We needed to assay a PEGylated RNA aptamer in multiple biological matrices utilizing oligonucleotide hybridization on Meso Scale Discovery (MSD) platform.
We completed method development and GLP validation to quantitate the sponsor’s aptamer in Rat, Rabbit, Human Plasma, and Human Urine.
This intricate method involved sandwich hybridization 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.
One of our sponsors needed to achieve a reproducible PK assay with high sensitivity for their oligonucleotide therapeutic. Here, we performed a Meso Scale Discovery based assay combined with oligonucleotide hybridization.
Our veteran team optimized every step of this oligonucleotide hybridization ECL method to improve the 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 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. Afterward, we washed off the unbound oligonucleotides and detected the ECL signal from hybridized oligonucleotide with streptavidin-SULFO-TAG on an MSD imager.
We developed a reliable and transferable 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.
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 assay for PK and TK studies can tremendously aid in accelerating the drug development for these novel therapeutics.
