生殖功能的調節主要由糖蛋白激素，卵泡刺激素（FSH）、促甲狀腺激素（TSH）、黃體生成素（LH）和絨毛膜促性腺激素（hCG）進行。糖蛋白激素包含α 亞基 &  β 亞基

 hCG、FSH 和 LH 有相同的α 亞基，包含 92 個氨基酸，

 hCG、FSH 和 LH 之 β 亞基對於所有三種激素是特異性和獨特的，可引發不同的生物學和免疫學特性。

聚醣glycans在促性腺激素與其各自受體的結合中發揮著重要作用，識別相關聚醣及其藥理學特性非常重要。

蛋白質糖基化Protein glycosylation 是一種複雜的翻譯後修飾post-translational modification (PTM)，涉及glycans在特定位點的附著，最常見的是天冬酰胺（N 連接）或絲氨酸/蘇氨酸（O 連接）殘基。糖基化glycosylation模式在蛋白質的溶解度、穩定性和生物活性中起著至關重要的作用。

新的聚醣 glycan是一種四觸角物種tetra antennary ， FSH 與受體的結合中發揮重要作用，具有更高的生物活性、更低的清除率和更長的半衰期。

由於糖基化的差異，糖蛋白的生物活性和半衰期可能不同。

質譜儀mass spectrometry MS技術的進步，可以鑑定微量的相關聚醣。MS 可進行定量和定性分析，例如分子量測定、結構闡明或序列測定。

 https://www.sciencedirect.com/science/article/abs/pii/S1642431X18300949

https://link.springer.com/article/10.1007/s00216-011-4923-5

Glycan mapping of recombinant human follicle stimulating hormone by mass spectrometry

Author links open overlay panel

• In humans, regulation of reproductive functions are carried out mainly by glycoprotein hormones namely follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH) and chorionic gonadotropin (CG). 
• Since glycans play an important role in binding of gonadotropins with their respective receptors, it is important to identify associated glycans and their pharmacological properties not only for the disease manipulation but also for making more efficacious and safer recombinant versions. 
• Protein glycosylation is a complex post-translational modification (PTM) involving enzyme mediated attachment of glycans at specific sites, most commonly at Asparagine (N-linked) or Serine/Threonine (O-linked) residues. The glycosylation pattern plays a critical role in the solubility, stability and bioactivity of proteins. 
• With the advancement of mass spectrometry, it is possible to identify minute quantity of associated glycans. 
• The new glycan was a tetra antennary species that may have important role in binding of FSH with receptor with higher biological activity as well as lower clearance rate and higher half-life.
• The α-subunit contains 92 amino acids and is identical in hCG, FSH and LH (Table 1) while β-subunit are specific and unique for all the three hormones for eliciting differential biological and immunological properties. 
• Due to differences in the glycosylation, glycoprotein may differ in bioactivities and half-life. 
• MS allows both quantitative and qualitative analysis like determination of molecular mass, structure elucidation or sequence determination. 

Analysis of recombinant human follicle-stimulating hormone (FSH) by mass spectrometric approaches

• Thorough analysis of the heterodimeric heavily glycosylated protein of rFSH is a prerequisite for the evaluation of production batches as well as for the determination of “essential similarity” of new biosimilars. 
• The concerted application of different liquid chromatography-mass spectrometry methods enabled the complete depiction of the primary structure of this pituitary hormone. 
• Sequence coverage of 100% for the α- as well as the β-chain was achieved with tryptic peptides. 
• Most of these peptides could be verified by tandem mass spectrometry. Site-specific analysis of all four glycosylation sites was, however, not possible with tryptic but with chymotryptic peptides. Quantification of the glycoforms of each glycopeptide was accomplished with the software MassMap®. Both protein subunits gave interpretable mass spectra upon S-alkylation and separation on a C5 reversed-phase column. Glycan isomer patterns were depicted by separation on porous graphitic carbon, using mass spectrometric detection for the evaluation of the glycopeptide liquid chromatography-electrospray ionization data. The currently marketed product Gonal-f™ and a potential biosimilar were compared with the help of these procedures.

新型卵泡素製劑FSH：聚醣結構差異如何影響生化和生物學功能以及臨床效果

• 人工合成知FSH變體的1級~4級結構，包括糖基化差異。對於FSH知生化效果均產生不同效果
• 這些糖苷形式包括糖基化位點的數量、糖苷鏈的複雜性以及唾液酸化和硫酸化方面均有差異，
• 高唾液酸化，末端 N-乙酰半乳糖胺或半乳糖的 2,3 唾液酸封端通過阻斷肝臟中脫唾液酸糖蛋白受體 (ASGPR) 的結合，導致更長的循環半衰期。
• FSH 糖基化主要在 β 亞基上，隨著女性年齡的增長而變化，已成為FSH作用的關鍵修飾因子，在動物模型體內具有深遠的生物學效應。
•  FSH 在垂體中以兩個亞基的形式產生，一個是所有糖蛋白激素共有的亞基，即 α 亞基，另一個是每種激素特有的亞基，即 β 亞基。
• 尿液中的促卵泡素與天然垂體促卵泡素的不同之處在於其聚醣的程度和復雜性以及相關糖蛋白激素（黃體生成素）和其他蛋白質的污染
• FSH Alpha類包括   Gonal-F®、Bemfola®、Ovaleap® 、Cinnal-f®. 它是CHO細胞系生產的
• FSH Beta 類包括    Follistim® 和 Puregon®。它是CHO細胞系生產的
• Follitropin Delta 包括促卵泡素 Rekovelle®，它是使用人類細胞系生產的 

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8018285/

New Human Follitropin Preparations: How Glycan Structural Differences May Affect Biochemical and Biological Function and Clinical Effect

:

1. Pituitary follitropin is secreted into the circulation as a mixture of variants, which differ not in primary structure but rather at the level of glycosylation. 
2. These glycosidic forms vary in the number of glycosylation sites filled, complexity of glycosidic chains, and sialylation and sulfation. 
3. high sialylation, 2,3 sialic acid capping of terminal N-acetyl galactosamine or galactose leads to longer circulating half-life, by blocking binding of asialoglycoprotein receptor (ASGPR) in the liver. 
4. FSH glycosylation, primarily on the β-subunit, which varies as women age, has emerged as a key modifier of follitropin action, with profound biological effects in vivo in animal models.
5.  FSH is produced in the pituitary gland as two subunits, one subunit common to all glycoprotein hormones, the α-subunit, and another subunit specific for each hormone, the β-subunit. 
6. . Follitropin in urine differed from naturally occurring pituitary follitropin in degree and complexity of their glycans and contamination with a related glycoprotein hormone (luteinizing hormone) and other proteins
7.  Recombinant follitropin Gonal-F®, and biosimilars Bemfola®, Ovaleap® and Cinnal-f® are grouped into one class called follitropin Alpha. 
8. The follitropin Beta class includes Follistim® and Puregon®. 
9. Follitropin Delta included the follitropin Rekovelle® which is produced using a human cell line and results in a product that possesses sialic acid2,6 as well as sialic acid2,3 capped Gal.

Follitropin delta 促卵泡素 delta (樂可孕 Rekovelle)相關臨床報告

https://www.youtube.com/watch?v=uKM0Bwz8giA

• 每日 10 µg 促卵泡素 delta (Rekovelle)劑量可提供與 150 IU/天促卵泡素 α (Gona-F) 相似的卵巢反應。
• Rekovelle與Gona-F& Puregon 相比，胚胎髮育和妊娠率相似。
• Rekovelle妊娠率為 6 μg/d 的 19%、9 μg/d 的 20% 和 12 μg/d 的 25%。
• Rekovelle 6 μg/d、9 μg/d 和 12 μg/d 取卵數量為 7.0 ± 4.1、9.1 ± 5.6 和 11.6 ± 5.6，
• Rekovelle與Gona-F的臨床妊娠率（35.4% vs. 31.5%，P = 0.239）和活產率（31.0% vs. 25.5%，P = 0.101）相當。
• AMH ≥ 15 pmol/L 的女性注射Rekovelle與Gona-F 治療相比，Rekovelle回收的卵母細胞較少（10.3 ± 6.2 與 12.5 ± 7.5，P < 0.001）  
• Rekovelle與Gona-F 治療相比，早期卵巢過度刺激綜合徵 (OHSS) 和/或預防性干預的發生率較低（6.1% vs. 11.0%，P = 0.013）。
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• 
•  A daily follitropin delta dose of 10 µg provides a similar ovarian response to 150 IU/day follitropin alfa in IVF/ICSI patients.
•  Similar embryo development and pregnancy rates compared with those of stimulation with follitropin alfa or beta.
• Vital pregnancy rate per started cycle with follitropin delta was 19% for 6 μg/d, 20% for 9 μg/d, and 25% for 12 μg/d.
• Oocytes retrieved in the 6 μg/d, 9 μg/d, and 12 μg/d follitropin delta groups was 7.0 ± 4.1, 9.1 ± 5.6, and 11.6 ± 5.6, 
• The clinical pregnancy rate (35.4% vs. 31.5%, P = 0.239) and live birth rate (31.0% vs. 25.5%, P = 0.101) were comparable between the follitropin delta group and the follitropin alfa group. 
• Overall, the individualized follitropin delta treatment resulted in fewer oocytes retrieved compared to follitropin alfa treatment (10.3 ± 6.2 vs. 12.5 ± 7.5, P < 0.001), which was mainly due to fewer oocytes (10.5 ± 6.4 vs. 13.9 ± 7.8) in women with AMH ≥ 15 pmol/L.
•  Accordingly there was a lower incidence of early ovarian hyper-stimulation syndrome (OHSS) and/or preventive interventions (6.1% vs. 11.0%, P = 0.013). 
• A daily follitropin delta dose of 10.2 µg (95% CI: 9.3-11.2 µg) was estimated to provide the same number of oocytes retrieved as a starting dose of 150 IU/d of follitropin

. 2020 Oct;41(4):616-622.

 doi: 10.1016/j.rbmo.2020.07.006. Epub 2020 Jul 15.Establishing the follitropin delta dose that provides a comparable ovarian response to 150 IU/day follitropin alfa

Research question: The objective of this investigation was to determine the daily follitropin delta dose (µg) providing a similar ovarian response to 150 IU/day follitropin alfa. Design: The study was a post-hoc analysis of ovarian response in 1591 IVF/intracytoplasmic sperm injection (ICSI) patients undergoing ovarian stimulation in a gonadotrophin-releasing hormone antagonist protocol in two recent randomized, assessor-blind, controlled trials in the development programme for follitropin delta: a phase II dose-response trial with a reference arm of a fixed daily dose of 150 IU follitropin alfa throughout stimulation, and a phase III efficacy trial with a comparator arm of 150 IU/day follitropin alfa as a starting dose. Results: Daily follitropin delta doses of 10.0 µg (95% confidence interval [CI] 7.9-12.8) and 10.3 µg (95% CI 9.7-10.8) yielded the same number of oocytes as 150 IU/day follitropin alfa for all patients participating in the phase II and III trials, respectively. When analysing patients with either normal or high ovarian reserve (based on serum anti-Mullerian hormone ≥15 pmol/l) and no dose changes, the same number of oocytes was obtained with 150 IU/day follitropin alfa and daily doses of follitropin delta of 9.7 µg (95% CI 7.5-12.4) and 9.3 µg (95% CI 8.6-10.1) in the two trials. Daily follitropin delta doses in the range 9.5-10.4 µg were consistently estimated to correspond to 150 IU/day follitropin alfa for serum oestradiol concentration and number of follicles ≥12 mm at the end of stimulation across analysis populations in the phase III trial.Conclusions: A daily follitropin delta dose of 10 µg provides a similar ovarian response to 150 IU/day follitropin alfa in IVF/ICSI patients.

F S Rep . 2020 Dec 14;2(1):30-35.  doi: 10.1016/j.xfre.2020.12.002. eCollection 2021 Mar.

In vitro fertilization cycles stimulated with follitropin delta result in similar embryo development and quality when compared with cycles stimulated with follitropin alfa or follitropin beta

Objective: To study the impact of follitropin delta for ovarian stimulation on embryo development and quality compared with that of follitropin alfa or beta in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) cycles.

Patients: A total of 403 IVF/ICSI cycles were conducted from September 1, 2018 to December 31, 2019. Cycles were grouped on the basis of stimulation with follitropin delta vs. follitropin alfa or beta.

Main outcome measures: Embryo parameters and clinical pregnancy and implantation rates.

Results: Ovarian stimulation using follitropin delta resulted in no statistically significant difference in day 3 embryo quality between the control group and follitropin delta group (median 0.50 vs. 0.54 for good quality embryos and median 0.25 vs. 0.20 for intermediate quality embryos). Although on initial analysis there was a lower proportion of good quality blastocysts in the follitropin delta group than in the control group (0.11 vs. 0.22), this difference was no longer present when day 3 after fertilization vitrification and transfer cycles were excluded (0.26 vs. 0.33 follitropin delta vs. control). The clinical pregnancy rates and clinical implantation rates were similar in both groups in fresh transfer cycles.

Conclusions: Stimulation with follitropin delta in IVF/ICSI cycles resulted in similar embryo development and pregnancy rates compared with those of stimulation with follitropin alfa or beta.

Clinical Trial

 

. 2021 Jun;115(6):1478-1486.

 doi: 10.1016/j.fertnstert.2020.10.059. Epub 2020 Dec 4. Randomized, assessor-blind, antimüllerian hormone-stratified, dose-response trial in Japanese in vitro fertilization/intracytoplasmic sperm injection patients undergoing controlled ovarian stimulation with follitropin delta

Objective: To establish the relationship between follitropin delta doses (recombinant follicle-stimulating hormone produced from the human cell line PER.C6) and ovarian response in Japanese women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment and to evaluate the influence of initial antimüllerian hormone (AMH) levels. 

Design: Randomized, controlled, assessor-blind, AMH-stratified (low 5.0-14.9 pmol/L; high 15.0-44.9 pmol/L) dose-response trial.

Patient(s): A total of 158 Japanese women (20-39 years of age).

Intervention(s): Controlled ovarian stimulation with 6, 9, or 12 μg/d of follitropin delta or 150 IU/d follitropin beta as a reference arm in a gonadotropin-releasing hormone antagonist cycle.

Main outcome measure(s): Number of oocytes retrieved.

Result(s): Among all women who started stimulation, the mean number (± standard deviation) of oocytes retrieved in the 6 μg/d, 9 μg/d, and 12 μg/d follitropin delta groups was 7.0 ± 4.1, 9.1 ± 5.6, and 11.6 ± 5.6, respectively, and a significant dose-relation was established, which also remained significant within each AMH strata. Significant dose-responses also were observed for serum estradiol, inhibin A, and progesterone at end-of-stimulation with follitropin delta. The vital pregnancy rate per started cycle with follitropin delta was 19% for 6 μg/d, 20% for 9 μg/d, and 25% for 12 μg/d. The rate of early moderate/severe ovarian hyperstimulation syndrome with follitropin delta was 8% for 6 μg/d, 8% for 9 μg/d, and 13% for 12 μg/d, with 82% of the cases in the high AMH stratum. Conclusion(s): This trial establishes the dose-response relationship between follitropin delta and ovarian response in Japanese women. 

Randomized Controlled Trial

 

. 2022 Oct 4;20(1):147.

 doi: 10.1186/s12958-022-01016-y.Comparative clinical outcome following individualized follitropin delta dosing in Chinese women undergoing ovarian stimulation for in vitro fertilization /intracytoplasmic sperm injection

Background: To compare the efficacy and safety of follitropin delta in its individualized fixed-dose regimen with follitropin alfa in a conventional adjustable dosing regimen in Chinese women. METHODS: This was a subgroup analysis of the randomized, multi-center, assessor-blind, non-inferiority trial (GRAPE) including 759 Chinese women (aged 20-40 years) recruited in 16 reproductive medicine clinics in China. Women were randomized in a 1:1 ratio to be treated with either follitropin delta dose based on anti-Müllerian hormone (AMH) and body weight or conventional dosing with follitropin alfa following a gonadotropin-releasing hormone (GnRH) antagonist protocol. The primary outcome was ongoing pregnancy rate assessed 10-11 weeks after embryo transfer in the fresh cycle (non-inferiority margin -10.0%). Results: 378 in the follitropin delta group and 381 in the follitropin alfa group were randomized and exposed. Non-inferiority was confirmed with respect to ongoing pregnancy with rates of 31.0% vs. 25.7% for follitropin delta compared to follitropin alfa, estimated mean difference of 5.1% (95% confidence interval (CI) -1.3% to 11.5%). The clinical pregnancy rate (35.4% vs. 31.5%, P = 0.239) and live birth rate (31.0% vs. 25.5%, P = 0.101) were comparable between the follitropin delta group and the follitropin alfa group. Overall, the individualized follitropin delta treatment resulted in fewer oocytes retrieved compared to follitropin alfa treatment (10.3 ± 6.2 vs. 12.5 ± 7.5, P < 0.001), which was mainly due to fewer oocytes (10.5 ± 6.4 vs. 13.9 ± 7.8) in women with AMH ≥ 15 pmol/L. Accordingly there was a lower incidence of early ovarian hyper-stimulation syndrome (OHSS) and/or preventive interventions (6.1% vs. 11.0%, P = 0.013). A daily follitropin delta dose of 10.2 µg (95% CI: 9.3-11.2 µg) was estimated to provide the same number of oocytes retrieved as a starting dose of 150 IU/d of follitropin alfa. Conclusion: Follitropin delta in its individualized fixed-dose regimen showed similar efficacy and improved safety compared with follitropin alfa in a conventional adjustable dosing regimen in Chinese women.

Randomized Controlled Trial

 

. 2023 Jan;43(1):37-44.

 doi: 10.1007/s40261-022-01232-9. Epub 2022 Dec 7.Pharmacokinetics and Safety of Follitropin Delta in Gonadotropin Down-Regulated Healthy Chinese Women

Background: Follitropin delta, a novel recombinant follicle-stimulating hormone (rFSH) preparation derived from a human cell line, has different pharmacokinetic and pharmacodynamic properties compared with existing rFSH preparations expressed by Chinese hamster ovary cells (CHO). Objectives: The objective of this study was to assess the pharmacokinetic characteristics, dose proportionality, and safety of follitropin delta in healthy Chinese women. Methods: This was a phase I, randomized, open-label study. Twenty-four healthy Chinese women were randomized (1:1:1) to receive a single subcutaneous administration of follitropin delta 12, 18, or 24 μg. The pharmacokinetic parameters (maximum observed serum concentration [Cmax], time to reach Cmax [tmax], area under the serum concentration-time curve from dosing to infinity [AUC∞], and elimination phase half-life [t½]) of follitropin delta were derived using noncompartmental analysis. Results: Following a single subcutaneous administration of follitropin delta 12, 18, or 24 μg, mean Cmax (0.388, 0.677, and 0.825 ng/mL, respectively) and AUC∞ (41.3, 62.9, and 83.1 h·ng/mL, respectively) increased in a dose-proportional manner. The median tmax was 24 h, and the mean t½ was in the range of 50.5-60.9 h. All treatment-related adverse events were categorized as mild, except for one case of urticaria from the follitropin delta 18-μg dose group which was considered moderate. Only one woman presented with elevation of alanine transaminase and aspartate aminotransferase at the follow-up visit, which was reported as a treatment-emergent adverse event. There were no injection-site reactions and none of the participants showed any confirmed presence of treatment-induced anti-FSH antibodies. Conclusions: The administration of single doses of follitropin delta to healthy Chinese women demonstrated dose-proportional pharmacokinetics over the dose range of 12-24 μg, and these doses were well tolerated.