Human thyroid-stimulating hormone synthesis in human embryonic kidney cells and related N-glycoprofiling analysis for carbohydrate composition determination.

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and ß-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t 1/2) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition.

Efficient Non-Invasive Plasmid-DNA Administration into Tibialis Cranialis Muscle of "Little" Mice.

BACKGROUND: An alternative treatment for growth hormone deficiency based on hGH-DNA administration, followed by electro gene transfer, was investigated by injecting the plasmid into surgically exposed or non-exposed quadriceps or tibialis muscle of immunodeficient "little" mice. METHODS: An optimization of electrotransfer conditions via a new combination of high/low voltage pulses is presented. After 3 days, serum hGH was determined and in a 28-day assay, the relative growth parameters were compared. RESULTS: Both groups exhibited similar results: 5.0 ± 2.2 (SD) and 3.5 ± 0.9 ng hGH/ml (P>0.05; n=7) for the exposed quadriceps and non-exposed tibialis treatments, respectively. The final body weight increases were 16.1% for the quadriceps and 18.9% for the tibialis group. The tail and nose-to-tail length increases were 4.5% and 7.1% for the quadriceps and 4.8 and 4.6% for the tibialis group. The right and left femur length increases, obtained from radiographic measurements, were 16.9% and 12.7% for the quadriceps and 19.4% and 12.3% for the tibialis, respectively. A non-significant difference between exposed quadriceps and non-exposed tibialis treatments (P=0.48) was confirmed via a completely integrated statistical analysis. Circulating mIGF-1 levels were 126 ± 47, 106 ± 93 (P>0.05) and 38 ± 15 ng/ml for the quadriceps, tibialis and saline treatments, respectively. CONCLUSION: These results show that hGH-DNA administration into non-exposed tibialis muscle followed by the new HV/LV electrotransfer protocol was an equally efficient, less traumatic treatment, much more suitable for pre-clinical testing than administration into exposed quadriceps.

High-yield purification of biosynthetic human growth hormone secreted in Escherichia coli periplasmic space.

A six-step, high-yield purification procedure for the preparation of clinical grade recombinant human growth hormone (rhGH) secreted in bacterial periplasmic space is described. Particular emphasis is given to hormone recovery yields and maximum contaminant host cell elimination. The strategy adopted, in addition to using one precipitation and five chromatographic steps in a particularly efficient sequence, was also based on running E. coli proteins - immunoradiometric assay profiles right after each chromatographic elution. Thus, an overall rhGH recovery higher than 40%, with a final concentration of E. coli proteins below 10 ppm is described for the first time. The accuracy of hGH and total protein quantification, especially in the early steps of the process, and the maximum elimination of hGH-related forms were also studied in detail. For these purposes size-exclusion and reversed-phase HPLC were found to be extremely valuable analytical tools.

Sensitive human thyrotropin immunoradiometric assay set up by the identification and minimization of nonspecific bindings.

An immunoradiometric assay (IRMA) of human thyrotropin (hTSH), based on magnetic solid phase separation, was studied especially in terms of its nonspecific bindings (B0) which were identified as a product of the interaction between an altered form of radioiodinated anti-hTSH monoclonal antibody (125I-mAB) and the uncoupled magnetizable cellulose particle (matrix). Preincubation with the same matrix, solid phase saturation with milk proteins, tracer storage at 4 degrees C and serum addition during incubation were found to be particularly effective in preventing their formation. These findings were used to reproducibly decrease nonspecific bindings to values < 0.1% (or < 70 cpm), thus increasing the signal-to-noise ratio (B60/B0) up to values of 300-500. This way hTSH radioassays were obtained with functional sensitivities of about 0.05 mIU/L and analytical sensitivities of the order of 0.02 mIU/L. Such sensitivities, and, more importantly, a general improvement in assay performance, were obtained in a highly reproducible manner and all over the useful tracer life.