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AAPS Interlaboratory Study

Tier 2 Method: Intrinsic Fluorescence Spectroscopy

Introduction

This protocol describes the procedure for monitoring protein tertiary structural changes using 

intrinsic fluorescence. Protein fluorescence spectroscopy is dependent on the presence of 

aromatic amino acids like phenylalanine, tyrosine, and tryptophan. Tryptophan fluorescence 

(with excitation wavelengths between 280 and 300 nm) is used to monitor structural changes in 

the protein as its emission maximum (ca. 300 – 350 nm) is highly dependent on the polarity of 

the environment. The intensity of the emission and the wavelength of maximum intensity for 

tryptophan fluorescence are indicators of the average residue microenvironment and will be 

monitored to provide information on protein tertiary structure.

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 

• Fluorescence Spectrophotometer with a temperature control unit

• UV spectrophotometer

• Quartz cuvette (path length depends on system being utilized)

• Pipets and pipette tips as recommended by the manufacturer.

• Cuvette cell washer

• High purity ethanol

• Milli-Q grade water or equivalent

• 5% Contrad 70 (or equivalent detergent)

• Protein samples and formulation buffers supplied by sample originators (see Table 1)

o Buffers, aliquots of unstressed, stir stressed, light stressed from Shipping #1 

• Appropriate dilution buffer for the mAbs

Reagents and Solutions 

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

Buffers, aliquots of unstressed, stir stressed and light stressed from Shipping #1 should be used 

for this analysis. All stressed material were generated at nominally 1 mg/mL. 
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Proteins Formulations Samples

Amgen IgG2 10 mM sodium acetate, pH 

5.0

Unstressed, 1 mg/mL

Stir stressed, 1 mg/mL

Light stressed, 1 mg/mL

NISTmAb IgG1

12.5 mM L-histidine, 12.5 

mMol L-histidine HCl, pH 

6.0

Unstressed, 1 mg/mL

Stir stressed, 1 mg/mL

Light stressed, 1 mg/mL

Procedures

Sample preparation

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

light stressed samples) 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 intrinsic fluorescence measurements

(prepared and frozen in #1) by bringing them to room temperature for no more than 15 

minutes. 

3. A proper mixing of the samples prior to analyzing is important to eliminate particle 

clumping. Prior to dilution of each sample, mix each aliquot (mentioned in #2) by pipetting 

the sample with a 200 uL tip near the bottom of the sample tube 10 times in different 

directions but not touching the bottom or creating bubbles. Remove sample from near the 

bottom of the vial, from about 3⁄4 of the depth of the tube from the top of the liquid before 

dispensing the sample into another container containing the buffer. 

4. Under laminar flow hoods, dilute all samples to 0.5 mg/mL with 0.22 μm filtered designated 

buffer. Use the UV Visible Spectroscopy protocol to determine the exact concentration of 

the diluted solution at 280 nm using the designated extinction coefficient. 

5. If the stressed sample is highly turbid, the sample may need to be briefly centrifuged. The 

“supernatant” of the centrifuged sample should be carefully separated from the vial and 

should be used for analysis. Here, we define the “supernatant” as 70% of the filling volume. 

Instrument Setup

1. Turn on the instrument and temperature controller. Set temperature to 20°C +/- 2°C. Warm 

the lamp for at least 30 min. 

2. Measure fluorescence of the background sample (buffer) prior to fluorescence of samples, 

using the parameters listed below in Table 2.

3. After collecting background, collect spectra of the sample. 

4. Between each sample, wash the cuvette thoroughly with water and dry with ethanol rinse, 

followed by vacuum drying.
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5. Turn off system in accordance with instrument manufacturer’s instructions.

Table 2: Intrinsic fluorescence parameters

Excitation Wavelength (nm) 295

Emission (nm) 300 to 550

Em/Ex Slit width (nm) Automatic, if possible 

Scan speed (nm/min) 100

Step size (nm) 1

Accumulations 3

Temperature 20◦C

# of Replicates 1

Sample Measurement

1. Review the Intrinsic Fluorescence Data reporting template for the requested information 

before beginning the measurements. 

2. Subtract the buffer spectrum from the sample spectrum. 

3. Buffer subtracted (corrected) value is then concentration-normalized by dividing the 

corrected fluorescence intensity value divided by the protein concentration determined by 

UV-Vis absorbance (A280).

4. Wavelengths of fluorescence peak maxima (λmax) are determined manually in Excel 

spreadsheet and confirmed visually. 

Understanding Results 

Enter instrument information and final data into the provided Intrinsic Fluorescence Data Excel 

sheet. Instrument biases can depend strongly on the instrument configuration. Document 

instrument type, model, and configuration and settings. Export the data as recommended by the 

instrument manufacturer. 

Normalized fluorescence spectrum: Results are plotted as Normalized Fluorescence Arbitrary 

Units (A.U.) vs.Wavelength (nm). 

Emission Maxima reporting: Round reported wavelength values (λmax) to the nearest whole 

number increment.

Troubleshooting 
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As with other samples of aggregated proteins, the proteins may adhere to the surface of the cell. 

Visually ensure no residual protein or residue streaks/smears remain on the cuvette. If the 

cuvette’s background scans appear to contain unusually high absorbance signal, soak the cuvette 

with 5% Contrad 70 (or similar detergent) for 30 minutes. Rinse with Milli-Q water to remove 

all detergent and wash cell thoroughly.

Further Information

For any specific questions regarding this method, please contact Sarita Mittal at 

sarita.mittal@merck.com. Please copy aapsinterlab@nist.gov on your email.