WO2009027284A1 - Purification de l'ostéopontine - Google Patents

Purification de l'ostéopontine Download PDF

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Publication number
WO2009027284A1
WO2009027284A1 PCT/EP2008/060886 EP2008060886W WO2009027284A1 WO 2009027284 A1 WO2009027284 A1 WO 2009027284A1 EP 2008060886 W EP2008060886 W EP 2008060886W WO 2009027284 A1 WO2009027284 A1 WO 2009027284A1
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WO
WIPO (PCT)
Prior art keywords
opn
osteopontin
buffer
resin
sodium phosphate
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PCT/EP2008/060886
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English (en)
Inventor
Thierry Ziegler
David Delvaille
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Laboratoires Serono Sa
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Publication of WO2009027284A1 publication Critical patent/WO2009027284A1/fr

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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of protein purification. More specifically, it relates to a process for the purification of osteopontin (OPN), in particular recombinant osteopontin.
  • OPN osteopontin
  • Proteins have become commercially important as drugs that are also generally called “biologicals”.
  • proteins are produced by cell culture, using either mammalian or bacterial cell lines engineered to produce the protein of interest by insertion of a recombinant plasmid containing the gene for that protein. Since the cell lines used are living organisms, they must be fed with a complex growth medium, containing sugars, amino acids, and growth factors. The standards set by health authorities for proteins intended for human administration are very high. Many purification methods for proteins known in the art contain steps requiring the application e.g. of low or high pH, high salt concentration or other extreme conditions that may irreversibly jeopardize the biological activity of the protein to be purified and are therefore not suitable.
  • Anionic exchange chromatography mediated by negatively charged amino acid side chains (e.g. aspartic acid and glutamic acid) interacting with positively charged surfaces
  • cationic exchange chromatography mediated by positively charged amino acid residues (e.g. lysine and arginine) interacting with negatively charged surfaces.
  • Anion exchangers can be classified as either weak or strong.
  • HIC hydrophobic interaction chromatography
  • RPLC reversed phase liquid chromatography
  • the salt in the buffer interacts with water molecules to reduce the solvation of the molecules in solution, thereby exposing hydrophobic regions in the sample molecules which are consequently adsorbed by the HIC column.
  • the more hydrophobic the molecule the less salt needed to promote binding.
  • a decreasing salt gradient is used to elute samples from the column. As the ionic strength decreases, the exposure of the hydrophilic regions of the molecules increases and molecules elute from the column in order of increasing hydrophobicity.
  • Sample elution may also be achieved by the addition of mild organic modifiers or detergents to the elution buffer.
  • Commonly used hydrophobic ligands are phenyl-, butyl- or octyl- residues.
  • Sepharose or Superose which are bothcrosslinked, beaded-forms of agarose, and treating it with a solution of one or more divalent metal ions such as Zn 2+ , Cu 2+ ,
  • OPN is highly acidic and exhibits variation in glycosylation, phosphorylation and sulfatation that give rise to different functional forms.
  • Human OPN is cleaved in vivo by thrombin between amino acids Arg 169 -Ser 170 (Kon et al. 2000). A further conserved thrombin cleavage site is at residues Arg 160 -Gly 161 (Smith et al. 1996). It has been well documented that OPN is very susceptible to proteolytic degradation leading to various fragments and truncated forms of OPN (Maeda et al. 2001 ;Maeda et al. 2001 ).
  • Human and rat OPN are cleaved by Matrix-Metalloproteinase-3 (Stomelysin-1 ) and Matrix-Metalloproteinase-7 (Matrilysin).
  • Human OPN cleavage sites for Stomelysin-1 and Matrilysin are between residues Gly 166 -Leu 167 (GIy 151 - Leu 152 in rat OPN), Ala ⁇ -Tyr 202 and Asp 210 -Leu 211 (Agnihotri et al. 2001 ).
  • OPN is a substrate for enterokinase (Giachelli et al. 1995).
  • OPN-a corresponds to the protein encoded by all known exons of the OPN gene
  • OPN-b is missing exon IV of the OPN gene (corresponding to residues 59-73 of the OPN-a protein)
  • OPN-c is missing exon III of the OPN gene (corresponding to residues 30-58 of the OPN-a protein) (He et al. 2006).
  • One embodiment of the invention is a process for the purification of OPN, preferably recombinant OPN, from cell culture supernatant.
  • Another embodiment of the invention relates to a process for purification of OPN, such as recombinant OPN, wherein before the process for purification according to the above embodiment a capture step is performed wherein the cell culture supernatant is subjected to IEC to produce an eluate, wherein the IEC comprises using a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE), preferably quaternary ammonium or DEAE, more preferably DEAE.
  • a capture step wherein the cell culture supernatant is subjected to IEC to produce an eluate
  • the IEC comprises using a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE), preferably quaternary ammonium or DEAE, more preferably DEAE.
  • Even another embodiment of the invention is a pharmaceutical composition comprising OPN, wherein the pharmaceutical composition is obtainable by a method of manufacturing according to the embodiments described herein, and wherein not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% of the OPN is truncated OPN.
  • Yet another embodiment of the invention is a pharmaceutical composition obtainable by a method of manufacturing according to the embodiments described herein, wherein at least 60%, or 65%, or 70%, or 75%, or 78% is full length osteopontin.
  • the invention relates to a process for the purification of OPN, such as recombinant OPN, comprising: a) subjecting a solution containing OPN, such as recombinant OPN, to immobilized metal ion affinity chromatography (IMAC) to produce a first eluate; b) subjecting the first eluate to hydrophobic interaction chromatography (HIC) to produce a second eluate; and c) subjecting the second eluate to ion exchange chromatography (IEC) to produce a third eluate.
  • IMAC immobilized metal ion affinity chromatography
  • HIC hydrophobic interaction chromatography
  • IEC ion exchange chromatography
  • the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the first, second, third or fourth embodiment of the invention, wherein the hydrophobic interaction chromatography (HIC) is performed on a resin comprising butyl or phenyl, preferably butyl, residues.
  • HIC hydrophobic interaction chromatography
  • the novel process for purification preferably features washing and elution during the different purification steps at a pH equal or above 3.0.
  • the invention relates to a process for the purification of OPN, such as recombinant OPN, according to the first, second, third, fourth, fifth or sixth embodiment of the invention, wherein the elution step during ion affinity chromatography (IMAC) is done in imidazol, or is done in a sodium phosphate, sodium acetate or sodium citrate buffer having a pH of 2.0 to 6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1 ; more preferred in a sodium phosphate buffer (50 mM) having a pH of 2.0 to 6.0, preferably 3.0 to 5.0, more preferred 3.5 to 4.5, and even more preferred 3.9 to 4.1.
  • IMAC ion affinity chromatography
  • step (b) comprises i) washing with an ammonium, sodium or kalium sulphate or TRIS buffer at pH of 6.0 to 8.0, preferably 6.9 to 7.1 ; more preferred with a buffer containing sodium phosphate at a molarity of 5 to 50.5 mM, preferably 19.8 to 20.2 mM, and ammonium sulphate at a molarity of 990 to 1500 mM, more preferred 990 to 1010 mM, the buffer having a conductivity of 130-140 m
  • the invention relates to a process for purification of OPN, such as recombinant OPN, wherein before the process for purification according to the first, second, third, fourth, fifth, sixth or eighth embodiment an IEC capture step is performed comprising subjecting cell culture supernatant, such as cell culture supernatant from mammalian cell culture, e.g.
  • a column comprising quaternary ammonium, diethylaminoehtyl (DEAE) or triethylaminoethyl (TMAE) coupled to a resin of suitable material known in the art, including but not limited to crosslinked, beaded-forms of agarose (e.g. Sepharose or Superose), wherein the beads preferably have a diameter of 20 to 100 ⁇ m, more preferred a diameter of 30 to 90 ⁇ m; modified methacrylate polymers (e.g. tentacle, hydroxylated); silica; ceramic and styrene divinylbenzene.
  • agarose e.g. Sepharose or Superose
  • modified methacrylate polymers e.g. tentacle, hydroxylated
  • silica ceramic and styrene divinylbenzene.
  • Even another embodiment of the invention is a pharmaceutical composition comprising osteopontin as active ingredient, wherein the pharmaceutical composition is obtainable by a method of manufacturing according to the ninth, tenth of eleventh embodiment, and wherein not more than 40%, more preferred not more than 35%, 30% or 25%, even more preferred not more than 22% and most preferred not more than 20% of the OPN is truncated OPN.
  • Even another embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising OPN as active ingredient, wherein at least 60%, or 65%, or 70%, or 75%, or 78% of OPN is full length OPN, and further comprising a pharmaceutically acceptable carrier, solvent or excipient.
  • purified denotes that the indicated molecule is present in the substantial absence of other biological macromolecules, e.g., polynucleotides, proteins, and the like.
  • solution containing OPN refers to a liquid composition of matter comprising OPN at least partially in solution.
  • the liquid composition may comprise substances other than OPN, wherein the substances are either in solution or not.
  • a solution containing OPN may be any body fluid, including but not limited to blood, serum, urine, liquor, synovial fluid or milk of an animal, preferably a mammal and most preferred a human.
  • One particular solution containing OPN is cell culture supernatant containing recombinant OPN, including but not limited to cell culture supernatant from yeast (e.g.
  • Cell culture supernatant containing OPN refers to cell culture supernatant, wherein OPN is at least partially in solution.
  • Cell culture supernatant containing OPN may be from mammalian cell culture.
  • Mammalian cell culture systems are known in the art and make use, e.g. of NSO, SP2.0, 3T3 cells, COS cells, human osteosarcoma cells, MRC-5 cells, BHK cells, VERO cells, CHO cells, rCHO-tPA cells, rCHO-Hep B Surface Antigen cells, CHO-S cells, HEK 293 cells, rHEK 293 cells, rC127-Hep B Surface Antigen cells, human fibroblast cells, Stroma cells, hepatocyte cells or PER.C6 cells.
  • the active protein(s) can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle (e.g. water, saline, dextrose solution) and additives that maintain isotonicity (e.g. mannitol) or chemical stability (e.g. preservatives and buffers).
  • a pharmaceutically acceptable parenteral vehicle e.g. water, saline, dextrose solution
  • additives that maintain isotonicity e.g. mannitol
  • chemical stability e.g. preservatives and buffers.
  • a “therapeutically effective amount” is amount of OPN that when administered to a patient in need of treatment with OPN, such as e.g. a patient suffering from a neurological disorder, the neurological disorder including without limitation multiple sclerosis, stroke, a neurodegenerative disorder or a peripheral nervous system such as peripheral neuropathy including without limitation diabetic neuropathy, the amount of OPN results in an improvement of the disorder un that patient vis-a-vis a patient who did not receive a therapeutically effective amount of OPN.
  • An improvement of the disorder can be measured by methods known in the art, the methods including the measurement of laboratory parameters taken from blood, urine or cerebrospinal fluid, the measurement of the functional status, pain, or disability; e.g. of a patient suffering from multiple sclerosis, stroke, a neurodegenerative disorder or a peripheral nervous system such as peripheral neuropathy including without limitation diabetic neuropathy; the methods also including imaging such as magnetic resonance imaging (MRI) or X-ray.
  • MRI magnetic resonance imaging
  • the dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including OPN pharmacokinetic properties, the route of administration, patient conditions and characteristics (sex, age, body weight, health, size), extent of symptoms, concurrent treatments, frequency of treatment and the effect desired. Adjustment and manipulation of established dosage ranges are well within the ability of those skilled in the art, as well as in vitro and in vivo methods of determining the effect of OPN in an individual.
  • OPN may be used in amounts in the ranges of 0.001 to 100 mg/kg or 0.01 to 10 mg/kg or body weight, or 0.1 to 5 mg/kg of body weight or 1 to 3 mg/kg of body weight or 2 mg/kg of body weight.
  • OPN may be administered daily or every other day or three times per week or once per week, at similar doses, or at doses increasing or decreasing with the time.
  • the daily doses are usually given in divided doses or in sustained release form effective to obtain the desired results.
  • Second or subsequent administrations can be performed at a dosage which is the same, less than or greater than the initial or previous dose administered to the individual.
  • a second or subsequent administration can be administered during or prior to onset of the disease.
  • OPN may be administered prophylactically or therapeutically to an individual prior to, simultaneously or sequentially with other therapeutic regimens or agents (e.g. multiple drug regimens), in therapeutically effective amounts.
  • OPN purified in accordance with the present invention may be used for preparation of a medicament for treatment and/or prevention of disorders, in particular human disorders .
  • OPN may be used for treatment and/or prevention of neurological disorders, including peripheral and central nervous system disorders, in particular multiple sclerosis, stroke and peripheral neuropathies such as diabetic neuropathy.
  • the level of truncated forms of OPN was assessed using a RP-HPLC technique.
  • the culture supernatant containing OPN was loaded on a Vydac 5 ⁇ m column and eluted with a gradient of Trifluoroacetic acid (TFA) in water and TFA in acetonitrile.
  • TFA Trifluoroacetic acid
  • the quantification of OPN was performed by running a standard composed of purified OPN-a at a known concentration (measured using a reference technique such as amino-acid analysis).
  • An example of RP-HPLC chromatogram of purified OPN according to Example 3 is shown in Fig. 2.
  • the truncated forms were eluted as the shoulder prior to the main peak which represents intact OPN.
  • the cell culture suspension of a high cell density culture of CHO cells transfected to express OPN that was grown in a stirred tank bioreactor was first clarified on a depth filter (Millipore Millistack or equivalent) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences).
  • Loading is done without pH or conductivity adjustment.
  • the column was washed with a sodium phosphate buffer (5OmM) the buffer with a conductivity of about 13 mS/cm and a pH of 7.2.
  • a sodium phosphate buffer (5OmM) the buffer with a conductivity of about 13 mS/cm and a pH of 7.2.
  • OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.
  • Table 1 Summary of 3 captures of OPN harvests performed at both small and production scale
  • the capture step used in this example has shown a reproducible yield in intact OPN of almost 100%. Furthermore, the level of truncated forms in the eluate is decreased from 49-52% to 34-43%.
  • This example describes the use of a weak anion exchange resin to capture OPN from cell culture supernatant comprising serum-free medium and cell-derived contaminants.
  • the chromatographic step presents several advantages: ⁇ a high capacity of at least 13 grams of OPN per liter of resin;
  • This example describes the purification of OPN from the capture step (as described in Example 2), OPN is purified to a degree that is suitable for injection into human.
  • 3 purification runs were performed at 150 mg scale using the eluate from the capture step.
  • the average of 6 runs performed at production scale is shown.
  • the purification of OPN is composed of 3 chromatographic steps and one concentration/ultrafiltration using tangential flow systems.
  • the eluate from the first step is further purified by hydrophobic interaction chromatography (HIC).
  • HIC hydrophobic interaction chromatography
  • the eluate from the IMAC step is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate then loaded into a column with 6.6 L of resin Super Butyl 550C (Tosoh Biosciences).
  • the column is washed with a sodium phosphate buffer (20 mM) containing ammonium sulphate (1 M) at pH 7.0.
  • OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0.
  • the eluate from the second step is further purified by ion exchange chromatography (IEC).
  • IEC ion exchange chromatography
  • the eluate from the Super Butyl 550C resin is loaded directly into an ion exchange resin Q Sepharose Fast Flow (G. E. Biosciences).
  • the column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0.
  • OPN is eluted in sodium phosphate buffer (50 mM) with 0.4 M NaCI at pH 7.0.
  • the eluate from the QSFF is concentrated and the buffer exchanged to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml.
  • Pre-treated milk will then be clarified on a depth filter (Millipore Millistack ) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences).
  • a depth filter Micropore Millistack
  • a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences).
  • Loading will be done with or without pH or conductivity adjustment.
  • the column was washed with a sodium phosphate buffer (5OmM) with a conductivity of about 13 mS/cm and a pH of 7.2.
  • 5OmM sodium phosphate buffer
  • OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.
  • the eluate from the capture step is loaded onto a column packed with Nickel
  • the eluate from the IMAC step is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate and then loaded onto a column with Super Butyl
  • OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0.
  • the eluted material is loaded directly onto a ion exchange resin Q Sepharose Fast Flow (G. E. Biosciences).
  • the column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0.
  • OPN is eluted in sodium phosphate buffer (50 mM) with 0.4 M NaCI at pH 7.0.
  • Post-QSFF is concentrated and buffer exchange to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml.
  • Example 5 Purification of OPN from urine
  • OPN is purified from human urine. OPN is first captured and stabilized by removing contaminants that may impact the integrity of the molecule and to reduce the volume. Peferably, a salt-precipitation step is performed to remove some major contaminant proteins (e.g. Tamm-Horsfall protein) prior to capture and purification.
  • contaminant proteins e.g. Tamm-Horsfall protein
  • Pre-treated urine will then be clarified on a depth filter (Millipore Millistack) and loaded onto a chromatographic column filled with a weak anion exchange resin (DEAE Sepharose Fast Flow, G. E. Biosciences). Loading will be done with or without pH or conductivity adjustment.
  • the column was washed with a sodium phosphate buffer (5OmM) the buffer with a conductivity of about 13 mS/cm and a pH of 7.2.
  • OPN is eluted from the column with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.
  • the eluate (treated or not) is then loaded onto a column packed with Nickel
  • Chelating Sepharose Fast Flow (Ni 6 SFF, G. E. Biosciences).
  • the column is washed with a sodium phosphate buffer (50 mM) containing 275 mM NaCI with a conductivity of about 31 mS/cm at pH 7.2.
  • OPN is then eluted in sodium phosphate buffer (50 mM) at acidic pH (e.g. pH 4.0).
  • the eluate is adjusted to a conductivity of 130-140 mS/cm using ammonium sulfate then loaded onto a column with SuperButyl 550C (Tosoh Biosciences).
  • the column is washed with a sodium phosphate buffer (20 mM) containing ammonium sulphate (1 M) at pH 7.0.
  • OPN is eluted in sodium phosphate buffer (20 mM) without salts at pH 7.0.
  • the eluted material is loaded directly onto a ion exchange resin Q
  • Sepharose Fast Flow G. E. Biosciences
  • the column is washed with a sodium phosphate buffer (50 mM) containing sodium chloride (200 mM) at pH 7.0.
  • OPN is eluted in sodium phosphate buffer (50 mM) with 0.4M NaCI at pH 7.0.
  • the eluate from the QSFF is concentrated and buffer exchange to remove NaCI. Typical final concentrations ranged from 10 to 100 mg/ml.

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Abstract

L'invention porte sur un procédé de purification de l'ostéopontine. Le procédé selon l'invention emploie trois principes de purification différents : une chromatographie d'affinité avec des ions métalliques immobilisés (IMAC), une chromatographie d'interactions hydrophobes (HIC) et une chromatographie d'échange d'ions (IEC). L'invention porte également sur un procédé de fabrication d'une composition pharmaceutique comprenant de l'ostéopontine à l'aide du procédé de purification de l'ostéopontine.
PCT/EP2008/060886 2007-08-31 2008-08-20 Purification de l'ostéopontine WO2009027284A1 (fr)

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EP07115465.2 2007-08-31
EP07115465 2007-08-31
US96749807P 2007-09-05 2007-09-05
US60/967,498 2007-09-05

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018098001A1 (fr) * 2016-11-27 2018-05-31 Triton Algae Innovations, Inc. Procédé de purification d'ostéopontine de recombinaison à partir de microalgues
US10548957B2 (en) 2012-09-28 2020-02-04 Dana-Farber Cancer Institute, Inc. Targeted expansion of Qa-1-peptide-specific regulatory CD8 T cells to ameliorate arthritis
US10596195B2 (en) 2010-10-22 2020-03-24 Dana-Farber Cancer Institute, Inc. Discovery of regulatory T cells programmed to suppress an immune response
AU2015308899B2 (en) * 2014-08-27 2020-05-28 Dana-Farber Cancer Institute, Inc. Intracellular osteopontin regulates the lineage commitment of lymphoid subsets
WO2024056840A1 (fr) 2022-09-16 2024-03-21 Univerza V Ljubljani Isolement de l'ostéopontine et du glycomacropeptide à partir du lactosérum

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001049741A2 (fr) * 2000-01-07 2001-07-12 Arla Foods Amba Procede d'isolation d'osteopontine a partir du lait

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WO2001049741A2 (fr) * 2000-01-07 2001-07-12 Arla Foods Amba Procede d'isolation d'osteopontine a partir du lait

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Title
AZUMA ET AL: "A rapid method for purifying osteopontin from bovine milk and interaction between osteopontin and other milk proteins", INTERNATIONAL DAIRY JOURNAL, ELSEVIER APPLIED SCIENCE, BARKING,, GB, vol. 16, no. 4, April 2006 (2006-04-01), pages 370 - 378, XP005239396, ISSN: 0958-6946 *
BAYLESS K J ET AL: "Isolation and Biological Properties of Osteopontin from Bovine Milk", PROTEIN EXPRESSION AND PURIFICATION, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 9, no. 3, April 1997 (1997-04-01), pages 309 - 314, XP004466987, ISSN: 1046-5928 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10596195B2 (en) 2010-10-22 2020-03-24 Dana-Farber Cancer Institute, Inc. Discovery of regulatory T cells programmed to suppress an immune response
US10548957B2 (en) 2012-09-28 2020-02-04 Dana-Farber Cancer Institute, Inc. Targeted expansion of Qa-1-peptide-specific regulatory CD8 T cells to ameliorate arthritis
US11590213B2 (en) 2012-09-28 2023-02-28 Dana-Farber Cancer Institute, Inc. Targeted expansion of Qa-1-peptide-specific regulatory CD8 T cells to ameliorate arthritis
AU2015308899B2 (en) * 2014-08-27 2020-05-28 Dana-Farber Cancer Institute, Inc. Intracellular osteopontin regulates the lineage commitment of lymphoid subsets
US11371989B2 (en) 2014-08-27 2022-06-28 Dana-Farber Cancer Institute, Inc. Intracellular osteopontin regulates the lineage commitment of lymphoid subsets
WO2018098001A1 (fr) * 2016-11-27 2018-05-31 Triton Algae Innovations, Inc. Procédé de purification d'ostéopontine de recombinaison à partir de microalgues
US10954280B2 (en) 2016-11-27 2021-03-23 Triton Algae Innovations, Inc. Method of purification of recombinant osteopontin from micro algae
WO2024056840A1 (fr) 2022-09-16 2024-03-21 Univerza V Ljubljani Isolement de l'ostéopontine et du glycomacropeptide à partir du lactosérum

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