Study of Hydrolytic Degradation of Heparin in Acidic and Alkaline Environments

The pharmaceutical heparin is a complex polysaccharide of the glycosaminoglycan family,
usually derived from porcine mucosa, and is a mainstay of anticoagulant therapy
worldwide. Its activity can be modified, most notably, through depolymerisation. Yet,
owing largely to the chain complexity, the progressive effects of environmental conditions
on the heparin structure have not been fully described. A systematic study of the
progressive effects of acidic and alkaline hydrolysis on heparin chain length and sulfate
substitution has therefore been conducted.

The initial analysis concerned the changes of weight-average molecular weight of heparin,
induced by applied conditions. In acidic environments, the relation between the molecular
weight loss and pH was inversely correlated, whilst the rate of degradation increased with
temperature. In alkaline environments, the molecular weight loss was proportional to pH
and temperature, although less effective.

Under milder acidic conditions, desulfation was the major factor affecting the molecular
weight of the polysaccharide, whilst in alkaline conditions, the hydrolytic sulfate scission
was not as prominent. Glycosidic scission was observed only after the prolonged hydrolytic
processing at pH 1, at 80 C. Stability studies confirmed the chain stability between pH 2
and pH 12.

To understand desulfation order and further investigate possible rearrangements, selected
acid and alkaline hydrolysates were monitored via 2D (HSQC) NMR. This study revealed
that in acidic environments all sulfate groups of heparin were altered. It was observed that
the sulfate groups were hydrolytically removed in the following order: N-sulfate (NS-),
then 2-O-sulfate (2OS-), and finally 6-O-sulfate (6OS-), although the selectivity of last two
was strongly dependent upon applied conditions. On the other hand, the NMR analysis of
alkali treated heparin demonstrated that applied environments catalysed only iduronate
desulfation (2OS-), followed by its exclusive rearrangements to a galacturonic residue.

The research discussed in this thesis investigated the behaviour of heparin in aqueous
systems altered by temperature and pH over time. The selected stressing factors closely
resembled conditions applied over the industrial and/or pharmacological processing.
Therefore, the contribution of presented findings reaches beyond theoretical knowledge
and extends to possible practical applications, i.e., optimisation of manufacturing, storage
and administration of pharmacologically active heparin products.