Evaluation of Capillary-Mediated Vitrification (CMV) as an Alternative to Traditional Cold Temperature Storage for Kinase Assay Reagents

Abstract

AssayQuant is a growing biotech company specializing in the development of sensor peptides that provide a quantitative picture of protein kinase activity over time via continuous measurement of chelation enhanced fluorescence. As a leader in kinome profiling and inhibitor potency and characterization, AssayQuant’s -80°C freezers hold thousands of samples of over 400 different protein kinases in addition to various other reagents utilized for assay execution and optimization. The prevalence of freezer-dependent storage exposes any organization to space, time, and cost concerns ranging from freezer procurement to placement and routine maintenance. Inadequate freezer storage conditions can lead to temperature fluctuations, potentially affecting sample integrity, or even complete equipment failure, risking total sample loss. This is especially true for smaller evolving companies where growth often outpaces infrastructure, and the high costs of reagent procurement and freezer storage can, at times, be prohibitive.

Although alternative methods to cold temperature storage exist, they rely largely on long sample preparation times, extensive product-specific optimization, and often employ high temperature or shear stress environments that are generally incompatible with the preservation of protein kinase activity. Capillary-mediated vitrification (CMV) is an emerging technique that offers a promising solution to eliminate dependence on coldtemperature reagent storage and shipment, and overcome its associated risks, while at the same time avoiding the concerns prevalent in other methods of sample stabilization.

AssayQuant Technologies has partnered with Upkara, a global leader in biomolecule stabilization, to assess the potential of capillarymediated vitrification as a viable alternative to its current cold-storage solutions. Assay reagents preserved via CMV were evaluated alongside conventionally stored samples in the context of a continuous enzymatic assay using AssayQuant’s Phosphosens® technology. A viable alternative sample stabilization approach and workflow is presented along with prospective applications geared towards both in-lab storage and product distribution.

Key Takeaways

• AssayQuant’s PhosphoSens® assay components vitrified, stored at ambient temperature, and eluted in aqueous media can be used to generate kinetic assay data. Upkara's CMV technology can further simplify assay execution by consolidating multiple temperature sensitive components onto a single scaffold that can be stored or shipped without cold chain concerns.
  
  
• Protein kinases can be stabilized via CMV while preserving enzymatic activity. Assays performed using kinase stabilized with Upkara’s Low Binding BioFixTM vitrification buffer, and subsequently stored at ambient temperatures, exhibit kinetic progress curves and reaction rates that are comparable to those generated with kinase stored at -80°C.
  
  
• Potency determinations performed with vitrified reagents yield IC50 values that are within 2-fold of traditionally cold stored reagents suggesting that potency and selectivity workflows are amenable to CMV stabilized reagents.