2D NMR peak profiling to compare chemical differences between batches of pentosan polysulfate sodium

Pentosan Polysulfate Sodium (PPS) is a semi-synthetic polysulfated xylan sourced from beechwood tree barks. PPS, which is mainly composed of a xylose chain with branched O-methyl-glucuronate (MGA), can have heterogeneity in monosaccharide species, sequence and chemical modifications including sulfation and acetylation. The monosaccharide composition in polysaccharide therapeutics is a frequently quoted quality attribute (QA), which has been assessed using two-dimensional (2D) ¹H-¹³C HSQC NMR. However, the sensitivity of 2D NMR for the assessment of PPS inter-lot variability from the same manufacturer was unclear and questions remained whether 2D NMR had sufficient sensitivity to distinguish normal batch to batch variations in this QA. Here, a 2D peak profile method was applied to compare high-resolution semi-quantitative (semi-q) HSQC spectra with the inclusion of intermediate precision spectra collected on two PPS drug lots released 29 months apart (where one of the lots was expired). The semi-q HSQC NMR confirmed the mass equivalence of total polysaccharides, O-Methyl and acetyl groups between the two lots. The 2D spectral peak profile results readily identified significant lot-to-lot differences (p < 0.05) in relative distribution among most monosaccharide species, in addition to heterogeneity in MGA distribution and acetyl transfer from PPS to free acetate in the expired lot. Precisely measured chemical QAs are prerequisites to establish normal batch variation in the innovator product, providing important reference ranges for complex generic drug developers. Overall, high-resolution semi-q HSQC NMR may provide a sensitive tool to measure fine chemical differences in polysaccharide therapeutics needed to establish chemical QAs and compare batches without concerns of intrinsic NMR method variation.