New Mass Spectrometry Methods to Characterise Virus Based Drug Products
Lead Research Organisation:
University of Manchester
Department Name: Chemistry
Abstract
AAV vectors have emerged at the forefront of gene therapy due to their lack of pathogenicity, relatively low immunogenicity and persistent gene expression in different tissue types. The analytics to support AAV production are very limited and to answer this challenge, this project will construct a new charge detection mass spectrometer - CDMS - that will significantly advance analytical capabilities for the manufacture and testing of AAV therapies
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Gentle electrospray ionisation coupled to mass spectrometry so called native mass spectrometry, provides a facile way of delivering large multimeric protein particles to the gas phase for analysis. Whilst ESI based mass spectrometry has proved hugely beneficial for biological and biomedical science, in particular in the study of large intact protein assemblies, still some assemblies are so massive that they cannot be resolved using conventional MS methods.
There has been considerable academic and commercial effort to development native mass spectrometry to investigate protein complexes and other assemblies including viruses, with masses into the mDa range. Despite extraordinary efforts and innovation in these areas, there remain challenges associated with the transmission and mass analysis of such large objects. The main issue is that the peaks in the m/z spectrum broaden and shift due to mass heterogeneity, either intrinsic or due to adduct formation whilst retaining the interactions that keep the assemblies intact.
Charge detection mass spectrometry (CDMS) bypasses the need to resolve charge states. CDMS is a single particle technique, where the m/z and z of individual ions are measured concurrently, thereby allowing direct determination of the mass of each ion. CDMS can analyse heterogeneous mixtures of protein complexes and other large assemblies that are intractable by conventional MS methods.
CDMS is highly simple and works by measuring the charge on a particle that passes through a chamber, along with the time it takes the particle to traverse the chamber. This charge will be some integer, z, multiplied by the fundamental unit of charge (e). The time it takes the particle to traverse the tube will be related to the m/z of the particle by Newton's equation of motion and these two measurements can be combined to provide M, the mass of the particle.
To date three academic groups in the world have developed CDMS (two in the USA (Martin Jarrold and Evan Williams) and one in France (Philippe Dugourd), we are not aware of any commercial CDMS instrument that has been developed to date. The highest resolution commercial mass spectrometer is unable to provide sufficient mass resolution in measurements on AAVs of this nature and also has very low throughput in the study of such massive particles due to the method of mass analysis and difficulties in transmitting these ions through conventional ion optics.
In this project we propose to build, install and test a new CDMS instrument with a novel ESI source for transmission of MDa ions and a different geometry to any previously reported, which will have better charge accuracy and hence higher throughput. We will use it to determine the DNA content of recombinant adenoassociated viral (AAV) vectors.
We propose to compare the capabilities of this instrument with that of commercially available mass spectrometry platforms and other methods. We will also build a stage to allow subsequent characterisation via microscopy.
We will develop standard operating procedures for the instrument using, large assembled protein standards (ferritin, Groel and IgEs) and subsequently develop its use for AAVs. The potential to study other large assemblies will also be explored especially heavily glycosylated protein assemblies. We will use the instrument to measure how many of a given AAV product have filled capsids versus empty and we will compare the data to the efficacy of the drug in animal models.
.
Gentle electrospray ionisation coupled to mass spectrometry so called native mass spectrometry, provides a facile way of delivering large multimeric protein particles to the gas phase for analysis. Whilst ESI based mass spectrometry has proved hugely beneficial for biological and biomedical science, in particular in the study of large intact protein assemblies, still some assemblies are so massive that they cannot be resolved using conventional MS methods.
There has been considerable academic and commercial effort to development native mass spectrometry to investigate protein complexes and other assemblies including viruses, with masses into the mDa range. Despite extraordinary efforts and innovation in these areas, there remain challenges associated with the transmission and mass analysis of such large objects. The main issue is that the peaks in the m/z spectrum broaden and shift due to mass heterogeneity, either intrinsic or due to adduct formation whilst retaining the interactions that keep the assemblies intact.
Charge detection mass spectrometry (CDMS) bypasses the need to resolve charge states. CDMS is a single particle technique, where the m/z and z of individual ions are measured concurrently, thereby allowing direct determination of the mass of each ion. CDMS can analyse heterogeneous mixtures of protein complexes and other large assemblies that are intractable by conventional MS methods.
CDMS is highly simple and works by measuring the charge on a particle that passes through a chamber, along with the time it takes the particle to traverse the chamber. This charge will be some integer, z, multiplied by the fundamental unit of charge (e). The time it takes the particle to traverse the tube will be related to the m/z of the particle by Newton's equation of motion and these two measurements can be combined to provide M, the mass of the particle.
To date three academic groups in the world have developed CDMS (two in the USA (Martin Jarrold and Evan Williams) and one in France (Philippe Dugourd), we are not aware of any commercial CDMS instrument that has been developed to date. The highest resolution commercial mass spectrometer is unable to provide sufficient mass resolution in measurements on AAVs of this nature and also has very low throughput in the study of such massive particles due to the method of mass analysis and difficulties in transmitting these ions through conventional ion optics.
In this project we propose to build, install and test a new CDMS instrument with a novel ESI source for transmission of MDa ions and a different geometry to any previously reported, which will have better charge accuracy and hence higher throughput. We will use it to determine the DNA content of recombinant adenoassociated viral (AAV) vectors.
We propose to compare the capabilities of this instrument with that of commercially available mass spectrometry platforms and other methods. We will also build a stage to allow subsequent characterisation via microscopy.
We will develop standard operating procedures for the instrument using, large assembled protein standards (ferritin, Groel and IgEs) and subsequently develop its use for AAVs. The potential to study other large assemblies will also be explored especially heavily glycosylated protein assemblies. We will use the instrument to measure how many of a given AAV product have filled capsids versus empty and we will compare the data to the efficacy of the drug in animal models.
Technical Summary
This project will develop a novel charge detection mass spectrometer CDMS to enable accurate, sensitive, and fast characterisation of AAV products by mass measurement of the fully packaged, partially full and empty viruses.
Of gene therapy products in development, recombinant AAV based vectors are the most widely used. A challenging feature of AAV vector generation in cell culture, like other viral vector systems, is the formation of an excess of "empty" capsids that lack the vector genome and are therefore unable to provide a therapeutic benefit. The regulation of differentiation of empty to full capsids is both a challenge for gene therapy process development and the separation, identification and quantification of empty, partially full and full AAV particles represents an analytical challenge which we seek to solve in this project with a new mass spectrometer.
An entirely novel geometry CDMS based on a paper from Poschenrieder (1972) where two opposing spherical field sectors are arranged to send ions in a figure of eight path. This has an advantage over existing CDMS designs due to its superior ion optical properties in terms of beam energy and spatial transverse acceptance, and due to its geometry, it should offer offering superior throughput and resolution.
We will couple to this novel mass analyser a highly transmitting ESI device and together this new instrument should provide a world first in terms of resolution and sensitivity. We will apply it to the study of AAVs that we will make in house. We will compare the differential state of the capsids as measured in the CDMS device with their efficacy when given to animal models.
The ability of the CDMS device to accurately determine the assembly state of AAVs will be compared to conventional MS and also to non-MS based methods including microscopy with the addition of a stage to land the assembles. We will also apply this instrument to the study of other massive glycosylated proteins.
Of gene therapy products in development, recombinant AAV based vectors are the most widely used. A challenging feature of AAV vector generation in cell culture, like other viral vector systems, is the formation of an excess of "empty" capsids that lack the vector genome and are therefore unable to provide a therapeutic benefit. The regulation of differentiation of empty to full capsids is both a challenge for gene therapy process development and the separation, identification and quantification of empty, partially full and full AAV particles represents an analytical challenge which we seek to solve in this project with a new mass spectrometer.
An entirely novel geometry CDMS based on a paper from Poschenrieder (1972) where two opposing spherical field sectors are arranged to send ions in a figure of eight path. This has an advantage over existing CDMS designs due to its superior ion optical properties in terms of beam energy and spatial transverse acceptance, and due to its geometry, it should offer offering superior throughput and resolution.
We will couple to this novel mass analyser a highly transmitting ESI device and together this new instrument should provide a world first in terms of resolution and sensitivity. We will apply it to the study of AAVs that we will make in house. We will compare the differential state of the capsids as measured in the CDMS device with their efficacy when given to animal models.
The ability of the CDMS device to accurately determine the assembly state of AAVs will be compared to conventional MS and also to non-MS based methods including microscopy with the addition of a stage to land the assembles. We will also apply this instrument to the study of other massive glycosylated proteins.