Validation and First results on the G185 long term incubator for embryos.

1. Validation of the G-185 incubator
Ronny Janssens – Romy Souffreau
29/06/2009

2. Validatie K-systems G185 Direct Heat
Incubator Introduction
2 18-8-2009

3. Validation
ISO 15189, §5.3.2
“Equipment shall be shown
(upon installation and in
routine use) to be capable of
achieving the performance
required and shall comply
with specifications relevant
to the examinations
concerned”
ISO 15189 §5.5.2
“The laboratory shall use only
validated procedures for
confirming that the
examination procedures are
suitable for the intended use”
3 18-8-2009

4. TOC
Technical validation
Clinical validation
3PN zygotes
Sibling oocyte study: G-185 vs Heracell240
Randomised Controled Trial
4 18-8-2009

5. Validation
User requirements specifications
Use as safe incubator for IVF
Space saving alternative for standard incubators
Functional specifications
Grow viable embryos
Good embryo quality
Easy to use
Alarm functions
Design specifications
37°C ± 0,5°C
6% CO2 ± 0,5%– 5% O2 ± 1%
Stable culture conditions
No humidification
Air quality (HEPA – VOC)
5 18-8-2009

6. TOC
Technical validation
Pre production model (2007 - 2008)
Final product (2008 - 2009)
Clinical validation (2009)
3PN zygotes
Sibling oocyte study: G-185 vs Heracell240
Randomised Controled Trial (started 26/05/2009)
6 18-8-2009

7. Technical validation: pre production model
User interface software: bugs removed – extra functionality
added
Minor changes in design: improvements on seals – design of
gas mixing chamber
Requirements on safety
Development of monitoring software
permanent monitoring and registration of critical parameters
(Temp – CO2 – O2)
Audit trail
E-mail messages
Alarm switch connected to modem
Independent monitoring
Installation of Vaisala CO2 sensor in gas mixing chamber
Temperature monitoring?
7 18-8-2009

8. Monitoring software
8 18-8-2009

9. 9 18-8-2009

10. Measurements
•Gas sampling in closed chamber
•Temperature: calibrated thermometer with
thermocouple fixed in culture dish
•pH: ISFET probes – suitable for small volumes
RI pH meter
10 18-8-2009

11. Temperature – closed chambers
11 18-8-2009

12. Temperature in Nunc 4well dish in opended
chamber
Time opening a chamber
37,4
37,2
Temperature (°C)
37
36,8
36,6
36,4
36,2
36
0 1 2 3 4 5 6
Tijd (min)
10 sec 20 sec 40 sec 60 sec 120 sec 180 sec
240 sec 150 sec
12 18-8-2009

13. Gas concentration (closed chambers)
Average (n=10) Stdev
[O2] [CO2] [O2] [CO2]
Chamber1 4,9 5,9 0,16 0,09
Chamber2 4,8 5,9 0,11 0,11
Chamber3 4,8 5,8 0,12 0,10
Chamber 4 4,9 5,9 0,14 0,13
Chamber 5 4,7 5,9 0,08 0,16
Chamber 6 4,8 6,0 0,13 0,13
Chamber 7 4,7 5,9 0,04 0,05
Chamber 8 4,8 5,9 0,04 0,09
Chamber 9 4,9 5,9 0,07 0,06
Chamber10 4,9 5,9 0,11 0,15
Average 4,8 5,9 0,10 0,11
t-test
O2 CO2
p-waarde 0,00 0,168
13 18-8-2009

14. Recovery time
CO2 en O2 after opening one chamber
18,0
Concentration O2 en CO2 (%)
16,0
14,0
12,0
10,0
8,0 1
6,0
4,0
2,0
0,0
1 5 9 13 17 21 25 29 33 37 41
Time(min)
O2 concentratie CO2 concentratie
Fast return to homeostatic conditions: 3 minutes
14 18-8-2009

15. Impact of opening one chamber on gas
concentrations
O2 en CO2 in closed chambers after opening
of one chamber
O2(%) CO2 (%)
8,0 7,0
7,5 6,5
7,0
6,0
6,5
↑ ↓ 5,5
6,0
5,0
5,5 not relevant
5,0 4,5
4,5 4,0
0 1 2 3 4 5
Tijd (min)
kamer 1 kamer 2 kamer 3 kamer 4 kamer 5 kamer 6
kamer 7 kamer 9 kamer 10 Serie1 Serie11 Serie12
15 18-8-2009

16. Impact of frequent opening
Measurement in 1 room and open 1 room every 5 minutes
pH
CO2 en O2 (%)
7,30 6,3
6,1
7,29
5,9
7,28 5,7
7,27 5,5
5,3
7,26
5,1
7,25 4,9
0 15 30 45 60 75 90 105 120 135
Tijd (min)
pH O2 concentratie (%) CO2 concentratie (%)
16 18-8-2009

17. Particle count
Maximum permitted number of particles per m3
equal to or greater than the tabulated size
At rest In operation
Grade 0.5 μm 5.0μm 0.5 μm 5.0μm
A 3520 20 3520 20
B 3520 29 352000 2900
C 352000 2900 3520000 29000
D 3520000 29000 Not Not
defined defined
Volume 4 - EU Guidelines for Good Manufacturing Practice Medicinal Products for Human and Veterinary Use – annex 1 (feb 2008)
17 18-8-2009

18. Validation G185 Direct Heat Incubator
Requirements
* [O2] ………………. 4,8% 5,0% ± 1%
* [CO2]……………… 5,9% 6,0% ± 0,5%
* Temperatuur…….. 37,1°C 37,0°C ± 0,5°C
* pH ………………... 7,28 ± 7,20 – 7,30
* Class A (GMP)
18 18-8-2009

19. Technical validation: conclusions
Closed incubator: homogeneous and stable gas and
temperature distribution
After opening:
1. Fast recovery of gas conditions in opened chamber
2. No significant impact on other chambers
Conclusions
Optimal culture parameters are maintained
Chambers can be considered as individual culture
19 18-8-2009

20. TOC
Technical validation
Preproduction model
Final product
Clinical validation
3PN zygotes (n=20)
Sibling oocyte study: G-185 vs Heracell240
Randomised Controled Trial (started 26/05/2009)
20 18-8-2009

21. Sibling oocyte study (n=24)
G-185 Heracell 240
N (%) N (%)
Injected 126 133
(inseminated)
Deg 11 10
1PN 9 2
2PN 86 (68.3) 90 (67.7) NS
3PN 3 2
21 18-8-2009

22. Embryo development day 3
Cleavage stage D3 G-185 Cleavage stage D3 Standard
Uncl Uncl
MNB MNB
2-4 2-4
5-7 5-7
8 8
>8 >8
Embryo quality D3 G-185 Embryo quality D3 Standard
A A
B B
C C
D D
22 18-8-2009

23. Embryo replacements - cyropreservation
G-185 Standard
Discard Discard
ET ET
Cryo Cryo
Cycles 24 +hCG
No replacement 2
Standard 4 1
Mixed 10 5
G-185 8 2
23 18-8-2009

24. RCT
Started: 27/05/2009
Cycles randomised: 100 (26/06/2009)
+ hCG: ?
24 18-8-2009

25. Conclusions
Design specifications:
37°C ± 0,5°C
6% CO2 ± 0,5%– 5% O2 ± 1%
Stable culture conditions
…
Functional specifications:
Grow viable embryos
Good embryo quality
Easy to use
Alarm functions
User requirements:
Use as safe incubator for IVF
Space saving alternative for standard incubators
25 18-8-2009