The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

Author:

Hotchkiss A1,Refino C J1,Leonard C K2,O'Connor J V2,Crowley C3,McCabe J1,Tate K4,Nakamura G2,Powers D4,Levinson A4,Mohler M1,Spellman M W4

Affiliation:

1. The Department of Pharmacological Sciences, Genentech, Inc., South San Francisco, CA, USA

2. The Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA, USA

3. The Department of Cell Genetics, Genentech, Inc., South San Francisco, CA, USA

4. The Department of Molecular Biology, Genentech, Inc., South San Francisco, CA, USA

Abstract

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

Publisher

Georg Thieme Verlag KG

Subject

Hematology

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