FSH and LH separation and purification process

A new process, particularly simple and economical, for FSH and LH separation and purification starting from crude HMG preferably urinary, comprising the following steps: PA1 1) optional exhaustion of crude HMG viral charge in aqueous EtOH PA1 2) ion-exchange chromatography on weakly basic anionic resins of DEAE type; PA1 3) affinity chromatography on resin having an antraquinone derivative as a ligand; PA1 4) optional ion-exchange chromatography on strongly basic anionic resins; Hormones obtained thereby, in particularly pure form and having high specific activity, may subsequently undergo a depyrogenation step.

The present application is the national stage filing of and claims priority 
to International Application No. PCT/EP97/06058, filed Nov. 3, 1997 and 
Italian Application Serial No. MI96A002313. 
FIELD OF THE INVENTION 
The present invention refers to a new process, particularly simple and 
economical, for FSH and LH separation and purification starting from crude 
HMG, and particularly from urine extracts of menopausal or post-menopausal 
women. 
STATE OF THE ART 
Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) are usually 
known as gonadotropic hormones or human fertility hormones. Such 
compounds, whose primary physiological effect is directed to the promotion 
of gametogenesis and/or the production of steroids involved in such 
biological process, are abundantly available in nature from pituitary 
glands and human and animal plasma, as well as of the urine of menopausal 
or post-menopausal women. FSH and LH are drawn from such natural sources 
in the form of a mixture, commonly known as HMG (Human Menopausal 
Gonadotropin), which is available on the market also as a crude extract; 
this mixture consists of FSH and LH in a ratio of about 1:1 in association 
with other urinary proteins. Nevertheless, these extracts have low purity 
degrees, that are incompatible with their administration in men for 
therapeutic purposes, principally because of the contamination from 
foreign proteins. 
Several FSH and LH purification processes starting from products of natural 
origin (pituitary glands, plasma or urine), essentially based on 
centrifugation, precipitation, chromatography and filtration techniques, 
have been developed in the state of the art with the aim of reducing the 
above mentioned protein contamination. 
The U.S. Pat. No. 3,674,865 describes FSH purification from urinary 
extracts such process, comprising 21 different steps in total, still 
results into a mixture of FSH and LH in a ratio ranging between 3:1 and 
6:1. 
The U.S. Pat. No. 3,973,004 (Derwent abstract) describes FSH separation 
from pituitary glands by means of selective precipitation with ammonium 
sulphate, while in U.S. Pat. No. 4,115,375 (Claims U.S. patent abstract), 
polyethylene glycol is used as precipitant agent. 
Nevertheless, the above mentioned processes suffer the disadvantage of 
leading to the obtainment of products having unsatisfactory specific 
activity and purity. In particular, the presence of protein contamination 
induces allergic reactions, so that the above mentioned hormones may be 
taken only by means of particular routes of administration, such as 
intramuscular injection, involving considerable difficulties in 
application. 
In the recent past, some purification processes using immuno-affinity 
chromatography and specific monoclonal anti-bodies (European patent EP 0 
322 438; European patent application EPA 0 328 248, Derwent abstract) or 
recombinant DNA techniques (international patent application WO 85/01958) 
have been set up. Though they allow to get products with high specific 
activity, these processes suffer the severe problem of possible 
contaminations from viruses, heterologous proteins and DNA residuals from 
the host cell; that is why products resulting from such processes have to 
be carefully purified and tested to exclude the presence of potential 
contaminants prior to their therapeutic use. 
Therefore, there is an obvious need of a process which allows the 
obtainment of higher purity and higher specific activity products, without 
meeting the disadvantages mentioned above. 
SUMMARY OF THE INVENTION 
It is the object of the present invention to provide a FSH and LH 
separation and purification process, starting from crude HMG, allowing the 
obtainment of these hormones with high purity and specific activity. Said 
process comprises the following steps: optional exhaustion of the viral 
charge of said crude HMG in a 80-90% v/v EtOH water solution; 
loading of the product obtained in step (1) on a ion-exchange 
chromatography column with weakly basic anionic resin of DEAE type, 
selectively eluting LH and FSH with 5-15% v/v EtOH aqueous solutions in a 
10-50 mM phosphate buffer, containing 0-70 mM NaCl with increasing ionic 
strength, pH 7,0 to 8,0; 
loading of the eluate obtained in step (2), containing LH or FSH, on an 
affinity chromatography column having a ligand consisting of an 
anthraquinone derivative on an inert support, selectively eluting 
contaminating proteins and FSH or LH with alkaline pH solutions, having an 
increasing ionic strength from 0 to 3 M of KCl; 
optional loading of the FSH hormone obtained in step (3) on a ion-exchange 
chromatography column with strongly basic anionic resins, containing 
quaternary ammonium groups, selectively eluting contaminating proteins and 
FSH with 0-400 M NaCl solutions with increasing ionic strength, at 
alkaline pH. 
Further to that, FSH and LH hormones, obtained in the above mentioned steps 
(3) and (4), may be depyrogened and lyophilized or frozen.

DETAILED DESCRIPTION OF THE INVENTION 
The characteristics and advantages of the process according to the present 
invention will be better reported in the following detailed description. 
By means of a simple extraction and easy purification steps on ion-exchange 
and affinity resins, the process of the invention allows the obtainment of 
FSH and LH having high specific activity and high purity, thus allowing 
their administration to men for therapeutic purposes, with no need of 
further purification treatments. This process is particularly simple and 
economical, and it also has the advantage of using very limited quantities 
of organic solvents, since it is mainly carried out in water. 
Starting crude HMG is preferably of an urinary concentrate obtained from 
the urine of menopausal or post-menopausal women; according to a preferred 
form of realization of the invention, said starting HMG has a specific 
activity in FSH lower than 10 I.U./mg, and more preferably ranging from 2 
and 10 I.U./mg, and a specific activity in LH ranging from 0.5 and 5 
I.U./mg. 
In step (1) of the process of the invention, said crude HMG is suspended in 
a 80-90% v/v EtOH water solution, at a temperature preferably comprised 
between -20.degree. C. and 5.degree. C., for a period of 1 to 4 hours. 
This treatment is carried out for the purpose of getting the exhaustion of 
crude HMG viral charge, which is essential to any use in the 
pharmaceutical field after purification. 
In step (2) of the process of the invention, the solid residue, whose viral 
charge has been optionally exhausted in step (1) is solubilized in a 5-15% 
v/v EtOH water so solution in a 5-50 mM phosphate buffer, preferably 
containing 10-30 mM sodium phosphate, the pH ranging from 7.3 to 7.6, at a 
temperature ranging from 0.degree. C. to 8.degree. C. and preferably from 
0.degree. C. to 6.degree. C. 
The thus obtained solution is loaded on a ion-exchange chromatography 
column with weakly basic anionic resin of DEAE type, at a temperature 
preferably ranging from 0.degree. C. to 8.degree. C., preferably using a 
conditioning buffer consisting of a 5-15% v/v EtOH water solution 
containing a 5-50 mM phosphate buffer, preferably containing 10-30 mM 
sodium phosphate, at a pH of 7.3-7.6. Said anionic resin preferably 
contains tertiary aminic groups, optionally in the presence of quaternary 
ammonium groups, on cellulose matrix, and more preferably it is 
DEAE-cellulose (for example DE52 Whatman). 
The preferred flow rate ranges from 10 to 30 ml/cm.sup.2.h, with a column 
diameter/length ratio ranging from 0.3 and 0.6, and with a total protein 
load ranging from 20 to 50 mg/ml of resin. 
LH hormon adsorbed on resin is preferably eluted with a 5-15% v/v EtOH 
water solution in a 10-50 mM phosphate buffer, preferably containing 10-30 
mM sodium phosphate, at a pH of 7.3 and 7.6. Said elution, that results 
into the recovery of LH only, is preferably carried out at the temperature 
between of 0-8.degree. C. 
FSH hormone adsorbed on resin is subsequently eluted with a 5-15% v/v EtOH 
water solution in a 10-50 mM phosphate buffer, preferably containing 10-30 
mM sodium phosphate, at a pH of 7.3-7.6, containing NaCl in concentration 
ranging from 30 to 50 mM. Said elution is preferably carried out the 
temperature of 0-8.degree. C. The presence of EtOH in eluting buffer 
solutions plays a determining role in this step, because it enables a 
better LH and FSH separation, as well as higher yelds in the recovery of 
the two hormones. 
In step (3) of the process of the invention, the eluates containing LH and 
FSH hormones, obtained from step (2), separately undergo a further 
purification procedure, by means of affinity chromatography containing an 
anthraquinone derivative as ligand, preferably CIBACRON.RTM. Blue, 
covalently linked to an inert support, preferably agarose, it is 
preferably used an affinity chromatography column consisting of agarose 
blue. 
Said eluates are preferably acidified up to a pH of 5.5 and 7.5 and loaded 
onto the to above mentioned column, previously conditioned in a 10-30 mM 
acetate buffer, preferably containing sodium acetate, at a pH of 5.5 to 
7.5, at a temperature ranging from 0.degree. C. to 8.degree. C. 
The preferred flow rate ranges from 7 to 14 ml/cm.sup.2.h, with a column 
diameter/length ratio ranging from 0.5 to 0.9, with a total protein load 
ranging from 5 to 15 mg/ml of resin. 
The FSH or LH hormone, adsorbed on the column, is then selectively eluted 
from contaminating proteins with one or more 40-60 mM glycine-NaOH 
buffers, containing KCl in concentrations ranging from 0 to 3 M, 
optionally of increasing growing ionic strength, changing the pH from 8.5 
to 11. 
The thus obtained hormones may then be concentrated and desalified for a 
subsequent freezing or lyophilization, with procedures known in the state 
of the art. 
According to a preferred form of realization of the process of the 
invention, desalified LH hormone is lyophilized and may subsequently 
undergo depyrogenation, with procedures known in the state of the art. 
According to step (4), the FSH hormone obtained in step (3), once 
desalified, undergoes a further purification stage carried out by means of 
ion-exchange chromatography on strongly basic anionic resin; such resin 
contains quaternary ammonium groups, optionally in association with 
tertiary amminic groups, on cellulose matrix, and it is preferably DE53 
Whatman. 
FSH is preferably balanced by dialysis in a 10-50 mM Tris-HCl buffer, pH 
9.0-9.5, and loaded on said column, conditioned with the same buffer. 
The preferred flow rate ranges from 20 to 40 ml/cm.sup.2.h, with a column 
diameter/lenght ratio ranging from 0.08 to 0.3, with a total protein load 
ranging from 0.5 to 10 mg/ml of resin. 
Initially the column is eluted with a 10-50 mM Tris-HCl buffer, pH 9.0-9.5, 
containing NaCl in a concentration preferably ranging from 40 to 80 mM, 
thus obtaining the elution of contaminating proteins. 
The FSH hormone bound to the resin is subsequently eluted using a linear 
NaCl concentration gradient, preferably ranging from 70-100 mM to 140-400 
mM, in a 10-50 mM Tris-HCl buffer, pH 9.0-9.5. 
The thus obtained, purified FSH hormone is then desalified by 
ultrafiltration, lyophilized and depyrogened with procedures known in the 
state of the art. According to a preferred embodiment of the invention, 
the process of depyrogenation of FSH and LH, obtained in steps (3) or (4), 
is carried out as follows: the purified hormone, in the form of 
Iyodhiilzed powder, is dissolved, at a temperature comprised between 
-15.degree. C. and 0.degree. C., in a 30-50% v/v EtOH water solution, 
containing 5-15% w/v ammonium acetate. 
To this solution are then added 3-8 mM tribasic sodium phosphate and 3-8 mM 
calcium acetate; the pH is then rised up to values ranging from 8 to 10 by 
means of sodium hydroxide. To the obtained supernatant, after 
centrifugation, 1,5-2,5 volumes of absolute EtOH for each supernatant 
volume are added at a temperature ranging between -20.degree. C. and 
0.degree. C. After having carried out a further centrifugation, the 
obtained precipitate is dissolved again in water and dialyzed, according 
to methods known in the state of the art. 
Finally, the solution containing purified LH or FSH may be frozen like it 
is or lyophilized according to methods known in the state of the art, thus 
keeping hormone activity unchanged for long-term storage. 
The process of the invention enables the achievement of very high purity 
FSH and LH, and respectively of FSH having a specific activity exceeding 
6,000 I.U./mg, free from LH, and of LH having a specific activity 
exceeding 500 I.U./mg. 
The following examples of the present invention are reported for 
illustrative but not limitative purposes. 
EXAMPLE 1 
FSH Purification from Crude Urinary HMG 
Exhaustion of the Viral Charge of Crude HMG in Aqueous EtOH 
All the operations mentioned hereinafter were carried out at a temperature 
ranging from 0 to 6.degree. C., unless differently specified. 
200 g of crude HMG, obtained from the urine of menopausal or 
post-menopausal women, were suspended in 10 l of an ethanol/water mixture 
(85:15 v/v), at the temperature of -10.degree. C.; the thus obtained 
suspension was maintained under stirring for 3 hours, at the temperature 
of -10.degree. C., and then centrifuged at 7,000 rpm for 20 minutes, using 
a GS3 Sorvall rotor; after removal of the supernatant, the precipitate was 
recovered. 
Ion-exchange Chromatoaraphy on DE52 
The precipitate obtained in step 1.1 was dissolved in 2 l of a 10% EtOH 
solution, containing 20 mM sodium phospate, with a pH of 7,3. The thus 
obtained solution was centrifuged at 7,000 rpm for 30 minutes, using a GS3 
Sorvall rotor, and the obtained supernatant was loaded on a DE52 Whatman 
ion-exchange column (1995 Catalogue Ref. 4057910), based on 
diethylaminoethylcellulose having a diameter of 14 cm and a height of 36 
cm, balanced in a 20 mM sodium phosphate buffer, at pH of 7.3, containing 
the 10% v/v of EtOH. In the course of chromatography, the column was 
eluted at a flow rate of 3 l/hour and the eluate was collected in 
fractions of about 700 ml. 
After the sample was loaded, the column was eluted with, in the order: 
10 l of 10% v/v EtOH, containing 20 mM sodium phosphate, pH=7.3; 
40 l of 10% v/v EtOH, containing 20 mM sodium phosphate and 40 mM sodium 
chloride, pH=7.3; 
After chromatography, the optical density at 280 nm (OD280) was determined 
for each eluate fraction, as well as LH and FSH activities. FIG. 1 shows 
the obtained chromatogram, where are reported LH and FSH specific 
activities (I.U./ml) and the optical density of the eluate fractions. 
Such chromatogram clearly shows that LH was eluted by buffer solution (a), 
while FSH was eluted by buffer solution (b), containing 40 mM NaCl. In 
particular, fractions from 6 to 20, containing LH, were collected and 
furtherly purified, as described in Example 2; fractions from 28 to 41, 
containing FSH, were collected and purified as described below. 
FSH Purification by Means of Affinity Chromatography on Agarose Blue 
The agarose blue 3 GA affinity resin (Sigma Chemical Co., St. Louis, Mo., 
1995 Catalogue Ref. C1410) was used; this resin, prior to be packed into 
the chromatography column, was washed with following reagents: 
10 l of a 2.5 M KCl solution in 0.5% Na.sub.2 CO.sub.3 for one hour; 
10 l of a 0.5% Na.sub.2 CO.sub.3 solution for 30 minutes; 
10 l of a 6 M urea solution for 2 hours; 
10 l of H.sub.2 O for 30 minutes; 
10 l of a 0.5% Na.sub.2 CO.sub.3 solution for 30 minutes; 
10 l of H.sub.2 O for 30 minutes; 
10 l of a 50 mM sodium acetate buffer, pH=6.5, for 30 minutes; 
10 l of a 20 mM sodium acetate buffer, pH=6.5, for 30 minutes. 
The resin, conditioned in a 20 mM sodium acetate buffer, pH=6.5, was packed 
in a chromatography column to obtain a resin bed with a diameter of 11 cm 
and a height of 16 cm. 
The pH of the sample containing FSH, obtained in step 1.2, was changed to 
6.5 by means of 1.7 M acetic acid and loaded into the column. 
Sample loading into the column and its further elutions were carried out at 
a flow rate of 1 l/h flow. The eluate recovered during the loading of the 
sample into the column was collected in one single fraction, while further 
fractions of about 700 ml were subsequently collected. 
After having loaded the sample, the column was first eluted with, in the 
order: 
1 l of a 20 mM sodium acetate buffer, pH=6.5; 
5 l of a 50 mM glycine-NaOH buffer, pH=10; 
3 l of a 50 mM glycine-NaOH, 0,2 M KCl buffer, pH=9; 
and then with a KCl linear gradient in a glycine-NaOH buffer, prepared as 
follows: 
chamber 1) 9 l of 0.3 M KCl in 50 mM glycine-NaOH buffer, pH=9; 
chamber 2) 9 l of 2.5 M KCl in 50 mM glycine-NaOH buffer, pH=9; 
After chromatography, the optical density at 280 nm (OD280) of every eluate 
fraction, as well as FSH specific activity (I.U./ml), were determined. 
FIG. 2 shows the obtained chromatogram. 
During sample loading (not reported in the graphic), FSH was retained in 
the column while the largest part of contaminating proteins was found in 
the eluate. Many of the contaminating proteins retained by the resin were 
eluted by the first washings and by the buffer containing 0.3 M KCl, while 
no FSH elution was observed under these conditions. 
The subsequent elution (starting from fraction 14) with KCl gradient 
resulted first into the elimination of other contaminating proteins (from 
fraction 15 to fraction 20 approximately); FSH elution occurred with a 
symmetrical peak at higher KCl concentrations (fractions from 22 to 29). 
After that, fractions from 23 to 28 were collected together; the pH of the 
obtained product was raised up to 7.5 with 1.7 M acetic acid; then the 
product itself way concentrated and desaiified by ultrafiltration, using 
an Amicon cell provided with an Amicon PM10 membrane. 
Ion-exchange Chromatography on DE53 
FSH sample, previously concentrated and desaiified as described in step 
1.3, was dialyzed for 18 hours against 5 l of a 25 mM Tris-HCl buffer, 
pH=9.3; after that, the sample was loaded on a DE53 Whatman ion-exchange 
chromatography column (1995 Catalogue ref. 4058910), having a diameter of 
2 cm and a height of 18 cm, balanced with a 25 mM Tris-HCl buffer, pH=9.3. 
The column was eluted with a flow rate of 100 ml/h and the eiuate was 
collected in fractions of about 18 ml. 
After sample loading, the column was first eluted, with 450 ml of a 25 mM 
Tris-HCl buffer, pH=9.3, containing 80 mM NaCl, and then with a NaCl 
linear gradient prepared as follows: 
chamber 1) 600 ml of 90 mM NaCl in 25 mM Tris-HCl buffer, pH=9.3; 
chamber 2) 600 ml of 160 mM NaCl in 25 mM Tris-HCl buffer, pH=9.3; 
FIG. 3 shows the obtained chromatogram, where are reported the optical 
density at 280 nm (OD280), as well as FSH specific activity (I.U./ml) for 
each eluate fraction. 
The chromatogram clearly shows that the washing with the first eluting 
solution, containing 80 mM NaCl, results into the elimination of some 
contaminating proteins, while FSH is still retained in the column. On the 
opposite, FSH is selectively eluted from contaminating proteins by means 
of the NaCl gradient, used from fraction 30 on. Fractions from 31 to 40 
have been collected together, concentrated, desalified by using an Amicon 
cell equipped with an Amicon PM10 membrane, and finally lyophilized. 
FSH Depyrogenation 
109 mg of lyophilized FSH, obtained in previous step, were dissolved in 55 
ml of a cold solution of 10% w/v ammonium acetate in 40% v/v ethanol. To 
the solution, maintained under magnetic stirring at a temperature of about 
-10.degree. C. by means of an ice and salt bath, the following ingredients 
were added: 
1.2 ml of a 7.6% tribasic sodium phosphate dodecahydrate water solution; 
1.4 ml of a 4.9% calcium acetate water solution. 
After having raised the pH up to about 8.5 by means of 20% w/v NaOH, the 
solution was maintained under stirring for 30 minutes, still at a 
temperature of about -10.degree. C., and then centrifuged at 8,000 rpm in 
a Sorvall SS34 rotor for 30 minutes. The precipitate was eliminated, while 
the collected supernatant (57 mm) was stirred and cooled down to about 
-10.degree. C. in an ice and salt bath. 
After the addition of 114 ml of 95% v/v EtOH, previously cooled down to 
-20.degree. C., the solution was maintained under stirring for about 30 
minutes and then left at rest for 12 hours, at the temperature of 
-20.degree. C. 
After a 30 minute centrifugation at 8,000 rpm, in a 0-4.degree. C. cooled 
centrifuge, the supernatant was eliminated and the-precipitate was 
dissolved again in apyrogen water (80 ml). The thus obtained solution was 
dialyzed for 18 hours against 10 l of apyrogen water and then lyophilized. 
FIG. 4 shows the absorption sprectrum of a solution containing 1.1 mg/ml of 
FSH, purified according to the process reported above. The activity of the 
thus purified FSH, corresponding to 6,870 I.U./mg of proteins, was 
determined with an immunoradiometric method, using the Serono FSH Maicione 
kit (Cat. Ref. 13101). The protein concentration in purified FSH was 
calculated considering that a water solution containing 1 mg/ml of FSH 
produces an optical density of 0.62 at 277 nm, in quartz cuvettes with 1 
cm optical path. Such coefficient was experimentally determined in the 
course of preparations, determining the absorption at 277 nm in solutions 
containing weighted quantities of previously lyophilized, salt-free 
hormone. Such coefficient proved to be more reliable than other protein 
determination methods in purified hormone samples with a specific activity 
in FSH ranging from 4,000 to 10,000 I.U./mg. The protein concentration of 
lower purity fractions (for example those obtained in step 1.2) were 
determined with BCA Protein Assay Pierce (Pierce catalogue ref. 23223 and 
23224), using bovine serum albumine as standard. 
Data referring to FSH purification, carried as described in steps 1.1-1.5, 
are shown in Table 1. 
__________________________________________________________________________ 
Total FSH specific 
Volume 
Proteins 
Total FSH 
Total LH 
activity 
FSH/LH 
Fraction (ml) 
(mg) (I.U.) 
(I.U.) 
(I.U./mg) 
ratio 
__________________________________________________________________________ 
Solution from 
2,200 
195,000 
1,384,000 
n.d. 7.1 n.d. 
Step 1.1 
FSH from DE52 
9,700 
18,516 
1,148,600 
27,000 
62 43 
Step 1.2 
FSH from Agarose Blue 
170 
229 961,300 
13,850 
4,198 69 
Step 1.3 
FSH from DE53 
92 
125 788,230 
2,513 
6,306 313 
Step 1.4 
Depyrogened FSH 
99 
109 748,780 
2,572 
6,870 291 
Step 1.5 
FSH from Step 1.5 
109 743,230 
n.d. 6,818 n.d. 
after 2 months (-20.degree. C.) 
FSH from Step 1.5 
109 741,640 
n.d. 6,804 n.d. 
after 4 months (-20.degree. C.) 
__________________________________________________________________________ 
Table 2 shows the results achieved in further FSH purification trials, 
carried out according to the methods described in Example 1. 
__________________________________________________________________________ 
Total FSH specific 
Volume 
Proteins 
Total FSH 
Total LH 
activity 
FSH/LH 
Fraction (ml) 
(mg) (I.U.) 
(I.U.) 
(I.U./mg) 
ratio 
__________________________________________________________________________ 
Solution from 
2,200 
205,000 
1.,50,000 
n.d. 6.1 n.d. 
Step 1.1 
FSH from DE52 
9,700 
19,800 
1,012,000 
21,360 
51 47 
Step 1.2 
FSH from Agarose Blue 
170 
232 805,000 
9,050 
3,470 90 
Step 1.3 
FSH from DE53 
90 
115 680,220 
2,305 
5,915 295 
Step 1.4 
Depyrogened FSH 
87 
97 631,620 
2,110 
6,511 299 
Step 1.5 
FSH from Step 1.5 
97 630,450 
n.d. 6,499 n.d. 
after 2 months (-20.degree. C.) 
FSH from Step 1.5 
97 629,880 
n.d. 6,494 n.d. 
after 4 months (-20.degree. C.) 
__________________________________________________________________________ 
EXAMPLE 2 
LH Purification from Crude Urinary HMG 
Steps 2.1 and 2.2, referring to the exhaustion of crude HMG viral charge in 
aqueous EtOH and to ion-exchange chromatography on DE52, are common for 
both FSH and LH, and were carried out as described in items 1.1 and 1.2 of 
Example 1. 
LH Purification by Affinity Chromatograohy on Agarose Blue 
The agarose blue 3 GA affinity resin was prepared as described in item 1.3. 
It was then balanced in 20 mM sodium acetate buffer, pH=6.5, and packed 
into a chromatography column to obtain a resin bed with a diameter of 11 
cm and a height of 16 cm. 
The pH of the sample containing LH, obtained as described in item 1.2, was 
changed to 6.5 by means of 1.7 M acetic acid and said sample was loaded 
into the column with a flow rate of 1 l/h. The eiuate obtained during the 
sample loading in the column was collected in one single fraction, while 
further fractions of about 600 ml were subsequently collected. 
After sample loading, the column was first eluted with, in the order: 
1 l of a 20 mM sodium acetate buffer, pH=6.5; 
5 l of a 50 mM glycine-NaOH buffer, pH=10; 
2 l of a 50 mM glycine-NaOH, 0.15 M KCl buffer, pH=10; 
and then with a KCl linear gradient in a glycine-NaOH buffer, prepared as 
follows: 
chamber 1) 8 l of 0.15 M KCl in a 50 mM glycine-NaOH buffer, pH=10; 
chamber 2) 8 l of 1.7 M KCl in a 50 mM glycine-NaOH buffer, pH=10. 
After chromatography, the optical density at 280 nm (OD280) was determined 
for each fraction, as well as LH specific activity (I.U./ml). 
FIG. 5 shows the resulting elution chromatogram, obtained as described 
above. During sample loading into the column, LH was retained into the 
column, while the largest part of contaminating proteins was found in the 
eluate (not reported in the graphic). Many of the contaminating proteins 
retained by the resin were eluted by means of washings carried out with 
the two buffers at pH 10, before the gradient elution. 
LH was eluted by the KCl gradient: low KCl concentrations resulted into the 
elution of further contaminating proteins, while LH elution occurred at 
higher ionic concentrations. 
Fractions from 17 to 25 were then collected together and the pH value was 
changed to 7.5 with 1.7 M acetic acid. Then the solution was concentrated, 
desalified by ultrafiltration on an Amicon PM10 membrane and finally 
lyophilized. 
LH Depyrogenation 
205 mg of lyophilized LH, obtained in the previous step, were dissolved in 
102 ml of a solution containing 40% v/v EtOH and 10% w/v ammonium acetate. 
To the solution, maintained under magnetic stirring at a temperature of 
about -10.degree. C. by means of an ice and salt bath, the following 
ingredients were added: 
2.25 ml of a 7.6% tribasic sodium phosphate dodecahydrate water solution; 
2.7 ml of a 4.9% calcium acetate water solution. 
After having raised the pH up to about 8.5 with 20% w/v NaOH, the solution 
was maintained under stirring for 30 minutes, at a temperature of about 
-10.degree. C., and then centrifuged at 8,000 rpm in a Sorvall SS34 rotor, 
for 30 minutes. The precipitate was eliminated, while the collected 
supernatant (107 ml) was stirred, maintaining the temperature at about 
-10.degree. C. in an ice and salt bath. 
After the addition of 214 ml of 95% v/v EtOH, previously cooled down to 
-20.degree. C., the solution was maintained under stirring for about 30 
minutes and then left at rest for 12 hours, at the temperature of 
-20.degree. C. 
After a 30 minute centrifugation at 8,000 rpm, in a 0-4.degree. C. cooled 
centrifuge, the supernatant was eliminated and the precipitate was 
dissolved again in apyrogen water (125 ml). The thus obtained solution was 
dialyzed for 18 hours against 10 l of apyrogen water and then lyophilized. 
FIG. 6 shows the U.V. spectrum of an aqueous solution containing 2.8 mg/ml 
of LH, purified according to the process reported above. Purified LH 
specific activity, corresponding to 521 I.U./mg of proteins, was 
determined with immunoradiometric method, using the Serono LH Maiclone kit 
(Cat. Ref. 13201). The protein concentration was determined with BCA 
Protein Assay Pierce (Pierce catalogue ref. 23223 and 23224), using bovine 
serum albumin as standard. 
Data corresponding to LH purification, carried out according to the above 
mentioned steps, are shown in Table 3. 
__________________________________________________________________________ 
Total LH specific 
Volume 
Proteins 
Total FSH 
Total LH 
activity 
FSH/LH 
Fraction (ml) 
(mg) (I.U.) 
(I.U.) 
(I.U./mg) 
ratio 
__________________________________________________________________________ 
Solution from 
2,200 
195,000 
1,384,000 
n.d. n.d. n.d. 
Step 1.1 
LH from DE52 
10,700 
8,970 30,400 
171,500 
19 0.18 
Step 1.2 
LH from Agarose Blue 
190 
307 21,280 
117,000 
381 0.18 
Step 2.3 
Depyrogened LH 
125 
205 19,600 
106,800 
521 0.18 
Step 2.4 
LH from Step 2.4 
205 n.d. 106,400 
519 n.d. 
after 2 months (-20.degree. C.) 
LH from Step 2.4 
205 n.d. 105,400 
514 n.d. 
after 4 months (-20.degree. C.) 
__________________________________________________________________________ 
Tabie 4 shows the results achieved in further LH purification trials, 
carried out according with methods described in Example 2. 
__________________________________________________________________________ 
Total LH specific 
Volume 
Proteins 
Total FSH 
Total LH 
activity 
FSH/LH 
Fraction (ml) 
(mg) (I.U.) 
(I.U.) 
(I.U./mg) 
ratio 
__________________________________________________________________________ 
Solution from 
2,200 
205,000 
1,250,000 
n.d. n.d. n.d. 
Step 1.1 
LH from DE52 
10,600 
11,785 
18,400 
165,000 
14 0.11 
Step 1.2 
LH from Agarose Blue 
110 
260 10,700 
105,600 
406 0.1 
Step 2.3 
Depyrogened LH 
120 
185 9,470 
97,800 
529 0.1 
Step 2.4 
LH from Step 2.4 
185 n.d. 96,800 
523 n.d. 
after 2 months (-20.degree. C.) 
LH from Step 2.4 
185 n.d. 96,450 
521 n.d. 
after 4 months (-20.degree. C.) 
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