Analytical ultracentrifugation for characterization of recombinant viral particles

Patent metadata

Field	Value
Publication number	US-10429288-B2
Application number	US-201615544498-A
Country	US
Kind code	B2
Filing date	Jan 19, 2016
Priority date	Jan 20, 2015
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

Provided herein are methods to characterize preparations of recombinant viral particles using analytical ultracentrifugation. Recombinant viral particles include recombinant adeno-associated viral particles, recombinant adenoviral particles, recombinant lentiviral particles and recombinant herpes simplex virus particles. Variant species of recombinant viral particles including empty capsids and recombinant viral particles with variant genomes (e.g., truncated genomes, aggregates, recombinants) can be identified and quantitated. The methods can be used to characterize preparations of recombinant viral particles regardless of the sequence of the recombinant viral genome or the serotype of the recombinant viral capsid.

First claim

What is claimed is:

1. A method of characterizing a preparation of recombinant adeno-associated viral (AAV) particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S), and
c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each peak, wherein each peak represents a species of recombinant AAV particle.

2. A method to assess vector genome integrity of recombinant AAV particles in a preparation of recombinant AAV particles comprising
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value C(s) versus the sedimentation coefficient in Svedberg units (S), and
c) identifying species of recombinant AAV particles in the preparation by presence of peaks on the plot corresponding to an S value, wherein the genome size of a particular species of recombinant AAV particles is calculated by comparing the S value of the species to a standard curve generated by S values of recombinant AAV particles comprising encapsidated AAV genomes of known nucleotide sizes.

3. The method of claim 2 further comprising integrating the area under each peak in the C(s) distribution to determine the relative concentration of each species of recombinant AAV particles.

4. A method to determine the presence of empty capsids or capsid particles comprising variant sized recombinant AAV genomes in a preparation of recombinant AAV particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals, and
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S), wherein the presence of one or more peaks other than the peak for full capsid particles comprising intact recombinant AAV genomes indicates that presence of capsid particles comprising variant sized genomes and/or empty capsids.

5. A method of measuring the relative amount empty capsids in a preparation of recombinant AAV particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S),
c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each species of recombinant AAV particles, and
d) comparing the amount of recombinant AAV particles having an S value corresponding to empty capsid particles to the amount of recombinant AAV particles having an S value corresponding to recombinant AAV particles comprising intact AAV genomes or the total amount of recombinant AAV particles in the preparation.

6. A method of measuring the relative amount of capsid particles comprising variant recombinant AAV genomes or empty AAV capsid particles in a preparation of recombinant AAV particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S),
c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each species of recombinant AAV particles,
d) comparing the amount of recombinant AAV particles having an S values that do not correspond to recombinant AAV particles comprising intact AAV genomes to the amount of recombinant AAV particles having an S value that corresponds to recombinant AAV particles comprising intact AAV genomes or to the total amount of recombinant AAV particles in the preparation.

7. A method of measuring the relative amount of capsid particles comprising variant recombinant AAV genomes in a preparation of recombinant AAV particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S),
c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each species of recombinant AAV particles,
d) comparing the amount of recombinant AAV particles having an S values that do not correspond to recombinant AAV particles comprising intact AAV genomes or empty capsid particles to the total amount of recombinant AAV particles in the preparation.

8. A method of measuring the relative amount of recombinant AAV particles comprising intact AAV genomes in a preparation of recombinant AAV particles comprising the steps of
a) subjecting the preparation to analytical ultracentrifugation under boundary sedimentation velocity conditions wherein the sedimentation of recombinant AAV particles is monitored at time intervals,
b) plotting the differential sedimentation coefficient distribution value (C(s)) versus the sedimentation coefficient in Svedberg units (S),
c) integrating the area under each peak in the C(s) distribution to determine the relative concentration of each species of recombinant AAV particles,
d) comparing the amount of recombinant AAV particles having an S values corresponding to recombinant AAV particles comprising intact AAV genomes to the amount of recombinant AAV particles having an S value corresponding to empty capsid particles, to capsid particles comprising variant recombinant AAV genomes, and/or to the total amount of recombinant AAV particles in the preparation.

9. A method of monitoring the removal of empty capsids and/or capsid particles comprising variant recombinant AAV genomes during the purification of a preparation of recombinant AAV particles, the method comprising removing a sample of the recombinant AAV particles from the preparation following one or more steps in the purification process and analyzing the sample for the relative amount of empty capsids and/or capsid particles comprising variant recombinant AAV genomes according to the method of claim 5 , wherein a decrease in the relative amount of empty capsids and/or capsids comprising variant genomes to full capsids indicates removal of empty capsids from the preparation of recombinant AAV particles.

10. The method of claim 4 , wherein the presence of a peak that corresponds to the S value of empty capsid particles indicates the presence of empty capsid particles; or the presence of one or more peaks other than the peak for full capsid particles comprising intact recombinant AAV genomes or empty capsid particles indicates that presence of capsid particles comprising variant sized genomes.

11. The method of claim 10 , wherein the presence of one or more peaks other than the peak for full capsid particles comprising intac

Assignees

• Genzyme Corp

Inventors

• O'Riordan Catherine R
• Burnham Brenda

Classifications

• G01N1/405

  by adsorption or absorption · CPC title

• C12N2750/14151

  Methods of production or purification of viral material · CPC title

• C12N7/00 Primary

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• G01N1/4077

  by other techniques involving separation of suspended solids · CPC title

• G01N2015/045

  by optical analysis · CPC title