BRAVE PARTNERS. PIONEERS. VISIONARIES.

Use Cases

Is your research or your production limited by your method of particle characterization? Do you want to get much deeper insights into the composition and behavior of your particles? OF2i is already used by the following partners in their groundbreaking research and for optimized production processes. Take a closer look at these early use cases to get a taste of the potential of OF2i and get in touch to talk about how we can help you raise the level of your particle analysis.

Online monitoring of the leaching of micro- and nanoplastics into water at ultra-low concentrations

Katharina Heider (a researcher at a leading Austrian university) was unable to obtain insights into leaching from polypropylene bottles until she performed OF2i measurements. There was no technology available to measure concentrations down to a few particles per milliliter. Before measuring these samples, they must typically be concentrated with other techniques to achieve the detection levels reached by conventional particle measuring technologies. The alternative was using BRAVE Analytics’ OF2i device for online monitoring at high flow throughputs. The sample was pumped through the flow cell and single particles were tracked for analysis.

The BRAVE solution

The BRAVE B-Curious nanoparticle analyzer enables high flow throughputs and detects nanoplastics and microplastics at concentrations down to a few particles per milliliter with single-particle sensitivity. The online measurement was performed at high throughputs to obtain high statistical relevance. Ultra-pure water samples in different plastic bottles were analyzed after cleaning cycles to determine cleaning efficiency and the results were compared to the reference (20 nm filtrated Ultra-pure H2O) in glass bottles.

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"The single-particle sensitivity of the BRAVE OF2i method meant we could finally detect the extremely low concentrations of nanoplastics in our samples and achieve a relevant and insightful comparison for our research."

Brave Analytics Testimonials - Professor Thomas Meisel

Prof. Thomas Meisel

Montan University Leoben

Application highlights

Monitor nanoplastics and PFAS at ultra-low concentrations (down to a few particles per mL):

Evaluate the cleaning, filtration and separation efficiency for drinking or waste water
Verify the occurrence of filter breakthrough, assess filter capacity

Specifications	OF2i
Detection limit	A few objects / mL
Object size range	50 to 3000 nm (sample-dependent)
Detected substances	Nanoplastics (e.g. SiO2, polystyrene, PP), PFAS
Measuring mode	Online (either coupled to the process or from a sample container)

Online monitoring of total parenteral nutrition products produced in high-pressure homogenization processes

One of the top-ten global pharmaceutical concerns was looking for a more efficient alternative to offline QC testing on their total parenteral nutrition products. These oil-in-water emulsions must comply with strict regulations regarding particle size and particle size distribution.
There was as yet no true PAT solution for monitoring this production process and the time lag inherent with manual quality control testing in the laboratory brings the possibility of producing out-of-spec formulations, with resulting high costs. The benchtop device the team were using for PSD determination was based on an ensemble-method technology which delivered unreliable results influenced by different parameters such as viscosity and concentration. The testing was a tedious procedure including manual operation and dilution, another possible source of error.

The best-case scenario would be a move to online monitoring and towards real-time release testing for these liquid formulations.
The challenges in this case are:

Emulsions with a particle size from 100 nm to 600 nm undergoing high-pressure homogenization processes.
Monitoring required 24/7 during the production process and providing direct communication with the process control system to enable immediate process regulation.

The BRAVE solution

The BRAVE B-Continuous online device (including continuous and automated online sample preparation unit) was installed in a pilot plant at the company premises. The homogenization pilot plant consists of two homogenization steps; by applying high pressure the particle size gradually decreases. After the first step particle size is reduced from 3 µm to approximately 0.3 µm. The process analyzer (BRAVE B-Continuous) is connected to the process connection positioned after the first step to measure the efficiency of this homogenization procedure. It continuously monitors PSD distribution and automatically evaluates and collects data such as D-values which are used to evaluate the efficiency of the process. The plug-and-play setup can also be easily moved to other measuring points to monitor the efficiency of other process steps.

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Benefits of OF2i in monitoring total parenteral nutrition products online during the production process:

Reduction of waste by eliminating the time lag inherent with manual quality control and instead monitoring production directly.
Gaining a better understanding of production processes based on data collected.
Single-particle sensitivity with high throughputs (up to 4000 particles/min), meaning detection of all the particle populations in a representative measurement and high statistical relevance.
The analyzing system can be easily moved to other measuring points, e.g. to evaluate the efficiency of different process units.

Understanding dynamic liquid-liquid phase separation (LLPS) processes

Professor Tobias Madl at Medical University of Graz, Austria, is researching (dys)regulation and the drug targeting of biomolecular condensate formation. Distortions in these condensates indicate age-related diseases such as cancers, Alzheimer’s and senile dementia. He was looking for alternative methods to study condensate size distributions and time evolution and came to BRAVE Analytics for advice. Without a thorough understanding of the molecular processes involved in early-stage condensate formation it is impossible to plan a strategy to improve existing models and discover how and when drugs are administered most effectively.

The BRAVE solution

Professor Madl used the BRAVE B-Curious module for time-resolved sizing to achieve dynamic particle characterization on only 100 µl sample volume. The sample composition was a buffer with 6 µM protein and various amounts (from 0.02 µM to 0.2 µM) of RNA added.

The measuring results clearly showed the kinetics of these LLPS processes (see excerpt) and allowed comparison of the formation of particles in the presence of different RNA concentrations.

The results of measurements will soon be published and will be part of a number of PhD thesis.

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"As the small condensates we are interested in cannot be detected with microscopy their study remains a blind spot. The OF2i technology promises to close this gap and to provide unprecedented insights into the processes driving early condensate formation and targeting of condensates. First exciting results obtained on the BRAVE B-Curious instrument demonstrate that the OF2i technology will be of outstanding interest for us and the entire field studying biomolecular condensates and their role in health and diseases. We are very much looking forward to the official release of the BRAVE B-Curious and purchasing it in the near future."

Brave Analytics Testimonials - Portrait Prof. Tobias Madl der Medizinischen Universität Graz

Univ. Prof. Dr. Madl

Medical University Graz & Gottfried Schatz Research Center

Benefits of OF2i in the study of biomolecular condensate formation:

Visualizing the formation and size distribution of proteins as they change over time in one seamless and complete measurement.
Single-particle sensitivity, even for condensate particles in the size range 10 nm to 2000 nm.
Results from low-volume samples of 80 µL to 100 µL.

Implementing OF2i in biomedical research and biotechnology

Are you frustrated by the limits of conventional particle characterization methods and what this means for your research? Unlike other light scattering methods, the OF2i method of particle characterization is particularly suited to:

- observing changes in particle size distribution and concentration as they happen over seconds, minutes, even hours
- delivering results sensitive to every particle trapped by the laser beam during measurement
- detecting ultra-low concentrations, rare large particles, anomalies and outliers
- providing concentration results based on a reliable, defined sample volume
  giving data with statistical relevance and representative of the actual particle populations

The BRAVE solution

The BRAVE B-Curious lab device is already at work in research into liquid-liquid phase separation processes. We believe BRAVE B-Curious would be a great asset in other biotechnological and biomedical fields. We look forward to hearing from you if you are interested in getting groundbreaking insights into particle composition and behavior on the following:

Microvesicles
Exosomes
Lipoproteins
Liposomes and lipid-based nanoparticles
Virus-like particles
Other protein complexes
And more

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“Within our collaborative project NanoPAT, the OF2i technology shows its great potential for applications in the biotech field.”

Brave Analytics Testimonials - Portrait Beatriz Alfonso Serrano BioNanoNet Forschungsgesellschaft

Beatriz Alfaro Serrano

Beatriz Alfaro Serrano, BioNanoNet Forschungsgesellschaft mbH (BNN), Austria

We see great potential for using OF2i in the following areas:

Observing changes in particle size distribution and concentration as they happen over seconds, minutes, even hours
Delivering results sensitive to every single particle in the trapped sample volume
Detecting ultra-low concentrations, rare large particles, anomalies and outliers
Providing concentration results based on a reliable, defined sample volume
Giving data with statistical relevance and representative of the actual particle populations
Future upscaling with an online device for production monitoring

Observing changes in particle size distribution and concentration as they happen over seconds, minutes, even hours

As OF2i delivers continuous and time-resolved results you gain insights into your particle behavior as it happens in one complete and seamless measurement. This continuous recording of your particle kinetics is a huge step forward from making several “snapshot” measurements, with gaps where the sample composition can only be inferred. Prof. Tobias Madl at the Medical University of Graz has gained valuable knowledge of the behavior of biomolecular condensates during early formation processes for his research into age-related diseases and drug targeting. This study involved measurements of particle size distributions during liquid-liquid phase separation (LLPS) to evaluate particle formation depending on the available RNA concentration.

Delivering results sensitive to every single particle in the trapped sample volume

BRAVE B-Curious records the size of every single particle that is trapped in the laser beam passing through the flow cell. The results protocol considers all measured particle sizes. Measurements on dynamic light scattering or laser diffraction instruments do not consider individual particles, providing instead an average size distribution curve calculated from all measured intensities (DLS) or volumes (LD). The results are therefore only representative of samples with very low polydispersity. These differing approaches to measurement can make the difference between understanding your sample by reading single-particle results and inferring the composition based on averaged ensemble values.

Detecting ultra-low concentrations, rare large particles, anomalies and outliers

The OF2i method is both sensitive to single particles and also the absence of particles. Take, for example, 20 nanometer filtrated water. OF2i measurements on nanofiltrated water will detect any particles above 20 nm remaining in the sample, even if the concentration is extremely low, e.g. only a few particles per milliliter. An interesting study in this field was carried out using OF2i to detect nanoplastic particles leached into filtered water with the results confirming how sensitive OF2i is when detecting ultra-low concentrations.

Providing concentration results based on a reliable, defined sample volume

The flow cell of BRAVE B-Curious has a defined volume and can be therefore used as the basis for reliable concentration measurements. In contrast, the measuring cell of NTA instruments is dependent on the particle size and therefore not adequately defined as the basis for reliable concentration values.

Giving data with statistical relevance and representative of the actual particle populations

BRAVE B-Curious scans approximately 2500 particles per minute and calculates the size of each one. This means the results are more representative than results from nanoparticle tracking analysis (NTA is another number-based method but with measurements restricted to around 100 particles per frame). With NTA it is also possible for the user to select the “best looking” frame for measurement, pressing sample through if the view is dominated by one oversized particle, for example, or selecting a frame in which enough particles are registered by the device. This does not happen with OF2i as the sample is measured as it flows through the flow cell (and is trapped by the laser beam). A high number of particles are evaluated in one measurement.

Future upscaling with an online device for production monitoring

By the beginning of 2024, BRAVE Analytics will launch its BRAVE B-Continuous online device with OF2i inside. This online sensor is for:

24/7 monitoring of particle size distributions, concentrations and
Detecting large-particle counts, large-diameter tails, anomalies and other outliers during production
Gathering data towards real-time release testing (RTRT) and
Process optimization, including monitoring the performance of dilution and mixing processes and
Giving a status report on the production plant, moving towards preventive maintenance

As BRAVE B-Continuous uses the OF2i method and provides the same measuring protocols as BRAVE B-Curious, you can easily upscale your research into production and always have compatible results.
BRAVE B-Continuous is already being tested by a global pharmaceutical concern in its pilot plant. This test installation is for monitoring the exact composition of nanoemulsion nutritional admixtures for quality control, process optimization and more efficient compliance with regulations.