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AAPS Interlaboratory Study

Tier 1 Method: UV-Vis spectroscopy

Introduction

This document will briefly describe a procedure to use ultraviolet (UV-Vis) spectroscopy to 

measure the optical density of a protein sample. The method aims at measuring the protein 

content (at 280 nm) and optical density as an indicator for the turbidity of the sample (at 320 and 

350 nm) of the undiluted mAb sample (ca. 1 mg/ml) in formulation buffer. 

Important Notes:

1) 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. 

2) Many biophysical methods, such as CD, fluorescence, etc., require accurate protein 

concentration information. If those methods are being performed to obtain structural 

information, the analyst can simply follow this protocol but does not need to fill out the 

accompanying data template. The analyst will be requested to record the measured 

concentration on those methods’ data templates. 

Equipment and Materials List 

● UV-Vis spectrometer

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

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

● UV-transparent cuvette (0.5-cm pathlength), well-plate, or other cell type with adjustable 

pathlength 

● 0.1 μm filters, syringes, needles for filtering buffers

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

Instrument selection

Select your preferred UV-Vis spectrophotometer that allows you to measure the undiluted mAb

samples according to the settings specified in the Table 2 below. Because the extinction 

coefficients of the mAb samples range between 1.45-1.7 ml mg-1cm-1, the absorbance of the 

sample ranges up to ca. 1.7 per cm. Please measure the samples undiluted by choosing 

appropriate instrumentation and pathlengths (e.g., 0.5-cm cuvettes, Solo-VPE, well plate reader 

with pathlength correction, etc.). 

Sample preparation

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

light stressed mAb 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 UV-Vis spectroscopy measurements

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

minutes. 

3. Prior to analysis, mix the aliquot in the following manner: pipet 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 before dispensing the sample into the cuvette or another container.

Sample Measurement

1. Review the UV Vis Data Template for the requested information before beginning the 

measurements and input the set-up information. 

2. Transfer material for UV-Vis analysis into the measurement cell, well plate, or into the 

measurement system by using a pipette. Avoid formation of air bubbles. Prevent 

evaporation of the sample by covering the sample and measuring promptly. 
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3. Measure the OD values of the sample as specified in Table 2 against highly purified water 

as blank. 

4. Perform a single measurement (n = 1) of both mAb samples and placebo buffer (in parallel 

or subsequently).

Table 2: Measurement parameters for UV-Vis spectroscopy

Wavelength of interest 280 to 1000 nm

Cell type UV-transparent cuvette (e.g., 0.5-cm 

pathlength), well plate, or other cell type with 

adjustable pathlength (e.g., Solo-VPE)

Blank Highly purified water

Extinction Coefficients (ε) (mL · mg−1 · cm−1) Amgen mAb = 1.5390; NISTmAb = 1.42

Measurement temperature 20 °C

Number of measurements per sample 1

Data Analysis

1. Use the instrument’s software or manually correct the measured absorbance/OD values for 

the pathlength used during analysis. Use the instrument’s software or manually subtract the 

buffer absorbance/OD values from the corresponding sample. 

2. Copy and paste the pathlength-corrected (1 cm) and buffer-subtracted absorbance/OD 

values into the provided UV-Vis Data Reporting Template. Enter the additional requested 

information, including the extinction coefficient of the protein provided with the samples. 

The UV-Vis Data Reporting Template will automatically calculate the following results:

- OD350 nm [turbidity] (AU)

- OD280 nm [raw] (AU)

- OD280 nm [scattering-corrected] (AU)

- Protein concentration [scattering-corrected] (mg/mL)

Understanding Results 

Record the various instrument parameters and the results in the UV-Vis Data Reporting 

Template. Provide the absorbance/OD values per cm (corrected for pathlength). The formulas in 

the UV-Vis Data Reporting Template will calculate the protein concentration based on the 

absorbance/OD at 280 nm, subtracted by the OD 350 nm to correct for scattered light 

(approximation). Furthermore, the OD values at 350 nm will be reported as a measure for the 

turbidity of the sample. 
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Troubleshooting 

● Diluted samples were measured -> measure undiluted samples in cells with appropriate 

pathlength

● Samples were not homogenized -> make sure that the sample is properly homogenized

● Absorbance/OD above linear range of instrument -> be sure to stay within the linear 

range of Beer-Lambert’s law during measurements by selecting the right instrument and 

settings (see above)

● Readouts were not corrected for the pathlength -> report pathlength-corrected absorbance 

values (per cm)

● Evaporation of samples during analysis -> prevent evaporation of the sample

● Artifacts from air bubbles, etc. -> prevent the formation of air bubbles, e.g., by careful 

and slow pipetting

● Artifacts from dirty cells -> follow cleaning procedures and/or use disposable 

consumables

Further Information

For any specific questions regarding this method, please contact Tim Menzen at 

tim.menzen@coriolis-pharma.com. Please copy aapsinterlab@nist.gov on all of your emails.