We needed to perform an oligonucleotide assay on a PEGylated RNA aptamer in multiple biological matrices utilizing oligonucleotide hybridization ELISA on Meso Scale Discovery (MSD) platform.
We completed oligonucleotide analysis method development and GLP validation to quantitate the sponsor’s aptamer in Rat, Rabbit, Human Plasma, and Human Urine through oligonucleotide hybridization analysis.
This oligonucleotide analysis involved sandwich hybridization ELISA of a capture complementary oligonucleotide and biotinylated detection complementary oligonucleotide, followed by streptavidin SULFO-TAG addition and detection of electrochemiluminescent (ECL) reaction on Meso Scale Discovery (MSD) platform for oligonucleotide quantification.
One of our sponsors needed to achieve a reproducible PK oligonucleotide assay with high sensitivity for their oligonucleotide therapeutic. Here, we performed a Meso Scale Discovery based assay combined with oligonucleotide hybridization analysis.
Our veteran team optimized every step of this oligonucleotide hybridization ECL method to improve the oligonucleotide assay performance and robustness.
To begin with, our team utilized a checkerboard approach to determine the optimal concentrations for coating and detection oligonucleotides, blocking conditions, temperature for oligonucleotide denaturation, annealing and hybridization, and duration of incubation. We also worked out appropriate sample dilution as this analyte posed a hook effect at higher concentrations.
MSD multi-array oligonucleotide analysis plate was coated overnight with the capture oligonucleotide. Standards and samples were prepared, heat-inactivated, and combined with biotinylated detection oligonucleotide. Next, this mixture was annealed on a thermocycler and incubated in the coated plate to allow oligonucleotide hybridization analysis. Afterward, we washed off the unbound oligonucleotides and detected the ECL signal from hybridized oligonucleotide with streptavidin-SULFO-TAG on an MSD imager for oligonucleotide quantification.
We developed a reliable and transferable oligonucleotide assay method for bioanalysis of RNA aptamer in multiple biological matrices. Furthermore, these PK assays demonstrated a dose-dependent profile and were submitted by the sponsor to the regulatory agencies for oligonucleotide quantification.
Since oligonucleotide aptamers have proven to be of high therapeutic and diagnostic value and particularly useful for cell-type specific delivery of other RNA therapeutics (like siRNA), there has been a greater impetus for the development of novel aptamers as therapeutics.
Consequently, a robust and reproducible oligonucleotide analysis for PK and TK studies can tremendously aid in accelerating the drug development for these novel therapeutics.
