Comparison of batch and perfusion culture in combination with pilot-scale expanded bed purification for the production of soluble recombinant β-secretase

E. Lüllau, A. Kanttinen, J. Hassel, M. Berg, A. Haag-Alvarsson, K. Cederbrant, Barry Greenberg, C. Fenge, F. Schweikart

Research output: Contribution to journalArticle

Abstract

β-Secretase is one of the prime targets for therapeutic intervention of Alzheimer's disease. For the development of a secretase inhibitor a steady supply of large quantities of a homogeneous and active recombinant β-secretase is a prerequisite. Therefore various culture modes were investigated using HEK-293 cells stably transfected with soluble recombinant βq-secretase. The coupling of the Fc part of human IgG1 to the ectodomain of β-secretase (residues 1-460) allowed a fast purification of the protein with rProtA expanded bed chromatography. Batch cultures of 5 to 50 L working volume run for 7 days showed reproducible cell growth and product yields of 3 mg/L purified protein. A 20 L perfusion culture was operated for 21 days, reaching a cell density of 30 × 106 cells/mL at a dilution rate of 2/d. The total product yield of the perfusion culture was 1.4 g of purified protein. The effect of different perfusion rates on cell growth, protein yield, and quality was investigated and compared to the results obtained in batch cultures. Protein quality was consistent as analyzed on 1D SDS-PAGE, and the final product contained both the mature and the pro form of β-secretase. Although the cell specific protein expression was slightly reduced in perfusion culture, a substantial increase in specific activity of over 75% was achieved. Some of the increase in activity can be explained by an increase in the percentage of the mature form of the recombinant protein.

Original languageEnglish (US)
Pages (from-to)37-44
Number of pages8
JournalBiotechnology Progress
Volume19
Issue number1
DOIs
Publication statusPublished - Jan 1 2003
Externally publishedYes

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ASJC Scopus subject areas

  • Biotechnology

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