NIST Stability Study Results for Ad5 RM

Introduction

NIST was asked to perform the final stability study measurements on the Adenovirus Serotype 5 

(Ad5) Reference Material distributed by ATCC. The measurements include particle count by 

OD260 nm in the presence of 0.1% SDS and TCID 50 assay to determine infectious particles/mL.

Detailed protocols were provided by Keith Carson from ISBiotech for both assays.

Materials were provided by ATCC (courtesy of Liz Kerrigan) at no charge to NIST to complete 

the stability study. The items received are listed below.

1) Two vials of HEK293 cells deposited by the Adenovirus Reference Material Working Group

(ATCC# DS-CELLS1, batch# 3553) 

2) Two vials of Ad5 RM (ATCC# VR-1516, lot# 001506)

Ad5 RM PARTICLE COUNT MEASUREMENT

Materials 

Ultra-pure water (Thermo Fisher# 10977015)

1 M Tris-HCL pH8 (Millipore Sigma# T3038)

NaCl (Millipore Sigma#71376-1KG)

Glycerol (Thermo Fisher# 15514011)

SDS, 10% (Millipore Sigma# 71736-500ml)

Hellmanex III (Millipore Sigma# Z805939)

Methanol (Fisher Scientific# A542-1)

Agilent Cary 3500 UV-Vis Spectrophotometer, double beam

Quartz cuvettes, 1cm pathlength

Procedure (provided by ISBiotech)

Title: Adenovirus reference material standard operating procedure for determination of particle 

concentration via spectrophotometric analysis.

Version 4 (Nov 7, 2001)
Date of analysis: Oct 19, 2021

Name of analyst: Jamie Almeida

Deviations made to SOP:

• The Particle Count SOP requires 0.85 mL of Ad5 RM to perform the measurements; 

however, there is only 0.5 mL of Ad5 RM in a single vial. The particle count and 

infectious titer should be performed on the same vial. Therefore, some of the volumes 

had to be reduced in the SOP. 

o Ad5 volumes for the 80% and 30% samples were cut in half. Ultimately, 200 μL of 

Ad5 RM was added to the 80% tube with 25 μL of excipient, and 75 μL of Ad5 RM 

was added to each of the 30% tubes with 150 μL of excipient. 

• 100 μL (instead of 200 μL) of the blank and Ad5 RM dilutions (80% and 30% samples) 

were added to the quartz cuvettes for OD260 nm measurements.

Results

Acceptability of the assay parameters as stated in the SOP were all met.
The assigned particle concentration of the Ad5 RM in 2001 was 5.8 E11 particles/mL with a 95% 

certainty range of 5.6 E11 to 6.0 E11 particles/mL. The NIST measured value of 5.781 E11 +/-

3.509 E10 particles/mL falls within the 95% certainty range.

Sample OD260nm AVG A260 Std Dev Dil Fact virus particle/mL AVG particle/mL Std Dev particle/mL

80% AD5 repA 0.43211496

80% AD5 repB 0.43181982 0.432 0.000197 0.8 5.93851E+11

80% AD5 repC 0.43174031

30% AD5 #1a 0.16026363

30% AD5 #1b 0.1600499 0.160 0.000148 0.3 5.87025E+11

30% AD5 #1c 0.15997997 5.781E+11 3.509E+10

30% AD5 #2a 0.14378244

30% AD5 #2b 0.14353283 0.144 0.000137 0.3 5.26624E+11

30% AD5 #2c 0.14355877

30% AD5 #3a 0.16513694

30% AD5 #3b 0.16489912 0.165 0.000151 0.3 6.0487E+11

30% AD5 #3c 0.16485761
Ad5 RM INFECTIOUS TITER MEASUREMENT

Materials 

HEK 293 cells deposited by the ARMWG (ATCC# DS-CELLS1, batch# 3553)

FBS, US origin (Thermo Fisher # 26140-079)

DPBS, -Ca, -Mg (Thermo Fisher# 14190144)

DMEM high glucose (Thermo Fisher# 11995065)

DMEM low glucose (Thermo Fisher# 11054020)

Sodium pyruvate, 100 mM (Thermo Fisher# 11360-070)

Glutamax, 100X ((Thermo Fisher# 35050061)

Bovine calf serum (Thermo Fisher# 10371-029)

Trypsin-EDTA, 0.05% (Thermo Fisher# 25300-054)

96 well plates (Costar# 3598)

T225 cm tissue culture flasks (Corning# 431082)

T75 cm tissue culture flasks (Corning# 430641)

Incubator, humidified (37°C, 5% CO2)

Isoton solution (Beckman Coulter# 8546719)

Beckman Multisizer 3 coulter counter

Olympus CKX53 inverted microscope

Laminar flow biosafety cabinet

Hermle Tabletop Centrifuge 

Procedure (provided by ISBiotech)

Title: Adenovirus reference material standard operating procedure for determination of 

infectious titer in 293 cells in a 96-well format

Version 3 (Nov 7, 2001)

Date of analysis: Oct 19, 2021 (Assay A) and Oct 20, 2021 (Assay B)

Name of analyst: Jamie Almeida
Assay A: HEK 293 cells were at passage 7

Assay B: HEK 293 cells were at passage 8

Deviations made to SOP:

• An initial plating of 40,000 cells per well of a 96 well plate resulted in HEK 293 cells that 

were over confluent with cells stacking on top of one another. A lower dilution was 

necessary to obtain a confluency of 60-80% at the time of infection. Cells were seeded 

at a density of 10,000 cells per well the day before infection and resulted in 

approximately 60% confluency on Day 0 for both A and B assay plates.

• The Particle Count SOP requires 0.85 mL of Ad5 RM to perform the measurements; 

however, there is only 0.5 mL of Ad5 RM in a single vial. The particle count and

infectious titer should be performed on the same vial. Therefore, the initial volume in 

the Ad5 dilution series had to be reduced in the SOP. 

o The volume for the 1:2 dilution was reduced to 70 μL so that there would be 

enough material for both the A and B assays. 

Results

Acceptability of the assay parameters as stated in the SOP were all met except for one 

condition.

• There was at least one sample dilution where CPE is evident in all 12 wells.

o At the highest dilution in Assay A, 11 out of 12 wells were positive for CPE.

o At the highest dilution in Assay B, 7 out of 12 wells were positive for CPE.

TCID50 Results for Assay A

Calculation for infectious titer per mL (IU/mL)

V=-([ln(1-(p/n))]*D/[A*C*I*√t]

Protocol section 6.2.5.3: Optimally, the number of positive wells should be between 20-80% of the total wells in the dilution (>2 and <10)

Assay A

Sample Dilution Positive Wells Confluency Time (sec) last part of the formula first part of formula Pos wells/total Full formula AVG SD

5.00E+07 11 0.6 3600 0.00274176 2.48E+00 0.083333333 4.53E+10 3.72E+10 6.63E+09

1.00E+08 8 0.6 3600 0.00274176 1.10E+00 0.333333333 4.01E+10

2.00E+08 4 0.6 3600 0.00274176 4.05E-01 0.666666667 2.96E+10

2.83E+08 4 0.6 3600 0.00274176 4.05E-01 0.666666667 4.19E+10

4.00E+08 2 0.6 3600 0.00274176 1.82E-01 0.833333333 2.66E+10

5.66E+08 2 0.6 3600 0.00274176 1.82E-01 0.833333333 3.76E+10

8.00E+08 3 0.6 3600 0.00274176 2.88E-01 0.75 8.39E+10

1.13E+09 1 0.6 3600 0.00274176 8.70E-02 0.916666667 3.59E+10

1.60E+09 2 0.6 3600 0.00274176 1.82E-01 0.833333333 1.06E+11

2.26E+09 1 0.6 3600 0.00274176 8.70E-02 0.916666667 7.17E+10

3.20E+09 1 0.6 3600 0.00274176 8.70E-02 0.916666667 1.02E+11

4.53E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

6.40E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

1.28E+10 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00
Average of plate A and B concentrations = 2.86 E10 IU/mL

Standard deviation of plate A and B = 1.15 E10 IU/mL

The assigned infectious titer for the Ad5 RM on HEK 293 cells in 2001 was 7 E10 IU/mL with a 

95% certainty range of 7 E10 to 8 E10 IU/mL. Previous data from the 17 laboratories that 

conducted initial TCID 50 assays for the Ad5 RM is also described in the RM product sheet and 

states that 99.7% of the time the result would fall within the range of 3 E10 and 18 E10 IU/mL 

(within 3 SD of the mean). The NIST measured value of 2.86 E10 +/- 1.15 E10 IU/mL falls within 

the 99.7% range that the previous 17 laboratories measured.

STR analysis on HEK 293 cells deposited by the ARMWG

STR analysis was performed on HEK 293 cells at passage 6 using the following protocol. 

One million cells were deposited into a sterile 1.5 mL microfuge tube and washed with DPBS 

and centrifuged (200 x g, 3 min) to pellet. The cell pellet was resuspended in 150 μL of DPBS 

and frozen at -80°C until ready for testing.

Cells were thawed, mixed well, and 15 μL was added to pre-cut sterile FTA punches. These 

punches were allowed to dry overnight in a 96 well plate in a laminar flow biosafety cabinet. 

Once dry, the FTA punches were added to tubes and given to Becky Steffen (NIST, DIV 645) to 

perform STR genotyping. 

Promega PowerPlex Fusion 6C kit was used (based on the manufacturers protocol) to 

determine STR allele calls for the HEK 293 cells. PCR products were separated on the Applied 

Biosystems Genetic Analyzer 3500xl and results analyzed using Applied Biosystems 

GeneMapper ID-X software.

TCID50 Results for Assay B

Assay B

Sample Dilution Positive Wells Confluency Time (sec) last part of the formula first part of formula Pos wells/total Full formula AVG SD

5.00E+07 7 0.6 3600 0.00274176 8.75E-01 0.416666667 1.60E+10 2.01E+10 8.22E+09

1.00E+08 4 0.6 3600 0.00274176 4.05E-01 0.666666667 1.48E+10

2.00E+08 4 0.6 3600 0.00274176 4.05E-01 0.666666667 2.96E+10

2.83E+08 1 0.6 3600 0.00274176 8.70E-02 0.916666667 8.98E+09

4.00E+08 1 0.6 3600 0.00274176 8.70E-02 0.916666667 1.27E+10

5.66E+08 2 0.6 3600 0.00274176 1.82E-01 0.833333333 3.76E+10

8.00E+08 1 0.6 3600 0.00274176 8.70E-02 0.916666667 2.54E+10

1.13E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

1.60E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

2.26E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

3.20E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

4.53E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

6.40E+09 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00

1.28E+10 0 0.6 3600 0.00274176 0.00E+00 1 0.00E+00
Results

HEK 293 cells (passage 6) deposited by the ARMWG have the following STR profile determined 

by Becky Steffen at NIST (see table below). Alleles in green show 100% concordance between 

NIST, ATCC, and Cellosaurus STR data for HEK 293 cells. Discrepancies between the STR profile 

of these cells and those from ATCC and Cellosaurus are highlighted in blue for human markers 

CSF1PO and D5S818. NIST data shows loss of heterozygosity (LOH) at these two loci which is not 

uncommon in cell lines after passaging. I was unable to locate STR profile data from the original 

characterization of these cells back in 2001. It is unclear if these cells obtained the LOH at these 

two loci while being adapted for adherence, or from passaging the cells at NIST. Either way, 

these cells are HEK 293 derived.

ATCC STR profile for HEK 293

STR Marker Allele 1 Allele 2 Notes

CSF1PO 12 12 11,12 (ATCC and Cellosaurus)

Penta D 9 10

TH01 7 9.3

vWA 16 19

D21S11 28 30.2

D7S820 11 12

D5S818 8 8 8,9 (Cellosaurus)

TPOX 11 11

D8S1179 12 14

D12S391 19 21

D19S433 15 18

SE33 17 20

D22S1045 16 16

FGA 23 23
Cellosaurus STR profile for HEK 293