The analytical characterization of biopharmaceuticals is a fundamental step in the early stages of development and prediction of their behavior in bioprocesses. Protein aggregation in particular is a common issue as it affects all stages of product development. In the present work, we investigate the stability and the aggregation kinetics of A33Fab, a therapeutically relevant humanized antibody fragment at a wide range of pH, ionic strength, and temperature. We show that the propensity of A33Fab to aggregate under thermally accelerated conditions is pH and ionic-strength dependent with a stronger destabilizing effect of ionic strength at low pH. In the absence of added salts, A33Fab molecules appear to be protected from aggregation due to electrostatic colloidal repulsion at low pH. Analysis by transmission electron microscopy identified significantly different aggregate species formed at low and high pH. The correlations between apparent midpoints of thermal transitions (Tm,app values), or unfolded mole fractions, and aggregation rates are reported here to be significant only at the elevated incubation temperature of 65 °C, where aggregation from the unfolded state predominates. At all other conditions, particularly at 4-45 °C, aggregation of A33 Fab was predominantly from a native-like state, and the kinetics obeyed Arrhenius behavior. Despite this, the rank order of aggregation rates observed at 45 °C, 23 and 4 °C still did not correlate well to each other, indicating that forced degradation at elevated temperatures was not a good screen for predicting behavior at low temperature.

The propensity of the prion protein (PrP) to adopt different structures is a clue to its pathological behavior. The determination of the region involved in the PrP(C) to PrP(Sc) conversion is fundamental for the understanding of the mechanisms underlying this process at the molecular level. In this paper, the polymerization of the helical H2H3 domain of ovine PrP (OvPrP) was compared to the full-length construct (using chromatography and light scattering). We show that the oligomerization patterns are identical, although the H2H3 domain has a higher polymerization rate. Furthermore, the depolymerization kinetics of purified H2H3 oligomers compared to those purified from the full-length PrP reveal that regions outside H2H3 do not significantly contribute to the oligomerization process. By combining rational mutagenesis and molecular dynamics to investigate the early stages of H2H3 oligomerization, we observe a conformationally stable beta-sheet structure that we propose as a possible nucleus for oligomerization; we also show that single point mutations in H2 and H3 present structural polymorphisms and oligomerization properties that could constitute the basis of species or strain variability.

Vibrio splendidus is a dominant Vibrio species in seawater presenting a remarkable genetic diversity; several strains have been linked to invertebrate's mortality. We report the complete genome sequence of V. splendidus LGP32, an oyster pathogen, and its comparison with partial genome sequences from related strains. As is typical for the genus, V. splendidus LGP32 contains two chromosomes (3.29 and 1.67 Mb) and most essential cellular processes are encoded by chromosome 1. Comparison with two other V. splendidus partial genome sequences (strains 12B01 and Med222) confirms the previously suggested high genotypic diversity within this species and led to the identification of numerous strain-specific regions that could frequently not be assigned to a specific mechanisms of recombination. Surprisingly, the chromosomal integron, the most variable genetic element in all other Vibrio species analysed to date, is absent from 12B01 and inactivated by a mobile element in Med222, while in LGP32 it only contains a limited number of cassettes. Finally, we found that the LGP32 integron contains a new dfrA cassette, related to those found in resistance integrons of gram-negative clinical isolates. Those results suggest that marine Vibrio can be a source of antibiotic resistance genes.