PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN THYROTROPIN (TSH) ISOFORMS PRODUCED BY CHINESE-HAMSTER OVARY CELLS - THE ROLE OF SIALYLATION AND SULFATION IN TSH BIOACTIVITY

The biological significance of glycosylation variants of pituitary gly coprotein hormones remains controversial because of the indirect metho ds usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to de termine bioactivity. We have previously characterized recombinant huma n TSH (rhTSH) secreted by Chinese hamster ovary cells attached to micr ocarrier beads in a large scale bioreactor after stable transfection o f hCGalpha and hTSHbeta minigenes. In the present study rhTSH has been used as a model to determine structure-function relationships of diff erent isoforms of glycoprotein hormones. We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor. The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rh TSH commercially produced in a large scale bioreactor (rhTSH-G) were q uantitated by immunoassays, receptor binding assay, and amino acid ana lysis and further characterized by a variety of physico-biochemical me thods, including chromatofocusing and carbohydrate analysis. rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with differ ent pI values. Compositional analysis of the fractions showed higher s ialic acid content in the more acidic rhTSH-G fractions. phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions. The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more ac idic fractions. In contrast to the in vitro data, highly sialylated an d acidic rhTSH-G isoforms showed longer plasma half-lives and higher i n vivo bioactivity than the basic forms. These results indicate that s ecreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of si alylation. The degree of sialylation, highly dependent on the bioreact or production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.