Quantification of Follicle Stimulating Hormone (follitropin alfa): Is In Vivo Bioassay Still Relevant in the Recombinant Age?

R. Driebergen, G. Baer

Disclosures

Curr Med Res Opin. 2003;19(1) 

In This Article

Summary and Introduction

Quantification of follicle-stimulating hormone (FSH) for clinical use has traditionally involved the use of in vivo bioassays, particularly the Steelman-Pohley bioassay. This assay has limited precision, requires large numbers of laboratory animals and involves cumbersome procedures for data generation and interpretation. Recent advances in manufacturing procedures for recombinant human FSH (r-hFSH) have resulted in a preparation (follitropin alfa; Gonal-F) that is highly consistent in both isoform profile and glycan species distribution. As a result, follitropin alfa can be reliably quantified using an optimised size exclusion high-performance liquid chromat-ography (SE-HPLC) method, and vials can be filled by mass. Preliminary clinical studies suggest that the fill-by-mass process results in a product that delivers a more consistent clinical response and is more effective than follitropin alfa vials filled by bioassay in women undergoing controlled ovarian stimulation. Non-bioassay methods such as SE-HPLC are likely to become increasingly important for quality testing and regulatory purposes, provided that the manufacturing process is well controlled and produces a protein of highly consistent physico-chemical properties.

Follicle stimulating hormone (FSH) is a glycoprotein produced and secreted by the anterior pituitary gland. It exists not as a single molecular structure, but rather as multiple, charged isoforms that result from variations in the composition of the four carbohydrate chains (in particular their sialic acid content) attached to the two (alpha and beta) inter-linked protein subunits.[1] These isoforms differ in their molecular weights, biological potency, elimination half-life and immunoreactivity.[2] Sialic acid content is the main determinant of half-life - the greater the sialic acid content, the higher the acidity of the isoform and the longer the biological half-life.[2] The amount and relative composition of FSH secreted, as well as its bioactivity, are influenced by the hormonal milieu.[3]

FSH plays a key physiological role in both males (spermatogenesis) and females (regulation of follicular growth). Over the past 40 years, injectable forms of FSH have established a leading role in the management of male and female infertility. In women, FSH is used in the treatment of ovulatory disorders and as part of controlled ovarian stimulation (COS) regimens for assisted reproductive technologies (ART). In men, it is most commonly used to treat hypogonadotrophic hypogonadism.

Prior to the advent of recombinant DNA technology, all therapeutic FSH preparations were urinary-derived. The rather crude manufacturing methods used during the mid-to-late 20th century required the collection of vast quantities of urine and led to FSH preparations with varying degrees of protein contamination (although the most recent urinary FSH product - highly purified urinary FSH - contains > 95% pure FSH). Nevertheless, given the highly variable composition of urinary gonadotrophins, there were (and continue to be) significant batch-to-batch inconsistencies in terms of contamination as well as FSH isoform profile. As a result, the quantification of FSH presented a serious problem, given that its expression by mass was meaningless. In order to quantify FSH content and standardise proprietary preparations, therefore, it was necessary to use an in vivo bioassay. Bioassays make use of internationally accepted standards (provided by the WHO) that enable samples of unknown biopotency to be estimated. The test results on the bulk preparations allow filling of FSH vials/ampoules according to the desired FSH bioactivity measured in international units (IU). This activity is confirmed by a final bioassay on the finished product just prior to its release; the result has to be within a specification range of 80-125% of the labelled potency.

Although a number of FSH bioassays have been developed, the one required by regulatory agencies is the classic Steelman-Pohley in vivo assay.[4] This is based on the fact that immature female rats (21-22 days old), pretreated with human chorionic gonadotrophin (hCG), are sensitive to exogenous FSH and that there is a linear relationship between administered FSH and ovarian weight. FSH is injected subcutaneously once daily for 3 days with an autopsy being performed after 72 h. One of the advantages of this assay is that it takes FSH clearance into account.

The manufacture of FSH using recombinant DNA technology now ensures a constant supply of the most biochemically pure FSH preparation (r-hFSH, > 10 000 IU FSH/mg protein) with high batch-to-batch consistency in isoform profile and glycan species distribution. The most significant advantage of this isoform and glycan species consistency is that it permits FSH to be reliably quantified by mass. This article considers the rationale and methodology behind this development and the advantages of non-bioassay-based quantification methods.

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