WO2023150820A1 - Procédé de synthèse de polymères biologiques radiomarqués - Google Patents

Procédé de synthèse de polymères biologiques radiomarqués Download PDF

Info

Publication number
WO2023150820A1
WO2023150820A1 PCT/AU2022/050088 AU2022050088W WO2023150820A1 WO 2023150820 A1 WO2023150820 A1 WO 2023150820A1 AU 2022050088 W AU2022050088 W AU 2022050088W WO 2023150820 A1 WO2023150820 A1 WO 2023150820A1
Authority
WO
WIPO (PCT)
Prior art keywords
process according
polycarboxylate
buffer
vessel
biological polymer
Prior art date
Application number
PCT/AU2022/050088
Other languages
English (en)
Inventor
Christian Werner WICHMANN
Stacey Erin RUDD
Andrew Mark Scott
Paul Stephen Donnelly
Original Assignee
The University Of Melbourne
Olivia Newton-John Cancer Research Institute
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
Application filed by The University Of Melbourne, Olivia Newton-John Cancer Research Institute filed Critical The University Of Melbourne
Priority to PCT/AU2022/050088 priority Critical patent/WO2023150820A1/fr
Publication of WO2023150820A1 publication Critical patent/WO2023150820A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
    • A61K51/0478—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group complexes from non-cyclic ligands, e.g. EDTA, MAG3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1027—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against receptors, cell-surface antigens or cell-surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1075—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody the antibody being against an enzyme
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/121—Solutions, i.e. homogeneous liquid formulation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/534—Production of labelled immunochemicals with radioactive label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/70—Mechanisms involved in disease identification
    • G01N2800/7023—(Hyper)proliferation
    • G01N2800/7028—Cancer

Definitions

  • the present disclosure relates generally to a process for the synthesis of radiolabelled biological polymers.
  • the disclosure relates to a process for the synthesis of 89 Zr radiolabelled biological polymer-chelate agent conjugates. Automation of the process is also described.
  • PET Positron Emission Tomography
  • 89 Zr Zirconium-89
  • 89 Zr has a useful, relatively long, half-life of 78.4 h.
  • Vosjan et al. describe a 89 Zr radiolabelling protocol which utilizes 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 6.8 - 7.2 to achieve radiochemical yields > 85% after incubation for 1 hour at ambient temperature (Conjugation and radiolabeling of monoclonal antibodies with zirconium-89 for PET imaging using the bifunctional chelate p-isothiocyanatobenzyl-desferrioxamine, Nature Protocols 2010, 5(4), 739-743). However the kinetics of the radiolabelling step were relatively slow.
  • HEPES 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid
  • the present disclosure is directed to processes for synthesising radiolabelled biological polymers.
  • the processes advantageously exhibit fast reaction kinetics enabling total process time to be reduced. Additionally, the processes can be automated which further reduces total process time.
  • the present disclosure provides a process for the synthesis of 89 Zr labelled biological polymers, wherein said process comprises at least the step of combining a biological polymer-chelating agent conjugate with a source of 89 Zr in the presence of a buffer solution comprising polycarboxylate, and wherein said polycarboxylate is capable of forming a chelate ring with 89 Zr, said chelate ring containing six or more ring atoms.
  • the buffer solution comprising polycarboxylate comprises one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.
  • the buffer solution comprising polycarboxylate consists of, or consists essentially of, one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.
  • the buffer solution comprising polycarboxylate consists of, or consists essentially of, succinate.
  • the concentration of polycarboxylate buffer is greater than the concentration of any non-carboxylate buffers.
  • the concentration of polycarboxylate buffer during the combining is between about 5mM and 1000mM, or between about 5mM and 500mM, or between about 5mM and 200mM, or between about 5mM and 200mM, or between about 5mM and about 150mM, or between about 5mM and about 100mM, or between about 5mM and about 80mM, or between about 5mM and about 50mM, or between about 5mM and about 30mM.
  • the concentration of polycarboxylate during the combining is greater than about 5mM, or greater than about 10mM.
  • the combining is performed in the substantial absence of 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES).
  • HEPES 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid
  • the buffer solution comprising polycarboxylate has a pH from about 4 to about 7, preferably from about 5.5 to about 6.5, more preferably about 6.
  • the 89 Zr source is derived from 89 Zr oxalate.
  • the 89 Zr source is produced by treating 89 Zr oxalate with a base in the presence of the buffer solution comprising polycarboxylate.
  • the base may be an alkali metal carbonate, such as sodium carbonate or potassium carbonate.
  • the resulting mixture is incubated for about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes.
  • the conversion to 89 Zr labelled biological polymer is greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, after 15 minutes at 25°C.
  • the conversion to 89 Zr labelled biological polymer is greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, after 5 minutes at 25°C.
  • the chelating agent is selected from DFO-squaramide, DFO*- squaramide, benzyl isothiocyanate-DFO, and benzyl isothiocyanate-DFO*, wherein DFO is desferrioxamine B and DFO* is desferrioxamine*.
  • the process comprises:
  • the biological polymer-chelating agent conjugate is combined with buffer solution comprising polycarboxylate prior to combining with the source of 89 Zr.
  • step (b) the desalting agent is pre-conditioned with a solution comprising radiolytic protectant.
  • any one or more vessels, transfer lines and transfer equipment exposed to 89 Zr prior to combining with biological polymer-chelating agent conjugate are treated with buffer solution comprising polycarboxylate and the resulting solution transferred to the first vessel.
  • any one or more vessels, transfer lines and transfer equipment exposed to 89 Zr after combining with biological polymer-chelating agent conjugate are treated with buffer solution comprising polycarboxylate and the resulting solution transferred to the second vessel.
  • the buffer solution comprising polycarboxylate further comprises one or more surfactants.
  • the solution comprising radiolytic protectant further comprises one or more surfactants.
  • the one or more surfactants comprise one or more non-ionic surfactants.
  • the one or more surfactants comprise one or more polysorbates.
  • the one or more polysorbates comprise polysorbate 80.
  • the one or more polysorbates comprise polysorbate 20.
  • the desalting agent in the second vessel comprises a bed of material through which the crude reaction product from the first vessel is passed.
  • the bed of material may comprise gel filtration resin.
  • radiation detectors are configured to measure radiation in one or both the first and second vessels.
  • the biological polymer comprises one or more of peptide, polypeptide, protein, and antibody.
  • the biological polymer is an antibody.
  • the biological polymer is Girentuximab.
  • the 89 Zr labelled biological polymer is 89 Zr-DFOSq- Girentuximab.
  • one or more of the process steps is automated.
  • the present disclosure provides a process for the synthesis of 89 Zr labelled biological polymers comprising:
  • the buffer solution comprising polycarboxylate comprises one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.
  • the buffer solution comprising polycarboxylate consists of, or consists essentially of, succinate.
  • the concentration of polycarboxylate buffer is greater than the concentration of any non-carboxylate buffers.
  • the concentration of polycarboxylate buffer during the combining is between about 5mM and 1000mM, or between about 5mM and 500mM, or between about 5mM and 200mM, or between about 5mM and about 150mM, or between about 5mM and about 100mM, or between about 5mM and about 80mM, or between about 5mM and about 50mM, or between about 5mM and about 30mM.
  • the concentration of polycarboxylate during combining is greater than about 5mM, or greater than about 10mM.
  • the combining is performed in the substantial absence of 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES).
  • HEPES 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid
  • the buffer solution comprising polycarboxylate has a pH from about 4 to about 7, preferably from about 5.5 to about 6.5, more preferably about 6.
  • the 89 Zr source is derived from 89 Zr oxalate.
  • the 89 Zr source is produced by treating 89 Zr oxalate with a base in the presence of the buffer solution comprising polycarboxylate.
  • the base may be an alkali metal carbonate, such as sodium carbonate or potassium carbonate.
  • the resulting mixture is incubated for about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes.
  • the conversion to 89 Zr labelled biological polymer is greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, after 15 minutes at 25°C.
  • the conversion to 89 Zr labelled biological polymer is greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, after 5 minutes at 25°C.
  • the chelating agent is selected from DFO-squaramide, DFO*- squaramide, benzyl isothiocyanate-DFO, and benzyl isothiocyanate-DFO*, wherein DFO is desferrioxamine B and DFO* is desferrioxamine*.
  • the biological polymer-chelating agent conjugate is combined with buffer solution comprising polycarboxylate prior to combining with the source of 89 Zr.
  • the desalting agent prior to step (b) is pre-conditioned with a solution comprising radiolytic protectant.
  • any one or more vessels, transfer lines and transfer equipment exposed to 89 Zr prior to combining with biological polymer-chelating agent conjugate are treated with buffer solution comprising polycarboxylate and the resulting solution transferred to the first vessel.
  • any one or more vessels, transfer lines and transfer equipment exposed to 89 Zr after combining with biological polymer-chelating agent conjugate are treated with buffer solution comprising polycarboxylate and the resulting solution transferred to the second vessel.
  • the buffer solution comprising polycarboxylate further comprises one or more surfactants.
  • the solution comprising radiolytic protectant further comprises one or more surfactants.
  • the one or more surfactants comprises one or more non-ionic surfactants.
  • the one or more surfactants comprise one or more polysorbates.
  • the one or more polysorbates comprise polysorbate 80.
  • the one or more polysorbates comprise polysorbate 20.
  • the desalting agent in the second vessel comprises a bed of material through which the crude reaction product from the first vessel is passed.
  • the bed of material may comprise gel filtration resin.
  • radiation detectors are configured to measure radiation in one or both the first and second vessels.
  • the biological polymer comprises one or more of peptide, polypeptide, protein, and antibody.
  • the biological polymer is an antibody.
  • the biological polymer is Girentuximab.
  • the 89 Zr labelled biological polymer is 89 Zr-DFOSq- Girentuximab.
  • one or more of the process steps is automated.
  • the present disclosure provides an 89 Zr labelled biological polymer formed by the process according to any one of the herein disclosed embodiments.
  • Advantages of the presently disclosed processes may include one or more of the following:
  • Figure 1 is a reaction scheme depicting an 89 Zr radiolabelling process according to embodiments of the present disclosure.
  • Figure 2 is a photograph of a radiosynthesiser used to conduct a process according to embodiments of the present disclosure.
  • Figure 3 is a plot of radiochemical yield (RCY) vs reaction time.
  • Figure 4 illustrates the influence of pH and buffer additives on radioactive residuals in isotope vessel (A) and reaction vessel (B).
  • Figure 5 compares radioactivity tracking during automated production using HEPES buffer with no additives and succinate buffer with added polysorbate 80 surfactant.
  • Figure 6 illustrates radioactivity traces during an automated radiolabelling process according to embodiments of the present disclosure.
  • Figure 7 is a plot of radiochemical yield (RCY) of 89 Zr-DFO-NCS-human I gG 1 vs reaction time in different reaction buffers.
  • Figure 8 is a plot of radiochemical yield (RCY) of 89 Zr-DFO*-NCS-humanised lgG1 vs reaction time in different reaction buffers.
  • Figure 9 is a plot of radiochemical yield (RCY) of 89 Zr-DFO-Sq-Durvalumab vs reaction time in different reaction buffers.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present disclosure relates to processes for synthesising radiolabelled biological polymers.
  • the processes advantageously exhibit fast reaction kinetics enabling total process time to be reduced. Additionally, the processes can be automated which further reduces total process time.
  • an 89 Zr source is treated with antibody-chelating agent conjugate in the presence of buffer solution comprising polycarboxylate. This results in chelation of the antibody-chelating agent to 89 Zr and formation of 89 Zr labelled antibody.
  • the labelled antibody is subsequently purified.
  • Figure 1 illustrates an overall reaction scheme in which, in a first reaction, an antibody-chelating agent conjugate is prepared by reacting an antibody with a chelating agent.
  • the antibody-chelating agent conjugate is subsequently reacted with an 89 Zr source in a succinate buffer (according to the present disclosure) or, alternatively, HEPES buffer (comparative) to form the labelled antibody.
  • Exemplary antibodies and antibody-drug conjugates include Trastuzumab, Cetuximab, Panitumumab, Nimotuzumab, Durvalumab, Atezolizumab, Girentuximab, Lintuzumab, Trastuzumab Emtansine, and Brentuximab Vedotin.
  • the 89 Zr source utilised in the presently disclosed processes is derived from, for example, zirconium oxalate.
  • Oxalate is used to assist in the purification of zirconium (IV) and stabilize the ion in solution, but this oxalate has to be removed prior to preparation of the radiolabelled biological polymer-chelating agent conjugate.
  • an aqueous solution of zirconium oxalate in oxalic acid is neutralised with base in the presence of a buffer comprising polycarboxylate.
  • the base is an alkali metal base such as sodium carbonate or potassium carbonate.
  • 89 Zr source may be derived from alternative zirconium complexes to zirconium oxalate.
  • Buffer solution comprising polycarboxylate (reaction buffer)
  • a key feature of the presently disclosed processes is performing the radiolabelling step in the presence of a buffer solution comprising polycarboxylate.
  • the buffer solution comprising polycarboxylate may also be referred to as ‘reaction buffer’, that is, the buffer in which the chelation of the biological polymer-chelating agent conjugate to 89 Zr occurs (see Figure 1).
  • Useful polycarboxylates comprise one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.
  • succinate is a preferred polycarboxylate.
  • the role of the polycarboxylate is to stabilize the zirconium (IV) ion and facilitate transfer chelation to the biological polymer-chelating agent conjugate.
  • Oxalate forms a stable five-membered chelate ring when bound to zirconium (IV).
  • polycarboxylates such as succinate, tartrate and malonate are likely to form less stable coordination complexes with zirconium (IV) based of their different chelate ring sizes - the less stable zirconium (IV) complexes will favour transfer chelation to the biological polymer-chelating agent conjugate. That is, oxalate will form complexes with stable five membered chelate rings - whereas succinate, tartrate and malonate, do not.
  • the chelation reaction occurs rapidly in polycarboxylate buffer. In embodiments, greater than 90% chelation occurred in 15 minutes at ambient temperature (about 25°C).
  • Non-limiting examples of chelators useful in the presently disclosed processes include DFO-squaramide, DFO*-squaramide, benzyl isothiocyanate-DFO, and benzyl isothiocyanate-DFO*, wherein DFO is desferrioxamine B and DFO* is desferrioxamine*.
  • the buffer solution comprising carboxylate may also comprise one or more surfactants.
  • useful surfactants include non-ionic surfactants such as polysorbates. Those skilled in the art will understand that other non-ionic surfactants can be used as long as they are pharmaceutically acceptable and suitable for administration to patients.
  • Embodiments of the present disclosure provide a process for synthesising radiolabelled biological polymers wherein one or more steps of the process is automated.
  • the automated process was performed in a disposable cassette based MultiSyn radiosynthesiser (iPHASE Technologies Pty Ltd, Australia).
  • FIG. 2 is a photograph of the radiosynthesiser.
  • the skilled person will appreciate that the depicted radiosynthesiser is in no way limiting and the presently disclosed processes could be performed in alternative equipment.
  • the radiosynthesiser comprised the following major components:
  • reactor vessel (3) containing antibody-chelate agent conjugate in polycarboxylate buffer
  • GMP-Durvalumab 50 mg/mL was provided by Vetter Pharma-Fert only GmbH & Co. KG, AstraZeneca AB (UK). Desferrioxamine B squaramide ester was provided by TELIX Pharmaceuticals (Australia). Boric acid and sodium chloride were purchased from VWR (Germany). Oxalic acid, 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid (HEPES), D-trehalose, sodium carbonate, sodium succinate, polysorbate 80 (Tween 80), DMSO, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (Sydney, Australia). Human serum albumin (20%) was purchased from CSL Behring (Australia). Methanol was purchased from Merck (Germany).
  • PD-10 desalting columns containing Sephadex G-25 resin were purchased from Cytiva (MA, USA). Sterile free-flex saline bags were purchased from Fresenius Kabi (NSW, Australia). Water-for-injection bags (WFI) were purchased from Baxter (Australia). Buffer solutions were prepared using WFI and stored in sterile vials at 2-8°C.
  • Bacterial endotoxin LAL testing kits were purchased from Charles River (Australia). Standard hospital issue 0.22 pm sterile filters, needles, and water-for- injection (WFI) were used. Sterile glass vials were purchased from Huayi Isotopes (Changshu, China). Sterile cassette kits were purchased from iPHASE Technologies (Melbourne, Australia).
  • Zirconium-89 was produced at Austin Health (Heidelberg, VIC) via the 89 Y(p,n) 89 Zr reaction using an IBA (Belgium) 18 MeV cyclotron and reconstituted in 0.05 M oxalic acid (Sigma Aldrich, USA, purified grade, 99.999% trace metal basis dissolved in Ultrapur water). Radioactivity was measured using either a Capintec CRC-55t PET dose calibrator (Mirion Technologies Inc., USA) or a Perkin Elmer (USA) Wizard2 automated gamma counter.
  • iTLC Instant thin layer chromatography
  • SE- HPLC Size-exclusion high performance liquid chromatography
  • Protein concentrations were determined via absorbance at 280nm using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, USA) in I gG 1 protein mode.
  • HEK293 cells transfected with human PD-L1 were cultured in DMEM:F12 media (Gibco) containing 10% fetal calf serum (FCS, Gibco) and 0.4 mg/mL Geneticin (Gibco, G418).
  • HCC827 were cultured in RPMI media (Gibco) containing 20% FCS.
  • A549 cells were cultured in DMEM:F12 media containing 10% FCS. Culture conditions for all cell lines were 5% CO2 at 37°C.
  • Buffer comprising polycarboxylate (reaction buffer)
  • Examples according to the present disclosure utilised a reaction buffer comprising polycarboxylate.
  • the polycarboxylate was succinate and was employed at concentrations of 20 mM and pH 6.
  • the reaction buffer may contain a surfactant.
  • the surfactant was polysorbate 20.
  • the surfactant was polysorbate 80. Comparative examples utilised HEPES buffer at 0.5 M concentration and pH 7.2. Formulation buffer
  • Formulation buffer was prepared using clinical grade saline bags (100 mL) by removing an appropriate amount of saline from the bag (20 mL) under sterile conditions and adding freshly dissolved sodium gentisate (450 mg) in ‘PBS concentrate’ (10 mL) consisting of 24.3 mM KCI, 90 mM Na2HPO4, 16.2 mM KH2PO4, and 0.18% polysorbate 80. This resulted in a sterile bag containing 0.5% sodium gentisate (w/v) and 0.02% polysorbate 80 (w/v) in PBS (90 mL).
  • GMP-Durvalumab 50 mg/mL was placed in 8x Amicon Ultra-15 centrifugal filter devices (MWCO 50 kDa) in 50 mg (1 mL) aliquots each. Buffer exchange into borate buffer (0.5 M, pH 9.0) was performed over 3 cycles (9 mL each, 15-25 minutes at 4000 ref) while ensuring the volume in each centrifugal filter did not drop below 1 mL. Following buffer exchange, aliquots were combined and diluted to approx. 10 mg/mL using borate buffer (0.5 M, pH 9.0).
  • DFOSqOEt (1018 pL, 10 mg/mL in DMSO, 6 molar equivalents) was then added (final DMSO concentration ⁇ 4%) and the reaction was allowed to stand at ambient temperature (24-26°C) for 22 hours. Equal aliquots of the reaction mixture were then applied to 8x Amicon Ultra-15 centrifugal filter devices (MWCO 50 kDa) and underwent three filtration cycles with buffer comprising polycarboxylate (20 mM sodium succinate, pH 6, 275 mM trehalose, 0.02% polysorbate 80) or HEPES (0.5 M, pH 7.2) (9 mL each, 15-25 minutes at 4000 ref).
  • buffer comprising polycarboxylate (20 mM sodium succinate, pH 6, 275 mM trehalose, 0.02% polysorbate 80) or HEPES (0.5 M, pH 7.2) (9 mL each, 15-25 minutes at 4000 ref).
  • Radiolabelling kinetics in HEPES (0.5 M, pH 7.2) and sodium succinate (20 mM, pH 6) buffer were compared by neutralizing 89 Zr-oxalate (3.7 MBq) using 0.4 equivalents (v/v) sodium carbonate (0.1 M, pH 10.8), diluting with reaction buffer to give a final reaction volume of 100 pL, followed by addition of varying amounts of DFOSq- Durvalumab. Reactions were incubated at ambient temperature and samples taken at 5, 15, 30, and 60 minutes were analysed by iTLC using citrate (20 mM, pH 5) as a mobile phase.
  • Figure 3 illustrates that reactions in HEPES buffer were consistently much slower than reactions in succinate buffer. At 1 pg/MBq the difference in radiochemical yield was most pronounced and quantitative reaction yields of 95.6% ⁇ 0.7% were observed after 15 minutes in succinate buffer compared to only 22.4% ⁇ 2.1 % in HEPES buffer.
  • Radioactivity residuals in the 89 Zr isotope vessel were investigated by incubating 89 Zr-oxalate (3 MBq) neutralized with 0.4 equivalents (v/v) sodium carbonate (0.1 M, pH 10.8) and diluted to a final volume of 100 pL using either HEPES (0.5 M, pH 7.2) or 20 mM sodium succinate buffer with varying pH between 4.4 - 7 in a 15 mL Falcon tube for 3 minutes at ambient temperature. Solutions were removed followed by rinsing with the respective formulation buffer (1 mL).
  • Radioactivity in the 89 Zr isotope vessels was recorded at the end of incubation, after removal of buffered 89 Zr-oxalate solution, and after the final rinsing step using a dose calibrator. Radioactivity residuals at each step were calculated as fractions of the initial activity in each isotope vessel.
  • Figure 4A shows the results of residual radioactivity analyses performed manually with respect to buffer pH and additives for the 89 Zr isotope vessel.
  • Residual radioactivity in the reactor vessel may arise from radiolabelled antibody sticking to plastic surfaces.
  • a range of different reactor vessel materials including PE, PET, PP, COC, and glass with either flat or conical shapes were investigated, however differences in residual 89 Zr-DFOSq-Durvalumab were minor, and these minor improvements were limited to vial types that were difficult to integrate into the radiosynthesiser flow path. Subsequently, buffer additives such as sodium chloride, polysorbate 80, and human serum albumin (HSA) were examined.
  • buffer additives such as sodium chloride, polysorbate 80, and human serum albumin (HSA) were examined.
  • Radioactivity residuals in the reactor vessel were investigated by incubating 89 Zr-DFOSq-Durvalumab (5 MBq, > 98% radiochemical purity) formulated in sodium succinate (20 mM, pH 6, 1 mL) containing combinations of 0.15 M NaCI, 0.02% polysorbate 80, and 1 % HSA in reactor vessels for 30 minutes at ambient temperature. Solutions were removed followed by rinsing with formulation buffer (1 mL) containing the respective additives. In each experiment, the radioactivity in the reactor vessel was recorded at the end of incubation, after removal of 89 Zr-DFOSq-Durvalumab solution, and after the final rinsing step using a dose calibrator. Radioactivity residuals at each step were calculated as fractions of the initial activity in each reactor vessel.
  • Figure 5 shows a summary of radioactivity residuals before and after optimisation of the automated protocol.
  • the total reaction volume was 1.8 mL and activity concentration was approximately 120 MBq/mL.
  • syringe (2) contained a mixture of sodium carbonate (0.1 M, pH 10.8) and buffer solution comprising polycarboxylate (reaction buffer; in this example succinate buffer (20 mM, pH 6) containing 0.02% polysorbate 80).
  • reaction buffer in this example succinate buffer (20 mM, pH 6) containing 0.02% polysorbate 80.
  • the volumes of Na2COs solution (V Na2CO3 ) and buffer solution (V reaction bu ⁇ er ) were determined by the volume of 89 Zr in oxalic acid via the following formulae:
  • the 89 Zr isotope vessel (1), syringe (2), and transfer lines were then rinsed with a total of 400 pL reaction buffer from the buffer vessel (7) and transferred to the reactor vessel (3).
  • the mixture was allowed to react for 15 minutes at 25°C during which time the desalting bed (4) was automatically conditioned with 30 mL of 0.5% sodium gentisate (w/v) in PBS pH 7.2 + 0.02% polysorbate 80 (w/v) from the formulation buffer vessel (8) using syringe (9).
  • the reactor vessel (3) was pressurised with inert gas and contents were loaded onto the desalting bed (4).
  • the reactor vessel (3) was rinsed with 400 pL reaction buffer and added to the desalting bed (4).
  • the procedure may be stopped if the radiopharmaceutical were to be shipped to other sites.
  • the intermediate vial was removed, and an appropriate patient dose dispensed manually into a separate sterile vial which was reconnected to the radiosynthesiser for automated sterile filtration into a patient syringe.
  • a vessel containing an appropriate patient dose (92.5 MBq) in 4 mL formulation buffer was reconnected to the intermediate vessel line.
  • the patient dose was drawn into syringe (9) and transferred into the patient syringe (7) via a 0.22 pm vented sterile filter followed by an inert gas flush.
  • the cassette was washed with formulation buffer and dried with inert gas to minimize cassette residual activity.
  • FIG. 6 shows representative radiation profiles of the reactor vessel (left trace) and the purification bed (right trace). Transfers of neutralised 89 Zr solution into the reactor vessel and crude reaction mixture out of the reactor vessel was followed via the reactor vessel radiation detector. Using the purification bed radiation detector, loading of the crude reaction mixture onto the purification bed, removal of leading waste fractions, and product collection was monitored. Based on the purification bed radiation profile, peak intensity was identified as the optimal point to trigger product collection. Appropriate steps were included in the synthesis program allowing for variable elution volumes to adjust for elution profile variability between syntheses. This feature also allowed for facile transfer of the procedure to suit radiolabelling of other conjugated antibodies.
  • Radionuclidic identity and purity were confirmed for every production. Sterility and bacterial endotoxin testing showed no positive results over the limit of detection. All productions fulfilled the quality control release criteria for clinical use.
  • Immunoreactive fraction was determined as described by Lindmo et al. (Journal of Immunological Methods 1984;72(1):77-89). Briefly, 89 Zr-DFOSq-Durvalumb (20 ng) was incubated with 0-5x10 6 HEK293/PD-L1 cells for 45 minutes at ambient temperature followed by centrifugation (2000 ref for 2 minutes). The supernatant was removed, and the cell pellet was washed with media (1 mL) followed by centrifugation (2000 ref for 2 minutes). Washing steps were repeated a further two times and the radioactivity in the cell pellet was determined using a gamma counter.
  • Non-specific binding was determined by incubating 89 Zr-DFOSq-Durvalumb (20 ng) together with Durvalumab (60 pg) following the above protocol. Immunoreactive fraction was calculated by dividing the radioactivity in the washed cell pellet by the average activity of triplicate standards containing 89 Zr-DFOSq-Durvalumb (20 ng).
  • 89 Zr in oxalic acid was neutralised using 0.4 eq v/v sodium carbonate (0.1 M, pH 10.8).
  • Neutralised 89 Zr (3.7 MBq, 10.4 pL) was added to DFO-NCS-human lgG1 conjugate (7.4 pg, 2 pg/MBq) formulated in either sodium succinate (20 mM, pH 6) or HEPES (0.5 M, pH 7.2) reaction buffer.
  • Reaction buffer was added to make up a total reaction volume of 100 pL. Reaction mixtures were incubated at ambient temperature (23-25°C).
  • 89 Zr in oxalic acid was neutralised using 0.4 eq v/v sodium carbonate (0.1 M, pH 10.8).
  • Neutralised 89 Zr (3.7 MBq, 11.3 pL) was added to DFO*-NCS- humanised lgG1 conjugate (7.4 pg, 2 pg/MBq) formulated in either sodium succinate (20 mM, pH 6) or HEPES (0.5 M, pH 7.2) reaction buffer.
  • Reaction buffer was added to make up a total reaction volume of 100 pL. Reaction mixtures were incubated at ambient temperature (23-25°C). Samples for determination of radiochemical yield were taken at 5, 15, 30, and 60 minutes and analysed via iTLC (20 mM citrate pH 5). Reactions were performed in triplicates and results are summarised in Figure 8.
  • 89 Zr in oxalic acid was neutralised using 0.4 eq v/v sodium carbonate (0.1 M, pH 10.8).
  • Neutralised 89 Zr (3.7 MBq, 4.16 pL) was added to DFO-Sq- Durvalumab conjugate (3.7 pg, 1 pg/MBq) formulated in either sodium succinate (20 mM, pH 6) or HEPES (0.5 M, pH 7.2) reaction buffer.
  • Relevant reaction buffer, or mixtures of reaction buffers were added to make up a total reaction volume of 100 pL. Reaction mixtures were incubated at ambient temperature (23-25°C). Samples for determination of radiochemical yield were taken at 5, 15, 30, and 60 minutes and analysed via iTLC (20 mM citrate pH 5). Reactions were performed in triplicates or as single points and results are summarised in Figure 9.
  • Example 8 Radiolabelling speed of different immunoconjugates in succinate buffer at clinical specific activities
  • 89 Zr in oxalic acid was neutralised using 0.4 eq v/v sodium carbonate (0.1 M, pH 10.8).
  • Neutralised 89 Zr was added to immunoconjugate (4.0 - 13.5 pg/MBq) formulated in sodium succinate (20 mM, pH 6) reaction buffer.
  • Reaction buffer was added (1.5 eq v/v relative to volume of 89 Zr in oxalic acid) and reaction mixtures were incubated at ambient temperature (23-25°C).
  • Samples for determination of radiochemical yield (RCY) were taken at 5 minutes and analysed via iTLC (20 mM citrate pH 5). Reactions were performed in triplicates and results are summarised in Table 2.
  • DFOSq-Girentuximab was prepared as per the procedure for DFOSq- Durvalumab in Example 1. Automated radiolabelling with 89 Zr was performed using a similar procedure as in Example 4 to yield 89 Zr-DFOSq-Girentuximab with a process yield of 60% and radiochemical purity >97%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Optics & Photonics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Dispersion Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des procédés de synthèse de conjugués polymère-chélate biologiques radiomarqués par du zirconium-89. Le radiomarquage est effectué en présence d'un tampon polycarboxylate conduisant à une cinétique de réaction améliorée et à des temps de traitement courts. Les procédés fournissent un degré élevé de reproductibilité, d'excellents rendements radiochimiques et peuvent être automatisés.
PCT/AU2022/050088 2022-02-10 2022-02-10 Procédé de synthèse de polymères biologiques radiomarqués WO2023150820A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/AU2022/050088 WO2023150820A1 (fr) 2022-02-10 2022-02-10 Procédé de synthèse de polymères biologiques radiomarqués

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/AU2022/050088 WO2023150820A1 (fr) 2022-02-10 2022-02-10 Procédé de synthèse de polymères biologiques radiomarqués

Publications (1)

Publication Number Publication Date
WO2023150820A1 true WO2023150820A1 (fr) 2023-08-17

Family

ID=87563325

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2022/050088 WO2023150820A1 (fr) 2022-02-10 2022-02-10 Procédé de synthèse de polymères biologiques radiomarqués

Country Status (1)

Country Link
WO (1) WO2023150820A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104722510A (zh) * 2015-03-24 2015-06-24 福建师范大学 一种太阳能电池板智能除尘装置
EP3865154A1 (fr) * 2018-10-10 2021-08-18 Astellas Pharma Inc. Composition pharmaceutique contenant un complexe de fragment fab d'anticorps au site anti-humain marqué

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104722510A (zh) * 2015-03-24 2015-06-24 福建师范大学 一种太阳能电池板智能除尘装置
EP3865154A1 (fr) * 2018-10-10 2021-08-18 Astellas Pharma Inc. Composition pharmaceutique contenant un complexe de fragment fab d'anticorps au site anti-humain marqué

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
C. WICHMANN, S. PONIGER, N. GUO, P. ROSELT, S. RUDD, P. S.DONNELLY, F. HEGI-JOHNSON, M. MACMANUS, A. M. SCOTT: "Fully Automated Radiosynthesis of [89Zr]Zr-DFOSq- Durvalumab for Clinical PET Imaging of PD-L1", EUROPEAN JOURNAL OF NUCLEAR MEDICINE, SPRINGER, BERLIN, HEIDELBERG, DE, vol. 48, Berlin, Heidelberg, DE , pages S220 - S221, XP009548217, ISSN: 0340-6997, Retrieved from the Internet <URL:https://link.springer.com/article/10.1007/s00259-021-05547-1> *
MEIJS, WE ET AL.: "Evaluation of desferal as a bifunctional chelating agent for labeling antibodies with Zr-89", INTERNATIONAL JOURNAL OF RADIATION APPLICATIONS AND INSTRUMENTATION. PART A. APPLIED RADIATION AND ISOTOPES, vol. 43, no. 12, 1992, pages 1443 - 1447, XP024706984, ISSN: 0883-2889, DOI: https://doi.org/10.1016/0883-2889(92)90170-J *

Similar Documents

Publication Publication Date Title
KR100729247B1 (ko) 방사선표지 키트 및 결합 분석
Choi et al. Astatine-211 labeled anti-HER2 5F7 single domain antibody fragment conjugates: radiolabeling and preliminary evaluation
CA3016901C (fr) Compositions d&#39;immunoglobulines anti-cd45 radio-marquees stabilisees
Behr et al. Reduction of the renal uptake of radiolabeled monoclonal antibody fragments by cationic amino acids and their derivatives
Sharkey et al. Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22
AU2011209441B2 (en) Radioimmunoconjugates and uses thereof
Behr et al. Radioiodination of monoclonal antibodies, proteins and peptides for diagnosis and therapy
KR20150092702A (ko) 방사성 제약 복합체
Kwon et al. 64Cu-labeled trastuzumab fab-PEG24-EGF radioimmunoconjugates bispecific for HER2 and EGFR: pharmacokinetics, biodistribution, and tumor imaging by PET in comparison to monospecific agents
Visser et al. Optimal quality 131I-monoclonal antibodies on high-dose labeling in a large reaction volume and temporarily coating the antibody with IODO-GEN
Price et al. A comparative evaluation of the chelators H4octapa and CHX-A ″-DTPA with the therapeutic radiometal 90Y
Li et al. cGMP production of astatine-211-labeled anti-CD45 antibodies for use in allogeneic hematopoietic cell transplantation for treatment of advanced hematopoietic malignancies
Hens et al. Labeling internalizing anti-epidermal growth factor receptor variant III monoclonal antibody with 177Lu: in vitro comparison of acyclic and macrocyclic ligands
Qiu et al. A pretargeted imaging strategy for immune checkpoint ligand PD-L1 expression in tumor based on bioorthogonal Diels-Alder click chemistry
Carpenet et al. Anti-CD20 immunoglobulin G radiolabeling with a 99mTc-tricarbonyl core: in vitro and in vivo evaluations
Wichmann et al. Automated radiosynthesis of [89Zr] Zr-DFOSq-Durvalumab for imaging of PD-L1 expressing tumours in vivo
WO2023150820A1 (fr) Procédé de synthèse de polymères biologiques radiomarqués
Nakashima et al. Development of Novel Trifunctional Chelating Agents That Enhance Tumor Retention of Radioimmunoconjugates
Mishra et al. Preparation and Comparative Evaluation of" Tc-labeled
Karczmarczyk et al. Influence of DOTA chelators on radiochemical purity and biodistribution of 177Lu-and 90Y-Rituximab in xenografted mice
Kameswaran et al. Clinical scale preparation and evaluation of 131I-Rituximab for Non-Hodgkin's Lymphoma
EP4317188A1 (fr) Complexe radioactif d&#39;anticorps anti-egfr, et produit radiopharmaceutique
EP4230637A1 (fr) Complexes radioactifs d&#39;anticorps anti-her2 et produit radiopharmaceutique
US20220378956A1 (en) Trivalent Radioisotope Bio-Targeted Radiopharmaceutical, Methods Of Preparation And Use
Carpenet et al. Anti-CD20 Immunoglobulin G Radiolabeling With a 99mtc-Tricarbonyl Core

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22925256

Country of ref document: EP

Kind code of ref document: A1