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AAPS Interlaboratory Study

Tier 2 Method: Asymmetric-Flow Field-Flow Fractionation

Introduction

Asymmetric flow field-flow fractionation (AF4) is a technique that allows for separation and 

quantification of monomers, dimers, trimers, and higher order aggregates.

This protocol briefly describes a method to employ asymmetric flow field flow fractionation 

(AF4) to characterize submicrometer particles. It can also be used to separate monomer, dimer, 

and soluble aggregates in protein solutions as an orthogonal technique to SEC, with the key 

difference being the reversed elution order of species compared to SEC. For this study, the 

primary method describes submicrometer characterization and the reported results should be 

geometric radius (hydrodynamic radius if in-line DLS is installed); this is required data from the 

participants. Participants may also report quantities such as quantities (%) of various species 

(fragment, monomer, dimer, and aggregated species), and weight average molecular mass but 

this is optional. 

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 Material List 

• AF4 system (including separation channel, membranes, etc.) including necessary fluidic 

system that may include pump(s) to run AF4, degasser, sample injection/autosampler, 

UV detector, MALS detector, DLS in-line (if equipped)

• Mobile phase (identical to the SEC mobile phase)

• Standards as recommended by the manufacturer 

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

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

• Pipets and pipette tips as recommended by the manufacturer.

• Particle free water/deionized ultra-filtered water

• Autosampler vials and caps
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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. 

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 

mM L-histidine HCl, pH 6.0

Unstressed, 1 mg/mL

Stir stressed, 1 mg/mL

Light stressed, 1 mg/mL

Procedures

The AF4 conditions to be used for each molecule are summarized in Table 2 below. The primary 

method describes the set-up to obtain information regarding the submicrometer content in each 

sample. The secondary, or optional, method describes the set-up to obtain information regarding 

the soluble aggregate content in each sample. Depending on the channel design, membrane, 

spacer, and flow conditions, it is possible to achieve suitable separation under a variety of 

conditions. The subsequent instructions assume the conditions have been optimized by the sample 

providers to achieve good separation between the species within the samples. 
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Table 2: Recommended AF4 conditions for each mAb(1)

Primary Method (Submicrometer Particles) Secondary Method (Soluble Aggregates)-Optional

NISTmAb Amgen mAb NISTmAb Amgen mAb

Mobile Phase 25 mM His/L-His 

HCl, pH 6.0

100 mM sodium 

phosphate + 250 mM 

NaCl, pH 6.8

25 mM His/L-His HCl, pH 

6.0

100 mM sodium phosphate 

+ 250 mM NaCl, pH 6.8

Channel Type Short Short Short Short

Spacer Thickness 250 or 190 μm 

Thick Mylar 

250 or 190 μm 

Thick Mylar 350 μm Thick Mylar 350 μm Thick Mylar

Membrane Type/MWCO 10 kDa Regenerated 

Cellulose

10 kDa Regenerated 

Cellulose

10 kDa Regenerated 

Cellulose

10 kDa Regenerated 

Cellulose

Detector Flow (mL/min) 1.0 1.0 1.0 1.0

Focus-Inject

Position 12% channel length 12% channel length 12% channel length 12% channel length

Flow-Rate (mL/min) 0.5 0.5 1.5 1.5

Duration (min) 3.0 3.0 5.0 5.0

Separation

Crossflow Initial (mL/min) 0.5 0.5 3 3

Crossflow Final (mL/min) 0 0 0 0

Ramp Type and Time (min) Linear, 45 min Linear, 45 min Linear, 45 min Linear, 45 min
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Prepare the AF4 system for experiments

This protocol assumes the HPLC/fluidics system driving the FFF separator is in good working order and 

equilibrated with the desired mobile phase. Ensure all inline filters, pump frits, and fluid lines are clean 

and free of microbial growth. Ensure that the channel membrane has been conditioned and the AF4

system pressures are normal as specified by the manufacturer.

1. Prepare at least 2 L mobile phase. Filter the mobile phase to 0.1 μm into a clean bottle using a 

bottle top filter (0.1 – 0.2 μm) that has been pre-flushed to a waste bottle with ~100 mL ultrapure 

water or mobile phase to remove particles. Maintain sterility but change mobile phase every few 

days and replacing the bottle instead of topping off with mobile phase. 

a. Mobile phase for NISTmAb: 25 mM Histidine buffer, pH 6

b. Mobile phase for Amgen mAb: 100 mM sodium phosphate + 250 mM NaCl, pH 

6.8

2. Install a new membrane in the channel, follow the manufacturer guidelines for disconnecting the 

channel from the system, disassembling the channel, installing new membranes or other 

consumables, reassembling the channel, and subsequently purging, conditioning, or otherwise 

preparing the channel for experiments. 

3. Membranes should be passivated by optimally running a large quantity of BSA in PBS buffer 

(Recommended: 7.5 mg BSA at 10 mg/mL) at a low detector flow rate (Recommended: 0.25 

mL/min) with a high constant cross flow (Recommended: 3.0 mL/min on 350 μm WS; 2.0 

mL/min on 250 μm WS) for 20-30 min to passivate the membrane. If an alternative membrane 

passivation procedure is implemented, please include details in your report. 

4. The crossflow should be terminated and the detector flow should be reduced to 1.0 mL/min (or 

whatever the run conditions will be) and the detectors allowed to equilibrate under the rinse.

5. Continue flowing to waste until detector signals are equilibrated, and air bubbles and other 

contaminants have been flushed out. The detector signals can be monitored to assess the current 

state of equilibration. 

6. (Optional) dRI detection may be added downstream for quantifying absolute molar mass using 

light scattering. Purge or flush all optional detectors according to manufacturer specifications. 

7. After setting up the system, conditioning the channel, and equilibrating the channel and detectors, 

perform at least one standard injection to validate the instruments. Determine normalization, 

alignment, and band broadening corrections per the manufacturer’s recommendation. Save these 

parameters to the method to be used for data analysis. Recommended quality control is BSA in 

PBS mobile phase at 10 mg/mL with a total injection mass of at least 50 μg to not more than 200 

μg to assess peak shape, quantitative recovery, and normalization constant accuracy. Quantitative 

recovery of the injected BSA standard should be > 80 % of injected mass. Following this, if all 

parameters look okay then the system can be purged and flushed with the corresponding buffer 

listed above for each mAb molecule. 
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Perform separations for analysis

1. Use the method created or the system parameters from “Prepare the AF4 system for experiments” 

for data collection. Follow the manufacturer recommendations for method creation, including 

appropriate flow equilibration steps, duration for focus or injection steps, and other method 

requirements for executing the AF4 method. The total experiment time should be long enough to 

ensure that all sample signals elute and may include additional flushing steps without cross flow 

to reduce carryover peaks into the next injection as per manufacturer recommendations.

2. Set up runs for each sample according to Table 2 for appropriate flow rates for focus or injection 

steps. Refer to your manufacturer’s instructions for setting up injections and sequences. 

3. Perform at least one blank (mobile phase) injection and at least one standard injection (e.g., BSA) 

at the beginning of the sequence to assess system suitability and identify possible system peaks. 

Ensure that the results obtained from these runs are consistent with expectations before 

proceeding to inject the mAb samples. Mobile phase or standard injections may be repeated at 

the end of the sequence to assess system drift.

4. Perform 3 injections of each experimental sample. Blank injections can also be acquired for 

baseline correction of the RI signal, if used, to improve the concentration determination. 

5. Process the data according to the analysis software manufacturer’s instructions. Include the 

following information listed below in the analysis. Ensure the boundaries of each peak extend 

from baseline-to-baseline or from inflection point to inflection point if baseline-resolution is not 

achieved. 

Primary method (required), see 1st tab of AF4 Data Reporting Template:

● geometric radius (nm)

Secondary method (optional), see the 2nd tab of the AF4 Data Reporting Template:

● High Molecular Weight-HMW (dimer and higher)

● Monomer

● Low Molecular Weight (LMW)

6. Apply baseline subtractions, despiking, and alignment to your sample runs using the blank runs 

with an identical method to improve the RI detector signal, using your manufacturer’s 

instructions. This can improve the accuracy of the molar mass calculations or concentration from 

RI detectors.

7. For each peak, determine the data listed below. Calculate the average and standard deviation (% 

CV) among the three injections of each sample and input the results in the appropriate cells 

included in the AF4 Data Template.

Primary method (required):

● Geometric radius (nm) at elution time (at peak max)

● UV Peak Area
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● UV % Peak Area (=Peak Area/Total Area)

Secondary method (optional):

● UV Peak Area

● UV % Peak Area (=Peak Area/Total Area)

● Weight-average molar mass (Mw) by MALS

● Optional: RI Peak Area and RI % Peak 

Understanding Results 

The concentration of eluting species can be determined using the UV and MALS detector. In the 

AF4 Data Reporting Template, specify the detection method. You may use the software 

available with your instrument for calculating the areas of the various peaks (hydrodynamic 

radius, fragment, monomer, and high molecular weight species, etc.). Ensure the buffer 

fractograms are subtracted from the sample (if they contribute to the signal) prior to calculating 

the peak areas. While you do not have to report mobile phase runs or standard BSA runs, please 

ensure that you perform them and that the results are consistent with expectations before running 

the protein samples. Be sure to save all raw data so they could be retrieved in the future, if 

needed. 

Further Information

For any specific questions regarding this method, please contact Sophia Kenrick at 

skenrick@wyatt.com and Sean Lehman at sean.lehman@nist.gov. 

Appendix 

Table 3: General recommendations for good AF4-MALS data quality

Settings Recommendations

General a) Change the HPLC pump frit according to the manufacturer’s specification.

b) Fully flush the sample loop, needle seat, and injection needle.

c) Make sure your sample is fully in solution. Sample filtration may be 

necessary.

Mobile Phase a) Use a clean solvent reservoir.

b) Filter mobile phase, particularly when containing salt.
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c) Suppress bacterial growth (antibacterial agent or frequent mobile phase 

change).

d) Clean contaminated HPLC system according to the manufacturer’s 

recommendation.

e) Use an inline filter between pump and autosampler/injector.

AF4 Separator 

& Channel 

Considerations

a) Follow manufacturer recommendations for replacing frits, filters, and other 

consumable components.

b) Observe system and channel pressures to assess the performance per 

manufacturer guidelines. 

c) Replace the membrane at least once a month but often more frequently 

depending on fouling or performance with standard. 

d) Condition new membranes with a suitable standard. 

e) Ensure adequate flushing, conditioning, and purging of the system for 

optimal signal baselines.

Reference(s)

1. Lehman SE, Karageorgos I, Filteau JR, Vreeland WN. Effect of Azide Preservative on 

Thermomechanical Aggregation of Purified Reference Protein Materials. J Pharm Sci. 2021;110(5):1948-

57. doi: 10.1016/j.xphs.2021.01.013.