Stability of Recombinant Human Thyrotropin Potency Based on Bioassay in FRTL-5 Cells

Introduction

R ecombinant human thyrotropin (rhTSH, Thyrogen^®, Genzyme 4–12 IU/mg) has been used extensively for the diagnosis of recurrence of differentiated thyroid cancer (1,2). The recommended dose is 0.9 mg intramuscular on 2 successive days; serum thyroglobulin is measured as a tumor marker 3 days after the second injection. rhTSH is also used for ablation of remnants of thyroid tissue after surgery for thyroid cancer (3). In addition, there have been several reports of the use of a single dose of 0.03–0.45 mg rhTSH, which is given to increase the thyroid uptake of a therapeutic dose of radioiodine-131 in patients with nodular goiter to reduce goiter size and compressive symptoms (4 –9). We have used 0.1 mg rhTSH to increase thyroid uptake of a therapeutic dose of ¹³¹I in patients with subclinical hyperthyroidism and relatively low 24-hour radioiodine uptake. The increased uptake of the therapeutic dose of radioiodine induced by rhTSH results in a greater effect of the therapeutic dose. This permits reduction of the amount of ¹³¹I that is administered. Reduction of the dose of radioiodine-131 may decrease side effects, such as damage to salivary glands and the lacrimal ducts.

Thyrogen is prepared in ampoules containing 1.1 mg rhTSH plus phosphate-buffered salt; it is diluted for injection in 1.2 mL sterile water so that 1.0 mL contains 0.9 mg. To determine whether the biologic activity of rhTSH can be preserved after dilution, we designed experiments to assess the biologic activity of rhTSH under different durations and temperatures of storage. The biologic activity was tested in vitro by measurement of ¹²⁵I-iodide uptake in FRTL-5 rat thyroid cells.

Materials and Methods

Two separate lots of Thyrogen were tested. The rhTSH was diluted in 1% bovine serum albumin in phosphate-buffered saline to a concentration of 0.9 mg/mL and further diluted to 0.1 mg/mL. Aliquots of 0.5 mL were stored at room temperature, 4°C, −11°C, and −60°C for various lengths of time. In addition, aliquots were subjected to several more extreme temperature conditions: incubation for 1 hour at 50°C; in another experiment, rhTSH was subjected to 10 cycles of freezing in dry ice alternating with thawing at 37°C.

The biologic activity of rhTSH was tested in FRTL-5 rat thyroid cells by a procedure reported previously (10). The FRTL-5 cells were generously provided by Dr. Leonard Kohn. FRTL-5 cells were cultured in Coon's modified Ham's F-12 medium supplemented with six hormones (bTSH, 2 U/L; insulin, 246 mU/L; somatostatin, 10 μg/L; hydrocortisone, 10 nM; transferrin, 5 mg/L; glycyl-histidyl-lysine, 2.5 μg/L), 5% calf serum, and antibiotics (6H medium). Cells were maintained in a 5% CO₂–95% air atmosphere at 37°C with a change of medium every second or third day, and passed every 7 days. To prepare cells for bioassay of TSH, they were grown in a TSH-free (5H) medium in 12-well plates containing about 10⁵ cells in 0.5 mL medium. rhTSH was tested in cells grown in the 5H medium for 5–7 days.

Chemicals, hormones, and reagents were purchased from Sigma (St. Louis, MO). Na¹²⁵I was purchased from Amersham Radiochemicals (Pascataway, NJ) and diluted for experiments in Hanks Balanced Salt Solution to 0.2 μCi/mL. Each well contained ∼100,000  counts/min (cpm) of Na¹²⁵I in 10 μM sodium iodide. The test substance was added, and cells were then incubated in a 37°C water bath for 60 min. To control for nonspecific uptake, each experiment included wells containing 40 μM perchlorate to block iodide transport.

The radioactive assay buffer was removed and cells in each well were rinsed twice, each with 1 mL of ice-cold Hanks' balanced salt solution as quickly as possible. After the last rinse, 0.5 mL of 0.5 M NaOH was added to each well and cells were incubated at room temperature for 30 min. The NaOH acts as a cell lysate, breaking cell membranes and releasing trapped radioiodide into the NaOH solution. The entire volume (0.5 mL) was placed in a sample tube, and the ¹²⁵I was counted in a gamma counter. Total radioactivity of cells in each well was expressed as cpm/well. After preliminary experiments showed that 5 ng/mL and 20 ng/mL rhTSH gave effective results, these concentrations were used in the bioassay. Dilution of the 0.1 mg/mL rhTSH aliquot was made to these concentrations on the day of each bioassay. Each concentration was run in triplicate and the ¹²⁵I uptake was calculated as an average of those three wells. The radioiodine uptake by cells incubated with perchlorate was subtracted as background from the cpm of the cells incubated in rhTSH without perchlorate. The results are expressed as mean percent of the cpm added to the media ± standard deviation.

Immunoassay of solutions of rhTSH was performed using the Advia Centaur XP instrument according to the manufacturer's protocol (Bayer HealthCare, Tarrytown, NY). The solutions were diluted so that they were in the linear range of the assay. Data are shown as mean for the aliquots stored at each temperature.

Statistical analysis of the bioassay data was performed using the program Instat3 (GraphPad Software, San Diego, CA). The data for each storage conditon of each lot were analyzed separately by analysis of variance; Dunnett multiple comparison test was used to compare the mean value with the mean at 0 day as control. The sensitivity of the assay was approximately 1 ng rhTSH/mL. The between-assay coefficient of variation was 25%.

Results

Figure 1 shows the results of the bioassay of the two lots of rhTSH maintained under various conditions before the bioassays: room temperature, −4°C (refrigerator), −11°C (freezer in refrigerator), or −60°C (ultra-low temperature freezer). Bioassay values of the 5 ng/mL and 20 ng/mL rhTSH at various days of storage were compared with the bioassays done at 0 time, the day of the initial dilution. When lot 1 was stored at −60°C, the activity of the 5 ng/mL dilution increased at 7 days, was slightly reduced at 30 days, and was unchanged at 36 and 59 days compared with the 0 day control; the activity of the 20 ng/mL dilution increased at 30 and 36 days (Fig. 1A). Storage of lot 2 at −60°C resulted in increased activity of the 5 ng/mL dilution at 7 and 185 days; the 20 ng/mL dilution had reduced activity at 36 days, increased activity at 185 days, and unchanged activity at 204 days (Fig. 1B). When lot 2 was stored at room temperature (usually 70–74°F), there was no loss of biologic activity up to 204 days (Fig. 1C). In addition, there were no significant changes in the activity of either dilution of lot 2 when it was stored at −11°C up to 204 days (Fig. 1D). When lot 1 was stored at 4°C up to 59 days, there was no loss of activity; in fact, the 20 ng/mL dilution showed increased activity at 7, 30, and 36 days (Fig. 1E). Storage of lot 2 at 4°C resulted in increased activity of the 5 ng/mL dilution at 185 days and reduced activity at 204 days; the activity of the 20 ng/mL dilution was increased at 185 days (Fig. 1F).

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FIG. 1.

Uptake of ¹²⁵I by FRTL-5 cells as percent of ¹²⁵I added to well, and mean ± standard deviation of triplicate wells corrected for nonspecific background uptake of cells incubated with 40 μM perchlorate. The upper figures show the uptake stimulated by rhTSH Lot 1 (A) and Lot 2 (B) stored for various periods of time at −60°C. The middle panels shows the ¹²⁵I uptake stimulated by rhTSH Lot 2 stored at room temperature (C) and at −11°C (D). The lower panel shows ¹²⁵I uptake by cells stimulated with rhTSH Lot 1 (E) and Lot 2 (F) stored at 4°C. Plus symbol indicates p < 0.01 compared with 0 time; star indicates p < 0.05 compared with 0 time. rhTSH, recombinant human thyrotropin.

Samples subjected to the freeze–thaw cycle 10 times in dry ice and then rapidly warmed in a 37°C water bath retained bioactivity similar to those of samples that were not frozen (data not shown). Samples heated at 50°C for 1 hour also stimulated iodide uptake as well as did samples that were not heated (data not shown).

Table 1 shows the immunoassay results of solutions of lot 2 stored in various ways and immunoassayed at day 185. The values were similar regardless of the condition of storage, indicating that the immunologic activity was preserved to a similar degree under the various conditions of storage.

Discussion

The data show that the bioactivity of rhTSH, when tested in a cell culture bioassay, is well preserved for many days when aliquots of 0.1 mg/mL are stored under four conditions: room temperature, −4°C (refrigerator), −11°C (freezer in refrigerator), and −60°C (ultra-low temperature freezer). Analysis of Figure 1 shows no pattern of loss of activity over time based on the condition of storage. In comparison with the control, there tended to be an increase of bioactivity. Although many of the comparisons are statistically significant as noted in Figure 1, this could be due to the variability of bioassays using cultured cells. The variability of cell-based bioassays and lack of precision of such assays does not completely exclude the possibility that there may be some loss of bioactivity. If this occurred, it might be expected to increase over time, but this was not found, making this consideration unlikely. It is possible that prolonged storage could alter the glycosylation of rhTSH in such a manner that it would have reduced activity in the patient; this could be determined by clinical studies.

One weakness of our study is that we did not compare the results with a reference standard in each assay. Bovine TSH is a known reference standard, but previous work with this bioassay showed that bovine TSH and human TSH do not give parallel responses in this assay (11). A human TSH was not used as standard because the conditions of its storage might have had the same impact on its bioactivity as the results we found for rhTSH bioactivity. In our experience and that of others, human TSH in serum and pituitary TSH are remarkably stable in regard to immunoassay and bioassay activity (12). Serum samples preserved at −11°C and thawed for repeat immunoassays give the same result within the variance of the assay.

One reservation is that the conditions of storage could have altered the sialylation of rhTSH, resulting in a change of half-life in vivo. However, this is not likely to have occurred spontaneously in solution, although variable sialylation of TSH has been documented in patients with TSH-secreting pituitary tumors (13).

Another reservation concerning our results is the use of 1% bovine serum albumin in phosphate-buffered saline for dilution of the samples of rhTSH. This was done to prevent the solutions of rhTSH from being lost by sticking to glass or plastic when diluted to the very low concentrations used in the cell bioassay.

In conclusion, the data show that the biologic activity of rhTSH is remarkably stable under the usual conditions of storage of materials for human use. The data suggest that storage in the cold for a few months does not result in loss of biologic activity. The use of stored rhTSH after dilution may result in financial savings in the clinical use of this important material in situations where less than a full ampoule is an appropriate dose. Clinical studies are needed to validate this idea.