Synthesis and characterization of poly(ethylene glycol)-insulin conjugates

Human insulin was modified by covalent attachment of short-chain (750 and 2 000 Da) methoxypoly (ethylene glycol) (mPEG) to the amino groups of either residue PheB1 or LysB29, resulting in four distinct conjugates: mPEG(750)-P heB1-insulin, mPEG(2000)-PheB1-insulin, mPEG(750)-LysB29-insulin, and mPEG( 2000)-LysB29-insulin. Characterization of the conjugates by MALDI-TOF mass spectrometry and N-terminal protein sequence analyses verified that only a single polymer chain (750 or 2000 Da) was attached to the selected residue of interest (PheB1 or LysB29). Equilibrium sedimentation experiments were p erformed using analytical ultracentrifugation to quantitatively determine t he association state(s) of insulin derivatives. In the concentration range studied, all four of the conjugates and Zn-free insulin exist as stable dim ers while Zn2+-insulin was exclusively hexameric and Lispro was monomeric. In addition, insulin (conjugate) self-association was evaluated by circular dichroism in the near-ultraviolet wavelength range (320-250 nm). This inde pendent method qualitatively suggests that mPEG-insulin conjugates behave s imilarly to Zn-free insulin in the concentration range studied and compleme nts results from ultracentrifugation studies. The physical stability/resist ance to fibrillation of mPEG-insulin conjugates in aqueous solution were as sessed. The data proves that mPEG(750 and 2000)-PheB1-insulin conjugates ar e substantially more stable than controls but the mPEG(750 and 2000)-LysB29 -insulin conjugates were only slightly more stable than commercially availa ble preparations. Circular dichroism studies done in the far ultraviolet re gion confirm insulin's tertiary structure in aqueous solution is essentiall y conserved after mPEG conjugation. In vivo pharmacodynamic assays reveal t hat there is no loss in biological activity after conjugation of mPEG(750) to either position on the insulin B-chain. However, attachment of mPEG(2000 ) decreased the bioactivity of the conjugates to about 85% of Lilly's Humul inR formulation. The characterization presented in this paper provides stro ng testimony to the fact that attachment of mPEG; to specific amino acid re sidues of insulin's B-chain improves the conjugates' physical stability wit hout appreciable perturbations to its tertiary structure, self-association behavior, or in vivo biological activity.