Proteolytic cleavage of recombinant two-chain factor VIII during cell culture production is mediated by protease(s) from lysed cells. The use of pulse labelling directly in production medium.

Cytotechnology
K HansenM Ezban

Abstract

During the production by mammalian cells of recombinant factor VIII from which the B domain was deleted (rFVIII), proteolytic cleavages in the C-terminal part of the heavy chain were observed (Kjalke et al., 1995). By radioactive pulse labelling it was investigated whether the cleavages took place inside the cells during protein synthesis or after release in the medium. The rFVIII-producing CHO (Chinese hamster ovary) cells were cultured in the presence of (35)S-methionine and then the cell lysate and the conditioned media were immunoprecipitated and analyzed by electrophoresis. By pulse labelling and chasing for various time periods, it was shown that the cleavages only took place after secretion of the protein from the cells. Adding cell lysate to uncleaved rFVIII caused cleavage of the heavy chain, as seen by loss of binding to a monoclonal antibody specific for intact rFVIII, indicating that the cleavage was performed by proteinase(s) released from the lysed cells. By incubating intact rFVIII with the multicatalytic proteinase (proteasome) present in cytoplasm and nucleus of eukaryotic cells, loss of binding to the monoclonal antibody was observed. This indicates that the multicatalytic proteinase, released from lysed rFVII...Continue Reading

References

Jun 29, 1992·FEBS Letters·N KobayashiE Munekata
Nov 1, 1986·Somatic Cell and Molecular Genetics·G UrlaubJ Hamlin
Jul 1, 1972·Proceedings of the National Academy of Sciences of the United States of America·P T VarandaniM A Nafz
Jan 1, 1982·Pharmacology & Therapeutics·W A Toscano, D R Storm
Jan 30, 1981·Biochemical and Biophysical Research Communications·R A Roth
Dec 15, 1995·European Journal of Biochemistry·M KjalkeM Ezban

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Citations

Jun 3, 2011·Biotechnology Letters·Susan M Browne, Mohamed Al-Rubeai
Dec 15, 2012·Cytotechnology·Mathilde MosserAnnie Marc
Dec 13, 2016·Journal of Biotechnology·Eric KarengeraOlivier Henry
Aug 26, 2010·Electrophoresis·Gian Maria D'AmiciLello Zolla
Jun 11, 2019·Biotechnology and Bioengineering·Alina SchneiderOliver Popp

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