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AAPS Interlaboratory Study

Tier 1 Method: Size Exclusion Chromatography with UV Detection (and optional MALS)

Introduction 

This protocol provides a brief procedure to measure size variants in mAb samples (unstressed 

control and stressed) using size-exclusion chromatography (SEC) with UV and an optional 

multi-angle light scattering (MALS) detection. The primary reported results are the percentages 

of the various size variants as calculated by UV detection (and mass of size variants, if MALS is 

used). 

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

● HPLC system with UV detectors (RI and MALS detectors, optional), degasser, pump, 

injector, guard column, column, column oven, data acquisition and analysis software

● Mobile phase recipes (provided by sample providers)

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

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

● Autosampler vials and caps and micro-inserts

● Pipette and pipette tips

● Bottle top filter (0.1 μm or 0.2 μm)

● Bovine serum albumin as suitability standard or for conditioning a new column, as 

required

● Molecular weight standards, if required
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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 

mMol L-histidine HCl, pH 

6.0

Unstressed, 1 mg/mL

Stir stressed, 1 mg/mL

Light stressed, 1 mg/mL

Procedures

Instrument Preparation

1. Set up HPLC with appropriate column and guard column and ramp up to the desired flow 

rate slowly. 

2. Input HPLC, detector, system suitability standards, and other information into the 

appropriate cells of the SEC Data reporting template.

Sample Preparation and SEC Separation

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 SEC/SEC-MALS measurements

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

minutes. It is recommended that these samples be analyzed within 2 hours of thawing. 

3. Prior to analysis, mix each aliquot in the following manner: pipet with a 1000 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. Review SEC Data reporting Template for the requested information before beginning the 

measurements and input set up information.

5. Set up runs for each sample according to Table 2 (flow rate, injection volume, run duration, 

etc.), which have been provided by the protein suppliers. Perform the runs according to the 

instrument manufacturer instructions and instrument familiarity. Avoid starting/stopping 
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pump between injections or sequences by recirculating mobile phase at constant flow rates. 

Set sample rack/autosampler temperature to (4 to 10)  ̊C. 

6. Perform at least one blank (mobile phase or formulation) 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. 

7. Depending on the column used, some samples for SEC injection may need to be 

concentrated if the starting concentration of 1 mg/mL is too low. If this is the case, Amicon 

Ultra 10K centrifugal filters or equivalent can be used for concentrating the sample. Refer 

to the manufacturer’s instructions on the use of these filters. After spinning the sample, 

remeasure the concentration of the filtrate and inject (50-100) μg depending on the column 

size. You can use theoretical extinction coefficient of 1.42. 

8. Perform 3 injections of each sample and corresponding buffer. 

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

following peaks 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. 

● High Molecular Weight (dimer and higher)

● Monomer

● Low Molecular Weight

10. For each peak, determine the data listed below. Calculate the average and standard 

deviation among the three injections of each sample and input the result in the appropriate 

cells of the SEC Data reporting template. 

a. UV Peak Area

b. Relative peak area (%) (peak area/total area) *100

c. Optional: weight-average molar mass (Mw) by MALS

11. Standard and mobile phase injections may be repeated at the end of the sequence to assess 

system drift. 

Updated Comment (10/18/2023):

“filtrate” above should be retentate

You may use theoretical extinction coefficient of 1.42 for NISTmAb and 1.539 for 

Amgen mAb.
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SEC Conditions

The SEC conditions for each molecule are summarized in the table below. This protocol 

assumes the conditions have been optimized to achieve good separation between monomer and 

aggregates and to reduce column interactions. 
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Table 2: SEC conditions previously used for each mAb (provided by the originator labs) from published papers. Your parameters 

might be a little different based on your column and instrument set-up. 

mAb Column Mobile Phase

UV 

Detection Column 

Temp. 

(°C)

Run 

Duration 

(min)

Flow 

Rate Injection 

wavelength (μg)

(nm) (mL/min)

NISTmAb(1)

UPLC Protein BEH 

1.7 μm particle size, 

200 Å pore size, 4.6 

mm × 150 mm 

length

100 mM sodium 

phosphate + 250 

mM NaCl, pH 

6.8

280 25 10 0.3 60

Amgen IgG2(2)

HPLC, Tosoh 

Bioscience TSK-Gel 

G3000SWxl (7.8 x 

300 mm, 5 μm 

particle size)

100 mM sodium 

phosphate + 250 

mM NaCl, pH 

6.8

280 25 35 0.5 100
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(Optional ) If using MALS detection in addition to UV detection, follow the steps below 

Addition of multi-angle light scattering (MALS) and differential refractive index (RI) detection

MALS and RI detection may be added downstream of virtually any SEC chromatograph for 

quantifying absolute molar mass using light scattering. 

1. After setting up the SEC-MALS system and equilibrating the column and detectors, perform 

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

alignment, and band broadening corrections according to manufacturer recommendation.

2. Introduce samples to perform SEC separation and collect MALS data using the appropriate 

software for the MALS detector. If available, RI may be used to quantify species.

3. Analyze data using instrument parameters established earlier. Define baselines and peaks to 

assess molar mass and quantify species (i.e. monomer and each oligomer population). Add 

measurement parameters and analyzed results to SEC Data reporting template.

Understanding Results

The concentration of eluting species can be determined using the UV detector (MALS detectors 

are optional). In the SEC Data reporting template, specify the detection method. You may use 

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

molecular weight, monomer, high molecular weight species). Ensure the buffer chromatograms 

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 of the raw data so they could be retrieved in the future, if 

needed. 

For integration, the main peak corresponds to the monomeric peak of the untreated sample. High 

molecular weight (HMW) peaks include everything in the range between the excluded volume 

and the start of the main peak, and low molecular weight (LMW) peaks include all peaks up to 

excipient buffer peak.

Troubleshooting

The protocol assumes the optimal method for resolving monomers and oligomers is being used 

for each molecule. An overview of common issues is given in Table 3, with additional details 

given below.

Table 3: Troubleshooting overview

Problem Probable Cause Resolution

Baseline Noise 

and Drift

Dirty detector flow cell Clean flow cell (try in-situ cleaning 

first).
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Dirty HPLC system Clean HPLC system per manufacturer’s 

recommendation.

Refractive index detector needs 

purging

Purge RI detector for at least 1 hour 

before running experiments.

Column shedding or sample 

adsorption on the column

Clean or replace column.

Excessive Peak 

Broadening

Aging or contaminated column Clean or replace column.

Mobile phase contamination or 

deterioration

Replace mobile phase, use a clean 

solvent reservoir and solvent filter, and 

clean HPLC and detectors if necessary.

Slow leaks in the HPLC system 

or inside detectors

Locate leaks, clean spills, and replace 

tubing and fittings as necessary.

Excessive tubing length or 

tubing I.D. too large

Reduce tubing length and use 

recommended tubing I.D.

References

1. Turner A, Yandrofski K, Telikepalli S, King J, Heckert A, Filliben J, et al. Development 

of orthogonal NISTmAb size heterogeneity control methods. Anal Bioanal Chem. 

2018;410(8):2095-110. doi: 10.1007/s00216-017-0819-3.

2. Joubert MK, Luo Q, Nashed-Samuel Y, Wypych J, Narhi LO. Classification and 

characterization of therapeutic antibody aggregates. J Biol Chem. 2011;286(28):25118-33. doi: 

10.1074/jbc.M110.160457.

Further Information

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

skenrick@wyatt.com and Katharina Yandrofski at katharina.yandrofski@nist.gov. Please copy 

aapsinterlab@nist.gov on your emails.