How was response factor (RF) obtained in equation 1 of the AAV empty vs full capsid application note using the Protein-Pak Hi Res Q column? - WKB252955

ENVIRONMENT

• Application notes
• Adeno-associated virus (AAV)
• Empty vs full capsid 
• Response factor (RF)
• Protein-Pak Hi Res Q 
• Ion exchange
• Anion exchange chromatography
• Fluorescence detector (FLR)
• UV detector (UV)

ANSWER

The response factor is the ratio of the full and the empty capsid's peak area, when you inject the same amount (concentration) of the full and empty capsid.

The actual FLR response is different when the DNA (full) is present in the capsid vs the FLR response when the capsid is empty, even if the concentrations of the empty and full are the same. The presence of DNA (full) results in slight changes of the FLR signal, as compared with the empty capsid. This difference in signal needs to be taken into account in the equation.

If the RF (empty) is 1.3 [full will be higher response], you need to divide the peak area of the full by 1.3 to normalize the signal.

For FLR, the RF should be close to 1.

ADDITIONAL INFORMATION

For additional information, see equation 1 in Anion-Exchange Chromatography for Determining Empty and Full Capsid Contents in Adeno-Associated Virus.

For additional references, see  Rapid AAV Concentration Determination Using Size-Exclusion Chromatography with Fluorescence and UV Dual Detection and the following journal article: Wang, C. et al. Developing an Anion Exchange Chromatography (AEX) Assay for Determining Empty and Full Capsid Contents in AAV6.2. Mol. Ther. Methods Clin. Dev. 2019, 15, 257–263.