A method for producing recombinant gonadotropins including corifollitropin ALFA (CFA), recombinant human chorionic gonadotropin (r-HCG), and recombinant human follicle-stimulating hormone (r-HFSH) in mammalian cell culture systems

Abstract

The invention provides optimized upstream processes for the production of recombinant gonadotropins including corifollitropin alfa (CFA), recombinant human chorionic gonadotropin (r-hCG), and recombinant human follicle- stimulating hormone (r-hFSH) in mammalian cell culture. The processes comprise revival of CHO cells from cryopreservation, expansion in shake flasks, seed generation in wave bioreactors, and production in stirred-tank bioreactors operated in fed-batch mode. Defined media supplemented with additives such as N-acetyl mannosamine, or stabilized glutamine enhance cell viability, productivity, and glycosylation quality. Cultures are terminated at defined viability thresholds or timepoints, and clarified by sequential depth and membrane filtration. The methods offer scalability, reproducibility, and superior product quality compared to conventional approaches, enabling consistent large-scale manufacture of therapeutic gonadotropins for fertility treatments and hormone therapies.

Description

[0001] A METHOD FOR PRODUCING RECOMBINANT GONADOTROPINS INCLUDING CORIFOLLITROPIN ALFA (CFA), RECOMBINANT HUMAN CHORIONIC GONADOTROPIN (R-HCG), AND RECOMBINANT HUMAN FOLLICLE- STIMULATING HORMONE (R-HFSH) IN MAMMALIAN CELL CULTURE SYSTEMS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biotechnology and biopharmaceutical manufacturing. More specifically, it concerns optimized upstream processes for the production of recombinant gonadotropins including corifollitropin alfa (CFA), recombinant human chorionic gonadotropin (r-hCG), and recombinant human follicle- stimulating hormone (r-hFSH). The invention utilizes mammalian cell culture systems, particularly Chinese Hamster Ovary (CHO) cell lines such as CHO-DG44 and CHO- DUXB11, to provide scalable, reproducible, and high-yield processes for therapeutic protein production.

[0004] BACKGROUND OF THE INVENTION

[0005] Recombinant gonadotropins such as CFA, r-hCG, and r-hFSH play a critical role in fertility management and hormone therapies. Conventional methods for producing these proteins rely heavily on downstream purification to address variability in upstream processes, often resulting in suboptimal yields, inconsistent glycosylation, and limited scalability.

[0006] Corifollitropin alfa (CFA), a hybrid molecule comprising the alpha subunit of hCG and the beta subunit of FSH, offers the advantage of prolonged half-life and reduced dosing frequency in controlled ovarian stimulation. Recombinant hCG is widely used for ovulation induction and luteal phase support, whereas recombinant hFSH is essential in stimulating follicle development and spermatogenesis.

[0007] Existing upstream processes for these gonadotropins lack uniformity across products, often requiring extensive optimization, and do not consistently provide reproducible high- density cultures with desired viability. Furthermore, current strategies may not adequately balance metabolic supplementation with process control, leading to premature loss of viability and reduced product quality.

[0008] Thus, there remains a need for robust and harmonized upstream processes that can reliably produce different gonadotropins at commercial scale, while maintaining high yield, purity, and bioactivity.

[0009] SUMMARY OF THE INVENTION

[0010] The invention addresses these challenges by providing a structured five-stage upstream process adaptable for multiple recombinant gonadotropins. The stages include:

[0011] REVIVAL OF CELLS: Thawing cryopreserved CHO cell lines under controlled conditions and resuspending in a chemically defined / protein-free medium supplemented with glutamine or derivatives.

[0012] EXPANSION IN SHAKE FLASKS: Culturing cells under controlled temperature, CO2 concentration, and agitation to achieve target densities and viability.

[0013] SEED GENERATION IN WAVE BIOREACTORS: Expanding cultures under defined rocking and aeration parameters until high viability and density are achieved.

[0014] PRODUCTION IN STIRRED-TANK SINGLE-USE BIOREACTORS: Fed-batch culture with a chemically defined / protein-free medium supplemented with additives such as N-acetyl mannosamine ( ManNAc), or glutamine, under controlled temperature, pH, dissolved oxygen, and agitation conditions.

[0015] TERMINATION AND HARVEST CLARIFICATION: Concluding the culture when viability thresholds are reached or by a set duration, followed by depth and membrane filtration to obtain clarified harvest for downstream purification.

[0016] The invention further provides specific embodiments for CFA, r-hCG, and r-hFSH, incorporating tailored media, supplements, feeding strategies, and process controls. These adaptations improve glycosylation fidelity, prolong cell viability, and enhance overall productivity. The methods described herein ensure scalability, reproducibility, and robustness, enabling consistent production of therapeutic-grade gonadotropins suitable for clinical applications in fertility and hormone replacement therapies.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure la: Upstream process steps details for production of Corifollitropin alfa (CFA) Figure lb: Flow chart illustrating the upstream process for production of corifollitropin alfa (CFA).

[0019] Figure 2a: Upstream process steps details for production of recombinant human chorionic gonadotropin (r-hCG).

[0020] Figure 2b: Flow chart illustrating the upstream process for production of recombinant human chorionic gonadotropin (r-hCG).

[0021] Figure 3 a: Upstream process steps details for production of recombinant human follicle- stimulating hormone (r-hFSH).

[0022] Figure 3b: Flow chart illustrating the upstream process for production of recombinant human follicle-stimulating hormone (r-hFSH).

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention relates to optimized upstream processes for the production of recombinant gonadotropins, namely corifollitropin alfa (CFA), recombinant human chorionic gonadotropin (r-hCG), and recombinant human follicle-stimulating hormone (r-hFSH). These processes broadly follow a five-stage framework — revival of a mammalian cell line from a cell bank, expansion in shake flasks, seed generation in a wave bioreactor, production in a stirred-tank bioreactor, and harvest clarification — while incorporating product-specific media, supplements, and optimized parameters to maximize viability, yield, and purity.

[0025] For the purposes of the present invention, the following terms are defined: “CHO-DG44” refers to a dihydofolate reductase (DHFR) -deficient Chinese hamster ovary (CHO) cell line widely used for recombinant protein production due to its adaptability to defined medium and high productivity after gene amplification.

[0026] “CH0-DUXB11” refers to a dihydrofolate reductase (DHFR)-defi cient CHO cell line, suitable for recombinant protein production due to its adaptability to defined medium and high productivity after gene amplification.

[0027] “Corifollitropin alfa (CFA)” refers to a long-acting recombinant follicle-stimulating hormone (FSH) analog with an extended half-life compared to conventional rFSH, designed for controlled ovarian stimulation.

[0028] “Recombinant human chorionic gonadotropin (r-hCG)” refers to a glycoprotein hormone produced in mammalian cells, structurally and functionally equivalent to natural hCG, and used clinically to trigger ovulation.

[0029] “Recombinant human follicle-stimulating hormone (r-hFSH)” refers to a recombinant glycoprotein hormone used in fertility treatments to stimulate ovarian follicle development or spermatogenesis.

[0030] “Defined medium” refers to a cell culture medium in which all chemical components are known and specified, typically free of animal-derived components. Examples include MAM-PF77, ProCHO5, CD OptiCHO, 4Cell XtraCHO, Dynamis, CDM4 MAb or equivalents.

[0031] “Glutamine” refers to a stabilized dipeptide derivative of glutamine, serving as a sustained source of L-glutamine in cell culture to minimize ammonia buildup.

[0032] “N-acetyl mannosamine (ManNAc)” refers to a metabolic precursor of sialic acid biosynthesis, enhancing terminal glycosylation and bioactivity of recombinant glycoproteins. “Wave bioreactor” refers to a rocking-motion bioreactor system designed for seed expansion, where cells are cultured in disposable bags with controlled rocking, aeration, gas exchange & other parameters like temperature, pH etc.

[0033] “Stirred-tank reactor (STR)” or “stirred-tank bioreactor” refers to a mechanically agitated, typically single-use, vessel employed for fed-batch production under controlled parameters of temperature, pH, dissolved oxygen, and agitation.

[0034] “Fed-batch” refers to a bioprocess mode in which nutrients are intermittently or continuously added during the culture to sustain productivity without complete medium replacement.

[0035] “Depth filtration” refers to the use of porous filter media (e.g., cellulose with diatomaceous resin) for harvest clarification by retaining particulates and host-cell debris within the filter matrix.

[0036] “Polyether sulfone (PES) filtration” refers to sterile membrane filtration using an absolute pore size, typically 0.22 pm, to remove particulates, microorganisms, and process impurities after depth filtration.

[0037] “Viability” refers to the percentage of live cells relative to total cells in culture, typically measured by trypan blue exclusion or equivalent viability assays.

[0038] “Air overlay” refers to the controlled introduction of sterile air above the culture surface in a bioreactor to aid in gas exchange and pH control.

[0039] Unless otherwise indicated, the singular forms “a,” “an,” and “the” include plural referents. The terms “comprising,” “including,” and “having” are used in an open-ended sense and do not exclude additional elements or steps. As illustrated in FIGURES 1-3, the overall process includes thawing and revival of the selected CHO cell line in defined medium; expansion under controlled conditions to achieve taigeted densities and viability; seed generation in a wave bioreactor; fed-batch production in a stirred-tank single-use bioreactor with nutrient supplementation; process termination at defined viability thresholds or timepoints; and harvest clarification by depth and membrane filtration. Each stage is controlled for temperature, pH, dissolved gases, and agitation to provide reproducible and scalable protein production.

[0040] In one embodiment, the invention provides a method for producing a recombinant gonadotropin selected from corifollitropin alfa (CFA), recombinant human chorionic gonadotropin (r-hCG), and recombinant human follicle-stimulating hormone (r-hFSH) in a mammalian cell culture.

[0041] The method comprises:

[0042] (i) reviving a mammalian CHO cell line selected from CHO-DG44 or CHO-DUXB 11 in a chemically defined protein free medium supplemented with glutamine;

[0043] (ii) expanding the revived cells in shake flasks under controlled temperature, CO2 concentration, and agitation conditions to achieve a viable cell density greater than 1.2 x 106cells / mL with >80% viability;

[0044] (iii) generating seed in a wave bioreactor under controlled process parameters including gas flow between 0. 1-0.3 L / min, rocking speed between 13-20 rpm, rocking angle of 7°-10°, and CO2 concentration of approximately 5%;

[0045] (iv) transferring the cells into a stirred-tank production bioreactor seeded at a density between 0.25-0.35 x 106and 0.45-0.55 x 106cells / mL, depending on the gonadotropin;

[0046] (v) producing the recombinant gonadotropin in the stirred-tank bioreactor using a defined medium supplemented with one or more additives selected from N-acetyl mannosamine (ManNAc), and glutamine, under process parameters comprising a temperature of 36.5-37.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, agitation of approximately 10 W / m3, and 5% air overlay;

[0047] (vi) feeding the culture during production with a nutrient feed selected from Feed 4 or equivalent, supplemented with additives like ManNAc, and maintaining glucose between 2.5-4.0 g / L and glutamine between 2.5-6.0 mM; and

[0048] (vii) harvesting the culture upon reduction of viability below a threshold between 60% and 80% or at a predetermined culture duration, followed by clarification using depth filtration and subsequent 0.22 pm polyether sulfone filtration.

[0049] The generalized framework described above provides a consistent and scalable upstream process. In certain embodiments, this framework is adapted to specific gonadotropins, as described in detail below with reference to FIGURES la-3b.

[0050] CORIFOLLITROPIN ALFA (CFA)

[0051] As illustrated in Figures la and lb, the upstream process for production of corifollitropin alfa (CFA) comprises similar steps to the generalized method, with product-specific conditions.

[0052] In one embodiment, CHO-DG44 cells are revived in a chemically defined, protein-free medium (such as MAM-PF77) supplemented with 8 mM L-glutamine. Thawing is performed at 37 °C for 1-2 minutes, optionally followed by centrifugation at 800 rpm for 8 minutes, and resuspension in fresh medium. Cells are expanded in shake flasks under controlled incubation parameters, including a temperature of 36.5-37.5°C, CO2 concentration of 4.5-5.5%, and agitation between 80-110 rpm, until a viable cell density greater than 1.2-1.5 x 106cells / mL with >80% viability is achieved.

[0053] Seed generation is performed in a wave bioreactor at 36.5-37.5 °C, with gas flow between 0.1-0.3 L / min, rocking speed of 13-20 rpm, rocking angle of 7-10°, and CO2 maintained at approximately 5%, until cell density exceeds 1.6 x 106cells / mL with >80% viability.

[0054] The production bioreactor is seeded at 0.25-0.35 x 106cells / mL using MAM-PF77 supplemented with 8 mM glutamine and 10 mM N-acetyl mannosamine. Process conditions comprise 37 ± 0.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, agitation 10 W / m3, and a 5% air overlay. A chemically defined nutrient feed containing 10 mM N- acetyl mannosamine is added at 3% on Days 3, 5, 7, 9, 10, and 11, while glucose is maintained at 2.5-4.0 g / L and glutamine between 2.5-4.0 mM. The culture is terminated at Day 12 or when viability falls below 80%, whichever occurs first. Clarification is performed by depth filtration with cellulose / diatomaceous resin followed by 0.22 pm PES filtration.

[0055] In another embodiment the present invention describes a method for producing corifollitropin alfa (CFA), comprising: (i) culturing CHO-DG44 cells in chemically defined / protein free medium supplemented with 8 mM L-glutamine and 10 mM N-acetyl mannosamine ; (ii) expanding the cells to a density greater than 1.5 x 106cells / mL with >80% viability in shake flasks and a wave bioreactor; (iii) producing CFA in a stirred- tank single-use bioreactor operated at pH 6.95-7.05, dissolved oxygen 30-40%, and agitation 10 W / m3, with periodic addition of Feed 4 containing N-acetyl mannosamine on Days 3, 5, 7, 9, 10 and 11; and (iv) terminating production at Day 12 or when viability falls below 80%, followed by harvest and clarification using cellulose / diatomaceous resin depth filtration and 0.22 pm polyether sulfone filtration.

[0056] The CFA produced is suitable for therapeutic use, including administration as a long- acting gonadotropin in fertility treatment.

[0057] RECOMBINANT HUMAN CHORIONIC GONADOTROPIN (r-hCG)

[0058] As illustrated in Figures 2a and 2b, the upstream process for r-hCG follows the same five- stage framework but incorporates specific supplements and conditions to optimize glycosylation and yield.

[0059] In another embodiment, CHO DUXB11 cell line is revived in a chemically defined, protein-free medium (such as MAM-PF77, ProCHO5, CD OptiCHO, 4Cell XtraCHO, Dynamis, CDM4 Mab ) supplemented with 6 mM L-glutamine. Cells are thawed at 37 °C for 1-2 minutes and cultured under conditions of 36.5-37.5 °C, CO2 4.5-5.5%, and 80-110 rpm until cell density exceeds 1.2-1.5 x 106cells / mL with >80% viability. Seed generation in the wave bioreactor is carried out at 36.5-37.5 °C, oxygen / air flow of 0.1-0.3 L / min, rocking speed of 13-20 rpm at a 7° angle, and 5% CO2 until cell density exceeds 1.6 x 106cells / mL and viability >80%.

[0060] Production is performed in a stirred-tank bioreactor seeded at 0.45-0.55 x 106cells / mL, using ProCHO5 medium supplemented with 6 mM glutamine. Process conditions include 37 ± 0.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, agitation 10 W / m3, and 5% air overlay. A chemically defined nutrient feed (such as Feed 4) supplemented with glutamine is added at 4% on Days 3, 5, 7, and 9. Glucose is maintained at 2.5-4.0 g / L and glutamine between 2.5-6.0 mM. Cultures are terminated at Day 12 or when viability falls below 80%. Harvest is clarified by cellulose / diatomaceous depth filtration followed by 0.22 pm PES filtration.

[0061] In another embodiment the present invention describes a method for producing recombinant human chorionic gonadotropin (r-hCG), comprising: (i) culturing CHO DUXB 11 cell line in chemically defined / protein free medium supplemented with 6 mM L-glutamine; (ii) expanding the cells in shake flasks and a wave bioreactor to >1.6 x 106cells / mL with >80% viability; (iii) producing r-hCG in a stirred-tank single-use bioreactor seeded at 0.25-0.35 x 106cells / mL, maintained at 37 ± 0.5 °C, pH 6.95-7.05, pCE 30- 40%, and agitation 10 W / m3; (iv) feeding with Feed 4 supplemented with glutamine at 4% on Days 3, 5, 7, and 9; and (v) clarifying the harvest using depth filtration with cellulose / diatomaceous resin followed by 0.22 pm polyether sulfone filtration.

[0062] The r-hCG produced demonstrates purity >95% and is suitable for therapeutic administration, including induction of ovulation and luteal support.

[0063] RECOMBINANT HUMAN FOLLICLE-STIMULATING HORMONE (r-hFSH)

[0064] As illustrated in Figures 3a and 3b, the upstream process for r-hFSH is generally consistent with the framework described above, but is distinguished by higher seeding densities and an earlier termination threshold. This difference reflects the need to prevent metabolite accumulation, thereby improving protein integrity.

[0065] In yet another embodiment, CH0-DUXB11 cells are revived in a chemically defined, protein-free medium (such as MAM-PF77, ProCHO5, CD OptiCHO, 4Cell XtraCHO, Dynamis, CDM4 Mab ) supplemented with 4 mM glutamine. Cells are thawed at 37 °C for 1-2 minutes and expanded in shake flasks and wave bioreactors at 36.5-37.5 °C, CO2 4.5-5.5%, and 95-105 rpm until cell density exceeds 1.5 x 106cells / mL with >80% viability.

[0066] Seed generation is performed in a wave bioreactor with gas flow between 0.1-0.3 L / min, rocking at 13-15 rpm, rocking angle of 7°, and 5% CO2 until cell density exceeds 1.5 x 106cells / mL. The production bioreactor is seeded at 0.45-0.55 x 106cells / mL using chemically defined / protein free medium supplemented with 4 mM glutamine. Process conditions include 36.5-37.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, agitation 10 W / m3, and 5% air overlay. The production process is terminated when viability falls below 60%. Harvest clarification is performed using cellulose-based depth filtration followed by 0.22 pm PES membrane filtration.

[0067] In another embodiment present invention describes a method for producing recombinant human follicle-stimulating hormone (r-hFSH), comprising: (i) culturing CHO-DUXB11 cells in a chemically defined, protein-free medium (such as MAM-PF77, ProCHO5, CD OptiCHO, 4Cell XtraCHO, Dynamis, CDM4 Mab ) supplemented with 4 mM glutamine; (ii) expanding the cells in shake flasks and a wave bio reactor until density exceeds 1.5 x I06cells / mL with >80% viability; (iii) producing r-hFSH in a stirred-tank single-use bioreactor seeded at 0.45-0.55 x 106cells / mL under controlled conditions of 36.5-37.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, and agitation 10 W / m3, with 5% air overlay; (iv) terminating production when viability falls below 60%; and (v) clarifying the harvest using cellulose-based depth filtration followed by 0.22 pm polyether sulfone filtration. The r-hFSH produced by the process is suitable for therapeutic use, including controlled ovarian stimulation and treatment of FSH deficiencies.

[0068] In some embodiments, the harvest clarified as described above may itself constitute a product. In other embodiments, the invention provides a pharmaceutical composition comprising CFA, r-hCG, or r-hFSH produced by the methods herein, together with pharmaceutically acceptable excipients.

[0069] In one embodiment, a method of treating a patient in need of fertility treatment is provided, comprising administering corifollitropin alfa produced by the methods described herein.

[0070] In another embodiment, a method of treating a condition associated with human chorionic gonadotropin deficiency is provided, comprising administering r-hCG produced by the methods described herein.

[0071] In a further embodiment, a method of treating a patient in need of fertility treatment is provided, comprising administering r-hFSH produced by the methods described herein.

[0072] In alternative process embodiments, CFA may be produced by culturing CHO-DG44 cells in chemically defined / protein free medium supplemented with 8 mM glutamine and 10 mM ManNAc, expanding to >1.5 x 106cells / mL with >80% viability, producing in a stirred-tank reactor under controlled parameters, and terminating at Day 12 or viability <80%. Similarly, r-hCG may be produced by culturing CHO-DUXB11 cells in chemically defined / protein free medium supplemented with 6 mM glutamine, expanding to >1.6 x 106cells / mL with >80% viability, producing in a stirred-tank reactor seeded at 0.25-0.35 x 106cells / mL, and feeding with Feed 4 containing glutamine. Likewise, r-hFSH may be produced by culturing CHO-DUXB 11 cells in chemically defined / protein free medium supplemented with 4 mM glutamine, expanding to >1.5 x 106cells / mL with >80% viability, producing in a stirred-tank reactor seeded at 0.45-0.55 x 106cells / mL, and terminating when viability falls below 60%. The processes of the present invention provide an optimized and reproducible upstream framework for the production of recombinant gonadotropins.

[0073] The processes described herein provide several significant advantages over conventional upstream systems for recombinant gonadotropin production. In particular, the invention:

[0074] (i) integrates defined media, optimized feed strategies, and robust bioreactor parameters, enabling consistent scale-up from laboratory flasks to large-volume stirred-tank reactors and ensuring reproducible performance across production campaigns;

[0075] (ii) ensures controlled revival, expansion, and seed generation steps that yield cell populations with higher viability (>80%) and density (>1.5 x 106cells / mL), supporting efficient inoculation of production bioreactors;

[0076] (iii) employs optimized feeding regimes to sustain growth and protein expression over extended culture durations;

[0077] (iv) enhances glycosylation, particularly sialylation, through supplementation with metabolic precursors such as N-acetyl mannosamine (ManNAc), thereby improving the biological activity, stability, and therapeutic efficacy of corifollitropin alfa, r-hCG, and r-hFSH;

[0078] (v) uses chemically defined, protein-free media such as MAM-PF77, ProCHO5, CD OptiCHO, 4Cell XtraCHO, Dynamis, CDM4 MAb to minimize variability and risk of adventitious contamination, facilitating regulatory compliance;

[0079] (vi) applies precise monitoring and control of temperature, pH, dissolved oxygen, CO2 concentration, and agitation to provide a stable culture environment that minimizes stress-induced apoptosis and maximizes protein yield; and

[0080] (vii) incorporates a two-step clarification strategy, combining depth filtration with absolute PES membrane filtration, to effectively remove cell debris, particulates, and host cell proteins, thereby streamlining downstream purification.

[0081] The recombinant gonadotropins produced using the disclosed processes exhibit superior purity, glycosylation fidelity, and bioactivity, making them highly suitable for clinical applications such as ovarian stimulation, ovulation triggering, follicular development, and treatment of gonadotropin deficiencies.

[0082] In summary, the present invention provides a robust, scalable, and versatile upstream bioprocess platform for recombinant gonadotropin production. The invention establishes a generalized five-stage framework adaptable to multiple gonadotropins, while preserving product-specific conditions that maximize yields. This unified manufacturing platform ensures consistent supply of high-quality CFA, r-hCG, and r-hFSH for therapeutic use.

Claims

CLAIMS1. A method for producing a recombinant gonadotropin selected from corifollitropin alfa (CFA), recombinant human chorionic gonadotropin (r-hCG), and recombinant human follicle-stimulating hormone (r-hFSH) in a mammalian cell culture, comprising:(i) reviving a mammalian CHO cell line selected from CHO-DG44 or CHO-DUXB11 in a chemically defined, protein-free medium supplemented with glutamine;(ii) expanding the revived cells in shake flasks under controlled temperature, CO2 concentration, and agitation conditions to achieve a viable cell density greater than 1.2 x 106cells / mL with >80% viability;(iii) generating seed in a wave bioreactor under controlled process parameters including gas flow between 0.1-0.3 L / min, rocking speed between 13-20 rpm, rocking angle of 7°- 10°, and CO2 concentration of approximately 5%;(iv) transferring the cells into a stirred-tank production bioreactor seeded at a density between 0.25 x 106and 0.55 x 106cells / mL, depending on the gonadotropin;(v)producing the recombinant gonadotropin in the stirred-tank bioreactor using a chemically defined, protein-free medium supplemented with one or more additives selected from N-acetyl mannosamine (ManNAc) and glutamine, under process parameters comprising a temperature of 36.5-37.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, agitation of approximately 10 W / m3, and 5% air overlay;(vi) feeding the culture during production with a nutrient feed selected from a chemically defined nutrient feed (such as Feed 4 or equivalent), supplemented with additives like N- acetyl mannosamine, and maintaining glucose between 2.5-4.0 g / L and glutamine between 2.5-6.0 mM; and(vii)harvesting the culture upon reduction of viability below a threshold between 60% and 80% or at a predetermined culture duration, followed by clarification using depth filtration and subsequent 0.22 urn polyether sulfone filtration.

2. The method of claim 1, wherein the recombinant gonadotropin is confolhtropin alfa (CFA).

3. The method of claim 1 , wherein the recombinant gonadotropin is recombinant human chorionic gonadotropin (r-hCG).

4. The method of claim 1 , wherein the recombinant gonadotropin is recombinant human follicle-stimulating hormone (r-hFSH).

5. A method for producing corifollitropin alfa (CFA) comprising the steps of claim 1, wherein the CHO-DG44 cell line is revived in MAM-PF77 medium supplemented with 8 mM L-glutamine.

6. The method of claim 5, wherein the cells are thawed at 37 °C for 1-2 minutes and optionally centrifuged at 800 rpm for 8 minutes prior to resuspension in fresh medium.

7. The method of claim 5, wherein cell expansion is performed in shake flasks at 36.5- 37.5 °C, CO2 4.5-5.5%, and agitation 80-110 rpm until cell density exceeds 1.2-1.5 x 106cells / mL with >80% viability.

8. The method of claim 5, wherein seed generation in the wave bioreactor is performed at gas flow 0. 1-0.3 L / min, rocking speed 13-20 rpm, angle 7-10°, and CO25%, achieving >1.6 x 106cells / mL with >80% viability.

9. The method of claim 5, wherein the production bioreactor is seeded at 0.25-0.35 x 106cells / mL in MAM-PF77 medium supplemented with 8 mM glutamine and 10 mM N- acetyl mannosamine.

10. The method of claim 5, wherein process conditions comprise pH 6.95-7.05, pC>2 30- 40%, agitation 10 W / m3, and 5% air overlay.

11. The method of claim 5, wherein Feed 4 containing 10 mM N-acetyl mannosamine is added at 3% on Days 3, 5, 7, 9, 10, and 11, with glucose maintained at 2.5-4.0 g / L and glutamine at 2.5-4.0 mM.

12. The method of claim 5, wherein the process is terminated when viability falls below 80% or on Day 12.

13. The method of claim 5, wherein clarification is carried out by depth filtration with cellulose and diatomaceous resin followed by 0.22 pm polyether sulfone filtration.

14. A method of treating a patient in need of fertility treatment by administering corifollitropin alfa produced by the method of claim 5.

15. A method for producing recombinant human chorionic gonadotropin (r-hCG) comprising the steps of claim 1, wherein the CH0-DUXB11 cell line is revived in ProCHO5 medium supplemented with 6 mM L-glutamine.

16. The method of claim 15, wherein thawing is carried out at 37 °C for 1-2 minutes without centrifugation.

17. The method of claim 15, wherein cell expansion in shake flasks is performed at 36.5-37.5 °C, CO2 4.5-5.5%, and 80-110 rpm agitation until density >1.2-1.5 x 106cells / mL with >80% viability.

18. The method of claim 15, wherein the wave bioreactor operates at 36.5-37.5 °C, oxygen / air flow 0. 1-0.3 L / min, rocking 13-20 rpm, rocking angle 7°, CO2 5%, expanding cells to >1.6 x 106cells / mL with >80% viability.

19. The method of claim 15, wherein the production bioreactor is seeded at 0.45-0.55 x 106cells / mL using ProCHO5 supplemented with 6 mM L-glutamine.

20. The method of claim 15, wherein process conditions comprise 37 ± 0.5 °C, pH 6.95-7.05, pCL 30-40%, agitation 10 W / m3, and 5% air overlay.

21. The method of claim 15, wherein Feed 4 supplemented with 6 mM glutamine is added at 4% on Days 3, 5, 7, and 9.

22. The method of claim 15, wherein glucose is maintained at 2.5-4.0 g / L and glutamine at 2.5-6.0 mM during production.

23. The method of claim 15, wherein production is terminated at viability below 80% or by Day 12.

24. The method of claim 15, wherein clarification is performed using cellulose / diatomaceous resin depth filtration followed by 0.22 pm poly ether sulfone filtration.

25. A method of treating a condition associated with human chorionic gonadotropin deficiency comprising administering r-hCG produced by the method of claim 15.

26. A method for producing recombinant human follicle-stimulating hormone (r-hFSH) comprising the steps of claim 1, wherein CHO-DUXB11 cells are revived in ProCHO5 medium supplemented with 4 mM Ultraglutamine-I.

27. The method of claim 26, wherein thawing is carried out at 37 °C for 1-2 minutes prior to culturing.

28. The method of claim 26, wherein expansion in shake flasks is performed at 36.5-37.5 °C, CO2 4.5-5.5%, agitation 95-105 rpm, with seeding density 0.40-0.60 x 106cells / mL.

29. The method of claim 26, wherein seed generation in the wave bioreactor is conducted at 36.5-37.5 °C, gas flow 0.1-0.3 L / min, rocking 13-15 rpm, rocking angle 7°, CO2 5%, until cell density exceeds 1.5 x 106cells / mL with >80% viability.

30. The method of claim 26, wherein the production bioreactor is seeded at 0.45-0.55 x 106cells / mL using ProCHO5 medium with 4 mM Ultraglutamine-I.

31. The method of claim 26, wherein the production bioreactor is operated at 36.5-37.5 °C, pH 6.95-7.05, pCh 30-40%, agitation 10 W / m3, and 5% air overlay.

32. The method of claim 26, wherein the working volume of the production bioreactor is initially 25% of maximum and expanded to 50% by Day 3 or 4 when density exceeds1.5 x 106cells / mL.

33. The method of claim 26, wherein the production process is terminated when viability falls below 60%.

34. The method of claim 26, wherein harvest clarification includes depth filtration with cellulose and binding resin followed by 0.22 pm polyether sulfone filtration.

35. A method of treating a patient in need of fertility treatment comprising administering r-hFSH produced by the method of claim 26.

36. A clarified harvest comprising a recombinant gonadotropin selected from CFA, r- hCG, and r-hFSH, produced by the method of any one of claims 1-35.

37. A pharmaceutical composition comprising a recombinant gonadotropin selected from CFA, r-hCG, and r-hFSH, produced by the method of any one of claims 1-35, together with a pharmaceutically acceptable excipient.

38. A method for producing corifollitropin alfa (CFA), comprising:(i) culturing CHO-DG44 cells in MAM-PF77 medium supplemented with 8 mM L- glutamine;(ii) expanding the cells to a density greater than 1.5 x 106cells / mL with >80% viability in shake flasks and a wave bioreactor;(iii) producing CFA in a stirred-tank single-use bioreactor operated at pH 6.95-7.05, dissolved oxygen 30-40%, and agitation 10 W / m3, with periodic addition of Feed 4 and N-acetyl mannosamine on Days 3, 5, 7, 9, 10 and 11; and(iv) terminating production at Day 12 or when viability falls below 80%, followed by harvest and clarification using cellulose / diatomaceous resin depth filtration and 0.22 pm polyether sulfone filtration.

39. A method for producing recombinant human chorionic gonadotropin (r-hCG), comprising:(i) culturing CHO-DUXB11 cells in ProCHO5 medium supplemented with 6 mM L- glutamine;(ii) expanding the cells in shake flasks and a wave bioreactor to > 1.6 x 106cells / mL with >80% viability;(iii) producing r-hCG in a stirred-tank single-use bioreactor seeded at 0.45-0.55 x 106cells / mL, maintained at 37 ± 0.5 °C, pH 6.95-7.05, pCh 30-40%, and agitation 10 W / m3;(iv) feeding with Feed 4 supplemented with glutamine at 4% on Days 3, 5, 7, and 9; and(v)clarifying the harvest using depth filtration with cellulose / diatomaceous resin followed by 0.22 pm polyether sulfone filtration.

40. A method for producing recombinant human follicle-stimulating hormone (r-hFSH), comprising:(i) culturing CHO-DUXB 11 cells in ProCHO5 medium supplemented with 4 mM Ultraglutamine-I;(ii) expanding the cells in shake flasks and a wave bio reactor until density exceeds 1.5 x 106cells / mL with >80% viability;(iii) producing r-hFSH in a stirred-tank single-use bioreactor seeded at 0.45-0.55 x 106cells / mL under controlled conditions of 36.5-37.5 °C, pH 6.95-7.05, dissolved oxygen 30-40%, and agitation 10 W / m3, with 5% air overlay;(iv) terminating production when viability falls below 60%; and5 (v) clarifying the harvest using cellulose and diatomaceous resin based depth filtration followed by 0.22 pm polyether sulfone filtration.

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