Manufacture and Composition

Alteplase is synthesized using the cDNA for native human tPA derived from a human melanoma cell line. The cDNA is cloned into a Chinese ovary cell (CHO) cell line, from which suitable master and working cell banks are produced. Product manufacture entails culture of the producer animal cell line in a nutrient medium containing the antibiotic gentamicin. The glycosylated protein is secreted by the producer cell line into the extracellular culture medium. While production protocols remain confidential, initial product recovery is likely to involve physical removal of the producer cells from the product-containing culture media by means of filtration or centrifugation. This is likely to be followed by initial product concentration via ultrafiltration. Product purification entails application of multiple high-resolution chromatographic purification steps, including anion-exchange, gel filtration, and lysine-affinity chromatography. The final product has been shown to be >99% pure by several analytical techniques, including high performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tryptic mapping, and N-terminal sequencing. After excipient addition, the product is filter-sterilized, aseptically filled into vials, and freeze-dried. The final product is presented in single-dose vials containing either 50 or 100 mg active ingredient. It is a sterile white to off-white powder that must be reconstituted with water for injections (WFI) prior to intravenous (IV) administration. The final composition of the freeze-dried product is Alteplase (580,000 IU/mg), arginine, phosphoric acid, and polysorbate 80. Upon reconstitution, the product displays a pH of 7.3 [17].

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