DNA impurities in a composition comprising a parvoviral virion
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.
1 . A method of determining whether a composition comprising a parvoviral vector is capable of human use in a clinical treatment, wherein the method comprises the steps of:
(i) identifying and quantifying an overrepresented nucleic acid impurity in the composition in a method comprising the steps of:
(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 the nucleotide sequence of a biological component identifies a nucleic acid impurity;
(c) determining an average number of reads per parvoviral vector; and
(d) determining a distribution of reads by calculating a number of reads per nucleotide of the identified nucleic acid impurity; wherein the identified nucleic acid impurity is identified as the overrepresented nucleic acid impurity when the distribution of reads is not random and the number of reads per nucleotide of the nucleic acid impurity is at least 0.001% the average number of reads per parvoviral vector, and
(ii) determining the composition as being capable of human use in the clinical treatment if the nucleic acid impurity is at least 10 times less present than a transgene as determined by the relative abundance of the overrepresented nucleic acid impurity.
2 . The method according to claim 1 , wherein the nucleic acid sequencing in step (a) comprises high-throughput sequencing.
3 . The method according to claim 1 , wherein the parvoviral vector is a recombinant adeno-associated virus (rAAV) vector.
4 . 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.
5 . The method according to claim 4 , wherein the nucleotide sequence of a biological component is a baculoviral vector.
6 . The method according to claim 5 , wherein the transgene is flanked by at least one parvoviral ITR.
7 . The method according to claim 4 , wherein the helper virus is a recombinant adenovirus and/or a recombinant herpes simplex virus.
8 . The method according to claim 1 , wherein the nucleotide sequence of a biological component comprises a nucleotide sequence encoding for a Rep, a Cap and/or the transgene.
9 . The method according to claim 1 , wherein the overrepresented nucleic acid impurity is quantified in a second or further composition.
10 . The method according to claim 1 , wherein the composition comprising the parvoviral vector comprises a parvoviral capsid, and wherein the parvoviral vector is packaged.
11 . The method according to claim 1 , wherein the composition comprising the parvoviral vector does not consist of a sample from a mammal.
12 . The method according to claim 1 , wherein the composition comprising the parvoviral vector is a pharmaceutical composition.