Inhomogeneous Diastereomeric Composition of Mongersen Antisense Phosphorothioate Oligonucleotide Preparations and Related Pharmacological Activity Impairment

Abstract

Mongersen is a 21-mer antisense oligonucleotide designed to downregulate Mothers against decapentaplegic homolog 7 (SMAD7) expression to treat Crohn's disease. Mongersen was manufactured in numerous batches at different scales during several years of clinical development, which all appeared identical, using common physicochemical analytical techniques, while only phosphorous-31 nuclear magnetic resonance (³¹P-NMR) in solution showed marked differences. Close-up analysis of 27 mongersen batches revealed marked differences in SMAD7 downregulation in a cell-based assay. Principal component analysis of ³¹P-NMR profiles showed strong correlation with SMAD7 downregulation and, therefore, with pharmacological efficacy in vitro. Mongersen contains 20 phosphorothioate (PS) linkages, whose chirality (Rp/Sp) was not controlled during manufacturing. A different diastereomeric composition throughout batches would lead to superimposable analytical data, but to distinct ³¹P-NMR profiles, as indeed we found. We tentatively suggest that this may be the origin of different biological activity. As similar manifolds are expected for other PS-based oligonucleotides, the protocol described here provides a general method to identify PS chirality issues and a chemometric tool to score each preparation for this elusive feature.

Keywords: 31P-NMR; Crohn's disease; antisense oligonucleotides; mongersen; phosphorothioate.

FIG. 1.

Schematic representation of phosphorothioate-modified oligonucleotides with Sp (red, left) and Rp (blue, right) configuration at the phosphorous atom.

FIG. 2.

(a) Western blots for SMAD7 protein expression in cells treated with mongersen from different batches. HCT-116 cells were either incubated with Lipofectamine 3000 only (Lipo) or transfected with the indicated batches of drug substance. Whole-cell extracts were prepared and analyzed for SMAD7 expression by Western blotting. β-actin was used as a loading control. One of at least three representative experiments for each set of samples is shown. (b) Quantitative analysis of SMAD7/β-actin protein ratio in total extracts of HCT-116 cells treated as indicated in (a), as measured by densitometry scanning of Western blots. Values are expressed in a.u. and are the mean ± SEM of at least three experiments for each set of samples. The dashed lines represent the SMAD7/β-actin protein ratio in cells transfected with the reference batch H. “Lipo” indicates the negative control, with cells treated with transfection reagent only. Reference batch H versus the indicated mongersen batches, *P < 0.05, **P < 0.01. (c) In vitro capability of mongersen batches to downregulate SMAD7 protein expression compared with batch H. An arbitrary pharmacology performance score, defined as the mean percentage difference in SMAD7 protein expression between treatment with the indicated batch and the reference batch H, was assigned to the mongersen batches based on their capability to downregulate the SMAD7 protein. The pharmacology performance of the batches is represented by colored bars and is defined as follows: green—similar to batch H (efficacy within 20% of H), yellow—marginal decrease in performance compared with batch H (decrease in efficacy of 20–40% vs. H); red—poor performance (>40% decrease of efficacy compared with H). “Lipo” indicates the negative control, with cells treated with transfection reagent only. For each batch, the bar represents the mean of at least three independent experiments. a.u., arbitrary units; HCT-116; SEM, standard error of the mean; SMAD7, Mothers against decapentaplegic homolog 7.

FIG. 3.

The ³¹P-NMR spectra of a subset of mongersen batches, revealing differences in the fine structures of the ³¹P resonances.

FIG. 4.

The score plot of the ³¹P spectra of the 10 batches of mongersen (M, C, L, S, U, G, W, Q, F, and H) used as a training set for the PCA. PCA, principal component analysis.

FIG. 5.

The score plot of the ³¹P spectra of the 27 batches of mongersen analyzed in this study. The filled circles (batch number reported in Fig. 4) represent samples from the training set. The open circles represent the external set. The colors correspond to SMAD7 expression inhibition (as defined in Fig. 2 legend): green—similar to batch H, yellow—marginal decrease in performance versus H; and red—poor performance.