EP4225769A1 - Method for purifying a cationic protein fraction and fraction thus obtained - Google Patents
Method for purifying a cationic protein fraction and fraction thus obtainedInfo
- Publication number
- EP4225769A1 EP4225769A1 EP21786507.0A EP21786507A EP4225769A1 EP 4225769 A1 EP4225769 A1 EP 4225769A1 EP 21786507 A EP21786507 A EP 21786507A EP 4225769 A1 EP4225769 A1 EP 4225769A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cationic
- proteins
- fraction
- less
- solution
- 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.)
- Pending
Links
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 88
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 88
- 125000002091 cationic group Chemical group 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 36
- 235000018102 proteins Nutrition 0.000 claims description 86
- 239000002158 endotoxin Substances 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 40
- 108010063045 Lactoferrin Proteins 0.000 claims description 23
- 102000010445 Lactoferrin Human genes 0.000 claims description 23
- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 claims description 23
- 229940078795 lactoferrin Drugs 0.000 claims description 23
- 235000021242 lactoferrin Nutrition 0.000 claims description 23
- 239000012528 membrane Substances 0.000 claims description 13
- 238000011026 diafiltration Methods 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 238000005349 anion exchange Methods 0.000 claims description 8
- 239000008267 milk Substances 0.000 claims description 8
- 210000004080 milk Anatomy 0.000 claims description 8
- 235000013336 milk Nutrition 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 5
- 238000001694 spray drying Methods 0.000 claims description 5
- 101800000263 Acidic protein Proteins 0.000 claims description 4
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000001471 micro-filtration Methods 0.000 claims description 4
- 108010023244 Lactoperoxidase Proteins 0.000 claims description 3
- 102000045576 Lactoperoxidases Human genes 0.000 claims description 3
- 102000006382 Ribonucleases Human genes 0.000 claims description 3
- 108010083644 Ribonucleases Proteins 0.000 claims description 3
- 229940057428 lactoperoxidase Drugs 0.000 claims description 3
- 239000012460 protein solution Substances 0.000 claims description 3
- 238000011013 endotoxin removal Methods 0.000 abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 19
- 101000798100 Bos taurus Lactotransferrin Proteins 0.000 description 18
- 229940072440 bovine lactoferrin Drugs 0.000 description 18
- 239000007788 liquid Substances 0.000 description 15
- 235000008504 concentrate Nutrition 0.000 description 10
- 239000012141 concentrate Substances 0.000 description 10
- 235000020247 cow milk Nutrition 0.000 description 10
- 239000013017 sartobind Substances 0.000 description 10
- 239000003729 cation exchange resin Substances 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 229940023913 cation exchange resins Drugs 0.000 description 7
- 229920006008 lipopolysaccharide Polymers 0.000 description 7
- 229920002684 Sepharose Polymers 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 239000012465 retentate Substances 0.000 description 4
- 241000239205 Merostomata Species 0.000 description 3
- 108010011756 Milk Proteins Proteins 0.000 description 3
- 238000005277 cation exchange chromatography Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000008030 elimination Effects 0.000 description 3
- 238000003379 elimination reaction Methods 0.000 description 3
- 150000004676 glycans Chemical group 0.000 description 3
- 238000012454 limulus amebocyte lysate test Methods 0.000 description 3
- 235000014666 liquid concentrate Nutrition 0.000 description 3
- 235000021239 milk protein Nutrition 0.000 description 3
- 229920001542 oligosaccharide Polymers 0.000 description 3
- 150000002482 oligosaccharides Chemical class 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 2
- 102000014171 Milk Proteins Human genes 0.000 description 2
- 102000016943 Muramidase Human genes 0.000 description 2
- 108010014251 Muramidase Proteins 0.000 description 2
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 2
- 108010046377 Whey Proteins Proteins 0.000 description 2
- 239000003011 anion exchange membrane Substances 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- GZQKNULLWNGMCW-PWQABINMSA-N lipid A (E. coli) Chemical compound O1[C@H](CO)[C@@H](OP(O)(O)=O)[C@H](OC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCCCC)[C@@H](NC(=O)C[C@@H](CCCCCCCCCCC)OC(=O)CCCCCCCCCCC)[C@@H]1OC[C@@H]1[C@@H](O)[C@H](OC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](NC(=O)C[C@H](O)CCCCCCCCCCC)[C@@H](OP(O)(O)=O)O1 GZQKNULLWNGMCW-PWQABINMSA-N 0.000 description 2
- 235000010335 lysozyme Nutrition 0.000 description 2
- 229960000274 lysozyme Drugs 0.000 description 2
- 239000004325 lysozyme Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000003134 recirculating effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101000798114 Homo sapiens Lactotransferrin Proteins 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 238000011050 LAL assay Methods 0.000 description 1
- 241000239218 Limulus Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 108010093965 Polymyxin B Proteins 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 239000005862 Whey Substances 0.000 description 1
- 102000007544 Whey Proteins Human genes 0.000 description 1
- 239000002253 acid Chemical group 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 238000011138 biotechnological process Methods 0.000 description 1
- -1 cationic amino acids Chemical class 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000011840 criminal investigation Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 102000050459 human LTF Human genes 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical group [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920000024 polymyxin B Polymers 0.000 description 1
- 229960005266 polymyxin b Drugs 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000036303 septic shock Effects 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/495—Transforming growth factor [TGF]
-
- 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
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01007—Peroxidase (1.11.1.7), i.e. horseradish-peroxidase
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a process for the purification of cationic protein fractions by elimination of endotoxins; it also relates to the purified fractions thus obtained.
- Endotoxins are the cell wall components of Gram-negative bacteria. Released during the lysis or destruction of these bacteria, they are responsible for systemic inflammatory manifestations, such as septic shock, during infections by this type of bacteria. It is for this reason that an endotoxin content limit is defined for drugs or injectable products (such as water) by authorities such as
- Endotoxins also called lipopolysaccharides (LPS)
- LPS lipopolysaccharides
- the glycan part is composed of two parts, one part called central oligosaccharide (core oligosaccharide) and the other part called O side chain polysaccharide (O antigen);
- Figure 1 illustrates their schematic representation (Maeshima & Fernandez 2013).
- Each molecule of LPS has multiple negative charges from the phosphate and acid groups of lipid A and the central oligosaccharide.
- Endotoxins are known to be thermally and chemically stable.
- the endotoxin content is expressed in IU (international unit) which is equivalent to one EU (endotoxin unit) (3.4 Test for bacterial endotoxins, The International Pharmacopoeia - 9th edition); as an indication, 1 ng of LPS corresponds to approximately 10 EU (WHO International Standard, 3rd IS for endotoxin), this may be different depending on the origin of the strains of bacteria.
- Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the LimulusAmebocyte Lysate Assay: Application for Endotoxin Removal from Cationic Proteins.Analytical Biochemistry 259, 42-47), ribonuclease A and lactoferrin (Elass-Rochard, E., Roseanu , A., Legrand, D., Trif, M., Salmon, V., Motas, C., Montreuil, J., Spik, G., 1995.
- Lactoferrinlipopolysaccharide interaction involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem J 312, 839-845) have strong interactions with LPS molecules which have several negative charges.
- a method for eliminating endotoxins bound to a cationic protein, in particular lactoferrin has been proposed in WO 2009/009706 (Glanbia Nutritionals); this method comprises the steps of a) binding the protein to a cation exchange resin; b) eluting the endotoxin using a low ionic strength solution in the absence of added surfactant; c) eluting the protein with a high ionic strength solution. This process makes it possible to obtain a lactoferrin isolate containing less than 1 IU/mg of endotoxins.
- the present invention proposes a method making it possible to effectively eliminate the endotoxins present in a fraction of cationic proteins or bound to a cationic protein or a fraction of cationic proteins regardless of the magnitude and location of their positive charges.
- the present invention thus relates to a method for purifying a cationic protein fraction comprising the following steps a) to d): a) obtaining a solution:
- such a solution can be chosen from:
- an egg white lysozyme powder reconstituted with a 0.5 M NaCl solution • an egg white lysozyme powder reconstituted with a 0.5 M NaCl solution.
- Figure 2 illustrates a schematic representation of the method according to the invention.
- Figures 3 A and 3B illustrate examples of diagrams allowing the implementation of step b) of the method according to the invention.
- the present invention relates to a process for purifying a cationic protein isolate comprising the following steps a) to d): a) obtaining a solution:
- this second method applies to the isolate obtained by the previous method.
- an anion exchange support preferably membrane (eg Sartobind Q, Sartorus Stedium Biotech) or monolithic (eg CIMmultus QA, BIA Separtions), the support being such that it has little steric exclusion effect, in order to adsorb and eliminate almost all of the endotoxins present in the solution; the solution is preferably passed several times through an anion exchange medium, preferably at least 3 times; c) optionally, microfiltration with a membrane having a cut-off threshold of between 0.2 and 1.4 ⁇ m in order to reduce the microbial load to an acceptable level for the conformity of the finished product; d) optionally, spray-drying or freeze-drying the solution to obtain a powdered cationic protein isolate.
- membrane eg Sartobind Q, Sartorus Stedium Biotech
- monolithic eg CIMmultus QA, BIA Separtions
- Figure 4 illustrates a schematic representation of a device allowing the implementation of the alternative method according to the invention.
- the present invention also relates to a fraction of cationic proteins capable of being obtained or such as it is obtained by the methods according to the invention and such that it has an endotoxin content of less than 5 IU/mg of protein, preferably less than 1 IU/mg protein, more preferably less than 0.1 IU/mg protein.
- the cationic proteins of the fraction come from milk; they can then consist mainly of lactoferrin or mainly consist of lactoperoxidase or mainly consist of ribonucleases or even contain TGF-P in a content greater than 20 pg/g, preferably greater than 50 pg/g, most preferably between 100 and 200 ⁇ g/g protein.
- a fraction comprising at least 50%, but also greater than 90% or 95% by weight of proteins relative to the weight of the dry matter.
- the fractions according to the invention can also mainly contain a mixture of cationic milk or whey proteins.
- Figure 1 schematic representation of an LPS (Maeshima & Fernandez 2013);
- FIG. 2 Schematic representation of the purification process of the cationic protein isolate
- Figure 3 Examples of Cationic Protein Isolate Purification Process Flow Chart
- Figure 4 Schematic representation of the alternative purification process of the cationic protein isolate.
- Example 1 bench test with a liquid concentrate of bovine lactoferrin
- a liquid concentrate of lactoferrin from cow's milk was obtained using an industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing the milk of pasteurized skimmed cow, then eluting successively with a solution of NaCl at 36 mS/cm and that at 110 mS/cm, finally the 2nd eluate was concentrated on an ultrafiltration of MWCO 20 kDa.
- the protein concentration is 13 mg/mL, the purity of bovine lactoferrin on proteins is 95%, the conductivity of this solution is 65 mS/cm, and the pH is 6.8 (“Concentrate LF 1”) .
- Example 2 bench-top test with a liquid isolate of bovine lactoferrin
- a liquid microfiltrate of lactoferrin from cow's milk was obtained using an industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing the milk pasteurized skimmed cow's milk concentrated by reverse osmosis to 130 g/L of dry matter, then eluting successively with a NaCl solution at 38 mS/cm and that at 10%, then the 2nd eluate was concentrated on an ultrafiltration of MWCO 20 kDa, then diafiltered on an ultrafiltration of MWCO 10 kDa with reverse osmosis water up to 1 mS/cm, finally the diafiltered retentate was microfiltered on a ceramic membrane at 0.8 ⁇ m in double layer (Membrarox®, Pali Corporation) .
- Example 3 bench-top test with a liquid isolate of bovine lactoferrin
- a liquid microfiltrate of lactoferrin from cow's milk was obtained using an industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing pasteurized skimmed cow's milk, then eluting successively with a NaCl solution at 36 mS/cm and that at 110 mS/cm, then the 2nd eluate was concentrated on an ultrafiltration of 20 kDa MWCO, then diafiltered on an ultrafiltration of 10 kDa MWCO with osmosed water up to 1 mS/cm, finally the diafiltered retentate was microfiltered on a ceramic membrane at 1.4 pm in double layer (Membrarox®, Pali Corporation).
- the protein concentration is 147 mg/mL, the purity of bovine lactoferrin on proteins is 95%, the conductivity of this solution is 0.8 mS/cm, and the pH is 6.8 ("Isolat LF 3 ").
- the recovered lactoferrin liquid isolate (60 mL) was passed through the recirculating Sartobind® Q nano 3 mL cartridge for 30 minutes at a flow rate of 6 mL/min for a total of 6 equivalent passes (“Isolat LF 6”) .
- the recovered lactoferrin liquid isolate (55 mL) was passed through the recirculating Sartobind® Q nano 3 mL cartridge for 37 minutes at a flow rate of 6 mL/min for a total of 10 equivalent passes (“Isolat LF 7”) .
- Example 4 bench-top test with a liquid cationic protein isolate containing
- a cationic protein fraction containing TGF-P from cow's milk was obtained according to the method described in Example 1 of Patent EP 1912513.
- the content of TGF-P2 analyzed by the ELISA kit (Quntikine TGF-2, R&D Systems) in the microfiltrate obtained before spray drying was 115 pg/g proteins.
- This liquid cationic protein isolate containing TGF-P was diluted with ultrapure deionized water prepared by Milli-Q (Millipore) to have a protein concentration of 2.6 mg/mg (w/v), the conductivity of this solution is 0.89 mS/cm, and the pH is 7.1 ("Cationic Milk Protein Isolate 1"), 50 mL of this liquid cationic milk protein isolate was recirculated through a cartridge of Sartobind® Q nano 3 mL (Sartorius Stedim) at a flow rate of 5 mL/min for 70 minutes.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Analytical Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Water Supply & Treatment (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR2010423A FR3115037A1 (en) | 2020-10-12 | 2020-10-12 | Process for purification of cationic protein fraction and fraction thus obtained |
PCT/EP2021/078094 WO2022078976A1 (en) | 2020-10-12 | 2021-10-12 | Method for purifying a cationic protein fraction and fraction thus obtained |
Publications (1)
Publication Number | Publication Date |
---|---|
EP4225769A1 true EP4225769A1 (en) | 2023-08-16 |
Family
ID=75108373
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP21786507.0A Pending EP4225769A1 (en) | 2020-10-12 | 2021-10-12 | Method for purifying a cationic protein fraction and fraction thus obtained |
Country Status (10)
Country | Link |
---|---|
EP (1) | EP4225769A1 (en) |
JP (1) | JP2023546382A (en) |
KR (1) | KR20230086688A (en) |
CN (1) | CN116323636A (en) |
AU (1) | AU2021359742A1 (en) |
CA (1) | CA3194989A1 (en) |
FR (1) | FR3115037A1 (en) |
MX (1) | MX2023004106A (en) |
TW (1) | TW202229308A (en) |
WO (1) | WO2022078976A1 (en) |
Family Cites Families (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BR9007970A (en) * | 1990-01-08 | 1992-11-17 | Nika Health Products Ltd | PROCESS FOR THE PREPARATION OF RIBONUCLEASE DIMERS |
JP3962427B2 (en) * | 1990-07-13 | 2007-08-22 | グロペップ リミテッド | Growth promoter |
US5861491A (en) * | 1994-02-16 | 1999-01-19 | Pharming B.V. | Isolation of lactoferrin from milk |
NL1005677C2 (en) * | 1997-03-27 | 1998-09-29 | Campina Melkunie Bv | Method for recovering growth factors, or a composition containing one or more growth factors, from milk or a derivative thereof. |
FR2827290B1 (en) * | 2001-07-13 | 2004-07-09 | Pierre Jouan Biotechnologies Sa | METHOD FOR OBTAINING A PROTEIN FRACTION ENRICHED IN ACTIVATED FORM TGF-BETA, PROTEIN FRACTION AND THERAPEUTIC APPLICATIONS |
FR2889068B1 (en) | 2005-07-29 | 2012-03-02 | Cie Laitiere Europeenne | NOVEL DAIRY PROTEIN FRACTIONS AND THEIR USE FOR THE PREVENTION OR TREATMENT OF CHRONIC INFLAMMATORY DISEASES |
JP2010533201A (en) | 2007-07-10 | 2010-10-21 | グランビア ニュートリショナルズ | Method for removing endotoxins from proteins |
NZ594212A (en) | 2009-01-28 | 2013-01-25 | Jean Paul Perraudin | Method for production of lactoferrin |
AU2013204858B2 (en) * | 2012-10-08 | 2015-06-25 | Saputo Dairy Australia Pty Limited | Improved process for purifying milk proteins and products thereof |
US20210388058A1 (en) * | 2018-11-06 | 2021-12-16 | Arhel Projektiranje In Inzeniring D.O.O. | Method for manufacturing highly purified lactoferrin and lactoperoxidase from milk, colostrum and acid or sweet whey |
-
2020
- 2020-10-12 FR FR2010423A patent/FR3115037A1/en active Pending
-
2021
- 2021-10-12 KR KR1020237012380A patent/KR20230086688A/en active Search and Examination
- 2021-10-12 MX MX2023004106A patent/MX2023004106A/en unknown
- 2021-10-12 WO PCT/EP2021/078094 patent/WO2022078976A1/en unknown
- 2021-10-12 JP JP2023521685A patent/JP2023546382A/en active Pending
- 2021-10-12 EP EP21786507.0A patent/EP4225769A1/en active Pending
- 2021-10-12 AU AU2021359742A patent/AU2021359742A1/en active Pending
- 2021-10-12 TW TW110137853A patent/TW202229308A/en unknown
- 2021-10-12 CA CA3194989A patent/CA3194989A1/en active Pending
- 2021-10-12 CN CN202180068653.1A patent/CN116323636A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
FR3115037A1 (en) | 2022-04-15 |
WO2022078976A1 (en) | 2022-04-21 |
KR20230086688A (en) | 2023-06-15 |
CA3194989A1 (en) | 2022-04-21 |
TW202229308A (en) | 2022-08-01 |
AU2021359742A1 (en) | 2023-05-25 |
CN116323636A (en) | 2023-06-23 |
MX2023004106A (en) | 2023-04-27 |
JP2023546382A (en) | 2023-11-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Galanakis | Separation of functional macromolecules and micromolecules: From ultrafiltration to the border of nanofiltration | |
EP0022696B2 (en) | Product enriched by alpha-lactalbumine, its production starting from lactoserum and applications of this product | |
Lee et al. | Protein fouling in carbon nanotubes enhanced ultrafiltration membrane: fouling mechanism as a function of pH and ionic strength | |
Yoshimura et al. | Purification and properties of Pseudomonas aeruginosa porin. | |
Ndiaye et al. | Electroseparation of bovine lactoferrin from model and whey solutions | |
CA1171723A (en) | Extraction process of lactoferrin and immunoglobulins from milk | |
Aravind et al. | Transport studies of BSA, lysozyme and ovalbumin through chitosan/polystyrene sulfonate multilayer membrane | |
US10729156B2 (en) | Methods of purifying exosomes | |
CA2723994C (en) | Angiogenin-enriched milk fractions | |
JP5677512B2 (en) | Reduction of endotoxin in polysialic acid | |
Gómez-Loredo et al. | Insights on the downstream purification of fucoxanthin, a microalgal carotenoid, from an aqueous two-phase system stream exploiting ultrafiltration | |
AU2019374409A1 (en) | Method for manufacturing highly purified lactoferrin and lactoperoxidase from milk, colostrum and acid or sweet whey | |
EP4225769A1 (en) | Method for purifying a cationic protein fraction and fraction thus obtained | |
US20030026845A1 (en) | Process for preparing protein isolate from milk, whey, colostrum, and the like | |
AU2013204858B2 (en) | Improved process for purifying milk proteins and products thereof | |
FR2631785A1 (en) | METHOD FOR FRACTIONING HUMAN MILK PROTEINS, DRIVING PRODUCTION, IN PARTICULAR LACTOFERRIN AND (ALPHA) -LACTALBUMIN, AND PRODUCTS OBTAINED | |
Bankar et al. | Purification and characterization of poly-ε-lysine from Streptomyces noursei NRRL 5126 | |
Acero-Lopez et al. | Characterization of lactoferrin oil-in-water emulsions and their stability in recombined milk | |
JP5261732B2 (en) | Method for producing oligosaccharide containing sialic acid | |
EP2451825A1 (en) | Use of a co-product from a method for extracting lysozyme from egg whites, in order to obtain at least one basic egg white protein | |
Gourley et al. | Identification of casein peptides interacting with polysulfone ultrafiltration membranes | |
KR102161280B1 (en) | Method of isolating extracellular vesicles using semi-dry size-exclusion chromatography | |
Smirnova | Ultrafiltration membranes based on interpolyelectrolyte complexes: Adsorption and mass-exchange properties | |
Hu et al. | Ion exchange adsorption and membrane filtration hybrid process for protein mixture separation | |
TW202328175A (en) | Lactoferrin compositions and methods of use |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: UNKNOWN |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE INTERNATIONAL PUBLICATION HAS BEEN MADE |
|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE |
|
17P | Request for examination filed |
Effective date: 20230315 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
REG | Reference to a national code |
Ref country code: HK Ref legal event code: DE Ref document number: 40089798 Country of ref document: HK |
|
DAV | Request for validation of the european patent (deleted) | ||
DAX | Request for extension of the european patent (deleted) |