FTIR P a g e | 1 1 November 2023

AAPS Interlaboratory Study

Tier 1 Method: FTIR

Introduction

This protocol briefly describes a method using Fourier-transform infrared spectroscopy (FTIR) 

to obtain information regarding the secondary structure of the protein in the unstressed and 

stressed monoclonal antibodies used in this study. This document describes sample handling, 

instrument set up, and data reporting of the results associated with this method. The primary 

reported results are the wavenumber versus absorbance for each sample. The measurements 

should be performed both in solution and on the particles present in the stressed samples. 

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 as it is not specific to the needs of each 

instrument/make/model. 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

● FTIR instrument and associated consumables for analysis of solution and analysis of 

particles in solution

● Protein samples and formulation buffers supplied by sample originators

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

● Pipette and pipette tips

● Liquid nitrogen (when LN2 cooled MCT detector is used)

● Detergent or cleaning solutions, deionized ultra-filtered water, acetone or ethanol and 

vacuum or compressed N2 for cleaning between runs

● PVDF syringe filters

● Pipets and pipette tips

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 1 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 FTIR measurements (prepared and 

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

3.) After thawing, particle concentration may change with time, even if the sample is 

maintained at (2-8) ̊C. This change may be mitigated by carefully planning the data 

acquisition to minimize the period of data acquisition, preferably to one day.

4.) 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. 

Instrument Setup

1.) Follow routine set-up procedures provided by the equipment manufacturer. Use the most 

recent software version available for collecting and analyzing data. Ensure the sample 

chamber and interferometer is purged with nitrogen to remove water vapor and CO2, as 

they can obscure important peaks in the spectrum. 

2.) The samples provided will not need any further dilution for analysis. FTIR analysis 

should be performed in solution for the unstressed and stressed materials as well as on the 

particles (isolated from the stressed material) to elucidate the secondary structure 

conformation of protein in solution or within the particle of interest, respectively. 

Comment added Nov 1, 2023: Disregard the highlighted text. This text is a copy error. FTIR samples should be run as 

is and should not be diluted. 
FTIR P a g e | 3 1 November 2023

a.) As these solutions have low protein concentration, the analyst can select 

appropriate FTIR procedures to collect the FTIR spectrum.

b.) For analysis of particles (proteinaceous or non-proteinaceous) in solution, a 

microscopic FTIR system in reflectance mode can be used to obtain spectra of 

isolated particles. 

Solution Sample Measurement

1.) Review the FTIR Data template for the requested information before beginning the 

measurements and input the set-up information. 

2.) All sample solutions including formulation buffer should be filtered using a 0.22 μm 

syringe filter to minimize the contribution from the particle to the measured spectrum.

3.) Since these solutions have low protein concentration, the analyst can select appropriate 

FTIR sampling method to collect the FTIR spectrum. For example, ATR cell can be 

loaded with excess volume of sample solution (30 to 50 μL) than the minimum required 

volume to minimize the impact of sample evaporation during measurement or in a 

transmission cell with a pathlength of ≤ 6 μm.

4.) Background measurements should be acquired before every sample measurement. Often 

instruments will allow you to specify a background measurement and will save the data 

accordingly. Subsequently, perform the sample measurement and collect the data using 

the parameters shown in Table 2. Some instruments will allow an automatic background 

subtraction and display the resulting sample absorbance spectrum. 

5.) If the protein is sticky, you may need to use detergents and/or additional cleaning 

procedures to clean the sample chamber prior to loading the next buffer blank. When a 

series of runs are being performed, it is always good practice to inspect your buffer blank 

spectra and ensure that it looks “clean” (i.e. spectrum doesn’t look like the protein 

spectrum of the previous protein sample). Only then load the corresponding protein 

sample. 

Table 2. Recommended instrument parameters for FTIR in solution

Number of Sample Scans 256

Number of Background Scans 256

Wavenumbers (cm-1) 600 to 4000

Result Spectrum Absorbance

Resolution (cm-1) 1

Temperature 20  ̊C

Replicates 1

Comment added Oct 5, 2023

Sample solutions should not be filtered before analysis. Only formulation buffers should be filtered. 
FTIR P a g e | 4 1 November 2023

Particle Sample Measurement using FTIR Microscopy in Reflectance Mode

1.) This analysis is appropriate for any particles (proteinaceous or non-proteinaceous) that 

are present in the samples and that can be collected by filtration. For example, particles 

can be filtered in a gold coated polycarbonate track edge filter (0.8 or 3 μm pore size) by 

the application of vacuum.

2.) Follow the filtering procedure employed in your lab to isolate particles for analysis, and 

apply washing procedures without compromising the integrity of the particles. 

3.) In addition to purging the detector, you must also purge the microscope with nitrogen. 

Set up the instrument as recommended by the manufacturer and check that the signal 

intensity is within the range prescribed by your manufacturer. 

4.) Select reflectance mode when collecting data. On a particle free surface (identical to the 

one where the particle is located on), start the reference measurement by selecting the 

background option on your software. Once the background region spectrum is acquired, 

move to the sample and focus using the binocular. Select the particle of interest by 

moving the stage in the x and y-direction. Adjust the focus and image contrast 

appropriately. Start the sample collection. Take a picture by optical microscope and 

collect an FTIR spectrum for each particle you observe. Be sure to save the particle 

image and corresponding FTIR second derivative spectrum of that particle using easily 

identifiable names. 

5.) Repeat for at least 10 particles, if available. 

Understanding Results 

Results from FTIR should inform us about the secondary structure of protein in solution or of the 

secondary structure of the protein and/or any non-protein component within the particles. 

After correcting your raw spectra, load the files and perform automatic atmospheric 

compensation (to eliminate the H2O and CO2 bands) and baseline correction (automatic 

preferred). Use the Savitzky-Golay algorithm (built into the software) to calculate the second 

derivative spectra and perform smoothing (use 9 as the number of smoothing points). Perform 

normalization of the spectra. 

Report the second derivative signal intensity over wavenumbers (from 1700 cm-1to 1500 cm-1) 

along with instrument parameters in the FTIR Data template or FTIR Microscopy Data template

for data acquired in solution (unstressed samples) or for data acquired on proteinaceous particles 

(stressed samples), respectively. If the particles imaged and analyzed are non-proteinaceous, the 

analyst should identify the particles as such and choose the appropriate spectral range for reporting 

these types of particles. For data acquired on particles, paste images of those particles above the 

corresponding fully analyzed FTIR data on the FTIR Data template. 

Further Information

For any specific questions regarding this method, please contact aapsinterlab@nist.gov.
FTIR P a g e | 5 1 November 2023