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Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur®) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

ABSTRACT: Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration operations. Studies on chromatography resin resource and ligand leakage showed that the immunoaffinity matrix employed was suitable for industrial use and stable for at least 40 full chromatography cycles, and the leaked single-domain antibody ligand was completely removed by subsequent purification steps. All chromatography resins employed withstood the same 40 cycles of use without significant changes in separation efficiency and product binding capacity. The resulting industrial purification process yielded batches of r-hFSH with consistent levels of purity and bioactivity.

SUBMITTER: Sinegubova M

PROVIDER: S-EPMC8781808 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur<sup>®</sup>) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

Sinegubova Maria M Vorobiev Ivan I Klishin Anatoly A Eremin Dmitry D Orlova Nadezhda N Orlova Natalya N Polzikov Mikhail M

Pharmaceutics 20220101 1

Recombinant human follicle stimulating hormone (r-hFSH) is widely used for infertility treatment and is subject to the development of biosimilars. There are different purification strategies that can yield r-hFSH of pharmaceutical quality from Chinese hamster ovary cell culture broth. We developed a purification process for r-hFSH centered on immunoaffinity chromatography with single-domain recombinant camelid antibodies. The resulting downstream process is simple and devoid of ultrafiltration o ...[more]

PMID: 35056992

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Project description:Recombinant human follicle-stimulating hormone (r-hFSH) is widely used in fertility treatment. Although biosimilar versions of r-hFSH (follitropin alfa) are currently on the market, given their structural complexity and manufacturing process, it is important to thoroughly evaluate them in comparison with the reference product. This evaluation should focus on how they differ (e.g., active component molecular characteristics, impurities and potency), as this could be associated with clinical outcome. This study compared the site-specific glycosylation profile and batch-to-batch variability of the in-vivo bioactivity of Bemfola, a biosimilar follitropin alfa, with its reference medicinal product GONAL-f. The focus of this analysis was the site-specific glycosylation at asparagine (Asn) 52 of the α-subunit of FSH, owing to the pivotal role of Asn52 glycosylation in FSH receptor (FSHR) activation/signalling. Overall, Bemfola had bulkier glycan structures and greater sialylation than GONAL-f. The nominal specific activity for both Bemfola and GONAL-f is 13,636 IU/mg. Taking into account both the determined potency and the nominal amount the average specific activity of Bemfola was 14,522 IU/mg (105.6% of the nominal value), which was greater than the average specific activity observed for GONAL-f (13,159 IU/mg; 97.3% of the nominal value; p = 0.0048), although this was within the range stated in the product label. A higher batch-to-batch variability was also observed for Bemfola versus GONAL-f (coefficient of variation: 8.3% vs 5.8%). A different glycan profile was observed at Asn52 in Bemfola compared with GONAL-f (a lower proportion of bi-antennary structures [~53% vs ~77%], and a higher proportion of tri-antennary [~41% vs ~23%] and tetra-antennary structures [~5% vs <1%]). These differences in the Asn52 glycan profile might potentially lead to differences in FSHR activation. This, together with the greater bioactivity and higher batch-to-batch variability of Bemfola, could partly explain the reported differences in clinical outcomes. The clinical relevance of the differences observed between GONAL-f and Bemfola should be further investigated.

Project description:BackgroundComplications of bone metastases such as skeletal-related events lead to the impaired functional status and quality of life including death in patients with bone metastasis from solid tumors. Denosumab (XGEVA®) is indicated for the prevention of skeletal-related events in bone metastasis patients with solid tumors. The biosimilar product LY01011, a fully human anti-receptor activator of nuclear factor kappa-B ligand monoclonal antibody, was developed to be compared with the reference product denosumab.Material and methodsA randomized, double-blind, single-dose, parallel-controlled phase 1 study was conducted in healthy Chinese subjects. A total of 168 enrolled subjects were randomly assigned in a 1:1 ratio to receive a single 120 mg dose of LY01011 (n = 85) or denosumab (n = 83) subcutaneously. The primary pharmacokinetic (PK) parameters, including maximum plasma concentration (Cmax) and area under the concentration-time curve from time zero to last quantifiable concentration (AUC0→t), were collected and measured for evaluation. Other secondary PK parameters included AUC0- ∞, Tmax, CL/F, λz, t1/2, Vd/F, etc. Pharmacodynamics (PD), safety and immunogenicity profiles were also accounted for data analysis.ResultsThe geometric mean ratios (GMRs) of LY01011 and denosumab for the primary PK parameters such as Cmax and AUC0→t were 98.13% and 100.32%. The 90% confidence intervals (CIs) were all within the acceptance range of 80%-125%. The GMRs of the PD parameters including AUEC0→t and Emax were 98.71% and 99.80%, which fell within the pre-defined acceptance range of 80%-125%. The results also demonstrated PK similarity even if Cmax and AUC0→∞ had been used as primary endpoints. Safety profiles were tolerable and similar between groups. 4 (4.7%) and 2 (2.4%) subjects had experienced Grade 3 or above treatment-emergent adverse events (TEAEs) in LY01011 group and denosumab group. 3 subjects were reported to have serious adverse events (SAEs). None of the Grade 3 or above TEAEs and SAEs were related to the study drug, LY01011. No subject was tested anti-drug antibody (ADA) positive in both groups prior to the study drug administration. Following the study drug administration, only one subject in denosumab group was tested ADA positive, whereas no subject with ADA positive was reported in LY01011 group. No neutralizing antibody (Nab) was detected in either group throughout the study.ConclusionsThe study demonstrated PK and PD similarity of LY01011, a denosumab biosimilar, to denosumab in healthy Chinese subjects, with comparable safety and immunogenicity profiles.

Project description:BackgroundHeifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments: non-implanted (n = 5), Revalor 200 for 28 d (n = 5), or Revalor 200 for 84 d (n = 6). Small follicle (1 to 5 mm) granulosa cells were isolated from each pair and incubated with phosphate buffered saline (n = 16) or 100 ng/mL follicle stimulating hormone (n = 16) for 24 h.ResultsGranulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar (P = 0.22) to non-implanted heifers, while medium estradiol concentrations were increased (P < 0.10) at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein (P = 0.05) mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 (P < 0.10) or 84 d (P < 0.05) compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase (P = 0.57) and aromatase (P = 0.23) were demonstrated in implanted or non-implanted heifers.ConclusionsThese results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

Dataset Information

Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur®) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

Publications

Purification Process of a Recombinant Human Follicle Stimulating Hormone Biosimilar (Primapur<sup>®</sup>) to Yield a Pharmaceutical Product with High Batch-to-Batch Consistency.

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