Finding Formulations Faster with SUPR-DSF

therapeutic.[ 1]

Analysis
The SUPR-DSF measures the intrinsic fluorescence of proteins, primarily from the Tryptophan or
Tyrosine residues. Analysing the fluorescence changes as a function of temperature allows you to
quantify protein stability by computing melting temperatures of the proteins. This technique is
very valuable in ranking the stability effects from many conditions.
The fluorescence waveforms generated in a thermal melt for the Infliximab control sample are
shown in Figure 1(a). Processing the waveforms with the BCM allows you to analyse the
wavelength shifts of the protein as it unfolds. The BCM thermal melt curve computed from the
Infliximab spectra is shown in Figure 1(b). The first derivative of the BCM melt curve was processed
to identify the melting temperatures. The first derivative analysis illustrated in Figure 1(c) shows
two distinct transitions at 68°C and 83°C, which matched differential scanning calorimetry (DSC)
results obtained by a collaborator.[2] All of the formulations in the screen were compared against
the control sample to determine which formulation provides the highest amount of stability.

Methodology
Microplate Preparation
The assay samples were made in a 96-well microplate by mixing 10 µL of stock FORMOscreen®
formulations with 33.75 µL water, and 6.25 µL of stock Infliximab. The samples were mixed and

Results
The melting temperatures for all formulations, except ones under patent protection, are shown in
Figure 2(a). Within this wide range of formulations, both increased and decreased stability of
Infliximab was observed. The formulations in the upper right-hand corner increased the stability
of both the first transition and second transition. The remaining formulations in other sectors of
the plot either stabilised one or the other transitions or destabilised both. The formulation screen
found the optimal buffer for Infliximab to be a salt-based buffer at a pH of 7.2, which is shown as a
red circle in Figure 2(a). This formulation increased the melting temperature of the first transition
by 2.3°C and the second transition by 1.3°C compared to the control sample. There were other
formulations that had a larger stability effect on the second transition, but in most cases, it is best
to pick the formulation that had the largest positive effect on the first transition because this is the
domain that will unfold first under normal operating conditions.
The optimal formulation is composed of 8.5 mg⁄mL sodium chloride, 100 mg⁄mL maltose monohydrate,
1 mg⁄mL potassium hydroxide, and 1 mg⁄mL sodium phosphate monobasic dihydrate. The normalized
fit curves for the first derivative of the optimal buffer compared to the control sample are shown in
Figure 2(b). This figure clearly illustrates the stability increase with the top stabiliser shifting the
peaks to higher temperatures for both transitions
Conclusion
The SUPR-DSF from Protein Stable demonstrates high data quality, capable of quantifying
multi-domain unfolding events of the therapeutic antibody Infliximab during a formulation screen
in a single 2-hour experiment. In this formulation experiment, we determined the optimal
formulation for Infliximab to be a salt-based buffer with a pH of 7.2. This formulation improved the
stability of Infliximab by increasing the melting temperature of the first transition by a 2.3°C and
the second transition by 1.3°C.
Pairing the easy connectivity and high-throughput capabilities of the SUPR-DSF with a pre-made
formulation plate allows you to determine the optimal formulation for your target in a single
experiment. This pairing greatly reduces the time it takes to complete formulation development
and allows you to proceed to the next steps in your pipeline quickly and confidently