WO1985005631A1 - Purifying recombinant dna products and checking product purity and efficiency of purification - Google Patents
Purifying recombinant dna products and checking product purity and efficiency of purification Download PDFInfo
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- WO1985005631A1 WO1985005631A1 PCT/SE1985/000218 SE8500218W WO8505631A1 WO 1985005631 A1 WO1985005631 A1 WO 1985005631A1 SE 8500218 W SE8500218 W SE 8500218W WO 8505631 A1 WO8505631 A1 WO 8505631A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
- B01D61/42—Electrodialysis; Electro-osmosis ; Electro-ultrafiltration; Membrane capacitive deionization
- B01D61/44—Ion-selective electrodialysis
-
- 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
- C07K14/62—Insulins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D2311/00—Details relating to membrane separation process operations and control
- B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
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- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
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- Immunology (AREA)
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Abstract
A method of purifying a contaminated product (protein/peptide) produced by rDNA technique, from hydrophobic contaminants which are charged and originate from the microbial cloning host employed, and the product thus purified. The contaminated product is subjected to electroseparation, such as electrodialysis, to remove the contaminants. A charge-providing pretreatment is resorted to when the contaminants themselves are not charged enough to enable them to be directly separated upon electroseparation. In addition, a method of checking the purity of a product (protein/peptide) produced by rDNA technique is disclosed. Moreover, a method of checking the efficiency of the purification process used for purifying a product is disclosed
Description
P /SE85/00218
Purifying recombinant DNA products and checking product purit andefficiency of purification.
T e present invention concerns the purification of biological material, more particularly the purification of contaminated products (peptides/proteins) which have been produced using recombinant DNA (rDNA) technique. In the preparation of products (proteins/peptides) by rDNA technique, use is made of microbial cloning host primarily bacteria or yeast.
During the wor ing-up and purification of these rDN produced products, one has mainly used permeation gel chromatography utilising e.g. Sephadex®, ion exchange chromatography and/or HPLC with different types of col¬ umns, including reverse phase columns. All of these puri fication methods have aimed at the removal of microbial contaminants originating from the cloning hosts (so-call E. coli peptides, EPC) (cf. "Journal n", Vol. 3, No. 12, 1983, pp. 331-334, published by Kabi-Vitrum Sverige AB). These methods have up to now been considered efficient enough to remove the above-mentioned contaminating host contaminants. Checking the purity of such products has most often been performed by different types of polyacry amide gel electrophoresis techniques including the use o dissociating agents such as SDS etc. Also, different typ of immuno-assays, such as ELISA and RIA, have been used. Moreover, one has frequently performed pyrogenicity test and Limulus amoebocyte lysate (LAL) tests.
In spite of the above-mentioned purification method the products produced by rDNA technique have still been shown to elicit antibodies directed against these prod- v ucts (proteins/peptides), even though this formation of antibodies has been deemed to be small and, perhaps, medi cally acceptable. Nevertheless, experts within this field have put forward that caution should be exercised in the registration of these products. ( "Lakartidningen", Vol. 81, No. 8, 1984, p. 636 "Proceed slowly in registering
2 biosynthetic drugs!"). However, in those instances where the protein is to be administered over long periods of time, such as in life-long diseases (e.g. diabetes), not even a slight antibody formation is acceptable. The basis of the present invention is the insight that also contaminants other than proteins/peptides from the microbial cloning host may give rise to immune re¬ actions.
The invention is based upon research results which show that, due to hydrophobic interaction, minor amounts of hydrophobic contaminants from the microbial cloning host may stick to the product (peptide/protein) during the working-up thereof from the rDNA production environment employed. These hydrophobic contaminants which firmly attach to the product will then constitute epitopes and create fusion epitopes which will elicit antibody for¬ mation, not only against the contaminant in question, but also against the product produced by rDNA technique.
The purification methods previously employed have been too "mild" to free the product produced by rDNA technique from these hydrophobic contaminants, and the contaminants which most often are smaller than the prod¬ uct (the peptide/protein) have therefore been carried along during the working-up and purification without being detected.
In the method according to the present invention, these hydrophobic contaminants which result in the for¬ mation of epitopes and fusion epitopes, are removed by electroseparation, such as electroseparation using gel-forming substances or electrodialysis, whereby the hydrophobic contaminants are separated from the product (the peptide/protein) such that the purified product will not give rise to immune reactions upon administra¬ tion to mammals, including man. The invention relates to a method of purifying a contaminated product (protein/peptide) which has been produced by rDNA technique in that the contaminated
3 product is purified from hydrophobic contaminants which are charged and which originate from the microbial cloning host employed, such that the product is sub¬ jected to electroseparation to remove the contaminants. In addition, the invention comprises that the hydrophobic contaminants may obtain at least part of their charge by a charge-providing pretreatment, such as mild alkaline treatment, dissociation by means of dissociating agents, treatment with kaotropic ions or chelate-forming sub- stances. The preferred electroseparation is electro¬ dialysis. According to one embodiment of the invention, the contaminated product is insulin produced by rDNA technique, and the hydrophobic contaminants are lipo- polysaccharides originating from Escherichia coli. The invention also comprises a method of checking the purity of a product (protein/peptide) produced by rDNA technique, in that the product is combined with antibodies directed against hydrophobic contaminants from the micro¬ bial cloning host utilised in the production, whereupon a possible binding of the antibodies to the product is detecte A method of checking the efficiency of the purifica¬ tion process used for purifying a product (protein/peptide) produced by rDNA technique involves combining the product, prior to purification, with a predetermined amount of ra- dioactively or otherwise labelled hydrophobic host con¬ taminant, whereupon the product is subjected to the puri¬ fication process and the amount of labelled contaminant in the purified product is determined.
Furthermore, the invention comprises the purified product (protein/peptide) which has been obtained after the product has been purified by the purification method according to the invention.
The hydrophobic contaminants from the microbial cloning host employed are frequently inherently charged enough and may therefore be directly separated from the contaminated product when this is subjected to electro¬ separation, such as electrodialysis. In those instances
4 where the hydrophobic contaminants are not sufficiently charged themselves to make them directly separable upon electroseparation, different charge-providing pretreat- ments may be resorted to, such as mild alkaline treat- ent, dissociation by means of dissociating agents, such as urea or guanidine hydrochloride, the use of kaotropic ions, such as potassium ions, or use of chelate-forming substances, such as EDTA.
There are two different methods of checking whether the product (protein/peptide) produced by rDNA technique is free from contaminating hydrophobic host contaminant. According to the first method, the product is combined with antibodies directed against hydrophobic contaminants from the microbial cloning host utilised in the produc- tion. If the antibodies bind to the product, the product is contaminated by host contaminants. Detecting the amount of antibodies that have been bound to the product is carried out by, for example, the ELISA test and is a measure of the degree to which the product has been purified. As a result, the efficiency of different puri¬ fication methods regarding the removal of contaminating host contaminants may be determined.
The other method of checking the purity of the product that has been produced by rDNA technique and/or when the product has been isolated from another source, involves combining the product, prior to purification, with a predetermined amount of radioactively or other¬ wise labelled hydrophobic host contaminant, whereupon the product contaminated by labelled host contaminant and, possibly, unlabelled host contaminant is purified, whereupon the amount of labelled host contaminant in the purified product is determined. If no labelled host contaminant is detected in the product, the product is pure. If a labelled host contaminant is detected in the purified product, purification has not been effi¬ cient enough to remove all host contaminants, i.e. if the product is contaminated by labelled contaminant, it
5 is also contaminated by unlabelle.d contaminant. This makes it possible to determine the efficiency of differ¬ ent purification methods.
The microbial cloning host which at present is most frequently employed is Escherichia coli K 12, but also other bacterial hosts, besides E. coli, are used for this purpose, for example Bacillus subtilis and Staphylococcus aureus. Experiments with yeast, such as Saccharomyces cerevisiae, have also been reported. rDNA technique is already being used in the produc¬ tion of many potential drugs, such as human growth hor¬ mone, insulin and interferon, but so far the only protein available on the Swedish market and produced by rDNA technique is insulin bearing the trade mark Humulin®. Humulin® has been produced using E. coli K 12 as cloning host. In the above-mentioned article in "Journalen", Vol. 3, No. 12, 1983, published by Kabi- Vitru Sverige AB, it is reported on p. 333 that it is possible, although with some difficulty, to obtain antibodies by means of complete Freund's adjuvant and to sensitise experimental animals against E. coli polypep- tides (ECP) (originating from the host employed in the production of Humulin® but without the genetic code for the A and B chains in insulin). In addition, it appears that Humulin®is not pyrogenic in rabbits and contained < 0.6 ng/ g of bacterial so-called endotoxin, which is equal to lipopolysaccharide (LPS). Neither could endo¬ toxin (LPS) be shown in the Limulus amoebocyte lysate (LAL) test which by many is regarded as the most sensi- tive test for endotoxin (LPS). The relevance of the LAL test has, however, been questioned, in particular when LPS is present in combination with other hydro- phobic substances.
From what has been stated above, one might conclude that it has not been possible with prior art technique to establish in vitro small amounts of microbial conta¬ minants. In accordance with what has been said above.
6 these products may nevertheless give rise to antibody formation, not only against the contaminants mentioned, but also against the product. Of particular interest in this context is that endotoxin (LPS) is a very potent immunological substance. LPS is, in fact, one of the most potent polyclonal B cell stimulating substances that are known.
The experiments described below show that small amounts of endotoxin from the cloning host E. coli which consists of lipopolysaccharides, will stick to Humulin® as is shown by means of radioactively labelled lipopoly- saccharide from E. coli K 12.
Moreover, it is shown that this radioactively labelled contaminant accompanies Humulin©upon gel chro- matography. By means of the experimentally contaminated
Humulin® it is shown that the radioactively labelled lipo- polysaccharide from E. coli K 12 (i.e. the host) de¬ taches itself and is removed from the Humulin® upon puri¬ fication by electrodialysis.
Description of the drawings Fig. 1 illustrates gel chromatography of 14C-labelled
E. coli K 12 LPS (curve A), Humulin® / n 0Ω~o r (curve B),
14 C-labelled E. coli K 12 LPS + Humulin_®» /in cpm/
(curve C), and 14C-labelled E. coli K 12 LPS + Humulin® /in 0D28n/ (curve D). The experimental conditions employed were: column Sepharose® G 75 (9 mm x 550 mm) eluted with
H-O-16 mg/ml; flow 4.0 ml/h; fraction volume 0.5 ml.
Fig. 2 illustrates electrodialysis purification of Humulin® from contaminating E. coli K 12 LPS, curve A indicating the percentage of remaining Humulin®, and curve B indicating the percentage of remaining 14C- labelled radioactive E. coli K 12 LPS.
Fig. 3 illustrates schematically an open dialysis- electrodialysis vessel 1 (which is used in Example 2) of, for example, plexiglass and a tubular dialysis chamber 3 closed by means of two dialysis membranes 2. 4 and 5 de note, respectively, a platinum anode and a platinum
7 cathode.
Materials utilised in the Examples
Humulin® regular for inject, sc. 40 IU/ml, (Kabi-
Vitrum, Sweden). U-14C-labelled E. coli K 12 LPS (1000 cpm/μg), labelled in vivo with U- 14C-galactose and ϋ-14C-glucose (NEC-520 and NEC-042, NEN, Great Britain) which has been purified by so-called PCP extraction and whose purity has been verified by SDS polyacrylamide gel electrophoresis with subsequent silver colouring and autoradiography. A study with H-NMR which involved comparison with nonradioactively labelled reference LPS from E. coli K 12, showed that this LPS was pure.
For the permeation gel chromatography experiments use was. made of Sepharose® G 75 (Pharmacia, Uppsala, Sweden), the eluent consisting of double-glass distilled water containing 16 mg/l of glycerol p.a. (Merck, Federal Republic of Germany).
In the electrodialysis experiment, use was made of an electrodialysis bath (see Fig. 3 (1)) and a power unit (LKB, type 2197 Power Supply, LKB, Sweden) using
160 V and 20 mA. The electrodialysis conditions included using the above-mentioned water-glycerol system and dialysis membranes (Union Carbide dialysis membrane 21 DM00C227 P7F20, Union Carbide, Chicago, USA). Example 1
Studying the binding of C-labelled E. coli K 12 LPS to Humulin®
2.5 g Humulin® (as determined by Bio-Rad protein analysis, Bio-Rad, Munich, Federal Republic of Germany) in 1.0 ml water/glycerol according to the above, were mixed with 100 μg of 14C-labelled E. coli K 12 LPS and incubated at room temperature (23°C) under slight agita¬ tion for 2 hours. After that, this mixture was applied to the G75 column (described above). The elution profile of this mixture is shown in Fig. 1 (see curves C and D). This experiment clearly shows that a major proportion of the
14 C-labelled LPS co-migrated with HumulinΛ®Γ,. Furthermore,
8 Fig. 1 shows that Humulin® alone has a retention time which is largely identical with the retention time of the experimentally contaminated Humulin® (see Fig. 1, curve B). In addition. Fig. 1 shows (curve A) that when only C-labelled LPS was applied, it was eluted out with the initial fraction (void) and that it therefore has a retention time which is completely and distinctly different from that of the mixed product. The reason why this last-mentioned elution profile with only 14C-labelled LPS is obtained is that this LPS is present in the form of micells. To sum up, it may be said that 14C-labelled E. coli
K 12 LPS binds strongly to Humulin® .
Example 2 Studying purification of experimentally contaminated Humulin® by electrodialysis
The Humulin® in fractions 48-63 "purified" by gel permeation chromatography on G75 in accordance with Example 1 was combined, and the total radioactivity was determined to 6,000 cpm (counts per minute) in a total of 7.53 ml. This radioactivity in Humulin® thus corre¬ sponds to an amount of LPS of 6 μg distributed on 2.5 mg Humulin®, i.e. each milligram of Humulin® carries 2.4 μg contaminating E. coli K 12 LPS. The present Example in- dicates the removal of this contaminant by electrosepa¬ ration, use being made of the above-mentioned material and the apparatus schematically illustrated in Fig. 3. This contaminated Humulin ® fraction was subjected to electrodialysis (160 V, 20 mA) , and samples were taken at different times. The amount of radioactivity in the dialysis membranes and the amount of Humulin® were de¬ termined. Fig. 2 shows the result, and it appears that during the entire time of the experiment all Humulin® remained within the dialysis membranes (see curve A),
14 but that the C-labelled E. coli LPS had decreased to
55% already after an electrodialysis time of 40 min
(see curve B). After 2 hours of electrodialysis, only
9 17% of the contaminating LPS remained.
To sum up, it may be said that electroseparation according to this experiment by electrodialysis shows that contaminating hydrophobic substances from the host (LPS) may be efficiently removed from rDNA-produced products (proteins/peptides). Example 3
Checking the purity of a product by mixing the product with antibodies directed against hydro- phobic contaminants.
Monoclonal antibodies of IgG and IgM classes were produced in Balb/c mice by hybridisation of lymphocytes isolated from the spleen of mice immunised with purified lipopolysaccharide (LPS) from E. coli K 12 with sp2 cells to yield hybridoma clones which by enzyme-linked immuno- sorbent-assay (ELISA) were screened for specific expres¬ sion of Ig with specificity for the coreoligosaccharide portion of said LPS using that LPS as coating antigen in the ELISA tests. After subsequent subcloning to yield monoclonal antibody producing hybridomas by dilution technique, these hybridomas were propagated both in vitro and in vivo (i.e. as ascites in Balb/c mice). The mono¬ clonal antibodies were isolated using either conventional gel permeation chromatography (IgM) or by using the protein A-affinity chromatography method for isolating IgG monoclonal antibodies.
The purity of each of the used monoclonal antibody preparations was assured by isoelectric focusing pattern, subclass and light chain identification using commerciall available light and heavy chain specific anti-mouse anti¬ bodies in double radial immune diffusion tests.
Purified pig insulin from Novo, Novo Industri A/S,
Novo Alle, 2880 Bagsvaerd, Denmark, was experimentally contaminated with 14-labelled purified E. coli LPS by mixing Actrapid 400 IU with 0.1 mg of LPS in a total volume of 10 ml. After incubation at 25°C for 5 hours the mixture was chromatographed on Sepharose® G75 (Pharmacia,
Sweden) and the insulin as judged by measurements of the optical density at 280 nanometers was collected.
This preparation was after extensive dialysis against distilled water, containing 1% glycerol and 0.7 M NaCl, used as coating antigen in ELISA and compared to the non- contaminated original product for specific binding of mouse-anti E. coli K 12 coreoligosaccharide specific monoclonal IgG antibodies prepared as described above. The coating doses of each of the two antigens were log- arithmic dilutions from 1000 micrograms protein (as measured by Lowry et al) to 0.001 micrograms per ml.
In the ELISA tests the experimentally contaminated product showed binding of the mouse monoclonal anti- E. coli coreoligosaccharide IgG antibodies down to a level of contamination of less than 0.01 microgram of E. coli K 12 LPS per mg of insulin.
This Example-shows that monoclonal antibodies directed against hydrophobic host contaminants (LPS) accompanying the product protein bind to the product protein if this is contaminated.
Claims
1. A method of purifying a contaminated product (protein/peptide) produced by rDNA technique, c h a r ¬ a c t e r i s e d in that the contaminated product is purified from hydrophobic contaminants which are charged and originate from the microbial cloning host employed, by subjecting the product to electroseparation in order to remove said contaminants.
2. A method as claimed in claim 1, c h a r ¬ a c t e r i s e d in that the hydrophobic contaminants are given at least a part of their charge by a charge- providing pretreatment.
3. A method as claimed in claim 2, c h a r ¬ a c t e r i s e d in that said pretreatment consists of mild alkaline treatment, dissociation by means of dissociating agents, treatment with kaotropic ions, or with chelate-forming substances.
4. A method as claimed in claim 1, c h a r ¬ a c t e r i s e d in that the said electroseparation is conducted by electrodialysis.
5. A method as claimed in claim 1, c h a r ¬ a c t e r i s e d in that the contaminated product consists of insulin produced by rDNA technique.
6. A method as claimed in claim 1, c h a r ¬ a c t e r i s e d in that the hydrophobic contaminants consist of lipopolysaccharides originating from Escheri- chia coli.
7. A method of checking the purity of a product (protein/peptide) produced by rDNA technique, c h a r ¬ a c t e r i s e d in that the product is combined with antibodies directed against hydrophobic contaminants from the microbial cloning host employed during the production, whereupon a possible binding of the antibodies to the product is detected. 12 8. A method of checking the efficiency of the purification process used for purifying a product produced by rDNA technique, c h a r a c t e r i s e d in that the product is combined, prior to purification, with a predetermined amount of radioactively or other¬ wise labelled hydrophobic host contaminant, whereupon the product is subjected to the purification process and the amount of labelled contaminant in the purified product is determined. 9. A purified product (protein/peptide), c h a r ¬ a c t e r i s e d in that it has been purified by the method as claimed in claim 1.
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
BR8506758A BR8506758A (en) | 1984-05-28 | 1985-05-24 | PURIFICATION OF RECOMBINANT DNA PRODUCTS AND VERIFICATION OF THE PURITY OF THE PRODUCT AND PURIFICATION EFFICIENCY |
HU853023A HUT39774A (en) | 1984-05-28 | 1985-05-24 | Process for purifying recombinant dns products, and for controlling the product's purity and the effectiveness of the purification |
FI860316A FI860316A0 (en) | 1984-05-28 | 1986-01-22 | RENING AV BIOLOGISKT MATERIAL. |
DK39786A DK39786A (en) | 1984-05-28 | 1986-01-27 | CLEANING OF REPLACEMENT PRODUCTS AND PRODUCT PURITY CONTROL AND CLEANING EFFICIENCY |
KR860700047A KR860700135A (en) | 1984-05-28 | 1986-01-28 | Method for purifying biological material and measuring its purity and efficiency |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
SE8402861A SE8402861L (en) | 1984-05-28 | 1984-05-28 | PURIFICATION OF BIOLOGICAL MATERIAL |
SE8402861-2 | 1984-05-28 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1985005631A1 true WO1985005631A1 (en) | 1985-12-19 |
Family
ID=20356050
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/SE1985/000218 WO1985005631A1 (en) | 1984-05-28 | 1985-05-24 | Purifying recombinant dna products and checking product purity and efficiency of purification |
Country Status (20)
Country | Link |
---|---|
US (1) | US4746647A (en) |
EP (1) | EP0185700A1 (en) |
JP (1) | JPS61502378A (en) |
KR (1) | KR860700135A (en) |
AU (1) | AU4435885A (en) |
BR (1) | BR8506758A (en) |
CS (1) | CS257789B2 (en) |
DD (1) | DD241748A5 (en) |
DK (1) | DK39786A (en) |
ES (3) | ES8609474A1 (en) |
FI (1) | FI860316A0 (en) |
HU (1) | HUT39774A (en) |
IL (1) | IL75258A0 (en) |
IN (1) | IN162119B (en) |
NO (1) | NO860295L (en) |
NZ (1) | NZ212155A (en) |
PL (1) | PL253664A1 (en) |
SE (1) | SE8402861L (en) |
WO (1) | WO1985005631A1 (en) |
ZA (1) | ZA853918B (en) |
Cited By (7)
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US4736020A (en) * | 1985-07-22 | 1988-04-05 | Basf Aktiengesellschaft | Purification of hTNF |
US4798886A (en) * | 1986-03-24 | 1989-01-17 | Takeda Chemical Industries, Ltd. | Mutual separation of proteins |
WO1989001035A1 (en) * | 1987-07-23 | 1989-02-09 | Europäisches Laboratorium Für Molekularbiologie (E | Process and device for purification of m-13 phage dna |
US4894439A (en) * | 1986-05-22 | 1990-01-16 | Cetus Corporation | N-terminal derivatives of tumor necrosis factor purified by microporous PTFE membranes |
US4902783A (en) * | 1986-06-18 | 1990-02-20 | The Research Foundation For Microbial Diseases Of Osaka University | Method for purifying a gene-expression product produced by recombinant DNA technique |
US5256769A (en) * | 1984-09-26 | 1993-10-26 | Takeda Chemical Industries, Ltd. | Mutual separation of proteins |
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US20050224355A1 (en) * | 1999-12-23 | 2005-10-13 | Brendon Conlan | Removal of biological contaminants |
AUPP790898A0 (en) | 1998-12-23 | 1999-01-28 | Life Therapeutics Limited | Renal dialysis |
AUPP790698A0 (en) | 1998-12-23 | 1999-01-28 | Life Therapeutics Limited | Separation of microorganisms |
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US7077942B1 (en) * | 1999-12-23 | 2006-07-18 | Gradipore Limited | Removal of biological contaminants |
AUPQ691400A0 (en) * | 2000-04-14 | 2000-05-11 | Life Therapeutics Limited | Separation of micromolecules |
AUPQ697300A0 (en) * | 2000-04-18 | 2000-05-11 | Life Therapeutics Limited | Separation apparatus |
EP1294472B1 (en) | 2000-04-18 | 2009-11-18 | Gradipore Limited | Electrophoresis separation and treatment of samples |
US6923896B2 (en) * | 2000-09-22 | 2005-08-02 | The Texas A&M University System | Electrophoresis apparatus and method |
ATE473040T1 (en) * | 2000-10-06 | 2010-07-15 | Arturus Capital Ltd | MULTI-PORT ELECTROPHORESIS SEPARATOR AND METHOD |
AUPR222300A0 (en) * | 2000-12-21 | 2001-01-25 | Life Therapeutics Limited | Electrophoresis device and method |
GB201204756D0 (en) * | 2012-03-19 | 2012-05-02 | Lewi Paulus J | Triazines with suitable spacers for treatment and/or prevention of HIV infections |
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US4426323A (en) * | 1980-01-10 | 1984-01-17 | Ionics, Incorporated | Selected recovery of proteins from fermentation broths |
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GB1188519A (en) * | 1966-10-27 | 1970-04-15 | Wellcome Found | Method for the Purification of Lipopolysaccharides |
US4185090A (en) * | 1972-02-02 | 1980-01-22 | Abbott Laboratories | Chemically modified endotoxin immunizing agent |
SE7804098L (en) * | 1977-04-20 | 1978-10-21 | R & Z Vermoegensverw Gmbh | PEPTIDE COMPLEX FROM DNA-CONTAINING ORGANISMS |
US4276140A (en) * | 1980-01-10 | 1981-06-30 | Ionics Inc. | Electrodialysis apparatus and process for fractionating protein mixtures |
US4322275A (en) * | 1980-01-10 | 1982-03-30 | Ionics Incorporated | Fractionation of protein mixtures |
US4321192A (en) * | 1980-01-10 | 1982-03-23 | Ionics Incorporated | Fractionation of protein mixtures by salt addition followed by dialysis treatment |
US4351710A (en) * | 1980-01-10 | 1982-09-28 | Ionics, Incorporated | Fractionation of protein mixtures |
SE8205892D0 (en) * | 1982-10-18 | 1982-10-18 | Bror Morein | IMMUNOGENT MEMBRANE PROTEIN COMPLEX, SET FOR PREPARATION AND USE THEREOF |
JPS6078999A (en) * | 1983-10-05 | 1985-05-04 | Chemo Sero Therapeut Res Inst | Purification of hbs antigen |
JPS61103895A (en) * | 1984-10-26 | 1986-05-22 | Green Cross Corp:The | Purification of hbsag |
-
1984
- 1984-05-28 SE SE8402861A patent/SE8402861L/en not_active Application Discontinuation
-
1985
- 1985-05-21 IL IL75258A patent/IL75258A0/en unknown
- 1985-05-22 CS CS853684A patent/CS257789B2/en unknown
- 1985-05-22 NZ NZ212155A patent/NZ212155A/en unknown
- 1985-05-22 IN IN389/CAL/85A patent/IN162119B/en unknown
- 1985-05-23 ZA ZA853918A patent/ZA853918B/en unknown
- 1985-05-24 BR BR8506758A patent/BR8506758A/en unknown
- 1985-05-24 EP EP85902715A patent/EP0185700A1/en not_active Withdrawn
- 1985-05-24 JP JP60502612A patent/JPS61502378A/en active Pending
- 1985-05-24 HU HU853023A patent/HUT39774A/en unknown
- 1985-05-24 US US06/834,336 patent/US4746647A/en not_active Expired - Fee Related
- 1985-05-24 WO PCT/SE1985/000218 patent/WO1985005631A1/en not_active Application Discontinuation
- 1985-05-24 AU AU44358/85A patent/AU4435885A/en not_active Abandoned
- 1985-05-24 DD DD85276680A patent/DD241748A5/en not_active IP Right Cessation
- 1985-05-27 ES ES543512A patent/ES8609474A1/en not_active Expired
- 1985-05-28 PL PL25366485A patent/PL253664A1/en unknown
-
1986
- 1986-01-22 FI FI860316A patent/FI860316A0/en not_active Application Discontinuation
- 1986-01-27 DK DK39786A patent/DK39786A/en not_active Application Discontinuation
- 1986-01-28 NO NO860295A patent/NO860295L/en unknown
- 1986-01-28 KR KR860700047A patent/KR860700135A/en not_active Application Discontinuation
- 1986-02-28 ES ES552519A patent/ES8802584A1/en not_active Expired
- 1986-02-28 ES ES552518A patent/ES8802583A1/en not_active Expired
Patent Citations (1)
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US4426323A (en) * | 1980-01-10 | 1984-01-17 | Ionics, Incorporated | Selected recovery of proteins from fermentation broths |
Non-Patent Citations (3)
Title |
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Biochemistry vol 22, p 2007-13published 1983 (Coughlin R T et al) "Physical properties of defined lipopolysaccharide salt" * |
European J Biochem vol 122 p 233-37 published 1982 (Brade H Galanos C) "Isolation, Purification and chemical analysis of the lipopolysaccharide and lipid A of Acinetobacter calcoaceticus NCTC 10305 * |
Pharm Ind Vol 38, p 827-831 published 1976 (Koppensteiner G et al) "Versuche zur Entpyrogenisierung parenteralen Arzneimittel" * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5256769A (en) * | 1984-09-26 | 1993-10-26 | Takeda Chemical Industries, Ltd. | Mutual separation of proteins |
US4736020A (en) * | 1985-07-22 | 1988-04-05 | Basf Aktiengesellschaft | Purification of hTNF |
US4798886A (en) * | 1986-03-24 | 1989-01-17 | Takeda Chemical Industries, Ltd. | Mutual separation of proteins |
US4894439A (en) * | 1986-05-22 | 1990-01-16 | Cetus Corporation | N-terminal derivatives of tumor necrosis factor purified by microporous PTFE membranes |
US4902783A (en) * | 1986-06-18 | 1990-02-20 | The Research Foundation For Microbial Diseases Of Osaka University | Method for purifying a gene-expression product produced by recombinant DNA technique |
WO1989001035A1 (en) * | 1987-07-23 | 1989-02-09 | Europäisches Laboratorium Für Molekularbiologie (E | Process and device for purification of m-13 phage dna |
EP1549759A1 (en) * | 2002-06-12 | 2005-07-06 | Bayer Healthcare LLC | Bacterial test method by glycated label binding |
EP1549759A4 (en) * | 2002-06-12 | 2006-11-02 | Bayer Healthcare Llc | Bacterial test method by glycated label binding |
Also Published As
Publication number | Publication date |
---|---|
BR8506758A (en) | 1986-09-23 |
ES8609474A1 (en) | 1986-08-01 |
JPS61502378A (en) | 1986-10-23 |
NZ212155A (en) | 1988-10-28 |
NO860295L (en) | 1986-01-28 |
ES552519A0 (en) | 1988-08-01 |
ES8802584A1 (en) | 1988-08-01 |
CS368485A2 (en) | 1987-11-12 |
SE8402861L (en) | 1985-11-29 |
HUT39774A (en) | 1986-10-29 |
DK39786D0 (en) | 1986-01-27 |
ES552518A0 (en) | 1988-08-01 |
ES8802583A1 (en) | 1988-08-01 |
DK39786A (en) | 1986-01-27 |
EP0185700A1 (en) | 1986-07-02 |
IN162119B (en) | 1988-04-02 |
AU4435885A (en) | 1985-12-31 |
US4746647A (en) | 1988-05-24 |
CS257789B2 (en) | 1988-06-15 |
ES543512A0 (en) | 1986-08-01 |
FI860316A (en) | 1986-01-22 |
FI860316A0 (en) | 1986-01-22 |
PL253664A1 (en) | 1986-02-11 |
DD241748A5 (en) | 1986-12-24 |
SE8402861D0 (en) | 1984-05-28 |
IL75258A0 (en) | 1985-09-29 |
KR860700135A (en) | 1986-03-31 |
ZA853918B (en) | 1986-01-29 |
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