. In a preferred embodiment of the present invention, the cell culture is a mammalian cell culture and is a batch or fed-batch culture. Generally, cell culture is performed in adherent or suspension culture under sterile, controlled temperature and atmospheric conditions. Cultures can be grown in shake flasks, small-scale bioreactors, and / or large-scale bioreactors. In certain embodiments of all aspects and embodiments herein, the methods of the invention can be used for large-scale mammalian cell culture, i.e., bioreactors of at least 10 L, preferably at least 50 L, more preferably at least 500 L, and particularly preferably at least 5000 L, and / or for commercial recombinant glycoprotein production. In one embodiment, the cell culture methods and compositions of the invention are suitable for large-scale CHO cell culture and glycoprotein production. In one embodiment of all aspects and embodiments herein, the recombinant glycoprotein production is for commercial purposes and / or is performed using large-scale bioreactors.

[0048] Once the glycoproteins described herein are produced by recombinant expression, they can be purified by any method known in the art, such as chromatography (e.g., ion exchange, affinity, and size-exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. For example, antibodies can be isolated and purified by appropriately selecting and combining affinity columns, such as Protein A columns, along with chromatography columns, filtration, ultrafiltration, salting-out, and dialysis procedures (see, e.g., *Antibodies: A Laboratory Manual*, Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988). Furthermore, as described herein, glycoproteins can be fused to heterologous polypeptide sequences to facilitate purification. Polypeptides bearing the desired glycosylation can be separated on lectin columns by methods known in the art.

[0049] As used herein, the term "cultivation medium" or "cultivation media" refers to a liquid solution used to provide nutrients (e.g., vitamins, amino acids, essential nutrients, salts, etc.) and properties (e.g., affinity, buffering) to maintain and support the growth of living cells, particularly mammalian cells. Preferably, mammalian cells are cultured at a neutral pH, e.g., about pH 6.5 to about pH 7.5, preferably about pH 6.6 to about pH 7.3, and more preferably about pH 7. Therefore, a buffering agent should be added to the culture medium. Commercially available culture media are known to those skilled in the art. In one embodiment of all aspects and embodiments, the culture medium is buffered using a carbonate buffer system. The culture medium used during the pre-culture step is referred to herein as the "pre-culture medium." It may have the same or different composition as the culture medium used for the main-stage culture.

[0050] As used herein, the term "glycoprotein" refers to a protein or polypeptide containing one or more covalently linked oligosaccharide chains. The oligosaccharide chains may consist of a single sugar residue, an unbranched chain of a single sugar residue, or a chain of sugar residues branched one or more times. In certain embodiments, the oligosaccharide chains are N-linked. In certain embodiments, the oligosaccharide chains are O-linked. As used herein, the term "glycoprotein composition" refers to a composition comprising at least one glycoprotein.

[0051] Glycoproteins include, for example, any of a variety of hematological agents (including, for example, erythropoietin, blood clotting factors, etc.), interferons, colony-stimulating factors, antibodies, enzymes, and hormones. The identity of the particular glycoprotein is not intended to limit the disclosure, and the therapeutic preparations described herein can include any glycoprotein of interest, e.g., a glycoprotein having an Fc region.

[0052] The glycoproteins disclosed herein can include a target-binding domain that binds to a target of interest (e.g., binds to an antigen). For example, a glycoprotein such as an antibody can bind to a transmembrane polypeptide (e.g., a receptor) or a ligand (e.g., a growth factor). Exemplary molecular targets (e.g., antigens) of the glycoproteins (e.g., antibodies) described herein include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52, and the like; members of the ErbB receptor family, such as the EGF receptor (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3), or HER4 (ErbB4) receptor; macrophage receptors, such as CRIg; tumor necrosis factors, such as TNFα or TRAIL / Apo-2; and integrins, such as LFA-1, MacI, p150,95, VLA-4, ICAM-1, VCAM, and αvβ3 integrin (including either its a or p subunits). These include cell adhesion molecules (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factors and receptors, such as EGF, FGFR (e.g., FGFR3), and VEGF; IgE; cytokines such as IL1; cytokine receptors such as IL2 receptor; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C; neutropilin; ephrins and receptors; netrins and receptors; slit and receptors; chemokines and chemokine receptors, such as CCL5, CCR4, and CCR5; amyloid beta; complement factors, such as complement factor D; lipoproteins, such as oxidized LDL (oxLDL); lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular targets include Tweak, B7RP-1, proprotein convertase subtilisin / kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

[0053] Exemplary glycoprotein products include abatacept (ORENCIA®, Bristol-Myers Squibb), abciximab (REOPRO®, Roche), adalimumab (HUMIRA®, Bristol-Myers Squibb), aflibercept (EYLEA®, Regeneron Pharmaceuticals), alefacept (AMEVIVE®, Astellas Pharma), alemtuzumab (CAMPATH®, Genzyme / Bayer), atezolizumab (TECENTRIQ®, Genentech), basiliximab (SIMULECT®, Novartis), belatacept (NULOJIX®, Bristol-Myers Squibb), belimumab (BENLYSTA®, GlaxoSmithKline), and others. SmithKline), bevacizumab (AVASTIN®, Roche), canakinumab (ILARIS®, Novartis), brentuximab vedotin (ADCETRIS®, Seattle Genetics), certolizumab (CIMZIA®, UCB, Brussels, Belgium), cetuximab (ERBITUX®, Merck-Serono), daclizumab (ZENAPAX®, Hoffmann-La Roche), denileukin-diftitox (ONTAK®, Eisai), denosumab (PROLIA®, Amgen;XGEVA®, Amgen), eculizumab (SOLIRIS®, Alexion Pharmaceuticals), efalizumab (RAPTIVA®, Genentech), etanercept (ENBREL®, Amgen-Pfizer), faricimab (VABYSMO®, Roche), gemtuzumab (MYLOTARG®, Pfizer), golimumab (SIMPONI®, Janssen), ibritumomab (ZEVALIN®, Spectrum Pharmaceuticals), infliximab (REMICADE®, Centocor), ipilimumab (YERVOY™, Bristol-Myers Squibb), muromomab (ORTHOCLONE OKT3I®, Janssen-Cilag), natalizumab (TYSABRI®, Biogen) Idee, Elan), oclizumab (OCREVUS®, Genentech), ofatumumab (ARZERRA®, GlaxoSmithKline), omalizumab (XOLAIR®, Novartis), palivizumab (SYNAGIS®, Medimmune), panitumumab (VECTIBIX®, Amgen), ranibizumab (LUCENTIS®, Genentech), rilonacept (ARCALYST®, Regeneron Pharmaceuticals), rituximab (MABTHERA®, Roche), tocilizumab (ACTEMRA®, Genentech; RoActemra, Hoffman-La Roche), tositumomab (BEXXAR®, GlaxoSmithKline), and trastuzumab (HERCEPTIN®, Roche). ;

[0054] As used herein, the term "erythropoietic composition" refers to a composition comprising at least one erythropoiesis-stimulating glycoprotein that contains a glycosylation site, in which at least a portion of the molecule bears a carbohydrate chain and, optionally, contains at least one terminal sialic residue (i.e., such a molecule is "sialylated"). Similarly, as used herein, the term "glycoprotein composition" refers to a composition comprising at least one glycoprotein molecule, in which at least a portion of the molecule is optionally sialylated.

[0055] As used herein, "erythropoiesis-stimulating glycoprotein" refers to a protein that directly or indirectly activates the erythropoietin receptor, for example, by binding to the receptor and causing dimerization. Erythropoiesis-stimulating glycoproteins include erythropoietin and its variants, analogs, or derivatives that bind to and activate the erythropoietin receptor; antibodies that bind to and activate the erythropoietin receptor; or peptides that bind to and activate the erythropoietin receptor. The erythropoietin variants, analogs, or derivatives referred to herein contain at least three N-glycosylation sites. In one embodiment, the N-glycosylation sites are Asn24, Asn38, and Asn83. Erythropoiesis-stimulating glycoproteins include, but are not limited to, epoetin alpha, epoetin beta, epoetin delta, epoetin omega, epoetin iota, epoetin zeta and analogs thereof, pegylated erythropoietin, carbamylated erythropoietin, mimetic peptides (including EMP1 / hematide), and mimetic antibodies. Exemplary erythropoiesis-stimulating glycoproteins include erythropoietin, darbepoietin, erythropoietin-agonistic variants, and peptides or antibodies that bind to and activate the erythropoietin receptor (including compounds reported in U.S. Patent Application Publication Nos. 2003 / 0215444 20 and 2006 / 0040858, the disclosures of each of which are incorporated herein by reference in their entirety), as well as the erythropoietin molecules or variants or analogs thereof disclosed in the following patents or patent applications, each of which is incorporated herein by reference in its entirety: U.S. Patent Nos. 4,703,008; 5,441,868; 5,547,933; 5,618,698; 5,621,080; 5,756,349; 5,767,078; 5,773,569; 5,955,422; 5,830,851; 5,856,298; 5,986,047; 6,030,086; 6,310,078; 6,391,633; 6,583,272; 6,586,398; 6,900,292; 6,750,369; 7,030,226; 7,084,245; 7,217,689; PCT Publication No. WO91 / 05867;WO95 / 05465;WO99 / 66054;WO00 / 24893;WO01 / 81405;WO00 / 61637;WO01 / 36489;WO02 / 014356;WO 02 / 19963;WO02 / 20034;WO02 / 49673;WO02 / 085940;WO03 / 029291;WO2003 / 055526;WO2003 / 0844 77;WO2003 / 094858;WO2004 / 002417;WO2004 / 002424;WO2004 / 009627;WO2004 / 024761;WO2004 / 0336 51;WO2004 / 035603;WO2004 / 043382;WO2004 / 101600;WO2004 / 101606;WO2004 / 101611;WO2004 / 1063 73;WO2004 / 018667;WO2005 / 001025;WO2005 / 001136;WO2005 / 021579;WO2005 / 025606;WO2005 / 032 460;WO2005 / 051327;WO2005 / 063808;WO2005 / 063809;WO2005 / 070451;WO2005 / 081687;WO2005 / 084 711; WO2005 / 103076; WO2005 / 100403; WO2005 / 092369; WO2006 / 50959; WO2006 / 02646; WO2006 / 29094; and U.S. Patent Application Publication Nos. US2002 / 0155998; US2003 / 0077753; US2003 / 0082749; US2003 / 0143202; US2004 / 000 9902;US2004 / 0071694;US2004 / 0091961;US2004 / 0143857;US2004 / 0157293;US2004 / 0175379;US2 004 / 0175824;US2004 / 0229318;US2004 / 0248815;US2004 / 0266690;US2005 / 0019914;US2005 / 00268 34;US2005 / 0096461;US2005 / 0107297;US2005 / 0107591;US2005 / 0124045;US2005 / 0124564;US2005 / 0137329;US2005 / 0142642;US2005 / 0143292;US2005 / 0153879;US2005 / 0158822;US2005 / 0158832;US2005 / 0170457;US2005 / 0181359;US2005 / 0181482;US2005 / 0192211;US2005 / 0202538;US2005 / 0227289;US2005 / 0244409;US2006 / 0088906;US2006 / 0111279。;

[0056] "Erythropoietin," "erythropoietin polypeptide," or "EPO" refers to a glycoprotein that belongs to the group of "erythropoiesis-stimulating glycoproteins" and directly or indirectly activates the erythropoietin receptor, for example, by binding to the receptor and causing dimerization. It refers to a glycoprotein having the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the term includes an amino acid sequence substantially homologous to the sequence of SEQ ID NO: 1, whose biological properties relate to stimulating red blood cell production and the division and differentiation of committed erythroid progenitor cells in the bone marrow. As used herein, these terms include such proteins that have been intentionally modified, for example, by site-directed mutagenesis, or accidentally by mutation. As used herein, "erythropoietin" includes erythropoietin and its variants, analogs, or derivatives that bind to and activate the erythropoietin receptor. As used herein, an erythropoietin variant, analog, or derivative contains at least three N-glycosylation sites, and in one embodiment, the N-glycosylation sites are Asn24, Asn38, and Asn83. Erythropoietins include, but are not limited to, epoetin alfa, epoetin beta, epoetin delta, epoetin omega, epoetin iota, epoetin zeta, and analogs thereof, pegylated erythropoietin, and carbamylated erythropoietin. In one embodiment, the term erythropoietin or EPO analog includes analogs with one to six additional sites for glycosylation, analogs with at least one additional amino acid at the carboxy terminus of the glycoprotein, where the additional amino acid comprises at least one glycosylation site, and analogs having an amino acid sequence that includes a rearrangement of at least one site for glycosylation. As used herein, "relocation" of a glycosylation site refers to the deletion of one or more glycosylation sites in naturally occurring EPO and the addition of one or more non-naturally occurring glycosylation sites. These terms include both natural and recombinantly produced human erythropoietin.

[0057] As used herein, the term "batch," "culture batch," or "fermentation batch" refers to a glycoprotein composition obtained by a single run of a process for recombinantly producing a glycoprotein composition in a single bioreactor, particularly a glycoprotein composition obtained by a single run of a process comprising the steps of: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells expressing the glycoprotein; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein.

[0058] The resulting glycoprotein composition can then be divided into smaller portions for further processing, which are generally referred to as "of / from the same batch" and are of comparable quality with respect to physical and chemical parameters, e.g., glycosylation profile, unless they are subjected to different subsequent processing steps.

[0059] As used herein, the terms "glycosylation" and "glycosylated" refer to the presence of carbohydrates (e.g., oligosaccharides or polysaccharides, also called "glycans") attached to a biological molecule (e.g., a protein or lipid). In certain embodiments, glycosylation refers to the presence of glycans (e.g., N-glycans) attached to a protein (e.g., an erythropoiesis-stimulating glycoprotein, particularly erythropoietin) or a portion of a protein of interest. As used herein, the term "glycan" refers to a polysaccharide, oligosaccharide, or monosaccharide. A glycan can be a monomer or polymer of sugar residues and can be linear or branched. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. O-linked glycosylation refers to the attachment of a sugar, N-acetylgalactosamine, galactose, or xylose, to one hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be involved in O-linked glycosylation. For a review of glycosylation, see, e.g., Varki et al., Essentials of Glycobiology, 3 rdSee, for example, "Aglycosylated" and "Non-glycosylated," which are used interchangeably herein, refer to a protein or portion of a protein of interest that is not glycosylated (e.g., not N-glycosylated). Glycans can contain natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6' sulfo-N-acetylglucosamine, etc.). Glycans are also used herein to refer to the carbohydrate moiety of glycoconjugates such as glycoproteins, glycolipids, glycopeptides, glycoproteomes, peptidoglycans, lipopolysaccharides, or proteoglycans. Glycans typically consist solely of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically, a glucan) composed of B-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be linear or branched. Glycans can be found attached to proteins, such as glycoproteins and proteoglycans. They are generally found on the exterior of cells. O- and N-glycans are very common in eukaryotes, but can also be found less commonly in prokaryotes. As used herein, "galactosylated glycan" refers to a glycan containing at least one galactose sugar residue. In some embodiments, the galactosylated glycan is a G1, G2, G1F, G2F, A1, and / or A2 glycan. In some embodiments, the galactosylated glycan contains one or more poly-N-acetyllactosamine repeats. In some embodiments, the galactosylated glycan is a galactose-alpha-1-3-galactose-containing glycan. In some embodiments, the galactosylated glycan is a triantennary glycan or a tetraantennary glycan.Non-galactosylated glycans include G0F or G0.

[0060] As used herein, the term "N-glycan" refers to an N-linked oligosaccharide, e.g., one attached to an asparagine residue of a polypeptide via an asparagine-N-acetylglucosamine linkage. N-glycans are found in sequons attached to the R-group nitrogen (N) of asparagine. The sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline. N-glycans have a common pentasaccharide core of Man3GlcNAc2 ("Man" refers to mannose; "Glc" refers to glucose; and "NAc" refers to N-acetyl; "GlcNAc" refers to N-acetylglucosamine). The pentasaccharide core may be fucosylated. The term "trimannose core" as used in reference to N-glycans also refers to the structure Man3GlcNAc2 ("Man3"). N-glycans differ with respect to the number of branches (arrangements) containing peripheral sugars (e.g., fucose [abbreviated herein as "Fuc"] and sialic acid) that are added to the Man3 core structure. N-glycans are classified according to their branching components (e.g., high mannose, complex, or hybrid). The abbreviations used herein, including sugar abbreviations, are commonly used in the art. Other common abbreviations include peptide N-glycans, ... "PNGase" refers to phosphosidase F (EC 3.2.2.18). The substrate UDP-GlcNAc is an abbreviation for UDP-N-acetylglucosamine. The intermediate ManNAc is an abbreviation for N-acetylmannosamine. The intermediate ManNAc-6-P is an abbreviation for N-acetylmannosamine-6-phosphate. The intermediate Sia-9-P is an abbreviation for sialate-9-phosphate. The intermediate cytidine monophosphate sialic acid is abbreviated as "CMP-Sia." Sialic acid is abbreviated as "Sia," "Neu5Ac," "NeuAc," or "NANA."

[0061] N-glycans on erythropoiesis-stimulating glycoproteins, such as erythropoietin, contain one or more N-acetyllactosamine units attached to an N-linked oligosaccharide core structure. As used herein, the number of oligosaccharide "branches" refers to the number of individual oligosaccharide chains attached to the pentasaccharide core structure. For example, as shown in Figure 2A, two individual oligosaccharide chains are attached to the pentasaccharide core structure, so the N-glycan contains two branches and is biantennary. For example, as shown in Figure 2B, three individual oligosaccharide chains are attached to the pentasaccharide core structure, so the N-glycan contains three branches and is triantennary. For example, as shown in Figure 2C, four individual oligosaccharide chains are attached to the pentasaccharide core structure, so the N-glycan contains four branches and is tetraantennary. N-glycans on erythropoiesis-stimulating glycoproteins, such as erythropoietin, contain poly-N-acetyllactosamine units attached to the N-linked oligosaccharide core structure. The term "repeat" or "poly N-acetyllactosamine repeat" as used herein refers to the number of N-acetyllactosamine units in an oligosaccharide branch minus one for the first N-acetyllactosamine unit. For example, as shown in Figure 2D, two N-acetyllactosamine units are present in an oligosaccharide branch, meaning that the oligosaccharide contains one poly N-acetyllactosamine repeat. The corresponding N-glycan structure herein is also referred to as a "tetraantennary + 1 repeat." For example, as shown in Figure 2E, four N-acetyllactosamine units are present in an oligosaccharide branch, meaning that the oligosaccharide contains three poly N-acetyllactosamine repeats. The corresponding N-glycan structure herein is also referred to as a "tetraantennary + 3 repeat."

[0062] As used herein, the term "glycosylation occupancy" refers to the probability that a protein is glycosylated at a particular glycosylation site (e.g., an Asn residue at a consensus glycosylation site), or the relative abundance of a protein in a population of proteins that is glycosylated at a particular glycosylation site. For example, an erythropoietin polypeptide may be glycosylated at amino acid residues Asn24, Asn38, and / or Asn83 of SEQ ID NO:1.

[0063] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Those skilled in the art are aware of methods for producing (erythropoiesis-stimulating) glycoprotein compositions, including culturing host cells containing the (erythropoiesis-stimulating) glycoprotein-encoding nucleic acid(s) provided above under conditions suitable for expression of the (erythropoiesis-stimulating) glycoprotein, and optionally recovering the (erythropoiesis-stimulating) glycoprotein from the host cells (or host cell culture medium). For recombinant production of (erythropoiesis-stimulating) glycoprotein compositions, for example, nucleic acids encoding the (erythropoiesis-stimulating) glycoproteins described above are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of an antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell. Mammalian cell lines adapted to grow in suspension may be useful.Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7), human embryonic kidney lines (e.g., 293 cells or 293T cells as described in Graham, FL et al., J. Gen. Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (e.g., Mather, JP et al., Annals NY Acad. Sci. 383 (1982) 44-68), MRC5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. In one embodiment of the present invention, the host cell is a CHO cell. In one embodiment of the present invention, the host cell is a CHO-K1 cell. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, A.M., Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0064] The term "in-line (pH) measurement" refers to measurements made directly in the bioreactor, typically using a process sensor installed in the bioreactor. As used herein, the term "in-line (pH) probe" or "in-line (pH) measurement device" refers to such a process sensor installed in the bioreactor. The measurements generated can be transmitted in real time to a system for automatic control of the measured parameters, such as pH. Cell culture process parameters, such as pH, dissolved oxygen, dissolved CO2, temperature, and conductivity, are generally measured in-line.

[0065] The terms "sialylation" and "sialylated" refer to the presence of sialic acid in a protein or portion of a protein of interest, particularly as a component of a glycan (e.g., N-glycan) chain attached to the protein. Sialic acid (also referred to herein as a "sialic acid moiety") generally refers to an N- or O-substituted derivative of neuraminic acid. N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-D-galactononulosonic acid; also known as NANA or Neu5Ac) is the most common sialic acid in mammals. Other exemplary sialic acids include, but are not limited to, 2-keto-3-deoxy-D-glycero-D-galactonononic acid (also known as Kdn), N-glycolylneuraminic acid (also known as Neu5Gc or NGNA), neuraminic acid (also known as Neu), and 2-deoxy-2,3-didehydro-Neu5Ac (also known as Neu2en5Ac). Free sialic acid (Sia) can be used for glycan synthesis after activation to the nucleotide donor CMP-Sia. The transfer of Sia from CMP-Sia to newly synthesized glycoconjugates (e.g., glycoproteins) in the eukaryotic Golgi system is catalyzed by a family of linkage-specific sialyltransferases (STs). Sialic acid is typically the terminal residue of a glycan (e.g., N-glycan) branch. In some embodiments, sialic acid can occupy an internal position within a glycan, most commonly when one sialic acid residue is linked to another. For a review of sialylation and sialic acids, see, e.g., Varki et al., Essentials of Glycobiology, 3 rd See Chapter 15 of the Cold Spring Harbor Laboratory Press, Cold Spring Harbor Edition, 2015-2017.

[0066] As used herein, the term "sialic acid content" refers to the level or amount of sialylation of a glycosylated protein or portion of a glycosylated protein of interest. The term "average sialic acid content" with respect to a composition containing a glycoprotein (e.g., a pharmaceutical composition or batch) refers to the total number of moles of sialic acid in the composition per mole of glycoprotein in the composition. Thus, for example, such a composition may contain a heterogeneous pool of glycoproteins, with individual glycoproteins within the composition having varying levels of sialylation (e.g., ranging from 0 to 14 moles of sialic acid per mole of erythropoiesis-stimulating glycoprotein). Table 1. Examples of common isoforms of erythropoietin TIFF2025536587000002.tif61148

[0067] With regard to sialylation, erythropoietin exists primarily in 10 isoforms. The term "isoform" refers to a group of erythropoietin molecules with the same amino acid sequence and the same number of attached sialic acid residues. Isoforms have the same isoelectric point and may differ in the degree, complexity, and branching of glycosyl residues attached to the amino acid sequence. For example, the term "erythropoietin isoform 2" encompasses a group of erythropoietin molecules with 14 sialic acid residues. Isoform 3 has 13 sialic acids, and so on. Furthermore, there are rare forms of erythropoietin with additional non-terminal sialic acids. Thus, isoform 1 has 15 sialic acids attached to glycosyl residues, and isoform 1' has 16 sialic acids attached to glycosyl residues.

[0068] As used herein, the term "sialic acid-free" refers to a population of glycoproteins that are substantially free of N-glycans containing terminal sialic acid moieties. In one embodiment, the term "sialic acid-free" as used herein refers to a population of glycoproteins that contain a relative frequency of N-linked glycans containing sialic acid moieties of 5% or less. In one embodiment, a sialic acid-free glycoprotein contains a relative frequency of N-linked glycans containing sialic acid moieties of about 0%.

[0069] As used herein, the term "glycosylated variant" or "glycosylation variant" refers to a glycoprotein, particularly an erythropoiesis-stimulating glycoprotein, e.g., erythropoietin, characterized by a defined carbohydrate moiety attached thereto. A "glycosylated variant" or "glycosylation variant" of a glycoprotein can be a glycoprotein having a specific N-linked oligosaccharide, particularly a Man3GlcNAc2 pentasaccharide core, and containing N-glycans with a different number of branches (antennary) attached to the pentasaccharide core structure. In certain embodiments of all aspects and embodiments, a "glycosylated variant" can be triantennary or tetraantennary. In another aspect, a "glycosylated variant" can contain a different number of poly-N-acetyllactosamine units ("repeats" / "poly-N-acetyllactosamine repeats") attached to the pentasaccharide core structure of the N-linked oligosaccharide. In particular, a "glycosylated variant" of a glycoprotein may have a "triantennary + 1 repeat," "tetraantennary + 1 repeat," "tetraantennary + 2 repeat," and / or "tetraantennary + 3 repeat" N-glycan structure. The term "glycosylated variant" or "glycosylation variant" herein also includes a "sialylated variant" of a glycoprotein. A "sialylated variant" or "sialylation variant" of a glycoprotein, as used herein, refers to a glycoprotein having a particular level or amount of sialylation (e.g., in the case of erythropoiesis-stimulating glycoprotein, this may range from 0 to 14 moles of sialic acid per mole of erythropoiesis-stimulating glycoprotein). Thus, a "glycosylated variant" or "glycosylation variant" of a glycoprotein may be a glycoprotein containing a particular amount of sialic acid moieties. In certain embodiments, the glycosylated variant of the erythropoietic glycoprotein can be one of the ten major isoforms of erythropoietin (isoforms 1-10), particularly one of the seven isoforms shown in Table 1. A glycoprotein composition can typically be composed of a mixture of different glycosylated variants of a glycoprotein with defined relative amounts of the different glycosylated variants present in the composition.In the case of an erythropoietic composition, this may be a mixture of different glycosylated variants of an erythropoiesis-stimulating glycoprotein with defined relative amounts of the different glycosylated variants present in the composition.

[0070] The term "fed-batch" as used herein refers to a cell culture in which cells are continuously or periodically fed with a nutrient-containing feed medium. Feeding can begin immediately after initiating the cell culture on day 0, or more typically 1, 2, or 3 days after initiating the culture. Feeding can also follow a preset schedule, such as daily, every 2, or 3 days. Alternatively, the culture can be monitored for cell growth, nutrients, or toxic by-products, and feeding can be adjusted accordingly. Typically, a batch or fed-batch culture is stopped at some point, and the cells and / or protein of interest in the medium are harvested and optionally purified.

[0071] The term "medium-specific correlation" refers to a mathematical description of the relationship between the pH value in a given culture medium in a bioreactor and the corresponding CO2 concentration determined in the headspace and / or exhaust of the bioreactor. The medium-specific correlation is universal for each medium and is applicable regardless of the location and vessel used. A medium-specific correlation for a particular culture medium can be determined by placing the culture medium in a vessel, determining the exhaust CO2 concentration at different pH values, and using this dataset to develop an appropriate model. Because the correlation is scale-independent, practically any vessel, such as a simple tank or bioreactor, can be used to collect such a dataset. Preferably, several measurements are performed to provide a more robust dataset. Those skilled in the art are familiar with methods for estimating correlations from such datasets. According to an embodiment, the medium-specific correlation is calculated using the formula FCO2 obtained by mathematically fitting multiple empirically determined pairs of pH values ​​of a sample of the culture medium used in the bioreactor and the respective measured fraction of CO2 gas in the headspace above the sample (which can be measured directly in the headspace or, more commonly, in the exhaust of the bioreactor). M1 (pH) = REL - M1 (pH). Those skilled in the art are familiar with methods for mathematically fitting empirically determined data sets, such as linear, quadratic, or logarithmic fitting. In one embodiment, the data set is fitted quadratically. The sample can be, for example, the entire cell-free culture medium in a bioreactor or a cell-free aliquot of the culture medium.

[0072] The quality of a model, i.e., the ability of the model to describe the modeled data points, can be measured, for example, by the regression coefficient R-squared (R 2 ) can be described by R 2A ρ of 1 means that all data points are perfectly described by the model. For all models prepared for this method, the R-squared was found to be greater than 0.98. The root mean square error was 0.02 for all models. Considering potential errors in calibration, environmental factors, etc., the accuracy of this method is considered to be within 0.03 pH units of the pH of the culture medium.

[0073] The term "offset" as used herein refers to the sum of errors compared to the actual pH value in the culture medium when determining pH values ​​from offline samples caused by multiple factors such as sampling procedure, equipment, sample retention time, sample temperature shifts, carbon dioxide degassing of the sample, differences between devices, general sample characteristics (such as cell density, dissolved carbon dioxide and medium buffering), and / or cross-site / scale pressure effects.

[0074] The term "pH measuring device" or "pH probe" refers to a device and / or substance used to measure the current pH value in a culture medium. In one embodiment, the pH measuring device is, for example, a potentiometric measuring device. According to a preferred embodiment, the pH measuring device is a pH meter. The pH meter can be, for example, a continuous pH meter, i.e., a pH meter that can continuously and repeatedly measure the pH of the culture medium in a bioreactor without the need to draw samples or insert the pH meter into the medium for each individual measurement. For example, the pH measuring device can be a precision voltmeter that contacts the culture medium, is connected to a reference electrode, and is scaled to display the ready pH value rather than the measured potential. Preferably, the pH measuring device is immersed in the medium and is used to repeatedly or continuously measure the current pH value in the culture medium over the entire time period while culturing cells in the bioreactor. For example, the pH measuring device can measure the current pH value every minute, every 30 minutes, or every hour. In typical modern pH meters used as pH measuring devices, the reference electrode is integrated into the pH electrode, making the device compact.

[0075] As used herein, the term "calibration" or "calibrating" refers to a procedure in which a measurement value, such as a pH value, of a measurement device, such as a pH measuring device, is compared to a reference measurement value or calibration standard of known accuracy and nominal (e.g., pH) value, and the measurement device is adjusted to that reference value. pH measuring devices are typically calibrated using so-called calibration buffers of defined pH values. Generally, one, two, or three buffers with different pH values ​​can be used (one-point calibration, two-point calibration, and three-point calibration, respectively). This procedure requires contacting the pH measuring device with the calibration buffer(s). Because this calibration method destroys the sterility of a sterilized bioreactor, a common alternative method for calibrating an in-line pH measuring device installed in a bioreactor is to remove a sample from the bioreactor, measure the pH of the sample offline using another pH measuring device of known accuracy, and then single-point calibrate the in-line pH measuring device to the offline-measured pH value ("offline measurement").

[0076] The terms "carbon dioxide-based pH reference method," "carbon dioxide-based calibration," or "carbon dioxide-based calibration" refer to a method disclosed herein that eliminates the need to sample and measure pH offline by calculating the pH value in a culture medium using a medium-specific correlation between the carbon dioxide concentration in the gas phase and the dissolved carbon dioxide, bicarbonate, and dependent proton concentrations, which directly affect the pH value in a carbonate buffer system. The carbon dioxide-based pH reference method is independent of scale and bioreactor configuration and can accurately determine the pH in the culture medium in a bioreactor without the need to remove samples.

[0077] The term "pH value of / in a culture medium" refers to a pH value determined by an in-line pH measurement device in direct contact with the culture medium, and therefore free from offsets introduced by, for example, sampling procedures, equipment, sample retention time, sample temperature and carbon dioxide degassing shifts, differences between devices, sample characteristics (cell density, dissolved carbon dioxide concentration, medium buffering, etc.), and / or cross-site / scale pressure effects, and calibrated according to accepted standards, for example, certified pH calibration standards 4.01, 7.00, and 9.21 pH. In one embodiment of all aspects, to determine the pH value of / in a culture medium with maximum accuracy, two independent pH measurement devices calibrated against said standards with a maximum allowable error of 0.01 pH can be simultaneously brought into direct contact with the culture medium. The maximum allowable difference between the values ​​measured in the culture medium using the two independent pH measurement devices described above can be set to 0.02 pH units, and the average value of the two measurements can be used.

[0078] As used herein, the term "relative content" of a glycosylation variant refers to the amount of a particular glycosylation variant in a glycoprotein composition. Typically, a glycoprotein composition may be heterogeneous and may contain two or more glycosylation variants, so a single glycosylation variant does not constitute 100% of the composition. Relative content can be expressed in any of a variety of ways known in the art. For example, relative content can be expressed as the relative content of the total amount of glycoprotein in the composition, or can be expressed relative to the amount of a particular type of glycosylation, such as the total amount of N-glycan variants or the total amount of sialylated isoforms. Methods and assays for determining the relative content of glycosylation variants are described herein.

[0079] As used herein, the term "sterilization" refers to any process that eliminates (removes) or kills (inactivates) all forms of life and other biological agents. Sterilization can be achieved with one or more of the following: heat, chemicals, irradiation, high pressure, and filtration. When systems or system elements cannot be moved (e.g., large stainless steel bioreactors), an in situ sterilization process called sterilization in place (SIP) is used. SIP processes can reduce or eliminate post-sterilization handling by providing sterile connections between sterilized equipment. Common sterilization modes for SIP are steam (moist heat), superheated water, dry heat, gas, liquid, and steam sterilization, with steam being the most common method. SIP methods are generally used in closed systems, such as large-scale biotechnology manufacturing in GMP environments, to maintain sterility during product manufacturing. The term "under sterile conditions," as used herein, refers to conditions that avoid and / or prevent the bioreactor (including all vessels and devices attached thereto), culture medium, and / or cell culture from becoming contaminated with any undesirable viable organisms, such as, but not limited to, any bacteria, pathogens, microorganisms (such as bacteria) (including their spores), and / or viruses (not including, of course, recombinant host cells expressing the glycoprotein of interest) after sterilization of the bioreactor has taken place. Methods for recombinantly producing glycoproteins "under sterile conditions" are well known to those skilled in the art and include the use of closed systems as described above, and appropriate measures to ensure that any material (whether they are solid, liquid, or gaseous) or any device (e.g., gas mixtures used for aeration, feed streams, sampling devices) that comes into contact with the culture medium and / or the interior of the bioreactor during the production process, particularly during the culturing process, is free of contaminating viable organisms (other than host cells).

[0080] As used herein, the term "batch-to-batch variability" refers to the difference in properties between an isolated glycoprotein composition obtained by one run of a recombinant production method and another isolated glycoprotein composition obtained by a subsequent run of the same recombinant production method. The batch-to-batch variability can be quantified with respect to any chemical or physical property of the glycoprotein composition, such as glycoprotein concentration, pH value, osmolality, glycosylated variant content, or protein modifications (e.g., deamidation, acidic variants). In one embodiment of the present invention, the batch-to-batch variability is determined with respect to at least the relative content of glycosylated variants, such as N-glycan variants or sialylated isoforms.

[0081] The term "yield" as used herein refers to the amount of glycoprotein harvested from a recombinant host cell culture. Yield can be expressed in terms of "mg protein / g biomass" of host cells (measured as dry cell weight or wet cell weight). The absolute yield of a fermentation batch can be expressed in terms of "g protein" and can be calculated by determining the concentration of the recombinant glycoprotein composition using an appropriate method, such as RP-HPLC, and multiplying the resulting concentration by the volume of the resulting glycoprotein composition. As used herein, the term "titer" similarly refers to the amount of a produced protein or model protein of interest, presented as "g protein / L culture medium."

[0082] 2. The method of the present invention In commercial production of recombinant (glyco)proteins, pH probes are typically placed inside bioreactors to measure pH inline. At the beginning of the cell culture process, the bioreactor is closed and sterilized before filling with culture medium. However, bioreactor sterilization affects inline pH probes placed inside the bioreactor because the temperatures used for SIP typically exceed the pH probe's operating range, potentially causing electrolytes in the probe to liquefy and potentially resulting in asymmetry changes. Therefore, recalibration is required after sterilization to ensure that measurements indicated by the inline pH probe can be relied upon. However, in GMP (Good Manufacturing Practice) manufacturing, after sterilization and filling, it is no longer possible to directly equilibrate or recalibrate the inline pH probe without destroying sterility and violating GMP principles. Therefore, the pH value of cell cultures is generally monitored and controlled using sample-based offline measurements. For this purpose, a sample is taken from the medium after bioreactor sterilization, the pH is measured offline (e.g., using a glass electrode or blood gas analyzer), and the inline pH probe is then recalibrated based on the offline pH value. During the cultivation, offline sampling continues frequently to detect any drift in the in-line pH probe: for this purpose, the pH values ​​measured offline are continuously compared with the pH values ​​measured in-line, and if they show a difference exceeding a certain tolerance limit, the in-line pH probe is recalibrated to the value measured in the offline sample.

[0083] However, this procedure can cause problems. Various factors such as measurement inaccuracies, incorrect handling of buffers (e.g., not using fresh alkaline buffers as CO2 absorbed from the ambient air can cause a decrease in pH), extreme pH values > pH 12 or < pH 2, sample holding time, sample temperature or degassing of carbon dioxide that cause a shift in pH, use of different offline methods, or different sampling procedures or equipment can affect the pH measured offline and thus deviate from the actual "true" pH within the bioreactor. All of these effects can together result in an offset of up to + / - 0.15 pH units. As a result, recalibrating the in-line pH electrode based on sample-based offline measurements results in a significant offset compared to the actual pH inside the bioreactor, i.e., the measured pH value does not accurately reflect the pH in the culture medium. This not only causes errors during readjustment of the pH electrode after sterilization and before inoculating the host cells into the culture medium, but can also lead to inconsistencies in the process where the pH changes. If offline measurements are used to continuously monitor and readjust the in-line pH probe during the cell culture process, the metabolism of the cells inside the bioreactor also adds a pH offset by changing the composition of the ground.

[0084] This can also affect the control of the cell culture process. Generally, a fixed pH setpoint is defined for the cell culture process for producing therapeutic proteins, and most commonly, the culture medium is controlled either via the partial pressure of dissolved carbon dioxide (pCO2) in the cell culture or by adding a base to the culture medium to keep the pH constant in the cell culture. If the measured pH does not reflect the pH in the culture medium, it can be incorrectly set to a pH value above or below the desired setpoint, resulting in undesired outcomes.

[0085] The present inventors have found that by using a method for calibrating an in-line pH measurement device in a closed bioreactor to a pH value that reliably reflects the pH value in the culture medium, the need for offline pH determination is avoided and the variability of specific glycosylation variants, as well as the variability of overall yield, is significantly reduced when recombinantly producing glycoproteins in bioreactors using mammalian host cell cultures. It has surprisingly been found that significant improvements can be achieved with respect to the variability of glycosylation variants in recombinantly produced glycoprotein compositions by replacing the pH value in the culture medium and adjusting the in-line pH probe to that pH value with a highly accurate carbon dioxide-based method, without changing any other parameters, and simply by addressing the variability imparted to the (large-scale) cultivation process using an offline, sample-based pH measurement / calibration method.

[0086] This can be achieved, for example, by using a non-invasive method for determining pH and pCO2 in sterile bioreactors that eliminates the need for offline sampling and measurement. This method utilizes the chemical correlation between carbon dioxide in the gas phase and the dissolved carbon dioxide, bicarbonate, and dependent proton concentrations, which directly affect the pH of the carbonate buffer system. This carbon dioxide-based pH reference method is independent of scale and bioreactor configuration and can accurately determine the true pH in a bioreactor without the need to remove samples.

[0087] For commercial manufacturing of therapeutic glycoproteins, reduced variability not only results in highly consistent product quality with respect to glycosylated variants, but also improves overall process yield and efficiency as it reduces the risk of not meeting required product specifications, thus avoiding the need to discard partial or entire production batches that were not within specifications.

[0088] Accordingly, one aspect of the present specification is a method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein, the method comprising the steps of: Culturing recombinant host cells that express a glycoprotein in a culture medium in a bioreactor having a pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium, (a) The culture is carried out under sterile conditions; (b) the pH value measured by the pH measuring device differs from the pH value of the culture medium by 0.05 units or less; and (c) the relative content of at least one glycosylated variant in the glycoprotein composition is wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. has reduced batch-to-batch variability compared to a process characterized by: Thereby producing a glycoprotein composition.

[0089] Another aspect of the present invention is a method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein in a recombinant host cell that expresses the glycoprotein, comprising: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein composition. and the calibrating step of step (d) comprises: (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). Includes:

[0090] In certain embodiments of all aspects and embodiments, the pH value measured by the pH measuring device after the calibration step differs from the pH value of the culture medium by no more than 0.05 pH units. In preferred embodiments of all aspects and embodiments, the pH measured by the pH measuring device after the calibration step differs from the pH value of the culture medium by no more than 0.03 pH units.

[0091] In certain embodiments of all aspects and embodiments, the carbon dioxide concentration is determined in the exhaust of the bioreactor.

[0092] The improvement of variability can be achieved by calculating the standard deviation of at least one glycosylated variant determined for at least two fermentation batches of a glycoprotein composition (i.e., from at least two different cultures carried out according to the method of the present invention) and comparing it with another method, i.e., a method in which the pH value measured by the pH measuring device differs from the pH value of the culture method by more than 0.05 pH units, preferably by more than 0.03 pH units; The inter-batch variability may be assessed by comparing the standard deviation calculated for the same glycosylated variant(s) determined for at least two batches of glycoprotein composition produced according to the method described above with the standard deviation calculated for the same glycosylated variant(s) determined for at least two batches of glycoprotein composition produced according to the method described above. In one embodiment, the inter-batch variability is compared by comparing the standard deviation between batches produced using essentially the same production method except for the method used to calibrate the pH measurement device. In certain embodiments of all aspects and embodiments, the standard deviation is calculated for at least one glycosylated variant from glycoprotein compositions from at least three fermentation batches. In another embodiment, the standard deviation is calculated for at least one glycosylated variant from glycoprotein compositions from at least five fermentation batches. In yet another embodiment, the standard deviation is calculated for at least one glycosylated variant from glycoprotein compositions from at least ten fermentation batches.

[0093] In certain embodiments of all aspects and embodiments, the glycoprotein composition comprises at least one glycosylated variant of the glycoprotein, and the standard deviation of the relative content of the glycosylated variant calculated for the glycoprotein composition from at least two fermentation batches is: wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0094] In one embodiment of all aspects and embodiments, the yield of the glycoprotein composition after culturing is: wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. In one embodiment of all aspects and embodiments, the absolute yield of the glycoprotein composition after culturing is: wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. The glycoprotein compositions produced using the PEG-4000-10000-10000-2 ...

[0095] In certain embodiments of all aspects and embodiments, the standard deviation of the (absolute) yields calculated for the glycoprotein composition from at least two fermentation batches is: wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. In certain embodiments of all aspects and embodiments, the standard deviation is calculated for glycoprotein compositions from at least three fermentation batches. In other embodiments, the standard deviation is calculated for glycoprotein compositions from at least five fermentation batches. In yet other embodiments, the standard deviation is calculated for glycoprotein compositions from at least ten fermentation batches.

[0096] In certain embodiments of all aspects and embodiments, the recombinant host cell is a mammalian cell. In preferred embodiments of all aspects and embodiments, the recombinant host cell is a CHO cell.

[0097] In certain embodiments, the method for recombinantly producing a glycoprotein composition is for commercial purposes and / or is carried out using a large-scale bioreactor. In a particular embodiment, the large-scale bioreactor has a volume of at least 10 L, preferably at least 50 L, more preferably at least 500 L, and particularly preferably at least 5000 L. In certain embodiments of all aspects and embodiments, the bioreactor is for commercial recombinant protein production. In one embodiment, the large-scale cell culture method of the present invention is suitable for CHO cell culture.

[0098] In certain embodiments of all aspects and embodiments, the glycosylation variant is selected from the group consisting of an N-glycan variant, an O-glycan variant, a sialylation variant, a mannosylation variant, a galactosylation variant, and a fucosylation variant.

[0099] In certain embodiments of all aspects and embodiments, the culture medium comprises a carbonate buffer.

[0100] In certain embodiments of all aspects and embodiments, the method for recombinantly producing a glycoprotein composition includes maintaining the pH in the culture medium at a desired set point, particularly during the cultivation of the host cells. In certain embodiments of all aspects and embodiments, the desired pH set point in the culture medium is adjusted (a) by increasing carbon dioxide influx if the pH is above the desired set point, or (b) by adding a basic component, preferably 1 mol / L NaOH, to the culture medium if the pH is below the desired set point.

[0101] In certain embodiments of all aspects and embodiments, the carbon dioxide is introduced into the bioreactor by sparging.

[0102] A further aspect of the present invention is a method for reducing the variability in the relative content of at least one glycosylated variant between batches of a recombinant glycoprotein composition, comprising the steps of: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells expressing the glycoprotein; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein, Here, the calibrating step of step (d) is (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH value calculated in step (iii); Including, Steps (a) to (g) result in a first batch of recombinant glycoprotein having a defined relative content of glycosylated variants; process, (h) repeating steps (a)-(g) to produce at least one subsequent batch of glycoprotein, wherein the relative content of the at least one glycosylated variant of the first batch and the at least one subsequent batch has reduced batch-to-batch variability.

[0103] To determine the batch-to-batch variability of a parameter, at least two glycoprotein compositions from different fermentation batches must be evaluated. Thus, in certain embodiments of all aspects and embodiments, the reduction in batch-to-batch variability due to the carbon dioxide-based calibration method is assessed by comparing the variability of glycoprotein composition from at least two fermentation batches with the variability from at least two fermentation batches obtained using a different method for calibrating the pH measurement device, preferably based on sample-based offline pH measurements. In certain embodiments of all aspects and embodiments, the reduction in batch-to-batch variability is assessed by comparing the variability of glycoprotein composition from at least three fermentation batches. In another embodiment, the reduction in batch-to-batch variability is assessed by comparing the variability of glycoprotein composition from at least five fermentation batches. In yet another embodiment, the reduction in batch-to-batch variability is assessed by comparing the variability of glycoprotein composition from at least ten fermentation batches.

[0104] In preferred embodiments of all aspects and embodiments, the standard deviation of the calculated relative glycoprotein content for said first and at least one subsequent batch is not more than 1%, in particular not more than 0.8%, most in particular not more than 0.5% of the median pH value.

[0105] Another aspect of the present invention is a method for producing a semiconductor device comprising: A method for measuring pH, wherein the measured pH value differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). In one embodiment, the use is for reducing the standard deviation of the relative content of glycosylated variants in a glycoprotein composition calculated between at least two fermentation batches, compared to a glycoprotein composition produced using a carbon dioxide-based method for calibrating a pH measuring device disposed within a bioreactor and configured to be in physical contact with the culture medium in the bioreactor. In a preferred embodiment of all aspects and embodiments, the carbon dioxide-based method includes the steps of: (i) introducing a gas mixture including carbon dioxide gas into a bioreactor containing a culture medium; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor; (iii) calculating the pH of the culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). In one embodiment, the use is for reducing the standard deviation compared to a produced glycoprotein composition, including calibrating the pH measuring device based on sample-based offline pH measurements.

[0106] In certain embodiments of all aspects and embodiments, the culture medium is buffered with carbonate. In certain embodiments of the aspects, carbon dioxide gas is introduced into the bioreactor by sparging.

[0107] In certain embodiments of all aspects and embodiments, the glycoprotein composition is an erythropoiesis composition. In preferred embodiments of the aspects, the glycoprotein is an erythropoiesis-stimulating glycoprotein. In certain embodiments of all aspects and embodiments, the glycoprotein composition is erythropoietin.

[0108] One aspect of the invention is an erythropoietic composition produced by the methods disclosed herein. In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises: (a) about 3.3 area % to about 3.8 area % of the N-glycans of the erythropoiesis-stimulating glycoprotein have a biantennary structure; (b) about 8.6 area% to about 9.5 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a triantennary structure; (c) about 5.6 area% to about 5.9 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a triantennary + 1 repeat structure; (d) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (e) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 1 repeat structure; (f) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 2 repeat structure; (g) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; (h) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; (i) about 23.5 area% to about 24.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 4; (j) about 17.1 area% to about 18.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 5; (k) about 9.4 area% to about 11.8 area% of the erythropoiesis-stimulating glycoprotein is isoform 6; (l) about 3.7 area% to about 5.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 7, and / or (m) about 0.9 area% to about 1.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 8. In certain embodiments of all aspects and embodiments, the area percent of N-glycans of the erythropoiesis-stimulating glycoprotein is determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein. In certain embodiments, the anion exchange chromatography is high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). In certain embodiments of all aspects and embodiments, the area percent of isoforms in the erythropoiesis composition is determined by capillary zone electrophoresis.

[0109] In-line pH measurement devices can experience undesirable pH drift, which can adversely affect process performance, product quality, and product yield in cell culture. Therefore, in certain embodiments of all aspects and embodiments, the bioreactor includes at least two pH measurement devices configured to be positioned within the bioreactor and physically contact the culture medium. In preferred embodiments of all aspects and embodiments, an alarm is triggered if the pH values ​​determined by the at least two pH measurement devices differ from each other by more than 0.05 units. This alarm indicates that the probe measurements may no longer be reliable and allows appropriate countermeasures to be taken to avoid any undesirable consequences, such as inadvertently adjusting the culture medium pH to an incorrect pH setpoint.

[0110] In certain embodiments of all aspects and embodiments, the medium-specific correlation is determined by collecting at least one medium-specific dataset for a culture medium, the dataset comprising: (a) filling a tank, preferably a bioreactor, with a culture medium, the tank having at least one pH measuring device configured to be disposed within the tank and in physical contact with the culture medium; (b) introducing a gas mixture comprising carbon dioxide into the tank; (c) measuring the pH in the culture medium using a pH measuring device, wherein the pH measuring device is calibrated (under non-sterile conditions) to measure the pH in the culture medium; (d) Any of the following: (i) varying the pH in the culture medium and measuring the carbon dioxide concentration in the headspace and / or exhaust air for at least two different pH values; or (ii) varying the carbon dioxide concentration in the gas mixture and measuring the pH value for at least two different carbon dioxide concentrations in the gas mixture; and (e) obtaining medium-specific correlations by mathematically fitting at least two pairs of headspace / exhaust carbon dioxide concentrations and corresponding pH values ​​in the culture medium; is collected using

[0111] It should be noted that the collection of the dataset is only possible in a process that is no longer under sterile conditions, since the pH probe used to measure the pH value of the culture medium is calibrated by removing the pH probe from the tank (bioreactor) for calibration, preferably by contacting the pH measuring device with calibration buffer(s) and returning it to the tank (bioreactor).

[0112] It may take some time for equilibrium to form between the carbon dioxide in the gas phase in the bioreactor and the dissolved carbon dioxide in the culture medium. Thus, in certain embodiments of all aspects and embodiments, the carbon dioxide in the headspace and / or exhaust air is determined after equilibrium has formed between the carbon dioxide in the gas phase in the bioreactor and the dissolved carbon dioxide in the culture medium.

[0113] The carbon dioxide-based pH calibration method can be used not only in main-stage fermentation, but also in seed trains, i.e., during the culturing step carried out to generate a sufficient number of host cells to inoculate the bioreactors of main-stage fermentation. Thus, in certain embodiments of all aspects and embodiments, the recombinant host cells have been cultured in a pre-culture medium in at least one pre-culture step in a bioreactor used for pre-culture prior to the inoculation step, the bioreactor used for pre-culture having at least one pH measuring device disposed therein and configured to be in physical contact with the pre-culture medium, and the pH measuring device in the bioreactor used for pre-culture is (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor used for pre-culture; (iii) calculating the pH of the pre-culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH calculated in step (iii). It is calibrated according to

[0114] In certain embodiments of all aspects and embodiments, the method for recombinantly producing a glycoprotein comprises maintaining the pH in the culture medium at a desired set point of pH 6.95.

[0115] In certain embodiments of all aspects and embodiments, the composition is an erythropoiesis composition. In preferred embodiments of all aspects and embodiments, the glycoprotein is an erythropoiesis-stimulating glycoprotein, particularly erythropoietin.

[0116] In certain embodiments of all aspects and embodiments, the erythropoiesis-stimulating composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having N-glycans with a biantennary structure, (b) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary structure, (c) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary +1 repeat structure, (d) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary structure, (e) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, (f) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure, (g) erythropoiesis-stimulating glycoprotein having 14 sialic acid residues (isoform 2), (h) erythropoiesis-stimulating glycoprotein having 13 sialic acid residues (isoform 3), (i) erythropoiesis-stimulating glycoprotein having 12 sialic acid residues (isoform 4), (j) erythropoiesis-stimulating glycoprotein having 11 sialic acid residues (isoform 5), (k) erythropoiesis-stimulating glycoprotein having 10 sialic acid residues (isoform 6), (l) erythropoiesis-stimulating glycoprotein having 9 sialic acid residues (isoform 7), and (m) erythropoiesis-stimulating glycoprotein having 8 sialic acid residues (isoform 8). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoiesis composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0117] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having N-glycans with a biantennary structure, (b) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary structure, (c) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary +1 repeat structure, (d) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary structure, (e) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, (f) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is the measured pH value differs from the pH value in the culture medium by more than 0.05 units, In certain embodiments of all aspects and embodiments, the erythropoiesis composition comprises a specified amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) erythropoiesis-stimulating glycoprotein having 14 sialic acid residues (isoform 2), (b) erythropoiesis-stimulating glycoprotein having 13 sialic acid residues (isoform 3), (c) erythropoiesis-stimulating glycoprotein having 12 sialic acid residues (isoform 4), (d) erythropoiesis-stimulating glycoprotein having 11 sialic acid residues (isoform 5), (e) erythropoiesis-stimulating glycoprotein having 10 sialic acid residues (isoform 6), (f) erythropoiesis-stimulating glycoprotein having 9 sialic acid residues (isoform 7), and (g) erythropoiesis-stimulating glycoprotein having 8 sialic acid residues (isoform 8). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0118] In certain embodiments of all aspects and embodiments, the erythropoiesis-stimulating composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary +1 repeat structure, (b) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, (c) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure, and (d) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure. (e) erythropoiesis-stimulating glycoprotein with N-glycans containing 14 sialic acid residues (isoform 2), (f) erythropoiesis-stimulating glycoprotein with 13 sialic acid residues (isoform 3), (g) erythropoiesis-stimulating glycoprotein with 11 sialic acid residues (isoform 5), (h) erythropoiesis-stimulating glycoprotein with 10 sialic acid residues (isoform 6), and (i) erythropoiesis-stimulating glycoprotein with 9 sialic acid residues (isoform 7). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoiesis composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is reduced compared to the standard deviation of the relative content of glycosylated variants calculated for the glycoprotein composition produced using the method of claim 1. In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having N-glycans with a triantennary +1 repeat structure, (b) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, (c) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, and (d) an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is reduced compared to the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0119] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) erythropoiesis-stimulating glycoprotein having 14 sialic acid residues (isoform 2), (b) erythropoiesis-stimulating glycoprotein having 13 sialic acid residues (isoform 3), (c) erythropoiesis-stimulating glycoprotein having 11 sialic acid residues (isoform 5), (d) erythropoiesis-stimulating glycoprotein having 10 sialic acid residues (isoform 6), and (e) erythropoiesis-stimulating glycoprotein having 9 sialic acid residues (isoform 7). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, In a preferred embodiment, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is reduced compared to the standard deviation of the relative content of glycosylated variants calculated for the glycoprotein composition produced using the measured pH value differs from the pH value in the culture medium by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0120] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary structure, (b) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +1 repeat structure, (c) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +2 repeat structure, (d) an erythropoiesis-stimulating glycoprotein having 14 sialic acid residues (isoform 2), and (e) an erythropoiesis-stimulating glycoprotein having 13 sialic acid residues (isoform 3). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary structure, (b) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +1 repeat structure, and (c) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +2 repeat structure. In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having 14 sialic acid residues (isoform 2), and (b) an erythropoiesis-stimulating glycoprotein having 13 sialic acid residues (isoform 3). In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, In a preferred embodiment, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is reduced compared to the standard deviation of the relative content of glycosylated variants calculated for the glycoprotein composition produced using the measured pH value differs from the pH value in the culture medium by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0121] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary structure. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of the glycosylated variant calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0122] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +1 repeat structure. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0123] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +2 repeat structure. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0124] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of erythropoiesis-stimulating glycoprotein (isoform 2) having 14 sialic acid residues. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0125] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises a defined amount of at least one glycosylated variant of erythropoiesis-stimulating glycoprotein (isoform 3) having 13 sialic acid residues. In certain embodiments of all aspects and embodiments, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.05 units, The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0126] In a preferred embodiment, the standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition between at least two fermentation batches is: the measured pH value differs from the pH value in the culture medium by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions produced using

[0127] In certain aspects and embodiments, the erythropoiesis-stimulating composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having from about 3.3 area% to about 3.8 area% of N-glycans with biantennary structures; (b) an erythropoiesis-stimulating glycoprotein having from about 8.6 area% to about 9.5 area% of N-glycans with triantennary structures; (c) an erythropoiesis-stimulating glycoprotein having from about 5.6 area% to about 5.9 area% of N-glycans with triantennary +1 repeat structures; (d) an erythropoiesis-stimulating glycoprotein having from about 42.2 area% to about 43.4 area% of N-glycans with tetraantennary structures; (e) an erythropoiesis-stimulating glycoprotein having from about 27.4 area% to about 28.1 area% of N-glycans with tetraantennary +1 repeat structures; (f) an erythropoiesis-stimulating glycoprotein having from about 10.7 area% to about 11.6 area% of N-glycans with tetraantennary +1 repeat structures; (g) from about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure; (h) from about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein of isoform 3; (i) from about 23.5 area% to about 24.6 area% of erythropoiesis-stimulating glycoprotein of isoform 4; (j) about 17.1 area% to about 18.6 area% of erythropoiesis-stimulating glycoprotein of isoform 5; (k) about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein of isoform 6; (l) about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein of isoform 7; and (m) about 0.9 area% to about 1.6 area% of erythropoiesis-stimulating glycoprotein of isoform 8. In certain embodiments of all aspects and embodiments, the area percent of the N-glycans of the erythropoiesis-stimulating glycoprotein is determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein. In certain embodiments, the anion exchange chromatography is high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). In certain embodiments of all aspects and embodiments, the area percent of the isoforms in the erythropoiesis composition is determined by capillary zone electrophoresis.

[0128] In certain aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) erythropoiesis-stimulating glycoproteins having from about 3.3 area% to about 3.8 area% N-glycans with biantennary structures; (b) erythropoiesis-stimulating glycoproteins having from about 8.6 area% to about 9.5 area% N-glycans with triantennary structures; (c) erythropoiesis-stimulating glycoproteins having from about 5.6 area% to about 5.9 area% N-glycans with triantennary +1 repeat structures; (d) erythropoiesis-stimulating glycoproteins having from about 42.2 area% to about 43.5 area% N-glycans with triantennary +1 repeat structures; (e) an erythropoiesis-stimulating glycoprotein having from about 27.4 area % to about 28.1 area % N-glycans with a tetraantennary + 1 repeat structure; and (f) an erythropoiesis-stimulating glycoprotein having from about 10.7 area % to about 11.6 area % N-glycans with a tetraantennary + 2 repeat structure (preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein). In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein of isoform 2; (b) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein of isoform 3; (c) about 23.5 area% to about 24.6 area% of erythropoiesis-stimulating glycoprotein of isoform 4; (j) about 17.1 area% to about 18.5 area% of erythropoiesis-stimulating glycoprotein of isoform 5; (k) about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein of isoform 6; (e) about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein of isoform 7; and (f) about 0.9 area% to about 1.6 area% of erythropoiesis-stimulating glycoprotein of isoform 8 (preferably determined by capillary zone electrophoresis).

[0129] In certain aspects and embodiments, the erythropoiesis-stimulating composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 8.6 area% to about 9.5 area% of N-glycans with triantennary structures; (b) an erythropoiesis-stimulating glycoprotein having about 5.6 area% to about 5.9 area% of N-glycans with triantennary +1 repeat structures; (c) an erythropoiesis-stimulating glycoprotein having about 42.2 area% to about 43.4 area% of N-glycans with tetraantennary structures; (d) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% of N-glycans with tetraantennary +1 repeat structures (preferably, an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% of N-glycans with tetraantennary +1 repeat structures). (e) from about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein of isoform 2; (f) from about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein of isoform 3; (g) from about 17.1 area% to about 18.6 area% of erythropoiesis-stimulating glycoprotein of isoform 5; (h) from about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein of isoform 6; and (i) from about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein of isoform 7 (preferably determined by capillary zone electrophoresis).

[0130] In certain aspects and embodiments, the erythropoiesis composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 8.6 area% to about 9.5 area% of N-glycans with triantennary structures; (b) an erythropoiesis-stimulating glycoprotein having about 5.6 area% to about 5.9 area% of N-glycans with triantennary +1 repeat structures; (c) an erythropoiesis-stimulating glycoprotein having about 42.2 area% to about 43.4 area% of N-glycans with tetraantennary structures; and (d) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% of N-glycans with tetraantennary +1 repeat structures (preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein). In certain embodiments of all aspects and embodiments, the erythropoiesis composition comprises one or more selected from the group consisting of: (a) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein of isoform 2; (b) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein of isoform 3; (c) about 17.1 area% to about 18.6 area% of erythropoiesis-stimulating glycoprotein of isoform 5; (d) about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein of isoform 6; and (e) about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein of isoform 7 (preferably as determined by capillary zone electrophoresis).

[0131] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) erythropoiesis-stimulating glycoproteins having from about 42.2 area % to about 43.4 area % N-glycans with tetraantennary structures; (b) erythropoiesis-stimulating glycoproteins having from about 27.4 area % to about 28.1 area % N-glycans with tetraantennary +1 repeat structures; (c) from about 10.7 area % to about 11.6 area % N-glycans with tetraantennary +2 repeat structures. and (d) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein of isoform 2; and (e) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein of isoform 3 (preferably determined by capillary zone electrophoresis).

[0132] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 42.2 area% to about 43.4 area% of N-glycans with a tetraantennary structure; (b) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% of N-glycans with a tetraantennary +1 repeat structure; and (c) an erythropoiesis-stimulating glycoprotein having about 10.7 area% to about 11.6 area% of N-glycans with a tetraantennary +2 repeat structure (preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein). In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) about 13.2 area% to about 16.0 area% erythropoiesis-stimulating glycoprotein of isoform 2; and (b) about 24.1 area% to about 26.5 area% erythropoiesis-stimulating glycoprotein of isoform 3 (preferably as determined by capillary zone electrophoresis).

[0133] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 42.2 area% to about 43.4 area% of N-glycans with tetraantennary structures; (b) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% of N-glycans with tetraantennary +1 repeat structures; and (c) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have tetraantennary +2 repeat structures (preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein).

[0134] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises about 26.5 area % to about 28.2 area % N-glycans of erythropoiesis-stimulating glycoproteins having N-glycans with tetraantennary +1 repeat structures, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein. In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises about 27.0 area % to about 28.1 area % N-glycans of erythropoiesis-stimulating glycoproteins having N-glycans with tetraantennary +1 repeat structures, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein. In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises about 27.4 area % to about 28.1 area % N-glycans of an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0135] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises about 42.2 area % to about 43.4 area % N-glycans of an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0136] In certain embodiments of all aspects and embodiments, the erythropoietic composition comprises about 10.7 area % to about 11.6 area % N-glycans of an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +2 repeat structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0137] In certain embodiments of all aspects and embodiments, the erythropoietic composition has an average sialic acid content of about 10.5 to 11.0 moles of sialic acid per mole of erythropoiesis-stimulating glycoprotein.

[0138] In a preferred embodiment of all aspects and embodiments, the erythropoiesis-stimulating glycoprotein is erythropoietin.

[0139] Also disclosed herein are glycoproteins, particularly erythropoiesis-stimulating glycoproteins, particularly erythropoietin, obtainable by the methods disclosed herein.Further disclosed are glycoproteins, particularly erythropoiesis-stimulating glycoproteins, particularly erythropoietin, obtainable by the methods disclosed herein.

[0140] The recombinant glycoproteins disclosed herein, particularly erythropoiesis-stimulating glycoproteins, particularly erythropoietin, may be incorporated (e.g., formulated) into pharmaceutical compositions. Accordingly, in another aspect, the present invention relates to a method for producing a pharmaceutical composition comprising a glycoprotein having reduced batch-to-batch variability with respect to one or more glycosylation variants, the method comprising performing a method for producing a glycoprotein composition and / or a method for reducing variability in glycosylation variants between batches of a recombinant glycoprotein described herein. Also an aspect is a pharmaceutical composition prepared using the glycoprotein disclosed herein.

[0141] In some embodiments, the method for producing a pharmaceutical composition further comprises combining the recombinant glycoprotein (i.e., obtained from the methods described herein) with a pharmaceutically acceptable carrier. Such pharmaceutical compositions are useful as alternative and / or improved compositions for preventing and / or treating one or more diseases compared to the corresponding reference glycoprotein. Pharmaceutical compositions containing glycoproteins can be formulated by methods known to those skilled in the art. Pharmaceutical compositions can be administered parenterally in the form of an injectable formulation comprising a sterile solution or suspension in water or another pharmaceutically acceptable liquid. For example, pharmaceutical compositions can be formulated by appropriately combining the glycoprotein with a pharmaceutically acceptable vehicle or medium, such as sterile water, saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring excipient, diluent, vehicle, preservative, or binder. The amount of active ingredient contained in the pharmaceutical formulation is such that an appropriate dosage within the specified range is provided.

[0142] Sterile compositions for injection can be prepared according to conventional pharmaceutical practice by using distilled water for injection as vehicle.For example, physiological saline or isotonic solution containing glucose and other supplements such as D-sorbitol, D-mannose, D-mannitol and sodium chloride can be used as aqueous solution for injection, optionally combined with suitable solubilizer, for example, alcohol such as ethanol and polyalcohol such as propylene glycol or polyethylene glycol, and nonionic surfactant such as polysorbate 80 (trademark).

[0143] Other ingredients that may be included include buffers such as phosphate buffer or sodium acetate buffer, sedatives such as procaine hydrochloride, stabilizers such as benzyl alcohol or phenol, and antioxidants. The formulated injection can be packaged in appropriate ampoules. The route of administration can be parenteral, e.g., via injection, nasal administration, pulmonary administration, or transdermal administration. Administration can be systemic or local, via intravenous, intramuscular, intraperitoneal, or subcutaneous injection. The appropriate administration method can be selected based on the patient's age and symptoms. A single dose of a pharmaceutical composition containing a modified glycoprotein can be selected from the range of 0.001 to 1,000 mg / kg body weight. The dosage can be selected from the range of 0.001 to 100,000 mg / kg body weight, although the present disclosure is not limited thereto. The dosage and administration method vary depending on the patient's weight, age, symptoms, etc., and can be appropriately selected by those skilled in the art.

[0144] Other technical features will be readily apparent to those skilled in the art from the following drawings, specifications, and claims.

[0145] 3. Analytical Assays Methods for analyzing and quantifying glycosylated variants of glycoproteins are well known to those skilled in the art.

[0146] Glycosylation variants can be determined directly from the culture medium or cell culture supernatant, i.e., from the culture medium once the host cells have been removed. The glycoprotein composition can be subjected to one or more purification steps before determining glycosylation variants by methods known to those skilled in the art, such as affinity chromatography (for antibodies, protein A chromatography), ion exchange chromatography, hydrophobic interaction chromatography, ultrafiltration, diafiltration, precipitation, centrifugation, and / or depth filtration. Those skilled in the art will be aware of suitable and useful methods for preparing glycoprotein samples for analytical assays. In certain embodiments of all aspects and embodiments, quantification of glycosylation variants is based on the relative area percentage of the detected peak corresponding to the glycosylated variant. The relative area percentage of a peak corresponds to the integrated area under the chromatogram peak measured by a detector relative to the total integrated peak area of ​​the entire chromatogram, e.g., a chromatogram generated by high-performance anion exchange chromatography or capillary zone electrophoresis using pulsed amperometric detection. In certain embodiments of all aspects and embodiments, the glycoprotein composition obtained by culturing the recombinant host cells is subjected to further isolation and / or purification steps before qualitatively and / or quantitatively analyzing glycosylated variants. In one embodiment, the glycoprotein is erythropoietin and has been purified using a downstream purification process comprising one or more steps selected from the group consisting of capture chromatography (e.g., Blue Sepharose chromatography), hydrophobic interaction chromatography (e.g., Butyl Toyopearl chromatography), adsorption chromatography (e.g., hydroxyapatite Ultragel chromatography), preparative reversed-phase high-performance liquid chromatography (RP-HPLC), and anion exchange chromatography (e.g., DEAE Sepharose chromatography). The process may further comprise a virus removal step (e.g., nanofiltration).

[0147] In certain embodiments of all aspects and embodiments, quantification of glycosylated variants of a glycoprotein characterized by a particular N-glycan structure is based on the relative area percent of detected peaks corresponding to the glycosylated variants as determined by anion exchange chromatography after enzymatic release of N-glycoside-linked oligosaccharides from the glycoprotein using the enzyme N-glycosidase F and, optionally, enzymatic desialylation. In certain embodiments, the anion exchange chromatography is high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). In certain embodiments of all aspects and embodiments, quantification of sialylated isoforms of a glycoprotein is based on the relative area percent of detected peaks corresponding to the glycosylated variants as determined by capillary zone electrophoresis.

[0148] The erythropoiesis-stimulating glycoproteins disclosed herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. In certain embodiments of all aspects and embodiments, quantification of glycosylated variants of erythropoiesis-stimulating glycoproteins, such as erythropoietin, characterized by specific N-glycan structures is based on the relative area percent of the detected peak corresponding to the glycosylated variant, determined by anion exchange chromatography after enzymatic release of N-glycoside-linked oligosaccharides from the glycoprotein, preferably using the enzyme N-glycosidase F, and desialylation, preferably using the enzyme neuraminidase. In certain embodiments, the anion exchange chromatography is high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). In certain embodiments, quantification is performed using the analytical assay for erythropoietin N-glycan profile described herein. In certain embodiments of all aspects and embodiments, quantification of sialylated isoforms of erythropoiesis-stimulating glycoproteins is based on the relative area percent of detected peaks corresponding to glycosylated variants as determined by capillary zone electrophoresis. In certain embodiments of all aspects and embodiments, quantification of sialylated isoforms of erythropoiesis-stimulating glycoproteins is performed using the analytical assays for determining erythropoiesis-stimulating glycoprotein isoform content described herein.

[0149] For example, the European Pharmacopoeia describes the N-glycan analysis of erythropoietin and the capillary zone electrophoresis (CZE) assay of erythropoietin isoforms (Ph.Eur.11.0,2673(01 / 2023)). Methods for CZE of erythropoietin isoforms are also described, for example, in Brinks et al. (Pharm Res (2011) 28:386-393; DOI10.1007 / s11095-010-0288-2) or Zhang et al. (J Pharm Biomed Anal 50(3):538-43; DOI:10.1016 / j.jpba.2009.05.007). In certain embodiments of all aspects and embodiments, the relative content of erythropoietin N-glycans is determined according to the method described in the European Pharmacopoeia (Ph.Eur.11.0,2673[01 / 2023]). In certain embodiments of all aspects and embodiments, the relative content of erythropoietin isoforms is determined according to the method described in the European Pharmacopoeia (Ph.Eur.11.0,2673[01 / 2023]). The following describes an exemplary method for analyzing erythropoiesis-stimulating glycoproteins.

[0150] Assay to Determine Erythropoiesis-Stimulating Glycoprotein Isoform Content Isoform distribution can be determined by capillary zone electrophoresis. In this method, separation is performed at a constant electric field strength in an uncoated glass capillary with a homogeneous buffer system, the pH of which is above the pI value of the EPO isoforms. Because all EPO isoforms are negatively charged and transported toward the cathode by inward osmotic flow, basic isoforms are detected first. The sample is diafiltered in water. The capillary is rinsed with electrolyte, and then the diluted sample is applied to the capillary. Separation is achieved by applying a high voltage of 25,000 V. A mobile buffer with excess cations is used, which results in electroosmotic flow. Proteins in the capillary can be detected photometrically using a quartz capillary and a diode array detector. Isoforms can be quantitatively determined by integrating the peaks corresponding to isoform 1 through isoform 9 to obtain the area under the peak [relative area - %].

[0151] Assay for determining erythropoiesis-stimulating glycoprotein N-glycan profile The relative distribution of N-glycans can be determined using an enzymatic test procedure coupled with a high-performance anion-exchange chromatography system with pulsed amperometric detection (HPAEC-PAD), essentially as described in WO 99 / 28346. In this procedure, the N-glycosidically linked oligosaccharides of erythropoietin are first cleaved by the enzyme N-glycosidase F. Terminal sialic acids are then removed from the oligosaccharides with the aid of the enzyme neuraminidase. After removal of the protein fraction by ultrafiltration, the resulting N-oligosaccharides are separated and analyzed using HPAEC-PAD and an appropriate data recording system. An erythropoietin reference standard, processed in exactly the same way as the samples, is measured during the test series. The area percentage of each glycosylated variant in a preparation can be calculated from the corresponding peak in the chromatogram of the produced glycan (e.g., biantennary, triantennary, triantennary + 1 repeat, tetraantennary, tetraantennary + 1 repeat, tetraantennary + 2 repeat).

[0152] Determination of the content of sialic acid residues Sialic acid content can be determined chromatographically by HPAEC-PAD after enzymatic cleavage of sialic acid with neuraminidase, essentially as described in WO 99 / 28346. For this purpose, erythropoietin samples are diluted with sodium phosphate buffer (pH 7.2). Half of each preparation is used to determine the exact amount of erythropoietin in the sample by RP-HPLC. Neuraminidase is added to the second half of each preparation and incubated overnight at 37°C. Subsequently, the digestion mixture is divided in half, diluted with water, and 50 μl of the mixture is applied to HPAEC-PAD. The amount of sialic acid in the applied sample can be determined using a calibration line obtained from the values ​​of sialic acid standards analyzed in the same test run. The sialic acid content (mol sialic acid / mol erythropoietin) can be calculated from the results of sialic acid determination and the determination of the amount of erythropoietin used by RP-HPLC.

[0153] 4. Embodiments of the present invention The present invention encompasses at least the following independent aspects and dependent embodiments.

[0154] 1. A method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein, comprising the steps of: Culturing recombinant host cells that express a glycoprotein in a culture medium in a bioreactor having a pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium, (a) The culture is carried out under sterile conditions; (b) the pH value measured by the pH measuring device differs from the pH value of the culture medium by 0.05 units or less; and (c) the relative content of at least one glycosylated variant in the glycoprotein composition is wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. has reduced batch-to-batch variability compared to a process characterized by: Thereby producing a glycoprotein composition.

[0155] 2. A method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein in a recombinant host cell that expresses the glycoprotein, comprising: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein composition. and the calibrating step of step (d) comprises: (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). A method comprising:

[0156] 3. The method according to embodiment 2, characterized in that after the calibration step, the pH measured using the pH measuring device differs from the pH value of the culture medium by no more than 0.05 units, preferably no more than 0.03 units.

[0157] 4. The method according to any one of embodiments 1 to 3, wherein the glycoprotein composition comprises at least one glycosylated variant of a glycoprotein; The standard deviation of the relative content of glycosylated variants calculated for glycoprotein compositions from at least two fermentation batches was wherein the pH value measured by the pH measuring device differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. The method is characterized in that the relative content of glycosylated variants is reduced compared to the standard deviation of the relative content of glycosylated variants calculated for a glycoprotein composition produced using

[0158] 5. The method of embodiment 4, wherein the standard deviation is calculated for glycoprotein compositions from at least 5, preferably at least 10, fermentation batches.

[0159] 6. The method of any one of embodiments 1 to 5, wherein the recombinant host cell is a mammalian cell.

[0160] 7. The method of any one of embodiments 1 to 6, wherein the recombinant host cell is a CHO cell.

[0161] 8. The method of any one of embodiments 4 to 7, wherein the glycosylated variant is selected from the group consisting of an N-glycan variant, an O-glycan variant, a sialylated variant, a mannosylated variant, a galactosylation variant and a fucosylated variant.

[0162] 9. The method of any one of embodiments 1-8, wherein the culture medium comprises a carbonate buffer.

[0163] 10. The method of any one of embodiments 1-9, comprising maintaining the pH in the culture medium at a desired set point of pH 6.95.

[0164] 11. The method according to any one of embodiments 1 to 10, wherein the desired pH set point in the culture medium is: (a) increasing carbon dioxide influx if the pH is above a desired set point; or (b) A method wherein if the pH is below a desired set point, it is adjusted by adding an alkaline component, preferably 1 mol / L NaOH, to the culture medium.

[0165] 12. The method of any one of embodiments 1-11, wherein the carbon dioxide is introduced into the bioreactor by sparging.

[0166] 13. The method of any one of embodiments 1-12, wherein the bioreactor comprises at least two pH measurement devices configured to be disposed within the bioreactor and in physical contact with the culture medium.

[0167] 14. The method of any one of embodiments 1-13, wherein an alarm is issued if the pH values ​​determined by the at least two pH measuring devices differ from each other by more than 0.05 units.

[0168] 15. The method according to any one of embodiments 1 to 14, wherein the medium-specific correlation is determined by collecting at least one medium-specific dataset for the culture medium, wherein the dataset comprises: (a) filling a tank, preferably a bioreactor, with a culture medium, the tank having at least one pH measuring device configured to be disposed within the tank and in physical contact with the culture medium; (b) introducing a gas mixture comprising carbon dioxide into the tank; (c) measuring the pH in the culture medium using a pH measuring device, wherein the pH measuring device is calibrated under non-sterile conditions to measure the pH in the culture medium; (d) Any of the following: (i) varying the pH in the culture medium and measuring the carbon dioxide concentration in the headspace and / or tank exhaust for at least two different pH values; or (ii) varying the carbon dioxide concentration in the gas mixture and measuring the pH value for at least two different carbon dioxide concentrations in the gas mixture; and (e) obtaining a medium-specific correlation by mathematically fitting at least two pairs of headspace and / or exhaust carbon dioxide concentrations and corresponding pH in the culture medium.

[0169] 16. The method according to any one of embodiments 1 to 15, wherein the carbon dioxide concentration in the headspace and / or exhaust air is determined after an equilibrium is formed between the carbon dioxide in the gas phase in the tank, preferably the bioreactor, and the dissolved carbon dioxide in the culture medium.

[0170] 17. The method according to any one of embodiments 1 to 16, characterized in that the recombinant host cells have been cultured in a pre-culture medium in at least one pre-culture step in a bioreactor used for pre-culture prior to inoculation, the bioreactor used for pre-culture having at least one pH measurement device arranged to be placed in the bioreactor and in physical contact with the pre-culture medium, and the pH measurement device in the bioreactor used for pre-culture is calibrated according to steps (i) to (iii) of embodiment 2.

[0171] 18. The method of any one of embodiments 1-17, wherein the glycoprotein composition is an erythropoietic composition.

[0172] 19. The method according to any one of embodiments 1 to 18, wherein the glycoprotein is an erythropoiesis-stimulating glycoprotein, preferably erythropoietin.

[0173] 20. The method of embodiment 18 or 19, further characterized in that the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) Erythropoiesis-stimulating glycoprotein with biantennary N-glycans; (b) erythropoiesis-stimulating glycoprotein with triantennary N-glycans; (c) erythropoiesis-stimulating glycoprotein with N-glycans with triantennary +1 repeat structures; (d) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary structure; (e) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +1 repeat structure; (f) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +2 repeat structure; (g) erythropoiesis-stimulating glycoprotein (isoform 2) with 14 sialic acid residues; (h) Erythropoiesis-stimulating glycoprotein (isoform 3) containing 13 sialic acid residues (i) erythropoiesis-stimulating glycoprotein (isoform 4) with 12 sialic acid residues; (j) erythropoiesis-stimulating glycoprotein (isoform 5) with 11 sialic acid residues; (k) erythropoiesis-stimulating glycoprotein (isoform 6) with 10 sialic acid residues; (l) erythropoiesis-stimulating glycoprotein (isoform 7) with nine sialic acid residues; and (m) Erythropoiesis-stimulating glycoprotein (isoform 8) with eight sialic acid residues.

[0174] 21. The method of any one of embodiments 18 to 20, further characterized in that the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) Erythropoiesis-stimulating glycoprotein with tetraantennary N-glycans; (b) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +1 repeat structure; (c) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +2 repeat structure; (d) erythropoiesis-stimulating glycoprotein (isoform 2) with 14 sialic acid residues; and (e) Erythropoiesis-stimulating glycoprotein (isoform 3) with 13 sialic acid residues.

[0175] 22. The method according to any one of embodiments 18 to 21, characterized in that the erythropoietic composition comprises one or more selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 3.3 area % to about 3.8 area % N-glycans with biantennary structures; (b) an erythropoiesis-stimulating glycoprotein having about 8.6 area % to about 9.5 area % N-glycans with triantennary structures; (c) erythropoiesis-stimulating glycoproteins having about 5.6 area % to about 5.9 area % N-glycans with triantennary +1 repeat structures; (d) an erythropoiesis-stimulating glycoprotein having about 42.2 area% to about 43.4 area% N-glycans with a tetraantennary structure; (e) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% N-glycans with a tetraantennary +1 repeat structure; (f) an erythropoiesis-stimulating glycoprotein having about 10.7 area% to about 11.6 area% N-glycans with a tetraantennary +2 repeat structure; (g) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein isoform 2; (h) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein isoform 3; (i) about 23.5 area% to about 24.6 area% of erythropoiesis-stimulating glycoprotein isoform 4; (j) about 17.1 area% to about 18.6 area% of erythropoiesis-stimulating glycoprotein isoform 5; (k) about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein isoform 6; (l) about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein isoform 7; and / or (m) about 0.9 area% to about 1.6 area% of erythropoiesis-stimulating glycoprotein isoform 8; Preferably, (a) to (f) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein, and (g) to (m) are determined by capillary zone electrophoresis.

[0176] 23. The method according to any one of embodiments 18 to 22, further characterized in that the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 42.2 area % to about 43.4 area % N-glycans with a tetraantennary structure; (b) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% N-glycans with a tetraantennary +1 repeat structure; (c) an erythropoiesis-stimulating glycoprotein having about 10.7 area % to about 11.6 area % N-glycans with a tetraantennary +2 repeat structure; (d) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein isoform 2; and (e) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein isoform 3; Preferably, (a) to (c) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein, and (d) to (e) are determined by capillary zone electrophoresis. 24. The method according to any one of embodiments 18 to 23, further characterized in that the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) an erythropoiesis-stimulating glycoprotein having about 42.2 area % to about 43.4 area % N-glycans with a tetraantennary structure; (b) an erythropoiesis-stimulating glycoprotein having about 27.4 area% to about 28.1 area% N-glycans with a tetraantennary +1 repeat structure; and (c) an erythropoiesis-stimulating glycoprotein having about 10.7 area % to about 11.6 area % N-glycans with a tetraantennary +2 repeat structure; Preferably, the method is determined by anion exchange chromatography after enzymatic release and desialylation of N-glycosidically linked oligosaccharides from the glycoprotein.

[0177] 25. The method of any one of embodiments 18 to 24, wherein the erythropoietic composition comprises about 27.4 area % to about 28.1 area % N-glycans of an erythropoiesis-stimulating glycoprotein having N-glycans with a tetraantennary +1 repeat structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0178] 26. The method of any one of embodiments 18-25, wherein the erythropoiesis composition has an average sialic acid content of about 10.5 to 11.0 moles of sialic acid per mole of erythropoiesis-stimulating glycoprotein.

[0179] 27. The method of any one of embodiments 18-26, wherein the erythropoiesis-stimulating glycoprotein is erythropoietin.

[0180] 28. A method for reducing the variability of the relative content of at least one glycosylated variant between batches of a recombinant glycoprotein composition, comprising the steps of: (a) providing a bioreactor comprising a pH measurement device configured to be disposed within the bioreactor and to be in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells expressing the glycoprotein; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing a glycoprotein composition, Here, the calibrating step of step (d) is (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH value calculated in step iii); Including, Steps (a) to (g) result in a first batch of recombinant glycoprotein having a defined relative content of glycosylated variants; (h) repeating steps (a)-(g) to produce at least one subsequent batch of glycoprotein, wherein the relative content of the at least one glycosylated variant of the first batch and the at least one subsequent batch has reduced batch-to-batch variability.

[0181] 29. The method according to embodiment 28, wherein the standard deviation of the relative glycoprotein content calculated for the first and at least one subsequent batch is not more than 1%, in particular not more than 0.8%, and most in particular not more than 0.5% of the median pH value.

[0182] 30. A method for measuring pH, wherein the measured pH value differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. and (c) use of a carbon dioxide-based method to calibrate a pH measuring device configured to be positioned within a bioreactor and in physical contact with the culture medium in the bioreactor to reduce the standard deviation of the relative content of glycosylated variants in a glycoprotein composition calculated between at least two fermentation batches compared to a glycoprotein composition produced using a carbon dioxide-based method.

[0183] 31. The use according to embodiment 30, wherein the carbon dioxide-based method for calibrating a pH measuring device comprises: (i) introducing a gas mixture comprising carbon dioxide gas into a bioreactor containing a culture medium; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust of the bioreactor; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). Including, use.

[0184] 32. The use according to embodiment 30 or 31, wherein carbon dioxide is introduced into the bioreactor by sparging.

[0185] 33. The use according to any one of embodiments 30 to 32, wherein the culture medium comprises a carbonate buffer.

[0186] 34. The use according to any one of embodiments 30 to 33, wherein the glycoprotein composition is an erythropoietic composition.

[0187] 35. The use according to embodiment 34, wherein the glycoprotein is an erythropoiesis-stimulating glycoprotein.

[0188] 36. The use according to embodiment 34 or 35, wherein the erythropoietic composition comprises a defined amount of at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: (a) Erythropoiesis-stimulating glycoprotein with biantennary N-glycans; (b) erythropoiesis-stimulating glycoprotein with triantennary N-glycans; (c) erythropoiesis-stimulating glycoprotein with N-glycans with triantennary +1 repeat structures; (d) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary structure; (e) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +1 repeat structure; (f) erythropoiesis-stimulating glycoprotein with N-glycans having a tetraantennary +2 repeat structure; (g) erythropoiesis-stimulating glycoprotein (isoform 2) with 14 sialic acid residues; (h) erythropoiesis-stimulating glycoprotein (isoform 3) with 13 sialic acid residues; (i) erythropoiesis-stimulating glycoprotein (isoform 4) with 12 sialic acid residues; (j) erythropoiesis-stimulating glycoprotein (isoform 5) with 11 sialic acid residues; (k) erythropoiesis-stimulating glycoprotein (isoform 6) with 10 sialic acid residues; (l) erythropoiesis-stimulating glycoprotein (isoform 7) with nine sialic acid residues; and (m) Erythropoiesis-stimulating glycoprotein (isoform 8) with eight sialic acid residues.

[0189] 37. The use according to any one of embodiments 34 to 36, wherein the erythropoietic composition comprises: (a) about 3.3 area% to about 3.8 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have biantennary structures; (b) about 8.6 area% to about 9.5 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have triantennary structures; (c) about 5.6 area% to about 5.9 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a triantennary + 1 repeat structure; (d) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (e) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 1 repeat structure; (f) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 2 repeat structure; (g) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; (h) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; (i) about 23.5 area% to about 24.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 4; (j) between about 17.1 area% and about 18.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 5; (k) between about 9.4 area% and about 11.8 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 6; (l) about 3.7 area% to about 5.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 7; and / or (m) about 0.9 area% to about 1.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 8; Preferably, (a) to (f) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein, and (g) to (m) are determined by capillary zone electrophoresis.

[0190] 38. The use according to any one of embodiments 34 to 37, wherein the erythropoietic composition comprises: (a) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (b) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 1 repeat structure; (c) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 2 repeat structure; (d) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; and / or (e) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; Preferably, (a) to (c) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein, and (d) to (e) are determined by capillary zone electrophoresis.

[0191] 39. The use according to any one of embodiments 34 to 38, wherein the erythropoietic composition comprises: (a) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (b) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary +1 repeat structure; and / or (c) use, wherein about 10.7 area % to about 11.6 area % of the N-glycans of the erythropoiesis-stimulating glycoprotein have tetraantennary +2 repeat structures, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0192] 40. The use of any one of embodiments 34-39, wherein about 27.4 area % to about 28.1 area % of the N-glycans of the erythropoiesis-stimulating glycoprotein in the erythropoiesis composition have a tetraantennary +1 repeat structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0193] 41. The use according to any one of embodiments 34 to 40, wherein the erythropoietic composition has an average sialic acid content of about 10.5 to 11.0 moles of sialic acid per mole of erythropoiesis-stimulating polypeptide.

[0194] 42. The use according to any one of embodiments 34 to 41, wherein the erythropoiesis-stimulating glycoprotein is erythropoietin.

[0195] 43. An erythropoietic composition comprising at least one erythropoiesis-stimulating glycoprotein, (a) about 3.3 area% to about 3.8 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have biantennary structures; (b) about 8.6 area% to about 9.5 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have triantennary structures; (c) about 5.6 area% to about 5.9 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a triantennary + 1 repeat structure; (d) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (e) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 1 repeat structure; (f) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 2 repeat structure; (g) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; (h) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; (i) about 23.5 area% to about 24.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 4; (j) between about 17.1 area% and about 18.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 5; (k) between about 9.4 area% and about 11.8 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein are isoform 6; (l) about 3.7 area% to about 5.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 7; and / or (m) about 0.9 area% to about 1.6 area% of the erythropoiesis-stimulating glycoprotein is isoform 8; Preferably, (a)-(f) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from glycoproteins, and (g)-(m) are determined by capillary zone electrophoresis.

[0196] 44. The erythropoietic composition according to embodiment 43, (a) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (b) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 1 repeat structure; (c) about 10.7 area% to about 11.6 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary + 2 repeat structure; (d) about 13.2 area% to about 16.0 area% of the erythropoiesis-stimulating glycoprotein is isoform 2; and / or (e) about 24.1 area% to about 26.5 area% of the erythropoiesis-stimulating glycoprotein is isoform 3; Preferably, (a)-(c) are determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from glycoproteins, and (d)-(e) are determined by capillary zone electrophoresis. 45. The erythropoietic composition according to embodiment 43 or 44, (a) about 42.2 area% to about 43.4 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary structure; (b) about 27.4 area% to about 28.1 area% of the N-glycans of the erythropoiesis-stimulating glycoprotein have a tetraantennary +1 repeat structure; and / or (c) an erythropoiesis-stimulating glycoprotein, wherein about 10.7 area % to about 11.6 area % of the N-glycans of said glycoprotein have a tetraantennary +2 repeat structure, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0197] 46. ​​The erythropoiesis-stimulating glycoprotein of any one of embodiments 43-45, wherein about 27.4 area% to about 28.1 area% of the N-glycans of the glycoprotein have tetraantennary +1 repeat structures, preferably as determined by anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from the glycoprotein.

[0198] 47. The erythropoietic composition of any one of embodiments 43 to 46, wherein the erythropoietic composition has an average sialic acid content of about 10.5 to 11.0 moles of sialic acid per mole of erythropoiesis-stimulating glycoprotein.

[0199] 48. The erythropoietic composition of any one of embodiments 43 to 47, wherein the erythropoietic composition is produced by a method according to any one of embodiments 1 to 20.

[0200] 49. A glycoprotein, in particular an erythropoiesis-stimulating glycoprotein, in particular erythropoietin, obtainable by a method according to any one of embodiments 1 to 29.

[0201] 50. A pharmaceutical composition comprising a glycoprotein produced by the method of any one of embodiments 1 to 29 or an erythropoietin according to any one of embodiments 43 to 48, together with pharmaceutical diluents, adjuvants and / or carrier agents. [Example]

[0202] The following are examples of methods and compositions of the present invention. Given the general description provided above, it will be understood that various other embodiments may be practiced.

[0203] Example 1 - Recombinant production of erythropoietin in CHO cells Erythropoietin (EPO) was produced in CHO cells in batch mode. For the main-stage fermentation, a carbonate-buffered serum-free culture medium consisting of basal medium DME(HG) HAM's F-12 modified (R5) (GRH Biosciences / Hazleton Biologics, Denver, USA, order number 57-736), sodium bicarbonate, L-(+) glutamine, D(+) glucose, recombinant insulin, sodium selenite, diaminobutane, hydrocortisone, iron(II) sulfate, asparagine, aspartic acid, serine, and polyvinyl alcohol was used as the culture medium. After sterilization according to GMP, the bioreactor was filled with culture medium and sparged with a defined gas mixture containing a defined fraction of carbon dioxide. During the sparging period, the pH control was set to inactive. The pH was determined by either a) withdrawing a sample through a sample port and transferring it to an offline analyzer to determine the sample's pH, or b) measuring the carbon dioxide concentration in the bioreactor exhaust and calculating the pH based on the applicable predetermined relationship between carbon dioxide concentration and the corresponding pH value. The bioreactor's pH probe signal was adjusted (by single-point calibration) to the pH determined in either procedure a) or b). The bioreactor was inoculated with an inoculum culture, and the pH control loop was set to active. During the fermentation phase, the pH was controlled and, if necessary, the culture pH was adjusted to a set point of pH 6.95 by adding 1 mol / L NaOH or increasing CO2 influx. After approximately 5 days, the fermenter contents were harvested. Intact CHO cells and cell fragments were removed from the fermentation supernatant by disk stack separation and discarded. The pH of the filtered cell-free culture supernatant was adjusted to pH 5.0–5.2 with acetic acid (2 mol / L) and incubated for several hours, followed by filtering the pH-adjusted solution at 1–9 °C.

[0204] Example 2 - Determination of medium-specific correlation between pH and corresponding exhaust carbon dioxide concentration for an EPO production process To empirically determine the medium-specific correlation between pH and corresponding exhaust carbon dioxide concentration for the fermentation process described in Example 1, a bioreactor was filled with the culture medium as described. Bioreactor pH was determined using an integrated online pH probe, initially calibrated at two points with buffer (4.00 and 7.00 at 25°C, Mettler Toledo). Carbon dioxide gas was used as an acidic pH corrector with pH control to maintain the pH in the upper deadband. To achieve constant carbon dioxide inflow, the pH controller was set as a proportional controller. Because no alkaline corrector was used, the pH naturally rose due to carbon dioxide removal by constant aeration with process air until the pH controller added carbon dioxide to maintain the pH in the upper deadband. After sterilization and medium filling, a hold step was defined to stabilize the pH, pressure, and temperature. To enable accurate pH readings, ports in the bioreactor lid were then opened, rendering the system non-sterile during the process. Two independent pH probes connected to respective pH meters were inserted into the bioreactor through the open ports to measure the pH in the liquid phase without sampling. Agitation, aeration, and temperature controls all remained active. Pressure control was set to disabled. The pH meter was independently calibrated at three points (calibration buffer pH 9.21, 7.00, and 4.00 at 25 °C) with active automatic temperature compensation (ATC). The average pH meter reading was used to adjust the online bioreactor pH probe signal (by single-point calibration). The maximum allowable difference in the pH meter signal was 0.02 units, and the maximum allowable difference from the buffer pH was 0.01 units. To detect unintended drift in the bioreactor probe signal, the same procedure was performed again after the experiment to check the bioreactor pH. After normalization of the bioreactor probe signal, the bioreactor lid was reclosed and pressure control was set to active. After establishing equilibrium (stable pH and carbon dioxide concentration in the exhaust), the first data point, online pH and the corresponding exhaust carbon dioxide concentration, were recorded.The correlation was determined in at least four technical replicates, meaning at least four independent bioreactors filled with the same medium, and a quadratic regression was fitted to obtain a final equation of the form: pH=ab*[CO2]+c*[CO2] 2

[0205] Four pH set points were determined in each bioreactor. After establishing equilibrium at each set point, the pH was also determined by sample-based offline measurements.

[0206] The medium-specific correlations for the production medium lots are shown in Figure 3 for three pressures: 20 mbar (top), 50 mbar (middle), and 135 mbar (bottom). Additionally, some data from the 10 L scale are shown (dots from the small scale, diamonds from the 10 L stainless steel bioreactor) that were used to demonstrate the scale independence of the determined pH.

[0207] Furthermore, the offset between sample-based offline pH values ​​and the pH in the bioreactor at production scale, as explained by the relationship shown in Figure 3, was determined to be 0.05 pH units in a large-scale validation study comparing bioreactor pH determined via exhaust carbon dioxide concentration with sample-based offline pH. A mean pH difference of 0.05 units was determined at all scales. To account for the determined offset, the pH setpoint for erythropoietin fermentation was lowered from pH 7.00 + / - 0.05 to 6.95 + / - 0.05. Therefore, the final equation for calculating pH based on exhaust carbon dioxide concentration yields the true pH, meaning a pH without any offset that may be introduced by sample-based offline measurements.

[0208] The quality of a model, i.e., the ability of the model to describe the modeled data points, can be measured, for example, by the regression coefficient R-squared (R 2 ) can be described by R 2A ρ of 1 means that all data points are perfectly described by the model. For all models, the R-squared was greater than 0.98. The root mean square error was 0.02 for all models. Taking into account potential errors in calibration, environmental factors, etc., the accuracy of the method is considered to be + / - 0.03 pH units.

[0209] Example 3 - Blue Sepharose Chromatography Erythropoietin polypeptides were purified essentially as described in WO 2010 / 34442. A chromatography column (Amicon P440x 500, Amicon, GB) was filled with approximately 500 L of Blue Sepharose and regenerated with 0.5 N NaOH. The column was then equilibrated with approximately 3 column volumes (CV) of equilibration buffer (20 mM Na acetate, 5 mM CaCl, 0.1 M NaCl, pH 5.0 ± 0.2). Erythropoietin binds to this support at low ionic strength and neutral to acidic pH values. The cell-free culture supernatant from Example 1 was absorbed onto the column, and the column was rewashed with approximately 1 CV of wash buffer 1 (20 mM Na acetate, 5 mM CaCl, 0.25 M NaCl, pH 5.0 ± 0.2), followed by approximately 2 CV of wash buffer 2 (20 mM TRIS-HCl, 5 mM CaCl, pH 6.9 ± 0.2). Erythropoietin was then eluted with approximately 2 CV of elution buffer (100 mM TRIS-HCl, 5 mM CaCl, 1 M NaCl, pH 9.0 ± 0.2) by increasing the ionic strength and pH. The entire protein peak was collected, adjusted to pH 6.9 with HCl, and stored for further processing.

[0210] Example 4 - Butyl Toyopearl Chromatography (Hydrophobic Chromatography) A chromatography column (Pharmacia BPG 300 / 500) was filled with approximately 200 liters of Butyl Toyopearl (Tosoh Haas) and regenerated with 4.6 M guanidine-HCl and 0.5 N NaOH. The column was then equilibrated with at least 3 CV of equilibration buffer (20 mM TRIS-HCl, 5 mM CaCl2, 0.75 M NaCl, 10% 2-propanol, pH 6.9 ± 0.2). The eluate from the Blue Sepharose column in Example 3 was adjusted to 10% 2-propanol and absorbed onto the column. The column was rewashed with approximately 1 CV of equilibration buffer and then with approximately 2 CV of wash buffer (20 mM TRIS-HCl, 5 mM CaCl2, 0.75 M NaCl, 19% 2-propanol, pH 6.9 ± 0.2). Erythropoietin was then eluted with approximately 2 CV of elution buffer (20 mM TRIS-HCl, 5 mM CaCl, 0.75 M NaCl, 27% 2-propanol, pH 6.9 ± 0.2). The entire protein peak was collected and immediately diluted 3-fold with dilution buffer (20 mM TRIS-HCl, 5 mM CaCl, pH 6.9 ± 0.2) and stored until further processing.

[0211] Example 5 - Hydroxyapatite Ultrogel Chromatography A chromatography column (Sartorius 100x50cm, Sartorius GB) was packed with approximately 200 L of hydroxyapatite Ultrogel and regenerated with regeneration buffer 1 (0.2 M potassium phosphate, 0.1 mM CaCl2, pH 6.9±0.2), followed by 0.5 N NaOH. The column was then equilibrated with approximately 3 CV of equilibration buffer (20 mM Tris-HCl, 5 mM CaCl2, 0.25 M NaCl, 9% isopropanol, pH 6.9±0.2). The eluate from Example 4 was adsorbed onto the column. The column was rewashed with approximately 1 CV of equilibration buffer and then with approximately 2 CV of wash buffer (10 mM Tris-HCl, 5 mM CaCl2, pH 6.9±0.2). It was then eluted with approximately 2.5 CV of elution buffer (10 mM Tris-HCl, 0.5 mM CaCl2, 10 mM potassium phosphate pH 6.9±0.2). The entire protein peak was collected and stored for further processing.

[0212] Example 6 - Reverse Phase HPLC (RP-HPLC) The eluate from Example 5 was filtered through a nanofilter to remove viruses. Preparative HPLC was performed using a Merck Prepbar 100 separation system (or equivalent). A separation column (30 × 50 cm) was packed with Vydac C4 material (Grace, USA). Prior to use, the column was regenerated by applying a gradient of Buffer A (0.1% trifluoroacetic acid in water) to 100% solvent several times, followed by equilibration with Buffer A. The eluate from the hydroxyapatite column was acidified to approximately pH 2.5 with trifluoroacetic acid and filter-sterilized. Erythropoietin was then absorbed onto the column at a temperature of 22 ± 4°C and a flow rate of 2700 ml / min. The column was eluted with a linear gradient from Buffer A to Buffer B (80% acetonitrile, 0.1% trifluoroacetic acid in water) at the same temperature and flow rate. The elution peak was collected in fractions. The eluate was immediately neutralized by adding 7 volumes of HPLC dilution buffer (10 mM Na / K phosphate, pH 7.5±0.2). Fractions with a purity of at least 99% by analytical HPLC were pooled (pool volume approximately 60 l).

[0213] Example 7 - DEAE Sepharose FF Chromatography A chromatography column (Merck Quickscale 25 x 50 cm) was filled with 7.5 L of DEAE Sepharose Fast Flow (Cytiva) gel per gram of erythropoietin in the applied sample and regenerated with 1 M NaOH. The column was then equilibrated with 100 mM NaKPO (pH 7.5 ± 0.2) and then equilibrated with at least 10 CV of 10 mM sodium / potassium phosphate (pH 7.5 ± 0.2). The eluate from the HPLC column from Example 6 was absorbed onto the column, and the column was washed with at least 5 CV of equilibration buffer and then approximately 10 CV of wash buffer (30 mM Na acetate, pH 4.5 ± 0.1). The column was then washed again with approximately 10 CV of equilibration buffer, and erythropoietin was eluted with approximately 4 CV of elution buffer (10 mM sodium / potassium phosphate, 80 mM NaCl, pH 7.5 ± 0.2). The entire protein peak was collected, and the conductivity and pH of the DEAE eluate was adjusted, sterilized by filtration, aliquoted, and stored.

[0214] Example 8 - Analytical methods used for product characterization sugar analysis An enzymatic test procedure was used to determine the relative content of N-glycans. In this procedure, N-glycosidically linked oligosaccharides of erythropoietin were cleaved by the enzyme N-glycosidase F. Furthermore, terminal sialic acids were removed from the oligosaccharides with the aid of the enzyme neuraminidase. After removing the protein fraction via ultrafiltration, the resulting N-oligosaccharides were separated and analyzed using a high-performance anion-exchange chromatography system with pulsed amperometric detection (HPAEC-PAD) and an appropriate data recording system. An erythropoietin reference standard, processed in exactly the same way as the samples, was measured during the series of tests. The area percentage of each glycosylated variant in the preparation was calculated from the corresponding peak in the chromatogram of the produced glycans (e.g., biantennary, triantennary, triantennary + 1 repeat, tetraantennary, tetraantennary + 1 repeat, tetraantennary + 2 repeat).

[0215] Determination of isoform distribution The relative content of isoforms was determined by capillary zone electrophoresis. In this method, separation was performed at a constant electric field strength in an uncoated glass capillary with a homogeneous buffer system, the pH of which was above the pI value of the EPO isoforms. All EPO isoforms are negatively charged and are transported toward the cathode by inward osmotic flow, resulting in the first detection of basic isoforms. For analysis, samples were diafiltered in water. The capillary was rinsed with electrolyte, and the diluted sample was then applied to the capillary. Separation was achieved by applying a high voltage of 25,000 V. The mobile buffer had an excess of cations, resulting in electroosmotic flow. The use of a quartz capillary allowed photometric detection of proteins in the capillary using a diode array detector and quantitative determination of the area under the peaks [relative area - %] by integrating the peaks corresponding to isoforms 1 through 9.

[0216] Determination of the content of sialic acid residues The sialic acid content was determined chromatographically by HPAEC-PAD after enzymatic cleavage of sialic acid with neuraminidase. For this purpose, erythropoietin samples were diluted with 5 mM Na phosphate buffer (pH 7.2). Half of each preparation was used to determine the EPO content by RP-HPLC. Neuraminidase was added to the second half of the preparation and incubated overnight at 37°C. Subsequently, the digestion mixture was divided in half, diluted with water, and 50 μl of the mixture was applied to HPAEC-PAD. The amount of sialic acid in the applied sample was determined using a calibration line obtained from the values ​​of similarly analyzed sialic acid standards. The sialic acid content (mol sialic acid / mol EPO) was calculated from the results of the sialic acid determination and the EPO usage amount determination by RP-HPLC.

[0217] Exclusion Chromatography (SE-HPLC) For SE-HPLC, before starting the separation, the chromatographic column was equilibrated with 3–5 column volumes (CV) of buffer to obtain a uniform baseline without additional peaks. The sample was diluted to a concentration of 0.2 mg / mL and injected into the SE-HPLC. Peaks were integrated according to standard methods. Peaks were separated from each other by dropping a perpendicular line.

[0218] Peptide Map Incompletely glycosylated erythropoietin species, such as de-O-EPO and de-N-EPO, can be quantitatively determined by peptide mapping. To this end, erythropoietin molecules were cleaved into peptides by endoproteinase Lys-C, and these peptides were separated by HPLC. The resulting peptide patterns were compared with reference standards. The results were compared with the standards in terms of peak size, peak appearance, and retention time.

[0219] Example 9 - Effect of pH determination on product quality To compare the effectiveness of different methods for pH calibration and determination, 10 batches of erythropoietin were produced as described in Examples 1-8 using the pH determination method according to the present invention. For standardization of the bioreactor pH-probe signal, the culture medium filled in the bioreactor after sterilization was then equilibrated with a defined gas mixture (93% process air and 7% CO2) until saturation. After the equilibration phase, the pH in the culture medium was calculated from the exhaust carbon dioxide concentration based on the medium-specific correlation determined according to the method described in Example 2. The bioreactor probe signal was then adjusted to the calculated pH value.

[0220] As a control, 10 batches produced according to the same manufacturing process were used, but the calibration of the pH probe in the fermenter was performed offline according to standard methods known in the art. For this, after sterilization of the fermenter, culture medium was added to the fermenter, a sample was removed, and the sample pH was determined offline using a three-point calibrated pH meter as described in Example 2. The in-line pH probe was then adjusted to the pH value measured offline. One of the control runs had to be terminated prematurely and sampled, and was therefore excluded for yield analysis. Table 2. Yields of erythropoietin products TIFF2025536587000003.tif32170

[0221] As can be seen in Table 2, the variability in product yield is reduced for runs in which the carbon dioxide-based calibration method was used to adjust the in-line pH probe. The standard deviation of the nine control runs with sample-based offline pH adjustments is much higher compared to the ten runs using the carbon dioxide-based method.

[0222] The results of the determination of sialylated isoforms 2 to 8 using capillary zone electrophoresis (analysis method described in Example 8) are shown in Table 3. Figure 4 shows an exemplary visual display of the results for isoform 2. The range between the measured minimum and maximum values, and therefore the standard deviation, is also significantly reduced for many parameters. Table 3. Sialylation of erythropoietin TIFF2025536587000004.tif92170

[0223] The results of the determination of desialylated N-glycans using anion exchange chromatography after enzymatic release and desialylation of N-glycoside-linked oligosaccharides from glycoproteins (analytical method described in Example 8) are shown in Table 4. Figure 5 shows an exemplary visual representation of the results for N-glycans with tetraantennary +1 repeats. Similar to the sialylated isoforms, the range between the measured minimum and maximum values, and therefore the standard deviation, is also significantly reduced for many parameters. Table 4. Glycosylation of erythropoietin TIFF2025536587000005.tif92170

[0224] The foregoing invention has been described in some detail by way of illustration and example, for purposes of clarity of understanding, but the illustrations and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entireties.

Claims

1. 1. A method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein, comprising the steps of: Culturing recombinant host cells expressing the glycoprotein in a culture medium in a bioreactor having a pH measuring device disposed within the bioreactor and configured to be in physical contact with the culture medium, (a) the culturing is carried out under sterile conditions; (b) the pH value measured by the pH measuring device differs from the pH value of the culture medium by no more than 0.05 units; and (c) the relative content of at least one glycosylated variant in said glycoprotein composition is wherein the pH value measured by said pH measuring device differs from the pH value in said culture medium by more than 0.05 units, preferably by more than 0.03 units. has reduced batch-to-batch variability compared to a process characterized by: thereby producing said glycoprotein composition.

2. 1. A method for recombinantly producing a glycoprotein composition comprising at least one glycoprotein in a recombinant host cell that expresses said glycoprotein, comprising: (a) providing a bioreactor comprising a pH measuring device configured to be disposed within the bioreactor and in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with the recombinant host cells; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein composition; and (g) thereby producing said glycoprotein composition. and the calibrating step of step (d) comprises: (i) introducing a gas mixture comprising carbon dioxide gas into the bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH value calculated in step (iii); A method comprising:

3. 3. The method of claim 2, wherein the pH value measured by the pH measuring device after the calibration step differs from the pH value of the culture medium by no more than 0.05 units, preferably no more than 0.03 units.

4. the glycoprotein composition comprises at least one glycosylated variant of the glycoprotein; and the standard deviation of the relative content of said glycosylated variants calculated for glycoprotein compositions from at least two fermentation batches is wherein the pH value measured by said pH measuring device differs from the pH value in said culture medium by more than 0.05 units, preferably by more than 0.03 units. The standard deviation of the relative content of the glycosylated variants calculated for the glycoprotein composition produced using The method according to any one of claims 1 to 3, characterized in that

5. The method according to any one of claims 1 to 4, wherein the recombinant host cell is a mammalian cell, preferably a CHO cell.

6. The method of claim 4 or 5, wherein the glycosylation variant is selected from the group consisting of an N-glycan variant, an O-glycan variant, a sialylation variant, a mannosylation variant, a galactosylation variant and a fucosylation variant.

7. The method of any one of claims 1 to 6, wherein the culture medium comprises a carbonate buffer system.

8. 8. The method of any one of claims 1 to 7, comprising maintaining the pH in the culture medium at a desired set point.

9. The method of any one of claims 1 to 8, wherein the glycoprotein composition is an erythropoietic composition and the glycoprotein is an erythropoiesis-stimulating glycoprotein.

10. The erythropoietic composition comprises a defined amount of: (a) an erythropoiesis-stimulating glycoprotein having an N-glycan with a biantennary structure; (b) an erythropoiesis-stimulating glycoprotein having a triantennary N-glycan; (c) an erythropoiesis-stimulating glycoprotein having an N-glycan with a triantennary +1 repeat structure; (d) erythropoiesis-stimulating glycoproteins having tetraantennary N-glycans; (e) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary +1 repeat structure; (f) an erythropoiesis-stimulating glycoprotein having an N-glycan with a tetraantennary + 2 repeat structure; (g) erythropoiesis-stimulating glycoprotein (isoform 2) with 14 sialic acid residues; (h) erythropoiesis-stimulating glycoprotein (isoform 3) with 13 sialic acid residues; (i) erythropoiesis-stimulating glycoprotein (isoform 4) with 12 sialic acid residues; (j) erythropoiesis-stimulating glycoprotein (isoform 5) with 11 sialic acid residues; (k) erythropoiesis-stimulating glycoprotein (isoform 6) with 10 sialic acid residues; (l) erythropoiesis-stimulating glycoprotein (isoform 7) having nine sialic acid residues, and / or (m) erythropoiesis-stimulating glycoprotein (isoform 8) with eight sialic acid residues at least one glycosylated variant of an erythropoiesis-stimulating glycoprotein selected from the group consisting of: the standard deviation of the relative content of said glycosylated variants calculated for the erythropoietic composition from at least two fermentation batches is: wherein the measured pH value differs from the pH value in the culture medium by more than 0.05 units, preferably by more than 0.03 units. The standard deviation of the relative content of glycosylated variants calculated for the erythropoietic composition produced using The method of claim 9 further characterized by:

11. 10. The erythropoietic composition comprising: (a) an erythropoiesis-stimulating glycoprotein having about 3.3 area % to about 3.8 area % N-glycans with biantennary structures; (b) an erythropoiesis-stimulating glycoprotein having about 8.6 area % to about 9.5 area % triantennary N-glycans; (c) an erythropoiesis-stimulating glycoprotein having about 5.6 area % to about 5.9 area % N-glycans with triantennary +1 repeat structures; (d) an erythropoiesis-stimulating glycoprotein having about 42.2 area % to about 43.4 area % N-glycans with tetraantennary structures; (e) an erythropoiesis-stimulating glycoprotein having about 27.4 area % to about 28.1 area % N-glycans with a tetraantennary +1 repeat structure; (f) an erythropoiesis-stimulating glycoprotein having about 10.7 area % to about 11.6 area % N-glycans with a tetraantennary + 2 repeat structure; (g) about 13.2 area% to about 16.0 area% of erythropoiesis-stimulating glycoprotein isoform 2; (h) about 24.1 area% to about 26.5 area% of erythropoiesis-stimulating glycoprotein isoform 3; (i) about 23.5 area% to about 24.6 area% of erythropoiesis-stimulating glycoprotein isoform 4; (j) about 17.1 area% to about 18.6 area% of erythropoiesis-stimulating glycoprotein isoform 5; (k) about 9.4 area% to about 11.8 area% of erythropoiesis-stimulating glycoprotein isoform 6; (l) about 3.7 area% to about 5.5 area% of erythropoiesis-stimulating glycoprotein isoform 7, and / or (m) about 0.9 area % to about 1.6 area % of erythropoiesis-stimulating glycoprotein isoform 8 11. The method according to claim 9 or 10, characterized in that it comprises one or more selected from the group consisting of:

12. The method of any one of claims 1 to 11, wherein the glycoprotein is erythropoietin.

13. 1. A method for reducing the variability in the relative content of at least one glycosylated variant between batches of a recombinant glycoprotein, comprising the steps of: (a) providing a bioreactor comprising a pH measuring device configured to be disposed within the bioreactor and in physical contact with the culture medium; (b) closing and sterilizing the bioreactor; (c) charging the culture medium into the bioreactor; (d) calibrating the pH measuring device; (e) inoculating the bioreactor with recombinant host cells expressing the glycoprotein; (f) culturing the recombinant host cell under conditions suitable for producing the glycoprotein; and (g) thereby producing said glycoprotein, Here, the calibrating step of step (d) is (i) introducing a gas mixture comprising carbon dioxide gas into the bioreactor; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust gas of the bioreactor; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; (iv) adjusting the pH measuring device to the pH value calculated in step (iii); Including, Steps (a) to (g) result in a first batch of said recombinant glycoprotein having a defined relative content of said glycosylated variants; process, (h) repeating steps (a)-(g) to produce at least one subsequent batch of said glycoprotein, wherein the relative content of at least one glycosylated variant of said first batch and at least one subsequent batch has reduced batch-to-batch variability.

14. Use of a carbon dioxide-based method for calibrating a pH measuring device, the pH measuring device being positioned within a bioreactor and configured to be in physical contact with a culture medium within the bioreactor, to reduce the standard deviation of the calculated relative content of at least one glycosylated variant in a glycoprotein composition between at least two fermentation batches.

15. The carbon dioxide-based method comprises: (i) introducing a gas mixture comprising carbon dioxide gas into the bioreactor containing the culture medium; (ii) determining the carbon dioxide concentration in the headspace and / or exhaust gas of the bioreactor; (iii) calculating the pH value of the culture medium based on a medium-specific correlation; and (iv) adjusting the pH measuring device to the pH value calculated in step (iii). The use according to claim 14, comprising:

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