Field of the disclosure

[0001] The present disclosure relates generally to a process for the synthesis of radiolabelled biological polymers. In particular, 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.

Background of the disclosure

[0002] Positron Emission Tomography (PET) using antibodies for targeting has become an important molecular imaging technique in cancer diagnosis and therapy. Zirconium-89 ( ⁸⁹ Zr) has emerged as one preferred radiometal for radiolabelling of antibodies because of its availability, cost, and relative ease of radiolabelling.

Additionally, ⁸⁹ Zr has a useful, relatively long, half-life of 78.4 h.

[0003] Clinical trials performed with ⁸⁹ Zr-labelled antibodies typically utilize manual production of radioimmunoconjugates. While this is suitable for preclinical and small clinical bioimaging trials, larger, and multicentre studies in particular, would benefit from a more consistent process. Another development that is slowly making its way into the clinic is the use bifunctional chelators with demonstrated improvement of complex stability.

[0004] Existing manual and automated processes for ⁸⁹ Zr radiolabelling often suffer in regard to process time and isolated yield. Quality control parameters including radiochemical purity, protein integrity and stability are also important factors to be considered.

[0005] 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. [0006] A published automated procedure describing ⁸⁹ Zr-labelling of monoclonal antibodies reported isolated yields between 60 - 75% and a process time of 77 minutes (Poot, A. J. et al., The Journal of Nuclear Medicine, 2019, 60(5), 691-695).

[0007] Beyond antibody radiolabelling, other biological polymers, such as peptides, polypeptides and proteins may be radiolabelled and such agents find use in diagnostic imaging and radionuclide therapy.

[0008] In view of the foregoing there is an ongoing need to develop improved processes for the synthesis of radiolabelled biological polymers.

[0009] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and/or combined with other pieces of prior art by a skilled person in the art.

Summary of the disclosure

[0010] 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.

[0011] In a first aspect 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.

[0012] In embodiments, the buffer solution comprising polycarboxylate comprises one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.

[0013] In embodiments, the buffer solution comprising polycarboxylate consists of, or consists essentially of, one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate. [0014] In embodiments, the buffer solution comprising polycarboxylate consists of, or consists essentially of, succinate.

[0015] In embodiments, during the combining, the concentration of polycarboxylate buffer is greater than the concentration of any non-carboxylate buffers.

[0016] In embodiments, 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.

[0017] In embodiments, the concentration of polycarboxylate during the combining is greater than about 5mM, or greater than about 10mM.

[0018] In embodiments, the combining is performed in the substantial absence of 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES).

[0019] In embodiments, 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.

[0020] In embodiments, the ⁸⁹ Zr source is derived from ⁸⁹ Zr oxalate.

[0021] In embodiments, 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.

[0022] In some embodiments of the process, after the combining, the resulting mixture is incubated for about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes.

[0023] In some embodiments of the process, 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.

[0024] In some embodiments of the process, 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. [0025] In embodiments, 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*.

[0026] In embodiments, the process comprises:

(a) in a first vessel, combining the biological polymer-chelating agent conjugate with the source of ⁸⁹ Zr in the presence of the buffer solution comprising polycarboxylate;

(b) transferring crude reaction product comprising ⁸⁹ Zr labelled biological polymer from the first vessel to a second vessel, said second vessel comprising a desalting agent; and

(c) removing desalted solution comprising ⁸⁹ Zr labelled biological polymer from the second vessel.

[0027] In embodiments, the biological polymer-chelating agent conjugate is combined with buffer solution comprising polycarboxylate prior to combining with the source of ⁸⁹ Zr.

[0028] In embodiments, prior to step (b) the desalting agent is pre-conditioned with a solution comprising radiolytic protectant.

[0029] In embodiments of any one of the herein disclosed processes, 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.

[0030] In embodiments of any one of the herein disclosed processes, 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.

[0031] In embodiments of any one of the herein disclosed processes, the buffer solution comprising polycarboxylate further comprises one or more surfactants.

[0032] In embodiments, the solution comprising radiolytic protectant further comprises one or more surfactants. [0033] In embodiments, the one or more surfactants comprise one or more non-ionic surfactants.

[0034] In embodiments, the one or more surfactants comprise one or more polysorbates.

[0035] In embodiments, the one or more polysorbates comprise polysorbate 80.

[0036] In embodiments, the one or more polysorbates comprise polysorbate 20.

[0037] In embodiments, 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.

[0038] In embodiments, radiation detectors are configured to measure radiation in one or both the first and second vessels.

[0039] In embodiments, the biological polymer comprises one or more of peptide, polypeptide, protein, and antibody.

[0040] In some preferred embodiments, the biological polymer is an antibody.

[0041] In embodiments, the biological polymer is Girentuximab.

[0042] In embodiments, the ⁸⁹ Zr labelled biological polymer is ⁸⁹ Zr-DFOSq- Girentuximab.

[0043] In embodiments of any one of the herein disclosed processes, one or more of the process steps is automated.

[0044] In another aspect the present disclosure provides a process for the synthesis of ⁸⁹ Zr labelled biological polymers comprising:

(a) in a first vessel, combining a biological polymer-chelating agent conjugate with a source of ⁸⁹ Zr in the presence of a buffer solution comprising polycarboxylate, wherein said polycarboxylate is capable of forming a chelate ring with ⁸⁹ Zr, said chelate ring containing six or more ring atoms; (b) transferring crude reaction product comprising ⁸⁹ Zr labelled biological polymer from the first vessel to a second vessel, said second vessel comprising a desalting agent; and

(c) removing desalted solution comprising ⁸⁹ Zr labelled biological polymer from the second vessel.

[0045] In embodiments, the buffer solution comprising polycarboxylate comprises one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate.

[0046] In embodiments, the buffer solution comprising polycarboxylate consists of, or consists essentially of, succinate.

[0047] In embodiments, during the combining, the concentration of polycarboxylate buffer is greater than the concentration of any non-carboxylate buffers.

[0048] In embodiments, 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.

[0049] In embodiments, the concentration of polycarboxylate during combining is greater than about 5mM, or greater than about 10mM.

[0050] In embodiments, the combining is performed in the substantial absence of 4- (2-hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES).

[0051] In embodiments, 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.

[0052] In embodiments, the ⁸⁹ Zr source is derived from ⁸⁹ Zr oxalate.

[0053] In embodiments, 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. [0054] In some embodiments of the process, after the combining, the resulting mixture is incubated for about 5 minutes to about 30 minutes, or from about 10 minutes to about 20 minutes.

[0055] In some embodiments of the process, 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.

[0056] In some embodiments of the process, 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.

[0057] In embodiments, 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*.

[0058] In embodiments, the biological polymer-chelating agent conjugate is combined with buffer solution comprising polycarboxylate prior to combining with the source of ⁸⁹ Zr.

[0059] In embodiments, prior to step (b) the desalting agent is pre-conditioned with a solution comprising radiolytic protectant.

[0060] In embodiments of any one of the herein disclosed processes, 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.

[0061] In embodiments of any one of the herein disclosed processes, 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.

[0062] In embodiments of any one of the herein disclosed processes, the buffer solution comprising polycarboxylate further comprises one or more surfactants.

[0063] In embodiments, the solution comprising radiolytic protectant further comprises one or more surfactants. [0064] In embodiments, the one or more surfactants comprises one or more non-ionic surfactants.

[0065] In embodiments, the one or more surfactants comprise one or more polysorbates.

[0066] In embodiments, the one or more polysorbates comprise polysorbate 80.

[0067] In embodiments, the one or more polysorbates comprise polysorbate 20.

[0068] In embodiments, 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.

[0069] In embodiments, radiation detectors are configured to measure radiation in one or both the first and second vessels.

[0070] In embodiments, the biological polymer comprises one or more of peptide, polypeptide, protein, and antibody.

[0071] In some preferred embodiments, the biological polymer is an antibody.

[0072] In embodiments, the biological polymer is Girentuximab.

[0073] In embodiments, the ⁸⁹ Zr labelled biological polymer is ⁸⁹ Zr-DFOSq- Girentuximab.

[0074] In embodiments of any one of the herein disclosed processes, one or more of the process steps is automated.

[0075] In another aspect the present disclosure provides an ⁸⁹ Zr labelled biological polymer formed by the process according to any one of the herein disclosed embodiments.

[0076] Advantages of the presently disclosed processes may include one or more of the following:

• fast process times, including an advantageously fast radiolabelling step; high radiochemical yields; • highly reproducible;

• reduced operator exposure to radiation.

[0077] Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

[0078] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and processes are clearly within the scope of the disclosure, as described herein.

[0079] Further aspects of the present disclosure and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.

Brief description of the drawings

[0080] Figure 1 is a reaction scheme depicting an ⁸⁹ Zr radiolabelling process according to embodiments of the present disclosure.

[0081] Figure 2 is a photograph of a radiosynthesiser used to conduct a process according to embodiments of the present disclosure.

[0082] Figure 3 is a plot of radiochemical yield (RCY) vs reaction time.

[0083] Figure 4 illustrates the influence of pH and buffer additives on radioactive residuals in isotope vessel (A) and reaction vessel (B).

[0084] Figure 5 compares radioactivity tracking during automated production using HEPES buffer with no additives and succinate buffer with added polysorbate 80 surfactant.

[0085] Figure 6 illustrates radioactivity traces during an automated radiolabelling process according to embodiments of the present disclosure.

[0086] 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. [0087] Figure 8 is a plot of radiochemical yield (RCY) of ⁸⁹ Zr-DFO*-NCS-humanised lgG1 vs reaction time in different reaction buffers.

[0088] Figure 9 is a plot of radiochemical yield (RCY) of ⁸⁹ Zr-DFO-Sq-Durvalumab vs reaction time in different reaction buffers.

Detailed description of the embodiments

[0089] It will be understood that the disclosure described and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the disclosure.

Definitions

[0090] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0091] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0092] "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instances ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0093] 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.

[0094] 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.

[0095] In an exemplary embodiment of the process, 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.

[0096] 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.

[0097] The skilled person would appreciate that a wide range of antibodies or antibody-drug conjugates could be utilised to prepare antibody-chelating agent conjugates suitable for use in the presently disclosed radiolabeling processes.

[0098] Exemplary antibodies and antibody-drug conjugates include Trastuzumab, Cetuximab, Panitumumab, Nimotuzumab, Durvalumab, Atezolizumab, Girentuximab, Lintuzumab, Trastuzumab Emtansine, and Brentuximab Vedotin.

[0099] The skilled person would further appreciate that other biological polymers, such as peptides, polypeptides and proteins could equally well form biological polymer- chelating agent conjugates suitable for use in the presently disclosed radiolabeling processes.

⁸⁹ Zr source

[0100] In embodiments, 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.

[0101] In embodiments, an aqueous solution of zirconium oxalate in oxalic acid is neutralised with base in the presence of a buffer comprising polycarboxylate.

[0102] In embodiments, the base is an alkali metal base such as sodium carbonate or potassium carbonate.

[0103] The skilled person will appreciate that the ⁸⁹ Zr source may be derived from alternative zirconium complexes to zirconium oxalate.

Buffer solution comprising polycarboxylate (reaction buffer)

[0104] 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).

[0105] Useful polycarboxylates comprise one or more of succinate, glutarate, tartrate, malonate, malate, fumarate, oxaloacetate and citrate. In some embodiments succinate is a preferred polycarboxylate.

[0106] Without wishing to be bound by theory it is postulated that the role of the polycarboxylate is to stabilize the zirconium (IV) ion and facilitate transfer chelation to the biological polymer-chelating agent conjugate.

[0107] Oxalate forms a stable five-membered chelate ring when bound to zirconium (IV). In contrast, 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. [0108] It was surprisingly discovered that the chelation reaction occurs rapidly in polycarboxylate buffer. In embodiments, greater than 90% chelation occurred in 15 minutes at ambient temperature (about 25°C).

[0109] This contrasted to typically utilized HEPES buffer where only 22% chelation was observed in 15 minutes at ambient temperature (about 25°C).

[0110] In one embodiment of the presently disclosed process the chelation reaction in a reactor vessel to form ⁸⁹ Zr-DFOSq-Durvalumab was performed under the following conditions

• Total volume in reactor vessel during chelation: about 1.8 mL

• ⁸⁹ Zr concentration in reactor vessel: from about 110 to about 140 MBq/mL

• Conjugate concentration in reactor vessel: about 0.5 mg/mL

• Succinate concentration in reactor vessel: from about 15 to about 20 mM.

[0111] It will be appreciated that based on the present disclosure, other concentrations of ⁸⁹ Zr, conjugate and succinate would also be effective.

Chelating agent

[0112] The skilled person would appreciate that a wide range of chelators may be utilised to prepare biological polymer-chelating agent conjugates suitable for use in the presently disclosed radiolabeling processes. A majority of useful chelators bear hydroxamate groups. Reference is made to Feiner et al., Cancers, 2021 , 13, 4466, which is incorporated by reference in its entirety and which describes both hydroxamate chelators and other classes of chelators.

[0113] 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*.

Surfactant

[0114] 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.

Process automation

[0115] Embodiments of the present disclosure provide a process for synthesising radiolabelled biological polymers wherein one or more steps of the process is automated.

[0116] In an embodiment, the automated process was performed in a disposable cassette based MultiSyn radiosynthesiser (iPHASE Technologies Pty Ltd, Australia).

Figure 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.

[0117] Referring to Figure 2, the radiosynthesiser comprised the following major components:

• isotope vessel (1) containing ⁸⁹ Zr source, for example ⁸⁹ Zr oxalate in an aqueous solution of oxalic acid;

• syringe (2) containing sodium carbonate in polycarboxylate buffer;

• reactor vessel (3) containing antibody-chelate agent conjugate in polycarboxylate buffer;

• purification bed (4);

• intermediate product vessel (5);

• patient syringe with sterile filter (6);

• vessel containing buffer comprising polycarboxylate (reaction buffer) (7);

• vessel containing formulation buffer (8)

• transfer syringe (9) vessel (10) for waste collection. Optimisation of Radioactivity Residuals

[0118] Initial testing of an automated process revealed significant residual activity in the ⁸⁹ Zr isotope vessel, the reactor vessel, and the purification bed, after transfer of solutions, which led to diminished overall process yields. It was discovered that yield losses could be addressed through appropriate rinsing of vessels and transfer lines resulting in a marked improvement in overall yield. Rinsing was aided significantly if the solution contained a surfactant or protein such as HSA.

Examples

General Methods

[0119] GMP-Durvalumab (50 mg/mL) was provided by Vetter Pharma-Fertigung 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).

[0120] 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.

[0121] 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).

[0122] 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.

[0123] Instant thin layer chromatography (iTLC) of labelled Durvalumab samples was performed using glass microfibre iTLC-SG chromatography paper strips (Agilent, CA, USA) and either methanol : water = 1 : 1 + 4% TFA or 20 mM citrate pH 5 as a mobile phase. Developed iTLC strips were counted either using an Elysia-Raytest miniGITA TLC reader with OFA probe and 20 mm tungsten collimator or an Elysia-Raytest Rita Star radio-TLC scanner. Size-exclusion high performance liquid chromatography (SE- HPLC) was performed on an Agilent 1260 Infinity II HPLC system equipped with a G7115A diode array detector and an Elysia-Raytest GABI-Nova radio-HPLC detector with mid-energy probe using a Phenomenex BioSep 5 pm SEC-s3000 column. Samples were analysed using an isocratic method running aqueous 50 mM phosphate buffer pH 7.2, 0.2 M sodium chloride, 5% isopropanol, and 0.02% sodium azide at 1 mL/min for 20 minutes.

[0124] Protein concentrations were determined via absorbance at 280nm using a NanoDrop Lite spectrophotometer (Thermo Fisher Scientific, USA) in I gG 1 protein mode.

[0125] HEK293 cells transfected with human PD-L1 (GenScript USA Inc, Piscataway, NJ, USA) 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)

[0126] Examples according to the present disclosure utilised a reaction buffer comprising polycarboxylate. In embodiments the polycarboxylate was succinate and was employed at concentrations of 20 mM and pH 6. In embodiments the reaction buffer may contain a surfactant. In embodiments the surfactant was polysorbate 20. In other embodiments the surfactant was polysorbate 80. Comparative examples utilised HEPES buffer at 0.5 M concentration and pH 7.2. Formulation buffer

[0127] 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).

Example 1 : Preparation of DFOSq-Durvalumab conjugate

[0128] Conjugation of DFOSqOEt to Durvalumab was investigated at different molar ratios between the chelator and antibody. It was found that a 6-fold molar excess of chelator resulted in optimal chelator-to-antibody ratio (CAR) of 3.02.

[0129] 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). Conjugate from each centrifugal filter was combined and the concentration adjusted to 2.22 mg/mL using either buffer comprising polycarboxylate or 0.5 M HEPES pH 7.2. The solutions were sterile filtered, and aliquots (450 pL, 1 mg) were stored at -80°C.

Example 2: Evaluation of radiolabelling kinetics

[0130] Reactions of desferrioxamine B conjugated antibodies with ⁸⁹ Zr are typically complete after incubation for 1 hour in HEPES buffer at ambient temperature (see, for example, Vosjan et al., 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). ⁸⁹ Zr-labelling of DFOSq- Durvalumab was investigated in an alternative reaction medium consisting of sodium succinate (20 mM, pH 6) and at varying ratios of DFOSq-Durvalumab to ⁸⁹ Zr.

[0131] 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.

[0132] 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.

Example 3: Evaluation of radioactivity residuals

[0133] 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). The 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.

[0134] Figure 4A shows the results of residual radioactivity analyses performed manually with respect to buffer pH and additives for the ⁸⁹ Zr isotope vessel.

[0135] It was found that residuals dropped from an initial 16% at pH 7 to 2.5% after rinsing at pH 6 with succinate buffer. Residuals at pH 4.4 were negligible. Antibodies for clinical use are commonly formulated and stored at pH 5-6. Durvalumab is formulated at pH 6 and ⁸⁹ Zr isotope vessel residuals are sufficiently low at this pH. In contrast, the use of HEPES buffer at pH 7.2 as rinsing solution only reduced isotope vessel ⁸⁹ Zr residuals from about 32% to about 25%.

[0136] 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. 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.

[0137] The results are shown in Figure 4B. Addition of 0.15 M sodium chloride resulted in no reduction of ⁸⁹ Zr-DFOSq-Durvalumab residual. Substantial reduction of residual radioactivity was achieved by addition of 0.02% v/v polysorbate 80 or 1 % v/v HSA. After rinsing, losses in the reactor vessel were approx. 1 %. Pre-treatment of the vessel with polysorbate 80 was found not to be necessary. As HSA can interfere with protein concentration measurements to determine the specific activity of radiolabelled antibody, polysorbate 80 was a preferred surfactant, and rinsing steps of the ⁸⁹ Zr isotope and reactor vessels were included in the automated process protocol.

[0138] Figure 5 shows a summary of radioactivity residuals before and after optimisation of the automated protocol. Changing the reaction buffer from HEPES (0.5 M, pH 7.2) to sodium succinate (20 mM, pH 6) reduced the amount of residual radioactivity in the ⁸⁹ Zr isotope vessel from 24% to 0.44% ± 0.18% (n=7). Further radioactivity losses observed in the reactor vessel which could not be removed by rinsing were practically eliminated by addition of 0.02% polysorbate 80 to the reaction buffer which reduced losses in the reactor vessel from 36% ± 6% (n=4) to 0.82% ± 0.75% (n=4).

Example 4: Automated production of ⁸⁹ Zr-DFOSq-Durvalumab

[0139] In a typical automated process, ⁸⁹ Zr-oxalate (approx. 222 MBq, 50-200 pL) was neutralized resulting in a pH of 5.80 ± 0.25 (n=3). pH variability is due to varying amounts of oxalic acid used which spans a range of 50-200 pL depending on the radioactivity concentration of ⁸⁹ Zr-oxalate stock solution. After transfer of the neutralized ⁸⁹ Zr mixture to the reactor vessel and rinsing of the isotope vessel with reaction buffer, the reaction pH was determined to be 6.02 ± 0.14 (n=3). The total reaction volume was 1.8 mL and activity concentration was approximately 120 MBq/mL. Quantitative reaction yields were recorded consistently after incubation for 15 minutes at ambient temperature. Purification of the crude reaction mixture was performed via gel filtration using a PD-10 desalting column packed with Sephadex G-25 resin. Automatic conditioning of the PD-10 column was implemented using final formulation buffer from a reservoir. pH of the final formulation buffer cocktail was confirmed to be 7.27 ± 0.01 (n=3). Integration of a sterile vent into the design of the custom PD-10 adapter allowed addition and removal of liquid from the PD-10 column without disturbing the column packing material.

[0140] After the recipe sequence (comprised as an Excel sequence step-list) was downloaded to the MultiSyn radiosynthesiser’s internal memory, purging of the cassette components with inert gas was performed. After successful cassette installation, reagent vials and syringes were installed as shown in Figure 2. The process was operated using a stepwise program sheet.

[0141] Referring to Figure 2, 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). 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:

^Na ₂ CO ₃ ⁼ ^89 _Zr ^X 0'4

^reaction buffer 1 TTtL ^89 _Zr ^Na ₂ CO ₃ [0142] This ensured reproducible neutralisation of ⁸⁹ Zr while keeping the volume of neutralised ⁸⁹ Zr mixture constant.

[0143] The contents of syringe (2) were transferred into the ⁸⁹ Zr isotope vessel (1) charged with approx. 222 MBq of ⁸⁹ Zr-oxalate in oxalic acid (0.05 M, 50-200 pL). The mixture was agitated by drawing up into syringe (2) and releasing back into the ⁸⁹ Zr isotope vessel (1). Neutralised ⁸⁹ Zr solution was then transferred into the reactor vessel (3) charged with DFOSq-Durvalumab (400 pL, 888 pg, 2.22 mg/mL) formulated in reaction buffer. 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). At the conclusion of the reaction, 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). Subsequently, a volume corresponding to the loaded reaction volume (2.2 mL) was removed from the desalting bed (4) via syringe (2). Formulation buffer (3.4 mL) was added to the desalting bed (4) using syringe (9). Additional fractions were discarded in 200 pL increments guided by the desalting bed radiation trace. When maximum intensity was reached, the cassette was washed with formulation buffer and purged, additional formulation buffer was added to the desalting bed (1.2 mL), and the product fraction was collected into syringe (9). Product was then transferred into the intermediate vessel (5) for quality control and syringe (9) and transfer lines were rinsed with additional 400 pL formulation buffer. At this point, the procedure may be stopped if the radiopharmaceutical were to be shipped to other sites. In this example, 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.

[0144] 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. [0145] Finally, the cassette was washed with formulation buffer and dried with inert gas to minimize cassette residual activity.

[0146] Automated synthesis including purification was monitored using in-built radiation detectors. Figure 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.

[0147] Interactive peak collection via the purification bed radiation profile (Figure 5) allowed reproducible collection of the product fraction with low amounts of radioactivity remaining on the bed (2.9% ± 0.95%, n=5). Residual on the sterile filter was 5.0% ± 2.7% (n=4) and the remaining kit components such as manifolds, transfer syringes, and tubing accounted for 3.3% ± 0.21% (n=3) of radioactivity losses. In summary, the total process yield was 75% ± 6.5% (n=5) and the total process time was only 40 minutes.

Quality control for clinical use

[0148] Quality control was performed on ⁸⁹ Zr-DFOSq-Durvalumab according to the criteria outlined in Table 1. The reported results were obtained from three and five clinical productions at two different sites.

[0149] Overall, production of ⁸⁹ Zr-DFOSq-Durvalumab proceeded in 75% ± 6% radiochemical yield providing approx. 148-183 MBq of product in a volume of 3.0 mL ± 0.3 mL of final formulation buffer at the end of synthesis (EOS). Radiochemical purity via iTLC was always > 99% and specific activity was 315 MBq/mg ± 34 MBq/mg (EOS). Protein integrity was determined via SE-HPLC showing 96.4% ± 0.19% intact monomer and 3.65% ± 0.19% aggregated protein at EOS. Immunoreactive fraction in HEK293/PD-L1 cells was 83.3% ± 9.02% at EOS.

[0150] Stability of bulk ⁸⁹ Zr-DFOSq-Durvalumab in final formulation buffer at ambient temperature was monitored over a period of 24 hours which showed very good retention of radiochemical purity and antibody integrity. The amount of free ⁸⁹ Zr was determined to be 0.41% ± 0.02%. Stability in human serum at 37°C was assessed over a period of 7 days to emulate the typical clinical imaging timeframe of an ⁸⁹ Zr-labelled antibody. Over this timeframe, radiochemical purity dropped slightly from 99.4% ± 0.20% at EOS to 97.5% ± 0.47% on day 7. Antibody integrity and immunoreactive fraction dropped to 64.7% ± 3.41% and 62.2% ± 20.9%, respectively. This is excellent compared to other ⁸⁹ Zr-labelled antibodies currently in clinical trials.

[0151] 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

[0152] Immunoreactive fraction (IRF) 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).

Example 5: Radiolabelling of DFO-NCS-human lgG1 in succinate versus HEPES buffer

[0153] ⁸⁹ Zr in oxalic acid (0.05 M) 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). Samples for determination of radiochemical yield were taken at 5, 15, and 30 minutes and analysed via iTLC (20 mM citrate pH 5). Reactions were performed in triplicates and results are summarised in Figure 7. [0154] Radiochemical yield of reactions in sodium succinate buffer showed significantly faster reaction kinetics and a higher average maximum radiochemical yield of 70.7% ± 6.4% (n = 3) compared to 9.7% ± 0.6% (n = 3) in HEPES buffer at 30 minutes reaction time.

Example 6: Radiolabelling of DFO*-NCS-humanised lgG1 in succinate versus HEPES buffer

[0155] ⁸⁹ Zr in oxalic acid (0.05 M) 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.

[0156] Reactions in sodium succinate buffer showed >90% radiochemical yield after 15 minutes. The average radiochemical yield in succinate buffer at 30 minutes was 98.1% ± 0.5% (n = 3) compared to 45.3% ± 1.9% (n = 3) in HEPES buffer.

Example 7: Radiolabelling of DFO-Sq-Durvalumab in succinate versus HEPES buffer

[0157] ⁸⁹ Zr in oxalic acid (0.05 M) 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.

[0158] Four different reaction buffers or combinations of reaction buffers were tested. Reactions in sodium succinate buffer (20 mM, pH 6) showed >95% radiochemical yield after 15 minutes. Succinate (0.5 M, pH 7.2) reached about 66% radiochemical yield after incubation for 60 minutes. HEPES buffer (0.5 M, pH 7.2) showed low average radiochemical yield at 60 minutes of 28.4% ± 1.5% (n = 3). This could not be improved by addition of 0.1 eq v/v (= 10mol%) succinate buffer (0.5M, pH 7.2) which showed 28.8% incorporation over the same timeframe. This shows that addition of low amounts of succinate to HEPES buffer has no effect on the radiochemical yield.

Example 8: Radiolabelling speed of different immunoconjugates in succinate buffer at clinical specific activities

[0159] ⁸⁹ Zr in oxalic acid (0.05 M) 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.

[0160] Reactions in sodium succinate buffer showed quantitative radiochemical yields after 5 minutes at protein amounts typically used for clinical scale production of radiolabelled I gG 1 antibodies. Radiochemical yields were consistently high, independent of the type of chelator conjugated to the antibodies.

Example 9: Automated production of ⁸⁹ Zr-DFOSq-Girentuximab

[0161] 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%.