Nanoparticle Characterization

NIS has established an internal Quality Unit, with the assistance of third party experts, to oversee quality systems and regulatory compliance to assist us in establishing cGMP compliant nanoparticle characterization testing services. These efforts are currently underway and are expected to be complete by the end of July 2023.

Cryo-TEM is a one of a kind analytical tool for nanoparticle characterization. A single cryo-TEM imaging study can visualize individual particles in your formulation, and can simultaneously provide information on particle size, morphology, lamellarity, drug encapsulation, homogeneity, and purity making cryo-TEM analysis essential for characterizing your nanoparticle therapeutic or vaccine.

Dynamic Light Scattering (DLS) is a powerful, non-destructive, fast technique to measure both particle size and evaluate the presence of aggregation. It also analyzes vastly more particles than cryo-TEM. However, DLS results are heavily affected by factors such as buffer, temperature and the presence of air bubbles, dust, or other impurities in the sample. In addition, DLS measures the hydrodynamic radius of the particles and doesn't do well with heterogeneous samples as the signal of the larger particles weigh more than that of smaller particles resulting in over-estimation of the particle size. Cryo-TEM captures the particles in their native state enabling the determination of true particle size. Cryo-TEM is also well-equipped to deal with heterogeneous samples. Cryo-TEM captures specimens in their natural hydrated state and is forgiving of heterogeneous mixtures of particles, making cryo-TEM an important analysis technique to measure particle size and size distribution, as well as lamellarity, drug encapsulation, and more, with as little as 3 microliters of sample. Cryo-TEM provides more answers than any other nanoparticle characterization technique in only one study.  

Here is a link to a paper describing the differences between lipid nanoparticle characterization done by DLS and cryo-TEM, including imaging of lipid nanoparticles (LNP’s) done by NIS: https://doi.org/10.1016/j.ijpharm.2010.10.025  

Here is a paper comparing of TEM and DLS methods to characterize size distribution of ceramic nanoparticles: https://iopscience.iop.org/article/10.1088/1742-6596/733/1/012039

Adeno-associated viruses (AAVs) are often used as vehicles for gene therapy. During the production process, many empty or partially filled capsids may be produced, which do not provide any benefits in the therapeutic process, but can impact immunogenicity and efficacy. The full/empty/partially full ratio of capsid is one of the most important Critical Quality Attributes (CQAs)of gene delivery vehicles to characterize. Analytical ultracentrifugation sedimentation velocity (AUC-SV) can measure the ratio of full and empty capsids by separation based on mass and buoyancy of full and empty particles. Cryo-TEM on the other hand can directly visualize capsids with various payloads. In addition, cryo-TEM allows you to also determine additional critical quality attributes such as particle size distribution, particle titer, and more. Another significant consideration is that cryo-TEM requires only 3μl of sample per experiment, whereas AUC-SV can require up to 400μl of sample.

Cryo-TEM refers to a technique using transmission electron microscopes where the sample will be frozen in vitreous ice in order to preserve its native hydrated state, allowing for deeper insight into your nanoparticle.

NIS has imaged 2,500+ liposomes and lipid nanoparticles (LNPs), characterized 880+ virus, vaccine, and virus-like particles (VLPs), and has imaged 200+ associated adeno-virus (AAV) as well as solved the structure of AAV to 1.8 Å.

We can image and analyze nanoparticles from 5 nm to 1 micron.

Each nanoparticle characterization project comes with its own set of challenges. NIS recommends scheduling a meeting with our experts to help determine if your project is suitable for a cryo-TEM study, and to discuss possible solutions to roadblocks that may appear during your project. NIS will always be fully transparent in regard to samples that might need more optimization or if a project might not be suitable for cryo-TEM.

Cryo-EM has an advantage over other methods in that only a small quantity of your sample is required. We typically request duplicates of ~50μl of material for each sample, at approximately ~1mg/mL protein, 10-30 mg/mL lipid, or E12 particles/mL. Your project manager will review this with you before sample submission.

Any standard buffer is allowed (i.e. PBS,Tris, HEPES, etc.). If specific additives are known to be necessary for sample stability, these should be present in the minimum amount possible. The dilution buffer composition can be your formulation buffer, but preferably without sucrose, glycerol, trehalose (or other cryo-protectants), or high salt. These components negatively affect the particle contrast In the images. If you cannot get around adding any of these, NIS would suggest keeping the cryo-protectant below 2% and the salt concentration below 500mM.

For nanoparticle characterization projects, you will typically receive your images and written report 4 weeks or less after sample submission & PO receipt. For rapid negative stain screening, images will be available to view in approximately 1 week from sample submissions & PO receipt. 5, 10, and 15 day rush services are available, ask your account manager for details.

There is usually a two week queue to begin imaging your project. It is best to ship your sample to us as soon as possible after your sample submission meeting, as the microscope schedule is fluid and we may be able to begin imaging your project earlier. If your sample is unstable, please contact your project manager for more details.

At NIS, we respect the confidentiality of our clients' intellectual property. Our internal contracts team collaborates closely with your legal team to complete all contracts. Typically, a CDA or NDA can be finalized within a week or less, while an MSA or MTA may take 2-3 weeks. In order to facilitate an open discussion of project-specific details during our introductory meeting, we strongly recommend having a CDA/NDA in place beforehand.