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Development of Serum‐Free Bioreactor Production of Recombinant Human Thyroid Stimulating Hormone Receptor
Author(s) -
Stiens Lars R.,
Büntemeyer Heino,
Lütkemeyer Dirk,
Lehmann Jürgen,
Bergmann Andreas,
Weglöhner Wolfgang
Publication year - 2000
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1021/bp000103l
Subject(s) - bioreactor , receptor , chemostat , fetal bovine serum , biology , cell culture , yeast , cell , chemistry , biochemistry , bacteria , botany , genetics
Abstract For the detection of autoantibodies to thyroid stimulating hormone receptors (TSH‐R) in Graves' disease based on a novel coated tube assay system, human TSH‐R is needed in large amounts. Whereas expression of TSH‐R in bacteria, yeast, or insect cells results in nonfunctional, denaturated receptor, mammalian cells such as COS, CHO, and HeLa are able to express functional TSH‐R, but only in very low amounts. Furthermore, for all of these cultivations expensive standard media containing 10% fetal calf serum are needed to obtain functional receptor. Here we report on the development of a serum‐free production‐scale process based on a stable transformed and highly productive human leukemia cell line K562 (1). Starting with K562‐TSH‐R cells growing in medium containing 10% fetal calf serum the cell line was adapted to serum‐free medium. The adaptation medium was optimized in regards to amino acid and protein concentrations, since the use of unadjusted medium caused cell death after 2 days. The adapted cells were stable and could be cultivated without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The process development was based on cultivations in a 2‐L bench‐scale bioreactor. Cultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin‐filter device were compared. After process optimization a continuous process using spin‐filter was set up and run in a 20 L‐pilot‐scale bioreactor. The presented results were the prerequisite for the production of the novel assay for the diagnosis of autoantibodies to TSH‐R in Graves' disease.