BIONANOPARTICLES,

i.e. viruses, virus-like particles (VLP) or extra-cellular particles such as exosomes, are the next generation of biopharmaceuticals used for vaccines, gene therapy and immunotherapy. A bottleneck in rapid development of these advanced medicines are the lack of understanding of the process mechanisms, the best choice of process units and the scale-up of up- and downstream production processes.

At acib a full set of instruments has been established to develop integrated bioprocesses and analytical methods for the production, purification and characterization of bionanoparticles. The infrastructure is located in a BSL2 facility at BOKU and can be made available to partners in collaborative projects and to external users.

For more information please contact Verena Beck

INSTRUMENTS

UPSTREAM PROCESS (USP) DEVELOPMENT:

 

Multibioreactorsystem with four parallel reactors

Multibioreactorsystem with four parallel reactors
DASGIP® system for cell culture, Eppendorf
  • Bioreactor system in a scalable benchtop format configured for cell culture and virus applications
  • Parallel processing and precise control of all relevant process parameters;
  • Highly suitable for process development and characterisation studies.
Lab-scale bioreactor systems

Lab-scale bioreactor systems
BioFlo® 320, Eppendorf
  • Lab-scale bioreactor system for cell culture and virus application for scales of up to 10 Litres;
  • Single-use bioreactor options available;
  • Test for upscale consistency and feed stock supply for downstream development studies.
Alternating Tangential Flow system

Alternating Tangential Flow system
XCell™ ATF 2, Repligen
  • Scalable cell retention system delivering high cell concentration and process intensification during cell culture;
  • Connects to any bioreactor and can be used in a variety of applications.

 

DOWNSTREAM PROCESS (DSP) DEVELOPMENT:

 

Preparative Chromatography
Preparative Chromatography:
ÄKTA Pure 25 and 150, Cytiva (formerly GE Healthcare Life Sciences) coupled to
MALS Detector (DAWN® Heleos, Wyatt)
  • On-line detection of particles during chromatographic experiments;
  • Accurate tracking of particle elution which is not possible by using only UV signals;
  • Faster and more efficient DSP development and optimization.

Continuous ultracentrifuge
Continuous ultracentrifuge:
pilot scale (Alfa Wassermann pKII) with
fluid handling system (Alfa Wassermann AFH)
  • Capture, concentration and primary purification of bionanoparticles directly from clarified harvest material in a single step;
  • Fully automated filling and fractionation of ultracentrifuge density gradient processes.

 

ANALYTICS:

 

Multibioreactorsystem

Field flow fractionation coupled to UV-MALS-DLS-RI detectors (Wyatt) &
Analytic HPLCs (Agilent and Thermo Fisher) coupled to UV-MALS-FLD-RI detectors

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  • FFF: high-resolution separation of bionanoparticles based on hydrodynamic radius in analytical and semi-preparative manner;
  • HPLC: SEC, AEX, HIC and affinity based analytical chromatography;
  • MALS and DLS: direct determination of particle number, size and shape by multi-angle and dynamic light scattering in the same flow cell;
  • RI: universal concentration measurement, determination of solvent absolute refractive index.
  • FLD: fluorescence detection
Nanoparticle Tracking Analysis

Nanoparticle Tracking Analysis:
NanoSight NS300 incl. Sample Assistant (Malvern Panalytical)
  • Analysis of the size and concentration of particles in solution by combining the properties of both Brownian motion and light scattering;
  • Particles are individually tracked, resulting in a size distribution of hydrodynamic diameters.
Microflow Cytometer (Apogee)

Microflow Cytometer (Apogee)
  • Flow Cytometer tuned for extremely small particle applications including extracellular vesicles (EVs), virus and protein aggregates;
  • Highest sensitivity and resolution from multiple light scattering and fluorescence detectors;
  • Able to measure biological particles down to about 100nm diameter by light scatter.

 

PUBLICATIONS

USP

Strobl, F., Ghorbanpour, S.M., Palmberger, D. et al. Evaluation of screening platforms for virus-like particle production with the baculovirus expression vector system in insect cells (2020) Scientific Reports 10, 1065
https://doi.org/10.1038/s41598-020-57761-w

Puente-Massaguer, E., Strobl, F., Grabherr, R., Striedner, G., Lecina, M., Gòdia, F., PEI-Mediated Transient Transfection of High Five Cells at Bioreactor Scale for HIV-1 VLP Production (2020) Nanomaterials 10(8), 1580
https://doi.org/10.3390/nano10081580

DSP

Strategies for the purification of enveloped bionanoparticles have been developed based on chromatographic methods including conventional ion exchange resins, core shell beads with multimodal functionality, ion exchange membrane adsorbers and affinity resins. Additionally, particle detection and characterization methods are a main focus of our research. Recent data can be found in the following references:

Pereira Aguilar, P., Reiter, K., Wetter, V., Steppert, P., Maresch, D., Ling, W.L., Satzer, P., Jungbauer, A. “Capture and purification of Human Immunodeficiency Virus-1 virus-like particles: Convective media vs porous beads” (2020) Journal of Chromatography A.
https://doi.org/10.1016/j.chroma.2020.461378

Pereira Aguilar, P., Schneider, T.A., Wetter, V., Maresch, D., Ling, W.L., Tover, A., Steppert, P., Jungbauer, A. “Polymer-grafted chromatography media for the purification of enveloped virus-like particles, exemplified with HIV-1 gag VLP” (2019) Vaccine.
https://doi.org/10.1016/j.vaccine.2019.07.001

Pereira Aguilar, P., González-Domínguez, I., Schneider, T.A., Gòdia, F., Cervera, L., Jungbauer, A. “At-line multi-angle light scattering detector for faster process development in enveloped virus-like particle purification” (2019) Journal of Separation Science.
https://doi.org/10.1002/jssc.201900441

Steppert, P., Burgstaller, D., Klausberger, M., Tover, A., Berger, E., Jungbauer, A. Quantification and characterization of virus-like particles by size-exclusion chromatography and nanoparticle tracking analysis(2017) Journal of Chromatography A, 1487, pp. 89-99.
https://DOI: 10.1016/j.chroma.2016.12.085

Steppert, P., Burgstaller, D., Klausberger, M., Kramberger, P., Tover, A., Berger, E., Nöbauer, K., Razzazi-Fazeli, E., Jungbauer, A. Separation of HIV-1 gag virus-like particles from vesicular particles impurities by hydroxyl-functionalized monoliths (2017) Journal of Separation Science, 40 (4), pp. 979-990.
https://DOI: 10.1002/jssc.201600765

Steppert, P., Burgstaller, D., Klausberger, M., Berger, E., Aguilar, P.P., Schneider, T.A., Kramberger, P., Tover, A., Nöbauer, K., Razzazi-Fazeli, E., Jungbauer, A. Purification of HIV-1 gag virus-like particles and separation of other extracellular particles (2016) Journal of Chromatography A, 1455, pp. 93-
https://DOI: 10.1016/j.chroma.2016.05.053

PARTNERS

Funding for the infrastructure was received by the Austrian Research Promotion Agency (FFG) through the “F&E Infrastrukturförderung”

University of Natural Resources and Life Sciences, Vienna (BOKU)

CONTACT


For more information on the infrastructure and its use
please contact Verena Beck
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