Process development for a flexible vaccine vector platform based on recombinant life virus

Vaccines are one of the most important, safe and efficient interventions to protect people from illness, disability and death. In recent years several new viral outbreaks where no vaccines are currently available were reported worldwide. Therefore, the development of flexible processes for the production of vaccines is urgently needed. This project aims at developing a platform process for the production of different viral vaccines. The core technology is based on the fact that large recombinant genes coding for selected, foreign antigens can be inserted into the genome of a well-established virus vaccination vector. The vaccine delivers the selected antigens directly to macrophages and dendritic cells, the most potent and effective antigen-presenting cells, thereby triggering a specific immune response to the selected antigens. As a replicating vector, the vaccine continuously expresses antigens even after immunization. This setup results in a powerful, antigen-focused immune response, which is expected to confer long-term immunity as shown for the measles vaccine. The challenges in production process design for such a vaccine are the establishment of a robust cell expansion and infection strategy as well the development of efficient downstream processing methods including several chromatography principals, ultra-diafiltration and employment of bio recognition principles. The implementation of a meaningful real-time process monitoring/characterization concept furthermore serves as a basis for reliable in-process control strategies (e.g. the prediction of the optimal infection/harvesting time point)

Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP

International audiencePolymer-grafted chromatography media, especially ion exchangers, are high performance materials for protein purification. However, due to the pore size limitation, conventional chromatography beads are usually not considered for the downstream processing of large biomolecules such as virus-like particles (VLPs). Contrariwise, since the outer surface of the chromatography beads provides satisfactory binding capacity for VLPs and impurities of smaller size can bind inside of the beads, conventional porous beads should be considered for VLP capture and purification. We used HIV-1 gag VLPs with a diameter of 100-200 nm as a model to demonstrate that polymer-grafted anion exchangers are suitable for the purification of bionanoparticles. The equilibrium binding capacity was 1 × 1013 part/mL resin. Moderate salt concentration up to 100 mM NaCl did not affect binding, allowing direct loading of cell culture supernatant onto the column for purification. Dynamic binding capacity at 10% breakthrough, when loading cell culture supernatant, was approximately 6 × 1011 part/mL column; only 1-log lower than for monoliths. Endonuclease treatment of the cell culture supernatant did not increase the dynamic binding capacity, suggesting that dsDNA does not compete for the binding sites of VLPs. Nevertheless, due to simultaneous elution of particles and dsDNA, endonuclease treatment is required to reduce dsDNA contamination in the product. Proteomic analysis revealed that HIV-1 gag VLPs contain different host cell proteins in their cargo. This cargo is composed of conserved proteins and other proteins that vary from one particle population to another, as well as from batch to batch. This process allowed the separation of different particle populations. HIV-1 gag VLPs were directly captured and purified from cell culture supernatant with a total particle recovery in the elution of about 35%. Columns packed with beads can be scaled to practically any dimension and therefore a tailored design of the process is possible

Complexation of Europium(III) by Bis(dialkyltriazinyl)bipyridines in 1-Octanol

The present work focuses on highly selective ligands for An^III/Ln^III separation: bis­(triazinyl)­bipyridines (BTBPs). By combining time-resolved laser-induced fluorescence spectroscopy, nanoelectrospray ionization mass spectrometry, vibronic sideband spectroscopy, and X-ray diffraction, we obtain a detailed picture of the structure and stoichiometry of the first coordination sphere of Eu^III-BTBP complexes in an octanolic solution. The main focus is on the 1:2 complexes because extraction studies revealed that those are the species extracted into the organic phase. The investigations on europium­(III) complexes of BTBP with different triazin alkylation revealed differences in the formed complexes due to the bulkiness of the ligands. Because of the vibronic sidebands in the fluorescence spectra, we were able to detect whether or not nitrate ligands are coordinated in the first coordination sphere of the Eu-BTBP complexes. In solution, less sterically demanding BTBP offers enough space for additional coordination of anions and/or solvent molecules to form 9-coordinated Eu-BTBP 1:2 complexes, while bulkier ligands tend to form 8-fold-coordinated structures. We also report the first crystal structure of a Ln-BTBP 1:2 complex and that of its 1:1 complex, both of which are 10-coordinated

Complexation of Europium(III) by Bis(dialkyltriazinyl)bipyridines in 1-Octanol

The present work focuses on highly selective ligands for An^III/Ln^III separation: bis­(triazinyl)­bipyridines (BTBPs). By combining time-resolved laser-induced fluorescence spectroscopy, nanoelectrospray ionization mass spectrometry, vibronic sideband spectroscopy, and X-ray diffraction, we obtain a detailed picture of the structure and stoichiometry of the first coordination sphere of Eu^III-BTBP complexes in an octanolic solution. The main focus is on the 1:2 complexes because extraction studies revealed that those are the species extracted into the organic phase. The investigations on europium­(III) complexes of BTBP with different triazin alkylation revealed differences in the formed complexes due to the bulkiness of the ligands. Because of the vibronic sidebands in the fluorescence spectra, we were able to detect whether or not nitrate ligands are coordinated in the first coordination sphere of the Eu-BTBP complexes. In solution, less sterically demanding BTBP offers enough space for additional coordination of anions and/or solvent molecules to form 9-coordinated Eu-BTBP 1:2 complexes, while bulkier ligands tend to form 8-fold-coordinated structures. We also report the first crystal structure of a Ln-BTBP 1:2 complex and that of its 1:1 complex, both of which are 10-coordinated

Capture and purification of Human Immunodeficiency Virus-1 virus-like particles: Convective media vs porous beads

International audienceDownstream processing (DSP) of large bionanoparticles is still a challenge. The present study aims to systematically compare some of the most commonly used DSP strategies for capture and purification of enveloped viruses and virus-like particles (eVLPs) by using the same staring material and analytical tools. As a model, Human Immunodeficiency Virus-1 (HIV-1) gag VLPs produced in CHO cells were used. Four different DSP strategies were tested. An anion-exchange monolith and a membrane adsorber, for direct capture and purification of eVLPs, and a polymer-grafted anion-exchange resin and a heparin-affinity resin for eVLP purification after a first flow-through step to remove small impurities. All tested strategies were suitable for capture and purification of eVLPs. The performance of the different strategies was evaluated regarding its binding capacity, ability to separate different particle populations and product purity. The highest binding capacity regarding total particles was obtained using the anion exchange membrane adsorber (5.3 × 1012 part/mL membrane), however this method did not allow the separation of different particle populations. Despite having a lower binding capacity (1.5 × 1011 part/mL column) and requiring a pre-processing step with flow-through chromatography, Heparin-affinity chromatography showed the best performance regarding separation of different particle populations, allowing not only the separation of HIV-1 gag VLPs from host cell derived bionanoparticles but also from chromatin. This work additionally shows the importance of thorough sample characterization combining several biochemical and biophysical methods in eVLP DSP