2024-12-12

Q: Tell us about Refeyn's product portfolio and how do their mass photometry solutions support drug discovery in the pharmaceutical industry?

Gabriella Kiss: Refeyn products are based on mass photometry technology, which enables the accurate mass measurement of single molecules and other particles in solution, in their native state and without the need for labels. It opens up new possibilities for bioanalytics and research into the functions of biomolecules.

Mass photometry builds on the principles of interference reflection microscopy and interferometric scattering microscopy. It was developed at Oxford University by a group led by Professor Philipp Kukura, who demonstrated that the minute amount of light scattered by single molecules can be reliably detected and correlates directly with molecular mass.

Refeyn’s product portfolio includes three main product lines, each providing an integrated analytics solution tailored for a specific range of applications. Each product line includes an instrument as well as associated software and consumables (e.g. calibrants and measurement slides). All three product lines share several features:

• Measurements are done in solution with little sample prep needed
• No labelling involved
• Measurements take just minutes
• Small quantities of sample are consumed
• By measuring single particles, this provides information on population distributions (and subpopulations) with single-particle resolution
• Easy-to-use benchtop instruments with intuitive custom software

The first two (TwoMP and SamuxMP) are mass photometry product lines optimized for different applications. They both measure the mass of each particle in solution individually, returning a mass histogram (the distribution of the masses of each particle measured). As mass is a universal property, it can provide information on anything that affects the mass, such as binding, aggregation or cargo loading.

The TwoMP product line can, in principle, measure any particle in the mass range 30 kDa – 5 MDa, including proteins - in general, as well as antibodies and membrane proteins embedded in membrane mimetics - and nucleic acids. In addition to standard software and consumables, this product line includes:

• The TwoMP mass photometer
• The TwoMP Auto automated mass photometer
• Launched this week (7 October): A new software module dedicated to measuring antibody aggregation 
• An optional microfluidics system, MassFluidix HC, which enables the measurement of samples at higher concentrations (up to the tens of micromolar) as compared to standard mass photometry (100 pM – 100 nM).

Key applications related to drug discovery:

• Analysis of antibody aggregation/fragmentation, informing stability studies
• Measurement of protein-protein (or protein-nucleic acid) binding – including determination of stoichiometry and affinity (Kd)
• Measurement of antibody-antigen affinity

The SamuxMP product line is optimized for adeno-associated virus (AAV) analytics. This line includes (in addition to standard software and consumables):

• The SamuxMP mass photometer
• The SamuxMP Auto automated mass photometer
• A software package for GMP-compliant environments

Key applications related to drug discovery:

• AAV empty/full measurement (partially filled and overfilled capsids can also be resolved and quantified)
• Titer estimation for AAV samples
• Measurement of transgene length in AAVs
• Assessment of AAV aggregation

The third product line, KaritroMP, uses a related technology developed by Refeyn: macro mass photometry. The main difference with macro mass photometry is that it measures two parameters: Particle size and contrast (a proxy for mass) and is optimized for larger vector particles (diameter 40 – 150 nm). (You can read more here: Macro Mass Photometry Technology | Refeyn ). It is particularly useful for checking the homogeneity and purity of viral vector preparations.

In terms of consumables, each product line has a dedicated calibrant (the mass calibrants MassFerence P1 for TwoMP line and MassFerence P1 for SamuxMP, and the size calibrant SizeFerence for Karitro). Clean glass sample carrier slides optimized for mass photometry measurements are also available. These products save users time, while ensuring reproducibility across experiments and labs. Refeyn has also developed software dedicated to mass photometry data acquisition (AcquireMP) and analysis (DiscoverMP).

Q: How does Refeyn’s mass photometry technology handle lentiviral vector characterization?

Gabriella Kiss: Refeyn developed macro mass photometry, a new technology that builds on the principles of mass photometry, for the analysis of large viral vectors. The size-contrast distribution reported by macro mass photometry provides a visual representation of sample heterogeneity. For lentivirus samples, we have shown that, once the size and contrast range of the particle of interest has been defined, quantification from macro mass photometry is proportional to physical titer. Therefore, macro mass photometry can provide a rapid way to compare relative differences in physical titer across samples.

Q: Can you explain how Refeyn’s tools adapt to high-throughput analytical workflows?

Gabriella Kiss: Refeyn’s instrument family includes two automated instruments, the TwoMP Auto and SamuxMP Auto. They can autonomously measure up to 24 samples in as little as 90 minutes.

In addition, the antibody stability software module (launched this week) streamlines the analysis of antibody aggregation, enabling straightforward comparison of data from many different samples (e.g. antibody types, stress conditions) and returning robust statistics for replicate groups.

Q: What role does Refeyn play in improving viral vector analytics and how do Refeyn solutions address antibody characterization needs?

Gabriella Kiss: In both cases, mass photometry is offering game-changing solutions in that it is much faster, cheaper and easier to use than the currently used technologies while offering comparable accuracy.

For antibody analytics, the main comparator technologies are SEC and AUC:

• Compared to SEC, mass photometry is about 10x faster, uses about 300x less sample and requires much less expertise to operate.
• Compared to AUC, mass photometry is about 100x faster (mass photometry takes minutes and AUC takes hours, and the process may be even more lengthy as AUC is often outsourced), takes 5,000-10,000x less sample, and again is much simpler to use.

For AAV analytics, AUC is a comparator as are cryoTEM, CDMS and ELISA/qPCR:

• Compared to cryoTEM, mass photometry is >10x faster, much cheaper and much easier to use, and enables quantification of AAV aggregation and fragmentation, which cannot be achieved with cryoTEM.
• Compared to CDMS, mass photometry is about is >10x faster and much simpler to use, and it offers label-free measurements in the native state whereas CDMS requires buffer exchange prior to measurement.
• Compared to ELISA/qPCR, mass photometry is >10x faster and has much better accuracy (error for MP is around 10% vs. >30% for ELISA/qPCR). Mass photometry is also serotype agnostic and can distinguish partially full capsids – two limitations of ELISA/qPCR.

Q: What potential areas for innovation are being explored by Refeyn’s Scientific Advisory Board in the near future and how do Refeyn’s products contribute to quality control in biomanufacturing?

Gabriella Kiss: In general, the advisory board is focused on exploring a range of potential applications across biologics and bioprocessing. With regards to quality control, we offer a software package that enables the use of the SamuxMP and SamuxMP Auto in GMP-regulated environments. This means mass photometry can be used within AAV-based gene therapy manufacturing facilities to measure empty/full ratios, while adhering to strict FDA and EMA regulatory requirements.

Q: How is Refeyn expanding the accessibility of molecular mass measurement technologies?

Gabriella Kiss: Mass photometry is a relatively young technology, having been developed in 2018 at the University of Oxford. For those who developed it, it became clear almost immediately that rapid, in-solution mass measurement would be a very useful analytical tool with a wide range of applications in the life sciences and drug discovery.  As a result, to make the technology accessible to the wide range of scientists who could likely benefit from it, a user-friendly mass photometer prototype was developed. It was commercialized through the founding of Refeyn that same year (2018).

That original vision of making a ground-breaking technology accessible is now being realized – hundreds of labs across the world now have easy-to-use mass photometry instruments on their benchtops. As further evidence of the rapid uptake of mass photometry, more than 500 scientific papers have now been published that use mass photometry. Although the technology inside our mass photometers took many years to develop, anyone who can pipette can operate a mass photometer after less than a day of training, and they can easily interpret their results.