Rapid sterility of ATMPs prior treatment - Validation of a qPCR-based test

Alexandra Mueller-Scholz, Sartorius Lab Instruments

NIST RMTM Workshop - Apr 19 , 2022
2

Safety of the Patient is a Risk

Introduction

Rapid QC-results:

• Mycoplasma

• Bacteria

• Fungi

The Challenge:

• Growth-based QC-release takes too long.

• How do you ensure non-contaminated ATMPs?
3

Strategies to Reduce False Results

Introduction

Reduce false-POSITIVES

 specificity of TaqMan ®

system

Reduce false-NEGATIVES

 control of PCR-inhibition

by integrated internal control

target-DNA

control-DNA

control-primer/probe

FAM Q

ROX Q

target-primer/probe
5

Reliable Results for Bacterial and Fungal Detection in ATMPs 

PRIOR Treatment

Validation study

Equivalency

Specificity

Sensitivity

Robustness

& 

Ruggedness

Guidance for validation of

rapid / alternativ methods: • PDA, TR 33

• USP <1223>

• USP <1071>

• EP 5.1.6 part 4-1-1 primary

validation by supplier

• EP 2.6.27

Including Guidance of the

German Governmental

Regulatory Agency (part

of EMEA) 

Challenge

To be accepted by 

regulatory bodies
6

In-silico prediction by sequence alignment and BLAST

Specificity

Acceptance criterion: ≤ 3 

nucleotides mismatch of 

primers and probe 

16S / 18S rDNA

http://www.biochem.umd.edu/biochem/kahn/bchm465-

01/ribosome/16SrRNA.html https://www.ncbi.nlm.nih.gov/
7

Detection Range - Bacteria

Specificity

Acceptance criterion: ≤ 3 

nucleotides mismatch of 

primers and probe 

Primer Mismatches

0 1 2 3

Bacteria 48.8 % 69.4 % 85.7 % 94.7 %

Archea n.a n.a 0.1% 40.4 %

Eukaryotes 0% 0% 0.1% 0.3 %
8

Detection Range - Fungi

Specificity

Acceptance criterion: ≤ 3 

nucleotides mismatch of 

primers and probe 

Strains of all Fungi species which were part of this study

Primer Mismatches

Forward Primer Probe Reverse Primer

Candida albicans (HQ876034.1) 0 0 0

Candida tropicalis (EU348785.1) 0 0 0

 

 

 

 

Candida glabrata (AB094140.2) 0 0 0

Candida guiliermondii (AJ508274.1) 0 0 0

Candida haemulonii (AB013572.1) 0 0 0

Candida parapsilosis (AY497758.1) 0 0 0

Aspergillus brasilliensis (D63697.1) 0 0 0

Aspergillus fumigatus (AF548061) 0 0 0

Penicillium crysogenum (M55628.1) 0 0 0

37 % of the whole kingdom fungi are putatively detected 

based on sequence alignments.

Challenge

Do we detect enough 

species?
9

Cross Reactivity to Cell Lines / Sample Matrix Effects

Specificity

Results

Hela

No Cq

0/2 No Cq

Vero

No Cq

0/2 No Cq

CHO-K1

No Cq

0/2 No Cq

RK13 No Cq

0/2 No Cq

CHO-DG44 No Cq

0/2 No Cq

CHO XM111-10 No Cq

0/2 No Cq

L-9296 (NCTC) No Cq

0/2 No Cq

Results

Hela

No Cq No Cq

0/8 No Cq No Cq

No Cq No Cq

No Cq No Cq

Vero

No Cq No Cq

0/8 No Cq No Cq

No Cq No Cq

No Cq No Cq

CHO-K1

No Cq No Cq

0/8 No Cq No Cq

No Cq No Cq

No Cq No Cq

HPBMC

No Cq No Cq

0/8 No Cq No Cq

No Cq No Cq

No Cq No Cq

Jurkat

No Cq No Cq

0/8 No Cq No Cq

No Cq No Cq

No Cq No Cq

Bacteria Fungi
10

26 species included in Validation

Sensitivity

Currently tested = 6

Bacillus subtilis

Clostridium sporogenes

Pseudomonas aeruginosa

Staphylococcus aureus

Candida albicans

Aspergillus brasiliensis

+

Streptococcus pyogenes

Bacteroides vulgatus

Escherichia coli

Pseudomonas protegens

Bacillus cereus

Enterococcus faecalis

Kocuria rhizophila

Staphylococcus epidermidis

Serratia marcescens

Propionibacterium acnes

Candida tropicalis

Candida glabrata

Candida krusei

Aspergillus fumigatus

Penicillium chrysogenum

Bacteroides fragilis

Enterobacter cloacae

Klebsiella pneumoniae

Clostridium perfringens

Yersinia enterocolitica

Colony Forming

Units (CFU)

Genome

Copies (GC)

Recommended Extension = 20

regulatory advice;

EP 2.6.27; user feedback

EP 2.6.1

USP<71>

Limit of Detection (LOD95): 99, 50, 25, 10, 5, 2.5, 1.25 CFU/ml 
11

Limit of Detection (LOD95) – Fungi

Sensitivity

CFU-based

Species (CFU-based) Acceptance

criterium

LOD95

(CFU/ml)

Acceptance

passed?

Candida albicans 23/24 50

Aspergillus brasiliensis 23/24 50

Candida tropicalis 8/8 10

Candida glabrata 8/8 25

Candida krusei 8/8 50

Aspergillus fumigatus 8/8 99

Penicillium chrysogenum 8/8 99

recommended

extension

currently tested

CFU/ml Mean

99

Run 1 31.32 31.90 32.78 32.80 32.20

24/24

Run 2 32.96 32.88 33.07 32.25 32.79

Run 3 32.29 32.81 36.03 32.09 33.30

Run 4 32.46 32.14 32.29 32.33 32.30

Run 5 32.32 32.55 32.71 32.36 34.48

Run 6 32.58 31.48 31.89 32.43 32.09

50

Run 1 33.15 33.79 34.11 34.44 33.87

23/24

Run 2 34.25 33.86 33.04 35.03 34.04

Run 3 33.59 33.90 32.63 33.89 33.50

Run 4 34.04 32.94 34.27 33.81 33.76

Run 5 No Cq 34.77 32.82 34.48 34.02

Run 6 32.58 32.57 32.82 34.81 31.19

25

Run 1 33.42 34.72 34.88 35.32 34.58

21/24

Run 2 34.67 37.19 33.28 36.08 35.30

Run 3 34.96 37.20 38.18 No Cq 36.78

Run 4 37.96 34.90 35.11 34.59 35.64

Run 5 33.55 No Cq 35.77 No Cq 34.66

Run 6 34.51 35.33 36.17 34.90 35.23

10

Run 1 35.85 36.48 37.27 37.38 36.74

14/24

Run 2 No Cq No Cq 37.00 34.65 35.82

Run 3 No Cq No Cq No Cq No Cq No Cq

Run 4 No Cq 35.93 No Cq 37.11 36.52

Run 5 35.04 No Cq No Cq 38.91 36.97

Run 6 35.54 36.26 35.84 37.52 36.29

Example: Candida albicans

Challenge

Detect 1 CFU/ml
Species (CFU-based) Classification Strain no. LOD95 (CFU/ml) Acceptance

passed?

Bacillus subtilis Gram pos. ATCC® 6633 25

Clostridium sporogenes Gram pos. ATCC® 19404 25

Pseudomonas aeruginosa Gram neg. ATCC® 9027 5

Staphylococcus aureus Gram pos. ATCC® 6538 10

Streptococcus pyogenes Gram neg. ATCC® 19615 99

Bacterioides vulgatus Gram neg. ATCC® 8482 2,5

Escherichia coli Gram neg. ATCC® 8739 10

Pseudomonas protegens Gram neg. ATCC® 17386 10

Bacillus cereus Gram pos. ATCC® 10876 5

Enterococcus faecalis Gram pos. ATCC® 29212 99

Kocuria rhizophila Gram pos. ATCC® 9341 10

Staphylococcus epidermidis Gram pos. ATCC® 12228 99

Serratia marcescens Gram neg. ATCC® 14756 50

Propionibacterium acnes Gram pos. ATCC® 11827 25

12

Limit of Detection (LOD95) – Bacteria

Sensitivity

recommended

extension

currently tested

CFU-based
13

Limit of Detection (LOD95) – Bacteria

Sensitivity

recommended

extension

GC-based

Species (GC-based) Classification Strain no. LOD95 10

GC/PCR

Acceptance

passed?

Bacteroides fragilis Gram neg. ATCC® 25285 10

Enterobacter cloacae Gram neg. ATCC® 13047 10

Klebsiella pneumoniae Gram neg. ATCC® 13883 10

Serratia marcescens Gram neg. ATCC® 13880 10

Clostridium perfringens Gram pos. ATCC® 13124 10

Yersinia enterocolitica Gram neg. ATCC® 27739 10
14

qPCR Device Comparability

Robustness and Ruggedness

Qiagen Rotor-Gene 6000

Spiking 99 CFU/ml of the species with the highest LOD95

Clostridium sporogenes (LOD95 = 25)

Candida albicans (LOD95 = 50)

Acceptance criterion 8/8 positive

Agilent Mx3005p

BioRad CFX96 touch

C l

5 10 15 20 25 30 35 40 45

0

5

0

5

0

Threshold

PC

NTC

NEC

Extracts

Thermo Fischer ABI Prism 7500
15

False-Positives due to Free Microbial DNA?

Robustness and Ruggedness

Low risk of falsepositive results

104 GC/ml 103 GC/ml 102 GC/ml 10GC/ml 0GC/ml

Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2 Run 1 Run 2

39.00 No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

39.93 39.89 No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

39.38 No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq No Cq

E. coli DNA spiked into DMEM

DNA-extraction

qPCR

failed passed

Challenge

Discriminate live and dead

microorganisms
16

Is the Cell Culture Medium DNA-free?

Robustness and Ruggedness

DMEM* without spike

acceptance criterion

> 95 % negative

Cell culture medium without spike

acceptance criterion

> 95 % negative

Fungi detection Negative 

Results

ROXTM % Negative 

results

DMEM 8/8 8/8 Correct 100%

Fetal bovine serum (FBS) 8/8 8/8 Correct 100%

DMEM high Glucose, GlutaMAX 8/8 8/8 Correct 100%

DMEM w/o Na-Pyruvat mit stable Glutamin 8/8 8/8 Correct 100%

RPMI 1640 8/8 8/8 Correct 100%

MEM (1) 8/8 8/8 Correct 100%

MEM (2) 8/8 8/8 Correct 100%

KnockOut DMEM 8/8 8/8 Correct 100%

DMEM/F-12 GlutaMAX 8/8 8/8 Correct 100%

Opti-MEM Reduced Serum GlutaMAX 8/8 8/8 Correct 100%

McCoys 5A Medium 8/8 8/8 Correct 100%

Leibovitz L-15 Medium 8/8 8/8 Correct 100%

Chrondrocyte Differentiation Medium 8/8 8/8 Correct 100%

Human Osteoblast Differentiation Medium 8/8 8/8 Correct 100%

ChondroMAX Differentiation Medium 8/8 8/8 Correct 100%

Tahara Lympho One + HS 8/8 8/8 Correct 100%

RPMI, penicillin/Streptomycin, Glutamax, 5% FBS 8/8 8/8 Correct 100%

Low risk of falsepositive results
17

Robustness and Ruggedness

Aspergillus brasiliensis 99 CFU (FAMTM)

106 cells/ml 105 cells/ml

Hela

37.54 31.26 33.72 32.10

35.24 31.84 33.34 32.07

4/4 4/4

Vero

33.30 31.43 32.78 31.39

34.45 32.76 33.48 31.60

4/4 4/4

CHO-K1

33.65 32.40 34.49 33.36

34.70 32.83 33.89 32.40

4/4 4/4

HPBMC

32.21 31.59 33.80 32.12

32.39 31.25 33.79 31.04

4/4 4/4

Jurkat

34.99 34.14 31.77 32.64

34.80 33.50 34.30 33.81

4/4 4/4

Tolerance of Eukaryotic Cell Background
18

Equivalence

recommended extension

Streptococcus pyogenes

Pseudomonas protegens

?

=

classical sterility test according

to EP 2.6.1. and USP<71>

all 6 mandatory species

Bacillus subtilis

Clostridium sporogenes

Pseudomonas aeruginosa

Staphylococcus aureus

Candida albicans

Aspergillus brasiliensis

2x LOD95 LOD95 1⁄2 LOD95

Sartorius L+S external contract lab

qPCR-based detection

Comparability to Compendial Sterility Test
19

Equivalence

Microsart® ATMP Bacteria Compendial culture method (External)

2x LOD95 LOD95 LOD95/2 2x LOD95 LOD95 LOD95/2

Bacillus subtilis 33.16 34.23 35.47 B. subtilis B. subtilis B. subtilis 33.23 34.32 34.38

Staphylococcus aureus 35.42 35.77 36.56 S. aureus S. aureus S. aureus 34.13 35.67 39.90

Clostridium sporogenes

34.20 34.87 35.45 C. sporogenes C. sporogenes C. sporogenes 34.10 33.43 35.61

Pseudomonas aeruginosa 36.40 36.74 37.22 P. aeruginosa P. aeruginosa Negative 36.22 37.96 No Cq

Streptococcus pyogenes 34.89 35.53 36.55 S. pyogenes S. pyogenes S. pyogenes 35.09 35.93 35.88

Pseudomonas protegens 34.14 34.38 36.52 Gram –

Oxidase + 

Gram – Oxidase 

+

Gram –

33.28 34.51 35.61 Oxidase +

Comparability to Compendial Sterility Test
20

Equivalence

Comparability to Compendial Sterility Test

Microsart® ATMP Fungi Compendial culture method (External)

2x LOD95 LOD95 LOD95/2 2x LOD95 LOD95 LOD95/2

Candida albicans 32.25 32.27 32.96 C. albicans C. albicans Negative 31.94 32.12 33.96

Aspergillus brasiliensis 34.38 37.06 34.94 A. brasiliensis A. brasiliensis A. brasiliensis 32.40 33.17 34.20
21

Validation Study

Equivalence

Specificity

Sensitivity

Robustness

& 

Ruggedness

high sequence homology

low cross reactivity

low risk of

false-positives

device

compatibility

LOD95 10-100 CFU/ml

tolerance to high 

cell background

separation of free 

microbial DNA

Validation Results in a Nutshell
22

Validation Study

Mycoplasma

Total 

Bacteria

Total 

Fungi

PCR@Sartorius.com

• DNA extraction

• qPCR reagents

• CFU Validation standards

prequantified and non-infectious
Alexandra Müller-Scholz

Manager PCR & Microbiology Applications

Lab Essentials Applications Development

Phone +49 551 3082503

Mobile +491713568400

Alexandra.Mueller-Scholz@Sartorius.com

Thank you.