DSF
(Differential scanning fluorimetry previously named also Thermofluor)
is a very simple tecniques that exploit the ability of a fluorescente probe, typically either SYPRO Orange or 1-anilino-8-naphthalenesulfonate (ANS) – that is quenched in an aqueous environment but becomes strongly fluorescent when bound to exposed hydrophobic groups of a protein. Therefore, the thermal unfolding of a protein of interest in the presence of such a dye, can be followed spectrophotometrically.
DSF is a 96 well based assay that could be performed on a RT-PCR thermocycler, it is inexpensive, fast, and require relatively little sample. All these advantages have made this approach attractive for screening applications in drug discovery and also for protein stability formulation
In the ProteoCool
n°24 in suggest as in my opinion the DSF may become an
essential assay todetermine lot to lot concistenciy in QC department of company producing recombinant proteins (e.g recombinant vaccine
and/or enzymes)
In this post i would like to share with you 2 examples that show as DSF may play an important role also in antibody screening, optimization and developability assesment.
DSF thanks to its excellent throughput and minimal material consumption (about 50ul of antibody at 0,3 mg/ml for each point) allow to readily compare the thermal stability of:
- different antibodies in the same buffer:
- different Fab mutants or Fc mutants in the same buffer;
- different antibody formulations; (optimize pH, salt concentration, excipients)
Example 1:
Comparision of thermal stability of different mabs
Full length human IgG1-Clk mabs in PBS may show 1 or 2 separate transitions on the basis of the Fab stability.
Mabs with lower Fab stability show in general a single transition while mabs with high stability show 2 separate transitions, 1 at about 69°C for the Fc and 1 at higher temperature for the Fab.
Example 2:
Comparision of thermal stability of different Fc mutants
In recent years the enhancement or elimination of the Fc-effector function has led to a growing interest in Fc-engineering, to give antibodies specific mechanisms of action and therapeutic properties.
Even if several Fc modification, as:
- LALANA, LALAPG, LALAGR mutations to remove effector function;
- SDIE, GAALIE mutations as well as afucosilation to enanche the effector function
were already reported, few data about developability of those solutions are still available and there is a lot work to do to select an enanched Fc combining the best effector function and good manufacturability, stability and pharmacokinetics.
Those are just 2 examples that show as in short time and using a limited amount od samples, thanks to DSF we can obtain essential informations to guide our decision in antibody during the screening, desing, formulation phases.
Of course, thermal stability itself do not Guarantee that the selected antibody has good developability profile but some of the following other tecniques as:
- DLS;
- PEG solubility;
- Self-interaction determined by BLI;
those provide informations about antibody aggregation propensity
- Hydrofobic interaction chromatografy (HIC)
those provide informations about antibody hydrofobicity
have to be peformed to complement the DSF data.
A great thanks to
Luca Sorrentino
