GB2173803A - Isolating pituitary glycoprotein hormones - Google Patents
Isolating pituitary glycoprotein hormones Download PDFInfo
- Publication number
- GB2173803A GB2173803A GB08609669A GB8609669A GB2173803A GB 2173803 A GB2173803 A GB 2173803A GB 08609669 A GB08609669 A GB 08609669A GB 8609669 A GB8609669 A GB 8609669A GB 2173803 A GB2173803 A GB 2173803A
- Authority
- GB
- United Kingdom
- Prior art keywords
- hormone
- buffer
- thyrotrophic
- antibody
- isolating
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 239000005556 hormone Substances 0.000 title claims abstract description 99
- 229940088597 hormone Drugs 0.000 title claims abstract description 99
- 230000001817 pituitary effect Effects 0.000 title claims abstract description 21
- 102000003886 Glycoproteins Human genes 0.000 title claims abstract description 19
- 108090000288 Glycoproteins Proteins 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 27
- 230000003488 thyrotrophic effect Effects 0.000 claims abstract description 26
- 230000001592 luteinising effect Effects 0.000 claims abstract description 17
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- 230000002378 acidificating effect Effects 0.000 claims abstract description 3
- 239000012062 aqueous buffer Substances 0.000 claims abstract description 3
- 239000000872 buffer Substances 0.000 claims description 20
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims description 3
- 102000018997 Growth Hormone Human genes 0.000 claims 1
- 108010051696 Growth Hormone Proteins 0.000 claims 1
- 239000000122 growth hormone Substances 0.000 claims 1
- 238000002955 isolation Methods 0.000 abstract description 4
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 abstract description 3
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 abstract description 3
- 229940028334 follicle stimulating hormone Drugs 0.000 abstract description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 16
- 239000004471 Glycine Substances 0.000 description 8
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000007983 Tris buffer Substances 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 238000002523 gelfiltration Methods 0.000 description 6
- 102000002265 Human Growth Hormone Human genes 0.000 description 5
- 108010000521 Human Growth Hormone Proteins 0.000 description 5
- 239000000854 Human Growth Hormone Substances 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 5
- 239000012507 Sephadex™ Substances 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 238000004166 bioassay Methods 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 235000002639 sodium chloride Nutrition 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 206010062767 Hypophysitis Diseases 0.000 description 3
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- 239000008351 acetate buffer Substances 0.000 description 3
- 230000004071 biological effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 2
- 102000003864 Human Follicle Stimulating Hormone Human genes 0.000 description 2
- 108010082302 Human Follicle Stimulating Hormone Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000005755 formation reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 235000003625 Acrocomia mexicana Nutrition 0.000 description 1
- 244000202285 Acrocomia mexicana Species 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000006877 Pituitary Hormones Human genes 0.000 description 1
- 108010047386 Pituitary Hormones Proteins 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical class [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000007910 cell fusion Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 239000000960 hypophysis hormone Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 208000000509 infertility Diseases 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 231100000535 infertility Toxicity 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000008174 sterile solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Endocrinology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Reproductive Health (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
A method is provided for isolating pituitary glycoprotein hormones by contacting an aqueous solution containing the hormones with immobilised monoclonal antibody to form a complex and then eluting the hormone with an acidic aqueous buffer of pH of from 3 to 4.0. The method of the invention enables the isolation of e.g. thyrotrophic hormone, follicle stimulating hormone and luteinising hormone in biologically active form.
Description
SPECIFICATION
Process for isolating pituitary glycoprotein hormones
This invention relates to a process for isolating pituitary glycoprotein hormones.
The production of purified pituitary glycoprotein hormones is desirable since these hormones, particularly thyrotrophic hormone, follicle stimulating hormone and luteinising hormone and potentially useful therapeutic agents. Thus, for example a purified preparation of human thyrothrophic hormone has potential use as a chemotherapeutic agent in the treatment of human thyroid cancer while human follicle stimulating hormone has a proven record in the treatment of infertility. The hormones to date have been purified by tedious multi-stage processes in poor yield with incomplete separation from one another. In particular, luteinising hormone is difficult to separate from the other pituitary glycoprotein hormones and hitherto has often been present as a contaminant of purified preparations of thyrotrophic hormone and follicle stimulating hormone.
Pituitary glycoprotein hormones are relatively unstable and procedures for isolating other pituitary hormones, for example human growth hormone, have proved to be unsuitable for separating glycoprotein hormones in biologically active form. For example attempts to apply to pituitary glycoprotein hormones the procedure described in UK Patent Specification No. 2091739 for the isolation of human growth hormone fail to yield any biologically active material.
We have now devised a method of isolating a pituitary glycoprotein hormone in biologically active form which comprises contacting an aqueous solution of the hormone with a solid support bearing immobilised monoclonal antibody to the hormone so as to form a complex between the immobilised antibody and the hormone and eluting the hormone with an acidic aqueous buffer having a pH of from 3 to 4.0, an ionic strength of at least 0.2M and wherein the buffer is substantially free of protein denaturing agents.
The contacting of the solution of pituitary glycoprotein hormone with the immobilised antibody is preferably carried out by passing the solution through a fixed bed of the support medium although it may also be carried out by suspending the support matrix in a solution of the hormone.
In order to obtain satisfactory yields of pituitary glycoprotein hormone in biologically active form the pH and ionic strength of the eluent need to lie within narrowly defined limits and optimum results have been found to be obtained using a eluent having a pH which is less than 3.8 and greater than 3.2. Preferably the ionic strength of the eluent is at least 0.3M, most preferably 0.5 to 1.OM.
Although it has been found possible to elute biologically active hormone at a relatively high pH, e.g. at pH 10.5 using an aqueous TRIS buffer, the use of eluents having such high pH's is not feasible in practice since it leads to rapid inactivation of the antibody. On the other hand the use of an eluent having a pH below 4.0 to dissociate the antibody/hormone complex allows the immobilised antibody to be re-used repeatedly.
The identity of the buffer has not been found to be particularly critical and satisfactory results have been obtained using amino acids and peptides as buffering ingredients as well as salts of weak organic and inorganic acids, e.g. acetates and phosphates. Similarly the identity of the salt used to provide the required ionic strength is not critical, provided that protein denaturing agents are not used. Thus for example physiologically acceptable inorganic salts such as sodium and potassium chlorides may be used, but protein denaturing agents such as urea and guanidine hydrochloride should be avoided. Optimum results have been achieved using as eluent glycine buffer at pH 3.5.
After being isolated as described above, the purified hormones may be separated from low molecular weight solutes and antibody fragments by known techniques, for example by gelfiltration using an appropriate molecular seive, e.g. Sephadex G-100 and then lyophilised.
The monoclonal antibodies used in carrying out the process of the invention may be produced using known techniques, for example by growing appropriate cell fusion clones as either rat or mouse ascites tumours. Suitable clones are available commercially, for example a clone capable of producing monoclonal antibody to human thyrotrophic hormone is available as cell line
W73/A from The Wellcome Foundation and clones capable of producing monoclonal antibody to human follicle stimulating hormone and to human luteinising hormone are available as cell lines ES13 and 44/2.7 from the Department of Surgery at Edinburgh University.
Similarly the monoclonal antibodies may be immobilised by binding them to a suitable support using known techniques.
The source of the aqueous solution of pituitary glycoprotein hormone used as starting material in the method of the invention is not critical and the invention is not limited to the isolation of pituitary glycoprotein hormone from any particular sources. Thus impure pituitary glycoprotein hormone of either natural or synthetic origin may be used. Impure pituitary glycoprotein hormones of natural origin used as starting material may, for example, be obtained as side-fractions from the production of human growth hormone from human pituitary glands. Side fractions from the production of human growth hormone from both acetone-treated and frozen pituitaries have been found to be suitable. Preferably the side fractions are substantially freed from human growth hormone as a preliminary step before carrying out the contacting and eluting steps of the invention.
Alternative natural sources of impure pituitary glycoprotein hormones include body fluids such as, for example urine. Synthetic sources can include the products of chemical, biochemical or microbiological syntheses including products derived from cultures of transformed microorganisms. It will be appreciated therefore that the term "pituitary" is intended to indicate the nature and not necessarily the source of the glycoprotein hormones isolated in accordance with the invention and the invention is not limited to isolation of hormones from by-products from the processing of pituitaries.
The following Examples illustrate the process of the invention:
EXAMPLE 1
A Binding of antibody to support
2 gm of cyanogen bromide-activated Sepharose 4B were washed exhaustively with 1 mM HCI.
The gel was then rapidly washed with 0.2 M HCO3 +0.5 M NaCI buffer pH 8.6, transferred to 20 ml of the same buffer and incubated at room temperature for 4-6 hours with 35mg of monoclonal antibody preparation. The reaction was continued overnight at 4"C and sampling the gel supernatant indicated greater than 90% coupling of protein to Sepharose. The gel was washed with the HCO3 buffer then treated at room temperature for 2 hours with 0.5 M
TRIS/HCI pH 8.5. Following this treatment the immobilised antibody gel was washed alternately with 0.1 M borate buffer pH 8.0 and 0.1 M acetate buffer pH 4 both containing 0.5 M NaCI.
Monoclonal antibodies to thyrotrophic, follitrophic and luteinising hormones were coupled by this method. Antibodies with a K for their antigen of the order of 5X 107 moles/l were found suitable and the preparation should contain more than 40% of the total protein as monoclonal antibody.
B Formations of antibody thyrotrophic hormone complex
A gel prepared as described in (A) above with monoclonal antibody to thyrotrophic hormone was formed as a column 1.6 cm in diameter and equilibrated with 50 mM TRIS/HCI pH 8.5. 5 ml of a solution containing 0.49 U/ml thyrotrophic hormone, 50 U/ml follitrophic hormone and 225 U/ml luteinising hormone equilibrated against the same buffer were applied to the column and all the thyrotrophic hormone bound under these conditions.
C Elution of thyrotrophic hormone
The column from stage B was washed with 50 mM TRIS/HCI pH 8.5 and then with 0.1 M glycine/HCI buffer containing 0.5 M NaCI pH 3.5 followed by 8 M urea in 0.1 M acetate buffer pH 4. 80% of the applied thyrotrophic hormone eluted as a sharp peak with the glycine/NaCI.
EXAMPLE 2
Thyrotrophic hormone prepared as described in Example 1 above was free of follitrophic hormone but still contained luteinising hormone in the ratio 1 U thyrotophic : 40 U luteinising hormone, i.e. about 3% of the totai protein. Formation of the antibody thyrotrophic hormone complex was carried out in 50 mM borate buffer plus 0.5 M NaCI pH 8.5.Elution of the thyrotrophic hormone was achieved by washing the column with borate/NaCI pH 8.5 followed by 50 mM borate buffer pH 8.5 then 0.1 M glycine/HCI buffer plus 0.5 M NaCI pH 3.5 and finally 8 M urea in 0.1 M acetate buffer pH 4.0. 80% of the applied thyrotrophic hormone eluted as a sharp peak in glycine/NaCI with no change in the capacity of the gel for antigen while the ratio of thyrotrophic to luteinising hormone fell to 1 U : 4 U, i.e. only 0.3% by weight of the protein present was identified as luteinising hormone by radio-immune assay. Bioassay of the product revealed a 1:1 relationship between thyrotrophic hormone biological activity and radioimmune assay values. The bioassay for luteinising hormone showed there to be little biologically active hormone suggesting that the radio-immune assay value may in fact be due to cross reaction between assay reagents and thyrotrophic hormone.
EXAMPLE 3 {Comparative)
Anti-thyrotrophic hormone monoclonal immunoglobulin was immobilised as described in
Example 1A and the antibody-antigen complex produced as described in Example 1B. After washing with TRIS/HCI buffer, the hormone was eluted with 1 M TRIS pH 10.5. Although the hormone was recovered with 60% yield the ability of the immobilised immunoglobulin to bind thyrotrophic hormone on subsequent applications was reduced and after 2 to 3 passages had lost its binding ability. In comparison, elution by glycine/NaCI and urea treatment resulted in no loss of binding capacity after 30 cycles of operation if sterile solutions are used.
EXAMPLE 4 (Comparative)
The procedure of Example 2 was repeated except that 0.5 M NaCI was omitted from the glycine buffer. No hormone was eluted from the complex.
EXAMPLE 5
The procedure of Example 2 was repeated except that the pH of the glycine/NaCI buffer was raised to 3.8. This resulted in a fall in yield from 80% to 35% recovery of hormone.
EXAMPLE 6
The procedure of Example 1A was repeated using antibody labelled with 1251. The column was then run as in Example 2 but without thyrotrophic hormone for several cycles. The solutions emerging from the column in the position one would expect to obtain the hormone were pooled and concentrated before gel filtration on Sephadex G-100. Radioactive counting had shown immunoglobulin to be eluted from the immunoaffinity column but gel filtration resolved this into two main components, both separated from the thyrotrophic hormone. Repetition in the presence of thyrotrophic hormone showed it could be freed of immunoglobulin or immunoglobulin fragements co-eluting from the immunoaffinity column by gel-filtration through Sephadex G-100.
EXAMPLE 7
Anti-follitrophic hormone monoclonal antibody was immobilised as described in Example 1 A.
The antibody complex was formed as described in Example 2. The gel had a capacity of 150 U follitrophic hormone per ml using as starting material a solution containing 2,260 U/ml follitrophic hormone, 10,950 U/ml luteinizing hormone and 2.67 U/ml thyrotrophic hormone. Elution of the hormone as described in Example 2 resulted in a 75% recovery of follitrophic hormone.
The product, following removal of immunoglobulin or immunoglobulin fragments by gel-filtration through Sephadex G-100 was found to be highly purified follitrophic hormone contaminated with only 0.1% by weight of luteinising hormone and 0.5% by weight of thyrotrophic hormone.
Bioassay of the product revealed a 1:1 relationship between in vivo biological activity and radioimmune assay potency.
EXAMPLE 8
Anti-luteinising hormone monoclonal antibody, 44/2.7, was immobilised as described in
Example 1A. The antibody-antigen complex was formed as described in Example 2. The gel had a capacity of 750 U luteinising hormone per ml using as starting material a solution containing 4300 U/ml luteinising hormone, 9.80 U/ml follitrophic hormone but free of the thyrotrophic hormone. Elution of the hormone as described in Example 2 resulted in a 52% recovery of luteinising hormone. The product, following removal of immunoglobulin or immunoglobulin fragments by gel-filtration through Sephadex G-100 was found to be highly purified luteinising hormone contaminated by less than 0.1% by weight of follitrophic hormone. Bioassay showed a 1:1 relationship between the biological activity of the product and its radioimmune assay potency.
Claims (9)
1. A method of isolating a pituitary glycoprotein hormone in biologically active form which comprises contacting an aqueous solution of the hormone with a solid support bearing immobilised monoclonal antibody to the hormone so as to form a complex between the immobilised antibody and the hormone and eluting the hormone with an acidic aqueous buffer having a pH of from 3 to 4.0, wherein the buffer is substantially free of protein denaturing agents.
2. A method according to Claim 1 wherein the pH of the buffer is less than 3.8.
3. A method according to Claim 1 or Claim 2 wherein the pH of the buffer is greater than 3.2.
4. A method according to any preceding claim wherein the ionic strength of the buffer is at least 0.3 M.
5. A method according to any preceding claim wherein the ionic strength of the buffer is from 0.5 to 1.0 M.
6. A method according to any preceding claim wherein thyrotrophic hormone is separated.
7. A method according to any of Claims 1 to 5 wherein follitrophic hormone is separated.
8. A method according to any of Claims 1 to 5 wherein luteinising hormone is separated.
9. A method according to any preceding claim wheren the aqueous solution of the hormone which is contacted with the solid support comprises of pituitary extract which has been substantially freed of growth hormone.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB858510177A GB8510177D0 (en) | 1985-04-22 | 1985-04-22 | Isolating pituitary glycoprotein hormones |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| GB8609669D0 GB8609669D0 (en) | 1986-05-29 |
| GB2173803A true GB2173803A (en) | 1986-10-22 |
| GB2173803B GB2173803B (en) | 1988-11-09 |
Family
ID=10577986
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB858510177A Pending GB8510177D0 (en) | 1985-04-22 | 1985-04-22 | Isolating pituitary glycoprotein hormones |
| GB08609669A Expired GB2173803B (en) | 1985-04-22 | 1986-04-21 | Process for isolating pituitary glycoprotein hormones |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| GB858510177A Pending GB8510177D0 (en) | 1985-04-22 | 1985-04-22 | Isolating pituitary glycoprotein hormones |
Country Status (1)
| Country | Link |
|---|---|
| GB (2) | GB8510177D0 (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU615930B2 (en) * | 1987-06-26 | 1988-06-24 | Industria Farmaceutica Serono S.P.A. | Urinary follicle stimulating hormone |
| EP0328248A2 (en) * | 1988-01-12 | 1989-08-16 | Bunge (Australia) Proprietary Limited | Monoclonal antibodies against a follicle-stimulating hormone |
| US4921808A (en) * | 1986-06-25 | 1990-05-01 | The Albany Medical College Of Union University | Method for determining follicle stimulating hormone |
| US5175255A (en) * | 1987-03-23 | 1992-12-29 | Amgen Inc. | Methods for purification of platelet-derived growth factor |
| WO1994009814A1 (en) * | 1992-11-05 | 1994-05-11 | B.R.A.H.M.S Diagnostica Gmbh | Purified tsh preparation, process for its production and its use for the production of tsh tracers for tsh receptor assays and in tsh receptor assays |
| US5317092A (en) * | 1989-11-20 | 1994-05-31 | Novo Nordisk A/S | Protein purification method |
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
| CN103030691A (en) * | 2011-09-29 | 2013-04-10 | 长春金赛药业有限责任公司 | Method for separating and purifying gonadotropin glycoprotein subunits |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2135676A (en) * | 1983-02-21 | 1984-09-05 | Snow Brand Milk Products Co Ltd | Erythropoietin by antibody affinity chromatography |
| GB2163751A (en) * | 1984-08-01 | 1986-03-05 | Amano Pharma Co Ltd | Human insulin-like growth factor II |
-
1985
- 1985-04-22 GB GB858510177A patent/GB8510177D0/en active Pending
-
1986
- 1986-04-21 GB GB08609669A patent/GB2173803B/en not_active Expired
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2135676A (en) * | 1983-02-21 | 1984-09-05 | Snow Brand Milk Products Co Ltd | Erythropoietin by antibody affinity chromatography |
| GB2163751A (en) * | 1984-08-01 | 1986-03-05 | Amano Pharma Co Ltd | Human insulin-like growth factor II |
Cited By (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4921808A (en) * | 1986-06-25 | 1990-05-01 | The Albany Medical College Of Union University | Method for determining follicle stimulating hormone |
| US5175255A (en) * | 1987-03-23 | 1992-12-29 | Amgen Inc. | Methods for purification of platelet-derived growth factor |
| JPH02500840A (en) * | 1987-06-26 | 1990-03-22 | イスティテュト ディ リチェルカ チェサレ セローノ ソチエタ ペル アツィオニ | follicle stimulating hormone |
| LT4018B (en) | 1987-06-26 | 1996-08-26 | Serono Cesare Ist Ricerca | Protein, biologically active hormone, method for protein production, pharmaceutical preparation |
| AU615930B2 (en) * | 1987-06-26 | 1988-06-24 | Industria Farmaceutica Serono S.P.A. | Urinary follicle stimulating hormone |
| US5840857A (en) * | 1987-06-26 | 1998-11-24 | Istituto Di Ricerca Cesare Serono S.P.A. | Urinary follicle stimulating hormone |
| GR880100418A (en) * | 1987-06-26 | 1989-03-08 | Serono Cesare Ist Ricerca | Hormone stimulating a gland |
| US5128453A (en) * | 1987-06-26 | 1992-07-07 | Istituto Diricerca Cesare Serono Spa | Urinary follicle stimulating hormone |
| WO1988010270A1 (en) * | 1987-06-26 | 1988-12-29 | Istituto Di Ricerca Cesare Serono Spa | Urinary follicle stimulating hormone |
| US5767067A (en) * | 1987-06-26 | 1998-06-16 | Istituto Di Ricerca Cesare Serono S.P.A. | Follicle stimulating hormone and pharmaceutical compositions containing same |
| JP2523843B2 (en) | 1987-06-26 | 1996-08-14 | イスティテュト ディ リチェルカ チェサレ セローノ ソチエタ ペル アツィオニ | Follicle stimulating hormone |
| EP0328248A2 (en) * | 1988-01-12 | 1989-08-16 | Bunge (Australia) Proprietary Limited | Monoclonal antibodies against a follicle-stimulating hormone |
| EP0328248A3 (en) * | 1988-01-12 | 1990-04-11 | Bunge (Australia) Proprietary Limited | Monoclonal antibodies against a follicle-stimulating hormone |
| US5317092A (en) * | 1989-11-20 | 1994-05-31 | Novo Nordisk A/S | Protein purification method |
| WO1994009814A1 (en) * | 1992-11-05 | 1994-05-11 | B.R.A.H.M.S Diagnostica Gmbh | Purified tsh preparation, process for its production and its use for the production of tsh tracers for tsh receptor assays and in tsh receptor assays |
| US5990288A (en) * | 1997-10-21 | 1999-11-23 | Vitro Diagnostics, Inc. | Method for purifying FSH |
| CN103030691A (en) * | 2011-09-29 | 2013-04-10 | 长春金赛药业有限责任公司 | Method for separating and purifying gonadotropin glycoprotein subunits |
Also Published As
| Publication number | Publication date |
|---|---|
| GB8510177D0 (en) | 1985-05-30 |
| GB2173803B (en) | 1988-11-09 |
| GB8609669D0 (en) | 1986-05-29 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PCNP | Patent ceased through non-payment of renewal fee |
Effective date: 19950421 |