1. Critical GMP Validations for Drugs/Biologics Ⅰ
Chung Keel Lee, Ph.D.
Special Advisor to the Minister, MFDS
Advisor, WHO
Adjunct / Invited Professor, KU / EWU
President, ISPE Korea
July 19, 2013

2. Validation
Definition: Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its pre-determined specifications and quality attributes.
Method:
Prospective Validation
Retrospective Validation
Concurrent Validation

3. Validation Elements of Validation Validation Report Revalidation
Equipment validation
Design Qualification
Installation Qualification
Operational Qualification
Process validation
Performance Qualification
Validation Report
Revalidation

4. Revalidation When there are changes in : Periodic revalidation
Critical processes
Equipment
Materials
Product characteristics
Periodic revalidation
Gradual process changes over a period of time
Wear of equipment

5. Validation Validation Steps Validation Master Plan Validation Protocol
Study
Validation
Report
Writing
SOP
Step by
At least
3 times
consecutively
Documented
plan
Easy to
follow
Conclusion

6. Validation Personnel qualification
Analyist performance in laboratories
Personnel following critical procedures
Personnel doing data entry in computerized systems

7. Validation Steam-sterilization process using autoclave
Calibration/certification
Empty chamber heat distribution studies
Heat penetration studies on various loading configurations
Challenge studies with live bacterial organisms
Depyrogenation process using hot-air oven
General validation study procedures are similar to the steam-sterilization process validation except the challenge study.
Endotoxin is used for the challenge study.
Depyrogenation process via washing/rinsing
All aseptic processes

8. Sterilization : Introduction
5 major methods for sterilization
Moist heat
Dry heat
Ethylene oxide gas
Ionizing radiation
Filtration

9. Sterilization : Introduction
Validation of sterilization – Biological Indicators
Sterilization method
Steam heat
Dry heat
Ethylene oxide
Ionizing radiation
Filtration
Biological indicator
Bacillus stearothermophilus
B. subtilis var. niger
B. subtilis var. globigii
B. pumilus
Brevundimonas diminuta

10. Sterilization : Introduction
Validation of sterilization
- Quantitation of indicator removal
Heat or ETO
Radiation
10 2
10 3
10 4
10 5
10 6
NO.
Survivors
(log)
Time(min)
10 2
10 3
10 4
10 5
10 6
NO.
Survivors
(log)
rads
- D value = the amount of time (for heat or ETO) or dose (for radiation) to reduce the organisms by one order of magnitude

11. Sterilization : Introduction
D Values
Biological indicators
B. Stearothermaphilus
B. subtilis var.niger
B. subtilis var.globigii
B. pumilus
Sterilization Process
Saturated steam at 121 ℃
Dry heat at 170 ℃
Ethylene oxide at 54 ℃
600mg/l, 50% RH
Gamma radiation : wet
Gamma radiation : dry
D value
1.5 min
1.0 min
3.0 min
0.2 Mrad
0.15 Mrad

12. Validation of Autoclave Steam Sterilization : General
Assurance of at least 10-6 probability of survival
2 basic approaches
The probability of survival approach
Heat labile products
Overkill approach
Heat stable products

13. Validation of Autoclave Steam Sterilization : Probability of Survival Approach
Bioburden & Heat Resistance studies
Calibration of biological indicators
Validation
Heat distribution
Heat penetration
Biological challenge
Reproducibility of the sterilization cycle

14. Validation of Autoclave Steam Sterilization : Overkill Approach
No need for bioburden & resistance studies
At least a 12-log reduction of microorganisms having a D value of at least 1 min
The most resistant isolates from production environment have D121 values ranging from 0.5 to 1.0 min.
Calibration of biological indicators
Validation

15. Validation of Autoclave Steam Sterilization : D value
Survivor curve (microbial death rate curve)
NO.
Survivors
(log)
10 2
10 3
10 4
10 5
10 6 1 2 3 4 5 6 7 8 9
D value at constant temp = 1
time (min)
Dt = time at a given temperature(t) required to reduce
the No.of microorganisms by 90%(1 logarithmic reduction)

16. Validation of Autoclave Steam Sterilization : Z value
Thermal Resistance Curve
100 10
1.0
0.1
110
115
120
125
130
135
Z = 10℃
D value (log)
Temperature
- Z = the No. of degrees temperature change to change the D value by a factor of ten

17. Validation of Autoclave Steam Sterilization : Z value
Steam sterilization : B. stearothermophilus
- Base temperature : 121 ℃
Z = 10 ℃ in the range of 110~135 ℃
Dry heat sterilization : B. subtilis
- Base temperature : 170 ℃
Z = 20 ℃ in the range of 150~190 ℃
Dry heat depyrogenation : Endotoxin
- Base temperature : 250 ℃
- Z = 50 ℃ in the range of 225~300 ℃

18. Validation of Autoclave Steam Sterilization : Typical Cycle Produced by a Sterilizer

19. Validation of Autoclave Steam Sterilization : Lethal Rate
where = temperature within the container(item)
being heate
= process temperature(reference temperature)
Lethal rate is the equivalent time for any specific temperature relative to another temperature.
ex) minutes at 121.1℃( ) per minute at 119℃( )

20. Validation of Autoclave Steam Sterilization : Lethal Rate
Example
The lethal rate for ℃ relative to ℃
(assuming a Z value of 10) :
This means that for every full minute of the process at a temperature of 117 ℃, the process is credited with the equivalent of min at ℃.

21. (minutes at 121.1℃ per minute at T℃)
Validation of Autoclave Steam Sterilization : Lethal Rate
Lethal Rate Table
(minutes at 121.1℃ per minute at T℃)
tempera -ture (℃)
Z value(℃) 7 8 9 10 11 12
100
0.001
0.002
0.005
0.008
0.012
0.017
101
0.003
0.006
0.010
0.015
0.021
102
0.004
0.018
0.026
103
0.023
0.031
104
0.007
0.013
0.019
0.028
0.038
105
0.016
0.025
0.034
0.046
106
0.042
0.055
107
0.027
0.039
0.052
0.067
108
0.035
0.049
0.064
0.081
109
0.045
0.062
0.079
0.098
110
0.041
0.058
0.078
0.119
111
0.036
0.075
0.121
0.144
112
0.050
0.073
0.097
0.123
0.149
0.174
113
0.070
0.126
0.155
0.184
0.211
114
0.130
0.163
0.195
0.226
0.256
114.5
0.114
0.150
0.185
0.219
0.251
0.282
115
0.134
0.173
0.210
0.245
0.279
0.310
115.5
0.158
0.200
0.239
0.275
0.341
116
0.187
0.230
0.271
0.309
0.344
0.376
116.5
0.220
0.266
0.308
0.347
0.382
0.414
117
0.260
0.307
0.350
0.389
0.424
0.455
117.5
0.306
0.355
0.398
0.437
0.471
0.501
tempera -ture (℃)
Z value(℃) 7 8 9 10 11 12
118
0.361
0.410
0.452
0.490
0.523
0.552
118.5
0.425
0.473
0.514
0.550
0.580
0.607
119
0.501
0.546
0.584
0.617
0.644
0.668
119.5
0.591
0.631
0.664
0.692
0.715
0.736
120
0.696
0.729
0.755
0.776
0.794
0.810
120.5
0.821
0.841
0.858
0.871
0.882
0.891
121
0.968
0.972
0.975
0.977
0.979
0.981
121.5
1.141
1.122
1.108
1.096
1.087
1.080
122
1.345
1.296
1.259
1.230
1.207
1.189
122.5
1.585
1.496
1.431
1.380
1.341
1.308
123
1.868
1.728
1.626
1.549
1.488
1.440
123.5
2.202
1.995
1.848
1.738
1.653
124
2.596
2.304
2.100
1.950
1.835
1.744
125
3.607
3.073
2.712
2.455
2.262
2.113
126
5.012
4.097
3.503
3.090
2.789
2.561
127
6.964
5.464
4.524
3.890
3.438
3.102
128
9.676
7.286
5.843
4.898
4.239
3.758
129
13.445
9.716
7.547
6.166
5.226
4.553
130
18.682
12.957
9.747
7.762
6.443
5.517

22. (minutes at 121℃ per minute at T℃)
Validation of Autoclave Steam Sterilization : Lethal Rate
Lethal Rate Table
(minutes at 121℃ per minute at T℃)
tempera -ture (℃)
Z value(℃) 7 8 9 10 11 12
100
.001
.002
.005
.008
.012
.018
101
.003
.006
.010
.015
.022
102
.004
.013
.019
.026
103
.016
.023
.032
104
.007
.020
.028
.038
105
.017
.025
.035
.046
106
.043
.056
107
.040
.053
.068
108
.014
.024
.036
.050
.066
.083
109
.063
.081
.100
110
.042
.060
.079
.121
111
.037
.077
.123
.147
112
.052
.075
.126
.152
.178
113
.072
.129
.158
.187
.215
114
.133
.167
.200
.231
.261
114.5
.118
.154
.190
.224
.257
.287
115
.139
.251
.285
.316
115.5
.164
.205
.245
.282
.348
116
.193
.237
.278
.351
.383
116.5
.228
.274
.355
.390
.422
117
.268
.359
.398
.433
.464
117.5
.365
.408
.447
.481
.511
tempera -ture (℃)
Z value(℃) 7 8 9 10 11 12
118
.373
.422
.464
.501
.534
.562
118.5
.439
.489
.527
.593
.619
119
.518
.599
.631
.658
.681
119.5
.611
.649
.708
.731
.750
120
.720
.774
.794
.811
.825
120.5
.848
.866
.880
.891
.901
.909
121
1.00
.100
121.5
1.18
1.16
1.14
1.12
1.11
1.10
122
1.39
1.33
1.29
1.23
1.21
122.5
1.64
1.54
1.47
1.41
1.37
123
1.93
1.78
1.67
1.59
1.52
123.5
2.28
2.05
1.90
1.69
1.62
124
2.68
2.37
2.15
2.00
1.87
125
4.39
3.16
2.78
2.82
2.31
126
5.18
4.22
3.59
2.85
2.61
127
7.20
5.62
4.64
3.98
3.51
128
10.0
7.50
6.00
5.01
4.33
3.83
129
13.9
7.74
6.31
5.34
130
19.3
13.3
7.94
6.58

23. Validation of Autoclave Steam Sterilization : F value
The accumulated lethality over the duration of the sterilization process (thermal death time)
where F = the accumulated lethality over the data collection period
t0 = the start of the data collection period
tE = the end of the data collection period
T = the temperature at each measurement point
Tb = the base temperature at which the Z was determined
Z = the z value within the process temperature range
dt = the time interval between temperature data

24. Validation of Autoclave Steam Sterilization : F0 value
For steam sterilization
where F0 = the accumulated lethality over the data collection period
t0 = the start of the data collection period
tE = the end of the data collection period
T = the actual temperature
121 = the usual base temperature for steam sterilization
10 = the z value usual for steam sterilization
t = the time interval between temperature data
It assumes that the temperature is measured every minute.

25. Accumulated Lethal Rates : F0 (in minute) : 14.22
Validation of Autoclave Steam Sterilization : F0 Calculation from Temperature Data (Temperature measured every minute)
Temperature
T-121
(T-121)/10
10(T-121)/10 ∑
101
-20 -2
0.01
111
-10 -1
0.10
0.11
121
1.00
1.11
124 3
0.3
2.00
3.11
131 10 1
10.00
13.11
14.11
14.21
14.22
Accumulated Lethal Rates : F0 (in minute) : 14.22

26. Validation of Autoclave Steam Sterilization : F0 Calculation from Temperature Data (Temperature measured every minute)

27. Validation of Autoclave Steam Sterilization : Probability of Nonsterile Unit (PNSU)
where B = probability of nonsterile unit
A = No. of presterilization bioburden(BB) organisms
F0 = the minimul F0 observed in the sterilization cycle
D121 = D value of the BB organism

28. Validation of Autoclave Steam Sterilization : Probability of Nonsterile Unit (PNSU)
Example from the previous Temperature Data : If the container has maximum of 100 organisms with D value of 0.5 min. at 121 ℃,
This means that the probability of a non-sterile unit is approximately 1 in 1026 units.

29. Validation of Autoclave Steam Sterilization : Spore Log Reduction(SLR)
where A = initial No. of spores
B = the No. of spores surviving the heat treatment

30. Validation of Autoclave Steam Sterilization : Minimum Required F0
Overkill approach requires the lethality sufficient to provide a 12-log reduction of microorganisms having a D value of 1.0
Minimum Required F0 should be 12 minutes or more

31. Validation of Autoclave Steam Sterilization
Example of Minute-by-Minute Temperature Observations of a Sterilizer Cycle with a 5-min Hold Time Along with the Incremental and Cumulative F (Lethal Rate) Values
Time (min)
Temperature (℃)
Lethal rate
Total lethal rate 1
25.0
0.000 2
55.0 3
85.0 4
100.0
0.008 5
110.0
0.079
0.087 6
115.5
0.282
0.369 7
119.0
0.631
1.000 8
121.1
1.00
2.000 9
3.000 10
4.000
Time (min)
Temperature (℃)
Lethal rate
Total lethal rate 11
121.1
1.00
5.000 12
6.000 13
116.0
0.316
6.316 14
108.0
0.200
6.516 15
96.0
0.000 16
80.0 17
65.0 18
42.0 19
30.0 20
25.0

32. Validation of Autoclave Steam Sterilization