Development of serum-free bioreactor production of recombinant human thyroid stimulating hormone receptor

For the detection of autoantibodies to thyroid stimulating hormone receptor s (TSH-R) in Graves' disease based on a novel coated tube assay system, hum an TSH-R is needed in large amounts. Whereas expression of TSH-R in bacteri a, yeast, or insect cells results in nonfunctional, denaturated receptor, m ammalian cells such as COS, CHO, and HeLa are able to express functional TS H-R, but only in very low amounts. Furthermore, for all of these cultivatio ns expensive standard media containing 10% fetal calf serum are needed to o btain functional receptor. Here we report on the development of a serum-fre e production-scale process based on a stable transformed and highly product ive human leukemia cell line K562 (1). Starting with K562-TSH-R cells growi ng in medium containing 10% fetal calf serum the cell line was adapted to s erum-free medium. The adaptation medium was optimized in regards to amino a cid and protein concentrations, since the use of unadjusted medium caused c ell death after 2 days. The adapted cells were stable and could be cultivat ed without antibiotics for more than 50 cell doublings without losing their productivity. The obtained receptor showed improved TSH binding. The proce ss development was based on cultivations in a 2-L bench-scale bioreactor. C ultivations in batch mode and chemostat mode and perfusion cultivation with the usage of an internal microfiltration device and a spin-filter device w ere compared. After process optimization a continuous process using spin-fi lter was set up and run in a 20 L-pilot-scale bioreactor. The presented res ults were the prerequisite for the production of the novel assay for the di agnosis of autoantibodies to TSH-R in Graves' disease.