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WO1999033860A1 - Procede de liaison d'albumine et systeme destine a etre utilise dans ledit procede - Google Patents

Procede de liaison d'albumine et systeme destine a etre utilise dans ledit procede Download PDF

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Publication number
WO1999033860A1
WO1999033860A1 PCT/SE1998/002468 SE9802468W WO9933860A1 WO 1999033860 A1 WO1999033860 A1 WO 1999033860A1 SE 9802468 W SE9802468 W SE 9802468W WO 9933860 A1 WO9933860 A1 WO 9933860A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
anyone
albumin
binding
conjugated partner
Prior art date
Application number
PCT/SE1998/002468
Other languages
English (en)
Inventor
Åke PILOTTI
Tor Regberg
Christel ELLSTRÖM
Charlotta Lindqvist
Ann Eckersten
Lars FÄGERSTAM
Original Assignee
Amersham Pharmacia Biotech Ab
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from US09/001,940 external-priority patent/US5994507A/en
Priority claimed from SE9800879A external-priority patent/SE9800879D0/xx
Application filed by Amersham Pharmacia Biotech Ab filed Critical Amersham Pharmacia Biotech Ab
Priority to EP98965360A priority Critical patent/EP1044214A1/fr
Priority to CA002316766A priority patent/CA2316766A1/fr
Priority to JP2000526535A priority patent/JP2001527090A/ja
Priority to AU20834/99A priority patent/AU2083499A/en
Publication of WO1999033860A1 publication Critical patent/WO1999033860A1/fr

Links

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
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA

Definitions

  • Albumin-binding ligands attached to a solid phase have been used for the removal of albumin from liquid samples mainly for two purposes: a) purification of albumin and b) further processing of the liquid samples in the absence of albumin.
  • the step involving binding to an albumin ligand has often been combined with other steps including ion exchange and binding based on hydrophobic interaction. Both batch-wise and chromatographic processes have been described.
  • Albumin-binding ligands in soluble form have been used for desorption of albumin adsorbed to a matrix via an albumin- binding ligand (e.g. regeneration of adsorbents).
  • a first aspect of the invention provides a method for binding albumin by contacting an aqueous liquid containing an albumin with an albumin binding compound which comprises the structure (scaffold)
  • m is zero or 1. represents that the conjugated partner is replacing a hydrogen in A, B, or -L-G.
  • This aspect of the invention may be used for the removal or purification of albumin from a liquid sample.
  • a liquid sample containing albumin is contacted with a conjugate according to formula II in which m is equal to 1 and the conjugated partner is a carrier (matrix) that is soluble, insoluble or insolubilizable in aqueous liquid media.
  • the conditions (pH, ionic strength, temperature, etc.) for adsorbing/desorbing should be non- denaturing for albumin with respect to irreversible denaturation in particular.
  • Soluble carriers may be insolubilized after the binding step in order to facilitate physical separation of the complex between albumin and the ligand-carrier conjugate from the medium. Insolubilization steps typically take place before any release step.
  • a subaspect of the present invention is a method of obtaining samples that are free of one or more of the albumins mentioned above, for instance for the purification of compounds other than the typical albumins as defined above. Release and washing steps may be included as in conventional purification of albumins in order to be able to reuse the ligand-carrier material.
  • the aromatic rings may typically comprise one, two or three heteroatoms providing at least one free electron pair and are selected from among oxygen, nitrogen or sulphur.
  • the aromatic rings may be fused to other aromatic or non-aromatic rings each of which may have heteroatoms as discussed above.
  • the two groups containing the aromatic rings are represented by parts A and B. These parts are often interchangeable .
  • the 5- or 6-membered aromatic ring may be represented by the formula:
  • the link from the aromatic ring to the scaffold I is through replacement of a hydrogen in D or of one of Ri and R 2 , or one of R 3 and R 4 .
  • a link to the scaffold I through replacement of one of R 3 and R 4 is only possible provided that R 3 and R 4 do not define a bivalent structure that is part of a ring fused to the aromatic ring of formula III.
  • the aromatic ring systems defined by formula III include phenyls, 1- and 2-naphthyls, 1- and 2-thienyls, 2-, 3- and 4-pyridyls, 2-, 3- and 4-quinolyls, 1-, 3- and 4- isoquinolyls, 2- and 3-indolyls, 2- and 3-furanyls, 1-, 2- and 3-pyrrolyls etc.
  • Ri and R 2 may be selected from: a. hydrogen (no replacement), alkyl, aryl, alkoxy, aryloxy and their thio analogues, typically a C]__]_g alkyl or 05-15 aryl groups optionally substituted with one or more halo groups, e.g. CF3-, CH3-, phenyl etc; b. halo, such as fluoro or chloro or bromo; c. nitro; d. cyano, carboxamido (-CONH2) and carboxy (-COOH) .
  • Groups such as N-substituted carboxamido (-CONH2) with one or two amino hydrogens replaced with hydrocarbyl and hydrocarbyl esters and salts of carboxy, are included in carboxamido and carboxy, respectively.
  • Typical hydrocarbyls are C]__]_Q alkyl, such as arylalkyl or unsubstituted alkyl, or alkylaryl or unsubstituted aryl, for instance containing 5-15 carbons.
  • Aryl groups may include phenyl, 1- and 2- naphthyls, 1-, 2- or 3-pyridyls etc. e.
  • L is an organic structure and may be - ⁇ (CH2) n ( )m' ( H2)n' _ where the left and right free valences bind to the right carbonyl group of the scaffold and to the group G, respectively.
  • X may be oxygen, sulphur or NH with the hydrogen preferably being replaced with a methyl group or a C2-10 alkyl.
  • n and n 1 are integers 0-3 and m' is an integer 0 or 1 with the proviso that n+n'+m 1 is 1, 2 or 3.
  • One or more of the hydrogen atoms in a CH2 _ group of the linker may be replaced with a C]__ ⁇ o alkyl group, or a hydroxy, a carboxy or an amino group or any other group containing a functional group enabling further derivatization and linking to a carrier .
  • albumin binders in which X is NH with the hydrogen being replaced as suggested in the preceding paragraph and/or one or more of the CH2 ⁇ groups being substituted with a methyl and/or some other group as suggested in the preceding paragraph.
  • a preferred linker chain of the invention has substituents on the linker L so that rotation around bonds in the linker chain is hindered. Other means for hindering rotation in this part of the molecule may have similar effects on the affinity for albumin, for instance divalent groups bridging a position in L-G with a position in A or B or in the scaffold.
  • m is 1.
  • the conjugated partner is linked to an albumin-binding ligand as defined in formula II via a bridge.
  • the bridge may derive wholly or partly from the ligand or from the conjugated partner. For the sake of simplicity the bridge will be discussed as an inherent part of the conjugated partner, unless otherwise specified.
  • the conjugated partner may be attached to the ligand at a position in group A, B or L-G. It is preferred to have the conjugated partner attached (a) at a functional group in the linker L as suggested above, or
  • the bridge may thus have: -CH 2 -CH 2 -, -CH 2 NH-, -NHCH 2 -, -CH 2 S-, -SCH 2 -, -CH 2 0-, or - OCH 2 - next to the aromatic ring of part A or B.
  • conjugate in organic chemistry and biochemistry is well known and encompasses two or more compounds which are linked together covalently so that properties from each compound are retained in the conjugate.
  • conjugate means that an albumin-binding ligand as defined in formula II
  • conjugated partner is covalently linked (conjugated) to a compound (conjugated partner) that has a property that is retained in the conjugate.
  • the conjugated partner may render the conjugate soluble, insoluble or insolubilizable in the media concerned, analytically detectable, reactive against a specified target such as a biospecific counterpart etc.
  • the conjugated partner (carrier) may be insoluble, insolubilizable or soluble in the liquid media concerned.
  • Typical media are aqueous, including water possibly containing water-miscible organic liquids, and other liquid media in which binding to albumin may take place.
  • Typical carriers are based on organic or inorganic polymers which may be of synthetic or biological origin (biopolymers) .
  • Insoluble carriers may be of the same kind as the carriers used as support in chromatography . Suitable insoluble carriers may be of various physical forms such as monoliths, particles, tube walls etc. The carriers may be porous or non-porous .
  • the carrier may contain density controlling filler material (particles) embedded in a polymer.
  • density controlling filler material particles embedded in a polymer.
  • Well known hydrophilic organic insoluble carriers are polymers which have on their liquid contact surface a plurality of hydrophilic groups, for instance hydroxy and/or amino and/or carboxy.
  • hydrophobic carriers that have been hydrophilized (e.g. coated with a hydrophilic compound) on outer and inner (pore) surfaces.
  • Typical hydrophobic insoluble carriers are based on styrene-divinyl-benzene polymer ' s, poly (alkyl methacrylates), polymers of perfluoro hydrocarbons (PFC) etc.
  • Inorganic variants of carriers may be based on materials such as glass, zeolites, silica, composites, zirconium oxide, etc.
  • Typical examples of carriers that are soluble in aqueous media as defined above are water soluble polymers, such as dextran.
  • the conjugated partner may contain an analytically detectable label, such as an enzymatically active moiety, a fluorophor/fluorogen and a chromophor/chromogen etc. a moiety giving the conjugate a predetermined reactivity, such as biotin, or a chemically reactive group.
  • Analytically detectable conjugates may be useful in an assay such as immunoassay methods.
  • Conjugates with a predetermined reactivity such as biotin or a chemically reactive group will allow introduction of albumin-binding structures containing the scaffold I onto various types of other carriers, for instance for use in the above-mentioned methods for removal of albumin.
  • a predetermined reactivity such as biotin or a chemically reactive group
  • conjugated partners normally result in soluble conjugates.
  • the conjugate of the second aspect of the invention has the formula (II):
  • the conjugated partner is a polymeric carrier.
  • the conjugated partner is linked to the ligand either at the A- or B-part or at L.
  • the preferred variants are those that are preferred for use in the first aspect of the invention.
  • a third aspect of the invention are new albumin binders having been obtained by changing one or more structural elements of the structure of an albumin binder complying with formula I, in particularly with formula II. This can be accomplished, for instance, by creating and screening chemical libraries in which the members differ around one structural elements of an albumin binder complying with formula I, in particular formula II. Structural elements to be changed may be the scaffold I, or part A, part B or L-G of the presently claimed albumin binders. Typically at least one of these elements is retained in an albumin binder according to this aspect of the invention.
  • a chemical library is contemplated a collection (an array) of two, three or more structurally different compounds.
  • the procedure comprises the steps:
  • the ligand to be assayed was dissolved in PBS and mixed with HSA (100 mM HSA in PBS) .
  • the volume of the solution was selected so that the ratio between the ligand and HSA was 5:1 with final concentrations were for ligand 50 ⁇ M in 10 ⁇ M HSA (human serum albumin) .
  • the free ligand not bound to HSA was then removed by rapid passage through a HITRAP desalting column (SEPHADEX G25; Pharmacia Biotech AB, Uppsala, Sweden) .
  • the void fraction from the desalting column containing HSA and possible ligand complexed to HSA were collected and analysed by reverse phase chromatography (RPC) on HISEP 4.6/50 (SUPELCO, U.S.A. ) .
  • the result from the RPC step may be influenced by factors such as variation in ligand concentrations in the original ligand sample and differences in extinction coefficient for different ligands.
  • FPLC System equipped with a HITRAP desalting column (SEPHADEX G25) (PHARMACIA BIOTECH AB, Uppsala) .
  • Sample dilution buffer and buffer A in FPLC PBS (0.05 M Phosphate, 0.15 M NaCl, pH 7.0).
  • Instant buffer for gel filtration MIKROKEMI AB, Uppsala, Sweden
  • Buffer B in FPLC Buffer A + 20 % (volume) acetonitrile . Buffer A was used for the gel filtration step and buffer B was used to regenerate the HITRAP column.
  • a screening library (Screening Library 1) was set up in order to screen for ligands that have affinity for IgG. No efficient IgG binding ligands were found. Since the library was at hand it was also checked for albumin binders.
  • Screening Library 1 was constructed from four different oxazolones :
  • the products in the library array were not further purified. Final yields were typically around 80%.
  • the crude products (ligand samples) thus sometimes consisted of a mixture of final and/or intermediate products and/or starting materials.
  • Reference Compound 1 is compound 23 in table 4.
  • the library members that were positive for binding to HSA were also checked for binding to human IgG, lysozyme and human insulin. Directed libraries and sublibraries were then constructed in order to map the Reference Compound 1-motif.
  • Compounds containing handles and attachment points for carriers were synthesized based on similar conditions to those used for the synthesis of the original library. Examples - Synthesis.
  • the particular ligand sample containing Reference Compound 1 was obtained by reaction of 3- (2-thienyl) -oxazolone with N-methyl-indole-3-aldehyde followed by subsequent ring- opening with ephedrine.
  • HSA human serum albumin
  • ligand sample contained at least four different compounds, one of which showed reactivity towards HSA.
  • the ligand sample as such was therefore subjected to preparative RPC and the four compounds were isolated and examined by mass spectrometry . It was determined that the molecular weight of the compound binding to HSA had a molecular weight of 473. In separate experiments two compounds with Mw 473, derivable from the reaction mixture and having NMR spectra suggesting they were the E and Z isomer, respectively were studied. Only Reference Compound 1 was active in binding to HSA. The results suggested that Z isomer was active in binding to albumin. No conclusive results have so far been obtained for the E-isomer.
  • [TL]t [TL(i; + tTL(2)] -kd ⁇ ss2*t
  • TL(1) and TL(2) denote the two types of complexes with the apparent dissociation rate constants kdissl and kdiss2, respectively, and t denotes the time from start of dissociation.
  • Reference Library AN1001 described in WO-A-9622529 was used as a reference library. It is an array based on oxazolones and contains 8000 compounds. It is a general library having members represented by formula II with known pharmacophore structures, usually aromatics, as groups A, B and L-G. Due to difficulties in solubilizing many of the members, it was never completely screened for albumin binders. The library was only used as a source for selecting interesting structures to be tested for binding to serum albumin.
  • the main objective of the synthetic design-work around the aromatic ring in the starting oxazolone (A-group) was to introduce a handle for attachment of the albumin ligand to a matrix.
  • A-group analogues see Examples - Synthesis.
  • Reference library Tested compounds that have affinity to serum albumin
  • A 2-naphthyl: 2,4-difluoro phenyl, 3-fluoro phenyl,
  • A 4-fluoro phenyl, 2-methyl phenyl.
  • A 2-thienyl: 3-fluoro phenyl, 4-fluoro phenyl, 1- naphthyl .
  • A 4-trifluoromethyl phenyl: 2,4-difluoro phenyl, 3- fluoro phenyl, 4-fluoro phenyl, 2-methyl phenyl,
  • A 3-methyl phenyl: 3-fluoro phenyl, 4-fluoro phenyl,
  • the characterization was performed on a JEOL ECLIPSE- 270MHz NMR. The samples were run in 5mm: s probes and the substances were dissolved in CDC1 3 or DMSO- 6 . TMS was used as an internal standard. TLC was run on MERCK KIESELGEL F 254 and was eluted with ethylacetate and then developed under UV-light (254 nm) . HPLC was performed on a SMART System on a SUPELCO HISEP column.
  • 2-Phenyloxazolone 2.5g (16mmol) was dissolved together with 2.42g (l ⁇ mmol) 1-naphthaldehyde in 15ml toluene in a screw- cap tube.
  • Triethylamine (1.0ml) was added and the closed tube placed on a heating block at 70°C over night (17h) .
  • the solvent was evaporated with heat and nitrogen and the reaction mixture was dissolved in hot ethyl acetate and a few drops of methanol and then cooled.
  • the crystals that grew from the solution were characterized to be the product by 1 H NMR.
  • the oxazolone is mixed with the amine and the solvent in a screw-cap tube.
  • the tube is placed on a heating block over night (18h) and then the solvent is evaporated with heat and/or nitrogen.
  • the synthesized products were not purified further, but used as they were.
  • the raw products were analyzed with HPLC, TLC and some of them with H NMR and ESMS.
  • the amines used are provided above, under the heading "Variations in the L-G-part".
  • the solvent, temperature and addition of triethyl amine are given in Tables 4-5.
  • A 2- (4-nitro-phenyl) oxazolones. This compound was prepared by acetic anhydride cyclization of 4-nitrohippuric acid.
  • Bl Introduction of l-methylindol-3-yl as ring system B and of structure -L-G by oxazolone ring opening with (-)-(lR, 2S)- ephedrine . These two steps were carried out as described above for other oxazolones, aldehydes and amines, the aldehyde now being l-methylindol-3-aldehyde .
  • Example 3B2 SnCl2 reduction of the ring-opened product of Example 3B2.
  • the compound obtained in Example 3B2 was reduced with SnCl2. Al .3.
  • the compound obtained in Example 3B3 was reduced with SnCl2.
  • the compound obtained in Example 3B1 was reduced with H 2 on Pd/C.
  • Example 4A1.1 Bl.Acylation of an 4-amino phenyl group in part A.
  • the product from Example 4A1.1 was acylated with acetic acid anhydride.
  • EAH SEPHAROSE 4B epoxy activated agarose that has been reacted with 1-6-diamino-hexane
  • ECH SEPHAROSE epoxy activated agarose that has been reacted with 1-6-diamino-hexane

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Un procédé de liaison d'albumine consiste à mettre en contact un liquide aqueux contenant une albumine avec un composé de liaison d'albumine sélectionné parmi des composés de liaison d'albumine contenant le squelette -CO-NH-C(=C-)-CO-, et des conjugués qui sont capables de lier l'albumine et qui comprennent le squelette -CO-NH-C(=C-)-CO-. On décrit également un composé d'albumine qui a été produit au moyen du changement de la structure du liant d'albumine comportant le squelette -CO-NH-C(=C-)-CO-.
PCT/SE1998/002468 1997-12-31 1998-12-30 Procede de liaison d'albumine et systeme destine a etre utilise dans ledit procede WO1999033860A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP98965360A EP1044214A1 (fr) 1997-12-31 1998-12-30 Procede de liaison d'albumine et systeme destine a etre utilise dans ledit procede
CA002316766A CA2316766A1 (fr) 1997-12-31 1998-12-30 Procede de liaison d'albumine et systeme destine a etre utilise dans ledit procede
JP2000526535A JP2001527090A (ja) 1997-12-31 1998-12-30 アルブミンを結合する方法およびその方法で使用すべき手段
AU20834/99A AU2083499A (en) 1997-12-31 1998-12-30 Method for binding albumin and means to be used in the method

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US09/001,940 US5994507A (en) 1997-12-31 1997-12-31 Method for binding albumin and means to be used in the method
US09/001,940 1997-12-31
SE9800879A SE9800879D0 (sv) 1998-03-17 1998-03-17 A method for binding albumin and the novel means to be used in the method
SE9800879-0 1998-03-17

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WO1999033860A1 true WO1999033860A1 (fr) 1999-07-08

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EP (1) EP1044214A1 (fr)
JP (1) JP2001527090A (fr)
AU (1) AU2083499A (fr)
CA (1) CA2316766A1 (fr)
WO (1) WO1999033860A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037501A1 (fr) * 1998-12-22 2000-06-29 Amersham Pharmacia Biotech Ab Elimination/purification d'albumines seriques
US9250211B2 (en) 2010-12-30 2016-02-02 Quest Diagnostics Investments Incorporated Magnetic separation of lipoproteins using dextran sulfate
US9791464B2 (en) 2007-06-08 2017-10-17 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104198690B (zh) * 2007-06-08 2016-09-07 奎斯特诊断投资公司 通过差分带电荷微粒迁移率进行脂蛋白分析
US9354200B1 (en) 2008-08-07 2016-05-31 Quest Diagnostics Investments Incorporated Detection apparatus for differential-charged particle mobility analyzer

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996002573A1 (fr) * 1994-07-20 1996-02-01 Pharming Bv Separation de l'albumine serique humaine
WO1996022529A1 (fr) * 1995-01-20 1996-07-25 Arqule, Inc. Procede de production de differents composes chimiques constituant un reseau spatial
US5663306A (en) * 1984-08-09 1997-09-02 Chiron Corporation Method of conjugating an activated ester to an amine-containing biological material

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995018972A1 (fr) * 1994-01-05 1995-07-13 Arqule, Inc. Production modulaire systematique de molecules a base d'aminimide et d'oxazolone ayant des proprietes choisies

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5663306A (en) * 1984-08-09 1997-09-02 Chiron Corporation Method of conjugating an activated ester to an amine-containing biological material
WO1996002573A1 (fr) * 1994-07-20 1996-02-01 Pharming Bv Separation de l'albumine serique humaine
WO1996022529A1 (fr) * 1995-01-20 1996-07-25 Arqule, Inc. Procede de production de differents composes chimiques constituant un reseau spatial

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000037501A1 (fr) * 1998-12-22 2000-06-29 Amersham Pharmacia Biotech Ab Elimination/purification d'albumines seriques
US9791464B2 (en) 2007-06-08 2017-10-17 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility
US10948503B2 (en) 2007-06-08 2021-03-16 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility
US11680949B2 (en) 2007-06-08 2023-06-20 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility
US12055554B2 (en) 2007-06-08 2024-08-06 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility
US9250211B2 (en) 2010-12-30 2016-02-02 Quest Diagnostics Investments Incorporated Magnetic separation of lipoproteins using dextran sulfate
US10308680B2 (en) 2010-12-30 2019-06-04 Quest Diagnostics Investments Incorporated Magnetic separation of lipoproteins using dextran sulfate

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JP2001527090A (ja) 2001-12-25
CA2316766A1 (fr) 1999-07-08
AU2083499A (en) 1999-07-19
EP1044214A1 (fr) 2000-10-18

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