Methods for purifying recombinant human gonadotropins using sequential chromatographic and filtration techniques

Abstract

The invention provides optimized processes for the production and purification of recombinant glycoprotein hormones, including recombinant human chorionic gonadotropin (r-hCG), recombinant human luteinizing hormone (r-hLH), recombinant follicle-stimulating hormone with extended half-life (FSH-CTP), and recombinant human follicle-stimulating hormone (r-hFSH). The processes employ sequential chromatography and filtration steps— affinity, hydrophobic interaction, ion exchange, mixed-mode, ultraf iltration / diaf iltration, nanofiltration, and final sterilizing filtration— with precisely controlled pH, conductivity, and buffer conditions. These strategies ensure robust viral clearance, high recovery yields, and effective removal of host cell proteins, DNA, aggregates, and other impurities, resulting in highly pure and stable drug substances. Analytical data from multiple batches confirm identity, consistency, and clinical suitability. The invention thus establishes a unified and scalable platform for large-scale manufacture of recombinant glycoprotein hormones for therapeutic use in fertility and reproductive healthcare.

Description

[0001] METHODS FOR PURIFYING RECOMBINANT HUMAN GONADOTROPINS USING SEQUENTIAL CHROMATOGRAPHIC AND FILTRATION TECHNIQUES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of biotechnology and biopharmaceutical production. More particularly, it relates to novel downstream purification methods for recombinant human glycoprotein hormones belonging to the gonadotropin family, including rFSH, FSH-CTP (CFA), rLH, and r-hCG.

[0004] BACKGROUND OF THE INVENTION

[0005] The downstream purification of recombinant human gonadotropins— including follicle- stimulating hormone (rFSH), corifol litropin alfa (CFA or FSH-CTP), luteinizing hormone (r- hLH), and chorionic gonadotropin (r-hCG)— presents significant challenges in achieving the required levels of purity, safety, and process efficiency. These glycoprotein hormones share structural similarities but differ in glycosylation patterns and isoforms, making their purification complex. Conventional processes often encounter issues such as crosscontamination, protein degradation, product loss, and inadequate removal of host cell proteins (HCPs), DNA, and viral contaminants. The demand for highly pure and biologically active gonadotropins for therapeutic use in assisted reproductive technologies (ART) and related indications underscores the need for improved purification methods.

[0006] A variety of approaches have been disclosed in the prior art. Processes such as those described in US20170029482A1 (Zydus Lifesciences), US8846344B2 (Glycotope GmbH), and US7754860B2 (Ares Trading SA) demonstrate the evolution of chromatographybased purification, combining affinity, ion-exchange, hydrophobic interaction, or reverse phase chromatography. Other disclosures, including JP4933439B2, W02007 / 065918A2, EP0974359B1, and US7741455B2, provide multi-step processes involving dye affinity, gel filtration, and multiple ion-exchange steps. While these methods achieve incremental improvements, they often lack integration of advanced modalities such as multimodal chromatography, structured sequences of orthogonal clearance, or dedicated virus safety measures such as nanofiltration.

[0007] Specific references for r-hLH (e.g., studies by Berg, DeMoll, Li, and Zhang) and r-hCG (e.g., US7297777B2, US9676835B2, and US8846344B2) describe purification methods incorporating ion exchange, hydrophobic interaction, gel filtration, hydroxyapatite chromatography, or reverse phase HPLC. While such approaches demonstrate bioactivity and some purity gains, they generally omit hormone-specific affinity matrices, multimodal purification, or virus clearance steps, thereby limiting their ability to achieve consistent therapeutic-grade purity.

[0008] Non-patent literature, including works by Sinegubova et al. (2022), Gurevich Messina et al. (2018), Butnev et al. (2015), and reviews by Ribela et al. (2003), Lee et al. (1993), and Shuo Tang et al. (2024), discuss immunoaffinity, synthetic peptide affinity, viral filtration, and single-use downstream systems. These highlight important advances, yet still do not provide a comprehensive, platform-level strategy tailored to recombinant gonadotropins.

[0009] Accordingly, there remains a clear unmet need for a robust, platform downstream purification process that integrates hormone-specific affinity capture, multimodal chromatography, ion exchange, hydrophobic interaction, and size exclusion steps, combined with ultrafiltration / diafiltration and dedicated virus removal measures. Such a process should deliver high yield, consistent quality, enhanced virus safety, and regulatory compliance across multiple recombinant gonadotropins.

[0010] The present invention addresses these gaps by introducing a structured downstream platform that leverages state-of-the-art resins, hormone-specific affinity matrices, and optimized process parameters to achieve superior purity, stability, and safety of rFSH, FSH-CTP, r-hLH, and r-hCG. SUMMARY OF THE INVENTION

[0011] The present invention provides a platform downstream purification process for recombinant human gonadotropins, including follicle-stimulating hormone (rFSH), corifollitropin alfa (CFA or FSH-CTP), luteinizing hormone (r-hLH), and chorionic gonadotropin (r-hCG). The platform integrates affinity capture, multimodal chromatography, ion exchange, hydrophobic interaction, ultrafiltration / diafiltration, and nanofiltration into structured sequences optimized for each hormone to achieve high yield, superior purity, viral safety, and product stability.

[0012] In one embodiment, purification of FSH-CTP employs multimodal chromatography in tandem with a highly selective CaptureSelect FSH affinity resin, providing orthogonal selectivity that enhances removal of host cell proteins, glycoform variants, and aggregates beyond what affinity alone can achieve. Subsequent first ultrafiltration / diafiltration (UF / DF), multimodal chromatography, cation exchange, anion exchange, size exclusion chromatography and second UF / DF. These steps further refine purity, while first and second ultrafiltration / diafiltration (UF / DF) ensures buffer exchange and concentration. Nanofiltration confers viral safety, resulting in a drug substance of exceptional purity and stability.

[0013] In another embodiment, purification of r-hLH begins with CaptureSelect hCG affinity chromatography, followed by ultrafiltration / diafiltration and sequential multimodal and anion exchange chromatography steps for protease removal, isoform separation, and impurity clearance. Viral filtration and sterile filtration complete the process, yielding a safe and stable drug substance.

[0014] In a further embodiment, purification of r-hCG employs Blue Sepharose affinity chromatography for capture, followed by sequential ultrafiltration / diafiltration, ion exchange, and hydrophobic interaction chromatography to separate isoforms and eliminate hydrophobic impurities. Nanofiltration and final sterile filtration ensure viral safety and regulatory compliance. In yet another embodiment, purification of r-hFSH begins with Blue Sepharose affinity chromatography, followed by hydrophobic interaction chromatography, ion exchange, and mixed mode chromatography for impurity clearance and isoform enrichment. Ultrafiltration / diafiltration and nanofiltration steps ensure viral safety, with final sterile filtration producing a highly pure drug substance.

[0015] Across these embodiments, the downstream platform is characterized by:

[0016] • Affinity chromatography using hormone-specific ligands for selective capture,

[0017] • Multimodal chromatography for orthogonal impurity clearance and isoform separation, including tandem use with affinity in the case of FSH-CTP,

[0018] • Ion exchange and hydrophobic interaction chromatography for removal of charge variants, proteases, and hydrophobic impurities,

[0019] • Ultrafiltration / diafiltration for buffer exchange and concentration, and

[0020] • Nanofiltration and 0.2 pm filtration for viral safety and final drug substance preparation.

[0021] The processes of the present invention provide a harmonized and scalable downstream platform adaptable to multiple gonadotropins. By integrating orthogonal chromatography modes with robust filtration and defined process parameters, the invention ensures consistent quality, enhanced therapeutic efficacy, and superiority over prior art purification methods.

[0022] BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1: Flow chart of the downstream purification process for recombinant human FSH-CTP.

[0024] Figure 2: SDS-PAGE (Non-reducing) profiles of FSH-CTP developmental batches compared with reference standard.

[0025] Figure 3: Capillary Electrophoresis (CZE) profiles of FSH-CTP developmental batches compared with reference standard.

[0026] Figure 4: Size-Exclusion HPLC chromatograms and purity data for FSH-CTP batches.

[0027] Figure 5: Step yields of various developmental batches of FSH-CTP during downstream processing.

[0028] Figure 6: Overall process recovery rates for FSH-CTP developmental batches.

[0029] Figure 7: Flow chart of downstream purification process for recombinant human luteinizing hormone (r-hLH).

[0030] Figure 8: SEC-HPLC profiles of r-hLH Drug Substance compared with reference standard.

[0031] Figure 9: SDS-PAGE (Non-reducing) analysis of r-hLH Drug Substance against reference standard.

[0032] Figure 10: Step yields and overall process recovery of r-hLH developmental batch.

[0033] Figure 11: Flow chart of downstream purification process for recombinant human chorionic gonadotropin (r-hCG).

[0034] Figure 12: Step-wise process efficiency and overall yield data for rhCG developmental batches.

[0035] Figure 13: Aggregate (HMW) content of rhCG Drug Substance batches.

[0036] Figure 14: Host Cell Protein (HCP) content in rhCG Drug Substance batches. Figure 15: Residual DNA content in rhCG Drug Substance batches.

[0037] Figure 16: Capillary Zone Electrophoresis (CZE) profile of rhCG Drug Substance compared with reference standard.

[0038] Figure 17: Flow chart of downstream purification process for recombinant human follicle-stimulating hormone (r-hFSH).

[0039] Figure 18: Isoelectric focusing (IEF) gel analysis of r-hFSH API compared with reference standards.

[0040] Figure 19: Follitropin oligomer (HMW) content across r-hFSH batches.

[0041] Figure 20: Oxidized follitropin content across r-hFSH batches.

[0042] Figure 21: Host Cell Protein (HCP) content across r-hFSH batches.

[0043] Figure 22: Host Cell DNA (HCD) content across r-hFSH batches.

[0044] Figure 23: Step yields and overall recovery rates of r-hFSH developmental batches.

[0045] DETAILED DESCRIPTION OF THE INVENTION

[0046] As used herein, unless expressly stated otherwise, the following terms have the meanings set forth below:

[0047] "Recombinant glycoprotein hormone" refers to a heterodimeric protein consisting of a common a-subunit and a hormone-specific p-subunit, produced using recombinant DNA technology in mammalian host cells, including but not limited to recombinant human follicle-stimulating hormone (r-hFSH), Corifollitropin alfa (CFA or FSH-CTP), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG).

[0048] "Corifollitropin alfa (CFA)" or "FSH-CTP" refers to a long-acting FSH analogue containing the carboxy-terminal peptide (CTP) of hCG fused to the p-subunit of FSH, conferring extended half-life and pharmacological duration. "CHO cells" or "Chinese Hamster Ovary cells" refers to immortalized mammalian cell lines widely used for recombinant protein expression in serum-free, chemically defined media.

[0049] "Affinity chromatography" refers to a purification technique utilizing specific binding interactions between the target glycoprotein hormone and a ligand immobilized on a solid support, including but not limited to Blue Sepharose 6 Fast Flow, CaptureSelect FSH resin, and CaptureSelect hCG resin.

[0050] "Multimodal chromatography (MMC)" refers to chromatography using resins that combine two or more interaction mechanisms (ionic, hydrophobic, hydrogen bonding), including Capto Adhere, Capto Adhere ImpRes, and Capto MMC ImpRes.

[0051] "Hydrophobic interaction chromatography (HIC)" refers to chromatography using resins bearing hydrophobic ligands, such as Phenyl Sepharose orToyopearl Butyl 600M, typically performed at elevated salt concentrations to promote binding.

[0052] "Ion exchange chromatography" refers to separation based on ionic interactions between charged groups on a resin and oppositely charged molecules, including cation exchange (e.g., Capto SP ImpRes) and anion exchange (e.g., Q. Sepharose, QHR resins). "Size-exclusion chromatography (SEC)" refers to separation based on molecular size, typically using resins such as Superdex 75 Prep Grade, to remove aggregates, multimers, or fragments.

[0053] "Ultrafiltration / Diafiltration (UF / DF)" refers to membrane-based processes using a defined molecular weight cut-off, such as a 10 kDa hollow fiber membrane, for buffer exchange and protein concentration under transmembrane pressure. UF / DF1 refers to the first ultraf iltration / d iaf iltration step, while UF / DF2 and UF / DF3 refer to subsequent repetitions of this process.

[0054] "Nanofiltration" refers to virus filtration using membranes with nominal pore sizes of about 15-25 nm, such as Planova 20N or Viresolve Pro filters, typically operated under controlled pressure not exceeding 1.5 bar or ~1.0 kg / cm2. "Viral inactivation" refers to a process of rendering viruses non-infectious, typically performed by low-pH incubation (e.g., pH 3.8-4.2 for >6 hours) or equivalent orthogonal measures.

[0055] "Drug substance (DS)" refers to the purified, bulk active pharmaceutical ingredient (API), prior to formulation into drug product.

[0056] "Pharmaceutical composition" refers to a preparation comprising a purified recombinant glycoprotein hormone in association with one or more pharmaceutically acceptable carriers, diluents, stabilizers, surfactants, antioxidants, preservatives, or excipients.

[0057] "Pharmaceutically acceptable carrier" means a non-toxic substance suitable for administration that does not adversely affect the biological activity of the glycoprotein hormone, including but not limited to buffers, sugars, amino acids, and surfactants.

[0058] "Stabilizers" include compounds such as mannitol, sucrose, trehalose, glycine, and L- arginine HCI.

[0059] "Surfactants" include polysorbate-20, polysorbate-80, and poloxamer 188.

[0060] "Antioxidants" include amino acids such as L-methionine and its pharmaceutically acceptable derivatives.

[0061] "Preservatives" include agents such as meta-cresol, phenol, and benzyl alcohol.

[0062] "Therapeutically effective amount" refers to an amount of recombinant glycoprotein hormone sufficient to elicit a desired clinical effect, including but not limited to induction of ovulation, luteal phase support, controlled ovarian stimulation, or stimulation of follicular development.

[0063] "About" as used in connection with numerical values (e.g., pH, conductivity, concentration, temperature) refers to a value ±10% of the recited value, unless a different margin is explicitly indicated.

[0064] "High molecular weight impurities (HMW)" refers to aggregates or multimers of the glycoprotein hormone, while "low molecular weight impurities (LMW)" refers to fragments or truncated forms of the protein. "Potency" for r-hCG is expressed in International Units (IU) and indicates biological activity as measured in validated in vitro or in vivo bioassays.

[0065] The present invention provides a platform process for producing recombinant glycoprotein hormones selected from recombinant human follicle-stimulating hormone (r-hFSH), follicle-stimulating hormone-CTP (FSH-CTP), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG). These hormones are essential in assisted reproductive technologies (ART) and other therapeutic applications, where product purity, potency, and viral safety are critical.

[0066] The process disclosed herein employs a sequential combination of chromatography, filtration, and virus removal steps to achieve high-purity recombinant hormones. The invention further provides pharmaceutical compositions comprising the purified hormones, and therapeutic methods of use.

[0067] In general, the process of the present invention comprises:

[0068] • Harvest clarification and capture by affinity chromatography, or in certain embodiments a multimodal step employed in tandem with affinity chromatography to achieve orthogonal selectivity;

[0069] • U Itraf iltration / diaf iltration (UF / DF1) for buffer exchange and protein concentration;

[0070] • Intermediate chromatographic purifications, including hydrophobic interaction chromatography (HIC), cation exchange, anion exchange, multimodal chromatography, and / or size exclusion chromatography (SEC);

[0071] • Optional low-pH viral inactivation;

[0072] • Second UF / DF (UF / DF2) for concentration and formulation adjustment;

[0073] • Nanofiltration for viral clearance; and

[0074] • Preparation of final drug substance (DS) by 0.2 pm filtration.

[0075] In one embodiment, purification of FSH-CTP is distinguished by the use of multimodal chromatography in tandem with CaptureSelect affinity chromatography. The multimodal resin provides an orthogonal mode of separation, combining ionic and hydrophobic interactions to remove mainly proteases, host cell proteins, aggregates, and structurally related glycoproteins that are insufficiently resolved by affinity binding alone. When coupled with the high specificity of CaptureSelect affinity chromatography, this tandem approach achieves superior clearance of impurities, charge variants, and aggregates, thereby ensuring a highly purified and stable product.

[0076] For other gonadotropins, including r-hLH, r-hCG, and r-hFSH, the process primarily employs affinity chromatography as the initial capture step, followed by orthogonal intermediate purification using ion exchange, hydrophobic interaction, multimodal, and / or size exclusion chromatography as required for each hormone. These combinations provide efficient impurity clearance, buffer exchange, and virus safety, while ensuring consistency, scalability, and regulatory compliance.

[0077] The protein downstream process begins with affinity chromatography, typically using Blue Sepharose 6FF or Capture Select FSH / hCG resins. This step operates in a bind- elute mode at pH 7.0-8.0 (most commonly between 7.4 and 7.6). Elution is performed under high ionic strength with conductivities between 300-400 mS / cm, using buffers such as 20 mM Tris containing salts like 0.1 M arginine HCI, 2.0 M magnesium chloride, or 15 mM sodium phosphate with 2.5 M sodium chloride. This capture step selectively binds the target protein and removes host cell proteins (HCP) and host cell DNA (HCD). In some cases, particularly for FSH-CTP, this step is combined with multimodal chromatography to enhance impurity clearance.

[0078] Following capture, the eluate undergoes ultrafiltration / diafiltration (UF / DF1) using a 10 kDa hollow fiber membrane. This step performs buffer exchange and protein concentration, operating at pH 7.0-8.0 and conductivity 3-7 mS / cm, typically requiring 7-10 diavolumes of buffer. The resulting retentate achieves a protein concentration of at least 0.3 mg / mL. Next, hydrophobic interaction chromatography (HIC) is performed using Phenyl Sepharose or Toyopearl Butyl resins in bind-elute mode at pH 7.0-8.0. Conductivity during this step ranges between 100-350 mS / cm. Elution is performed with 1 M urea and 10% isopropanol in phosphate buffer, enabling the removal of hydrophobic impurities.

[0079] Cation exchange chromatography (CEX) is then applied, typically with Capto SP ImpRes resin in bind-elute mode. Washing is done at pH 4.9-5.1, while elution occurs at pH 7.0-8.0. This step helps eliminate HCP and charge variants. In parallel, anion exchange chromatography (AEX) can be used in a flow-through mode, utilizing Capto Q, or Q. Sepharose FF, resins. Conductivity is maintained between 1-20 mS / cm in 20 mM Tris buffer at pH 6.9-7.6, effectively removing negatively charged impurities, including HCP and DNA.

[0080] Multimodal chromatography using Capto Adhere Impres or Capto MMC Impres resins is commonly applied in gradient mode over 30 column volumes. The process begins with sodium acetate buffer at pH 5.9-6.1 and conductivity around 1.5 mS / cm, and transitions to a Tris buffer with 0.5 M NaCI at pH 7.9-8.1 and conductivity of about 45 mS / cm. This step is useful for isoform enrichment, and for FSH-CTP, is often performed in tandem (flow through mode) with affinity capture for enhanced impurity clearance, mainly for removal of proteases.

[0081] Size exclusion chromatography (SEC) with Superdex 75 prep grade resin follows, operated at pH 7.0 ± 0.1 and conductivity 0.5-4.0 mS / cm, which removes aggregates and low molecular weight fragments. For viral safety, an optional low-pH viral inactivation step may be included by adjusting the pH to 3.9-4.1 with acetate buffer and holding for at least six hours on column.

[0082] The process continues with UF / DF2, another ultrafiltration / diafiltration step using a 10 kDa hollow fiber membrane. This concentrates the protein to >1.0 mg / mL and exchanges into the final formulation buffer. Conditions are maintained at pH 6.5-7.5 and conductivity 0.5-10 mS / cm.

[0083] To ensure viral safety, nanofiltration is employed using Planova 20N or equivalent filters with a pore size of 15-25 nm. The step is operated in histidine or phosphate buffer or sodium citrate buffer at pH 6.0-8.0, conductivity 0.1-10 mS / cm, and under nitrogen pressure not exceeding 1.0 kg / cm2, effectively removing viral particles and adventitious agents.

[0084] Before formulation, the protein undergoes final filtration through a 0.2 pm PES capsule filter at pH 6.0-8.0 and conductivity 0.1-10 mS / cm, yielding drug substance ready for formulation.

[0085] Finally, formulation and stabilization are carried out using excipients such as mannitol, sucrose, trehalose, glycine, and L-arginine HCI, with antioxidants (e.g., L-methionine), surfactants (such as polysorbate-20, polysorbate-80, or Poloxamer 188), and preservatives including meta-cresol, phenol, or benzyl alcohol to ensure stability and shelf life.

[0086] Analytical characterization of the final drug substance (DS) is performed to assess its purity and structural integrity using techniques such as SDS-PAGE, capillary zone electrophoresis (CZE), size-exclusion HPLC, and western blotting. The typical acceptance criteria for a purified preparation include high molecular weight (HMW) impurities of less than 1%, low molecular weight (LMW) impurities of less than 5%, and potency that falls within pharmacopeial limits, such as at least 30,000 lU / mL for recombinant human chorionic gonadotropin (r-hCG) & between 0.4 to 0.8 mg / ml for recombinant human follicle stimulating hormone

[0087] Therapeutic use of these pharmaceutical compositions lies in the treatment of infertility, hypogonadism, and gonadotropin deficiencies. Recombinant hCG (r-hCG) is typically administered as a single dose of 3250-6500 IU. Recombinant LH (r-hLH) is given at doses ranging from 3.0-6.0 pg, often in combination with recombinant FSH (r- hFSH). Recombinant FSH or its long-acting variant, FSH-CTP, is used for controlled ovarian stimulation in women and for the treatment of hypogonadotropic hypogonadism in men. Administration of these preparations is preferably carried out via subcutaneous injection.

[0088] The present invention is further described by the following embodiments and examples.

[0089] The process of the invention may be adapted as follows:

[0090] • Selection of capture matrix depending on hormone expressed;

[0091] • Use of different combinations of intermediate chromatography (HIC, ion exchange, multimodal, SEC) to address product-specific impurities;

[0092] • Inclusion or exclusion of viral inactivation step depending on regulatory requirements;

[0093] • Adjustments in UF / DF to tailor protein concentration for final formulation.

[0094] In a specific embodiment of the platform process, the production of r-hFSH-CTP (CFA) is described as follows.

[0095] In one embodiment, the downstream purification process for Corifollitropin alfa (CFA) or recombinant human Follicle Stimulating Hormone-C terminal peptide (FSH-CTP) is organized into several key steps, each contributing to the progressive enhancement of purity and safety of the final drug substance:

[0096] MULTIMODAL CHROMATOGRAPHY (CAPTO ADHERE) FOLLOWED BY AFFINITY CHROMATOGRAPHY (CAPTURE SELECT FSH MATRIX):

[0097] In an embodiment of the present invention, Multimodal Chromatography using Capto Adhere Resin followed by affinity chromatography with a Capture Select FSH matrix is employed to purify the target protein. The Multimodal Chromatography resin is used to bind the protease related impurities while target protein is passed through flowthrough for further process. The affinity resin binds the target protein under specific conditions, followed by sequential washing and elution steps to isolate the protein with high purity.

[0098] In an embodiment of present invention , Multimodal Chromatography I , resin / column is washed using 0.5M NaOH solution to ensure bound impurities are washed off followed by IM NaCI solution wash before column equilibration with 20mM Tris with 0.18 M NaCI (pH 7.5) for 5 CV , with a pH range of 7.4 to 7.6 , and conductivity range of 9 ± 3 mS / cm.

[0099] In another embodiment, an affinity column cleaning-in-place (CIP) step is performed using 0.1 M Glycine (pH 2.0), ensuring the column is prepared for protein binding. The preferred pH is 2.0, with a pH range of 1.9 to 2.1, and a conductivity range of 9 ± 3 mS / cm.

[0100] In another embodiment, the affinity column is equilibrated with 20 mM Tris buffer (pH 7.5). The preferred pH range is 7.4 to 7.6, with a conductivity of 1.5 ± 0.5 mS / cm. The column volume used is 5 CV, and the pH range for this step is 7.0 to 8.0.

[0101] In another embodiment, equilibrated multimodal I column is attached in tandem to equilibrated affinity column

[0102] In another embodiment, the target protein harvest is loaded onto the series of column. The dynamic binding capacity is maintained at NMT 1.6 mg / ml resin for Affinity column whereas dynamic binding capacity of multimodal I column is kept at 5 mg / ml resin, with a residence time of 5-6 minutes.

[0103] In another embodiment, wash 1 is given to series of columns for 5CV using Buffer A (20mM Tris with 0.18 M NaCI) followed by detaching multimodal column and further purification is carried out on affinity column.

[0104] In another embodiment, affinity column is washed with 2 buffers: Buffer B (20 mM Tris) for 5CV and Buffer C (containing 20 mM Tris, 0.1% Triton X 100, 5% Isopropyl alcohol along with 0.1M Arginine HCI) for 15 CV to remove non-specifically bound proteins. Buffer B (20mM Tris) wash is given to affinity column before target protein elution for 5CV. Elution is performed using Buffer D (20 mM Tris with 0.1M Arginine HCI and 2M MgCI2), ensuring the eluted protein is highly purified. The preferred pH range is 7.0 to 8.0, with a target pH of 7.5 ± 0.1, and the eluate exhibits a conductivity range of 300 to 400 mS / cm.

[0105] In another embodiment, after elution, the column undergoes another CIP step with 0.1 M Glycine, followed by a cleaning step with 0.5 M Acetic Acid and storage in 20% ethanol in 20 mM Tris (pH 7.5).

[0106] Ultrafiltration / diafiltration (UF / DF 1):

[0107] In another embodiment, ultrafiltration / diafiltration (UF / DF) is utilized wherein the process includes a concentration step using a 10 kDa hollow fiber ultrafiltration membrane. This step concentrates the eluted protein and diafilters it against a specified buffer to achieve the desired concentration and buffer exchange.

[0108] In another embodiment, the Hollow Fiber Membrane is cleaned with 0.5 M NaOH, followed by rinsing with purified water until the permeate pH reaches 5-7.

[0109] In another embodiment, diafiltration is performed using Buffer H (20 mM Tris buffer with 50mM L Arginine HCI) within a pH range of 7.0 to 8.0, with a target pH of 7.5 ± 0.1, and a preferred conductivity of 5 ±2 mS / cm. The diafiltration process is carried out over 7 to 10 diavolumes, ensuring effective buffer exchange.

[0110] In another embodiment, the protein concentrate is adjusted to a target concentration of 1 -3 mg / mL.

[0111] In another embodiment, post-diafiltration, the membrane is stored in 0.1 M NaOH.

[0112] Multimodal Chromatography II: In another embodiment, a purification step is used to separate desired isoforms using bu passing on Capto Adhere ImpRes Resin with dynamic binding capacity of NMT 20 mg / ml and residence time of 5 minutes.

[0113] In another embodiment, the column is washed with 0.5 M NaOH solution for Cleaning in place (CIP) purpose followed by equilibration by passing 5 CV of Buffer H ( 20mM Tris with 50mM L Arginine HCI) (pH 7.5) keeping pH range of 7.4 to 7.6 and conductivity range of 5 ±2 mS / cm.

[0114] In another embodiment, the diafiltrate protein solution is loaded on the column followed by wash using Buffer H of 15 CV followed by eluting protein by employing step gradient of 20 % B with Buffer H (Port A) and Buffer I (100 mM sodium citrate)( pH 6.0 - 7.5) (Port B) with pH at range of 7.0 to 5.5 to 6.5 (preferable pH range of 5.9 to 6.1) and conductivity range of 15 ±3 mS / cm .

[0115] In another embodiment, Post elution; column is washed with 0.5 M NaOH solution.

[0116] Cation Exchange Chromatography:

[0117] In another embodiment, the process of the present invention involves a Cation Exchange Chromatography step conducted in flow through mode, using Capto SP ImpRes resin for further purification of the protein.

[0118] In another embodiment, the Cation Exchange Chromatography step includes equilibrating the column with Buffer J (10 mM Sodium Citrate buffer) at a pH range of 5.9 to 6.1 and a conductivity range of 1 to 2 mS / cm to remove impurities.

[0119] In another embodiment, the process involves loading the multimodal chromatography elute, with pH adjusted to 5.9 to 6.10 using Buffer L (0.2 M Acetic Acid) and conductivity checked to be NMT 9 mS / cm and loaded onto the column and collection of flowthrough / elute. In another embodiment, the elution in the Cation Exchange Chromatography step is continued by passing Buffer J and the column post elution is regenerated by passing 3CV of 0.5 M NaOH solution.

[0120] Anion Exchange Chromatography:

[0121] In another embodiment, the process includes an Anion Exchange Membrane Chromatography step to further purify the protein and remove any remaining impurities.

[0122] In another embodiment, the Anion Exchange Chromatography step is performed in non-binding flow-through mode using Capto Q. resin. The column is equilibrated with 0.13 M NaCI in 20 mM Tris (pH 7-8, preferably 7.5 ± 0.1) with a conductivity of 10-20 mS / cm, allowing FSH or FSH-CTP to pass through while removing anionic impurities.

[0123] In another embodiment, the protein from the Cation Exchange step is loaded onto the Capto Q. resin in the Anion Exchange Chromatography step, with adjustments made to the target pH (7.5 ± 0.1) and conductivity 10-20 mS / cm.

[0124] In another embodiment, the column is washed with the equilibration buffer, and an optional elution step with 1 M NaCI is performed to ensure maximum protein recovery.

[0125] Ultrafiltration / diafiltration (UF / DF2):

[0126] In another embodiment, the process includes a second UF / DF step to concentrate the protein to the 5.0 ± 1 mg / ml concentration and buffer is exchanged to ensure loading onto next step of purification.

[0127] In another embodiment, the second UF / DF step involves using a 10 kDa molecular weight cutoff membrane or hollow fiber membrane, cleaned with 0.5 M NaOH and rinsed with purified water until the permeate pH reaches 5-7.

[0128] In another embodiment, the second UF / DF step includes diafiltration using Buffer M ( 25 mM Sodium Citrate buffer with 0.5 mg / ml L Methionine) (pH 7.0) with a conductivity of 5.5 ± 1 mS / cm, over 8 to 10 diavolumes. In another embodiment, the protein concentrate is adjusted to a target concentration of 5.0 ± 1.0 mg / mL. and the hollow fiber membrane, cleaned with 0.5 M NaOH and rinsed with purified water until the permeate pH reaches 5-7. And stored in 0.1M NaOH solution.

[0129] Size Exclusion Chromatography:

[0130] In another embodiment, the process includes a Size Exclusion Chromatography step targeting the final polishing of the protein, ensuring high purity and minimal aggregates.

[0131] In another embodiment, the Size exclusion Chromatography step is performed using a Superdex 75 Prep grade resin. The column is equilibrated with Buffer M (25 mM Sodium Citrate buffer with 0.5 mg / ml L Methionine) (pH 7.0, preferably 6.5 to 7.5) with a conductivity of 5.5 ± 1 mS / cm.

[0132] In another embodiment, the diafiltrate and concentrated protein is loaded onto the column ensuring linear velocity of 30 cm / hr. and Postload wash of equilibration buffer for 1 CV is given with peak collected in fractions. The theoretical pool of fractions are prepared and analysed for purity (HMW / LMW content) by analytical method of by SEC HPLC method for purity content and on based on the result; final pooling of fractions is performed done for further step of purification.

[0133] In another embodiment, wash of 0.5 M NaOH for v3CV is given to column and stored in 20% Ethanol solution.

[0134] Nanofiltration:

[0135] In another embodiment, the process includes a Nanofiltration step to ensure viral clearance, providing an additional safety layer in the purification process by removing particles of approximately 20 nm or larger, including viral particles and other adventitious agents. In another embodiment, the Nanofiltration step involves equilibrating the nanofiltration unit with 20 mM Tris buffer with 0.5 mg / ml L Methionine (pH 6.5-7.5, preferably 7.0 ± 0.1) with a conductivity of 4.5-6.5 mS / cm.

[0136] In another embodiment, the protein eluate from the Multimodal Chromatography pooled fractions from the Size exclusion chromatography step is loaded onto the nanofiltration unit.

[0137] Excipient addition and DS Preparation followed by 0.2um filtration:

[0138] Excipient addition is performed using 50% Sucrose and 1% Tween 20 stock solutions in 25 mM Sodium Citrate buffer with 0.5 mg / ml L-Methionine to achieve Sucrose concentration at 70 mg / ml and Tween 20 concentration at 0.2 mg / ml post stock solution spiking..

[0139] In another embodiment, the final nanofiltrate is diluted with respective excipient addition, achieving a target protein concentration of 0.5 to 1.5 mg / mL ,with a pH range of 6.5 to 7.5; a target pH of 7.0 ± 0.1, and a conductivity range of 6.5 to 7.5 mS / cm.

[0140] In another embodiment, the process involves purifying recombinant follicle- stimulating hormone-CTP (FSH-CTP) from a liquid containing these proteins by:

[0141] - (I) Subjecting FSH-CTP-containing liquid to Multimodal chromatography using Capto Adhere Resin followed by affinity chromatography using a Capture Select FSH matrix, where FSH-CTP binds to the matrix and is subsequently eluted;

[0142] - (II) Performing ultrafiltration / diafiltration (UF / DF1) to concentrate and bufferexchange FSH-CTP eluate;

[0143] - (Ill) Passing FSH-CTP eluate through Multimodal chromatography using Capto Adhere ImpRes resin mainly for active isoforms enrichment of the target protein and their subsequent separation in eluate. ; - (IV) Subjecting FSH-CTP eluate to cation exchange chromatography using Capto SP Impres resin to further purify the FSH by binding impurities with opposite charge properties;

[0144] - (V) Subjecting FSH-CTP eluate to anion exchange chromatography using Capto Q to remove anionic impurities, including viruses, host cell DNA (HCDs), and endotoxins;

[0145] - (VI) Performing ultrafiltration / diafiltration (UF / DF2) to achieve the final concentration and buffer exchange of the purified FSH-CTP;

[0146] - (VII) Subjecting FSH-CTP eluate to Size exclusion chromatography using Superdex 75 prep grade resin for further purification of high and low molecular weight contaminants,

[0147] - (VIII) Performing nanofiltration to remove viral particles from FSH-CTP eluate followed by excipient addition and sterile filtration for bulk generation;

[0148] In another embodiment, the process begins with a tandem operation of Capto Adhere and Capture Select FSH matrix designed for FSH-CTP, ensuring highly selective and efficient capture of the target proteins. The Capto Adhere resin ensures proteases related contaminant binding as flow through contains protein of interest. This affinity matrix binds and elutes only FSH-CTP, enhancing initial purity and minimizing impurities. Following affinity chromatography, UF / DF1 is performed to concentrate the eluate and conduct a buffer exchange, making it compatible with subsequent purification steps. The eluate is then passed through Capto Adhere ImpRes resin, ensuring enrichment of desired isoforms. The process continues with cation exchange chromatography using Capto SP Impres resin to target impurities with specific charge properties, primarily removing host cell proteins (HCPs) and refining the protein further. Next, the eluate undergoes anion exchange chromatography using Capto Q, crucial for removing anionic impurities, including viruses and host cell DNA. The eluate is subjected to the UF / DF-2 step to concentrate and buffer exchange target protein for Size exclusion chromatography operations. . The Size Exclusion chromatography using Superdex 75 prep grade resin helps in separating low as well as high molecular weight contaminants. Nanofiltration follows to physically remove adventitious viruses, ensuring product safety. Finally, required excipients and added as per desired formulation and concentration of the purified FSH-CTP.

[0149] In another embodiment, a pharmaceutical composition is formulated comprising the purified recombinant follicle-stimulating hormone-CTP (FSH-CTP) obtained by the above process, in combination with a pharmaceutically acceptable carrier or excipient.

[0150] In another embodiment, the process of the present invention ensures that the final drug substance (DS) material has a target protein concentration of no less than 0.5 mg / mL and an overall process recovery of no less than 40%.

[0151] In another embodiment, the process further includes analyzing the purity of the final FSH-CTP product using techniques such as SDS-PAGE, Capillary Electrophoresis (CZE), Size-Exclusion HPLC, or Western blotting.

[0152] In another embodiment, the process ensures that the target protein concentration has less than 0.5% high molecular weight (HMW) impurities and low molecular weight (LMW) impurities less than 5%.

[0153] In another embodiment, a pharmaceutical composition is formulated comprising the purified recombinant follicle-stimulating hormone-CTP (FSH-CTP) obtained by the process of the present invention, in combination with a pharmaceutically acceptable carrier or excipient.

[0154] In another embodiment, a pharmaceutical composition is formulated for subcutaneous injection.

[0155] In another embodiment, an effective amount of the pharmaceutical composition is used for treating infertility or conditions related to endogenous FSH deficiency in a subject in need thereof. In further embodiments, the present invention provides for the medical use of Corifollitropin alfa (CFA) or recombinant follicle-stimulating hormone-CTP (FSH-CTP) purified according to the processes described herein in the manufacture of a medicament for controlled ovarian stimulation.

[0156] FSH-CTP (CORIFOLLITROPIN ALFA (CFA)) PROCESS STEPS: MULTIMODAL CHROMATOGRAPHY I FOLLOWED BY AFFINITY CHROMATOGRAPHY (IN

[0157] TANDEM): 5 UF DF 1 :

[0158] MULTIMODAL CHROMATOGRAPHY II: 5 CATION EXCHANGE CHROMATOGRAPHY:

[0159] ANION EXCHANGE CHROMATOGRAPHY:

[0160] UF DF2: EXCIPIENT ADDITION AND DS PREPARATION:

[0161] As illustrated in Figure 1, the downstream purification process for FSH-CTP is outlined in a step-wise flow chart.

[0162] The SDS-PAGE profiles in Figure 2 demonstrate the purity of FSH-CTP developmental batches compared with the reference standard.

[0163] As shown in Figure 3, the CZE profiles confirm the expected charge heterogeneity of FSH-CTP across batches, consistent with the reference standard.

[0164] The SEC-HPLC chromatograms in Figure 4 indicate the absence of high molecular weight aggregates and confirm the monomeric nature of FSH-CTP.

[0165] Step yields of various developmental batches are summarized in Figure 5, highlighting consistent performance across the downstream steps.

[0166] The overall process recovery for FSH-CTP batches is shown in Figure 6, with recoveries maintained within the targeted range.

[0167] Analysis of multiple developmental batches demonstrated that the downstream process reproducibly yields FSH-CTP of high purity and recovery. Non-reducing SDS- PAGE profiles for all batches were consistent with the reference standard, confirming identity and purity. Capillary Electrophoresis (CZE) and Size-Exclusion HPLC further substantiated these findings, with minimal high and low molecular weight species observed. Specifically, Size-Exclusion HPLC showed a main peak percentage in the range of 97.48-97.92%, with low molecular weight (LMW) content maintained between 2.03-2.46%.

[0168] Process yields across critical purification steps— including multimodal chromatography, affinity chromatography, UF / DF, cation exchange, anion exchange, and size-exclusion chromatography— were high, typically within 60-100%. Overall process recovery remained consistently robust across batches.

[0169] These results demonstrate that the described purification process ensures reproducible, pharmaceutical-grade FSH-CTP with stringent control of impurities and strong process efficiency, meeting regulatory requirements for therapeutic applications.

[0170] Building upon the principles demonstrated for CFA, in another specific aspect of the platform process, the production of r-hFSH is described as follows.

[0171] In one aspect, a downstream process is provided for the purification of recombinant human luteinizing hormone (r-hLH). The process comprises the following sequential steps:

[0172] In another embodiment a process for producing recombinant human luteinizing hormone (r-hLH), comprising:

[0173] - subjecting a harvested cell culture to affinity chromatography using a Capture Select HCG matrix;

[0174] - performing ultrafiltration and diafiltration (UF / DF1) using a 10 kDa hollow fiber for buffer exchange with simultaneous concentration of the target protein;

[0175] - carrying out Multimodal Chromatography I using Capto Adhere ImpRes selectively targeting removal of proteases and protease related impurities;

[0176] - conducting anion exchange chromatography using Q. Sepharose Fast Flow for removal of negatively charged impurities, including host cell proteins (HCPs) and host cell DNA (HCD); 1 - employing multimodal chromatography II using Capto MMC ImpRes matrix for active isoform enrichment and separation in eluate;

[0177] - carrying out a second ultrafiltration and diafi Itration (UF / DF2) using a 10 kDa hollow fiber for buffer exchange and concentrating protein for next step;

[0178] - conducting size exclusion chromatography using Superdex 75 prep grade resin for separating out low and high molecular weight impurities if present by fractionating protein peak and pooling further based on purity results;

[0179] - performing nanofiltration using a Planova 20N filter for removal of viruses and adventitious agents;

[0180] - preparing and filtering the final drug substance (DS) using a 0.2 pm capsule filter.

[0181] In an embodiment of the present invention the affinity chromatography step is conducted using a Capture Select HCG matrix with a residence time of 6 minutes.

[0182] In another embodiment of the present invention the affinity chromatography step is performed with a dynamic binding capacity of not more than 1.2 mg / mL of resin.

[0183] In yet another embodiment of the present invention the affinity chromatography step utilizes a 20mM Tris buffer at a pH of 7.5 and a conductivity of 1.5 ± 0.5 mS / cm.

[0184] In an embodiment of the present invention the affinity chromatography step involves washing the column with 20mM Tris buffer at pH 7.5 as an after load wash to remove bound impurities.

[0185] In other embodiment of invention , column is washed with 20mM Tris with 0.2M Arginine HCI buffer having pH of 7.5 , range of 7.0 to 8.0 and a conductivity of 15 ± 3 mS / cm (range of 10 to 20 mS / cm)for 15 CV targeted for removal of non-specific impurities for 5CV followed by wash of equilibration buffer containing 20mM Tris buffer at a pH of 7.5 and a conductivity of 1.5 ± 0.5 mS / cm.

[0186] In an embodiment of the present invention the affinity chromatography step uses a buffer consisting of 20 mM Tris, 0.2 M Arginine HCI, and 2 M MgCI2 for elution having pH of 1.5, range of 7.0 to 8.0 and a conductivity of 350 ± 50 mS / cm (range of 300 to 400 mS / cm) passed for 5CV under cold condition.

[0187] In an embodiment of the present invention the ultrafiltration / diafiltration (UF / DF1) step is carried out within a pH range of 7.5 to 8.5, with a target pH of 8.0 ± 0.1, using a 10 kDa molecular weight cutoff membrane, and the eluate has a conductivity range of 0.5 to 1.5 mS / cm.

[0188] In an embodiment of the present invention the ultrafiltration / diafiltration (UF / DF1) step achieves a target protein concentration not less than 0.3 mg / ml.

[0189] In an embodiment of the present invention the UF / DF 1 step is performed using a 20mM Tris buffer at a pH of 8.0 with range of 7.5 to 8.5 and a conductivity of 0.5 to 1.5 mS / cm.

[0190] In another embodiment of the present invention the UF / DF 1 step involves using 8 to 12 diavolumes (DV) of d iafi Itration buffer to remove impurities.

[0191] In another embodiment of the present invention the UF / DF 1 step is performed at a transmembrane pressure of 0.3 to 0.7 bar.

[0192] In yet another embodiment of the present invention the UF / DF 1 step is performed under cold conditions to minimize protein degradation.

[0193] In an embodiment of the present invention the Multimodal chromatography I step utilizes Capto Adhere ImpRes resin that combines hydrophobic interaction and anionic exchange operated at 5 minutes residence time; where resin is regenerated using 3CV of 0.5 M NaOH solution followed by charging of resin using 3CV of IM NaCI solution followed by equilibration using buffer containing 20mM Tris buffer at a pH of 8.0 with range of 7.5 to 8.5 and a conductivity of 0.5 to 1.5 mS / cm which is passed for 5CV.

[0194] In another embodiment; diafiltrate material is loaded followed by afterload wash using same equilibration buffer containing 20mM Tris buffer at a pH of 8.0 with range of 7.5 to 8.5 and a conductivity of 0.5 to 1.5 mS / cm which is passed for 10 CV. In an embodiment of the present invention 10CV length of gradient between Buffer F (20mM Tris buffer at a pH of 8.0 with range of 7.5 to 8.5 and a conductivity of 0.5 to 1.5 mS / cm) and Buffer G containing lOOmM Citrate buffer having preferable pH of 5.5 with range of 5.0 to 6.0 and conductivity of 10 to 20 mS / cm , preferable 13± 3 mS / cm where gradient includes 0%B to 50% B at end of 10CV gradient length.

[0195] In an embodiment of the present invention protein peak is collected in fractions and main peak is pooled for further processing and optionally 3 CV of IM NaCI solution is passed post elution to ensure complete recovery of protein.

[0196] In an embodiment of the present invention the anion exchange chromatography step is performed in a non-binding flow-through mode, adjusting the pH of the multimodal chromatography I step eluate within a pH range of 6.0 to 8.0; with a target pH of 7.0 ± 0.1, and a conductivity targeted at 6± 2 mS / cm and range of 1 to 10 mS / cm, allowing r- hLH to pass through while removing anionic impurities.

[0197] In an embodiment of the present invention the anion exchange chromatography step utilizes 50mM NaCI in 20 mM Tris as the flow-through buffer.

[0198] In an embodiment of the present invention the anion exchange chromatography step is performed using a 20mM Tris buffer with 50mM NaCI at a pH of 7.0 and a conductivity of 6 ± 2 mS / cm.

[0199] In an embodiment of the present invention the anion exchange chromatography step employs a Q. Sepharose Fast Flow for capturing impurities.

[0200] In an embodiment of the present invention the anion exchange chromatography step involves washing the resin after loading with a 20mM Tris buffer containing 50mM NaCI at least 5 CV and protein peak of 5mAU ascending to 5mAU descending is collected as flow through / eluate followed by 3CV wash of IM NaCI solution to ensure protein recovery. In an embodiment of the present invention the multimodal chromatography II step is performed using a Capto MMC ImpRes resin that combines hydrophobic interaction and cation exchange, with the anion exchange eluate loaded with buffer condition using a 20mM Tris buffer with 50mM NaCI at a pH of 7.0 and a conductivity of 6 ± 2 mS / cm followed by Afterload wash with same buffer for 15CV.

[0201] In an embodiment of the present invention the multimodal chromatography II step further utilizes linear gradient of 30CV having Buffer I : 20mM Tris buffer with 50mM NaCI at a pH of 7.0 and a conductivity of 6 ± 2 mS / cm for Port A and Buffer J: 20mM Tris buffer with 500 mM NaCI at a pH of 7.0 ± 0.1 with range of 6.0 to 8.0 and a conductivity of 45 ± 5 mS / cm in Port B with target protein peak collection in second peak.

[0202] In an embodiment of the present invention the multimodal chromatography step employs a residence time of 5 minutes overall with after elution, optional step utilizes wash of 3CV of IM NaCI solution to ensure complete protein recovery

[0203] In an embodiment of the present invention the multimodal chromatography step is performed with a dynamic binding capacity of 10 mg / mL of resin.

[0204] In an embodiment of the present invention the multimodal chromatography step utilizes Capto MMC ImpRes resin for enhanced impurity removal and as an active isoform enrichment step.

[0205] In an embodiment of the present invention the Ultrafiltration / Diafiltration 2 (UF / DF 2) step is performed using a 10 mM Sodium Phosphate buffer with 11.85 mg / mL L Arginine HCI at a pH of 7.0 with range of 6.0 to 8.0 and a conductivity of 5 ± 2 mS / cm and range of 3.0 to 7.0 mS / cm.

[0206] In an embodiment of the present invention the UF / DF 2 step involves concentrating the product to the desired final concentration along with buffer exchange. In an embodiment of the present invention the final ultrafiltration / diafiltration (UF / DF2) step is conducted using a 10 kDa molecular weight cutoff membrane, achieving a target protein concentration of 5.0 ± 1 mg / mL, and buffer exchange with the eluate having a pH range of 6.0 to 8.0, with a target pH of 7.0 ± 0.1, and a conductivity range of 3 to 7 mS / cm.

[0207] In an embodiment of the present invention the UF / DF 2 step is performed with a transmembrane pressure of 0.3 to 0.7 bar.

[0208] In an embodiment of the present invention the UF / DF 2 step is conducted under cold conditions to preserve the stability of the protein.

[0209] In an embodiment of the present invention the UF / DF 2 step uses 8 to 12 diavolumes of diafiltration buffer to achieve the desired buffer exchange.

[0210] In another embodiment, the process includes a Size Exclusion Chromatography step targeting the final polishing of the protein, ensuring high purity and minimal aggregates.

[0211] In another embodiment, the Size exclusion Chromatography step is performed using a Superdex 75 Prep grade resin. The column is equilibrated with Buffer K ( 10 mM Sodium Phosphate buffer with 11.85 mg / mL L Arginine HCI) (pH 7.0, preferably 6.5 to 7.5) with a conductivity of 5 ± 2 mS / cm.

[0212] In another embodiment, the diafiltrate and concentrated protein is loaded onto the column ensuring linear velocity of 30 cm / hr. and Postload wash of equilibration buffer for 1 CV is given with peak collected in fractions. The theoretical pool of fractions are prepared and analysed by analytical method of SEC for purity content on based on result; final pooling of fractions is done for further step of purification.

[0213] In another embodiment, wash of 0.5 M NaOH for 3CV is given to column and stored in 20% Ethanol solution. In an embodiment of the present invention the nanofiltration step is performed using a Buffer K (10 mM Sodium Phosphate buffer with 11.85 mg / mL L Arginine HCI) (pH 7.0, preferably 6.5 to 7.5) with a conductivity of 5 ± 2 mS / cm.

[0214] In an embodiment of the present invention the nanofiltration step employs a Planova 20N filter for virus removal.

[0215] In an embodiment of the present invention the nanofiltration step is carried out at a flow rate of 50 to 100 L / m2 / h.

[0216] In an embodiment of the present invention the nanofiltration step is performed to remove particles approximately 20 nm or larger, including viral particles and other adventitious agents.

[0217] In an embodiment of the present invention wherein the process, further comprises analyzing the purity of the final r-hLH product using techniques such as SDS-PAGE, Capillary Electrophoresis (CZE), Size-Exclusion HPLC.

[0218] In an embodiment of the present invention the drug substance is prepared with 38.47 g / L Sucrose + 0.15 g / L L-Methionine + 0.25 g / L Poloxamer 188 + 11.85 g / L L-Arginine HCI in Buffer K ( 10 mM Sodium Phosphate buffer with 11.85 mg / mL L Arginine HCI) (pH 7.0, preferably 6.5 to 7.5) with a conductivity of 5 ± 2 mS / cm.

[0219] In an embodiment of the present invention the drug substance preparation involves adjusting the pH to 7.0 using IM NaOH or 5M Ortho phosphoric acid.

[0220] In an embodiment of the present invention the drug substance is filtered using Polyethersulphone (PES) based filters with a pore size of 0.2 pm targeting final concentration of 0.3 to 1.0 mg / mL.

[0221] In an embodiment of the present invention the drug substance filtration is performed at a pressure of 1 to 1.5 bar to ensure efficient filtration without damaging the protein.

[0222] In an embodiment of the present invention the final drug substance is filled into sterile containers like PETG under aseptic conditions and stored at -20°C. In an embodiment of the present invention the final drug substance (DS) material has a target protein concentration of 0.1 mg / ml to 1.0 mg / ml, and the overall process recovery is no less than 20%.

[0223] One embodiment of the present invention provides a pharmaceutical composition comprising recombinant human luteinizing hormone (r-hLH), produced by the process of present invention, in combination with a gonadotropin such as r-hFSH, wherein the composition is in liquid form.

[0224] Another embodiment of the present invention offers a method for treating luteinizing hormone deficiency in a patient. This method involves administering a therapeutically effective amount of recombinant human luteinizing hormone (r-hLH), produced by the process of present invention, in combination with a gonadotropin such as r-hFSH.

[0225] In further embodiments, the present invention provides for the medical use of recombinant human luteinizing hormone (r-hLH) purified according to the processes described herein in the manufacture of a medicament for stimulation of follicular development or ovulation induction.

[0226] In a further embodiment, the combination of r-hLH and a gonadotropin like r-hFSH is administered via subcutaneous injection.

[0227] Additionally, another embodiment provides that the therapeutically effective dose ranges from 3.0 to 6.0 pg of r-hLH per administration, with the dosage determined based on the patient's response and treatment plan as advised by a fertility expert

[0228] RECOMBINANT HUMAN LUTEINIZING HORMONE (R-HLH) PROCESS STEPS DETAILS :

[0229] AFFINITY CHROMATOGRAPHY

[0230] UF / DF 1 MULTIMODAL CHROMATOGRAPHY I :

[0231] SIZE EXCLUSION CHROMATOGRAPHY:

[0232] DS PREPARATION / 0.2 UM FILTRATION: The downstream purification process for r-hLH is presented in the flow chart of Figure 7.

[0233] The SEC-HPLC profile in Figure 8 demonstrates the purity and monomeric nature of the r-hLH drug substance compared to the reference standard.

[0234] As depicted in Figure 9, the SDS-PAGE (non-reducing) analysis confirms the identity and purity of r-hLH relative to the reference standard.

[0235] Step yields and overall process recovery of r-hLH are shown in Figure 10, reflecting consistent recovery across purification stages.

[0236] The production process for recombinant human luteinizing hormone (r-hLH) demonstrated consistent and efficient recovery across each step, achieving an overall process recovery of 14.99%. The final drug substance (DS) met high purity standards, as confirmed by the SEC HPLC profile, which showed a monomer content of 96.23%, with minimal high molecular weight (HMW) and low molecular weight (LMW) impurities. SDS-PAGE analysis (non-reducing) demonstrated a clear match between the drug substance and the reference standard, affirming the integrity of the r-hLH protein. Additionally, capillary zone electrophoresis (CZE) confirmed the product's consistency against the reference standard.

[0237] Each step of the process, from affinity chromatography to ultrafiltration / diafiltration (UF / DF) and multimodal chromatography (MMC), contributed to maintaining product quality and yield. The step yields ranged from 82% in the affinity step to 113.88% in the size exclusion chromatography step, reflecting the process's robustness and scalability. The final drug substance, after excipient addition and 0.2 pm filtration, achieved an overall yield above 10%, meeting the target for large-scale production.

[0238] This innovative process not only ensures the clinical-grade quality of r-hLH but also improves its yield and stability. By integrating various purification techniques optimized for parameters such as pH, conductivity, and buffer composition, the method addresses the challenges associated with r-hLH production. The process is designed to meet stringent regulatory standards, making it valuable for therapeutic applications in fertility treatments, ultimately advancing reproductive health and benefiting both patients and healthcare providers.

[0239] Building upon the principles demonstrated for r-hLH, in a specific aspect of the platform process, the production of r-hCG is described as follows

[0240] In one aspect, the present invention provides a process for the purification of recombinant human chorionic gonadotropin (r-hCG), a heterodimeric glycoprotein hormone. The process comprises the following sequential steps:

[0241] The process is further defined by the following embodiments.

[0242] In an embodiment of the present invention, a process for producing recombinant human chorionic gonadotropin (r-hCG) comprises the steps of:

[0243] (i) AFFINITY CHROMATOGRAPHY of a harvest containing r-hCG on a suitable affinity resin, wherein the conditions include equilibrating the resin at a specific pH and conductivity range, and applying a residence time and dynamic binding capacity sufficient to capture r-hCG;

[0244] (ii) DIAFILTRATION of the eluate from the affinity chromatography using a membrane with a defined molecular weight cutoff to remove impurities, wherein the conditions include a specific pH and conductivity range;

[0245] (iii) MULTIMODAL CHROMATOGRAPHY utilizing Capto Adhere ImpRes resin, for separation of active isoform of rhCG , wherein the conditions include a specific pH and conductivity range;

[0246] (iv) DIAFILTRATION of the eluate from the multimodal chromatography using a membrane with a defined molecular weight cutoff to remove impurities, wherein the conditions include a specific pH and conductivity range;

[0247] (vi) ION EXCHANGE CHROMATOGRAPHY of the diafiltered retentate on an ion exchange resin, in a flow through mode wherein the conditions include applying a residence time and dynamic binding capacity, and equilibrating at a specific pH and conductivity range to further purify r-hCG;

[0248] (vii) HYDROPHOBIC INTERACTION CHROMATOGRAPHY of the ion exchange chromatography eluate on a hydrophobic interaction resin, wherein the conditions include a defined residence time, dynamic binding capacity, and specific pH and conductivity range;

[0249] (viii) DIAFILTRATION / CONCENTRATION of the eluate from the hydrophobic interaction chromatography using diafiltration, wherein the conditions include a specific pH and conductivity range;

[0250] (ix) NANOFILTRATION of the diafiltered concentrate using a suitable nanofiltration filter, wherein the conditions include a defined pH and conductivity range;

[0251] (x) FILTRATION of the nano filtrate through a filter to obtain a final drug substance with a target protein concentration of not less than a defined amount.

[0252] In an embodiment of the present invention, a process for producing recombinant human chorionic gonadotropin (r-hCG) comprises the steps of:

[0253] 1. Affinity Chromatography Using Blue Sepharose 6 Fast Flow Resin:

[0254] - Preparation and Equilibration: The column is prepared by rinsing with Water for Injection (WFI) and performing a Clean-in-Place (CIP) using NaOH. The column is then equilibrated with a sodium phosphate buffer, ensuring the optimal pH and conductivity for subsequent protein binding.

[0255] This step selectively captures r-hCG, significantly reducing impurities and setting the stage for high-purity extraction.

[0256] - Loading and Post-Load Washes: The harvested culture is loaded onto the equilibrated column. The column is then washed with sodium phosphate buffer and a low-pH buffer to remove unbound and weakly bound impurities.

[0257] This step efficiently removes unwanted proteins and contaminants, ensuring that only the desired r-hCG is retained on the column.

[0258] - Virus Inactivation and Post-Hold Washes: A low-pH hold of not less than 6 hrs. is applied to inactivate potential viral contaminants, followed by additional washes to remove inactivated viral particles and other residuals.

[0259] This step is critical for ensuring viral safety and compliance with regulatory standards.

[0260] - Elution: The bound r-hCG is eluted using a high-salt buffer, which releases the protein from the column. This step yields a concentrated r-hCG solution with reduced contaminants, ready for further purification.

[0261] 2. First Ultrafiltration / Diafiltration (UF / DF1) Using Merck Pellicon 3 Cassette:

[0262] - Preparation and Diafiltration: The hollow fiber membrane is prepared through rinsing and CIP. The r-hCG elution from the affinity chromatography is then concentrated and diafiltered using a sodium phosphate buffer.

[0263] This step concentrates the r-hCG and facilitates buffer exchange, which is crucial for subsequent purification steps.

[0264] - Loading, Concentration, and Recirculation Washes: The product is loaded onto the hollow fiber, concentrated, and washed with buffer to further refine the product.

[0265] It removes additional impurities and prepares the r-hCG for further chromatographic purification.

[0266] 3. Multimodal Chromatography Using Capto Adhere ImpRes Resin:

[0267] - Preparation and Equilibration: The column is prepared and equilibrated with sodium acetate buffer to set the conditions for multimodal binding.

[0268] It provides additional refinement by selectively binding active acidic isoforms of r-hCG under specific pH and conductivity conditions.

[0269] - Loading, Post-Load Wash, and Elution: The concentrated product from UF / DF1 is loaded onto the column, washed, and eluted with sodium citrate buffer.

[0270] Further reduces contaminants, particularly host cell proteins and DNA, enhancing the overall purity of the r-hCG.

[0271] 4. Second Ultrafiltration / Diafiltration (UF / DF2) Using Merck Pellicon 3 Cassette:

[0272] - Preparation and Diafiltration: Similar to UF / DF1, this step involves the preparation and diafiltration of the product using sodium phosphate buffer.

[0273] It prepares the r-hCG for ion exchange chromatography by ensuring the proper buffer conditions.

[0274] - Loading, Concentration, and Recirculation Washes: The product is loaded, concentrated, and further washed to achieve the desired purity level.

[0275] It reduces ionic impurities and prepares the product for ion exchange purification. 5. Ion Exchange Chromatography Using DEAE Sepharose:

[0276] - Preparation and Equilibration: The column is prepared and equilibrated to bind charged impurities like HCPs under specific pH & ionic conditions.

[0277] This step is essential for removing charged impurities, further enhancing the purity of the r-hCG.

[0278] - Loading and flow-through wash collection: The product is loaded, and collected in flow-through wash (non-binding conditions) using same equilibration buffer, achieving a high-purity r-hCG solution.

[0279] 6. Hydrophobic Interaction Chromatography (HIC):

[0280] Preparation and Equilibration: The Hydrophobic Interaction Chromatography (HIC) step begins with thorough preparation of the chromatography column. The column is first cleaned using a pre-use cleaning protocol with 0.5 M NaOH to ensure that any residual contaminants or previous substances are removed. This step is crucial for maintaining the integrity and performance of the column.

[0281] Next, the column is equilibrated with Buffer, which consists of 15 mM Sodium Phosphate and 1.0 M NaCI. The purpose of this equilibration step is to establish the optimal conditions for protein binding. The high salt concentration in Buffer promotes strong hydrophobic interactions between the target protein (recombinant human chorionic gonadotropin, r-hCG) and the hydrophobic resin. This setup ensures that the column environment is ideal for the subsequent loading of the protein sample.

[0282] Loading and Flow-Through Wash Collection: The loading phase involves introducing the pre-treated and concentrated sample (AEX Elute) into the equilibrated column. The high salt concentration in the loading buffer facilitates the binding of r-hCG to the hydrophobic resin, while allowing other less hydrophobic contaminants to pass through.

[0283] Following the loading step, a flow-through wash is performed using Buffer. This wash collects proteins and impurities that do not strongly bind to the resin and thus flow through the column. The impact of this wash is twofold: it helps in removing non- specifically bound contaminants, and it ensures that only the proteins with the strongest hydrophobic interactions remain attached to the resin. This step is critical for achieving a high purity of r-hCG as it ensures that the proteins that elute later are predominantly the target protein with minimal contamination.

[0284] 7. Third Ultrafi Itration / Diafiltration (UF / DF3) Using Merck Pellicon 3 Cassette: - Preparation and Diafiltration: The final ultrafiltration and diafiltration steps prepare the r-hCG for viral clearance.

[0285] Ensures the product is concentrated and in the correct buffer for final filtration steps.

[0286] 8. Nanofiltration Using Planova 20N Filter:

[0287] - Virus Filtration: The r-hCG is passed through a Planova 20N filter to remove any remaining viral particles.

[0288] Ensures the viral safety of the final drug substance (DS), which is critical for patient safety and regulatory approval.

[0289] 9. Final Drug Substance Preparation:

[0290] - Formulation and Filtration: The purified r-hCG is formulated in suitable buffer and filtered to ensure sterility.

[0291] Produces a good quality, stable, and with high-purity r-hCG product ready for therapeutic applications.

[0292] In one embodiment, the process for producing recombinant human chorionic gonadotropin (r-hCG) begins by subjecting the harvested cell culture to affinity chromatography using a column packed with Blue Sepharose 6 Fast Flow resin. The column is equilibrated with a buffer comprising 15 mM Sodium Phosphate at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 1.0 to 4.0 mS / cm. The chromatography is conducted with a residence time of not less than 4 minutes and a dynamic binding capacity of 150,000 lU / mL.

[0293] Following affinity chromatography, the eluate is subjected to ultrafiltration and diafiltration (UF / DF1) using a 10 kDa molecular weight cutoff membrane. The diafiltration is performed with 4 mM Sodium Phosphate buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 0.7 to 1.7 mS / cm. This step is conducted to both concentrate the protein and perform buffer exchange.

[0294] Next, the diafiltered retentate undergoes purification by Multimodal Chromatography using a column packed with Capto Adhere ImpRes resin. The column is equilibrated with 20 mM Sodium Acetate buffer at a pH range of 5.5 to 6.5, preferably at pH 6.0, and a conductivity range of 1.0 to 2.0 mS / cm. The chromatography is conducted with a residence time of not less than 4 minutes and a dynamic binding capacity of 260,000 lU / mL, corresponding to 10 mg of protein per mL of resin.

[0295] The eluate from the multimodal chromatography is again subjected to ultrafiltration and diafiltration (UF / DF2) using a 10 kDa molecular weight cutoff membrane. This diafiltration is carried out using a buffer comprising 4 mM Sodium Phosphate and 0.1M NaCI at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 10 to 20 mS / cm. Subsequently, the diafi Itered retentate is purified by ion exchange chromatography on a column packed with Diethylaminoethyl (DEAE) Sepharose resin. The column is equilibrated with 4 mM Sodium Phosphate and 0.1 M NaCI buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 10 to 20 mS / cm. The target protein is collected in the flow-through (non-binding mode).

[0296] Following ion exchange chromatography, the eluate is further purified by hydrophobic interaction chromatography (HIC) using a column packed with Toyopearl Butyl 600 M resin. The column is equilibrated with 15 mM Sodium Phosphate and 1.0 M NaCI buffer at a pH range of 7.0 to 8.0 and a conductivity range of 100 to 200 mS / cm. The target protein is eluted in bind-and-elute mode using IM Urea + 10% Isopropyl Alcohol (IPA) in 15 mM Sodium Phosphate as the elution buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 0.5 to 3.0 mS / cm.

[0297] The eluate from HIC is then concentrated using a 10 kDa molecular weight cutoff membrane in diafiltration (UF / DF3), with conditions maintained at a pH range of 6.3 to 6.7 and a conductivity range of 0.2 to 0.6 mS / cm. This step further concentrates the r- hCG and prepares it for the final purification steps.

[0298] The concentrated retentate is subjected to nanofiltration using a Planova 20N filter, under conditions comprising a pH range of 6.3 to 6.7 and a conductivity range of 0.2 to 0.6 mS / cm. This step ensures viral safety by removing any potential viruses and adventitious agents.

[0299] Finally, the nanofiltrate is filtered through a 0.2 pm filter to obtain the final drug substance (DS) with a target potency is not less than 30,000 lU / mL. The purified r-hCG exhibits a purity of not less than 95%, making it suitable for pharmaceutical compositions.

[0300] In another embodiment, the r-hCG produced by this process is formulated into a pharmaceutical composition comprising a therapeutically effective amount of r-hCG combined with a pharmaceutically acceptable carrier or excipient. This composition may include stabilizers such as mannitol, sucrose, or trehalose, and is presented in a liquid dosage form.. The pharmaceutical composition is intended for use in treating infertility, hypogonadism, and assisted reproductive technologies (ART), with the r-hCG administered via subcutaneous or intramuscular injection at a dosage ranging from 3250 III per dose to 6500 III per dose.

[0301] In a further embodiment process for producing recombinant human chorionic gonadotropin (r-hCG) involves several sequential steps. First, the harvested cell culture is subjected to affinity chromatography using a column packed with Blue Sepharose 6 Fast Flow resin. The column is equilibrated with a buffer comprising 15 mM Sodium Phosphate at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 1.0 to 4.0 mS / cm. The column operates with a residence time of not less than 4 minutes and a dynamic binding capacity of 150,000 lU / mL. Following affinity chromatography, ultrafiltration and diafiltration (UF / DF1) are performed using a 10 kDa hollow fiber membrane for buffer exchange and simultaneous concentration of the target protein. The process continues with multimodal chromatography using a column packed with Capto Adhere Impres resin, equilibrated with 20 mM Sodium Acetate buffer at a pH range of 5.5 to 6.5, preferably at pH 6.0, and a conductivity range of 1 to 2 mS / cm. The column has a residence time of not less than 4 minutes and a dynamic binding capacity of 260,000 lU / mL, corresponding to 10 mg protein per mL of resin. A second ultrafiltration and diafiltration (UF / DF2) using a 10 kDa hollow fiber membrane is then carried out for further buffer exchange. Ion exchange chromatography is performed using a column packed with Diethylaminoethyl (DEAE) Sepharose resin, equilibrated with 4 mM Sodium Phosphate and 0.1 M NaCI buffer at a pH range of 7.5 to 8.5, preferably at pH 7.5, and a conductivity range of 10 to 20 mS / cm, with the target protein collected in flow-through (non-binding mode). The process also includes hydrophobic interaction chromatography (HIC) using a column packed with Toyopearl Butyl 600M resin, equilibrated with 15 mM Sodium Phosphate and 1.0 M NaCI buffer at a pH range of 7.0 to 8.0 and a conductivity range of 100 to 200 mS / cm, with the target protein collected in bind & elute mode using 1 M Urea + 10% Isopropyl alcohol (IPA) in 15 mM Sodium Phosphate as elution buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 0.5 to 3.0 mS / cm. Subsequently, a third ultrafiltration and diafiltration (UF / DF3) is performed using a 10 kDa hollow fiber membrane for buffer exchange. Nanofiltration using a Planova 20N filter is conducted to remove viruses and adventitious agents, followed by the preparation and filtration of the final drug substance (DS) using a 0.2 pm capsule filter.

[0302] In another embodiment the affinity chromatography step includes an additional elution step where the r-hCG is eluted with a buffer comprising 15 mM Sodium Phosphate and 2.5 M NaCI at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 300 to 400 mS / cm.

[0303] In another embodiment, following the affinity chromatography step, a low pH wash is performed using 25 mM Sodium Acetate buffer at a pH range of 3.5 to 4.5, preferably at pH 4.0. This is followed by a hold period of not less than 6 hours for virus inactivation. In another embodiment, the ultrafiltration and diafiltration (UF / DF1) step is carried out using 4 mM Sodium Phosphate buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 0.5 to 2.5 mS / cm. The process includes 6 diavolumes, with final retentate volume adjusted between 10.5 L to 12.5 L by giving 3 recirculation washes with the same equilibration buffer and addition to retentate.

[0304] In another embodiment, the multimodal chromatography step includes performing washes with a buffer comprising 20 mM Sodium Acetate at a pH range of 5.5 to 6.5 and a conductivity range of 1 to 2 mS / cm. The r-hCG is then eluted with a buffer comprising 100 mM Sodium Citrate at a pH range of 4.0 to 5.0, preferably at pH 4.5, and a conductivity range of 5 to 15 mS / cm.

[0305] In another embodiment, the second ultrafiltration and diafiltration (UF / DF2) step is carried out using 4 mM Sodium Phosphate and 0.1 M NaCI buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 10 to 20 mS / cm. The process includes 6 diavolumes, with final retentate volume adjusted between 10.5 L to 12.5 L by giving 3 recirculation washes with the same equilibration buffer and addition to retentate.

[0306] In another embodiment, the ion exchange chromatography step includes performing flow-through washes with 4 mM Sodium Phosphate and 0.1 M NaCI buffer at a pH range of 7.3 to 7.7 , preferably at pH 7.5, and a conductivity range of 10 to 20 mS / cm, with a residence time of 8 minutes and a dynamic binding capacity of 1,000,000 lU / mL.

[0307] In another embodiment, the hydrophobic interaction chromatography step includes eluting the r-hCG with a buffer comprising 1 M Urea and 10% Isopropyl Alcohol (IPA) in 15 mM Sodium Phosphate at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 0.5 to 3.0 mS / cm.

[0308] In another embodiment, the final ultrafiltration and diafiltration (UF / DF3) step is performed using 20 mM Histidine buffer at a pH range of 6.3 to 6.7 and a conductivity range of 0.1 to 1.0 mS / cm. The process includes 8 diavolumes, with final retentate volume adjusted between 24 L to 40 L by giving 3 recirculation washes of 20 mM Histidine buffer and addition to retentate.

[0309] In another embodiment, the total retentate volume after ultrafiltration / diafiltration (UF / DF3) is adjusted such that the ELISA assay indicates a concentration of not less than 30,000 lU / mL.

[0310] In another embodiment, the nanofiltration step is performed under conditions where the pH is maintained between 6.3 to 6.7, the conductivity is maintained between 0.2 to 0.6 mS / cm, and the nitrogen pressure limit is set between 0.9 to 1.0 kg / cm2.

[0311] In another embodiment, the nanofiltration step is conducted to remove particles approximately 20 nm or larger, including viral particles and other adventitious agents.

[0312] In another embodiment the process further comprises analyzing the purity of the final r-hCG product using techniques such as SDS-PAGE, Size-Exclusion HPLC, or Western blotting. In another embodiment, the target potency is not less than 30,000 lU / mL, with an overall process recovery of not less than 30% and high molecular weight (HMW) impurities below 3%.

[0313] In another embodiment, the final drug substance is prepared using 20 mM L-Histidine at a pH range of 6.3 to 6.7, preferably at pH 6.5, and a conductivity range of 0.1 to 1.0 mS / cm.

[0314] In another embodiment, the final drug substance is filtered using Regenerated Cellulose Acetate (RCA) or Polyethersulfone (PES) based filters.

[0315] In another embodiment a pharmaceutical composition is prepared comprising the purified recombinant human chorionic gonadotropin (r-hCG) obtained by the process of present invention, in combination with a pharmaceutically acceptable carrier or excipient.

[0316] In another embodiment the pharmaceutical composition further comprises an antioxidant selected from the group consisting of L-methionine and variants thereof.

[0317] In another embodiment the pharmaceutical composition further comprises a surfactant selected from the group consisting of polysorbate-20, polysorbate-80, poloxamer 188, and variants thereof.

[0318] In another embodiment the pharmaceutical composition the r-hCG is present in an amount of 3250 III per vial or 6500 III per vial.

[0319] In another embodiment the pharmaceutical composition the formulation is in the form of liquid dosage.

[0320] In another embodiment the pharmaceutical composition is used for the treatment of infertility or other related reproductive disorders in humans.

[0321] In further embodiments, the present invention provides for the medical use of recombinant human chorionic gonadotropin (r-hCG) purified according to the processes described herein in the manufacture of a medicament for ovulation induction or luteal phase support. Recombinant Human Chorionic Gonadotropin hormone (r-hCG) Process steps details:

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328] The downstream purification process for r-hCG is shown schematically in Figure 11.

[0329] The step-wise process efficiency and overall yield of rhCG batches are presented in Figure 12, highlighting batch-to-batch consistency.

[0330] Aggregate (high molecular weight) content across r-hCG batches is illustrated in Figure 13, demonstrating values well below acceptable thresholds.

[0331] As shown in Figure 14, host cell protein (HCP) levels across r-hCG batches were effectively minimized, far below regulatory limits. The residual DNA content across r-hCG drug substance batches is presented in Figure 15, with all values consistently below defined limits.

[0332] The capillary zone electrophoresis (CZE) profile of rhCG, compared with the reference standard, is illustrated in Figure 16, confirming product identity and purity.

[0333] The analytical evaluation of recombinant human chorionic gonadotropin (r-hCG) drug substance (DS) batches demonstrated strong overall process performance and product consistency. Bioassay data confirmed high biological activity across all batches, with Batch 2 showing the highest potency at 52,259 lU / mL, indicating enhanced biological activity relative to the other batches.

[0334] Aggregate content was consistently well controlled, remaining below the 3.0% threshold in all batches, thereby confirming the stability of the final DS. Host cell protein (HCP) clearance was robust, with levels far below the acceptance criterion of 100 ng / 6500 IU. Among the evaluated batches, Batch 1 showed the lowest HCP content at 1.55 ng / 6500 IU, highlighting the efficiency of impurity removal.

[0335] Residual DNA levels were also maintained at extremely low levels, consistently meeting the acceptance criterion of <100 pg / 6500 IU. Batch 3 demonstrated the lowest DNA content at 0.34 pg / 6500 IU, reinforcing the effectiveness of the purification strategy.

[0336] Yield and process recovery data indicated controlled and reproducible performance across batches. Batch 2 achieved the highest recovery at 81.86%, reflecting improved product retention and purification efficiency.

[0337] Overall, the process consistently delivered r-hCG drug substance of high purity, stability, and potency, with impurity levels (aggregate, HCP, residual DNA) maintained well within defined acceptance limits. These results confirm that the disclosed process is highly effective for producing therapeutic-grade r-hCG with reproducible yield and robust biological activity. In another specific aspect of the platform process, the present invention provides a process for the purification of recombinant human follicle-stimulating hormone (r- hFSH), a heterodimeric glycoprotein hormone expressed in mammalian cells. The process comprises the following sequential steps:

[0338] Affinity Chromatography:

[0339] In one embodiment of the present invention the process begins with affinity chromatography utilizing Blue Sepharose 6 Fast Flow resin, a critical step for selectively binding and eluting recombinant human follicle-stimulating hormone (r-hFSH). This resin is specifically chosen for its affinity to r-hFSH, ensuring effective binding and facilitating the removal of unwanted proteins and impurities early in the purification process.

[0340] In another embodiment the Blue Sepharose 6 Fast Flow resin is equilibrated with a 15 mM sodium phosphate buffer at a pH range of 7.0 to 8.0 and a conductivity range of

[0341] 2.2 to 3.2 mS / cm. These conditions are meticulously controlled to optimize the binding of r-hFSH to the resin while maintaining the structural integrity of the protein.

[0342] In another embodiment after equilibration, the harvested cell culture containing r-hFSH is loaded onto the column. This step ensures that the r-hFSH binds efficiently to the Blue Sepharose resin, allowing for the subsequent removal of contaminants through sequential wash steps.

[0343] In yet another embodiment following the loading of the cell culture, a post-load wash is conducted using the same 15 mM sodium phosphate buffer to remove unbound proteins and other impurities. This wash step is essential to ensure that only specifically bound r-hFSH remains on the resin for further purification.

[0344] In another embodiment a low pH wash is performed using a 25 mM sodium acetate buffer at pH range of 3.8-4.2, preferably at pH 4.0, with a conductivity range of 1.7 to

[0345] 2.3 mS / cm. This step includes a virus inactivation hold period of no less than 6 hours, which is critical for ensuring the safety of the final drug substance (DS) by eliminating potential viral contaminants.

[0346] In another embodiment after virus inactivation, the column is further washed with a 15 mM sodium phosphate + 250 mM NaCI buffer at pH 7.0 to 8.0 and a conductivity range of 25 to 35 mS / cm. This high salt wash step is crucial for removing any weakly bound impurities from the resin, further purifying the bound r-hFSH.

[0347] In another embodiment finally, r-hFSH is eluted using a 15 mM sodium phosphate + 2.5 M NaCI buffer at pH range of 7.0 to 8.0, preferably at pH 7.5 and a conductivity range of 300 to 400 mS / cm. This elution step is designed to release the r-hFSH from the resin in a highly concentrated and purified form, ready for the next stage of the purification process.

[0348] Hydrophobic Interaction Chromatography (HIC)

[0349] In another embodiment the process includes a hydrophobic interaction chromatography (HIC) step using Phenyl Sepharose Fast Flow resin. This resin is chosen for its strong hydrophobic interactions with the r-hFSH protein, facilitating the separation of r-hFSH from hydrophilic impurities and enhancing the purity of the product.

[0350] In another embodiment the Phenyl Sepharose Fast Flow resin is equilibrated with a 15 mM sodium phosphate + 2.5 M NaCI buffer at a pH range of 7. O to 8.0 and a conductivity range of 300 to 400 mS / cm. These equilibration conditions are optimized to promote strong binding of the r-hFSH to the hydrophobic resin, setting the stage for effective impurity removal.

[0351] In another embodiment the eluate from the affinity chromatography step is loaded onto the HIC column, where the r-hFSH binds to the hydrophobic resin. This loading step ensures that r-hFSH is efficiently captured by the resin, allowing for further purification through the selective retention of hydrophobic molecules. In another embodiment a post-load wash is performed using the same 15 mM sodium phosphate + 2.5 M NaCI buffer, ensuring that loosely bound impurities are removed from the column while the r-hFSH remains bound to the resin. This step is essential for enhancing the purity of the final drug substance (DS) by removing non-specifically bound contaminants.

[0352] In another embodiment the r-hFSH is eluted from the Phenyl Sepharose Fast Flow resin using a 15 mM sodium phosphate + 1.0 M NaCI buffer at pH range of 7.0 to 8.0, preferably at pH 7.5 and a conductivity range of 100 to 200 mS / cm. This elution step is designed to release r-hFSH from the hydrophobic interactions with the resin, yielding a highly purified product.

[0353] Ultrafiltration / Diafiltration

[0354] In another embodiment following HIC, ultrafiltration / diafiltration is conducted using a 10 kDa MWCO Hollow Fiber membrane. This membrane selection is critical for concentrating the r-hFSH while removing low molecular weight impurities, ensuring the retention of the desired protein while allowing smaller molecules to pass through.

[0355] In another embodiment diafiltration is performed using a 15 mM sodium phosphate buffer at a pH range of 7.8 to 8.2 and a conductivity range of 1.5 to 3.5 mS / cm. These conditions are carefully controlled to maintain the stability and activity of r-hFSH while removing salts and other small molecules.

[0356] In another embodiment the retentate is concentrated until a final volume of 1.0 to 1.5 liters is achieved. This step is crucial for reducing the volume of the solution, thereby increasing the concentration of r-hFSH in preparation for subsequent purification steps.

[0357] In another embodiment the three recirculation washes of 1.5 liter each are conducted using a 15 mM sodium phosphate + 0.3 M NaCI buffer at pH 7.8 to 8.2, preferably at pH 8.0 with a conductivity range of 30 to 50 mS / cm, totaling a volume of 5.8 to 6.2 liters. These washes are performed to further remove any remaining impurities while maintaining the desired ionic environment for r-hFSH for the next process step. Ion Exchange Chromatography (IEC)

[0358] In another embodiment the diafiltered retentate is subjected to ion exchange chromatography (IEC) using Quaternary High Resolution (QHR) resin. This resin is selected for its ability to separate r-hFSH based on its charge, providing a high- resolution separation that enhances the purity of the final drug substance (DS).

[0359] In another embodiment the QHR resin is equilibrated with a 15 mM sodium phosphate + 0.3 M NaCI buffer at a pH range of 7.8 to 8.2, preferably at pH 8.0 and a conductivity range of 30 to 50 mS / cm. These conditions are optimized to facilitate the binding of negatively charged impurities, as well as basic isoforms of r-hFSH to the ion exchange resin while allowing active acidic isoforms of r-hFSH collection in flow-through wash using the same equilibration buffer.

[0360] In another embodiment the diafiltered retentate is loaded onto the IEC column, where r-hFSH and other charged impurities interacts with the QHR resin. This step is critical for capturing the inactive basic isoforms of r-hFSH, while setting the stage for selective flow-through of active acidic isoforms of r-hFSH protein.

[0361] In another embodiment a flowthrough wash is performed using the same equilibration buffer until a final volume of 24.5 ± 0.1 liters is reached.

[0362] Nanofiltration

[0363] In another embodiment Nanofiltration is conducted using a Viresolve Pro Modus 1.2 filter, selected for its ability to remove viruses and other large contaminants from the r-hFSH solution, ensuring the safety and purity of the final drug substance (DS).

[0364] In another embodiment the filter is equilibrated with a 15 mM sodium phosphate + 0.3 M NaCI buffer at a pH range of 7.8 to 8.2, preferably at pH 8.0and a conductivity range of 30 to 50 mS / cm. These conditions are maintained to ensure optimal filtration performance and prevent the loss of r-hFSH during the nanofiltration process. In another embodiment the nitrogen pressure during filtration is carefully controlled, not exceeding 1.9 kg / cm2. This pressure limit is essential for maintaining the integrity of the filter and preventing damage to the r-hFSH protein during the nanofiltration process.

[0365] Concentration

[0366] In another embodiment the 10 kDa concentration step using Hollow Fiber membrane is performed with the following conditions:

[0367] - Concentration with 15 mM sodium phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 , preferably at pH 7.5 and a conductivity range of 30 to 50 mS / cm, until the retentate volume is reduced to approximately 0.8 liters and a protein concentration not less than 1.0 mg / ml.

[0368] - Three Recirculation washes of 0.25 Liters each with 15 mM sodium phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 , preferably at pH 7.5 and a conductivity range of 30 to 50 mS / cm, , followed by addition in the above retentate volume and making up a total volume of not less than 1.6 liters with buffer

[0369] Mixed-Mode Chromatography (MMC)

[0370] In another embodiment Mixed-mode chromatography (MMC) is performed using Capto Adhere resin, chosen for its ability to interact with r-hFSH through a combination of ionic, hydrophobic, and hydrogen bonding interactions. This multi-modal approach enhances the selectivity and purity of the final drug substance (DS). This purification step is carefully designed to bring down r-hFSH oligomer content, as well as target protein concentration to a pharmacopeial acceptable level.

[0371] In another embodiment the Capto Adhere resin is equilibrated with a 15 mM sodium phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 and a conductivity range of 30 to 50 mS / cm. In another embodiment the concentrated retentate is loaded onto the MMC column, where r-hFSH and oligomeric impurities interacts with the resin's mixed-mode functionalities These conditions are carefully selected to maximize the binding of r- hFSH oligomeric impurities while allowing target protein to be collected in flow-through wash with same equilibration buffer and providing a high level of purification of r-hFSH.

[0372] In another embodiment, a Flowthrough wash is performed by passing not more than 2.25 liter of same equilibration buffer at a pH range of 7.3 to 7.7, and a conductivity range of 30 to 50 mS / cm, with a flowthrough is collected until a final volume of 3.0 to 4.0 liters is reached This volume range is targeted to achieve a protein concentration between 0.4 and 0.8 mg / mL after collection of buffer wash in flow-through.

[0373] In another embodiment, the pH of the final MMC flow through volume is adjusted between 7.65 to 7.85, preferably at pH 7.75 and a conductivity range of 30 to 50 mS / cm, to finally yield purified r-hFSH in a form suitable for final filtration.

[0374] Final Filtration

[0375] In another embodiment the final filtration step involves filtering the mixed-mode chromatography eluate using a 0.2 pm regenerated cellulose acetate (RCA) filter. This filter is selected for its ability to remove particulate contaminants while maintaining the integrity and concentration of r-hFSH.

[0376] In another embodiment the final drug substance is obtained with a target protein concentration of 0.4 to 0.8 mg / mL. This concentration range is carefully controlled to ensure the efficacy and stability of the r-hFSH in the final drug substance formulation.

[0377] In another embodiment the final drug substance (DS) is formulated to maintain a pH range of 7.0 to 8.0. This pH range is optimized to ensure the stability and bioactivity of r-hFSH in the final injectable formulation, making it suitable for therapeutic use.

[0378] In yet another embodiment:

[0379] - The process begins with Affinity Chromatography using Blue Sepharose 6 Fast Flow resin to selectively bind and elute recombinant human follicle-stimulating hormone (r- hFSH). This step involves multiple buffer conditions to equilibrate, load, wash, and elute the protein. Key parameters include:

[0380] - Equilibration with 15 mM Sodium Phosphate buffer at a pH range of 7.0 to 8.0 and a conductivity range of 2.2 - 3.2 mS / cm.

[0381] - Loading of the harvested cell culture onto the column, followed by a post-load wash using 15 mM Sodium Phosphate buffer at a pH range of 7.0 to 8.0 and a conductivity range of 2.2 - 3.2 mS / cm.

[0382] - A unique low pH wash using 25 mM Sodium Acetate buffer at a pH range of 3.8 to 4.2, preferably at pH 4.0, and a conductivity range of 1.7 to 2.3 mS / cm, with a hold of not less than 6 hours for virus inactivation.

[0383] - Column washing with 15 mM Sodium Phosphate + 250 mM NaCI buffer at a pH range of 7.0 to 8.0 and a conductivity range of 25 to 35 mS / cm.

[0384] - Elution with 15 mM Sodium Phosphate + 2.5 M NaCI buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 300 to 400 mS / cm.

[0385] - Following Affinity Chromatography, Hydrophobic Interaction Chromatography (HIC) with Phenyl Sepharose Fast Flow resin is employed to further purify the eluted r-hFSH, including removal of host cell proteins (HCPs) and endotoxins. Key parameters include:

[0386] - Equilibration and post-load wash with 15 mM Sodium Phosphate + 2.5 M NaCI buffer at a pH range of 7.0 to 8.0 and a conductivity range of 300 to 400 mS / cm.

[0387] - Elution with 15 mM Sodium Phosphate + 1.0 M NaCI buffer at a pH range of 7.0 to 8.0, preferably at pH 7.5, and a conductivity range of 100 to 200 mS / cm.

[0388] - The Diafiltration step utilizes a 10 kDa MWCO Hollow Fiber membrane to concentrate and exchange the buffer for the next process step. Key parameters include:

[0389] - Diafiltration with 15 mM Sodium Phosphate buffer at a pH range of 7.8 to 8.2 and a conductivity range of 1.5 to 3.5 mS / cm, followed by concentration until a final retentate volume of 1.0 to 1.5 liters is achieved. - Recirculation washes with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.8 to 8.2 and a conductivity range of 30 to 50 mS / cm, with three washes of 1.5 liters each, making up a total volume of 5.8 to 6.2 liters.

[0390] - Ion Exchange Chromatography (IEC) with Quaternary High Resolution (QHR) resin is used to further purify the product, refining the purity of r-hFSH and achieving active acidic isoform separation from inactive basic ones. Key parameters include:

[0391] - Equilibration with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.8 to 8.2 and a conductivity range of 30 to 50 mS / cm.

[0392] - Loading the 10 kDa diafi Itered retentate, followed by a flowthrough wash with the same buffer at a pH range of 7.8 to 8.2 and a conductivity range of 30 to 50 mS / cm, collecting the flowthrough until a final volume of 24.5 ± 0.1 liters is reached.

[0393] - Nanofiltration is performed using Viresolve Pro Modus 1.2 membrane, focusing on buffer equilibration and filtration of the eluate under controlled nitrogen pressure. Key parameters include:

[0394] - Equilibration with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.8 to 8.2 and a conductivity range of 30 to 50 mS / cm.

[0395] - Loading the IEC elute with a nitrogen pressure limit not exceeding 1.9 kg / cm2.

[0396] - A second concentration step is carried out with a 10 kDa MWCO Hollow Fiber membrane to concentrate the nanofiltrate. Key parameters include:

[0397] - Concentration with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 and a conductivity range of 30 to 50 mS / cm, until the retentate volume is reduced to approximately 0.8 liters and a protein concentration not less than 1.0 mg / ml.

[0398] - Recirculation washes with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 and a conductivity range of 30 to 50 mS / cm, with three washes of 0.25 liters each, making up a total volume of not less than 1.6 liters.

[0399] - The final purification step involves Mixed Mode Chromatography (MMC) using Capto Adhere resin, featuring: - Equilibration with 15 mM Sodium Phosphate + 0.3 M NaCI buffer at a pH range of 7.3 to 7.7 and a conductivity range of 30 to 50 mS / cm.

[0400] - Loading the 10 kDa concentrated retentate, followed by a flowthrough wash with not more than 2.25 liters of the same buffer at a pH range of 7.3 to 7.7 and a conductivity range of 30 to 50 mS / cm, with flowthrough collection until a final volume of 3.0 to 4.0 liters is reached.

[0401] - Adjustment of the pH of the final MMC flow-through to between 7.65 to 7.85, preferably at pH 7.75, and a conductivity range of 30 to 50 mS / cm, to finally yield purified r-hFSH in a form suitable for final filtration.

[0402] - The final step involves 0.2 pm filtration using a regenerated cellulose acetate (RCA) filter to ensure the final drug substance (DS) is free from particulate matter, with a target protein concentration of 0.4 to 0.8 mg / ml at a pH range of 7.65 to 7.85.

[0403] In yet another embodiment the purified r-hFSH is free from detectable levels of host cell proteins (HCPs), endotoxins, and aggregates.

[0404] In yet another embodiment the purified r-hFSH can be formulated into pharmaceutical compositions suitable for injection. These compositions include a pharmaceutically acceptable carrier and are formulated to contain r-hFSH at a concentration within pharmacopeial limits, with a pH of 6.5 to 7.5, preferably between 7.1 to 7.3.

[0405] In another embodiment he pharmaceutical composition is free from detectable levels of host cell proteins (HCPs), Host cell DNA (HCD), endotoxins, Oxidised follitropins and Fol litropin Oligomers (HMW / aggregates).

[0406] In another embodiment the pharmaceutical compositions are used for treating infertility in subjects in need thereof. This includes human females undergoing controlled ovarian stimulation and human males undergoing treatment for hypogonadotropic hypogonadism. The compositions can be administered via subcutaneous or intramuscular injection. Another embodiment covers a method of treating infertility in a subject in need thereof, comprising administering an effective amount of these pharmaceutical compositions.

[0407] Another embodiment covers a method, wherein the subject is a human female undergoing controlled ovarian stimulation. Another embodiment covers a method, wherein the subject is a human male undergoing treatment for hypogonadotropic hypogonadism.

[0408] In further embodiments, the present invention provides for the medical use of recombinant human follicle-stimulating hormone (r-hFSH) purified according to the processes described herein in the manufacture of a medicament for controlled ovarian stimulation.

[0409] Another embodiment covers a method, wherein the pharmaceutical composition is administered via subcutaneous or intramuscular injection.

[0410] Recombinant Human Follicle Stimulating hormone (r-hFSH) Process steps details :

[0411]

[0412]

[0413]

[0414]

[0415] The downstream purification process for r-hFSH is summarized in the flow chart of Figure 17.

[0416] Isoelectric focusing (IEF) gel analysis in Figure 18 demonstrates the isoelectric point (pl) consistency between r-hFSH and the reference standard. As shown in Figure 19, follitropin oligomer (HMW) content across r-hFSH batches remained consistently low, below defined limits.

[0417] The oxidized follitropin content, presented in Figure 20, demonstrates minimal oxidation across batches, ensuring product stability.

[0418] As illustrated in Figure 21, host cell protein (HCP) content was reduced to trace levels in all r-hFSH batches.

[0419] Residual host cell DNA (HCD) levels are shown in Figure 22, with all values far below the acceptable pharmacopeial limit.

[0420] Step yields and overall process recovery for r-hFSH batches are summarized in Figure

[0421] 23, confirming robustness and reproducibility of the purification process. The comprehensive analysis of the recombinant human follicle-stimulating hormone (r- hFSH) production process demonstrates consistent high purity, yield, and product quality across multiple batches. Analytical characterization confirmed that the isoelectric focusing (IEF) profile of the produced r-hFSH was fully aligned with the reference standard, with all samples exhibiting the expected bands, thereby affirming protein identity and charge consistency.

[0422] The impurity profile across batches was tightly controlled. Foil itropin oligomer content was consistently maintained at or below 0.06%, and oxidized follitropin species were kept below 1.2%, reflecting minimal degradation and excellent product stability. Host cell proteins (HCPs) were reduced to trace levels, with concentrations consistently below 0.01 ppm, while host cell DNA (HCD) was effectively removed to levels below 0.005 ng per 300 III of r-hFSH. These results confirm the robustness of the purification process in eliminating process- and product-related impurities.

[0423] Process recovery and yield data further validated the efficiency of the method. Step recoveries across chromatography and filtration stages were consistently above 80%, with overall downstream process (DSP) yields ranging from 57% to 84%. Final drug substance (DS) recovery exceeded 60% in most batches, confirming both scalability and reproducibility of the process.

[0424] Distinctive process features, including high dynamic binding capacity of ~8000 lU / mL in the affinity chromatography step, incorporation of a low pH wash followed by a six-hour viral inactivation hold, efficient removal of hydrophobic impurities by HIC, selective separation of active acidic isoforms via IEC, and robust viral clearance by nanofiltration, collectively ensured the quality and safety of the final drug substance (DS). The integration of mixed-mode chromatography (MMC) and final sterile filtration further optimized purity and ensured compliance with pharmacopeial specifications, yielding r-hFSH at a concentration of 0.4-0.8 mg / mL within a pH range of 7.0-8.0.

[0425] Overall, the results confirm that the present purification process consistently delivers a high-purity, stable, and biologically active r-hFSH product with process robustness suitable for large-scale therapeutic manufacturing, thereby ensuring compliance with stringent regulatory standards.

[0426] While specific examples have been provided, the invention further extends to related process variations and formulations, which are detailed in the following embodiments. In one embodiment, the present invention provides a platform process for producing recombinant glycoprotein hormones selected from recombinant human follicle- stimulating hormone (r-hFSH), Corifollitropin alfa (CFA) / follicle-stimulating hormone- CTP (FSH-CTP), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG). The process may comprise, in certain embodiments, sequential steps of affinity chromatography, optionally in tandem with multimodal chromatography, ultrafiltration / diafiltration (UF / DF), intermediate chromatographic purifications, nanofiltration, and final sterile filtration.

[0427] Affinity chromatography may employ matrices such as Blue Sepharose 6 Fast Flow resin, CaptureSelect FSH resin, or CaptureSelect hCG resin, operated at pH 6.5-8.0 with elution at a conductivity of 250-400 mS / cm. Buffers may include 20 mM Tris containing 0.1-0.2 M L-arginine HCI and 2.0 M MgCI2for r-hFSH, FSH-CTP, or r-hLH, or 15-20 mM sodium phosphate containing 2.0-2.5 M NaCI for r-hCG. In certain embodiments, a low- pH wash (25 mM sodium acetate, pH 3.8-4.2) may be applied, followed by a viral inactivation hold of at least six hours, particularly for r-hCG processes.

[0428] Following affinity chromatography, UF / DF1 may be performed using a 10 kDa hollow fiber membrane for buffer exchange and concentration. Intermediate purification steps may include hydrophobic interaction chromatography (HIC), cation exchange, anion exchange, multimodal chromatography, or size-exclusion chromatography (SEC). HIC may be carried out using Phenyl Sepharose or Toyopearl Butyl resins at conductivities of 100-200 mS / cm, with elution by 0.5-1.0 M urea and 5-15% isopropyl alcohol in phosphate buffer. Cation exchange may employ Capto SP ImpRes resin in bind-elute mode, anion exchange may be conducted in flow-through mode using Q-based resins or membranes, and multimodal chromatography may employ Capto Adhere or Capto MMC ImpRes, optionally for isoform enrichment or protease removal. SEC may be performed using Superdex 75 resin to remove aggregates and fragments.

[0429] In certain embodiments, the eluate may be subjected to a low-pH viral inactivation step at pH 3.9-4.1 for at least six hours, particularly for r-hCG. Following viral inactivation or its alternatives, UF / DF2 may be performed to achieve a protein concentration of at least 0.5 mg / mL, with buffer exchange at pH 6.8-7.5 and conductivity 0.5-10 mS / cm. Target concentrations may vary by molecule: 0.4-0.8 mg / mL for r-hFSH, >1.0 mg / mL for FSH- CTP, 5.0 ± 1.0 mg / mL for r-hLH, and >0.9 mg / mL for r-hCG, with r-hCG further exhibiting potency >30,000 lU / mL.

[0430] Nanofiltration may be performed using filters of 15-25 nm pore size, such as Planova 20N, under nitrogen pressure not exceeding 1.0 kg / cm2. The final drug substance (DS) may then be prepared and sterile-filtered through a 0.2 pm capsule filter. In certain embodiments, the DS has a protein concentration >0.4 mg / mL, overall recovery >20- 80%, high molecular weight impurities <1.0%, and low molecular weight impurities <5%. For r-hCG, aggregates may be <3.0%, host cell protein <100 ng per 6500 IU, and residual DNA <100 pg per 6500 IU. For r-hFSH, protein concentration may range from 0.4-0.8 mg / mL at pH 7.0-8.0.

[0431] Pharmaceutical compositions comprising r-hFSH, CFA / FSH-CTP, r-hLH, or r-hCG prepared by the processes described herein may be combined with pharmaceutically acceptable carriers, stabilizers (mannitol, sucrose, trehalose, glycine, or L-arginine HCI), surfactants (polysorbate-20, polysorbate-80, or poloxamer 188), antioxidants (L- methionine or its variants), or preservatives (meta-cresol, phenol, or benzyl alcohol). Such compositions may be administered subcutaneously or intramuscularly for treating infertility, hypogonadism, or gonadotropin deficiency. In specific applications, r-hCG may be used for ovulation induction or luteal phase support, r-hFSH or CFA / FSH-CTP for controlled ovarian stimulation, and r-hLH for treating hypogonadotropic hypogonadism. Collectively, these processes establish a unified platform for downstream purification of recombinant glycoproteins, incorporating a structured sequence of affinity, intermediate, and multimodal chromatography, ultrafiltration / diafiltration, nanofiltration, and viral inactivation under controlled buffer, pH, and conductivity conditions. The orthogonal strategies and process controls described herein, enable scalable, reproducible, and regulatory-compliant production of clinical-grade recombinant glycoprotein hormones for therapeutic use in reproductive medicine.

Claims

CLAIMS:

1. A process for purifying a recombinant glycoprotein hormone selected from recombinant human follicle-stimulating hormone (r-hFSH), Corifol litropin alfa (CFA), recombinant follicle-stimulating hormone-CTP (FSH-CTP), recombinant human luteinizing hormone (r-hLH), and recombinant human chorionic gonadotropin (r-hCG), comprising sequentially:(i) subjecting a harvested cell culture to affinity chromatography using a matrix selected from Blue Sepharose 6 Fast Flow, CaptureSelect FSH resin, or CaptureSelect hCG resin, operated at pH 6.5-8.0, with conductivity not less than 200 mS / cm;(ii) performing a first ultrafiltration / d iafiltration (UF / DF1) using a 10 kDa membrane for buffer exchange and concentration;(iii) optionally subjecting the eluate to a viral inactivation step at pH 3.8-4.2 for not less than 6 hours;(iv) subjecting the eluate to one or more intermediate chromatographic steps selected from hydrophobic interaction chromatography (HIC), cation exchange chromatography, anion exchange chromatography, multimodal chromatography (MMC), or size-exclusion chromatography (SEC);(v) performing a second u Itrafi Itration / diafiltration (UF / DF2) with a 10 kDa membrane, achieving buffer exchange and protein concentration between 0.4-1.0 mg / mL;(vi) subjecting the protein solution to nanofiltration using a filter with nominal pore size 15-25 nm operated under nitrogen pressure <1.5 bar; and(vii) preparing the drug substance and filtering through a 0.2 pm filter2. The process of claim 1, wherein the host cell is a Chinese Hamster Ovary (CHO) cell cultured in chemically defined, serum-free medium.

3. The process of claim 1, wherein the affinity chromatography eluate is subjected to a viral inactivation step at pH 3.9-4.1 for >6 hours.

4. The process of claim 1, wherein UF / DF is performed with a 10 kDa membrane, conductivity 1-2 mS / cm, achieving protein concentration between 1.0-3.0 mg / mL.

5. The process of claim 1, wherein multimodal chromatography uses Capto Adhere, Capto Adhere ImpRes, or Capto MMC ImpRes resins to separate active isoforms or remove proteases.

6. The process of claim 1, wherein nanofiltration is carried out with a Planova 20N or Viresolve Pro filter between 0.5-1.5 bar pressure.

7. The process of claim 1, wherein the recombinant glycoprotein hormone is r- hFSH, comprising affinity capture on Blue Sepharose 6 FF (15 mM phosphate pH 7.0-8.0, 2-3 mS / cm), with viral inactivation at pH 3.9-4.1 (>6 h), high-salt wash (250 mM NaCI, 25-35 mS / cm), and elution at 2.5 M NaCI, pH ~7.5 (300- 400 mS / cm).

8. The process of claim 7, wherein hydrophobic interaction on Phenyl Sepharose FF is performed (equilibration 2.5 M NaCI, elution 1.0 M NaCI, pH 7.0-8.0, 100-200 mS / cm).

9. The process of claim 7, wherein UF / DF with a 10 kDa hollow fiber is conducted in 15 mM phosphate pH 7.8-8.2 (1.5-3.5 mS / cm) to 1.0-1.5 L retentate with >6 L washes at 0.3 M NaCI (30-50 mS / cm).

10. The process of claim 7, wherein ion exchange on QHR resin is carried out in 15 mM phosphate + 0.3 M NaCI pH 7.8-8.2 (30-50 mS / cm) in flowthrough to separate isoforms.

11. The process of claim 7, wherein nanofiltration is performed on Viresolve Pro Modus 1.2 in 15 mM phosphate + 0.3 M NaCI pH 7.8-8.2 at <1.9 kg / cm2.

12. The process of claim 7, wherein UF / DF is repeated (10 kDa, phosphate + 0.3 M NaCI pH 7.3-7.7, 30-50 mS / cm) to "'0.8 L retentate at >1.0 mg / mL with >1.6 L washes.

13. The process of claim 7 , wherein mixed-mode chromatography on Capto Adhere is employed (15 mM phosphate + 0.3 M NaCI pH 7.3-7.7, 30-50 mS / cm) with 3-4 L flowthrough adjusted to pH 7.65-7.85.

14. The process of claim 7 , wherein final filtration is performed through a 0.2 pm filter at pH 7.0-8.0, protein concentration between 0.4-0.8 mg / mL.

15. The process of claim 1, wherein the recombinant glycoprotein hormone is CFA or FSH-CTP, comprising tandem chromatography wherein harvest is passed through Capto Adhere resin to bind proteases (flow-through containing protein of interest), followed by CaptureSelect FSH affinity matrix binding at pH 7.4-7.6, 20 mM Tris buffer, conductivity 1.5 ± 0.5 mS / cm, and elution with 20 mM Tris + 0.1 M Arginine HCI + 2 M MgCI2at pH 7.5 ± 0.1, 300-400 mS / cm.

16. The process of claim 15, wherein the affinity eluate is concentrated and buffer- exchanged using a 10 kDa hollow fiber membrane against 20 mM Tris + 50 mM Arginine HCI, pH 7.5 ± 0.1, conductivity 5 ± 2 mS / cm, over 7-10 diavolumes, to reach 1-3 mg / mL protein.

17. The process of claim 15, wherein UF / DF1 material is loaded onto Capto Adhere ImpRes resin, equilibrated in Tris + Arginine buffer, pH 7.4-7.6, conductivity 5 ± 2 mS / cm, and eluted by step gradient using sodium citrate buffer at pH 5.9- 6.1, conductivity 15 ± 3 mS / cm.

18. The process of claim 15, wherein Capto SP ImpRes resin is equilibrated with 10 mM sodium citrate, pH 5.9-6.1, conductivity 1-2 mS / cm; the protein is loaded at <9 mS / cm adjusted with 0.2 M acetic acid, and flowthrough fractions collected as purified eluate.

19. The process of claim 15, wherein Capto Q. resin is used in non-binding mode with 20 mM Tris + 0.13 M NaCI, pH 7.5 ± 0.1, conductivity 10-20 mS / cm, allowing FSH-CTP to pass through while removing anionic impurities, DNA, endotoxins, and viruses.

20. The process of claim 15, wherein protein is concentrated to 5.0 ± 1 mg / mL using a 10 kDa membrane, diafiltered against 25 mM sodium citrate + 0.5 mg / mL L-methionine, pH 7.0, conductivity 5.5 ± 1 mS / cm, over 8-10 diavolumes.

21. The process of claim 15, wherein UF / DF2 concentrate is passed through Superdex 75 Prep resin, equilibrated with citrate + methionine buffer, pH 6.5- 7.5, conductivity 5.5 ± 1 mS / cm, at linear velocity ~30 cm / hr, fractions pooled to remove HMW / LMW contaminants.

22. The process of claim 15, wherein pooled SEC fractions are filtered through Planova 20N, equilibrated in Tris + methionine buffer, pH 7.0 ± 0.1, conductivity 4.5-6.5 mS / cm, to remove viral particles >20 nm.

23. The process of claim 15, wherein the nanofiltrate is mixed with sucrose (70 mg / mL) and Tween-20 (0.2 mg / mL) in sodium citrate buffer with methionine, adjusted to pH 7.0 ± 0.1, conductivity 6.5-7.5 mS / cm, sterile-filtered (0.2 pm PES), yielding drug substance at 0.5-1.5 mg / mL, with <0.5% HMW and <5% LMW impurities, formulated for subcutaneous injection to treat infertility.

24. The process of claim 1, wherein the recombinant glycoprotein hormone is r- hCG, comprising affinity chromatography on Blue Sepharose (15 mM Sodium Phosphate, pH 7.0-8.0, ~7.5; conductivity 1-4 mS / cm; DBC >150,000 lU / mL; residence time >4 min), UF / DF1 using 10 kDa hollow fiber membrane (6 DV, 4 mM Sodium Phosphate, pH 7.0-8.0), multimodal chromatography on Capto Adhere ImpRes (20 mM Sodium Acetate, pH 5.5-6.5; DBC >260,000 lU / mL), UF / DF2 (4 mM Sodium Phosphate + 0.1 M NaCI, pH 7.0-8.0), DEAE Sepharose flow-through (4 mM Sodium Phosphate + 0.1 M NaCI, pH 7.0-8.0, conductivity 10-20 mS / cm), HIC on Toyopearl Butyl 600M (1 M NaCI load, elution with 1 M Urea + 10% IPA), UF / DF3 (20 mM Histidine, pH 6.3-6.7), nanofiltration (Planova 20N, ~20 nm cutoff), and final 0.2 pm sterile filtration.

25. The process of claim 1, wherein the recombinant glycoprotein hormone is r- hLH, comprising affinity chromatography on CaptureSelect HCG (residence time ~6 min; DBC <1.2 mg / mL; Tris buffer pH 7.5; elution with Tris / Arg / MgCI2, conductivity 300-400 mS / cm).

26. The process of claim 25, wherein the eluate is subjected to UF / DF using a 10 kDa hollow fiber (8-12 DV; pH 8.0 ±0.1; conductivity 0.5-1.5 mS / cm; TMP 0.3- 0.7 bar; protein >0.3 mg / mL).

27. The process of claim 25, wherein the product is purified by multimodal chromatography on Capto Adhere ImpRes (pH 8.0; conductivity 0.5-1.5 mS / cm; 10 CV gradient to citrate pH 5.0-6.0; resin regenerated with NaOH / NaCI).

28. The process of claim 25, wherein the eluate is further treated by anion exchange chromatography on Q. Sepharose FF in flow-through mode (20 mM Tris + 50 mM NaCI; pH 7.0 ±0.1; conductivity 6 ±2 mS / cm).

29. The process of claim 25, wherein multimodal chromatography on Capto MMC ImpRes is employed (DBC ~10 mg / mL; 30 CV gradient between 50-500 mM NaCI; conductivity 6-45 mS / cm; 5 min residence).

30. The process of claim 25, wherein a second UF / DF is performed (10 kDa cutoff; Buffer K with 11.85 mg / mL Arg HCI; pH 7.0 ±0.1; conductivity 3-7 mS / cm; protein 5.0 ±1 mg / mL).

31. The process of claim 25, wherein size exclusion chromatography on Superdex 75 Prep Grade is conducted (Buffer K; pH 6.5-7.5; conductivity 5 ±2 mS / cm; linear velocity 30 cm / hr).

32. The process of claim 25, wherein nanofiltration is performed using Planova 20N (Buffer K; pressure <0.98 kg / cm2; flux 50-100 L / m2 / h; viral clearance >20 nm).

33. The process of claim 25, wherein drug substance is formulated with sucrose38.47 g / L, L-Met 0.15 g / L, Poloxamer 188 0.25 g / L, and L-Arg HCI 11.85 g / L in Buffer K, pH adjusted to 7.0.

34. The process of claim 25, wherein the drug substance (0.3-1.0 mg / mL protein; recovery >20%) is sterile-filtered through 0.2 pm PES, aseptically filled into PETG containers, and stored at -20 °C.

35. A pharmaceutical composition comprising a recombinant glycoprotein hormone purified by the process of any one of claims 1-34, together with a pharmaceutically acceptable carrier.

36. The pharmaceutical composition of claim 35, further comprising one or more stabilizers, surfactants, antioxidants, or preservatives.

37. A method of treating infertility, hypogonadism, or gonadotropin deficiency, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 35 or 36.

38. The method of claim 37, wherein the recombinant glycoprotein hormone is r- hCG and is administered for ovulation induction or luteal phase support.

39. The method of claim 37, wherein the recombinant glycoprotein hormone is r- hFSH and is administered for controlled ovarian stimulation.

40. The method of claim 37, wherein the recombinant glycoprotein hormone is CFA or FSH-CTP and is administered for controlled ovarian stimulation.

41. The method of claim 37, wherein the recombinant glycoprotein hormone is r- hLH and is administered for stimulation of follicular development or ovulation induction.

Images