Water-soluble dry solid containing proteinaceous bioactive substance

Proteinaceous bioactive substances (including lymphokine and peptide hormone) in dry solid are extremely stabilized by the presence of the specific polysaccharide mainly composed of repeating maltotriose units. Pullulan, elsinan, and their partial hydrolysates are feasible as the polysaccharide. The weight ratio of the polysaccharide to the substance which effectively stabilizes the latter substance is at least 0.5 on the basis of dry solids. The dry solid is advantageously usable as a test reagent, injection, granule, tablet, suppository or ointment.

FIELD OF THE INVENTION 
The present invention relates to a stabilized water-soluble dry solid 
containing a proteinaceous bioactive substance along with a polysaccharide 
mainly composed of repeating maltotriose units. 
ABBREVIATIONS 
Interferon is abbreviated herein as "IFN"; human interferon, "HuIFN"; tumor 
necrosis factor, "TNF"; lymphotoxin, "LT"; growth hormone, "GH"; 
erythropoietin, "EPO"; epidermal growth factor, "EGF"; and 
thyroid-stimulating hormone, "TSH". 
The "h" prefixed to some of these abbreviations means that the specific 
substance is human-specific (i.e. hGH, hEGF, hEPO, and hTSH). 
BACKGROUND OF THE INVENTION 
Proteinaceous bioactive substances usable as test reagent and/or 
pharmaceutical, e.g. lymphokines and peptide hormones, have been 
extensively studied and developed as the recent remarkable advances in 
biochemistry and medical science. Some of these substances are 
commercialized or readily for commercialization. 
Since proteinaceous bioactive substances are, in general, relatively 
unstable, a stabilizer must be added for their commercialization. The 
stabilizer most frequently used is human serum albumin (HSA). The use of 
HSA is, however, disadvantageous because: 
(1) The stabilization effect on proteinaceous bioactive substances is 
unsatisfactory; 
(2) The addition of HSA obscures the specific activity of the substance. A 
specific activty is represented by activity/mg protein, and used for 
determination of purification degree; 
(3) The use of HSA has a fear of mediating human infectious diseases since 
HSA is a protein derived from human serum; 
(4) HSA tends to form a water-insoluble solid on drying. 
In order to avoid these demerits of HSA, various stabilizers have been 
proposed. As a stabilizer for IFN, Japan Patent Kokai No. 92,691/83 
proposes cyclodextrin, and Japan Patent Kokai No. 25,333/84 proposes 
saccharides (excluding polysaccharides) such as mono- and 
oligo-saccharides, and polyols such as glycerine and ethylene glycol. For 
TNF stabilization, Japan Patent Kokai No. 39,829/84 proposes non-ionic 
surface active agents, and Japan Patent Kokai No. 59,625/84 proposes 
D-glucose, D-galactose, D-xylose, D-glucuronic acid, dextran, hydoxyethyl 
starch, and, preferably, trehalose. The stabilization effects attained 
with these stabilizers have proved insufficient. In addition, trehalose is 
relatively expensive. Thus, these stabilizers have not been in practical 
use. 
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
We investigated various stabilizers, especially polysaccharides, to obtain 
a highly-stable water-soluble dry solid containing a proteinaceous 
bioactive substance. As the result, we found that a polysaccharide, mainly 
composed of repeating maltotriose units, unexpectedly satisfies the 
objects of the present invention. More particularly, we found that a dry 
solid obtained by drying an aqueous solution, containing the substance 
along with the specific polysaccharide, by evaporation has a desirable 
water-solubility, as well as that the dry solid much more stably retains 
the activity of the substance. This is the present invention. 
The term "proteinaceous bioactive substance" as used herein means 
proteinaceous substances which exhibit a bioactivity in vivo, such as 
simple proteins and protein conjugates, in particular, lymphokines, such 
as IFN, LT, TNF, macrophage migration inhibitory factor, transfer factor, 
T cell growth factor, and colony stimulating factor; and peptide hormones, 
such as insulin, GH, prolactin, chorionic gonadotropin, EPO, 
follicle-stimulating hormone, luteinizing hormone, EGF, 
adrenocorticotropic hormone, placental lactogen, TSH, and parathyroid 
hormone, which have a molecular weight within the range from about 10,000 
daltons to about 200,000 daltons. 
The proteinaceous bioactive substances usable in the invention include 
those which may be isolated from body fluid, cell, tissue or organ wherein 
such substance naturally occurs; those which may be recovered from an in 
vitro or in vivo culture of any of the above; those which may be recovered 
from a culture of either human cell, animal cell or microorganism wherein 
the producibility of such substance is engineered by conventional means, 
e.g. cell fusion, gean recombinant technique, etc.; but are not restricted 
to those which may be prepared by particular procedures. 
The polysaccharides usable in the invention are those which are mainly 
composed of maltotriose units polymerized in .alpha.-fashion. Examples of 
the polysaccharides are pullulan, elsinan, and their partial hydrolysates 
having a molecular weight within the range of 10,000-10,000,000 daltons, 
preferably within then range of 20,000-2,000,000 daltons. The weight ratio 
of the polysaccharide to the substance is at least 0.5, desirably, from 
1.0 to 10,000, on the basis of dry solids. 
The aqueous solution containing a proteinaceous bioactive substance along 
with the polysaccharide is dried under conditions appropriate to give a 
water-soluble dry solid without substantial decrease in the activity of 
the substance. Although conventional drying procedures carried out at a 
reduced pressure and a temperature below 30.degree. C. are feasible in the 
invention, freeze-drying is desirable. In addition to the specific 
polysaccharide, one or more additional substances, e.g. mineral, buffer, 
amino acid, saccharide, etc., may be desirably incorporated into the 
aqueous solution, prior to its drying. The dry solid thus obtained is 
readily dissolvable in water, and very stably retains the activity of a 
proteinaceous bioactive substance. Thus, the dry solid is advantageously 
usable for, e.g. test reagent, injection, medicine for external or 
internal administration, etc., to prevent and/or treat human diseases. 
The following experiments will further explain the present invention.

EXPERIMENT 1 
Stabilization test on IFN 
EXPERIMENT 1-A 
Preparation of IFN 
Newborn hamsters were injected with an antiserum, prepared with rabbit in 
conventional manner, to weaken their immunoreaction, subcutaneously 
implanted with BALL-1 cell, and fed for three weeks in usual way. The 
tumor masses, formed subcutaneously in the animals, were extracted, 
minced, and disaggregated in saline. The cell so obtained was washed with 
serum-free RPMI 1640 medium (pH 7.2), suspended in a fresh preparation of 
the same culture medium to give a cell density of about 2.times.10.sup.6 
cells/ml, and kept at 35.degree. C. The cell suspension was added with a 
partially purified HuIFN in a dose of 200 U/ml, incubated for about two 
hours, added with Sendai virus (about 300 hemagglutination titers/ml), and 
incubated for additional twenty hours to induce HuIFN production. The 
resultant culture was centrifuged to about 1,000.times.g and about 
4.degree. C. to remove the solid, and the supernatant was 
membrane-filtered. The filtrate was chromatographed with a column of 
conventional immobilized anti-HuIFN antibody, and the non-adsorbed 
fractions were removed. The adsorbed HuIFN was eluted from the column, and 
concentrated with a membrane to obtain an about 0.01 w/v % concentrate 
with a specific activity of about 1.5.times.10.sup.8 U/mg protein in a 
yield of about 4 ml/hamster. 
The activity of HuIFN was assayed by the conventional plaque reduction 
method using FL cell. The hemagglutination titers were assayed in 
accordance with the method as described by J.E. Salk, The Journal of 
Immunology, Vol. 49, pp. 87-98 (1944). 
EXPERIMENT 1-B 
Comparison of stabilization effects of several stabilizers on IFN 
One-half ml of an HuIFN concentrate, obtained by the method in Experiment 
1-A, and 1 ml aqueous solution of a stabilizer were placed in a glass 
vial, admixed, freeze-dried, and stored at either 4.degree. C. or 
37.degree. C. for two months. The solid was added with 30.degree. C. 
saline to dissolve or elute HuIFN. The HuIFN was assayed, and the 
retension ratio (%) to the activity obtained before the freeze-drying was 
calculated with the following equation: 
##EQU1## 
The results are listed in Table I. 
These results evidently confirmed that the dry solids using as the 
stabilizer the specific polysaccharide, i.e. pullulan or elsinan, were 
excellent in HuIFN stability and water-solubility, and, therefore, 
handleable with ease. 
TABLE I 
______________________________________ 
Retension 
Solubility in 
ratio (%) 
Stabilizer saline 4.degree. C. 
37.degree. C. 
Remark 
______________________________________ 
None Readily soluble 
60.9 0 Control 
Phosphate buffer 
Readily soluble 
68.3 15.2 Control 
Maltose Readily soluble 
72.4 41.2 Control 
.beta.-Cyclodextrin 
Readily soluble 
78.1 45.6 Control 
HSA Soluble 86.2 67.3 Control 
Amylopectin Soluble 80.1 59.2 Control 
Hydroxyethyl starch 
Readily soluble 
78.3 56.6 Control 
Dextran Readily soluble 
81.5 65.3 Control 
Pullulan Readily soluble 
100.0 100.0 Present 
invention 
Elsinan Readily soluble 
100.0 99.1 Present 
invention 
Gum arabic Readily soluble 
76.2 54.6 Control 
Gum tragacanth 
Readily soluble 
77.3 52.1 Control 
Carrageenan Readily soluble 
75.4 48.7 Control 
Agar Scarcely soluble 
33.1 21.6 Control 
Pectin Readily soluble 
50.3 25.3 Control 
______________________________________ 
Note: 
In the column of "stabilizer", "none", only deionized water; "phosphate 
buffer", 0.01 M phosphate buffer (pH 7.2); and the others, 0.5 w/v % 
aqueous solution of the specified stabilizer. 
EXPERIMENT 2 
Stabilization test on TNF 
EXPERIMENT 2-A 
Preparation of TNF 
Newborn hamsters were injected with an antiserum, prepared with rabbit in 
conventional manner, to weaken their immunoreaction, subcutaneously 
implanted with an SV-40 virus-transformed human monocyte line, fed for one 
week in usual way, intraperitoneally injected with crude BCG cell in a 
dose of 10.sup.7 cells/hamster, and fed for an additional two weeks. The 
tumor masses, formed subcutaneously in the animals, about 15 g each, were 
extracted, minced, and disaggregated in saline containing trypsin. The 
cell so obtained was washed in Eagle's minimal essential medium (pH 7.2) 
supplemented with 5 v/v % human serum, suspended in a 37.degree. C. fresh 
preparation of the same culture medium to give a cell density of about 
5.times.10.sup.6 cells/ml, added with Escherichia coli endotoxin (about 10 
.mu.g/ml), and incubated at this temperature for sixteen hours to induce 
TNF production. 
The resultant culture was centrifuged at about 1,000.times.g and about 
4.degree. C. to remove the solid, after which the supernatant was dialyzed 
against saline containing 0.01 M phosphate buffer (pH 7.2) for twenty-one 
hours, and membrane-filtered. The filtrate was concentrated, and 
freeze-dried to obtain a powder with TNF activity. The powder was purified 
by adsorption/desorption using ion exchange, molecular weight 
fractionation using gel filtration, concentration, and 
membrane-filtration, in accordance with the procedures as reported in G. 
Bodo, Symposium on Preparation, Standardization and Clinical Use of 
Interferon (11-th International Immunological Symposium, 8 & 9 June 1977, 
Zagreb, Yugoslavia). The resultant HuIFN-free preparation was salted out 
with ammonium sulfate, purified by affinity-chromatography using Con 
A-Sepharose, and concentrated to obtain an about 0.01 w/v % concentrate 
containing a high-purity TNF in a yield of about 30 ml/hamster: TNF was 
characterized in that it effected hemorrhagic necrosis on Meth A sarcoma, 
but did not affect on normal cells. The TNF thus obtained was a 
glycoprotein with a specific activity of about 3.5.times.10.sup.5 U/mg 
protein, and free of the inducer used. 
The activity of TNF was assayed in conventional manner as reported in 
Lymphokines, Vol. 2, edited by E. Pick, pp. 235-272, "Tumor Necrosis 
Factor", published by Academic Press, Inc. (1981): After exposing a 
culture of L-929 cell (TNF-susceptive normal cell line) to dilutions of 
TNF for the prescribed time, the number of residual treated cells was 
counted. 
EXPERIMENT 2-B 
Comparison of stabilization effects of several stabilizers on TNF 
Similarly as in Experiment 1-B, 0.5 ml of a TNF concentrate, obtained by 
the method in Experiment 2-A, and 1 ml aqueous solution of a stabilizer 
were placed in a glass vial, freeze-dried, and stored, followed by 
determination of the retension ratio (%). 
The results are listed in Table II. 
TABLE II 
______________________________________ 
Retension 
Solubility in 
ratio (%) 
Stabilizer saline 4.degree. C. 
37.degree. C. 
Remark 
______________________________________ 
None Readily soluble 
43.8 0 Control 
Phosphate buffer 
Readily soluble 
46.2 6.7 Control 
Maltose Readily soluble 
62.5 22.3 Control 
.beta.-Cyclodextrin 
Readily soluble 
60.3 25.7 Control 
HSA Soluble 76.1 40.9 Control 
Amylopectin Soluble 64.3 41.7 Control 
Hydroxyethyl starch 
Readily soluble 
70.9 36.9 Control 
Dextran Readily soluble 
78.2 43.6 Control 
Pullulan Readily soluble 
100.0 99.4 Present 
invention 
Elsinan Readily soluble 
100.0 98.2 Present 
invention 
Gum arabic Readily soluble 
63.8 28.6 Control 
Gum tragacanth 
Readily soluble 
70.4 30.1 Control 
Carrageenan Readily soluble 
62.6 25.2 Control 
Agar Scarcely soluble 
25.1 10.5 Control 
Pectin Readily soluble 
28.8 16.3 Control 
______________________________________ 
Note: 
In the column of "stabilizer", "none", only deionized water; "phosphate 
buffer", 0.01 M phosphate buffer (pH 7.2); and the others, 0.5 w/v % 
aqueous solution of the specified stabilizer. 
These results evidently confirmed that the dry solids using as the 
stabilizer the specific polysaccharide, i.e. pullulan or elsinan, were 
excellent in TNF stability and solubility in water, and, therefore, 
handleable with ease. 
EXPERIMENT 3 
Ratio of polysaccharide 
The stabilization effect on proteinaceous bioactive substances was studied 
with different weight ratios of the polysaccharide: One-half ml of either 
an HuIFN concentrate, obtained by the method in Experiment 1-A, or a TNF 
concentrate, obtained by the method in Experiment 2-A, was placed in a 
glass vial along with either pullulan or elsinan having an appropriate 
concentration to give a weight ratio of the polysaccharide to the 
substance of either 0, 0.01, 0.1, 0.5, 1.0, 5.0, 10.0, or 100.0 on the 
basis of dry solids, mixed, freeze-dried similarly as in Experiment 1-B, 
and stored at 37.degree. C. for one month, followed by determination of 
the retension ratios (%). 
The results are listed in Table III. 
All the dry solids were readily dissolvable in saline. 
As is evident from these results, the weight ratio which effectively 
stabilizes a proteinaceous bioactive substance is 0.5 or higher, 
preferably, 1.0 or higher, on the basis of dry solids. 
TABLE III 
______________________________________ 
Weight ratio Retension of 
Retension of 
Polysaccharide 
to protein (folds) 
HuIFN (%) TNF (%) 
______________________________________ 
Pullulan 0 0 0 
0.01 12.3 0 
0.1 34.5 28.4 
0.5 87.2 83.5 
1.0 94.6 89.9 
5.0 98.3 96.2 
10.0 100.0 97.8 
100.0 100.0 99.4 
Elsinan 0 0 0 
0.01 10.5 0 
0.1 27.7 21.2 
0.5 83.5 81.6 
1.0 91.6 87.3 
5.0 96.1 93.7 
10.0 97.7 96.5 
100.0 98.9 98.2 
______________________________________ 
As described above, the dry solid of the invention is characterized in that 
it is freely soluble in water, and that it stably retains the activity of 
a proteinaceous bioactive substance over a long period of time under 
vigorous conditions, e.g. at room temperature. Unlike conventional 
stabilizer such as HSA, the specific polysaccharide has no fear of 
mediating human infectious diseases, and does not obscure the specific 
activity of the substance which is used for determination of purification 
degree. 
Thus, the dry solid of the invention is advantageously usable for test 
reagent, injection, and medicine for internal or external administration, 
to prevent and/or treat human diseases. 
The merits of the present invention will be explained with reference to the 
following examples. 
EXAMPLE 1 
HuIFN 
A concentrate containing a purified HuIFN, obtained by the method in 
Experiment 1-A, was added with an aqueous solution containing 100 parts of 
a purified pullulan based on the weight of HuIFN solid, filtered under 
sterile conditions, distributed into 2 ml-glass vials to give an HuIFN 
content of 3.times.10.sup.6 U/vial, and freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent, as well as for intramuscular- or 
intravenous-injection. 
EXAMPLE 2 
TNF 
A concentrate containing a purified TNF, obtained by the method in 
Experiment 2-A, was added with an aqueous solution containing 200 parts of 
a purified pullulan based on the weight of TNF solid, filtered under 
sterile conditions, distributed into 2 ml-glass vials to give a TNF 
content of 2,000 U/vial, and freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent, as well as for intramuscular- or 
intravenous-injection. 
EXAMPLE 3 
LT 
Non-adsorbed fractions from a column of immobilized anti-HuIFN antibody, 
obtained by the method in Experiment 1-A, were purified by 
affinity-chromatography using phytohemagglutinin-Sepharose, and 
concentrated with a membrane. The concentrate containing a high-purity LT 
was admixed with an aqueous solution containing 50 parts of elsinan based 
on the weight of LT solid, distributed into 5 ml-glass vials to give an LT 
content of 1,000 U/vial, and freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent, injection, granule, tablet, ointment, etc. 
The activity of LT was assayed in conventional manner as reported in In 
Vitro Methods in Cell-Mediated Immunity, edited by B.R. Blood & P.R. 
Glade, published by Academic Press, Inc. (1971), wherein a culture of 
mouse L cell is exposed to dilutions of LT, and the number of residual 
treated L cells is counted. 
EXAMPLE 4 
GH 
An acidophilic adenoma cell was extracted from acidophilic adenoma patient, 
minced, disaggregated, and subcutaneously implanted into adult nude mice, 
followed by three week-bleeding. The tumor masses, formed subcutaneously 
in the animals, about 10 g each, were extracted, minced, and disaggregated 
in saline containing trypsin. The cell so obtained was washed with 
glucose-free Earle's 199 medium (pH 7.2) supplemented with 10 v/v % fetal 
calf serum, suspended in a fresh preparation of the same culture medium, 
additionally containing 30 mM L-arginine as GH inducer, to give a cell 
density of about 10.sup.5 cell/ml, and incubated at 37.degree. C. for six 
hours to induce hGH production. The cell in the resultant culture was 
ultrasonically disintegrated, and the hGH in the supernatant was assayed. 
The hGH production was about 500 ng/ml cell suspension. The supernatant 
was purified and concentrated in conventional manner to obtain an hGH 
concentrate which was then admixed with an aqueous solution containing 
10,000 parts of pullulan based on the weight of hGH solid, distributed 
into 2 ml-glass vials to give an hGH content of 10 ng/vial, and 
freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent or injection. 
The activity of hGH was assayed by conventional radioimmunoassay as 
described in S.M. Gilick et al., Nature, Vol. 199, page 784 (1963). 
EXAMPLE 5 
EGF 
A submaxillary gland tumor cell was extracted from submaxillary gland tumor 
patient, minced, disaggregated, and inoculated on Earle's 199 medium (pH 
7.2), supplemented with 10 v/v % fetal calf serum, to give a cell density 
of about 1.times.10.sup.5 cells/ml. The tumor cell was cultured at 
37.degree. C. in a closed system for one week while periodically 
refreshing the culture medium. When the proliferated cell formed a 
monolayer, the culture was washed in saline containing a phosphate along 
with trypsin. The proliferated cell was suspended in a fresh preparation 
of the same culture medium to give a cell density of about 
1.times.10.sup.5 cells/ml, and cultured in suspension at 37.degree. C. and 
pH 7.2 for an additional one week. The cell was then ultrasonically 
disintegrated, and the hEGF in the supernatant was assayed. The hEGF 
production was about 1.3 .mu.g/ml cell suspension. After purifying and 
concentrating the supernatant in conventional manner, the resultant hEGF 
concentrate was admixed with an aqueous solution containing 200 parts of 
elsinan based on the weight of hEGF solid, distributed into 5 ml-glass 
vials to give an hEGF content of 0.5 .mu.g/vial, and freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent, injection, granule, tablet, suppository, etc. 
The activity of hEGF was assayed by conventional radioreceptor assay as 
described in R.L. Ladda et al., Analytical Chemistry, Vol. 93, pp. 286-294 
(1979). 
EXAMPLE 6 
EPO 
A human kidney tumor cell was extracted from kidney tumor patient, minced, 
disaggregated, and suspended in a flask along with Namalwa (a human 
lymphoblastoid line), with a salt solution, containing 140 mM NaCl, 54 mM 
KCl, 1 mM NaH.sub.2 PO.sub.4 and 2 mM CaCl.sub.2, to give respective cell 
density of about 10.sup.3 cells/ml. The cell suspension was added under 
ice-chilled conditions with a fresh preparation of the same salt solution 
but additionally containing Sendai virus preinactivated with 
uv-irradiation. After a lapse of five minutes, the cell suspension was 
placed in a 37.degree. C. incubator for about thirty minutes to effect 
cell fusion. Thus, the hEPO producibility was introduced into the Namalwa 
cell. Adult nude mice were intraperitoneally implanted with the Namalwa 
cell, and fed for five weeks in usual way. The tumor masses, formed 
subcutaneously in the animals, about 15 g each, were extracted, and 
disaggregated in saline containing trypsin. The cell so obtained was 
washed with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % fetal 
calf serum, to give a cell density of about 10.sup.6 cells/ml, and 
incubated at 37.degree. C. for twenty hours under a reduced pressure of 
700 mmHg to induce hEPO production. The cell was ultrasonically 
disintegrated, and the hEPO in the supernatant was assayed. The hEPO 
production was about 170 U/ml cell suspension After purifying and 
concentrating the supernatant in conventional manner, the resultant hEPO 
concentrate was admixed with an aqueous solution containing 500 parts of 
pullulan based on the weight of hEPO solid, distributed into 5 ml-glass 
vials to give an hEPO content of 10 U/vial, and freeze-dried. 
The product is stable over a long period of time even at room temperature, 
and readily dissolvable in water. Thus, the product is advantageously 
usable for test reagent or injection. 
The activity of hEPO was assayed by conventional bioassay using 
incorporation of .sup.59 Fe as reported in P.M. Cotes and D.R. Bangham, 
Nature, No. 4793, pp. 1065-1067 (1961). One Unit (U) of hEPO activity is 
equal to one-tenth of the hEPO activity in one vial distributed from the 
WHO. 
EXAMPLE 7 
TSH 
A human basophilic adenoma cell was extracted from basophilic adenoma 
patient, minced, disaggregated, and suspended along with Namalwa cell in a 
salt solution, containing 140 mM NaCl, 54 mM KCl, 1 mM NaH.sub.2 PO.sub.4 
and 2 mM CaCl.sub.2, to give respective cell density of about 10.sup.4 
cells/ml. The cell suspension was added under ice-chilled conditions with 
a fresh preparation of the same salt solution but additionally containing 
Sendai virus preinactivated with uv-irradiation. After a lapse of five 
minutes, the cell suspension was placed in a 37.degree. C. incubator for 
about thirty minutes to effect cell fusion. Thus, the hTSH producibility 
was introduced into the Namalwa cell. Adult nude mice were 
intraperitoneally implanted with the Namalwa cell, and fed for five weeks 
in usual way. The tumor masses, formed in the animals, about 15 g each, 
were extracted, minced, and disaggregated in saline containing trypsin. 
The cell so obtained was washed with Earle's 199 medium (pH 7.2) 
supplemented with 10 v/v fetal calf serum, suspended in a fresh 
preparation of the same culture medium, additionally containing 30 mM 
L-arginine as TSH inducer, to give a cell density of about 10.sup.5 
cells/ml, and incubated at 37.degree. C. for thirty-five hours to induce 
hTSH production. The cell was ultrasonically disintegrated, and the hTSH 
in the supernatant was assayed. The hTSH production was about 180 mIU/ml 
cell suspension. After purifying and concentrating the supernatant in 
conventional manner, the resultant hTSH concentrate was admixed with an 
aqueous solution containing 100 parts of elsinan based on the weight of 
hTSH solid, distributed into 5 ml-glass vials to give an hTSH content of 
10 mIU/vial, and freeze-dried. 
The product is stable over a long period of storage even at room 
temperature. Thus, the product is advantageously usable for test reagent 
or injection. 
The activity of hTSH was assayed by conventional radioreceptor assay as 
reported in S.W. Manley et al., Journal of Endrocrinology, Vol. 61, pp. 
419-436 (1974), and represented by International Unit (IU) with reference 
to the standard hTSH specimen distributed from the National Institute for 
Medical Research, England. 
While the preferred forms of the present invention have been described, it 
is to be understood that modifications will be apparent to those skilled 
in the art without departing from the spirit of the invention. The scope 
of the invention, therefore, is to be determined solely by the following 
claims.