Pragmatic mAb lead molecule engineering from a developability perspective.

As the number of antibody drugs being approved and marketed increases, our knowledge of what makes potential drug candidates a successful product has increased tremendously. One of the critical parameters that have become clear in the field is the importance of mAb "developability." Efforts are being increasingly focused on simultaneously selecting molecules that exhibit both desirable biological potencies and manufacturability attributes. In the current study mutations to improve the developability profile of a problematic antibody that inconsistently precipitates in a batch scale-dependent fashion using a standard platform purification process are described. Initial bioinformatic analysis showed the molecule has no obvious sequence or structural liabilities that might lead it to precipitate. Subsequent analysis of the molecule revealed the presence of two unusual positively charged mutations on the light chain at the interface of VH and VL domains, which were hypothesized to be the primary contributor to molecule precipitation during process development. To investigate this hypothesis, straightforward reversion to the germline of these residues was carried out. The resulting mutants have improved expression titers and recovered stability within a forced precipitation assay, without any change to biological activity. Given the time pressures of drug development in industry, process optimization of the lead molecule was carried out in parallel to the "retrospective" mutagenesis approach. Bespoke process optimization for large-scale manufacturing was successful. However, we propose that such context-dependent sequence liabilities should be included in the arsenal of in silico developability screening early in development; particularly since this specific issue can be efficiently mitigated without the requirement for extensive screening of lead molecule variants.

Oldest known Eucalyptus macrofossils are from South America.

The evolutionary history of Eucalyptus and the eucalypts, the larger clade of seven genera including Eucalyptus that today have a natural distribution almost exclusively in Australasia, is poorly documented from the fossil record. Little physical evidence exists bearing on the ancient geographical distributions or morphologies of plants within the clade. Herein, we introduce fossil material of Eucalyptus from the early Eocene (ca. 51.9 Ma) Laguna del Hunco paleoflora of Chubut Province, Argentina; specimens include multiple leaves, infructescences, and dispersed capsules, several flower buds, and a single flower. Morphological similarities that relate the fossils to extant eucalypts include leaf shape, venation, and epidermal oil glands; infructescence structure; valvate capsulate fruits; and operculate flower buds. The presence of a staminophore scar on the fruits links them to Eucalyptus, and the presence of a transverse scar on the flower buds indicates a relationship to Eucalyptus subgenus Symphyomyrtus. Phylogenetic analyses of morphological data alone and combined with aligned sequence data from a prior study including 16 extant eucalypts, one outgroup, and a terminal representing the fossils indicate that the fossils are nested within Eucalyptus. These are the only illustrated Eucalyptus fossils that are definitively Eocene in age, and the only conclusively identified extant or fossil eucalypts naturally occurring outside of Australasia and adjacent Mindanao. Thus, these fossils indicate that the evolution of the eucalypt group is not constrained to a single region. Moreover, they strengthen the taxonomic connections between the Laguna del Hunco paleoflora and extant subtropical and tropical Australasia, one of the three major ecologic-geographic elements of the Laguna del Hunco paleoflora. The age and affinities of the fossils also indicate that Eucalyptus subgenus Symphyomyrtus is older than previously supposed. Paleoecological data indicate that the Patagonian Eucalyptus dominated volcanically disturbed areas adjacent to standing rainforest surrounding an Eocene caldera lake.