Orthogonal high-throughput thermal scanning method for rank ordering protein formulations

Area of Science:

• Biochemistry
• Pharmaceutical Science
• Protein Chemistry

Background:

• Therapeutic proteins require stable formulations for effective storage and delivery.
• Assessing protein unfolding and aggregation is crucial for formulation development.
• Existing methods for evaluating protein stability can be time-consuming and require sample manipulation.

Purpose of the Study:

• To develop and validate a high-throughput thermal-scanning method for ranking protein formulation conditions.
• To compare the efficacy of SYPRO Orange and thioflavin T (ThT) dyes in thermal scanning assays.
• To assess the method's applicability for high-concentration protein solutions and mutant screening.

Main Methods:

• A high-throughput thermal-scanning approach was employed to monitor protein unfolding and aggregation.
• Two fluorescent dyes, SYPRO Orange and thioflavin T (ThT), were utilized to detect these events.
• Apparent transition temperatures were determined for four different therapeutic proteins across various buffer conditions.

Main Results:

• The thioflavin T (ThT)-based thermal scanning method demonstrated advantages over the SYPRO Orange method.
• The ThT method allowed for rapid rank ordering of solution conditions relevant to long-term therapeutic molecule storage.
• The assay is suitable for high protein concentrations, requires no sample dilution, and is amenable to mutant screening.

Conclusions:

• The ThT-based thermal scanning assay provides an efficient and robust method for optimizing therapeutic protein formulations.
• This high-throughput technique accelerates the development of stable protein therapeutics.
• The parallel use of SYPRO Orange and ThT offers a versatile tool for protein stability assessment and characterization.