The C-terminal end of engineered albumin variants and fusion proteins after exposure to carboxypeptidase A

Albumin has a long plasma half-life due to engagement of the neonatal Fc receptor (FcRn), which prevents intracellular degradation. However, its C-terminal end can be cleaved by carboxypeptidase A, and removal of the last leucine residue (L585) weakens receptor binding, reducing its half-life from 20 days to 3.5 days in humans. This biology is important to consider when designing human albumin-fused biologics. Here, we explore engineering strategies to secure favorable FcRn binding and pharmacokinetic properties, and we use LC-MS/MS to analyze the amino acid sequence of the C-terminal end of engineered albumin variants and albumin fusion proteins, following incubation with or without carboxypeptidase A.