UV and X-ray structural studies of a 101-residue long Tat protein from a HIV-1 primary isolate and of its mutated, detoxified, vaccine candidate.

The 101-residue long Tat protein of primary isolate 133 of the human immunodeficiency virus type 1 (HIV-1), wt-Tat(133) displays a high transactivation activity in vitro, whereas the mutant thereof, STLA-Tat(133), a vaccine candidate for HIV-1, has none. These two proteins were chemically synthesized and their biological activity was validated. Their structural properties were characterized using circular dichroism (CD), fluorescence emission, gel filtration, dynamic light scattering, and small angle X-ray scattering (SAXS) techniques.

Identification and characterization of asparagine deamidation in the light chain CDR1 of a humanized IgG1 antibody.

Despite technological advances, detection of deamidation in large proteins remains a challenge and the use of orthogonal methods is needed for unequivocal assignment. By a combination of cation-exchange separation, papain digestion, and a panel of mass spectrometry techniques we identified asparagine deamidation in light chain complementarity determining region 1 (CDR1) of a humanized IgG1 monoclonal antibody. The reaction yields both Asp and isoAsp, which were assigned by Edman degradation and by isoAsp detection using protein isoaspartate methyltransferase.

Trends in glycosylation, glycoanalysis and glycoengineering of therapeutic antibodies and Fc-fusion proteins.

Monoclonal antibodies (MAbs) are the fastest growing class of human pharmaceuticals. More than 20 MAbs have been approved and several hundreds are in clinical trials in various therapeutic indications including oncology, inflammatory diseases, organ transplantation, cardiology, viral infection, allergy, and tissue growth and repair. Most of the current therapeutic antibodies are humanized or human Immunoglobulins (IgGs) and are produced as recombinant glycoproteins in eukaryotic cells.

The way forward, enhanced characterization of therapeutic antibody glycosylation: comparison of three level mass spectrometry-based strategies.

Glycosylation which plays a crucial role in the pharmacological properties of therapeutic monoclonal antibodies (MAbs) is influenced by several factors like production systems, selected clonal population and manufacturing processes. Efficient analytical methods are therefore required in order to characterize glycosylation at different stages of MAbs discovery and production. Three mass spectrometry (MS)-based strategies were compared to analyze N-glycosylation of MAbs either expressed in murine myeloma (NS0) or Chinese Hamster Ovary (CHO) cell lines, the two current main production systems used for therapeutic MAbs.

Peptides as tools and drugs for immunotherapies.

Peptides are essential tools for discovery and pre-clinical and pharmaceutical development of viral and cancer vaccines ('active immunotherapies') as well as for therapeutic antibodies ('passive immunotherapies'). They help to trigger and analyze immune responses at a molecular level (B-cell, T-helper and CTL epitopes). They contribute largely to the design of new vaccine candidates and to the generation of monoclonal antibodies.