A covalently linked recombinant albumin dimer is more rapidly cleared in vivo than are wild-type and mutant C34A albumin

Abstract

Mammalian albumins are abundant plasma proteins that exhibit a relatively slow terminal clearance. For this reason they have been fused to potentially therapeutic proteins with rapid terminal clearance to produce fusion proteins with more desirable clearance profiles. A disulfide-linked albumin dimer has been described, but its abundance and stability in plasma are uncertain. To determine whether an obligatory albumin dimer incapable of dissociation would clear less rapidly than monomeric albumin, we expressed 3 recombinant rabbit serum albumin (RSA) polypeptides: H 6RSA, RSA modified by the addition of an N-terminal hexahistidinyl tag; H 6RSA(C34A), H 6RSA with a single cysteine (Cys) 34–to–alanine (Ala) substitution (C34A); and DiRSA, H 6RSA(C34A) joined by way of its C-terminus to RSA(C34A) through an intervening hexaglycine spacer. The C34A mutation was introduced to eliminate the possibility of disulfide bond–mediated dimerization. We expressed the proteins with the use of the yeast Pichia pastoris and purified them using nickel-chelate, ion exchange, and gel-filtration chromatography. After radioiodination and injection into rabbits, H 6RSA and H 6RSA(C34A) exhibited indistinguishable terminal catabolic half-lives (4.9 ± 0.7 and 4.8 ± 0.5 days, mean ± SD), whereas that of DiRSA was reduced to 3.0 ± 0.3 days (p < .05). The three proteins circulated in intact form, and their distributions in liver, lung, kidney, heart, and spleen did not differ 24 hours after injection. Although more DiRSA than H 6RSA(C34A) was present in urine, in both cases it was in acid-soluble form. Ethyl palmitate treatment reduced the relative acceleration of DiRSA clearance compared with that of H 6RSA(C34A), suggesting a role for the reticuloendothelial system in the differential clearance of the larger protein. Our results suggest that an albumin fusion protein should include only a single copy of albumin; that if the fusion protein exceeds a certain size, it may not acquire the slow clearance profile of native albumin; and that albumin dimerization through Cys34 probably does not contribute substantially to albumin metabolism in vivo.