BioChem - DOS

Production and characterization of a novel human recombinant alpha-1-antitrypsin in PER.C6 cells

Journal of biotechnology

Ross, D.; Brown, T.; Harper, R.; Pamarthi, M.; Nixon, J.; Bromirski, J.; Li, C.M.; Ghali, R.; Xie, H.; Medvedeff, G.; Li, H.; Scuderi, P.; Arora, V.; Hunt, J.; Barnett, T.

Alpha-1-antitrypsin (A1PI) is a proteinase inhibitor of the serpin superfamily and circulates in plasma at about 1-2g/L. A1PI deficiency in humans often results in organ damage, particularly to the lungs and liver. Current augmentation therapies rely entirely on A1PI isolated from human plasma, thus prompting an evaluation of alternate sources. We have co-expressed recombinant A1PI and α-2,3-sialyltransferase in the human cell line, PER.C6. The requirement for sialyltransferase overexpression in PER.C6 and the essential contribution of sialic acid glycan capping on pdA1PI and recA1PI to prevent rapid A1PI plasma elimination is shown. Using assays to predict high levels of A1PI production and sialylation, stably transfected PER.C6 cells were screened through two rounds of cell cloning to ensure monoclonality. Fed-batch culturing was used to evaluate recA1PI production and cell line characteristics, identifying subclones expressing over 2.5g/L recA1PI. Cell stability was assessed over 50 generations, verifying subclone stability during continuous culture. Finally, data are presented showing that recA1PI and pdA1PI are equivalent in their ability to block elastase activity in functional cell-based assays and their pharmacokinetic properties. These data show that recombinant human A1PI recovered from PER.C6 cells offers a reliable source of functionally active A1PI for augmentation therapies and, potentially, other diseases.

2012

Elsevier Science Publishers

0168-1656

1873-4863

162

2

pp.262-273

10.1016/j.jbiotec.2012.09.018

http://click.ndsl.kr/servlet/OpenAPIDetailView?keyValue=05787966&target=NART&cn=NART65040381

Alpha-1-antitrypsin; Sialyltransferase; Recombinant protein; PER.C6