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AAPS Interlaboratory Study

Tier 2 Method: Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC)

Introduction

This protocol describes measurement of size distribution by sedimentation velocity analytical 

ultracentrifugation for samples diluted to within the linear absorbance range (0.2-1.2 O.D.) of the 

instrument. The results of the measurements are reported as relative abundances and 

sedimentation coefficients for each species present. 

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 Materials List 

● Beckman Coulter XL-A, XL-I, or Optima AUC

● An50Ti (8 hole) or An60Ti (4 hole) titanium rotor

● Assembled analytical sample cell

a. Centerpiece

i. 12 mm epon charcoal-filled centerpiece for low sample concentrations 

(customary)

b. Window assembly

i. Quartz or sapphire (recommended)

ii. Liner

iii. Window gasket

iv. Window holder

c. Cell housing

d. Hex screws

e. Neoprene gaskets

● Alignment tool

● Pipets and pipette tips

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

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

Reagents and Solutions 
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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

Procedure

1. Prior to sample preparation ensure the AUC chamber and rotor are equilibrated to the run 

temperature (20 °C). This will take approximately 1 hour.

2. Thaw the samples from Shipping #1 and aliquot the Tier 2 vials (for the unstressed, stir, 

and light stressed mAb samples) as described in the instructions into smaller aliquots for 

the various methods. Be sure to follow the procedure for vigorously mixing the sample 

prior to aliquoting. 

3. For this analysis, thaw the aliquots designated for SV-AUC measurements (prepared and 

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

4. Prior to analysis, mix the aliquot in the following manner: pipet with 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 into the appropriate 

container for analysis. 

5. If the sample is not transparent/clear visually, use adequate centrifugation to remove any 

suspended particles. 

6. Prepare approximately 450-500 μL of sample for each replicate (three recommended) and 

have identical volumes of the appropriate buffer/diluent. For absorbance optics, a target 

O.D. of 0.5-0.8 is recommended, although O.D. of 0.2-1.2 are acceptable. For OD 

measurements, analysts can refer to the UV Visible Spectroscopy protocol. 

7. Obtain a clean assembled AUC cell with a double sector centerpiece. Verify the 

assembled cell has been torqued to at least 120-inch pounds. For absorbance detection 

either sapphire windows or quartz windows may be used. 

8. Load approximately 480 μL of buffer and sample into the appropriate sectors. 

a. For absorbance optics, it is recommended that approximately 20 μL less sample 

volume be loaded to offset the meniscus. 
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9. Seal the sector filling holes with a neoprene gasket and hex screw.

10. Obtain a pre-temperature equilibrated rotor with counterbalance. Verify a counterbalance 

weighs within ± 0.5 g of the sample cell directly across.

11. Ensure that the assembled cells have balanced weight such that the rotor will be balanced 

once all cells are loaded.

12. Load the assembled cells into the rotor and align cells to the center of rotation using an 

alignment tool.

13. Place the rotor into the AUC chamber and turn on the vacuum. Once the vacuum is below 

100 μm Hg, allow the sample loaded rotor to equilibrate for at least 30 min.

14. If desired, a check at 3000 rpm may be performed prior to the actual run to verify proper 

sample loading and cell assembly. This is to check for proper sample absorbance and to 

ensure that none of the cells are leaking. 

15. Instrument run parameters used are as follows:

Parameter Value

Angular velocity (rpm) 40,000

Temperature (°C) 20

Detection (nm) Absorbance at 280

Rmin (cm) 5.8

Rmax (cm) 7.05

Radial scan increment (cm) 0.003

Replicates 1

Mode Continuous

Delayed start 0

Time between scans 4 min

Number of scans per cell 100

16. After the run is complete the cells must be removed and cleaned manually

a. Disassemble cells and carefully place centerpiece and windows in a small plastic 

beaker with 1% Hellmanex to cover the materials. Warm the solutions in a 

microwave, then sonicate for 30 min.

b. Remove centerpiece and windows from the beaker and rinse with copious water. 

Final rinse must be with high purity water (such as Milli-Q, HPLC grade).

c. Dry centerpiece and windows.

d. Reassemble the clean dry cell. Optionally add Spincote to the screw ring. Do not 

tighten the screw ring with the torque wrench at this time.
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17. Data analysis is performed in SEDFIT v.16.1c software package using the following 

parameters:

Parameter Value

Model Continuous c(s)

Meniscus (from center of rotation) (cm) Flot from max sample of side spike (cm)

Bottom (from center of rotation) (cm) 7.2 (fixed)

Top fitting limit (from center of rotation) (cm) +0.1 from meniscus

Bottom fitting limit (from center of rotation) (cm) 7.0 or 6.7

Frictional ratio 1.6 (float)

Baseline Float

RI noise Disabled

TI noise Enabled

Resolution 200, log spaced

Smin (S) 0.1

Smax (S) 40

Confidence level (F-ratio) 0.68

Scans included ~60 for full 8-hole rotor

Protein specific volume (mL/g) 0.73

Buffer density (g/cm3) 1.00000

Buffer viscosity (P) 0.01002

Data should be entered in the provided SV-AUC template. If data was not obtained for any 

peaks listed on the templates, enter “NA” in the cells for that peak.

Most users will report c(s) distributions as generated by the data analysis software to be used as 

xy data for visual fitting. 

Further Information

For any specific questions regarding this method, please contact Nikki Machalek at 

NMachalek@KBIBiopharma.com. Please copy aapsinterlab@nist.gov on your email.