Improved particle counting and size distribution determination of aggregated virus populations by asymmetric flow field-flow fractionation and multiangle light scattering techniques.

A method using a combination of asymmetric flow field-flow fractionation (AFFFF) and multiangle light scattering (MALS) techniques has been shown to improve the estimation of virus particle counts and the amount of aggregated virus in laboratory samples. The method is based on the spherical particle counting approach given by Wyatt and Weida in 2004, with additional modifications. The new method was tested by analyzing polystyrene beads and adenovirus samples, both having a well-characterized particle size and concentration. Influenza virus samples were analyzed by the new AFFFF-MALS technique, and particle size and aggregate state were compared with results from atomic force microscopy analysis. The limitations and source of possible errors for the new AFFFF-MALS analysis are discussed.

Biophysical characterization of influenza virus subpopulations using field flow fractionation and multiangle light scattering: correlation of particle counts, size distribution and infectivity.

Adequate biophysical characterization of influenza virions is important for vaccine development. The influenza virus vaccines are produced from the allantoic fluid of developing chicken embryos. The process of viral replication produces a heterogeneous mixture of infectious and non-infectious viral particles with varying states of aggregation. The study of the relative distribution and behavior of different subpopulations and their inter-correlation can assist in the development of a robust process for a live virus vaccine. This report describes a field flow fractionation and multiangle light scattering (FFF-MALS) method optimized for the analysis of size distribution and total particle counts. The FFF-MALS method was compared with several other methods such as transmission electron microscopy (TEM), atomic force microscopy (AFM), size exclusion chromatography followed by MALS (SEC-MALS), quantitative reverse transcription polymerase chain reaction (RT Q-PCR), median tissue culture dose (TCID(50)), and the fluorescent focus assay (FFA). The correlation between the various methods for determining total particle counts, infectivity and size distribution is reported. The pros and cons of each of the analytical methods are discussed.

Identification of a single tryptophan residue as critical for binding activity in a humanized monoclonal antibody against respiratory syncytial virus.

We have identified a single tryptophan (Trp) residue responsible for loss of binding and biological activity upon ultraviolet (UV) light irradiation in MEDI-493, a humanized monoclonal antibody (MAb) against respiratory syncytial virus (RSV). This finding provides a better understanding of structure-function relationship in a 150-kDa protein. Irradiation of MEDI-493 with UV light resulted in spectral changes typical of Trp photoproducts and in a progressive loss of MEDI-493 binding and biological activity as measured by ELISA, Biacore, and cell-based assays. Mass spectrometric characterization of the proteolytic peptides generated from the UV irradiated MEDI-493 confirmed that most methionine (Met) and a few Trp residues were oxidized to various extents upon exposure to UV light. Among Trp residues, only Trp-105, containing the most solvent-exposed indole moiety in MEDI-493 and residing in a complementary-determining region (CDR) of the heavy chain, was significantly oxidized. When bound to a synthetic antigenic peptide, MEDI-493 showed significant resistance toward binding activity loss during UV irradiation. A second MAb (MEDI-524) with Trp-105 replaced by phenylalanine (Phe) showed a similar pattern of Met oxidation, but no loss of binding and biological activity following irradiation. Treatment of both MAbs with Met- and Trp-specific oxidizing reagents showed that oxidation of Trp-105 correlated with the activity loss, whereas Met oxidation did not affect the activity. These results demonstrate that Trp-105 in MEDI-493 is responsible for the UV light-induced effects.

Characterization of a novel modification to monoclonal antibodies: thioether cross-link of heavy and light chains.

A novel, nonreducible thioether bridge between the light and heavy chains of different IgG1 monoclonal antibodies has been characterized. An additional band with an apparent molecular weight of 92 kDa was detected when monoclonal antibodies were analyzed by reducing capillary gel electrophoresis (rCGE) and reducing SDS-PAGE. To further investigate this observation, an early-eluting peak in the size exclusion chromatogram of a reduced and alkylated monoclonal antibody was collected and characterized by liquid chromatography, mass spectrometry, and gel electrophoresis. The reduced and alkylated Mab was shown to be a cross-linked adduct with a molecular weight of 75 kDa. In the adduct, the heavy and light chains of the antibody were cross-linked by a nonreducible thioether bond between Cys-223 of the heavy chain and the C-terminal Cys residue of the light chain. The thioether bond modification was confirmed in the Fab fragment of a monoclonal antibody by LC-MS and nonreduced Lys-C peptide mapping with tandem mass spectrometry. The data show that the disulfide bond modification occurred under nonreducing conditions and was not an artifact of sample preparation for the rCGE analysis. The thioether bond modification was observed in several IgG1 monoclonal antibody products. Structural characterization of this novel modification is important in understanding the mechanism of thioether bond formation.