C-Terminal Lysine Processing of IgG in Human Suction Blister Fluid: Implications for Subcutaneous Administration.

ABSTRACT

C-terminal lysine (CTK) is often classified as a potential critical quality attribute for therapeutic antibodies being developed for subcutaneous (SC) administration because of its potential to impact antibody SC bioavailability/pharmacokinetics (PK). This classification both inflates development costs and increases comparability risks for SC administration of antibodies. However, prior risk assessments of CTK have not fully considered biotransformation of CTK in the SC space, which may play an important role given that circulating carboxypeptidases in humans rapidly process CTK on intravenously administered antibodies. Here, CTK biotransformation in biofluid derived from human SC space was investigated. The representative fluid from the human SC space was sampled from 10 healthy human subjects using the suction blister method. Glycosylated antibody containing high levels of CTK (expressed using a carboxypeptidase D CRISPR/Cas9 knockout CHO cell line) was incubated in the collected suction blister fluids (SBFs), recovered using cognate antigen pulldowns, and characterized for remaining CTK levels using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analysis. CTK processing (i.e., carboxypeptidase activity) was evident in all SBF and exhibited first-order kinetics with rate constants of 2.18 ± 0.57 d-1 (at 37 °C). PK simulations that integrated CTK processing pathways and their associated rate constants were subsequently performed using a range of clinically observed PK parameters for therapeutic antibodies, including atezolizumab- and pertuzumab-specific parameters. The impact of CTK content (even up to 100%) on SC PK outcomes such as bioavailability and Ctrough were modest (<14%) for all combinations of PK parameters tested in the sensitivity analysis. This study forms the cornerstone data package for derisking CTK as a PK liability for antibody SC programs and highlights the usefulness of fully considering biotransformation during product quality criticality assessments.