Abstract
BackgroundAnimal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. While recombinant insulin and albumin already exist to replace their human counterparts in cell culture media, until recently there has been no equivalent for serum transferrin.ResultsThe first microbial system for the high-level secretion of a recombinant transferrin (rTf) has been developed from Saccharomyces cerevisiae strains originally engineered for the commercial production of recombinant human albumin (Novozymes' Recombumin® USP-NF) and albumin fusion proteins (Novozymes' albufuse®). A full-length non-N-linked glycosylated rTf was secreted at levels around ten-fold higher than from commonly used laboratory strains. Modification of the yeast 2 μm-based expression vector to allow overexpression of the ER chaperone, protein disulphide isomerase, further increased the secretion of rTf approximately twelve-fold in high cell density fermentation. The rTf produced was functionally equivalent to plasma-derived transferrin.ConclusionsA Saccharomyces cerevisiae expression system has enabled the cGMP manufacture of an animal-free rTf for industrial cell culture application without the risk of prion and viral contamination, and provides a high-quality platform for the development of transferrin-based therapeutics.
Highlights
Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications
Removal of N-linked glycosylation sites in recombinant transferrin (rTf) reduces product heterogeneity and allows visualization by SDS-PAGE Our initial transferrin expression vector contained the mature human transferrin (C1 variant) sequence derived from a cDNA
The high expression level (> 2 g/L) achieved in high cell density fed-batch fermentation has enabled an economically viable process to be developed for production of a high quality transferrin analogue for cell culture purposes (Novozymes’ CellPrimeTM rTransferrin AF)
Summary
Animal-free recombinant proteins provide a safe and effective alternative to tissue or serum-derived products for both therapeutic and biomanufacturing applications. Transferrin (Tf) is the major iron binding protein in human plasma, responsible for the regulated delivery of iron to cells. It is a monomeric glycoprotein (~80 kDa) with the capacity to bind two ferric ions very tightly, but reversibly. A current major use for plasma-derived transferrin is in the preparation of media for the culture of mammalian cells. The use of such media for the production of pharmaceutical products requires stringent attention to the source materials to control the risk of viral and prion disease transmission. A recombinant microbial source avoiding the use of mammalian-derived materials provides an obvious advantage
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