The Uchiyama Laboratory in the Department of Biotechnology at Osaka University, Japan, is using Bruker’s timsTOF Pro for high-sensitivity peptide mapping to research immune-related proteins and the formulation of therapeutic proteins and gene therapy products.
Head of the Uchiyama Lab, Prof. Uchiyama, is a biophysical chemist with over 25 years of experience. For the last 10 years, Prof. Uchiyama’s research has centered around the final formulation and production of gene therapy products.
The Uchiyama Lab uses reliable biophysical and biochemical methods to develop a comprehensive understanding of proteins, including the study of the protein aggregates and higher-level protein structures that play an important role in disease. Expanding on Prof. Uchiyama’s 2016 study of viral vector characterization, and the use of Adeno-Associated Virus (AVV) for in vivo gene therapy, the Uchiyama lab is currently manufacturing AVV using human culture cells and adding plasmid to force cells to produce viral vectors which can then be purified using a combination of chromatography and centrifugation. For quality control, mass spectrometry (MS) is used to identify the host cell protein (HCP) and the stoichiometry of the viral proteins as VP1, VP2 and VP3 have different functions. A higher number of VP1 commonly results in higher efficacy, so the VP1 number need to be precisely determined.
Although MS is an effective tool for proteomics research, it cannot comprehensively reveal the sequence coverage of proteins alone, nor can it provide precise measurements of the molecular interactions and downstream effects of protein complexes. Trapped ion mobility spectrometry (TIMS) has come to the forefront as a new technique for the separation of peptides and proteins because it adds new levels of sensitivity, selectivity and speed to proteomics research. It is especially useful for the low abundant, difficult-to-isolate species involved in many diseases.
Bruker’s innovative timsTOF technology helps accelerate gene therapy research. Through the incorporation of collision cross section (CCS) measurement, scientists are able to move from 3D-proteomics (retention time, mass to charge (m/z) and tandem MS (MS/MS) fragment ion spectra) to 4D-Proteomics™. CCS values also provide greater sensitivity, improving the reliability of quantitation in complex samples.
The Bruker timsTOF has facilitated the efficient and complete characterization of antibodies and viral vector capsid proteins in the Uchiyama Lab. While the timsTOF is typically used for the identification of proteins, Prof. Uchiyama and his team have been using it for peptide mapping, confirming protein sequences and revealing the post-translational modification (PTM) state of capsid proteins. It has vastly improved the lab’s production of viral vectors due not only to its ability to identify impurity proteins simultaneously but also its high sensitivity.
Prof. Uchiyama explains “With viral vectors we can only obtain 1 mg from 1 L of culture in the best-case scenario. That’s a much smaller yield than that for therapeutic antibodies. With viral vectors being expensive and the amount produced limited, having a highly sensitive instrument is crucial. The Bruker timsTOF is the ideal solution for this. With a limited quality of material, analysis was almost impossible before we had the timsTOF Pro.”
Next, Prof. Uchiyama will collaborate in a project focusing on the development of characterization and quality control of virus vectors for gene therapy, with the goal of supplying clinical grade viral vectors for hospital trials. The timsTOF Pro 2 from Bruker will formulate viral vector methods that can be manufactured by contract development and manufacturing organizations (CDMO) under good manufacturing practice (GMP) conditions.
For Research Use Only. Not for use in clinical diagnostic procedures.