Methods and compositions for resolving components of a virus preparation
The present invention provides a method of identifying components present in a preparation of virus particles, comprising: a) analyzing the preparation of virus particles with single molecule mass spectrometry to obtain a mass histogram; and b) interpreting the mass histogram of (a) to identify different components present in the preparation.
1. A method of identifying components present in a preparation of virus particles, the method comprising:
a) ionizing virus particles in the preparation of virus particles;
b) subjecting the ionized virus particles to single-particle mass spectrometry to determine a mass of each of the ionized virus particles; and
c) identifying the components based on the determined masses of the ionized virus particles;
wherein the identified components comprise an impurity present in the preparation or each of full genome-containing virus particles, partial genome-containing virus particles and genome-free virus particles.
2. The method of claim 1 , wherein the single-particle mass spectrometry comprises charge detection mass spectrometry.
3. The method of claim 1 , wherein the genome comprises a single-stranded genome or a self-complementary genome.
4. The method of claim 1 , further comprising heating the virus particle;
wherein identifying the components further comprises monitoring thermally-induced capsid disassembly of at least one of the full genome-containing virus particles, the partial genome-containing virus particles and the genome-free virus particles.
5. The method of claim 4 , wherein the virus particles are heated to at least 35° C.
6. The method of claim 4 , wherein the virus particles are heated to at least 50° C.
7. The method of claim 1 , further comprising producing a mass histogram of the ionized virus particles subjected to the single-particle mass spectrometry and identifying the components based on the mass histogram.
8. The method of claim 1 , wherein the virus particles are selected from the group consisting of adeno-associated virus (AAV), adenovirus, lentivirus, retrovirus, herpesvirus, poxvirus (vaccinia or myxoma), paramyxovirus (measles, RSV or Newcastle disease virus), baculovirus, reovirus, alphavirus, flavivirus, and any combinations thereof.
9. The method of claim 1 , wherein one or more of the virus particles are complexed with an exogenous entity.
10. The method of claim 9 , wherein the exogenous entity is selected from the group consisting of a protein, a nucleic acid, a carbohydrate molecule, and combinations thereof.
11. The method of claim 1 , further comprising distinguishing the components based on differences in the determined masses of the ionized virus particles.
12. The method of claim 1 , wherein the identified components comprise the impurity present in the preparation;
and wherein the method further comprises detecting the impurity based on differences in the determined masses of the ionized virus particles.
13. The method of claim 1 , wherein the method does not comprise electron microscopy.
14. The method of claim 1 , wherein the components present in the preparation of virus particles are selected from the group consisting of full genome-containing virus particles, partial genome-containing virus particles, genome-free virus particles, empty virus capsids and fragments thereof, genomic components and fragments thereof, packaged genomes and fragments thereof, unpackaged nucleic acid, contaminants, and any combination thereof.
15. The method of claim 1 , wherein the single particle mass spectrometry is performed by time of flight mass spectrometry, charge detection mass spectrometry, quadrupole ion trap mass spectrometry, Fourier transform ion cyclotron resonance, Orbitrap mass spectrometry or carried out using a micromechanical/nanomechanical oscillator.
16. The method claim 1 , wherein the single particle mass spectrometry is carried out on a commercial mass spectrometer retro-fitted for single particle measurements.
17. The method of claim 1 , wherein the preparation of virus particles is a research grade preparation.
18. The method of claim 1 , wherein the preparation of virus particles is a GMP grade preparation.
19. The method of claim 1 , wherein the preparation of virus particles is a commercial preparation.
20. The method of claim 1 , wherein the method is carried out in about 20 minutes.