Biological
Applications
Extracellular Vesicles and Exosomes

Extracellular Vesicles and Exosomes

Extracellular vesicles (EVs) are biological particles ranging in size from about 30 – 10,000 nanometers in diameter. Extracellular vesicles comprise diverse types of particles having a broad range of physical properties and biological origin, and include exosomes, ectosomes, microvesicles, microparticles, oncosomes, and apoptotic bodies.

Extracellular vesicles have roles associated with an equally broad set of biological processes, including immune response, transfer of functional proteins and nucleic acid, elimination of unwanted materials, nutrition, surface-receptor mediated cell signaling, and numerous conditions of disease including cancer metastasis. Because of their involvement in so many physiological processes, extracellular vesicles hold enormous potential as therapeutics and biomarkers of health and disease. In these applications, accurate quantification of extracellular vesicles is critical for performing well controlled science, as well as ensuring safety and efficacy of extracellular vesicle-based products.

Accurate quantification with MRPS and simultaneous single-particle fluorescence

Spectradyne’s Arc delivers single-particle fluorescence combined with MRPS measurements, yielding size and phenotype for each individual nanoparticle, providing unparalleled individualized characterization not available with any other instrument. The Arc is fast and easy to use, and requires only 3 microliters of your sample to help you do better EV science.

Subpopulation analysis with fluorescence

Almost all biological nanoparticle samples contain heterogeneous populations of many different particle types. ARC can measure the size and concentration of the entire distribution using MRPS, and simultaneously measure and display only those particles in the sample that are positive for fluorescence in up to 3 different channels.

The example above shows that for an EV sample containing 1.26E+10 particles/ml overall, only 1.03E+09 particles/ml, or 8.1%, are tagged FITC- positive. In the same way, one can easily measure subpopulations in a virus sample, for instance.

Accurate quantification with microfluidic resistive pulse sensing (MRPS)

Spectradyne’s nCS1 delivers the most accurate concentration and size measurements of extracellular vesicles available in a practical bench top solution. The nCS1 is fast and easy to use, and requires only 3 microliters of your sample to help you do better EV science.

“The nCS1 is fast, accurate, and easy to use for routine EV quantification. We looked at other nanoparticle measurement solutions and the nCS1 is the only instrument that could measure the size of every nanoparticle of interest in as little as 3 uL of volume. Moreover, their customer service is superb and have went out of their way to ensure our nCS1 is always functioning well.”

-Joseph Sedlak, Co-Founder at Mercy BioAnalytics

Accurate concentrations yield better science

For measuring the biological activity of exosomes, characterizing vesicles of different biological origin and exploring vesicle biomarker applications, accurate concentration measurements are critical for performing well-controlled experiments.

The figure below shows a comparison of Spectradyne’s nCS1 to NTA and the gold standard, Cryo-TEM. The nCS1 measurements are in excellent agreement with those of TEM, and indicate that the exosome concentration increases to smaller particle size down to 50 nm. In contrast, NTA reports misleading results: A loss of counting efficiency is apparent for particles as large as 200 nm in diameter, leading to a 10,000-fold error by 65 nm. Critically, NTA reports a prominent peak in the exosome size distribution near 130 nm diameter that does not in fact exist. Read more about why this happens.

More researchers are choosing Spectradyne’s instruments for extracellular vesicle quantification because these are fast and easy to use and deliver a more accurate analysis of their vesicles than they can obtain using any other method.

Industrial and Biological
Applications
Other Therapeutics

Particle Characterisation in Biotherapeutics

Particle content is a critical quality attribute for many biologics that must be monitored to meet regulatory requirements like USP <788> and mitigate product safety risks.

Flow imaging microscopy is an established technique recommended by USP <1788> for subvisible and submicron particle analysis, revealing particle count, size, and shape, indicating their type and source.

Monitor the aggregation of adjuvants, nano-drug delivery systems, and other small particles to larger, potentially concerning, submicron and subvisible particles.

Use FlowCam to:

• Detect aggregation and agglomeration of the active pharmaceutical ingredient (API) and drug delivery vehicles to improve product stability
• Obtain images and particle morphology information not obtainable from orthogonal techniques to assess product degradation
• Differentiate between inherent particles, intrinsic particles like glass flakes and silicone oil droplets, and extrinsic contaminants
• Optimize and control particulates in your formulation

FlowCam – A Flexible Particle Analysis Solution

Apply flow imaging microscopy techniques to therapeutics including aggregates of drug delivery systems like liposomes, exosomes, and gold nanoparticles, and vaccine components like virus-like particles and adjuvants.

FlowCam is ideally suited to analyze samples containing larger particles like CHO cells, cell cultures and associated particles like Dynabeads™ and Tentagel™ beads, and hydrogel spheres.

Obtain size and morphology information of these particles that is related to product quality issues such as cell viability, misshapen drug delivery vehicles, and the form of any aggregates present.

• Improving vaccine formulations by monitoring API and adjuvant aggregation
• Characterising large liposome, exosome, and other drug delivery platform morphology with an automated microscopy technique
• Optimizing cell concentrations and viability during biotherapeutic manufacturing
• Observing Dynabead binding and measuring unbound bead concentrations in cell culture applications

Biological
Applications
Protein Therapeutics

FlowCam for Protein Therapeutics Development and Manufacturing

Discover a high-throughput flow imaging microscopy platform for characterizing API aggregates and other particulates in your protein, monoclonal antibody, or antibody-drug conjugate formulation.

Monitor your formulation for protein aggregates, intrinsic particles including silicone oil, degraded polysorbate, and glass flakes, as well as extrinsic contaminants.

FlowCam provides quality assurance for your parenteral drug product to give you peace of mind about the stability and safety of your formulation.

Use FlowCam to:

• Count and size protein aggregates as small as 300 nm with industry-leading image quality
• Obtain complementary particle image data recommended by USP <1788> to verify orthogonal particle size measurements by light obscuration
• Utilize image-based analytics including artificial intelligence tools to classify subvisible and submicron particles

FlowCam Provides Confidence in Protein Formulation Quality

Capture high-resolution images for the identification of particle type, allowing you to detect and mitigate undesirable and potentially harmful particle formation at the source.

Improve formulation design based on knowledge obtained by using FlowCam in accelerated protein stability studies.

• Use FlowCam LO to obtain USP <787> compendial particle sizing information and images in a single instrument in quality control monitoring
• Improve lab productivity and data reproducibility with ALH for FlowCam automated liquid handling.
• Employ VisualAI™ to classify images of protein biotherapeutics automatically with higher than 90% accuracy

Biological
Applications
Plant Cells

Moniotoring Plant Cell Cultures with BioLector and Multisizer 4e Instruments

Cell cultures can be used to grow plant cells. Typically, this technique is performed in an aseptic and controlled environment with a variety of nutrient solutions.

Plant cell cultures have a wide range of applications from basic research (e.g., studies on plant growth and differentiation) to mass production of plant-derived metabolites (e.g. production of the anti-cancer agent paclitaxel). Therefore, they have become increasingly attractive and cost-effective alternatives to classical approaches for the mass production of plant-derived metabolites. However, it is crucial to monitor culture conditions to optimise the rate of cell prol
iferation and maximize productivity levels. Likewise, the cells must be characterised to
detect changes in the cell culture, such as the formation of cell aggregates or cellular lysis. This application note will demonstrate how the BioLector XT microbioreactor and the Multisizer 4e Coulter Counter can be used to optimise plant cell culture conditions and therefore cell growth.

Biological
Applications
Probiotic Bacteria

Anaerobic cultivation processes of probiotic bacteria in the BioLector XT microbioreactor

Probiotics are living bacteria that are said to have a health-promoting benefit and biofunctional effects on the human organism. They are commonly used to increase the number of desirable bacteria in the intestine and to regenerate the intestinal flora, for example after antibiotic treatments.

That is one reason why the market for probiotics or probiotic nutritional supplements has greatly increased in value. The research field of the human intestinal microbiome and its health-promoting benefits is particularly important for the nutrition industry. Therefore, scientific research on anaerobic or microaerophilic cultivation techniques, such as the cultivation of probiotics under microbiome-like conditions, is essential. Probiotics include a whole range of anaerobic bacteria such as Lactobacillus or Bifidobacterium. Among the various probiotic bacteria, Bifidobacterium spp. is one of the most widely used and studied probiotic bacterium species. They are classified as strict anaerobes due to the incapability of oxygen respiration and growth under aerobic cultivation conditions an they are a major member of the dominant human gut microbiota.

They play a significant role in controlling the pH through the release of lactic and acetic acids, which restrict the growth of many potential pathogenic bacteria. In the intestinal tract of breast-fed infants, Bifidobacterium is the predominant cell species. It accounts for more than 80% of microorganisms in the intestine. There are more than 200 known species of Lactobacillus, the largest and most diverse genus within the lactic acid bacteria that is generally recognized as safe (GRAS) by the US Food and Drug Administration (FDA). Lactobacillus spp. have been deployed and studied extensively as fermentation starter cultures for dairy products or probiotics due to their applied health potential.

In this application note we present anaerobic cultivation experiments using the BioLector XT microbioreactor in combination with the gassing lid.

Biological
Applications
E. coli

Monitoring E. coli Cultures with the BioLector and Multisizer 4e Instruments

Escherichia coli (E. coli) is a facultative anaerobic bacterium that lives in the lower intestine of warmblooded
animals, including humans

E. coli can be cultured easily and inexpensively in a laboratory setting
and has become an important model organism in genetics, microbiology and biotechnology. E. coli is
the most common organism used for the large-scale production of therapeutic proteins. Indeed, 30%
of approved therapeutic proteins are currently being produced using E. coli. This application note will
demonstrate how the BioLector microbioreactor and the Multisizer 4e Coulter Counter can be used to
optimize E. coli culture conditions and characterise cell growth

Particle Size and Count
Proteins

Optimised procedures for running protein samples

Beckman Coulter’s Multisizer 4 provides analysts with an easy-to-use, technologically-advanced system that can solve most particle sizing and counting challenges. Implementing the Coulter Principle with Smart Technology, the Multisizer 4 ensures the repeatability and uniformity of sample analysis conditions and therefore produces reliable results.

Although the following procedures were developed to address common issues encountered when analysing subvisible particles in protein formulations, the general guidelines can also be used for applications requiring small aperture tubes. Specifically, the cleaning procedures are valuable when characterising very small particles at low concentrations.

Particle Size Distribution
Nanocellulose

Grading of nanocellulose using a Centrifuge

Nanocellulose is a nanomaterial that is garnering attention recently, and it is obtained from cellulose or a cellulose derivative through wet-type high-pressure dispersion and pulverisation or wet-type grinding and pulverisation. We will introduce the results of particle size measurements made using a Laser diffraction / scattering method particle size distribution measurement apparatus LS 13 320 XR to measure nanocellulose that was obtained by performing wet-type high-pressure dispersion and pulverization on low-substituted hydroxy propylcellulose, which is one type of cellulose derivative, as well as the results for separation of only the component to be measured in the nanosize region through centrifugation.

A Shin-Etsu Chemical Co., Ltd. L-HPC (low-substituted hydroxy propylcellulose) LODICEL LDC-H 2 wt% dispersion was passed through a Sugino Machine Ltd. Starburst test apparatus 10 times at a pressure of 150 MPa for use as the dispersion in the experiment.

The results obtained by measuring the nanoized dispersion using LS 13 320 XR are shown.

At the same time as the detection of the nanosize particles generated as a result of the nanoization processing, we also detected microsize particles that remained without undergoing nanoization.
These results indicate that, even after nanoization processing, many particles remaining at the microsize will be present.

By using LS 13 320 XR, it will be possible to perform a quantitative comparative study by simultaneously measuring micro and nanosize particles.

After using a high-speed refrigerated centrifuge (Avanti JXN-26) and a fixed angle rotor (JA-14.50) to centrifuge the material at 14,000 rpm (35,000 xg) for 1 hour, the supernatant was collected and the particle diameter was measured. As a result of
the centrifugation processing, it was possible to isolate and recover only the component consisting of the nanosize particles. The particles measured to be 188 nm prior to centrifugation were measured as 117 nm after centrifugation. This could be because, when there is polydispersion, the smaller particle diameter values will tend to approach the larger particle diameter values.