Elucidating the Mechanism of Multispecific Antibody Aggregation through Subunit Analysis.

Multispecific antibody constructs are quickly becoming more common constructs in biopharmaceuticals to improve specificity and efficacy. While the advent of this technology has led to improved therapeutics, its development has challenged the analytical tools through which these therapeutics are characterized. Moreover, new critical quality attributes, such as aggregation, have challenged the approaches to characterization even further.

"Lab of the Future"─Today: Fully Automated System for High-Throughput Mass Spectrometry Analysis of Biotherapeutics.

Here we describe a state-of-the-art, integrated, multi-instrument automated system designed to execute methods involved in mass spectrometry characterization of biotherapeutics. The system includes liquid and microplate handling robotics and utilities, integrated LC-MS, along with data analysis software, to perform sample purification, preparation, and analysis as a seamless integrated unit. The automated process begins with tip-based purification of target proteins from expression cell-line supernatants, which is initiated once the samples are loaded onto the automated system and the metadata are retrieved from our corporate data aggregation system.

A fully integrated multi-column system for abundant protein depletion from serum/plasma.

This work details the transformation of a conventional HPLC system to a low back pressure liquid chromatography set-up for automated serum/plasma depletion and fractionation. A Dionex U3000 HPLC was converted to low back pressure operation (125 psi max) by replacing all narrow-bore lines to larger inner-diameter tubing. The system was configured to use two immunoaffinity columns, first for depletion of the top 14 most abundant proteins (Seppro IgY14), then for the next 200-300 proteins (Seppro SuperMix).

Probing the solution structure of tumor necrosis factor-α homotrimer and heterotrimer after complex perturbation using electrospray ionization mass spectrometry.

There are a number of proteins whose active forms are non-covalent multichain complexes. Therapeutic intervention involving such complexes has been proposed through the use of muteins to form heterostructures. These resulting structures would either not be recognized by receptors or would be inactive competitive inhibitors to wild-type (wt) proteins.

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain.

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3.

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells.

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production.

Characterization of the epitope for anti-human respiratory syncytial virus F protein monoclonal antibody 101F using synthetic peptides and genetic approaches.

Chimeric 101F (ch101F) is a mouse-human chimeric anti-human respiratory syncytial virus (HRSV) neutralizing antibody that recognizes residues within antigenic site IV, V, VI of the fusion (F) glycoprotein. The binding of ch101F to a series of peptides overlapping aa 422-438 spanning antigenic site IV, V, VI was analysed. Residues 423-436 comprise the minimal peptide sequence for ch101F binding.