U.S. patent application number 11/287341 was filed with the patent office on 2006-04-13 for purified lh.
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 | 20060079460 11/287341 |
Document ID | / |
Family ID | 8167926 |
Filed Date | 2006-04-13 |
United States Patent
Application |
20060079460 |
Kind Code |
A1 |
Paradisi; Gianfranco ; et
al. |
April 13, 2006 |
Purified LH
Abstract
Recombinant human luteinizing hormone (LH) having a specific
bioactivity of from 20,522 to 31,229 IU/mg and obtainable from a
process for the purification of recombinant LH from a sample of
crude recombinant LH in the supernatant of CHO cells which
comprises the combined use of ion-exchange chromatography and
reverse phase HPLC. The ion-exchange chromatography and the reverse
phase HPLC are performed twice and the final use of a gel
permeation column allows the purification from any residual traces
of contaminants. The recombinant LH can be formulated into a
pharmaceutical composition and used for treating a fertility
disorder.
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
AN
|
Family ID: |
8167926 |
Appl. No.: |
11/287341 |
Filed: |
November 28, 2005 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10204554 |
Dec 11, 2002 |
|
|
|
PCT/EP01/00666 |
Jan 22, 2001 |
|
|
|
11287341 |
Nov 28, 2005 |
|
|
|
Current U.S.
Class: |
514/10.1 ;
514/53; 514/9.9; 530/313 |
Current CPC
Class: |
A61K 9/19 20130101; A61K
9/0019 20130101; A61P 5/24 20180101; A61P 5/34 20180101; A61K 47/26
20130101; A61P 5/06 20180101; C07K 14/59 20130101; A61P 15/08
20180101; A61K 38/00 20130101 |
Class at
Publication: |
514/015 ;
530/313; 514/053 |
International
Class: |
A61K 38/09 20060101
A61K038/09; A61K 31/7012 20060101 A61K031/7012 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 22, 2000 |
EP |
00103692.0 |
Claims
1. The recombinant LH of claim 10 having a specific bioactivity of
from 20,522 to 31,229 IU/mg and obtainable by a process which
comprises: subjecting said sample to ion-exchange chromatography
with an anion exchange resin; subjecting said sample to reverse
phase HPLC; and isolating the recombinant LH.
2. The recombinant LH of claim 10, having a specific bioactivity of
about 25,000 IU/mg.
3. A pharmaceutical composition, comprising a therapeutically
effective amount of the recombinant LH of claim 2 and suitable
excipients thereof.
4. The pharmaceutical composition of claim 3, wherein the excipient
is sucrose.
5. A pharmaceutical composition of claim 3, for subcutaneous
administration.
6. A pharmaceutical composition, comprising a therapeutically
effective amount of the recombinant LH of claim 10 and suitable
excipients thereof.
7. The pharmaceutical composition of claim 6, wherein the excipient
is sucrose.
8. A pharmaceutical composition of claim 6, for subcutaneous
administration.
9. A method for treating a fertility disorder, comprising
administering to a patient in need thereof an effective amount of
the recombinant LH of claim 1.
10. A recombinant LH having a specific bioactivity of from 20,522
to 31,229 IU/mg.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a division of 10/204,554, which
is a 371 national stage application of PCT EP01/00666, filed Jan.
22, 2001. The entire content of both applications are incorporated
herein by reference.
FIELD OF INVENTION
[0002] The present invention relates to a process for the
purification of Luteinising Hormone (LH), in particular the
purification of recombinant LH (r-LH) from a sample of crude
recombinant LH, comprising the combined use of ion-exchange
chromatography and reverse phase HPLC. Luteinising Hormone (LH) is
a gonadotropin secreted by the anterior lobe of the pituitary
together with another gonadotropin, follicle-stimulating hormone
(FSH). These hormones are heterodimers consisting of non-covalently
bound .alpha. and .beta. subunits.
[0003] These gonadotropins stimulate the normal functioning of the
gonads and the secretion of sex hormones in both men and women. In
women, follicle-stimulating hormone stimulates the development and
maturation of the follicles and ova. As the follicle develops it
produces oestrogen in increasing amounts which at mid-cycle
stimulates the release of LH. This causes rupture of the follicle
with ovulation and converts the follicle into the corpus luteum
which secretes progesterone. In men, luteinising hormone stimulates
the interstitial cells of the testis to secrete testosterone, which
in turn has a direct effect of the seminiferous tubules.
Gonadotrophic substances with luteinising or follicle-stimulating
activity or both are used in the treatment of fertility disorders,
chiefly in females but also in males. Such substances include
chorionic gonadotropin which possesses LH activity and human
menopausal gonodotropins which possess both LH and FSH activity. A
recombinant DNA-derived human luteinising hormone (rechLH) is being
investigated as an alternative to chorionic gonadotropin or for
administration in conjunction with FSH.
[0004] Various methods have been used to isolate and purify LH,
such as ion-exchange, gel-filtration and immunoaffinity
chromatography (Jack, G. W., Blazek, R., James, K.,Boyd, J. E.
& Micklem, L. R. The automated production by immunoaffinity
chromatography of the human pituitary glycoprotein hormones
thyrotropin, follitropin and lutropin. Journal of Chemical
Technology and Biotechnology 39, 45-58, 1987).
[0005] Ion-exchange chromatrography has been used for the isolation
of these hormones, however, this method appears to have several
interrelated problems caused by the considerable charge
heterogeneity of LH in pituitary tissue. First, because these
glycoproteins and FSH have overlapping charges, their complete
separation is difficult and laborious. Secondly, the purification
of these hormones as single fractions may be difficult (Stockell
Hartree, A., Thomas, M., Furnival, B. E., Burns, T. W. &
Langley, P. Thyroid-stimulating and lipolytic activities of
purified preparation s of human thyroid-stimulating hormone.
Journal of Endocrinology 53, 95-100, 1972). As a result, certain
charged forms of the hormone may be selected during purification as
suggested in the case of LH (Storring, P. L. Zaidi, A. A., Mistry,
Y. G. Lindberg, M., Stenning, B. E. & Diczfalusy, E. A
comparison of preparations of highly purified human pituitary
luteinising hormone: differences in the luteinising hormone
potencies as determined by in vivo bioassays, in vitro bioassay and
immunoassay. Acta Endocrinologica 101, 339-347, 1982). Selective
purification will further complicate the characterization of these
heterogeneous forms, including the structural analysis of their
carbohydrate components. Variation in the content of anionic
oligosaccharides that contain sialyl and sulphate groups may be the
major cause of charge heterogeneity in LH.
[0006] Conventional fractionation methods have been described for
the preparation of human urinary luteinising hormone (LH) with a
potency of 982 i.u./mg by biological assay and 1166 i.u. by
radioimmunoassay (Donini S. & Donini P. Acta endocr., Copenh.
63, Suppl. 142, 257-277, 1969). An immunoabsorbent of rabbit
antiserum to purified human chorionic gonadotropin (HCG) was used
to purify LH from the main and side fractions obtained during the
preparation of follicle-stimulating hormone (FSH) from menopausal
urine (van Hell, H., Schuurs A. H. W. M. & den Hollander, F. C.
In Symposium on gonadotrophins, New York, 17 Jun. 1971. Eds B. B.
Saxena, C. G. Beling & H. M. Gandy. New York: John Wiley &
Son, Inc, 1972). The preparation obtained had higher LH potencies,
but also higher FSH:LH ratios than those prepared by Donini &
Donini (1969).
[0007] Recombinant LH has the advantage of being devoid of other
gonadotropin hormones, such as FSH and TSH. The crude preparation
of recombinant LH 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 Luteinising
hormone is highly desirable.
SUMMARY OF THE INVENTION
[0008] We have now found that a crude preparation of LH, derived
from a sample of the culture medium obtained after the recombinant
process or from a crude concentrate of post-menopausal urine can be
purified to such a degree that the resulting LH is practically free
from proteins and/or other contaminants contained in the crude LH
preparation. Depending on the starting material, the protein and
other contaminants are from human origin (starting material: human
menopausal gonadotropins) or from host cell origin, e.g. CHO in
case of a CHO host cell.
[0009] The purification process is based on the use of ion-exchange
chromatography and reverse phase HPLC. The optional further use of
a gel permeation column allows the removal of any residual traces
of contaminants from the pure LH preparation. Optimum results are
obtained when two steps of ion-exchange chromatography and two
steps of reverse phase HPLC are performed.
[0010] The process of the invention can be used for the
purification of recombinant LH, starting from a sample of a culture
medium obtained after the recombinant process, such that the
resulting highly purified LH is practically free, for example, from
FBS proteins often contained in the culture medium, nucleic acids
or other contaminants present in the host cells used for the
recombinant process
[0011] The process of the invention can be used as well for the
purification of urinary LH, starting from a crude concentrate of
post-menopausal urine, and for the purification of LH from other
species, particularly mammalian, including, for example, bovine,
equine, porcine, ovine, rat, mouse and monkey.
[0012] It is, therefore, an object of the present invention to
provide a process for purification of LH from a sample comprising
the combined use of ion-exchange chromatography and reverse phase
HPLC. The process comprises the steps of subjecting the sample (if
necessary concentrated) to ion-exchange chromatography and
subjecting the eluate to reverse phase HPLC. A further step of
applying the eluate to a gel permeation column may additionally be
carried out.
[0013] Depending on the prutiy of the starting preparation, the
ion-exchange chromatography and the reverse phase HPLC are
preferably performed twice in order to obtain optimum results from
the purification process. Such a process may comprise the steps of:
[0014] (a) eluting the sample through a DEAE Sepharose ion-exchange
chromatography column; [0015] (b) eluting through a Q-Sepharose
ion-exchange chromatography column; [0016] (c) eluting through a
Silica C18 reverse phase HPLC column; [0017] (d) again eluting
through a Silica C18 reverse phase HPLC column [optionally with a
different eluent from step (c)]; and [0018] (e) eluting through a
gel permeation column.
[0019] In a preferred embodiment of the invention, elution through
the DEAE Sepharose ion-exchange chromatography is carried out in
sodium phosphate buffer at pH 8. Elution through the Q-Sepharose
ion-exchange chromatography is preferably carried out in ammonium
acetate buffer at pH 7.5.
[0020] The reverse phase HPLC step (c) is preferably carried out
with 2-propanol/ammonium acetate as mobile phase.
[0021] The reverse phase HPLC step (d) is preferably carried out
with 2-propanol/Tris-HCl as mobile phase.
[0022] The LH of the present invention is preferably human LH and
most preferably is recombinant human LH, deriving from the culture
medium of mammalian cells (preferably CHO cells) used in the
recombinant process.
[0023] It is a further object of the present invention to provide a
pharmaceutical composition comprising a therapeutically effective
amount of the recombinant LH as prepared by the recombinant process
as described above, together with suitable excipients, such as
sucrose, necessary for the stabilisation of the lyophilised
product. The pharmaceutical composition of the recombinant LH is
particularity suitable for subcutaneous administration.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention provides a method for the purification of LH,
in particular for the purification of recombinant LH from a crude
preparation in the culture medium of the recombinant process. r-hLH
is obtained with a high degree of purity and high specific
activity, 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.
[0025] The invention is intended for use with biological materials,
particularly crude mixtures containing LH and other contaminating
proteins referred to herein as starting material samples. The
examples described in detail below use starting material samples
containing r-hLH, obtained from culture supernatant medium from a
bioreactor. Alternatively, the sample is human Menopausal
Gonadotropin (hMG), a crude concentrate of post-menopausal urine.
The sample is constituted by freshly collecting cell culture
supernatant medium perfused through a bioreactor. It is preferably
clarified by filtration. The crude solution can then be
concentrated, if necessary, and subjected to ultrafiltration to
remove material having molecular weights lower than 10.
Ultrafiltration also permits the buffer to be changed to sodium
phosphate, pH 8.
[0026] After the preliminary steps, the sample is then subjected to
ion-exchange chromatography and to reverse phase HPLC, which are
preferably each performed twice. The first ion-exchange step is
preferably carried out with DEAE Sepharose. This is essentially an
LH "flow-through" step in which a large part of the non-LH proteins
are eliminated. The second ion-exchange step is preferably carried
out with a Q-Sepharose column. This is also an LH "flow through"
step and is designed to remove potential DNA and host cell or
medium protein contaminants. In a preferred embodiment this step is
performed at about 5.degree. C. eluting with ammonium acetate
buffer at pH 7,5.
[0027] Reverse phase chromatography on Silica C18 is also
preferably performed twice and is effective in removing trace
amounts of FBS, cell protein and endotoxin contaminants. The first
HPLC step is preferably carried out with 2-propanol/ammonium
acetate as mobile phase. The second reverse phase HPLC step is
preferably performed using 2-propanol/Tris-HCl as mobile phase. The
retentate solution is then concentrated and can be recovered with
ammonium hydrogen carbonate, pH 8. The concentrated product is
preferably subjected to Gel permeation chromatography on Sephacryl
S100 HR. In this step, a separation based on molecular size is
achieved eluting with ammonium hydrogen carbonate pH 8 and the
eluate then undergoes preferably a filtration to remove viral
contaminants, then an ultrafiltration on membranes with 10 KD
cut-off in sodium phosphate buffer, pH 8. After filtration, the
purified LH bulk is preferably stored in sterile bottles at low
temperature.
EXAMPLE 1
[0028] Reagents [0029] Acetic acid (glacial), analytical grade (Ph.
Eur.) [0030] Ammonium acetate, analytical grade [0031] Ammonium
hydrogen carbonate, analytical grade (B.P.) [0032] Dibasic sodium
phosphate, analytical grade [0033] Hydrochloric acid, analytical
grade (Ph. Eur.) [0034] Phosphoric acid, analytical grade (Ph.
Eur.) [0035] 2-propanol, analytical grade (Ph. Eur.) [0036] Sodium
chloride, analytical grade (Ph. Eur.) [0037] Monobasic sodium
phosphate, analytical grade [0038] Sodium hydroxide pellets,
analytical grade (Ph. Eur.) [0039] Trifluoroacetic acid (TFA), HPLC
grade [0040] Tris-(hydroxymethyl) aminomethane, analytical grade
[0041] Water for Injection (WFI) (Ph. Eur.)
[0042] Purification Process Summary Flow Diagram
[0043] Table 1 is a flow diagram summarising the r-hLH purification
process, outlining the principles of operation of each of the
intermediate steps. TABLE-US-00001 TABLE 1 ##STR1## ##STR2##
[0044] Clarification, Concentration, Dialysis and Filtration of
Harvests (Step I)
[0045] In this step (Step I) the buffer is changed to be of
controlled composition and a preliminary concentration is achieved.
This step, is carried out at about +5.degree. C. and is repeated
individually for each harvest during the production cycle of the
bioreactor. A preferred range of temperature is 5.+-.3.degree.
C.
[0046] (i) Clarification of Harvests [0047] Upon receipt of freshly
collected culture medium from a bioreactor the material is
preferably processed starting with clarification of the supernatant
solution by filtration.
[0048] (ii) Concentration/dialysis of Harvests [0049] The
membranes, stored in 0.05 M sodium hydroxide between batches, are
rinsed with WFI until the pH descends to approximately 8. [0050]
The equilibration buffer, 0.025 M sodium phosphate pH 8, replaces
the water. Once conditioned the crude r-hLH solution from the
bioreactor is concentrated and dialysed to remove material having
molecular weights lower than 10 kD (membrane cut-off 10 kD). [0051]
The resulting concentrate is stored at about -15.degree. C.
[0052] Ion Exchange Chromatography on DEAE Sepharose CL-6B (Step
II)
[0053] The chromatography step is an r-hLH "flow-through" step in
which a large part of the non r-hLH proteins are eliminated and the
solution is further concentrated and dialysed. The chromatography
stages where product passes through the column, is carried out in a
cold room.
[0054] (i) Ion Exchange Chromatography on DEAE Sepharose CL-6B
[0055] The column is packed with a weakly charged anion-exchange
resin, diethyl amino ethane (DEAE) Sepharose, equilibrated in the
first instance with 0.15 M sodium phosphate pH 8. A preferred pH
range is 8.+-.0.3. [0056] The column is then conditioned with the
running buffer, 0.025 M sodium phosphate pH 8. A preferred pH range
is 8.+-.0.3. [0057] The r-hLH solution is loaded onto the column
through a filter apparatus, which is located on the column as a
guard. [0058] The column is fed with 0.025 M sodium phosphate pH 8.
A preferred pH range is 8.+-.0.3. The chromatographic process is
monitored by spectrophotometry at 280 nm. [0059] The leading
effluent is discarded until the baseline passes the 5% absorbance
mark. [0060] The unbound fraction containing the r-hLH is collected
until the baseline has descended to 10%.
[0061] (ii) Ultrafiltration [0062] An ultrafiltration apparatus,
equipped with a 100 kD cut-off membrane, stored in NaOH 0.05 M is
rinsed with WFI until the pH of the permeate is approximately 8.
[0063] The water is replaced by the equilibration buffer 0.08 M
ammonium acetate pH 7.5. A preferred pH range is 7.5+0.3. [0064]
The r-hLH solution obtained from the ion exchange chromatography
step is ultrafiltered through the 100 kD membrane and the permeate
fraction is collected. [0065] The ultrafilter is washed with
aliquots of 0.08 M ammonium acetate pH 7.5 and all of the washing
fractions are collected into the permeate solution.
[0066] (iii) Concentration/Dialysis [0067] An ultrafiltration
device equipped with a 10 kD cut-off membrane, stored in 0.05 M
NaOH, is rinsed with WFI until the pH of the permeate fraction is
approximately 8. The water is replaced by the equilibration buffer,
0.08 M ammonium acetate pH7.5. [0068] The r-hLH solution is
concentrated. Ammonium acetate 0.08 M pH 7.5 is added to the
retentate and the solution concentrated. The dialysis is continued
until the pH and conductivity of the retentate are the same as
those of the incoming buffer. The resulting retentate is
recovered.
[0069] Ion Exchange Chromatography on Q Sepharose Fast Flow (Step
III)
[0070] This step, is also an r-hLH "flow-through" step and is
designed to remove potential DNA and host cell or medium protein
contaminants.
[0071] (i) Column Equilibration [0072] Conditioning of the column
is performed with running buffer, 0.08 M ammonium acetate buffer,
pH 7.5. A preferred pH range is 7.5.+-.0.3.
[0073] (ii) r-hLH Purification Step on Q-Sepharose FF [0074] The
r-hLH solution is loaded through a filter apparatus which is
located on the Q Sepharose column as a guard. [0075] The column is
further washed with 0.08 M ammonium acetate pH 7.5. [0076] The
leading effluent is discarded until the baseline passes the 5%
absorbance mark. [0077] The unbound fraction containing the r-hLH
is collected until the baseline has descended to 10%. [0078] The
r-hLH solution from Step III can be stored frozen for subsequent
use. If stored at a temperature of -15.degree. C. or below, the
r-hLH intermediate is thawed at +5.+-.3.degree. C., typically over
a period of 24.+-.8 hours before undertaking the reverse phase HPLC
(Step IV).
[0079] First Preparative Reverse Phase HPLC (Step IV)
[0080] This step, performed at room temperature, is effective in
removing trace amounts of FBS/CHO protein and endotoxin
contaminants.
[0081] (i) Column Packing and Resin Activation [0082] The column is
packed with C18 wide-pore silica and, if new, the C18 resin is
conditioned with 2-propanol.
[0083] (ii) Column Equilibration [0084] The column is equilibrated
with 12.4% .sup.w/.sub.w 2-propanol in 0.05 M ammonium acetate
buffer, pH 7. A preferred pH range is 7.+-.0.2.
[0085] (iii) pH and Volume Adjustments of r-hLH Solution From Step
III [0086] The r-hLH solution is adjusted to pH 7 with concentrated
acetic acid. A preferred pH range is 7.+-.0.2. [0087] The volume of
the r-hLH solution is then adjusted by the addition of 2-propanol
in order to obtain a final concentration of 2-propanol equal to
12.4% .sup.w/.sub.w.
[0088] (iv) Filtration of Adjusted r-hLH Solution [0089] The
filtration apparatus equipped with a 0.22 .mu.m filter is washed
with 12.4% .sup.w/.sub.w 2-propanol in 0.05 M ammonium acetate
buffer, pH 7. A preferred pH range is 7.+-.0.2. [0090] The adjusted
r-hLH solution is filtered. [0091] The recipient is rinsed with
aliquots of 12.4% .sup.w/.sub.w 2-propanol in 0.05 M ammonium
acetate buffer, pH 7, filtered and the rinses pooled with the r-hLH
solution. A preferred pH range is 7.+-.0.2.
[0092] (v) r-hLH Purification Step on the First C 18 RP-HPLC Column
[0093] The r-hLH solution is loaded onto the column and the
chromatography is monitored by UV spectrophotometry at 280 nm.
[0094] The column is fed with 12.4% .sup.w/.sub.w 2-propanol in
0.05 M ammonium acetate buffer, pH 7 until the A.sub.280 returns to
baseline whereupon the unbound fraction is discarded. [0095]
Elution of the r-hLH is subsequently performed with a
2-propanol/ammonium acetate 0.05 M mobile phase across a linear
gradient from 14.7% to 20.7% .sup.w/.sub.w 2-propanol. [0096] The
r-hLH is fractioned when the A.sub.280 starts to increase. All
fractions of the r-hLH peak whose heights are greater than 20% of
full scale are pooled.
[0097] Second Preparative Reverse Phase HPLC Column (Step V)
[0098] This step, performed at room temperature, is effective in
removing trace amounts of FBS/CHO protein and endotoxin
contaminants.
[0099] (i) Column Packing and Resin Activation [0100] The column is
packed with C18 wide-pore silica and, if new, the C18 resin is
conditioned with 2-propanol.
[0101] (ii) Column Equilibration [0102] The column is equilibrated
in 14.7% .sup.w/.sub.w 2-propanol in 0.5 M Tris-HCl buffer, pH 7. A
preferred pH range is 7.+-.0.2.
[0103] (iii) Volume and pH Adjustments of r-hLH Solution from Step
IV [0104] 2M Tris-HCl buffer, pH 7 is added to the r-hLH sample in
order to bring the 2-propanol concentration down to approximately
the same as that in the column equilibration buffer (14.7%
.sup.w/.sub.w). [0105] The r-hLH solution is adjusted to pH 7 with
HCl 12 M. A preferred pH range is 7.+-.0.2.
[0106] (iv) r-hLH Purification Step on the Second C18 RP-HPLC
Column [0107] The r-hLH solution is loaded onto the column and the
chromatography is monitored by UV spectrophotometry at 280 nm.
[0108] The column is fed with 14.7% .sup.w/.sub.w 2-propanol in 0.5
M Tris-HCl buffer, pH 7. A preferred pH range is 7.+-.0.2. The
unbound fraction is discarded. [0109] Elution of the r-hLH is
subsequently performed with a 2-propanol/0.5 M Tris-HCl mobile
phase across a linear gradient from 14.7% to 20.7% .sup.w/.sub.w
2-propanol. [0110] The r-hLH is fractioned when the A.sub.280
starts to increase. All fractions of the r-hLH peak whose heights
are greater than 20% of full scale are pooled.
[0111] (v) Dialysis [0112] The r-hLH solution from the second C18
RP-HPLC step is diluted with WFI. Preferably 8 volumes of WFI are
used. [0113] The diluted r-hLH solution is dialysed by
ultrafiltration on a 10 kD membrane (see page 7, step VI) against
WFI. Aliquots of 0.5 M ammonium hydrogen carbonate pH 8 are
subsequently added and the dialysis continued until the
characteristics of the ammonium hydrogen carbonate buffer are met.
[0114] The retentate solution is concentrated to a final volume of
approximately 1 L and recovered. The ultrafilter is washed with 0.5
M ammonium hydrogen carbonate pH 8 and the ultrafiltration washes
are pooled with the retentate and optionally further concentrated.
This further concentration is dependent on the size of the column
used in the next step, i.e. Step VI.
[0115] Gel Permeation Chromatography on Sephacryl S100 HR and
Ultrafiltration (Step VI)
[0116] In this step, a separation based on molecular size is
achieved and the solution undergoes ultrafiltration. All operations
performed in this step are carried out at about +5.degree. C. A
preferred temperature range is +5.degree. C..+-.3.
[0117] (i) Gel Permeation Chromatography on Sephacryl S100 HR
[0118] The column is packed with Sephacryl S100 HR and is
equilibrated in the first instance with WFI. [0119] The column is
then equilibrated with 0.5 M ammonium hydrogen carbonate pH 8.
[0120] The column is fed with 0.5 M ammonium hydrogen carbonate pH
8. The chromatographic process is monitored by spectrophotometry at
280 nm. [0121] The r-hLH is fractioned when the A.sub.280 starts to
increase. All fractions of the r-hLH peak whose heights are greater
than 20% of full scale are pooled. [0122] The r-hLH solution,
eluted from the Sephacryl S100 HR column, is then preferably passed
through a filter, e.g. Virosolve.TM., to remove viral
contaminants.
[0123] (ii) Dialysis and Concentration of r-hLH [0124] The
membranes (ultrafiltration membranes 10 kD), stored in 0.05 M
sodium hydroxide between purification runs, are rinsed with WFI
until the pH descends to approximately 8. [0125] The diluted r-hLH
solution is dialysed (by ultrafiltration membranes 10 kD) against
WFI. Aliquots of 0.01 M sodium phosphate buffer, pH 8 are
subsequently added and the dialysis continued until the
characteristics of the sodium phosphate buffer are met. [0126] If
necessary the retentate solution is concentrated to a final volume
of approximately 500 ml and recovered. The ultrafilter is washed
with 0.01 M sodium phosphate buffer, pH 8 and the ultrafilter
washes are pooled with the retentate. [0127] A further optional LH
concentration step can be performed depending on the condition
selected for storage. [0128] The r-hLH solution is filtered and the
filtrate collected into a sterile vessel. [0129] The purified r-hLH
bulk is preferably stored in sterile bottles at about -15.degree.
C.
[0130] Reagents, Buffers, Eluants and Chemicals
[0131] Chromatographic Resins
[0132] The following chromatographic resins are currently employed
in the purification process. Equivalent resins can be employed as
well in the purification process. TABLE-US-00002 Step II: DEAE
Sepharose CL-6B (Pharmacia) Step III: Q-Sepharose Fast Flow
(Pharmacia) Step IV: C18 Silica RP-HPLC (Waters) Step V: C18 Silica
RP-HPLC (Waters) Step VI: Sephacryl S100 HR (Pharmacia)
[0133] The suppliers are: TABLE-US-00003 Amersham Pharmacia
Biotech, Waters Corporation Bjorkgatan 30 34 Maple Street S-751 84,
Uppsala Milford, MA 01757 Sweden USA
RESULTS
[0134] Biological Activity
[0135] Biological Activity of different batches of r-LH after
purification with the method of the present invention is reported
in Table 2. The protein concentration (mg of LH protein/ml) has
been determined by spectrophotometry at 276.5 nm, using the
experimentally derived absorptivity based on amino acid sequence
analysis a=0.812
[0136] The average specific activity of the r-LH preparation is
particularly high, amounting to about 25.000 IU/mg (of protein of
LH). TABLE-US-00004 TABLE 2 Specific activity of r-hLH bulk batches
Spec act N.sup.o lot [IU/mg] BLCA 9802 28173 BLCA 9803 25819 BLCA
9804 27472 BLCA 9805 31229 BLCA 9806 26995 BLCA 9808 26279 BLCA
9809 20522 BLCA 9810 22275 BLCA 9811 27642 BLCA 9812 29941 BLCA
9813 28345 BLCA 9814 27581 BLCA 9815 24541
[0137] Formulations
[0138] Freeze dried formulations have been developed with highly
purified recombinant LH of the present invention.
[0139] As a typical example, a freeze dried formulation at 75 IU
strength was prepared in vials DIN 2R using sucrose as excipient
(Table 3), which resulted stable at 4.degree. C. for several
months. TABLE-US-00005 TABLE 3 Name of ingredients Unit formula
Active ingredient Recombinant human LH 3.4 mcg (75 IU) Other
ingredient Sucrose 47.75 mg Tween 20 0.05 mg Disodium phosphate
dihydrate 0.825 mg Monosodium phosphate monohydrate 0.052 mg
* * * * *