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AAPS Interlaboratory Study

Tier 1 Method: Nanoparticle Tracking Analysis

Introduction

This protocol describes Nanoparticle Tracking Analysis (NTA) of sub-micron particles present 

in various protein solutions. This document briefly describes sample handling, instrument set up, 

and data reporting of the results associated with NTA analysis. The primary reported results are 

the particle size distributions of particles in the size range between 100 nm and 1000 nm. 

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. 

These samples are time sensitive with regards to particles but depending on the time 

requirement, analyst, and instrument availability, it might be possible to do some measurements 

simultaneously (i.e. run the samples prepared at the same time on light obscuration and MFI/ 

FlowCam simultaneously). Please coordinate if it is a general practice to do these measurements 

concurrently. 

Equipment and Materials List

● NTA equipment and associated consumables (specify laser wavelengths, magnification, 

syringe pump information, autosampler, if available, etc.)

● Protein samples and formulation buffers supplied by sample originators

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

○ Stir-stressed samples from Shipping #2

● Control sample typically used by lab to verify the instrument’s performance

● Particle free/de-ionized ultrafiltered water

● Disposable 1 mL silicone-free syringes

● 0.02 μm filters, syringes, needles for filtering buffers

● Detergent or cleaning solutions for cleaning between runs

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 and stir-stressed samples 

from Shipping #2 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 Setup

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

software version available for collecting and analyzing data. Cleaning solutions and 

buffer blanks can be run to ensure the instrument is free of particles. Follow general lab 

practices to confirm the sizing and counting accuracy of the instrument (e.g. 200 nm 

polystyrene beads). 

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 NTA measurements (prepared and 

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

1 vial each of the Amgen mAb and NISTmAb pre-diluted stir stressed samples from 

Shipping #2 by bringing them to room temperature for 30 minutes.

3.) These samples are time sensitive with respect to particle concentration. After 

thawing, particle concentrations may change with time, even if the sample is maintained 

at (2-6)°C. This change may be mitigated by analyzing the samples immediately (within 

2 hours) of thawing. It is recommended to perform all control measurements before the 

protein samples are fully thawed.

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

clumping. Prior to dilution or analysis, mix each 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. 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. 
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5.) Filter the formulation buffers with a 0.02 μm filters before making any dilutions. 

6.) Under laminar flow hoods, prepare 3 mL of each sample into standard PP-tubes for NTA 

analysis by diluting the samples 100-fold into the diluent provided for each protein. Take 

care to avoid formation of air bubbles and prevent evaporation of the sample by closing 

the tubes. The pre-diluted stir stressed samples do not need to be diluted. 

Sample Measurement

1.) Review the NTA Data Reporting Template for the requested information before 

beginning the measurements. 

2.) Inject samples into the sample chamber using silicone oil-free syringes. It may be 

necessary to make further dilutions using filtered buffer to optimize the measurement 

counts for your instrument. Aim for an optimal number of particles per frame depending 

on your instrument. Too few or too many particles may prevent accurate particle 

tracking. 

3.) Measurements should be taken at room temperature (25 ̊C) with an assumed viscosity of 

8.90 x 10-4 Pa·s. Whenever possible, use automatic settings to adjust shutter, minimum 

track length, gain, and camera levels. Appropriately set the detection threshold to include 

as many particles as possible for analysis. During injection, avoid formation of air 

bubbles. 

4.) Measure each sample in triplicates (n=3) by loading new sample aliquots for every 

replicate. Between each mAb sample, run cleaning/detergent solutions to ensure there 

are no particles stuck in the sample chamber and being carried over into the next run. 

Once the sample chamber is verified to be clean, insert the next sample for analysis. 

Record all instrument parameters in the accompanying NTA Data Collection Template

Excel sheet. Adjust the final counts or concentrations based on the dilution factor. 

Understanding Results 

Results from NTA should inform us about the particle size distribution of proteinaceous particles 

spanning the size range of 100 nm to 1 μm in each of the stressed and unstressed mAb samples. 

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

type, model, and configuration (e.g., cell thicknesses, illumination, laser, etc), and settings (blur, 

MEPS, minimum track length, etc.). Export the data as recommended by the instrument 

manufacturer. 

Report instrument parameters and the average size and concentration with standard deviations into 

the NTA Data Collection Template. The final data should be adjusted for the dilution factors.

Troubleshooting 

Comment added 10/5/2023 regarding #2: A centrifugation step might be needed if large particles are 

interfering with the measurement.
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Reference: M. Hubert et al./ Journal of Pharmaceutical Sciences 109 (2020): 830-844

Particles adhering to the walls of the sample chamber can cause increases in apparent counts. 

Follow manufacturer’s recommendations for removal of stuck particles. It is important to ensure 

that the instrument is cleaned sufficiently between runs to prevent carryover from an earlier 

sample. Always confirm sample cell cleanliness with blank runs, and then recommence sample 

measurements.

Further Information

For any specific questions regarding this method, please contact Kurt Benkstein at 

kurt.benkstein@nist.gov. Please also copy aapsinterlab@nist.gov on this email.