New GMP manufacturing processes to obtain thermostable HIV-1 gp41 virosomes under solid forms for various mucosal vaccination routes.

The main objective of the MACIVIVA European consortium was to develop new Good Manufacturing Practice pilot lines for manufacturing thermostable vaccines with stabilized antigens on influenza virosomes as enveloped virus-like particles. The HIV-1 gp41-derived antigens anchored in the virosome membrane, along with the adjuvant 3M-052 (TLR7/8 agonist) on the same particle, served as a candidate vaccine for the proof of concept for establishing manufacturing processes, which can be directly applied or adapted to other virosomal vaccines or lipid-based particles. Heat spray-dried powders suitable for nasal or oral delivery, and freeze-dried sublingual tablets were successfully developed as solid dosage forms for mucosal vaccination.

Immuno-PCR with digital readout.

Immuno-PCR (IPCR) combines the versatile ELISA antigen detection with ultrasensitive PCR signal amplification, thereby enabling the highly sensitive detection of a broad range of targets with a typically very large dynamic detection range. The quantification of the antigen is usually achieved by real-time PCR, which provides a correlation between the target concentration and amplified DNA marker. We here report on the implementation of digital droplet PCR as a means for direct quantification of DNA copies to enable the highly sensitive detection of protein biomarkers.

Highly sensitive ligand-binding assays in pre-clinical and clinical applications: immuno-PCR and other emerging techniques.

Recombinant DNA technology and corresponding innovations in molecular biology, chemistry and medicine have led to novel therapeutic biomacromolecules as lead candidates in the pharmaceutical drug development pipelines. While monoclonal antibodies and other proteins provide therapeutic potential beyond the possibilities of small molecule drugs, the concomitant demand for supportive bioanalytical sample testing creates multiple novel challenges. For example, intact macromolecules can usually not be quantified by mass-spectrometry without enzymatic digestion and isotopically labeled internal standards are costly and/or difficult to prepare.

Emerging technologies to increase ligand binding assay sensitivity.

Ligand binding assays (LBAs) have been the method of choice for protein analyte measurements for more than four decades. Over the years, LBA methods have improved in sensitivity and achieved larger dynamic ranges by using alternative detection systems and new technologies. As a consequence, the landscape and application of immunoassay platforms has changed dramatically.

Immuno-PCR assays for immunogenicity testing.

The administration of therapeutic proteins often induces immunogenic response and thus formation of anti-drug antibodies (ADA), which can neutralize the drug's therapeutic effect and may even cause serious health problems. We here report on the employment of the ultra-sensitive immuno-PCR (IPCR) method to facilitate immunogenicity testing using two established assay formats. In a "bridging assay", in which ADA forms a bridge to immobilize a signal-generating drug reporter probe, IPCR detection enabled an at least 1000-fold increase in sensitivity, as compared to the analogous ELISA, along with a high drug tolerance value.

Screening for cytochrome p450 reactivity by harnessing catalase as reporter enzyme.

Cytochrome P450 enzymes are known to catalyze a variety of reactions that are difficult to perform by standard organic synthesis, such as the oxidation of unactivated C--C bonds. Cytochrome P450 enzymes can also be used in artificial systems in which organic peroxides act as cosubstrates. To find substrates that are converted by a certain P450 catalyst in the presence of an organic peroxide, various screening assays have been established, however, most of them are limited to one or only a few specific substrates.

Engineering and assaying of cytochrome P450 biocatalysts.

Cytochrome P450s constitute a highly fascinating superfamily of enzymes which catalyze a broad range of reactions. They are essential for drug metabolism and promise industrial applications in biotechnology and biosensing. The constant search for cytochrome P450 enzymes with enhanced catalytic performances has generated a large body of research.

Synthesis of covalent DNA-protein conjugates by expressed protein ligation.

Semisynthetic DNA-protein conjugates are versatile tools for many applications in bioanalytics and nanobiotechnology. We here report a method based on expressed protein ligation (EPL) for the site-specific coupling of cysteine-modified DNA oligomers with recombinant intein-fusion proteins. The latter contain a C-terminal thioester, enabling the mild and highly specific reaction with N-terminal cysteine compounds.