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

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.

In Vivo Reoxidation Kinetics of Free Thiols in Multiple Domains of IgG1 Antibodies in Rats.

While free thiols in monoclonal antibodies (mAbs) have been extensively characterized by in vitro studies to probe its effect on antibody function and stability, their in vivo biotransformation has not been comprehensively studied. In this study, a panel of five recombinant IgG1 mAbs with elevated free thiols in the VH, VL, and CH2 domains were intravenously administered into Wistar rats. In vivo biotransformation of thirty-five free thiol sites in total (7 disulfide pairs in VL, CL, VH, CH1, HH, CH2, CH3 domains across the 5 mAbs) were monitored using a denaturing differential isotopic tagging procedure on immunopurified timepoints followed by LC-MS of tryptic digests.

Identification and quantitation of hinge cysteinylation on endogenous IgG2 antibodies from human serum.

Cysteinylation is a post-translational modification (PTM) that occurs when a cysteine residue on a protein forms a disulfide bond with a terminal cysteine molecule. This PTM has been found in the hinge region of several recombinant therapeutic IgG2 antibodies, but the impact of cysteinylation on the safety and immunogenicity of therapeutics remains unclear. In this study, we characterized recombinant and endogenous IgG2 antibodies to quantify their levels of hinge cysteinylation, if present.

Stress Temperature Studies in Small Scale Hastelloy® Drug Substance Containers Lead to Increased Extent of and Increased Variability in Antibody-Drug Conjugate and Monoclonal Antibody Aggregation: Evidence for Novel Oxidation-Induced Crosslinking in Monoclonal Antibodies.

Health authorities require that suitable stability of the drug substance be shown in relevant materials of construction. ICH Q1A(R2) explicitly states that "stability studies should be conducted on drug substance packaged in a container closure system that is the same as or simulates the packaging proposed for storage and distribution". Stainless steel containers are commonly used for the long-term storage of frozen bulk drug substances (DSs).

Dentate granule cell recruitment of feedforward inhibition governs engram maintenance and remote memory generalization.

Memories become less precise and generalized over time as memory traces reorganize in hippocampal-cortical networks. Increased time-dependent loss of memory precision is characterized by an overgeneralization of fear in individuals with post-traumatic stress disorder (PTSD) or age-related cognitive impairments. In the hippocampal dentate gyrus (DG), memories are thought to be encoded by so-called 'engram-bearing' dentate granule cells (eDGCs).

Structural Characterization of Cross-Linked Species in Trastuzumab Emtansine (Kadcyla).

The antibody-drug conjugate, trastuzumab emtansine (Kadcyla), is produced by attachment of the antitubulin drug, DM1, to lysine amines via a heterobifunctional linker, SMCC (succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate). Following the reaction of the N-hydroxysuccinimide activated linker with antibody lysines to produce a linker-modified intermediate (Tmab-MCC), DM1 is added to yield the desired product. In addition to the expected distribution of drug-linked forms (from 0 to 8), mass spectrometry also demonstrates the presence of a second distribution shifted by about +222 Da.

Statistical modeling of the drug load distribution on trastuzumab emtansine (Kadcyla), a lysine-linked antibody drug conjugate.

Trastuzumab emtansine (Kadcyla) is a recently approved antibody-drug conjugate produced by attachment of the anti-tubulin drug, DM1, to lysine amines via the SMCC linker. The resulting product exhibits a drug load distribution from 0 to 8 drugs per antibody that can be quantified using mass spectrometry. Different statistical models were tested against the experimental data derived from samples produced during process characterization studies to determine best fit.