A generic approach for simultaneous measurements of total antibody and cleavable antibody-conjugated drug by LC/MS/MS.

The current industry practice for antibody-drug conjugate (ADC) bioanalysis includes quantification of total antibody and antibody-conjugated drug. Here, we report a novel 2-in-1 approach for measuring total antibody and protease-cleavable conjugated drug Monomethyl Auristatin E (MMAE) concurrently. This allows for the determination of the DAR (Drug Antibody Ratio) for in vivo samples, with a 3-orders linear range based on total antibody concentration from 0.1 to 100 µg/mL. Our generic, concurrent method has been cross-validated with the previously established methods in an animal study. This novel approach is applicable to all human IgG1 ADCs with papain cleavable conjugated drug in preclinical studies.

Rapid quantification of a cleavable antibody-conjugated drug by liquid chromatography/tandem mass spectrometry with microwave-assisted enzymatic cleavage.

Antibody-drug conjugates (ADCs) play an increasingly important role for targeted cancer treatment. One class of ADCs has attracted particular interest in drug development. These ADCs employ a cleavable chemistry linkage for drugs and utilize the reduced interchain disulfide cysteine residues for conjugation. In this work, a novel bioanalytical method for the quantification of a cleavable antibody-conjugated drug in plasma was developed, qualified, and implemented. This novel method significantly improves throughput by combining a microwave-assisted, enzymatic cleavage of conjugated drugs from ADCs with a 96-well based sample preparation procedure to immunocapture ADCs in plasma. The released drug is subsequently quantified using a LC/MS/MS method. Our results represent a high-throughput, generic, and sensitive quantification method for antibody-conjugated microtubule inhibitors (such as MMAE) for preclinical PK/PD studies. The linear range of the standard curve for antibody conjugated drug (MMAE) was from 2.01 to 2010ng/mL with an excellent linearity (r(2)>0.997). The intra-run precision was below 8.14% and accuracy was from -7.71% to -1.08%. No matrix effect or carryover was observed for this method. This method was successfully used to measure the level of conjugated drug in a preclinical PK/PD study in mice.

Surface tensiometry of apolipoprotein B domains at lipid interfaces suggests a new model for the initial steps in triglyceride-rich lipoprotein assembly.

Apolipoprotein B (apoB) is the principal protein component of triacylglyceride (TAG)-rich lipoproteins, including chylomicrons and very low density lipoprotein, which is the precursor to LDL (the "bad cholesterol"). TAG-rich lipoprotein assembly is initiated by the N-terminal ßα1 superdomain of apoB, which co-translationally binds and remodels the luminal leaflet of the rough endoplasmic reticulum. The ßα1 superdomain contains four domains and is predicted to interact directly with lipids. Using drop tensiometry, we examined the interfacial properties of the α-helical and C-sheet domains and several subdomains to establish a detailed structure-function relationship at the lipid/water interface. The adsorption, stress response, exchangeability, and pressure (Π)-area relationship were studied at both triolein/water and triolein/1-palmitoyl, 2-oleoylphosphatidylcholine/water interfaces that mimic physiological environments. The α-helical domain spontaneously adsorbed to a triolein/water interface and formed a viscoelastic surface. It was anchored to the surface by helix 6, and the other helices were ejected and/or remodeled on the surface as a function of surface pressure. The C-sheet instead formed an elastic film on a triolein/water interface and was irreversibly anchored to the lipid surface, which is consistent with the behavior of amphipathic ß-strands. When both domains were adsorbed together on the surface, the C-sheet shielded a portion of the α-helical domain from the surface, which retained its globular structure. Overall, the unique secondary and tertiary structures of the N-terminal domains of apoB support the intrinsic capability of co-translational lipid recruitment. The evidence presented here allows the construction of a detailed model of the initiation of TAG-rich lipoprotein assembly.

Competition between intradomain and interdomain interactions: a buried salt bridge is essential for villin headpiece folding and actin binding.

Villin-type headpiece domains are ∼70 residue motifs that reside at the C-terminus of a variety of actin-associated proteins. Villin headpiece (HP67) is a commonly used model system for both experimental and computational studies of protein folding. HP67 is made up of two subdomains that form a tightly packed interface. The isolated C-terminal subdomain of HP67 (HP35) is one of the smallest autonomously folding proteins known. The N-terminal subdomain requires the presence of the C-terminal subdomain to fold. In the structure of HP67, a conserved salt bridge connects N- and C-terminal subdomains. This buried salt bridge between residues E39 and K70 is unusual in a small protein domain. We used mutational analysis, monitored by CD and NMR, and functional assays to determine the role of this buried salt bridge. First, the two residues in the salt bridge were replaced with strictly hydrophobic amino acids, E39M/K70M. Second, the two residues in the salt bridge were swapped, E39K/K70E. Any change from the wild-type salt bridge residues results in unfolding of the N-terminal subdomain, even when the mutations were made in a stabilized variant of HP67. The C-terminal subdomain remains folded in all mutants and is stabilized by some of the mutations. Using actin sedimentation assays, we find that a folded N-terminal domain is essential for specific actin binding. Therefore, the buried salt bridge is required for the specific folding of the N-terminal domain which confers actin-binding activity to villin-type headpiece domains, even though the residues required for this specific interaction destabilize the C-terminal subdomain.