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Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for PET or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in several malignancies, including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce 89Zr-radiolabeled onartuzumab (a monovalent, antihuman c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous 89Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing 89Zr-oxalate, the photoactive chelate desferrioxamine B (DFO)-aryl azide (DFO-ArN3), and MetMAb to give 89Zr-DFO-azepin-onartuzumab. As a control, 89Zr-DFO-benzyl Bn-isothiocyanate Bn-NCS-onartuzumab was prepared via a conventional two-step process using prepurified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum, and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0-72 h) was performed on female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image was obtained. The tumor specificity of 89Zr-DFO-azepin-onartuzumab was assessed in vivo by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced 89Zr-DFO-azepin-onartuzumab in less than 15 min, with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity of approximately 90%, and a molar activity of approximately 1.5 MBq nmol-1 Reaction optimization improved the radiochemical conversion of 89Zr-DFO-azepin-onartuzumab to 56.9% ± 4.1% (n = 3), with isolated RCYs of 41.2% ± 10.6% (n = 3) and radiochemical purity of more than 90%. Conventional methods produced 89Zr-DFO-Bn-NCS-onartuzumab with an isolated RCY of more than 97%, radiochemical purity of more than 97% and molar activity of approximately 14.0 MBq nmol-1 Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake (percentage injected dose [%ID]) reached 15.37 ± 5.21 %ID g-1 and 6.56 ± 4.03 %ID g-1, respectively, for 89Zr-DFO-azepin-onartuzumab (n = 4) and 21.38 ± 11.57 %ID g-1 and 18.84 ± 6.03 %ID g-1, respectively, for 89Zr-DFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34 ± 0.47 %ID g-1) of 89Zr-DFO-azepin-onartuzumab (n = 4). Conclusion: The experiments demonstrated that photoradiosynthesis is a viable alternative approach for producing 89Zr-radiolabeled antibodies directly in protein formulation buffer, reducing protein aggregation and liver uptake.

The creation of discrete, covalent bonds between a protein and a functional molecule like a drug, fluorophore, or radiolabeled complex is essential for making state-of-the-art tools that find applications in basic science and clinical medicine. Photochemistry offers a unique set of reactive groups that hold potential for the synthesis of protein conjugates. Previous studies have demonstrated that photoactivatable desferrioxamine B (DFO) derivatives featuring a para-substituted aryl azide (ArN

Most experimental work in the space of bioconjugation chemistry focuses on using new methods to construct covalent bonds between a cargo molecule and a protein of interest such as a monoclonal antibody (mAb). Bond formation is important for generating new diagnostic tools, yet when these compounds advance to preclinical

The synthesis, characterisation and application of radiolabelled compounds for use in diagnostic and therapeutic medicine requires a diverse skill set. This article highlights a selection of our ongoing projects that aim to provide new synthetic methods and radiochemical tools for building molecular imaging agents with various radionuclides.

Methods that provide rapid access to radiolabeled antibodies are vital in the development of diagnostic and radiotherapeutic agents for PET or radioimmunotherapy. The human hepatocyte growth factor receptor (c-MET) signaling pathway is dysregulated in several malignancies, including gastric cancer, and is an important biomarker in drug discovery. Here, we used a photoradiochemical approach to produce (89)Zr-radiolabeled onartuzumab (a monovalent, antihuman c-MET antibody), starting directly from the fully formulated drug (MetMAb). Methods: Simultaneous (89)Zr-radiolabeling and protein conjugation was performed in one-pot reactions containing (89)Zr-oxalate, the photoactive chelate desferrioxamine B (DFO)–aryl azide (DFO-ArN(3)), and MetMAb to give (89)Zr-DFO-azepin-onartuzumab. As a control, (89)Zr-DFO-benzyl Bn-isothiocyanate Bn-NCS-onartuzumab was prepared via a conventional two-step process using prepurified onartuzumab and DFO-Bn-NCS. Radiotracers were purified by using size-exclusion methods and evaluated by radiochromatography. Radiochemical stability was studied in human serum, and immunoreactivity was determined by cellular binding assays using MKN-45 gastric carcinoma cells. PET imaging at multiple time points (0–72 h) was performed on female athymic nude mice bearing subcutaneous MKN-45 xenografts. Biodistribution experiments were performed after the final image was obtained. The tumor specificity of (89)Zr-DFO-azepin-onartuzumab was assessed in vivo by competitive inhibition (blocking) studies. Results: Initial photoradiosynthesis experiments produced (89)Zr-DFO-azepin-onartuzumab in less than 15 min, with an isolated decay-corrected radiochemical yield (RCY) of 24.8%, a radiochemical purity of approximately 90%, and a molar activity of approximately 1.5 MBq nmol(−1). Reaction optimization improved the radiochemical conversion of (89)Zr-DFO-azepin-onartuzumab to 56.9% ± 4.1% (n = 3), with isolated RCYs of 41.2% ± 10.6% (n = 3) and radiochemical purity of more than 90%. Conventional methods produced (89)Zr-DFO-Bn-NCS-onartuzumab with an isolated RCY of more than 97%, radiochemical purity of more than 97% and molar activity of approximately 14.0 MBq nmol(−1). Both radiotracers were immunoreactive and stable in human serum. PET imaging and biodistribution studies showed high tumor uptake for both radiotracers. By 72 h, tumor and liver uptake (percentage injected dose [%ID]) reached 15.37 ± 5.21 %ID g(−1) and 6.56 ± 4.03 %ID g(−1), respectively, for (89)Zr-DFO-azepin-onartuzumab (n = 4) and 21.38 ± 11.57 %ID g(−1) and 18.84 ± 6.03 %ID g(−1), respectively, for (89)Zr-DFO-Bn-NCS-onartuzumab (n = 4). Blocking experiments gave a statistically significant reduction in tumor uptake (6.34 ± 0.47 %ID g(−1)) of (89)Zr-DFO-azepin-onartuzumab (n = 4). Conclusion: The experiments demonstrated that photoradiosynthesis is a viable alternative approach for producing (89)Zr-radiolabeled antibodies directly in protein formulation buffer, reducing protein aggregation and liver uptake.

The ability to modify biologically active molecules such as antibodies with drug molecules, fluorophores or radionuclides is crucial in drug discovery and target identification. Classic chemistry used for protein functionalisation relies almost exclusively on thermochemically mediated reactions. Our recent experiments have begun to explore the use of photochemistry to effect rapid and efficient protein functionalisation. This article introduces some of the principles and objectives of using photochemically activated reagents for protein ligation. The concept of simultaneous photoradiosynthesis of radiolabelled antibodies for use in molecular imaging is introduced as a working example. Notably, the goal of producing functionalised proteins in the absence of pre‐association (non‐covalent ligand‐protein binding) introduces requirements that are distinct from the more regular use of photoactive groups in photoaffinity labelling. With this in mind, the chemistry of thirteen different classes of photoactivatable reagents that react through the formation of intermediate carbenes, electrophiles, dienes, or radicals, is assessed.

Summary A method for the simultaneous (one-step) photochemical conjugation and 89Zr-radiolabeling of antibodies is introduced. A photoactivatable chelate based on the functionalization of desferrioxamine B with an arylazide moiety (DFO-ArN3, [1]) was synthesized. The radiolabeled complex, 89Zr-1+, was produced and characterized. Density functional theory calculations were used to investigate the mechanism of arylazide photoactivation. 89Zr-radiolabeling experiments were also used to determine the efficiency of photochemical conjugation. A standard two-step approach gave a measured conjugation efficiency of 3.5% ± 0.4%. In contrast, the one-step process gave a higher photoradiolabeling efficiency of ∼76%. Stability measurements, cellular saturation binding assays, positron emission tomographic imaging, and biodistribution studies in mice bearing SK-OV-3 tumors confirmed the biochemical viability and tumor specificity of photoradiolabeled [89Zr]ZrDFO-azepin-trastuzumab. Experimental data support the conclusion that the combination of photochemistry and radiochemistry is a viable strategy for producing radiolabeled proteins for imaging and therapy., Graphical Abstract, Highlights • Photochemistry is combined with radiochemistry for radiosynthesis in a flash • Simultaneous photoradiochemistry is achieved with high radiolabeling efficiency • Photoradiochemistry produces viable 89Zr-radiolabeled antibodies • Density functional theory calculations elucidate the photoactivation mechanism, Density Functional Theory (DFT); Medical Imaging; Radiochemicals

Most products are developed in generations. This needs to be considered with regard to development methods and processes to make existing knowledge available to achieve increased efficiency. To realize this, the approach of PGE – product generation engineering – is formulated. Product generation engineering is understood as the development of products based on reference products (precursor or competitor products). The subsystems are either adapted to the new product generation by means of carryover or they are newly developed based on shape variation or principle variation. Validation is considered as the central activity in the product engineering process and is a major challenge, especially for complex mechatronic systems. Therefore, it is important to understand validation as an ongoing activity during product development. The pull principle of validation describes the definition and development of validation activities, including models and validation environments based on specific validation objectives. In order to have effectiveness within validation of subsystems, it is necessary to map the interactions with the overall system, namely the super-system. The relevant subsystems can be connected under consideration of functional and energetic aspects by means of virtual, physical or mixed virtual–physical modeling applied by the holistic IPEK-X-in-the-Loop approach within the integrated Product engineering Model (iPeM).

According to interviews with experts from automotive industry, one of the main changes in the future automotive development will be the entry of new disciplines. This leads to more and closer cooperation between company departments at different locations. In addition location-dependent influencing factors due to different driver types and environmental situations have an increasing significance. For supporting the automotive development in the future environment like mentioned above, the IPEK has implemented a new validation concept based on its X-in-the-Loop-Framework. “X” is the substitution for the system under development (SUD), which is validated under consideration of the influences from the remaining system, user and environmental situation. The new validation concept is characterized by a “Distance Loop”, which means the SUD and other elements can be located across the globe. The advantages of the new validation concept are demonstrated in this paper with concrete application examples based on an international collaborative project.

The automotive industry, like many other sectors, is significantly changing. Besides emobility, light weight construction and smart materials, digitalization is one major driver of change. Online services like real-time traffic information are already a major differentiating factor in the automotive industry. According to a forecast from Gartner, there will be over 250 million cars with wireless internet connection by 2020 [15]. The Connected Car will more and more be connected to its smart environment and become part of a complex system-of-systems, which will cause new challenges in development.

Developing electric vehicles is very important in reducing greenhouse gas (GHG). Since one of the main changes in automotive industry is the entry of new disciplines, the development of electric vehicle as a multidisciplinary product requires more and closer cooperation between company departments at different locations, for example between OEMs and globally distributed suppliers.

Most experimental work in the space of bioconjugation chemistry focuses on using new methods to construct covalent bonds between a cargo molecule and a protein of interest such as a monoclonal antibody (mAb). Bond formation is important for generating new diagnostic tools, yet when these compounds advance to preclinical in vitro and in vivo studies, and later for translation to the clinic, understanding the fate of potential metabolites that arise from chemical or enzymatic degradation of the construct is important to obtain a full picture of the pharmacokinetic performance of a new compound. In the context of designing new bioconjugate methods for labeling antibodies with the positron-emitting radionuclide 89Zr, we previously developed a photochemical process for making 89Zr-mAbs. Experimental studies on [89Zr]ZrDFO-PEG3-azepin-mAb constructs revealed that incorporation of the tris-polyethylene glycol (PEG3) linker improved the aqueous phase solubility and radiochemical conversion. However, the use of a PEG3 linker also has an impact on the whole-body residence time of the construct, leading to a more rapid excretion of the 89Zr activity when compared with radiotracers that lack the PEG3 chain. In this work, we investigated the metabolic fate of eight possible metabolites that arise from the logical disconnection of [89Zr]ZrDFO-PEG3-azepin-mAb at bonds which are susceptible to chemical or enzymatic cleavage. Synthesis combined with 89Zr-radiolabeling, small-animal positron emission tomography imaging at multiple time points from 0 to 20 h, and measurements of the effective half-life for whole-body excretion are reported. The conclusions are that the use of a PEG3 linker is non-innocent in terms of its impact on enhancing the metabolism of [89Zr]ZrDFO-PEG3-azepin-mAbs. In most cases, degradation can produce metabolites that are rapidly eliminated from the body, thereby enhancing image contrast by reducing nonspecific accumulation and retention of 89Zr in background organs such as the liver, spleen, kidney, and bone.