Instructions for AAPS Interlab Study Page | 1 27 August 2023

AAPS Interlaboratory Study-Instructions

Main Contact: Srivalli Telikepalli (aapsinterlab@nist.gov) 

1. Overall Objective

The goal of this study is to characterize protein degradants of two stressed monoclonal 

antibodies, an IgG1 and an IgG2, in multiple labs using particle counting/characterization, liquid 

chromatography, mass spectroscopy, electrophoresis, and spectroscopy tools and examine assay 

variability. The originator labs will generate and distribute the samples and provide a 

harmonized set of analytical protocols to the participants. 

2. Participants’ Roles 

Participants will receive samples of unstressed and stressed mAbs supplied by Amgen (Shipment 

#1) and NIST (Shipment #2), aliquot them based on the methods their organization will perform 

for Shipment #1 samples or analyze them directly for Shipment #2 samples. They will provide 

the results back to NIST using the data reporting templates provided. NIST will anonymize the 

data and assess variability of the data among the labs. 

Participant responsibilities:

1. Participants should read and understand the goals of the study. Please reach out to

Srivalli Telikepalli (aapsinterlab@nist.gov) for any questions or clarifications. 

2. Upon receival of the two packages, notify Srivalli Telikepalli (aapsinterlab@nist.gov) 

and Kimya Nourbakhsh (at knourbak@amgen.com) the condition of the packages and 

samples (thawed, broken, leaking vials, etc.) when received. Shipment #1 will arrive 

from Amgen and Shipment #2 will arrive from NIST. 

3. If the samples are received in good condition, participants can thaw and aliquot the 

samples (as shown in Figure 1) or directly analyze the samples as appropriate for the 

sample type. There are two sets of samples, and the working lifetime of the samples 

when thawed is limited—please read the instructions carefully before aliquoting and 

planning your measurements. 

4. Participants will evaluate the samples by multiple methods, (see Table 1 for the methods 

for each lab), as previously determined by their organization/PI. They will be requested 

to follow the method protocols supplied to them (if available) and record method 

parameters and results as fully as possible in the accompanying data collection templates. 

5. Once all the data has been collected, finalized, and reviewed, 1 participant from each lab 

can collate the data collection sheets for all the methods and email them to 

aapsinterlab@nist.gov. It is strongly recommended that the data entry is cross-checked 
Instructions for AAPS Interlab Study Page | 2 27 August 2023

by a second person before submitting the data. The data will be anonymized after it has 

been received by NIST. Data should be returned 3 months after receipt of the samples. 

6. Participants are requested to retain all raw data for the duration of this study. 

7. Some participants have volunteered to be the subject matter experts (SME) for specific 

methods (see the bottom of each protocol). Those participants may be contacted if a 

question arises regarding a specific method. Anonymized questions and SME answers 

will be distributed to all participants.

3. Important Considerations When Planning Experiments

1. Samples are time sensitive. Time the data acquisition accordingly so that once samples 

are thawed, they can be immediately aliquoted or analyzed. It is important to set up the 

instrument and perform any required control measurements before the samples are 

thawed. In general, to save time, run all buffers and controls before the protein samples 

are fully thawed. Record all method parameters and data in the accompanying data 

collection templates. 

2. Perform Tier 1 methods first. Tier 1 methods are the top priority of the study. 

Complete the Tier 2 methods once all the Tier 1 data has been acquired successfully. 

3. Balance guidelines provided in protocols with general, routine practices in your 

organization. If any step in the protocols is inconsistent with your routine practices, 

reach out to aapsinterlab@nist.gov. Remember, the protocols are not meant to be 

Standard Operating Procedures but aim at aligning key measurement parameters and data 

output while allowing the researcher to follow their routine workflow and operate with 

compatible instrumentation available in their laboratory. 
Instructions for AAPS Interlab Study Page | 3 27 August 2023

Table 1: Participating labs and the methods they will complete. The first row shows the number of replicates suggested for each 

method. The approximate amount of material allotted (in μL) is shown for each method and per lab (for a 1 mg/mL mAb). All 

methods will be run on unstressed and stressed material; CG-MALS, DSC, and DSF will only be performed on unstressed material. 

Abbreviations: VI - visual inspection; LO – light obscuration; NTA – nanoparticle particle tracking analysis

VI DLS LO MFI FlowCam

(SEC)/SECMALS NTA UV-Vis

Near 

UV CD FTIR

rCE-SDS 

and/or 

nrCESDS

cIEF or 

IEC

batch 

SLS 

AF4/H

F5

Intrinsic 

fluor.

Extrinsic 

fluor. RMM SV-AUC

CGMALS DSC DSF

# of replicates 1 3 2 3 3 3 3 1 1 1 1 3 3 3 1 1 3 1 1 1 1

Participating Labs

AstraZeneca 1 vial 1500 150 180 180 750 75 1000 75 500 100

Amgen 1 vial 1500 150 750 75 500 1000 300 1500 500 100

BMS 1 vial 1500 150 180 750 500 1000 300 1500 1000

Compassion 180 3000 100

Coriolis 1 vial 1500 150 180 180 750 75 500 1000 300 600 900 1500 75 1000 400 100

FDA 1500 180 3000

GSK 1500 180 750 1000 1500 75

Lonza 1 vial 1500 150 180 180 750 75 600 1500

Merck 1 vial 1500 150 180 750 1000 900 500 100 100

NIST 1 vial 1500 150 180 180 750 75 500 300 600 900 1500 75 500

Novartis 1 vial 1500 150 180 180 75 75

Pfizer 150 180 300

Sanofi-Aventis 1 vial 1500 150 180 750 75 500 600 900 100

Leiden University 1500 150 180 180 750 75 500 1000 1500 500 500 75

KBI Biopharma 1 vial 1500 150 180 750 75 600 75 1000 500

Univ of Colorado 1500 180 180 75

Univ of Copenhagen 500 1000 300 500 1000

Wyatt 1500 750 1500 1500 5000

Tier 1. Highest Priority Methods Tier 2: Sizing/Characterization

Clarification added Sept 20th regarding number of replicates for LO and Flow Imaging Methods:

Shipment #1: 2 replicates for LO, and 3 replicates for Flow Imaging; both degassed and non-degassed (follow MFI protocol for sample dispensed volume) – No Change

Shipment #2: 1 replicate for LO, and 3 replicates for Flow Imaging; both degassed and non-degassed (follow MFI protocol for sample dispensed volume)

Clarification added Oct. 4th:

Perform triplicates for non-reduced/reduced CE-SDS and DSF if possible. 
Instructions for AAPS Interlab Study Page | 4 27 August 2023

4. Samples

Participants will receive 2 separate shipments of samples for analysis; both shipments will be 

sent frozen. Please store at −70 °C or below upon receipt. 

 Shipment # 1 

Shipment 1 will contain approximately 14 vials (some labs might receive more vials 

depending on how much material they initially requested) containing unstressed and stressed. 

NISTmAb IgG1 and Amgen IgG2 (see Table 2) along with corresponding buffers. Each 

sample type will have 2 vials, filled with nominally 0.2 mL to 4.5 mL of sample in a 5 mL 

cryovial. The 2 vials will be for Tier 1 methods (highest priority methods) or for Tier 2 and 

other methods (see Table 1 for the methods that fall under the Tier 1 or Tier 2 categories). 

The values in Table 1 represents the maximum amount of material allotted for each method. 

These vials will be labeled with the following information: protein, type of stress, Tier #, 

nominal volume, nominal concentration of 1 mg/mL, and lab name. 

Table 2: Samples in Shipment 1

 Shipment # 2 

The second shipment will contain a second, prediluted set of stir stressed samples only for 

the Amgen mAb and NISTmAb along with 8 sterile 2 mL glass vials and stoppers. Ten 15 

mL conical tubes will contain approximately 5 mL of prediluted (100 fold diluted) stir 

stressed samples (5 tubes for stirred NISTmAb and 5 tubes for stirred Amgen mAb). These 

samples are intended to be used primarily for the Tier 1 particle methods (visual 

inspection, light obscuration, 2 types of flow imaging, nanoparticle tracking analysis). They 

can also be used for RMM, if selected by labs (see Table 1 above). These vials will be 

NISTmAb Amgen mAb

Buffer Buffer

Unstressed, Tier 1 Unstressed, Tier 1

Unstressed, Tier 2 Unstressed, Tier 2

Stress 1-Tier 1 Stress 1-Tier 1

Stress 1-Tier 2 Stress 1-Tier 2

Stress 2-Tier 1 Stress 2-Tier 1

Stress 2-Tier 2 Stress 2-Tier 2

Clarification added Sept 20th regarding naming of stress conditions:

Stressed samples are labeled as Stress 1 or Stress 2

Stress 1 refers to the stir stressed samples

Stress 2 refers to the light stressed samples
Instructions for AAPS Interlab Study Page | 5 27 August 2023

labeled with the protein, type of stress, 100-fold dilution, methods it can be used for, 

preparation date, and nominal volume of 5 mL. 

Participants may perform multiple assays from the same thawed vial (i.e. perform flow 

imaging simultaneously as light obscuration) or thaw multiple vials for multiple assays, but 

this needs to be coordinated based on analyst and instrument availability and the general 

practices of the lab. Regardless, because of the time sensitivity of the samples, analysts 

should consider time as an important factor when performing these particle measurements on 

these samples. 

Analysts will need to prepare samples for visual inspection using the 2 mL vials provided. 

5. Formulations 

• Buffers supplied have been filtered through 0.22 μm PVDF filters prior to being filled in 

clean vials. The participants are encouraged to re-filter the buffers prior to use.

• The NISTmAb formulation buffer is composed of 25 mM histidine buffer, pH 6. 

• The Amgen mAb dilution buffer is composed of 10 mM Acetate buffer, pH 5. 

6. Aliquoting of Samples

Shipment 1 - After receiving the 1st shipment, thaw the Tier 1 and Tier 2 samples by placing the 

vials upright at room temperature for no more than 30 minutes, aliquot, and refreeze. Aliquot the 

samples in a laminar airflow hood according to the scheme shown in Figure 1 based on the 

assays being performed by your lab (see Table 1). 

Important notes regarding Figure 1: 

1) Some participants will receive vials that are filled with 4.5 mL sample in a 5 mL capacity 

vial. Quick introduction of the pipet into these filled vials will cause the sample to spill 

over so be careful when inserting the pipet into these vials for mixing. Be sure to depress 

the plunger while placing it into the vial so the sample can be pipetted up just as the pipet 

tip goes into the vial. 

2) Remember to thaw the samples right before aliquoting or before analysis because of the 

tendency of the particles to reverse. Samples sitting in the formulation buffer for more 

than 2 hours reverse, resulting in a decrease in particle concentration. 

3) A proper mixing of the samples prior to aliquoting or analyzing is important to eliminate 

particle clumping. To “mix” the sample, do the following: gently invert vial 10 times, 

pipet with 1 mL tip (or 200 uL tip if removing the sample from a 1 mL vial) near the 

bottom of the sample tube 10 times (see note #1) 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. 
Instructions for AAPS Interlab Study Page | 6 27 August 2023

Shipment 2 - The samples in the 2nd shipment do not need to be aliquoted and should remain 

frozen until ready for analysis. Prior to analysis, these samples should be mixed in the same way 

as described above in #3. 

Clarification added Sept 20th regarding unavailable analytical protocols:

Not all protocols are provided (i.e cIEF). For the methods that do not have a written protocol, participants should 

follow standard methods used in their labs to perform the measurements. Since there is also no data collection 

form associated with these measurements, participants should report important instrument parameters and final 

data by using one of the data collection forms as a template. 
Instructions for AAPS Interlab Study Page | 7 27 August 2023

Figure 1. Aliquoting scheme for Tier 1 and Tier 2 vials from Shipment 1. The approximate volume of the sample to be aliquoted for 

each assay is shown below each method (the range shown for some methods are provided by the labs, see Table 1). CG-MALS, DSC, 

and DSF will only be performed on unstressed material. Based on the sample and method, some aliquots may require dilution (100 

fold) or may require a brief centrifugation after thawing for analysis. 

Abbreviations: LO – light obscuration; VI – visual inspection; IF – intrinsic fluorescence; EF – extrinsic fluorescence. 

Clarification added Sept 20th regarding sample preparation of NTA:

After thawing and dilution, NTA samples may need to be briefly 

centrifuged to remove large particles prior to analysis. 
Instructions for AAPS Interlab Study Page | 8 27 August 2023

7. Data Collection 

Follow the method protocols, if available, to collect the data. Perform Tier 1 methods first with 

the sample allotted for Tier 1, then follow with Tier 2 methods. Measure both aliquots from 

Shipment 1 and samples from Shipment 2. Shipment 2 samples should only be analyzed with 

the Tier 1 particle methods (light obscuration, Microflow Imaging, Flow Imaging, Nanoparticle 

Tracking Analysis, and RMM, if selected). 

8. Data Reporting 

Report all data in the appropriate template accompanying the method, if available. Include 

method parameters and any notes for future reference. If a template is not available, record the 

measurement parameters and the final, analyzed data on an Excel file. 

In general, save all raw data files in the following format, for later retrieval, if necessary: 

“Protein_Stress_Method_Replicate#_Date” 

Ex: NISTmAb_Stir_DLS_3_21082023 

 

Return all completed Excel files saved in the following format:

“Lab_Method_Date” 

Ex: NIST_DLS_21082023 

Perform all routine data analysis (i.e. averaging, standard deviation, etc.) and include the final 

data in the templates. Controls and standards should be run to ensure optimum performance of 

the instrument but there is no need to share the data. Please retain all raw data for the 

duration of this study. It is strongly recommended that a second analyst cross-check data 

entered into the templates before submitting any data. 

Data is due three months after receipt of the material. Submit all data to Srivalli Telikepalli at 

aapsinterlab@nist.gov.