Methods for quantitating viral capsids
AssigneeSartorius BioAnalytical Instruments, Inc.
InventorsYuanyuan Zhang, Ling Zhang
Life of this patent
Estimating the term…
Abstract
Methods for quantitating viral capsids and/or polynucleotides contained therein using a charged biopolymer-loaded biosensor are provided. The signals indicative of binding of molecules (such as polynucleotides or viral capsids) to the biosensor can be measured by bio-layer interferometry (BLI). The amount of viral capsids can be calculated based on signals indicative of binding of the viral capsids to the biosensor. The empty/full viral capsid ratio in a viral sample can be calculated based on the amount of the viral polynucleotides and/or the binding kinetics of the biosensor to the viral capsids or the polynucleotides.
Claims22
A method for quantitating one or more polynucleotides contained within one or more viral capsids in a sample, the method comprising:
- subjecting the sample containing one or more viral capsids to a separation process to separate the one or more polynucleotides from the one or more viral capsids, the sample being a subject sample or a reference sample;
- contacting the sample with a biosensor such that the one or more polynucleotides in the sample are bound to a positively charged biopolymer of the biosensor, the biosensor containing a core component, a negatively charged biopolymer bound to an external surface of the core component, and the positively charged biopolymer bound to an external surface of the negatively charged biopolymer;
- contacting the one or more polynucleotides bound to the biosensor with one or more signal development elements to generate signals indicative of an amount of the one or more polynucleotides bound to the biosensor;
- and
- determining a ratio of empty and full viral capsids in the subject sample based at least partially on the amount of polynucleotides in the reference sample bound to the biosensor and an amount of polynucleotides in the subject sample bound to the biosensor.
(canceled)
A method for quantitating one or more polynucleotides contained within one or more viral capsids in a sample, the method comprising:
- subjecting the sample containing one or more viral capsids to a separation process to separate the one or more polynucleotides from the one or more viral capsids;
- contacting the sample with a biosensor such that the one or more polynucleotides in the sample are bound to a positively charged biopolymer of the biosensor, the biosensor containing a core component, a negatively charged biopolymer bound to an external surface of the core component, and the positively charged biopolymer bound to an external surface of the negatively charged biopolymer, different full/empty viral capsid ratios in the sample resulting in different binding kinetics of the biosensor to the one or more polynucleotides contained in the sample;
- and
- contacting the one or more polynucleotides bound to the biosensor with one or more signal development elements to generate signals indicative of an amount of the one or more polynucleotides bound to the biosensor.
A method for quantitating one or more viral capsids of adeno-associated virus (AAV) in a sample, the method comprising:
- contacting the sample with a biosensor such that the one or more viral capsids in the sample are bound to a positively charged biopolymer of the biosensor and generate a first set of signals indicative of binding of the viral capsids to the biosensor, the biosensor containing a core component, a negatively charged biopolymer bound to an external surface of the core component, and the positively charged biopolymer bound to an external surface of the negatively charged biopolymer;
- and
- quantitating the first set of signals generated by binding of the viral capsids to the biosensor.
A method for quantitating polynucleotides contained within one or more viral capsids of adeno-associated virus (AAV) in a sample, the method comprising:
- contacting the one or more viral capsids with a first biosensor to isolate and/or purify the viral capsids from a crude sample, the first biosensor comprising an affinity biosensor;
- subjecting the one or more viral capsids bound to the first biosensor to a separation process to separate the polynucleotides from the one or more viral capsids;
- contacting the polynucleotides with a second biosensor such that the polynucleotides are bound to the second biosensor;
- contacting the polynucleotides bound to the second biosensor with one or more signal development elements to generate signals indicative of an amount of the polynucleotides bound to the biosensor, wherein the one or more signal development elements comprise a dye;
- and
- determining a ratio of empty and full viral capsids based at least partially on the amount of polynucleotides bound to the second biosensor and/or binding kinetics of the second biosensor to the polynucleotides.
Dependencies are inferred from the claim text.