# US 11,021,762 B2 · DNA impurities in a composition comprising a parvoviral virion

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- Number: US 11,021,762 B2
- Document ID: US11021762B2
- Status: Granted
- Kind: B2
- Application number: 15/531,095
- Filed: 2015-11-27
- Published: 2017-11-09
- Granted: 2021-06-01
- Earliest claimed: 2014-11-28
- Assignees: uniQure IP B.V.
- Applicants: uniQure IP B.V.
- Inventors: Jacek Lubelski; Wilhelmus Theodorus Johannes Maria Christiaan Hermens
- Web page: https://patentgrep.com/patent/US11021762B2

## CPC

- C12Q 1/701: {Specific hybridization probes}
- C12Q 1/6851: Quantitative amplification
- C12Q 1/6869: Methods for sequencing
- C12N 2710/14021: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12N 2710/14111: Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
- C12N 2750/14111: Dependovirus, e.g. adenoassociated viruses
- C12N 2750/14121: Viruses as such, e.g. new isolates, mutants or their genomic sequences
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The current invention relates to nucleic acid impurities in a composition comprising a parvoviral vector. In particular, the current invention shows that DNA impurities are not randomly encapsulated within a parvoviral virion. The invention therefore relates to a method for identifying and quantifying a nucleic acid impurity in a composition comprising a parvoviral vector. Finally, the current invention relates to method of determining whether a composition comprising a parvoviral vector is regarded as clinically pure.

## Claims

### Claim 1

A method for identifying and quantifying a nucleic acid impurity in a composition comprising a parvoviral vector, comprising:
(a) subjecting the composition to nucleic acid sequencing to obtain at least a thousand random reads of nucleotide sequences;
(b) comparing, using a processor, the at least a thousand random reads from step (a) with a nucleotide sequence of a biological component used in a process for producing the composition, whereby a match between a random read and a nucleotide sequence of a biological component identifies a nucleic acid impurity;
(c) determining an average number of reads per parvoviral vector;
(d) determining a distribution of reads by calculating a number of reads per nucleotide of an identified nucleic acid impurity;
wherein an identified nucleic acid impurity is identified as an overrepresented impurity when the distribution of reads is not random and the number of reads per nucleotide of a nucleic acid impurity is at least 0.001% the average number of reads per parvoviral vector.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the nucleic acid sequencing in step (a) comprises high-throughput sequencing.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the parvoviral vector is a recombinant adeno-associated virus (rAAV) vector.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the nucleotide sequence of a biological component is selected from a group consisting of nucleotide sequences of: a host cell, a plasmid, a vector other than the recombinant parvoviral vector, and a helper virus.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the nucleotide sequence of a biological component is a baculoviral vector.

### Claim 6 (depends on 4)

The method according to claim 4, wherein the helper virus is a recombinant adenovirus and/or a recombinant herpes simplex virus.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the nucleotide sequence of the biological component comprises a nucleotide sequence encoding for Rep, Cap and/or a transgene.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the transgene is flanked by at least one parvoviral ITR.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the overrepresented nucleic acid impurity is quantified in a second or further composition.

### Claim 10 (depends on 1)

The method according claim 1, wherein the composition comprising the parvoviral vector comprises a parvoviral capsid wherein the parvoviral vector is packaged.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the composition comprising the parvoviral vector does not consist of a sample from a mammal.