Developing AUC Standards

For more information, see:

Savelyev, A., Gorbet, G. E., Henrickson, A., Demeler, B. (2020). Moving analytical ultracentrifugation software to a good manufacturing practices (GMP) environment. PLOS Computational Biology, 16(6), e1007942. https://doi.org/10.1371/journal.pcbi.1007942

The requirements of a standard to be used in validating an instrument are:

  1. The standard must be sufficiently stable, with a sufficient shelf life during which all measurable properties are not changed.

  2. The standard must be able to be characterized using orthogonal methods that are already in a GMP environment.

  3. The standard must exhibit properties that address all the parameters that are measurable and detectable.

  4. The standard must be able to have its parameters characterized over the entire dynamic range of the instrument.

  5. The standard must be available with sufficient purity, and it must be possible to manufacture with great reproducibility.

Proposal for dsDNA Standards

We propose using double-stranded DNA (dsDNA) for a standard for AUC instruments.

[✓] Stable, with long shelf life.

[✓] Characterize with GMP orthogonal methods.

[✓] Addresses all measurable AUC parameters.

[✓] Utilized AUC's entire dynamic range.

[✓] Sufficient purity.

dsDNA is a very stable biopolymer, and can remain unchanged for years, if placed under the proper storage conditions. The dsDNA molecules can be characterized by multiple methods outside of AUC, such as sequencing, size-exclusion chromatography, dynamic and static multi-angle light scattering, fluorescence correlation spectroscopy, and gel electrophoresis.

By changing the length of the strand, discretely sized molecules can be created. This allows for modulation of their molar mass, diffusion coefficient, hydrodynamic radius, and frictional ratio. The discrete molecules will also exhibit a strong anisotropy signal as a function of length and molar mass. Additionally, the dsDNA's partial specific volume, anisotropy, and non-ideality properties can be easily modulated using buffers of different ionic strengths.

Sets of dsDNA with different lengths and buffer conditions can be utilized alone or in combination at different ratios to generate standards at different rotor speeds. dsDNA can also be stained with fluorphores (such as SybrGreen), which could impact the absorbance of the molecule to extend the utility of the standard for wavelengths outside the UV range.

Vectors containing different lengths of dsDNA fragments can be mass produced with bacterial cultures. Recombinant cloning technologies can be used to ensure reproducible sequences. Size-exclusion chromatography and gel electrophoresis can be used to obtain ultra-pure preparations.

Finally, dsDNA molecules can be mixed at varying ratios to produce standards to validate an instrument's limit of detection and limit of quantification.