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

Paper ID Volume ID Publish Year Pages File Format Full-Text
23628 43459 2012 12 PDF Available
Title
Production and characterization of a novel human recombinant alpha-1-antitrypsin in PER.C6 cells
Abstract

Alpha-1-antitrypsin (A1PI) is a proteinase inhibitor of the serpin superfamily and circulates in plasma at about 1–2 g/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 2.5 g/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.

► Recombinant A1PI and a sialyltransferase were co-expressed in a human cell line. ► The essential contribution of sialic acid glycan capping on A1PI is shown. ► Screens and fed-batch studies isolated a stable subclone expressing 2.5 g/L recA1PI. ► Functional assays and pharmacokinetics show that recA1PI and pdA1PI are equivalent.

Keywords
Alpha-1-antitrypsin; Sialyltransferase; Recombinant protein; PER.C6
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Production and characterization of a novel human recombinant alpha-1-antitrypsin in PER.C6 cells
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Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 162, Issues 2–3, 31 December 2012, Pages 262–273
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us