U.S. patent application number 11/846118 was filed with the patent office on 2007-12-27 for purified hcg.
This patent application is currently assigned to APPLIED RESEARCH SYSTEMS ARS HOLDING N.V.. Invention is credited to Gianfranco PARADISI, Mara Rossi, Laura Scaglia.
Application Number | 20070299001 11/846118 |
Document ID | / |
Family ID | 8167925 |
Filed Date | 2007-12-27 |
United States Patent
Application |
20070299001 |
Kind Code |
A1 |
PARADISI; Gianfranco ; et
al. |
December 27, 2007 |
PURIFIED hCG
Abstract
A process for the purification of recombinant human Chorionic
Gonadotropin (hCG) from a sample of crude recombinant hCG in the
supernatant of CHO cells comprises the combined use of ion-exchange
chromatography and reverse phase HPLC. The ion-exchange
chromatography is performed twice and the final use of a size
exclusion chromatography allows the purification from any residual
traces of contaminants. The specific bioactivity of the highly
purified hCG obtained form the process is particularly high,
amounting to about 25,000 IU/mg.
Inventors: |
PARADISI; Gianfranco;
(Monterotondo, IT) ; Rossi; Mara; (Rome, IT)
; Scaglia; Laura; (Rome, IT) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
APPLIED RESEARCH SYSTEMS ARS
HOLDING N.V.
Curacao
NL
|
Family ID: |
8167925 |
Appl. No.: |
11/846118 |
Filed: |
August 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10204630 |
Dec 9, 2002 |
|
|
|
PCT/EP01/00665 |
Jan 22, 2001 |
|
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11846118 |
Aug 28, 2007 |
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Current U.S.
Class: |
530/399 ;
514/9.7; 514/9.8 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 15/08 20180101; A61P 5/00 20180101; C07K 14/59 20130101 |
Class at
Publication: |
514/002 ;
530/399 |
International
Class: |
A61K 38/24 20060101
A61K038/24; A61P 15/00 20060101 A61P015/00; C07K 14/59 20060101
C07K014/59 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2000 |
EP |
00103690.4 |
Claims
1. A recombinant human chorionic gonadotropin (hCG) having a
specific bioactivity in the range of 23,000 to 28,000 IU/mg.
2. A pharmaceutical composition comprising the recombinant hCG of
claim 1, and suitable excipients.
3. The pharmaceutical composition according to claim 2 for
subcutaneous administration.
4. A pharmaceutical composition according to claim 2, wherein the
excipient is sucrose.
5. The pharmaceutical composition according to claim 4 for
subcutaneous administration.
6. A pharmaceutical composition according to claim 4, which is a
freeze-dried composition.
7. The pharmaceutical composition according to claim 6 for
subcutaneous administration.
8. A pharmaceutical composition according to claim 2, wherein the
excipient is mannitol.
9. The pharmaceutical composition according to claim 8 for
subcutaneous administration.
10. A pharmaceutical composition according to claim 8, which is a
liquid composition.
11. The pharmaceutical composition according to claim 10 for
subcutaneous administration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of application Ser.
No. 10/204,630, filed Dec. 9, 2002, which is a 371 national stage
of PCT/EP01/00665 filed Jan. 22, 2001, which claims priority to EP
00103690.4 filed Feb. 22, 2000. The entire contents of these
applications are incorporated herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to a process for the
purification of Chorionic Gonadotropin, in particular the
purification of recombinant human Chorionic Gonadotropin (hCG) from
a sample of crude recombinant hCG. The method comprises the use of
ion-exchange chromatography and reverse phase HPLC.
[0003] Chorionic gonadotropin is a hormone produced by the placenta
and traditionally obtained from the urine of pregnant women.
[0004] The hormone is a heterodimer consisting of non-covalently
bound .alpha. and .beta. subunits.
[0005] Its effects are predominantly those of the gonadotropin
luteinising hormone.
[0006] Chorionic gonadotropin is given to women to induce ovulation
after follicular development has been stimulated with
follicle-stimulating hormone or human menopausal gonadotropins in
the treatment of anovulatory infertility due to absent or low
concentrations of gonadotropins. A single dose of 5000 to 10000
units is given by intramuscular injection to mimic the midcycle
peak of luteinising hormone which normally stimulates ovulation.
Chorionic gonadotropin is also given in conjunction with
menotrophin and sometimes also clomiphene citrate as an adjunct to
in vitro fertilization procedures and other assisted conception
techniques involving superovulation and oocyte collection. In males
it has been used in the treatment of prepubertal cryptorchidism.
Regimens vary widely, but doses usually range from 500 to 4000
units three times weekly by intramuscular injection.
[0007] It is also given for male infertility associated with
hypogonadotrophic hypogonadism. Again, there is considerable
variation in the dosage regimen, and doses have varied from 500 to
4000 units two to three times weekly. An agent with
follicle-stimulating activity such as menotrophin is often added to
enable normal spermatogenesis. For oligospermia, doses of up to
3000 units of chorionic gonadotropin weekly with menotrophin or
another follicle-stimulating preparation may be employed. In the
treatment of delayed puberty associated with hypogonadism in males,
a dose of 500 to 1500 units is given twice weekly; the dose should
be titrated against plasma-testosterone concentration.
[0008] Different methods have been used to isolate and purify hCG
from raw urine samples (Birken et al., Endocrinology, 133(3):
1390-7, 1993; Sakakibara et al., Chem. Pharm. Bull., 35(5): 1414-6,
1990; Donini et al., Acta Endocrinol., 73(1): 133-45, 1973).
Recently, a different method of affinity chromatography, termed
membrane filtration affinity chromatography, has been developed and
applied to purify hCG from urine (Xu et al., Protein expression and
purification, 16: 221-3, 1999). The method avoids the use of BrCN
activated Sepharose as a solid-phase for the affinity
chromatography column and represents a variation of the usual
methods of purification of hCG by affinity chromatography from
urine samples. Immunoactivity of the purified hCG according to this
method is 8554 IU/mg.
[0009] Recombinant hCG has the advantage of being devoid of other
gonadotropin hormones and contaminants of human origin and more
specifically of those present in human urine. The crude preparation
of recombinant hCG contains, however, all other proteins and
contaminants of the cell used in its recombinant production and a
method for achieving an absolute purity of recombinant Chorionic
Gonadotropin is highly desirable.
SUMMARY OF THE INVENTION
[0010] We have now found that a crude preparation of hCG, deriving
from a concentrated sample of a culture medium obtained after the
recombinant process or from a crude concentrate of urine of
pregnant women, can be purified such that the resulting hCG is
practically free from proteins or other contaminants contained in
the crude hCG preparation.
[0011] The purification process is based on the use of ion-exchange
chromatography and reverse phase HPLC. The possible further use of
a size exclusion column allows the removal of any residual traces
of contaminants. Optimum results are obtained when at least two
steps of ion-exchange chromatography are performed.
[0012] The process of the invention can be used for the
purification of recombinant hCG from a crude preparation of the
culture medium derived from the recombinant process. The r-hCG is
obtained with a high degree of purity and high specific bioactivity
(in the range of 23,000-28,000 IU/mg), practically free from Foetal
Bovine Serum (FBS) proteins, if present in the culture medium, and
from nucleic acids or other contaminants contained in the host
cells used in the recombinant process.
[0013] The process of the invention can be used as well for the
purification of urinary hCG, starting from a crude concentrate of
urine of pregnant women, and for the purification of CG from other
mammalian species including, for example, bovine, equine, porcine,
ovine and monkey.
[0014] It is an object of the present invention to provide a
process for purification of hCG from a sample comprising the use of
ion-exchange chromatography and reverse phase HPLC.
[0015] The process comprises the steps of subjecting the sample to
ion-exchange chromatography and subjecting the eluate to reverse
phase HPLC. A further step of applying the eluate to a size
exclusion column may additionally be carried out.
[0016] The two ion-exchange chromatography steps are preferably
performed under different conditions in order to obtain optimum
results from the purification process. A preferred embodiment of
the process of the invention comprises the steps of:
[0017] (a) eluting the sample through a silica chromatography
column;
[0018] (b) eluting through a DEAE SEPHAROSE (cross-linked agarose
matrix with diethylaminoethyl weak anion exchanger) ion-exchange
chromatography column;
[0019] (c) eluting through a CM-SEPHAROSE (cross-linked agarose
matrix with carboxymethyl weak anion exchanger) ion-exchange
chromatography column;
[0020] (d) eluting through a Silica C18 reverse phase HPLC column;
and
[0021] (e) eluting through a SEPHACRYL (spherical allyl dextran and
N,N'-methylenebisacrylamide) size exclusion chromatography
column.
[0022] In a preferred embodiment of the invention, elution through
the DEAE SEPHAROSE ion-exchange column is carried out in sodium
phosphate buffer at about pH 7.5. Elution through the CM-SEPHAROSE
(cross-linked agarose matrix with carboxymethyl weak anion
exchanger) ion-exchange column is preferably carried out in sodium
phosphate buffer at about pH 6. The reverse phase HPLC step (d) is
preferably carried out with 2-propanol/Tris-phosphate buffer as
mobile phase.
[0023] The CG of the present invention is preferably human CG and
most preferably is recombinant hCG, deriving from the culture
medium of CHO cells used in the recombinant process.
[0024] It is a further object of the present invention to provide a
pharmaceutical composition comprising a therapeutically effective
amount of purified recombinant hCG as prepared by the recombinant
process as described above, together with suitable excipients. An
example of a suitable excipient is sucrose, which aids in the
stabilization of the lyophilized product. The pharmaceutical
composition of recombinant hCG is particularity suitable for
subcutaneous administration.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention provides a method for the purification of hCG,
in particular for the purification of recombinant hCG from a crude
preparation of the culture medium of the recombinant process. The
r-hCG is obtained with a high degree of purity and high specific
bioactivity ( in the range of 23,000-28,000 IU/mg), practically
free from Foetal Bovine Serum (FBS) proteins which are present in
the culture medium and from nucleic acids or other contaminants
contained in the host cells used in the recombinant process.
[0026] The invention is intended for use with biological materials,
particularly crude mixtures containing hCG and other contaminating
proteins referred to herein as starting material samples. The
examples described in detail below use starting material samples
containing r-hCG obtained from cell culture supernatant medium from
a bioreactor.
[0027] Alternatively, the sample is crude concentrated urine from
pregnant women.
[0028] The sample is constituted by freshly collecting cell culture
supernatant medium perfused through a bioreactor over two days.
Preferably the supernatant is clarified by filtration.
[0029] If necessary, the crude solution is concentrated and
subjected to C4 silica chromatography to remove contaminants
derived from the cell culture.
[0030] The semi-purified harvest, after ultrafiltration, is then
subject to ion-exchange chromatography, which is preferably
performed twice, and preferably under different conditions, and to
reverse phase HPLC. A first DEAE SEPHAROSE ion-exchange step may be
performed, essentially acting as an hCG "flow through" step, in
which a large part of the non-hCG proteins and DNA are eliminated.
A second ion-exchange step, preferably through a CM-SEPHAROSE
column, acts as an hCG binding step, and removes residual DNA and
host cell or medium protein contaminants. In a preferred embodiment
this step is performed at about 5.degree. C. eluting with sodium
phosphate buffer at about pH 6.
[0031] Reverse phase chromatography on a Silica C18 column is
effective in removing trace amounts of nucleic acids and cell
culture derived contaminants. The column is preferably eluted with
2-propanol/Tris-phoshate buffer as mobile phase. The retentate
solution is preferably then subjected to 10 kD cut-off
ultrafiltration, concentrated and can be recovered with ammonium
hydrogen carbonate pH 8. The concentrated product can then be
applied to a size exclusion chromatography column on SEPHACRYL S200
HR. In this step, a separation based on molecular size is achieved
eluting with ammonium hydrogen carbonate pH 8 to remove still
possible trace amounts of cell culture derived contaminants,
potential aggregates and free hCG sub-units. The eluate can then
undergo a dialysis by ultrafiltration on membranes with 10 kD
cut-off, preferably in sodium phosphate buffer, pH 7. After
filtration, the purified hCG bulk is preferably stored in sterile
bottles at low temperature.
EXAMPLE 1
[0032] Reagents:
[0033] Ammonia, analytical grade
[0034] Ammonium hydrogen carbonate, analytical grade (B.P.)
[0035] Di-sodium hydrogen phosphate, analytical grade
[0036] Absolute denatured Ethanol,
[0037] Phosphoric acid, analytical grade (Ph.Eur.)
[0038] 2-propanol, analytical grade (Ph.Helv.)
[0039] Sodium chloride, analytical grade (Ph.Eur.)
[0040] Sodium di-hydrogen phosphate, analytical grade
[0041] Sodium hydroxide pellets, analytical grade (Ph.Eur.)
[0042] Trifluoroacetic acid (TFA), HPLC grade
[0043] Tris-(hydroxymethyl) aminomethane, analytical grade
[0044] Purification Process Summary Flow Diagram
[0045] Harvest material derived from the cell culture process is
purified and concentrated by a series of five chromatographic
steps.
[0046] The following flow diagram (Table 1) summarizes a preferred
embodiment of the r-hCG purification process, outlining the
chromatographic column resins and the principles of operation of
each of the intermediate steps. TABLE-US-00001 TABLE 1 Flow diagram
summarising the r-hCG purification process. Step I CULTURE MEDIUM
FROM BIOREACTOR | C4 silica chromatography (Eluate contains r-hCG)
| Ultrafiltration 10 kD cut-off (Retentate contains r-hCG) |
CONCENTRATED CRUDE r-hCG HARVEST | Step II DEAE SEPHAROSE FF
(Unbound fraction contains r-hCG) | Step III CM SEPHAROSE FF
(Eluate contains r-hCG) | Step IV RP-HPLC ON SILICA C18 (Eluate
contains r-hCG) | Ultrafiltration (10 kD) Step V SEPHACRYL S-200 HR
(Eluate contains r-hCG) | r-hCG BULK SOLUTION
[0047] A detailed flow diagram and process description are provided
below. The conditions given for the capture step (step I) are those
which are normally applied when the crude material is of
recombinant origin.
[0048] Step I (Capture Step)
[0049] In this step (Step I), a preliminary concentration is
achieved and the buffer is changed to be of controlled composition.
This step is initiated at room temperature (Silica C4
chromatography) and then continued at about +5.degree. C. A
preferred temperature range is 5.+-.3. It is repeated individually
for each harvest during the production cycle of the bioreactor.
[0050] (i) Clarification of Harvests [0051] The freshly collected
culture medium from the bioreactor is usually first clarified by
filtration.
[0052] (ii) Silica C4 Chromatography [0053] After clarification,
the harvests are loaded onto a C4 silica chromatography column,
which has been previously equilibrated in sodium phosphate 25 mM,
pH 7. A preferred pH range is from 6.6 to 7.7. The column is washed
with sodium phosphate 25 mM until the UV monitor signal returns to
baseline. The product is then eluted with 34.2% (w/w) 2-propanol in
sodium phosphate 25 mM.
[0054] (iii) Ammonia Treatment [0055] Ammonia is then added to the
solution to reach a final concentration of 1 M. This mixture is
incubated for 6. hours. Then the solution is 2 fold diluted with
water, and the pH is adjusted to 7.5 using phosphoric acid 85%. A
preferred pH range is 7.5.+-.0.2.
[0056] (iv) Concentration and Dialysis [0057] The 10 kD cut-off
membranes stored in 0.05 M sodium hydroxide between batches are
rinsed with purified water until the pH descends to approximately
8. [0058] The product is concentrated and dialysed (by
ultrafiltration on the 10 kD membrane) to remove material having
molecular weight lower than 10 kD and to eliminate traces of
2-propanol and to change the ammonia solution to sodium phosphate
40 mM pH 7.5. A preferred pH range is 7.5.+-.0.2 [0059] The final
retentate is recovered from the membranes with sodium phosphate 40
mM in order to achieve a target protein concentration of 3 to 15
mg/ml. [0060] The solution is then filtered and the resulting
concentrate is stored frozen at about -15.degree. C.
[0061] Step II (Filtration and Ion Exchange on DEAE SEPHAROSE FF
Chromatography)
[0062] This chromatography step is an r-hCG "flow-through" step in
which a large part of the non r-hCG proteins and nucleic acids are
eliminated. Whilst the filtration is carried out at room
temperature, the chromatography stage where product passes through
the column, is carried out in a cold room.
[0063] (i) Thawing and Pooling of the r-hCG Concentrated Crude
Harvests [0064] The frozen concentrates are thawed and pooled. A
batch of purified bulk r-hCG is processed from a pool of a variable
number of r-hCG crude concentrates produced from the same working
cell bank. The criteria for the number of r-hCG crude concentrates
pooled is based on the maximum protein binding capacity of the next
chromatographic step in the purification process (4 mg total
protein/mg of resin).
[0065] (ii) Clarification by Filtration [0066] The r-hCG solution
is preferably passed through a filter apparatus and the filters
washed with 40 mM sodium phosphate pH 7.5. [0067] The filtered
solution and washes are pooled.
[0068] (iii) Ion Exchange Chromatography on DEAE SEPHAROSE FF
[0069] The column, packed with a weakly charged anion-exchange
resin, diethyl amino ethane (DEAE) SEPHAROSE Fast Flow, is
equilibrated with 40 mM sodium phosphate (pH 7.5). [0070] The r-hCG
solution is loaded onto the column. [0071] The column is fed with
40 mM sodium phosphate pH 7.5. The chromatographic process is
monitored by spectrophotometry at 280 nm. [0072] The leading
effluent is discarded until the peak starts eluting. The unbound
fraction containing the r-hCG is then collected.
[0073] Step III (CM SEPHAROSE FF Chromatography)
[0074] In this chromatographic step, a large part of the host cells
contaminants are removed. The chromatographic step is carried out
about +5.degree. C. A preferred temperature range is 5.+-.3.
[0075] (i) Dilution of the DEAE SEPHAROSE FF Eluate [0076] Water
for injection is added to the DEAE SEPHAROSE FF eluate and the pH
adjusted to 6 using phosphoric acid 85%. A preferred pH range is
6.+-.0.1.
[0077] (ii) Ion Exchange Chromatography on CM SEPHAROSE FF [0078]
The column, packed with a weakly charged cation-exchange resin,
Carboxymethyl (CM) SEPHAROSE Fast Flow, is equilibrated with 20 mM
sodium phosphate buffer (pH 6). A preferred pH range is 6.+-.0.1.
[0079] The r-hCG solution is loaded onto the column. [0080] The
column is washed with 20 mM sodium phosphate buffer pH 6. The
chromatographic process is monitored by spectrophotometry at 280
nm. [0081] The product is eluted using 130 mM sodium phosphate
buffer pH 6. The leading effluent is discarded until the peak
starts eluting. [0082] The entire peak containing the r-hCG is
collected. The product can be optionally filtered at this stage to
remove viral contaminants.
[0083] Step IV (RP-HPLC on Silica C18)
[0084] This RP-HPLC chromatographic step is effective in removing
trace amounts of cell culture contaminants, nucleic acid residues
and endotoxins. It is followed by a 10 kD cut-off ultrafiltration
and optional filtration.
[0085] (i) Preparation of the Aliquots [0086] The pH of the
aliquots is adjusted to 5 and 2-propanol is added to a final
concentration of 15% (v/v).
[0087] (ii) RP-HPLC Chromatography on Silica C18. [0088] The
column, packed with a Silica C18 resin is first equilibrated in 15%
(v/v) 2-propanol in Tris-phosphate 0.5 M buffer. [0089] The first
aliquot is loaded onto the column and the chromatography is
monitored by UV spectrophotometry. [0090] The column is washed with
the same equilibration buffer. [0091] Elution of the r-hCG is
subsequently performed with a linear gradient of
2-propanol/Tris-phosphate 0.5 M buffer mobile phase from 15% to 25%
(v/v). [0092] The r-hCG is fractionated when the corresponding peak
is detected by spectrophotometry (A.sub.280). The fractions whose
absorbance is greater than 65% of the maximum peak height at the
ascending part and higher than 20% of the maximum peak height at
the descending part are pooled. [0093] The four r-hCG containing
pools are then pooled and diluted in an equivalent volume of Water
For Injection (WFI). [0094] The product is concentrated and
dialysed ( by ultrafiltration on a 10 kD membrane) against WFI to
remove material having molecular weight lower than 10 kD and to
eliminate 2-propanol. [0095] The product is then dialysed by
ultrafiltration against ammonium hydrogen carbonate buffer 0.1 M,
pH 8. [0096] The resulting intermediate is stored at about
+5.degree. C. or frozen if required. Preferred storage temperatures
are 5.+-.3.degree. C. and equal or below -15.degree. C.
respectively.
[0097] Step V (Size Exclusion Chromatography on SEPHACRYL S-200
HR)
[0098] This size exclusion chromatographic step is effective in
removing trace amounts of cell culture derived contaminants,
potential aggregates and/or free sub-units. It is followed by a 10
kD cut-off ultrafiltration. The Sephacryl S-200 HR and the 10 kD
cut-off ultrafiltration steps are carried at about +5.degree. C. A
preferred temperature range is 5.+-.3.degree. C.
[0099] (i) Size Exclusion on Sephacryl S-200 HR. [0100] The column
packed with Sephacryl S-200 HR resin is equilibrated with Ammonium
hydrogen carbonate 0.5M (pH 8). A preferred pH range is 8.+-.0.2.
[0101] The r-hCG solution is loaded onto the column, and the
elution initiated using Ammonium hydrogen carbonate 0.5 M, pH 8. A
preferred pH range is 8.+-.0.2. [0102] The collection of the r-hCG
fraction is initiated from the beginning of the peak and lasts
until the 50% mark of the maximum peak height at the descending
part of the peak is reached.
[0103] (ii) 10 kD Cut-Off Ultrafiltration. [0104] 10 kD cut-off
membranes stored in 0.05 M sodium hydroxide between batches are
rinsed with WFI until the pH descends to approximately 8. [0105]
The product is concentrated and dialysed (by ultrafiltration)
against WFI. [0106] The product is then dialysed (by
ultrafiltration) against Sodium phosphate buffer 0.01 M, pH 7, and
the final protein concentration adjusted to reach a target final
concentration of 3.5 mg/ml. [0107] The resulting r-hCG final bulk
solution is preferably stored frozen at about -15.degree. C.
[0108] Chromatographic Resins
[0109] The following chromatographic resins may be employed in the
purification process: Equivalent resins can also be employed.
[0110] Step I: Silica C4, 250 {umlaut over (.LAMBDA.)}ngstrom--50
.mu.m (Matrex.RTM., Millipore)
[0111] Step II: DEAE SEPHAROSE FF (Pharmacia)
[0112] Step III: CM SEPHAROSE FF (Pharmacia)
[0113] Step IV: Silica C18, 300 angstrom--15-20 .mu.m (Vydac)
[0114] Step V: SEPHACRYL S-200 HR (Pharmacia)
[0115] The current suppliers are:
[0116] Amersham Pharmacia Biotech, Millipore Corporation
[0117] Bjorkgatan 30 17 Cherry Hill Drive
[0118] S-751 84, Uppsala Danvers, Mass. 01923
[0119] Sweden USA
[0120] Vydac, The Separations Group,
[0121] 17434 Mojave St.
[0122] Hesperia, Calif. 92345
[0123] USA
[0124] Results
[0125] Molecular Weight and Size [0126] SDS-PAGE [0127] The
relative molecular weight of r-hCG obtained following the
purification method of the present invention has been determined by
SDS-PAGE against standard proteins of known molecular weight.
[0128] Coomassie brilliant blue staining after non-reducing
SDS-PAGE revealed a single broad band for the r-hCG heterodimer at
approximately molecular weight 70 kD (range 65-75 kD). The identity
of the band was confirmed by Western blotting.
[0129] Biological Activity [0130] Biological Activity of different
batches of r-hCG after purification with the method of the present
invention is reported in Table 2. The protein concentration has
been determined by spectrophotometry at 276.5 nm, a=0.616.
[0131] The average specific activity of the r-hCG preparation is
particularly high, amounting to about 25.000 IU/mg. TABLE-US-00002
TABLE 2 Specific bioactivity r-hCG batch (IU/mg) BCEA 9901 24427
BCEA 9902 26868 BCEA 9903 25636 BCEA 9904 27152 BCEA 9905 23729
[0132] Formulations [0133] Both liquid and freeze dried
formulations have been developed with the highly purified
recombinant hCG of the present invention. [0134] Liquid Formulation
[0135] Two liquid formulations at 10000 IU/ml were prepared in
vials DIN 2R using mannitol or sucrose as excipient and submitted
to stability tests at 50, 40, 25 and 4.degree. C.
[0136] The composition is reported in tables 3 and 4.
TABLE-US-00003 TABLE 3 Ingredients UNIT r-hCG IU/ml 10000 SUCROSE
mg/ml 102.6 O. PHOSPHORIC ACID mg/ml 0.98 SODIUM q.s. to pH 7.0
HYDROXIDE Filling volume: 0.5 ml
[0137] TABLE-US-00004 TABLE 4 Ingredients UNIT r-hCG IU/ml 10000
MANNITOL mg/ml 54.6 O. PHOSPHORIC ACID mg/ml 0.98 SODIUM q.s. to pH
7.0 HYDROXIDE Filling volume: 0.5 ml
[0138] The results of the stability tests, carried out by Bioassay,
SE/HPLC and RP-HPLC, showed that the mannitol formulation was more
stable with respect to the sucrose formulation. Refrigerated
storage conditions were preferably required to minimize the protein
oxidation and free subunit formation.
[0139] Freeze Dried Formulation
[0140] A freeze dried formulation at 5000 IU strength was prepared
in vials DIN 2R for stability tests at 50, 40, 25 and 4.degree. C.
using sucrose as excipient.
[0141] The composition is reported in table 5. TABLE-US-00005 TABLE
5 Ingredients UNIT r-hCG IU 5000 SUCROSE mg 30 O-PHOSPHORIC ACID mg
0.98 SODIUM q.s. to pH 7.0 HYDROXIDE
[0142] The results of the stability tests, carried out by Bioassay,
SE/HPLC and RP-HPLC, showed that this freeze dried formulation was
stable at 40 and 50.degree. C. at least for 19 weeks.
[0143] The stability tests at 25 and 4.degree. C. were performed up
to 6 months indicating no degradation of the active substance.
* * * * *