Direct Affinity Purification of Long-Acting PASylated Proteins with Therapeutic Potential Using L-Prolinamide for Mild Elution.

Both insufficient plasma half-life (circulation for only few hours or less) and laborious downstream purification can be bottleneck for biological drug development. We report a novel strategy for the efficient and gentle affinity purification of pharmacologically relevant proteins modified by PASylation for prolonged action in vivo. We previously described antibodies specific for Pro/Ala-rich sequences (PAS) covering a range of binding characteristics. Our present approach relies on a chromatography matrix functionalized with a low-affinity PAS-specific antibody Fab fragment for specific adsorption of the PASylated protein from a macromolecular mixture. With the complete absence of hydrophobic/aromatic or ionic groups in the PAS sequence epitope, binding is mediated by Van der Waals contacts and distinct hydrogen bonds only. Surprisingly, selective competitive elution is achieved by application of the highly soluble and biologically inactive imino acid derivative L-prolinamide. Based on the specific but strongly dynamic biomolecular interaction, our procedure allows the direct one-step purification of PASylated proteins from a cell extract or culture supernatant while avoiding harsh elution conditions as they are often needed for conventional affinity chromatography.

Increased Vaccination Diversity Leads to Higher and Less-Variable Neutralization of TBE Viruses of the European Subtype.

Tick-borne encephalitis (TBE) is an infectious disease of the central nervous system. The causative agent is the tick-borne encephalitis virus (TBEV), which is most commonly transmitted by tick bites, but which may also be transmitted through the consumption of raw dairy products or, in rare instances, via infected transfusions, transplants, or the slaughter of infected animals. The only effective preventive option is active immunization. Currently, two vaccines are available in Europe-Encepur® and FSME-IMMUN®. In Central, Eastern, and Northern Europe, isolated TBEV genotypes belong mainly to the European subtype (TBEV-EU). In this study, we investigated the ability of these two vaccines to induce neutralizing antibodies against a panel of diverse natural TBEV-EU isolates from TBE-endemic areas in southern Germany and in regions of neighboring countries. Sera of 33 donors vaccinated with either FSME-IMMUN®, Encepur®, or a mixture of both were tested against 16 TBEV-EU strains. Phylogenetic analysis of the TBEV-EU genomes revealed substantial genetic diversity and ancestry of the identified 13 genotypic clades. Although all sera were able to neutralize the TBEV-EU strains, there were significant differences among the various vaccination groups. The neutralization assays revealed that the vaccination using the two different vaccine brands significantly increased neutralization titers, decreased intra-serum variance, and reduced the inter-virus variation.