Production and characterization of mono-PEGylated alpha-1 antitrypsin for augmentation therapy.

Alpha-1 antitrypsin (AAT) is an endogenous inhibitor of serine proteases which, in physiological conditions, neutralizes the excess of neutrophil elastase and other serine proteases in tissues and especially the lungs. Weekly intravenous infusion of plasma-purified human AAT is used to treat AAT deficiency-associated lung disease. However, only 2 % of the AAT dose reach the lungs after intravenous infusion. Inhalation of AAT might offer an alternative route of administration. Yet, the rapid clearance of AAT from the respiratory tract results in high and frequent dosing by inhalation and limited efficacy. In the present study, we produced and characterized in vitro a PEGylated version of AAT which could offer a prolonged body residence time and thereby be useful for augmentation therapy by the intravenous and inhalation routes. Two PEGylation reactions - N-terminal and thiol PEGylation - and three polyethylene glycol (PEG) chains - linear 30 kDa, linear 40 kDa and 2-armed 40 kDa - were used. The yields of mono-PEGylated AAT following purification by anion exchange chromatography were 40-50 % for N-terminal PEGylation and 60-70% for thiol PEGylation. The PEG-AAT conjugates preserved the ability to form a protease-inhibitor complex with neutrophil elastase and proteinase 3 as well as the full inhibitory capacity to neutralize neutrophil elastase activity. These results open up interesting prospects for PEGylated AAT to achieve a prolonged half-life and an improved therapeutic efficacy in vivo.