| 1. |
- Tran, Thuy A., 1980-, et al.
(author)
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Design, synthesis and biological evaluation of a multifunctional HER2-specific Affibody molecule for molecular imaging
- 2009
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In: European Journal of Nuclear Medicine and Molecular Imaging. - : Springer Science and Business Media LLC. - 1619-7070 .- 1619-7089. ; 36:11, s. 1864-1873
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Journal article (peer-reviewed)abstract
- Purpose: The purpose of this study was to design and evaluate a novel platform for labelling of Affibody molecules, enabling for both recombinant and synthetic production and for site-specific labelling with 99mTc or trivalent radiometals. Methods: The HER2-specific Affibody molecule PEP05352 was made by peptide synthesis. The chelator sequence SECG (serine-glutamic acid-cysteine-glycine) was anchored on the C-terminal to allow 99mTc-labelling. The cysteine can alternatively serve as a conjugation site of the chelator DOTA for indium-labelling. The resulting 99mTc- and 111In-labelled Affibody molecules were evaluated both in vitro and in vivo. Results: Both conjugates retained their capacity to bind to HER2 receptors in vitro and in vivo. The tumour-to-blood ratio in LS174T xenografts was 30 at 4 h p.i. for both conjugates. Biodistribution data showed that 99mTc-labelled Affibody molecule had 4-fold lower kidney accumulation compared with 111In-labelled Affibody molecule while the accumulation in other organs was similar. Gamma-camera imaging of the conjugates could clearly visualise the tumours 4 h after injection. Conclusions: Incorporation of C-terminal SECG sequence in Affibody molecules provides a general multifunctional platform for site-specific labelling with different nuclides (technetium, indium, gallium, cobalt, or yttrium) and for a flexible production (chemical synthesis or recombinant).
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| 2. |
- Tran, Thuy, 1980-, et al.
(author)
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Glycosylation profiling of therapeutic antibodies in serum samples using a microfluidic CD platform and MALDI-MS
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Other publication (other academic/artistic)abstract
- The serum clearance rate of therapeutic antibodies is important as it affects the clinical efficacy, required dose and dose frequency. The glycosylation of antibodies has in some studies been shown to have an impact on the elimination rates in vivo. Monitoring changes to the glycan profiles in pharmacokinetics studies can reveal whether the clearance rates of the therapeutic antibodies depend on the different glycoforms, thereby providing useful information for improvement of the drugs. In this paper a novel method for glycosylation analysis of therapeutic antibodies in serum samples is presented. A microfluidic CD platform in combination with MALDI-MS was used to monitor changes to the glycosylation profiles of samples incubated in vitro. Antibodies were selectively purified from serum using immunoaffinity capture on immobilized target antigens. The glycans were enzymatically released, purified and finally analyzed by MALDI-TOF-MS. To simulate changes to glycan profiles after administration in vivo, a therapeutic antibody was incubated in serum with the enzyme α1-2,3 mannosidase to artificially reduce the amount of the high mannose glycoforms. Glycan profiles were monitored at specific intervals during the incubation. The relative abundance of the high mannose 5 glycoform was clearly found to decrease and simultaneously, that of high mannose 4 increased over the incubation period. The method can be performed in a rapid, parallel and automated fashion for glycosylation profiling consuming low amounts of samples and reagents. This can contribute to less labor work and reduced cost of the studies of therapeutic antibodies glycosylation in vitro and in vivo.Keywords:
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| 3. |
- Tran, Thuy, 1980-
(author)
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Molecular Imaging of HER2 Expression using Synthetic Affibody Molecules : Design, Synthesis and Biological Evaluation
- 2009
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Doctoral thesis (other academic/artistic)abstract
- Molecular imaging is an emerging multidisciplinary field that addresses the visualisation of diseases at the cellular and molecular levels. This thesis focuses on the development of a synthetic Affibody molecule-based imaging tracer for the detection of HER2 expression in malignant tumours. Papers I-IV report the development of the HER2-specific Affibody molecule, ZHER2:342 by peptide synthesis and the use of different chelators attached to the N-terminus to allow 99mTc-labelling. Paper I described the optimisation of labelling of Affibody molecules using cysteine-based chelator sequences, in which the direct labelling method under alkaline conditions was the most suitable one. Papers II-IV report the development and optimisation of the in vivo properties of the HER2-specific Affibody molecule for high-contrast imaging. By using an array of mercaptoacetyl-based chelators, it was found that the substitution of a single amino acid in a 60 amino acid-long Affibody molecule can dramatically change the pharmacokinetics of the tracer. Strategic approaches that utilised hydrophilic amino acids, such as serine, glutamate and lysine, changed the excretion pathway from hepatobiliary to renal excretion. Problems with the high accumulation of radioactivity in the abdomen area and restricted imaging were resolved by the use of mercaptoacetyl-triglutamyl, maEEE or mercaptoacetyl-seryl-lysyl-seryl, maSKS chelators. Paper V reports the re-engineering of the HER2-specific Affibody molecule to provide a C-terminal SECG sequence using peptide synthesis. Incorporation of this sequence provided a multifunctional platform for labelling (with technetium or trivalent radiometals) and a flexible production route (recombinant or chemical synthesis). Combination of a serine, a glutamic acid and a thiol-bearing group in the chelating sequence reduced the renal accumulation of Affibody molecules. Altogether, the in vivo efficiency of Affibody molecules to target tumours and their biodistribution properties can be improved by strategic design and suitable chemistry. Hopefully, these observations will be applicable to other small peptide and protein scaffold-based tracers.
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| 4. |
- Tran, Thuy, 1980-, et al.
(author)
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Nanoplasmonic Avidity-Based Detection and Quantification of IgG Aggregates
- 2022
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In: Analytical Chemistry. - : AMER CHEMICAL SOC. - 0003-2700 .- 1520-6882. ; 94:45, s. 15754-15762
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Journal article (peer-reviewed)abstract
- Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein Afunctionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 mu g/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 mu g/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.
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| 5. |
- Tran, Thuy, 1980-, et al.
(author)
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Real-Time Nanoplasmonic Sensor for IgG Monitoring in Bioproduction
- 2020
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In: Processes. - : MDPI. - 2227-9717. ; 8:10
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Journal article (peer-reviewed)abstract
- Real-time monitoring of product titers during process development and production of biotherapeutics facilitate implementation of quality-by-design principles and enable rapid bioprocess decision and optimization of the production process. Conventional analytical methods are generally performed offline/at-line and, therefore, are not capable of generating real-time data. In this study, a novel fiber optical nanoplasmonic sensor technology was explored for rapid IgG titer measurements. The sensor combines localized surface plasmon resonance transduction and robust single use Protein A-modified sensor chips, housed in a flexible flow cell, for specific IgG detection. The sensor requires small sample volumes (1-150 mu L) and shows a reproducibility and sensitivity comparable to Protein G high performance liquid chromatography-ultraviolet (HPLC-UV). The dynamic range of the sensor system can be tuned by varying the sample volume, which enables quantification of IgG samples ranging from 0.0015 to 10 mg/mL, without need for sample dilution. The sensor shows limited interference from the sample matrix and negligible unspecific protein binding. IgG titers can be rapidly determined in samples from filtered unpurified Chinese hamster ovary (CHO) cell cultures and show good correlation with enzyme-linked immunosorbent assay (ELISA).
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| 6. |
- Tran, Thi Thuy, 1980-
(author)
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Compact-disc microfluidic methods for characterization of therapeutic antibodies : Analysis of post-translational modifications
- 2012
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Doctoral thesis (other academic/artistic)abstract
- Characterization of post-translational modifications (PTMs) of therapeutic proteins is very important during the bioprocess development to maintain desired product quality and during the submission process to regulatory authorities for product approval. Monitoring glycosylation in pharmacokinetic studies can be useful to evaluate the dependence of clearance rates on different glycoforms. The cost and efficiency of characterization affect the speed to market of biopharmaceutical proteins. A reduction in the number of manual processing steps, cost of reagents and consumption of sample, as well as the time required for chemical analysis, is therefore necessary.The research presented in this thesis is focused on the potential of using microfluidic discs for automated, miniaturized, parallel and rapid sample preparation for PTM characterization of therapeutic monoclonal antibodies. Paper I describes the method development for N-linked glycosylation profiling. Several sample preparation steps have been performed in an integrated process in the microfluidic compact disc (CD). Paper II demonstrates the use of the method presented in paper I in combination with multivariate statistics for discrimination of glycosylation profiles of different therapeutic antibodies and simulation of a real case of quality control. Paper III is focused on a method for monitoring changes in glycosylation profiles of therapeutic antibodies in serum over time by incubation with an exoglycosidase enzyme. Paper IV describes the method for peptide mapping of therapeutic antibodies. In addition, recent work (unpublished results) assesses the potential of this method for methionine oxidation detection.The developed methods were fast, robust with low sample/reagent consumption. Generation of glycosylation profile data for one sample was established in approximately 2 h. The amount of samples and antigens loaded into the CD platform for one replicate was less than 0.3 μg and approximately 0.06 μg, respectively. Furthermore, considering the parallel function of the CD, conducting the analysis for 54 samples can be completed within a day.
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