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

Physico-Chemical Consistency of r-hFSH (Gonal-F)

Human pituitary FSH can be separated into at least 20 isoform fractions with the endocrine milieu strongly influencing the overall isoform profile.[2,3] For this reason, urinary gonadotrophins have a highly variable composition and a highly variable level of bioactivity, necessitating the quantification of FSH by bioassay. By contrast, the manufacturing processes used to produce Gonal-F lead to a product with a highly consistent glycosylation profile resulting in a consistent isoform pattern and bioactivity. Glycan mapping and isoelectric focusing (IEF) have both shown that Gonal-F has a high physico-chemical consistency that is reflected in a consistent specific bioactivity. Analysis of the vial filling and finishing operations (including lyophilisation) has confirmed the consistent biological integrity of the r-hFSH molecule (Serono, data on file).

Glycan mapping produces a 'fingerprint' of the glycan (polysaccharide) species of r-hFSH and an estimation of the degree of sialylation. The glycan species are separated by charge - a function of their sialic acid content - and the results are expressed as the relative percentage of neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated glycan species and as a hypothetical charge number 'Z' calculated from the different proportions of the different species. The Z number is a very precise estimation of the degree of sialylation and has been assessed with a CV of just 2%.

Evaluation of the batch data on a year-by-year production basis (1998-2000) shows that the glycoform distribution has been highly consistent over the years (Figure 1), reflecting the high consistency of the molecular profile of the Gonal-F product (Serono, data on file).

Glycan mapping - glycan species distribution comparison of control sample ((a), left) (51 independent values) and 121 batches of r-hFSH (Gonal-F) drug substance ((b), right) produced in 1998 (n = 60), 1999 (n = 41) and 2000 (n = 20). (Serono, data on file)

Glycan mapping - glycan species distribution comparison of control sample ((a), left) (51 independent values) and 121 batches of r-hFSH (Gonal-F) drug substance ((b), right) produced in 1998 (n = 60), 1999 (n = 41) and 2000 (n = 20). (Serono, data on file)

The IEF of Gonal-F is performed in acrylamide gels across a pH range of 3.5-7.0. The reference standard produces seven major bands - one band above pI 5.20; three bands between pI 5.20 and 4.55 and three bands below pI 4.55. The isoform distribution consistency of the commercial manufacturing process over the years has been confirmed by evaluating the relative intensity of each of the major and minor bands on the IEF gel, measured by its integrated optical intensity (IOD) (Figure 2). The distribution of the main bands is very similar to that of the reference standard; the mean %IOD obtained for each of the major bands is within 2-4% of the mean %IOD of the same band of the standard (Serono, data on file).

Isoelectric focusing - isoform distribution comparison of reference standard (top) (26 independent values) and 63 batches of r-hFSH (Gonal-F) drug substance produced in 1999 (n = 41) and 2000 (n = 22). (Serono, data on file)

Specific activity is a ratio of Gonal-F bioactivity (measured using the Steelman-Pohley assay) and protein content (measured by SE-HPLC) and expressed in IU/mg protein. Detailed statistical analysis has been performed on specific activity data for 100 Gonal-F batches of drug substance manufactured in 1997, 1998 and part of 1999 from nine different bioreactor runs. These demonstrated that the specific activity of Gonal-F was normally distributed, stable, and that there was no bioreactor run effect. At an average specific activity of 13 745 IU/mg, a target mass of 5.5 µg was judged to be equivalent to 75 IU. Detailed evaluation of the drug substance production data from 1998 to 2000 confirmed, within process and analytical variability, the well-controlled behaviour and consistency of the process (Figure 3) and supported this conversion factor (Serono, data on file).

Specific activity (bioassay/assay by SE-HPLC) of 120 r-hFSH (Gonal-F) drug substance batches, manufactured in 1998, 1999 and 2000. (Serono, data on file)

The highly consistent physico-chemical and biological properties of the product now permit FSH quantification by SE-HPLC and vials/ampoules to be reliably filled by mass (FbM) rather than by specific bioactivity - this product is referred to as Gonal-F FbM.

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