Updated: 10-Aug-23 CE-SDS Protocol Page | 1

AAPS Interlaboratory Study

Tier 1 Method: Non-reduced and reduced CE-SDS

Introduction 

CE-SDS can be used to quantify monomeric purity, heavy and light chain relative abundance, 

and glycan occupancy of a monoclonal antibody. 

Samples can be reduced or nonreduced prior to analysis. For the non-reduced analysis, the 

native protein is denatured with SDS prior to separation to mask the protein native charges. An 

alkylating agent such as iodoacetamide (IAM) may be added to the sample to minimize disulfide 

shuffling. The non-reduced sample can help quantify fragmentation levels (partial mAbs lacking 

one or both heavy or light chains) and monomeric purity. 

For reduced analysis, the sample is treated with SDS, and either dithiothreitol (DDT) or betamercaptoethanol (BME) to reduce the native protein structure (disrupt disulfide bonds). A 

recombinant mAb can be reduced to glycosylated heavy chain (HC), non-glycosylated heavy 

chain (NGHC), and a light chain (LC). The reduced form can help to quantify heavy and light

chains, heavy chain glycan occupancy, and to determine the content of non-reducible size 

variants. Peak assignments to LMW combination of heavy (H) and light (L) chains are often 

based on their apparent molecular weight and migration relative to the monomer. 

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 (ensure chemicals are molecular biology grade and are suitable 

for electrophoresis)

1. CE-SDS separation system with a UV detector

2. SDS-MW Analysis Kit or equivalent 

3. Reducing agent (i.e. 2-mercaptoethanol (BME))

4. Alkylating agent (i.e. Iodoacetamide (IAM))

5. Protein samples and formulation buffers supplied by sample originators

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

6. Pipets and pipette tips as recommended by manufacturer.

7. Cleaning solutions or detergents to run between samples, as recommended by the 

manufacturer 

8. Particle free water/deionized ultra-filtered water or equivalent

9. 0.5 mL Micro-Centrifuge capped vials
Updated: 10-Aug-23 CE-SDS Protocol Page | 2

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 Set Up

1. Prepare the instrument and components as described by the manufacturer. 

2. Cut the bare fused silica capillary to desired length before installing it use and install it 

according to the manufacturer’s instructions. 

3. Set up the instrument with the parameters from Table 1. This table is modified from the 

paper by Turner et al.(1).

4. Prepare and run an instrument qualification standard and ensure the results are as 

expected.

Table 2: Initial conditions for CE-SDS methods, adapted from Turner et al. (Ref 1)

Parameter Setting

Auxillary data channels Current; max = 300.0 μA

Temperature (cartridge & sample 

storage) 25.0 °C

Peak detect threshold 2

Peak detect peak width 9

Electropherogram scan data rate 2 Hz

Scan range 190 to 400 nm
Updated: 10-Aug-23 CE-SDS Protocol Page | 3

Filter Normal; Peak width: 16 to 25 points

Electropherogram channel Wavelength = 220 nm; Bandwidth = 10 nm

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 CE-SDS 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. A proper mixing of the sample prior to analyzing is important to eliminate particle 

clumping. Prior to analysis, mix the aliquots in the following manner 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.

4. The mixed samples can be used for analysis but it might be necessary to spin down a few 

samples. If additional sample preparation is needed, detail it in the data sheet. 

5. The final concentration of the protein should be optimally between 0.5 mg/mL and 1 

mg/mL and the final volume for each sample should be near 100 μL (it may be a little 

higher after addition of the internal standard protein). Be sure to prepare all stocks on the 

same day as analysis. 

6. Using the reducing and alkylating agent of choice, prepare the reduced and non-reduced 

samples keeping in mind the above concentration and sample requirement. 

7. Be sure to document this information in the CE-SDS Data template. 

Sample Measurement

1. Review the CE-SDS Data template for the requested information before beginning the 

measurements.

2. Load samples in triplicates (3 injections from 1 sample prep).

3. Run all the non-reduced samples separately from the reduced samples (see sequence of 

runs on data collection sheet). 
Updated: 10-Aug-23 CE-SDS Protocol Page | 4

Understanding Results 

Data analysis

1. Analyze the electropherograms using the software that accompanies the instrument. 

Migration times and corrected peak areas should be recorded for the non-reduced and 

reduced sample analysis. 

2. See Table 3 below that describes the data that should be recorded and calculated. 

Additional data can be recorded, as needed. 

Table 3: Measured and calculated assay results for CE-SDS analyses (modified from paper by 

Turner et al.)

Sample Measurand Needed Calculated

Quality Parameters

Nonreduced 

samples

10 kDa internal standard migration time (min) 

Monomer migration time (min)

Corrected area of monomer

Corrected area of fragment peaks: L, H, H:L, H:H, 

H:H:L, clip species

Monomeric purity (%)

Reduced 10 kDa internal standard migration time (min) 

Migration time (min) of L and H

Corrected area of L, NGH, H, and thioether

Heavy chain 

relative abundance 

(%) Light chain 

relative abundance 

(%)

Glycan occupancy (%) 

*Thioether relative 

abundance (RA) (%)

L = light chain; H = heavy chain; H:L = heavy chain:light chain fragment; H:H = heavy 

chain:heavy chain fragment; H:H:L = heavy chain:heavy chain:light chain fragment; 

NGH = aglycosylated/non-glycosylated heavy chain

Data Reporting

Instrument parameters used, measured, and calculated data should be entered in the CE-SDS 

Data reporting template. If any corrections to any data are performed, report the magnitude and 

basis of the corrections.

Troubleshooting 

For any errors associated with failed system suitability tests, low or unsteady current, broad 

peaks and poor resolution, check the instruction manual of your CE-SDS system. 
Updated: 10-Aug-23 CE-SDS Protocol Page | 5

Appendix 

Use the equations below for the calculations:

Monomeric purity (%) = 

CA(monomer)

CA(monomer)+ ƩCA(fragments)

× 100%

Glycan Occupancy (%) of heavy chain = CA(Heavy)

CA(Heavy )+ CA(Nonglycosylated Heavy)

× 100%

Heavy Chain RA (%) = CA(Heavy)

CA(Light )+CA(Heavy)+ CA(Nonglycosylated Heavy)+CA(Thio)

× 100%

Light Chain RA (%) = CA(Light)

CA(Light )+CA(Heavy)+ CA(Nonglycosylated Heavy)+CA(Thio)

× 100%

Thioether RA (%) = CA(Thio)

CA(Light )+CA(Heavy)+ CA(Nonglycosylated Heavy)+CA(Thio)

× 100%

Further Information

For any specific questions regarding this method, please contact Katharina Yandrofski at 

katharina.yandrofski@nist.gov. Please copy aapsinterlab@nist.gov on your email. 

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.