For over 20 years, the mainstay of female infertility treatment was human menopausal gonadotropins (hMG), an injectable medication extracted from human urine with FSH and LH activity. During the 1980–90s, a host of technological improvements led to greater purity of urinary products and the availability of recombinant gonadotropins. The recombinant products differed from hMG in several significant ways: (1) purity, (2) mode of administration, e.g. self-administered by subcutaneous injection versus patient-assisted intramuscular injection, and (3) the presence of a single, well-characterized gonadotropin protein (r-hFSH, r-hLH, or r-hCG) . The purity and characterization of recombinant gonadotropins paved the way for novel research on the roles of FSH and LH in follicular development and facilitated the independent titration of FSH and LH for the benefit of infertile patients .
Infertility treatment protocols are individualized according to a variety of factors, including the patient's age and diagnosis, ovarian reserve, history of prior response to COS, and co-administered medications. Due to the complexity of treatment, patients prefer the fewest number of steps to prepare each injection . Accordingly, the need for administration of mixed doses of recombinant FSH and LH is becoming more apparent.
The results of the current study demonstrated that the biological potency of a mixture of 75 IU of follitropin alfa RFF (freeze-dried formulation) and 75 IU lutropin alfa in a 1:1 ratio, after reconstitution in SWFI using a plastic syringe, was similar to the biological potency of the individual respective recombinant hormones when administered within 1 h of injection and stored at 6 ± 2°C. As a result, this study has shown that follitropin alfa RFF and lutropin alfa may be mixed, without any significant alterations in either the resulting FSH or LH bioactivity, if administered within 1 h. It is important to note, however, that the results of this study cannot be extrapolated to other FSH or FSH-containing products, such as follitropin beta injection (a recombinant-derived product), urofollitropin for injection, purified or menotropins for injection, USP (urine-derived products). The urine-derived products are reconstituted with 2 ml 9% sodium chloride injection, USP. Follitropin beta injection is a 0.5 ml vial containing the active ingredient, excipients, and water for injection, USP.
The inherent lack of precision is a widely recognized shortcoming of the in vivo bioassays used to quantify gonadotropin content prior to release of commercial gonadotropin products [2, 21]. The Steelman-Pohley in vivo bioassay used to determine FSH content is highly specific but it lacks precision, as the coefficient of variation (CV) in a single determination is 10–20% . The USP, the official authority that establishes standards for the quality of all prescription products sold in the United States, describes the methods used to identify gonadotropin biopotency. The USP monograph specifies that these products contain ≥ 80% and ≤ 125% of the hormones listed on the products' labels . As noted by Driebergen and Baer, realistically, a 75 IU vial of FSH could contain between 60 and 94 IU (80–125%) based on the bioassay, and between 48 and 117 IU based on the same bioassay's CV (10–20%) .
In the present study, the mean and 95% CI for follitropin alfa RFF alone was 84.2 IU (75.5–92.7), while that of the follitropin alfa RFF/lutropin alfa mixture was 87.6 IU (79.1–96.5). These results are consistent with the assay limitations previously discussed. Therefore, regardless of the product source (urine- or recombinant-derived), the FSH biopotency will vary because of the assay and not necessarily the FSH product itself. Because a highly consistent glycosylation profile is produced batch-to-batch, follitropin alfa RFF may be assessed through physico-chemical means .
The ovarian ascorbic acid depletion assay was used to assess the biopotency of LH in the current study. Although the Van Hell seminal vesicle weight gain bioassay is used by regulatory authorities to assess the LH content of gonadotropin products, the ovarian ascorbic acid depletion assay remains the most widely used in vivo bioassay for quantifying LH . The ovarian ascorbic acid depletion assay is easy to perform and, furthermore, results correlate better than those of the seminal vesicle weight assay with in vitro LH assays [25, 26]. However, the precision of the ovarian ascorbic acid depletion assay is limited by heterogeneity in the structure of LH, endogenous interferences, and inter-laboratory variability .
As previously described, le Cotonnec et al. evaluated the PK and PD interactions between 150 IU follitropin alfa and 150 IU lutropin alfa in a prospective, randomized crossover study in 12 healthy women . No statistically significant differences were observed for tmax, Cmax, or AUC between the single and combined doses for either r-hLH or r-hFSH. PD markers that measured the response to r-hFSH (serum estradiol and inhibin concentrations, and follicular number and size) were not markedly affected by r-hLH when both products were administered together. However, the formulations of follitropin alfa and lutropin alfa used by le Cotonnec et al. in this Phase I study differed from those currently marketed in the USA; the current formulations of both products include methionine and polysorbate 20 [12, 17].
Reassuringly, the results of this study show that currently marketed formulations of follitropin alfa RFF and lutropin alfa may be mixed without significant alterations in the resulting bioactivity of either FSH or LH.