PRODUCTION OF RECOMBINANT FSH
The genes coding for the human FSH α-subunit and β-subunit were inserted in cloning vectors (plasmids)to enable efficient transfer into recipient cells. These vectors also contained promoters that could direct transcription of foreign genes in recipient cells. CHO cells were selected as recipient cells since they wereeasily transfected with foreign DNA, and are capable of synthesizing glycoproteins. Furthermore they could be grown in cell cultures on a large scale. To construct a FSH-producing cell line NV Organon, the manu-facturer of Puregon /Follistim , used one single vector containing the coding sequences for both subunit genes (Olijve, 1996). Merck Serono S.A., the manufacturer of Gonal-F , used two separate vectors, one for each subunit gene (Howles, 1996). Following transfection, a genetically stable transformant produ-cing biologically active recombinant FSH was iso-lated. For the CHO cell line used for manufacturing Puregon /Follistim it was shown that approxi-mately 150 to 450 gene copies were present.
To establish a master cell bank (MCB) identical cell preparations of the selected clone are stored in individual vials and cryopreserved until needed. Subsequently a working cell bank (WCB) is established by the expansion of cells derived from a single vial of the MCB and aliquots are put in vials and cryopre-served, as well. Each time a production run is started cells from one or more vials of the WCB are cultured.
Both recombinant FSH products are isolated from cell culture supernatant. This supernatant is collected from a perfusion-type bioreactor containing recombi-nant FSH-producing CHO cells grown on microcarriers. This is because CHO cells are anchorage-dependent cells, which implies that a proper surface must be provided for cell growth. The reactor is perfused with growth-promoting medium during a period that may continue for up to three months. The down-stream purification processes for the isolation of the two recombinant FSH products are different. For Puregon /Follistim a series of chromatographic steps, including anion and cation exchange chromatography, hydrophobic chromatography and size-exclusion chro-matography is used. Recombinant FSH in Gonal-F is obtained by a similar process of five chromatographic steps, but also includes an immunoaffinity step using a murine FSH-specific monoclonal antibody. In both production processes, each purification step is rigor-ously controlled in order to ensure the batch-to-batch consistency of the purified product.