Formulation screen of Trastuzumab using the SUPR-DSF

In this report we show the analysis of a thermal denaturation-based formulation screen of the
commercially available therapeutic antibody Trastuzumab, in 96 different conditions with the
SUPR-DSF instrument. Along with screening the stabilising agents, confidence in the results is
gained as there is consistency with both Differential Scanning Calorimetry, and the formulation
used for the commercial drug Herceptin®. This screen was directly measured in a single 384-well
microplate in less than 1.5 hours. This high-throughput can be leveraged further through lab
automation integration to screen thousands of samples per day

Results
The denaturation curves measured (which Figure 1 and 2 are exemplars) exhibit two separate
transition regions. Through Least-Squared fitting of the denaturation data, via a standard
three-state model[1], the Tm values, van 't Hoff enthalpy (ΔHm) and Tonset values were determined
and are listed in Table 1. The table lists all the excipients that improved the stability of the antibody
(compared to the control sample).
The three-state behaviour is consistent with DSC data for Trastuzumab[2] which reports two Tm
values around 70°C and 82°C. Acknowledging sample and solvent variability, the Tm values are
consistent with values reported elsewhere.[2] Along with similar Tm values, the SUPR-DSF data
established that both histidine and trehalose dihydrate either did not hinder or improved the
conformational stability of Trastuzumab, which are formulation constituents in the Trastuzumab
containing drug Herceptin®.[3] These two points help demonstrate SUPR-DSF’s measurement
consistency 。all while offering superior throughput capabilities.



Conclusion
The SUPR-DSF from Protein Stable demonstrates high data quality, capable of quantifying
multi-domain unfolding events of the biotherapeutic antibody Trastuzumab under 96 formulation
conditions in a single 1.5-hour measurement. The results are consistent with those reported by
other lower throughput methods, and correctly identified excipients that are used in the marketed
biotherapeutic formulation of Trastuzumab.
In addition, the intrinsic fluorescence based Differential Scanning Fluorimetry method of the
SUPR-DSF can achieve these outcomes all while increasing throughput, convenience, and
lowering consumables cost and sample consumption.
This already data rich and high throughput method can be leveraged further through integration
with lab automation to screen well over a thousand conditions per day.

Methodology
Microplate Preparation
Trastuzumab was prepared at 1 mg⁄mL. The 96 different solvent conditions were obtained
pre-formulated in a deep well 96-well plate. Samples were prepared in the wells of a 384-well,
commercial polypropylene PCR microplate. The final antibody concentration of 0.1 mg⁄mL was
achieved by dispensing 2 µL of antibody stock into 18 µL formulation solution to a final well volume
of 20 µL. After dispensing, solution mixing was achieved by shaking the plate for 5 mins at
1500 rpm before being sealed with an optically clear adhesive film.
Thermal Denaturation Measurement
The SUPR-DSF was set-up to measure the fluorescence spectra of Trastuzumab samples from
20°C to 100°C, with a 1 °C per minute ramp rate. Quantification of the spectral shift, that is indicative
of protein denaturation, was determined via the barycentric mean (BCM) calculation.
The thermal denaturation curves were fitted to a three-state model[1] via a Least-Squared
regression algorithm, the fitted parameter values of which are listed in Table 1. Identification of
better performing excipients was achieved by identifying samples with Tm1 and Tm2 values that
were higher than the control sample. Methods and results for all samples are available on request.