Cell and Gene Therapy
Custom-Built Cell and Gene Therapy Research
Oligonucleotide-based drugs (antisense RNAs, aptamers, siRNAs, mRNAs, vectors) are receiving a great deal of attention since the approval of Ionis’ Vitravene and Spark Therapeutics’ LUXTURNA™. QPS has supported gene therapy development since 2005 and can use that experience to help you navigate PK, immunogenicity, biodistribution and viral clearance of these novel molecules in this rapidly expanding field.
As this is still a relatively new area, the quantitation of oligonucleotide-based drugs in any biological matrix is not as well established as small organics or proteins and vaccines.
QPS uses both chromatographic and ligand binding methods for quantitation of oligonucleotide-based drugs. For chromatographic methods, we have used the traditional LC-MS/MS, as well as LC/UV assays to examine plasma and tissues exposure. We are also using LC-TOF-MS for quantitation of double stranded (ds) oligonucleotides. This high resolution (LC-HRMS) methodology can simultaneously determine both the sense and the anti-sense strands using a “N-in-1” methodology where the “N”s are the exact mass of the individual isotopic peaks of the different charge envelopes. We have quantitated up to 20,000 Daltons ds oligonucleotides with LLOQ at 5 ng/mL per the latest FDA Bioanalytical Method Validation guidance.
QPS scientists will work with your development team to determine specific study parameters. The choice of detection and quantitation and whether to use chromatographic or LBA is based on the primary structure, the number of monomeric units and the desired study design.
OLIGONUCLEOTIDES QUANTITATION BY
HIGH-RES MS OR HYBRIDIZATION PLUS CHROMATOGRAPHY
We use UPLC-HRMS to simultaneously quantify both the antisense and the sense strands of double stranded oligos using a “N in 1” methodology. Here, the “N”s are the exact mass of the individual isotopic peaks of the different charge envelopes.
Depending on the nature of the oligonucleotide drug, we may instead employ a probe-hybridization approach. QPS designs complementary fluorescence-tagged probes to track the antisense strand or other relevant portion of the drug entity. The fluorescence-tagged antisense drug and “N-1, 2, 3…” length metabolites are assessed by LC fluorescence in a single run allowing both the parent drug and its metabolites to be distinguished and quantitated. Additionally, QPS has experience with PEGylated aptamers, which are best studied using LC/UV assay systems for quantitation.
QPS scientists will work with your development team to determine specific study parameters. The choice of platform for detection and quantitation – chromatography or probe hybridization – is based on the primary structure of the target, the number of monomeric units and the desired study design.
mRNA THERAPEUTIC AND GENE THERAPY VECTOR QUANTITATION BY qPCR
Nucleic acid drug targets that are long enough and offer sufficient sequence complexity are quantitated using real-time, quantitative PCR (typically probe hydrolysis) with reverse-transcription, if necessary. Assays are custom built to detect only your drug target, while additional assays can be developed to assess secondary targets or drug response. Multiple automated extraction devices and microfluidic plate assembly robots are used for high throughput production. The staff have more than fifty cumulative years of nucleic acid assay design and quantitation in regulated environments. QPS is ready to work with you to provide complete, fully-compliant data packages and reports, ready for submission.