nanoDSF Page | 1 25 September 2023

AAPS Interlaboratory Study

Tier 2 Method: Nano Differential Scanning Fluorimetry

Introduction

DSF is a valuable tool for conformational stability assessment. NanoDSF uses intrinsic 

fluorescence, mainly tryptophan and tyrosine residues, to monitor protein unfolding. A 

temperature ramp is applied to a protein solution to measure the temperature at which the first 

conformational changes start to occur (Tonset) and the temperature at which 50% of the protein or 

most temperature-sensitive protein domain is unfolded, i.e. the melting temperature (Tm). In 

nanoDSF, upon unfolding, the environment of those residues will alter because they become 

exposed to the solvent and thus, their fluorescence intensity will change. The relationship between 

fluorescence intensity changes and temperature gradient can be used to obtain the apparent Tm and 

Tonset. MAbs have different Tm values attributed to their different domains, but the most 

temperature sensitive one is normally the CH2 domain, which is the one that we will be focusing 

on for the Tm values reported.

This protocol briefly describes a method to measure the apparent melting temperature (Tm) and 

onset temperature (Tonset) by nano differential scanning fluorimetry (nanoDSF) to assess the 

conformational stability of unstressed proteins. The stir and light stressed samples will not be 

analyzed with DSF. 

Important Notes: This protocol is not a Standard Operating Protocol and assumes the required 

instrument is in good working order and the analysis is performed by experienced user(s). 

Required calibration or check standards should be run according to the manufacturer 

instructions. This protocol does not contain all the details of the analysis. The analyst should 

rely on their best judgment, routine practices, and knowledge of the technique to conduct the 

study; this protocol should be used as a guideline for the analysis. Samples should be analyzed 

immediately after thawing and preparation.

Equipment and Materials List 

• NanoDSF device

• Single use sample containers (e.g. capillaries for Prometheus, UNi for UNcle) 

• Protein samples and formulation buffers supplied by sample originators 

o Buffers and aliquots of unstressed samples ONLY from Shipping #1 

• Pipets and pipette tips

Reagents and Solutions 
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Table 1: The following samples and their buffers will be provided by sample originators. Buffers 

and aliquots of unstressed samples from Shipping #1 should be used for this analysis. They are 

nominally at 1 mg/mL. 

Proteins Formulations Samples

Amgen IgG2 10 mM sodium acetate, pH 

5.0 Unstressed, 1 mg/mL 

NISTmAb IgG1

12.5 mM L-histidine, 12.5 

mMol L-histidine HCl, pH 

6.0 

Unstressed, 1 mg/mL 

Procedures

Instrument Set up and Sample Preparation 

1. Thaw the samples from Shipping #1 and aliquot the Tier 2 vials (for the unstressed only) 

as described in the instructions into smaller aliquots for the various methods and freeze 

until ready to use. Be sure to follow the procedure for vigorously mixing the sample 

prior to aliquoting. 

2. For this analysis, thaw the aliquots designated for nano-DSF measurements (prepared and 

frozen in #1) by bringing them to room temperature for no more than 15 minutes. 

3. Using a pipette, fill the sample container with the volumes suggested by the manufacturer 

(e.g. 9μL for Uni, 10μL for capillaries of Prometheus). No dilution is required. Use a 

maximum of 8 samples in triplicate per run, i.e. up to 24 wells/capillaries per run.

NOTE: Maximum of 24 wells/capillaries per run to ensure a good and constant data point density. 

Make sure that there are no bubbles and that the sample order matches the one inserted in the 

software.

4. Insert the sample container in the support provided by the manufacturer and insert it in 

the nanoDSF device 

5. When setting up the method, use the parameters described in Table 2 and start the 

measurements. 

Data Acquisition 

1. Review the nanoDSF Data template for the requested information before beginning the 

measurements and input the set-up information. 
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2. Use the following experimental parameters to collect the data (default of the UNcle – 

quick start mode): 

Table 2. Data parameters for NanoDSF 

Number of samples Up to 8 samples in triplicate per run, i.e. up to 24 

wells/capillaries per run

Temperature ramp Linear: 1°C/min

Equilibration time 5 min

Temperature range 20-95°C

Sample concentration range 1-150 mg/ml – no dilution required

Fluorescence settings Ex: 266 nm (or equivalent device default) Em: 300 - 450nm 

(or equivalent device default)

2. Perform a discovery scan if necessary. 

3. Attribute the sample names in the software and proceed to the melting scan. 

Understanding Results

1. Data and selected instrument parameters should be entered in the NanoDSF Data 

reporting Template.

2. Use the software default method to calculate the Tonset and Tm automatically (e.g. 

barycentric mean [BCM] in range 300-450nm for the UNcle) 

3. Average the temperature of the triplicates to obtain the final values and us the standard 

deviation to report the measurement variability 

Troubleshooting 

1) Ensure that there are no bubbles in the UNi/capillaries.

2) Ensure that the sample holder is properly placed in the instrument. 

Further Information

For any specific questions regarding this method, please contact Vasco Filipe at 

Vasco.Filipe@sanofi.com. Please copy aapsinterlab@nist.gov on your email.