Manufacture and Composition

During product development, a cDNA library from a Bowes melanoma cell line was screened and a full-length tPA cDNA clone produced. The sequence coding for the F, K1, and EGF domains were removed [29]. The Reteplase coding sequence was then introduced into the vector plasmid pKK223-3 and the resulting construct introduced into E. coli by transformation [30]. Master and working cell banks have been generated. An overview of the manufacturing procedure is presented in Figure 3.4. The product accumulates intracellularly in E. coli in the form of inclusion bodies [31,32]. Cell harvesting follows fermentation, with subsequent cellular disruption and inclusion body (IB) recovery. IBs are solubilized and denatured under reducing conditions, followed by in vitro product folding. Four primary steps characterize downstream processing: acidification and filtration, affinity chromatography using Erythrina trypsin inhibitor-Sepharose, and two ion-exchange steps. This is followed by a concentration step and removal of low molecular mass species via diafiltration. Excipient addition is followed by sterile filtration, aseptic filling, and lyophilization. Each 20-mL single-use glass vial contains Reteplase (active ingredient) as well as tranexamic acid, dipotassium hydrogen phosphate, phosphoric acid, and polysorbate as excipients. It appears as a white powder, which is reconstituted with WFI before administration. The solution pH following reconstitution is 6.0.

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