Characterisation of recombinant factor IX before and after GlycoPEGylation.

The effect of the GlycoPEGylation process used for prolonging the half-life of recombinant factor IX (rFIX) has no impact on the primary and higher order structure of activated factor IX. Characterisation work performed on recombinant factor IX and on the GlycoPEGylated form of rFIX (N9-GP), confirm that the primary structure as well as the post translational modifications (PTMs) (disulphide bonds, γ-carboxylation, ß-hydroxylation, sulphation and O- and N-linked glycan structures) were comparable for rFIX and N9-GP. Three O-linked glycan sites were identified in the activation peptide (Thr159, Thr163 and Thr169), where Thr163 has not been reported previously. For N9-GP, the mono GlycoPEGylation is directed toward one of the two N-linked glycans present at Asn157 and Asn167 in the activation peptide in a one to one ratio. Spectroscopic techniques, such as far and near UV Circular Dichroism studies show comparable secondary and tertiary structures of rFIX and N9-GP. The thermally induced unfolding of rFIX and N9-GP shows that the unfolding temperature is approximately 1 °C higher for N9-GP than that of the rFIX. Furthermore, the pH dependent degradation was reduced due to the GlycoPEGylation of rFIX. GlycoPEGylated rFIX (N9-GP) is used for the manufacturing of Refixia® (nonacog beta pegol, Rebinyn®, Novo Nordisk A/S, Bagsvaerd, Denmark).

Effect of ethylenediamine on chemical degradation of insulin aspart in pharmaceutical solutions.

PURPOSE: To examine the effect of different amine compounds on the chemical degradation of insulin aspart at pharmaceutical formulation conditions. METHODS: Insulin aspart preparations containing amine compounds or phosphate (reference) were prepared and the chemical degradation was assessed following storage at 37 degrees C using chromatographic techniques. Ethylenediamine was examined at multiple concentrations and the resulting insulin-ethylenediamine derivates were structurally characterized using matrix assisted laser desorption ionization time-of-flight mass spectroscopy. The effects on ethylenediamine when omitting glycerol or phenolic compounds from the formulations were investigated. RESULTS: Ethylenediamine was superior in terms of reducing formation of high molecular weight protein and insulin aspart related impurities compared to the other amine compounds and phosphate. Monotransamidation of insulin aspart in the presence of ethylenediamine was observed at all of the six possible Asn/Gln residues with Asn(A21) having the highest propensity to react with ethylenediamine. Data from formulations studies suggests a dual mechanism of ethylenediamine and a mandatory presence of phenolic compounds to obtain the effect. CONCLUSIONS: The formation of high molecular weight protein and insulin aspart related impurities was reduced by ethylenediamine in a concentration dependant manner.