Monclonal antibdies

Human Antibody Repertoire following Ebola Virus Infection and Vaccination.

What is the Ebola anti-body response?

Limited knowledge exists on the quality of polyclonal antibody response generated following Ebola virus (EBOV) infection compared with vaccination. Polyclonal antibody repertoire in plasma following EBOV infection in survivors was compared with ChAd3-MVA prime-boost human vaccination.

Higher antibody binding and affinity to GP was observed in survivors compared with vaccinated plasma that correlated with EBOV neutralization.

IgG, IgD, IgA, IgE, IgM

Surprisingly, a predominant IgM response was generated after prime-boost vaccination, whereas survivors demonstrated IgG-dominant antibody response.

Lieven Gevaert, Bio-ir. Genprice Inc.

EBOV infection induced more diverse antibody epitope repertoire compared with vaccination.

A strong binding to antigenic sites in the fusion peptide and another in the highly conserved GP2-HR2 domain was preferentially recognized by EBOV survivors than vaccinated individuals that correlated strongly with EBOV neutralization titers. These findings will help development and evaluation of effective Ebola countermeasures including therapeutics and vaccines.

How many classes of main heavy chain C domains antibodies are there?

5 classes: Each class defines the IgM, IgG, IgA, IgD, and IgE isotypes. IgG can be split into four subclasses, IgG1, IgG2, IgG3, and IgG4, each with its own biologic properties; and IgA can similarly be split into IgA1 and IgA2.

Djoumana Ounas, Pharamcist, Gentaur Bvba
Isotypes of Ebov antibodies

Characterization of the acidic species of a monoclonal antibody using free flow electrophoresis fractionation and mass spectrometry.

Antibody charge heterogeneity is one of the major product-related variants in recombinant biopharmaceuticals, which has been commonly monitored by imaged capillary isoelectric focusing (icIEF). Due to the challenges with sample recovery and fractionation, other charge-based analytical approaches have been explored as complementary methods allowing for further detailed charge variant characterization.

This study describes the utilization of free flow electrophoresis (FFE) fractionation in combination with other analytical techniques, such as mass spectrometry for monoclonal antibody acidic variants characterization. The preparative FFE technique allowed for continuous sample separation and fluid phase fractionation of antibody charge isoforms. The monoclonal antibody starting material was fractionated by FFE, followed by purification and characterization. icIEF analysis demonstrated the purity of the fractions and comparability of the charge profiles between these two techniques.

The intact molecular mass analysis revealed that glycation modification was highly enriched in the acidic fractions. SEC UV/Fluorescence method was developed to assess the levels of aggregation and fluorescent advanced glycation end-products (AGEs). Detailed peptide map was performed and revealed that acidic fractions were enriched in AGEs, methionine, tryptophan, histidine oxidation, asparagine deamidation, lysine glycation, carboxymethyl lysine, glycine to aspartic acid substitution compared to the main peak and starting material. The results indicate that acidic variants can account for a variety of low-level modifications present as very heterogeneous forms.

Coupling non-denaturing chromatography to mass spectrometry for the characterization of monoclonal antibodies and related products.

The hyphenation of non-denaturing liquid chromatographic (LC) modes (ion exchange (IEX), size exclusion (SEC) and hydrophobic interaction chromatography (HIC)) with mass spectrometry (MS) has attracted significant attention in the last few years. The inherent problem of these couplings is that non-denaturing LC separations have tended to use non-volatile mobile phase additives. Indeed, classical methods have not been directly compatible with MS. Therefore two approaches can be used to address this challenge: finding innovative volatile mobile phases.