Bottle Freezing and Thawing Rate Controlled Validation (9-L Bottle Study...
9
1 phone: (740) 374-8300 fax: (740) 374-8310 30765 State Route 7, Marietta, OH 45750 e-mail: [email protected] website: www. farrarscientific.com Freezing and Thawing Rate Controlled Validation 9-L Bottle Study Introduction: It was previously shown that controlled freezing and thawing rates were validated using 16-L single use bags. 1 This effectively refuted prior claims made by other authors. 2 This study shows that a consistent method is also available for controlled rate freezing and thawing of bottles. It also includes the effect of load and container’s position on freeze rates. The freeze/thaw controlled rate chamber used in this study was a Model 4002 manufactured by Farrar Scientific. It permits rapid, uniform bulk freezing and/or thawing of products with temperature ranges from +40 o C to -80 o C. It has a minimum of 1.7 kw of net cooling and heating capacity over the entire temperature range. It is also equipped with programmable profiles, which allow flexibility to meet unique product conditioning. Experimental Procedure: The effect of the program temperature profile on the rate of product freezing and thawing were explored. Various temperature profiles were tested in order to determine the best for uniformly controlling the freezing rate to the 1 st phase transition (0 o C), and the cooling rate from the 1 st phase transition to -30 o C. The target time to break from the first phase transition was 11- 21 hours. The target product cooling rates from -5 o C to -30 o C were 0.2 o C/min to 0.9 o C/min. The target thaw rate was as fast as possible without overshooting a particular temperature. Tap water was used to simulate a water-based biological formulation. Bottles (10L capacity with 9L fill) were used in this study. For the 1 bottle test, the bottle was arranged in the middle of the top shelf. The objective was to maximize air flow to the entire outer surface as uniformly as possible (Figure 1). The bottle contained 9L of water. The bottle had a thermocouple placed through the middle of the closure with the tip located half way between the bottom and the top surface of the liquid. The end of the thermocouple is also equal distance from all bottle sides (geometric center). This is the expected last point to freeze.
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Transcript of Bottle Freezing and Thawing Rate Controlled Validation (9-L Bottle Study...
1
phone: (740) 374-8300 fax: (740) 374-8310 30765 State Route 7, Marietta, OH 45750 e-mail: [email protected]
website: www. farrarscientific.com
Freezing and Thawing Rate Controlled Validation
9-L Bottle Study
Introduction:
It was previously shown that controlled freezing and thawing rates were validated using 16-L
single use bags.1 This effectively refuted prior claims made by other authors.2
This study shows that a consistent method is also available for controlled rate freezing and
thawing of bottles. It also includes the effect of load and container’s position on freeze rates.
The freeze/thaw controlled rate chamber used in this study was a Model 4002 manufactured by
Farrar Scientific. It permits rapid, uniform bulk freezing and/or thawing of products with
temperature ranges from +40oC to -80oC. It has a minimum of 1.7 kw of net cooling and heating
capacity over the entire temperature range. It is also equipped with programmable profiles,
which allow flexibility to meet unique product conditioning.
Experimental Procedure:
The effect of the program temperature profile on the rate of product freezing and thawing
were explored. Various temperature profiles were tested in order to determine the best for
uniformly controlling the freezing rate to the 1st phase transition (0oC), and the cooling rate from
the 1st phase transition to -30oC. The target time to break from the first phase transition was 11-
21 hours. The target product cooling rates from -5oC to -30oC were 0.2oC/min to 0.9oC/min.
The target thaw rate was as fast as possible without overshooting a particular temperature.
Tap water was used to simulate a water-based biological formulation. Bottles (10L capacity
with 9L fill) were used in this study. For the 1 bottle test, the bottle was arranged in the middle
of the top shelf. The objective was to maximize air flow to the entire outer surface as uniformly
as possible (Figure 1). The bottle contained 9L of water. The bottle had a thermocouple placed
through the middle of the closure with the tip located half way between the bottom and the top
surface of the liquid. The end of the thermocouple is also equal distance from all bottle sides
(geometric center). This is the expected last point to freeze.
2
Figure 1. Standard 1 Bottle Position in Chamber.
Before meeting the freezing requirements, the freezing cooling rate maximums were
established at a small and large load. Thus, the controlled rate chamber was set up with the
following program (Test 1).
Step 1. Timed Event to -80oC in 10 seconds
Step 2. Wait for process
Step 3. Soak for 12 hours
Step 4. Wait for event
Controlled Freeze Rate Results and Discussion: Controlled Freeze Test 1 results are shown in Figure 2. It only took 317 minutes to break
from the phase change and 332 minutes to reach -30oC. The cooling rates from -5oC to
-30oC are shown in Table 1. The cooling rates are 1.7oC/min to 2.3oC/min. The cooling rate is
at its slowest rate from -20oC to -30oC because the difference in air temperature
(-80oC) and product temperature is less.
Figure 2.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 100.0 200.0 300.0 400.0 500.0 600.0 700.0 800.0 900.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Freeze Test 1
1 9-L Bottle
Bottle 1 Control
3
Table 1. Test 1 Cooling Rates.
Temp. Range (oC)
-5 to -10
-10 to -20
-20 to -30
2.31
2.22
1.71
Cooling Rate (oC/min)
Test 2 was conducted with the same temperature profile using 15 bottles. The 15 bottles were
arranged in order to maximize air flow to the surface of all bottles as uniformly as possible
(Figure 3). The freezing and cooling rates are shown in Figure 4 and Tables 2 and 3. The time it
took for every bottle to break from the phase change (freeze time) ranged from 369 minutes to
544 minutes. The cooling rates from -5oC to -30oC ranged from 0.3oC/min to 1.6oC/min. Since
both the small load (1 bottle) and large load (15 bottles) had cooling rates faster than our target,
Test 3 (1 9-L Bottle) was conducted with the following adjusted temperature profile.
Step 1. Timed Event to -25oC in 10 seconds
Step 2. Wait for process
Step 3. Soak for 9 hours
Step 4. Timed Event to -45oC in 10 seconds
Step 5. Wait for process
Step 6. Soak for 13 hours
Step 7. Timed Event to -80oC in 10 seconds
Step 8. Wait for process
Step 9. Soak for 8 hours
Step 10. Wait for event
Figure 3. Standard 15 Bottle Positions in Chamber.
Test 3 cooling rate results are shown in Figure 5 and Table 4. The time for the product to break
from the phase change (767 minutes) was within the desired time range. In addition, the cooling
rates from -5oC to -30oC were within the desired range.
1
4 3
2
5
6 7 8
9 10
11 12
13 14
15
4
Figure 4.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Freeze Test 2
15 9-L Bottles
Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 Bottle 6 Bottle 7 Bottle 8
Bottle 9 Bottle 10 Bottle 11 Bottle 12 Bottle 13 Bottle 14 Bottle 15 Control
Table 2. Test 2 Cooling Rates for Bottles 1-8.
Temp. Range (oC) 1 2 3 4 5 6 7 8
-5 to -10 0.36 0.75 0.77 0.86 1.00 1.36 0.64 0.73
-10 to -20 0.38 0.81 0.72 0.90 1.30 1.25 0.76 0.74
-20 to -30 0.45 0.79 0.61 0.79 1.62 0.91 0.81 0.70
Cooling Rate (oC/min)
Bottle #
Table 3. Test 2 Cooling Rates for Bottles 9-15.
Temp. Range (oC) 9 10 11 12 13 14 15
-5 to -10 0.42 0.40 0.33 0.53 1.00 0.39 0.88
-10 to -20 0.46 0.50 0.33 0.53 1.33 0.41 1.20
-20 to -30 0.51 0.61 0.41 0.50 1.46 0.44 1.28
Cooling Rate (oC/min)
Bottle #
Figure 5.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0 1800.0 2000.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Freeze Test 3
1 9-L Bottle
Bottle 3 Control
5
Table 4. Test 3 Cooling Rates.
Temp. Range (oC)
-5 to -10
-10 to -20
-20 to -30
0.53
0.55
0.29
Cooling Rate (oC/min)
Since the correct temperature profile was determined with 1 bottle, the next test (Test 4) was
conducted using the same temperature profile with 6 bottles. For the 6 bottle test, bottles were
arranged side by side on each shelf as shown in Figure 6. Figure 7 and Table 5 show the cooling
rates of each bottle. The 6 bottles break from the phase change in the range of 779 minutes to
922 minutes. The 1 bottle (Test 3) broke from the phase change at 767 minutes, within 12
minutes of the 6 bottle temperature range. Additionally, all six bottle cooling rates from -5oC to
-30oC fell in the desired range just as the 1 bottle test. These results show that the freezing and
cooling rates are independent of product load or bottle position.
Figure 6. Standard 6 Bottle Positions in Chamber.
Figure 7.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 500.0 1000.0 1500.0 2000.0 2500.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Freeze Test 4
6 9-L Bottles
Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 Bottle 6 Control
1 2
3 4
5 6
6
Table 5. Test 4 Cooling Rates.
Temp. Range (oC) 1 2 3 4 5 6
-5 to -10 0.77 0.28 0.37 0.51 0.28 0.50
-10 to -20 0.63 0.34 0.35 0.50 0.35 0.49
-20 to -30 0.34 0.25 0.26 0.23 0.25 0.20
Cooling Rate (oC/min)
Bottle #
Since the correct temperature profile was determined with 1 bottle and 6 bottles, the next test
(Test 5) was conducted using the same temperature profile with 12 bottles. For the 12 bottle test,
bottles were arranged as shown in Figure 8. Figure 9 and Tables 6 and 7 show the cooling rates
of each bottle. All 12 bottles break from the phase change in the range of 769 minutes to 977
minutes. This range encompasses that of the 6 bottle range, and is within 2 minutes of the 1
bottle test. This shows that the freezing rate is independent of product load. Additionally, all 12
bottle cooling rates from -5oC to -30oC were in the desired range, just like the 6 bottle and 1
bottle tests. These results show that the cooling rate is not dependent on bag position in the
chamber or load.
Figure 8. Standard 12 Bottle Positions in Chamber.
All the odd numbered bottles, positioned on the left side of the chamber, broke from the phase
transition in less time than each even numbered bottle positioned beside them. This is to be
expected since the air flow enters the chamber from the top left corner and flows down and out
of the left duct toward the right side.
Bottle 4 had the fastest cooling rate from -5oC to -30oC. This was expected because the other
3 bottles on the top shelf were ≤ -20oC when bottle 4 broke from the phase change. Bottle 9 had
the slowest cooling rate from -5oC to -30oC and Bottle 10 was the last to break from the phase
transition. These results are again expected since the bottom floor of the cabinet has the least
amount of air flow in contact with the bottles.
1 2
3 4
5 6
7 8
9 10
11 12
7
Figure 9.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 500.0 1000.0 1500.0 2000.0 2500.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Freeze Test 5
12 9-L Bottles
Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 Bottle 6 Bottle 7
Bottle 8 Bottle 9 Bottle 10 Bottle 11 Bottle 12 Control
Table 6. Test 5 Cooling Rates for Bottles 1-8.
Temp. Range (oC) 1 2 3 4 5 6 7 8
-5 to -10 0.47 0.41 0.48 0.68 0.27 0.33 0.33 0.24
-10 to -20 0.51 0.38 0.44 0.86 0.30 0.37 0.30 0.32
-20 to -30 0.33 0.27 0.29 0.28 0.28 0.23 0.26 0.22
Cooling Rate (oC/min)
Bottle #
Table 7. Test 5 Cooling Rates for Bottles 9-12.
Temp. Range (oC) 9 10 11 12
-5 to -10 0.24 0.41 0.25 0.61
-10 to -20 0.28 0.50 0.28 0.60
-20 to -30 0.24 0.19 0.27 0.22
Bottle #
Cooling Rate (oC/min)
Controlled Thaw Rate Results and Discussion: It has been reported that fast thaw rates are best for avoiding damage to biological
formulations.3 However, most biological formulations are temperature sensitive. Therefore, this
study shows how the maximum temperature of the product could be controlled as well as
controlled thaw rates. The thaw test temperature profile starts with product temperatures at
-80oC and ramps to +20oC as fast as possible. For the purpose of this study, the time it took for
each bottle to reach to +2oC is recorded as the thaw time. Test 6 thaw results are shown in
Figure 10. The 12 bottles reached +2oC in the range of 807 minutes (Bottle 1) to 1549 minutes
(Bottle 8). As was the case with odd numbered bottles, positioned on the left side of the
chamber, freezing faster, they also thaw faster than each even numbered bottle positioned beside
them. The odd numbered bottles show relatively tight thaw rate control with 479 minutes
separating the thaw time of the 1st odd numbered bottle from the last odd numbered bottle. The
8
Figure 10.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 500.0 1000.0 1500.0 2000.0 2500.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Thaw Test 6
12 9-L Bottles
Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 Bottle 6 Bottle 7
Bottle 8 Bottle 9 Bottle 10 Bottle 11 Bottle 12 Control
even numbered bottles also show relatively tight thaw rate control with 446 minutes separating
the thaw time of the 1st even numbered bottle from the last even numbered bottle.
In order to test the effect of load, thaw Test 7 was conducted with the same conditions using 6
bottles (Figure 11). The 6 bottles reached +2oC in the range of 892 minutes (Bottle 1) to 1412
minutes (Bottle 6). These thaw times are within the range of the 12 bottles in Test 6. Thus thaw
rates are independent of load. Again, the odd and even numbered bottle thaw rates were
controlled independent of the bag position in the chamber.
Figure 11.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0 500 1000 1500 2000 2500
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Thaw Test 7
6 9-L Bottles
Bottle 1 Bottle 2 Bottle 3 Bottle 4 Bottle 5 Bottle 6 Control
To further test the effect of load, thaw Test 8 was conducted with the same conditions using 1
bottle (Figure 12). The bag reached +2oC in 836 minutes, which is within the range of all 12
bottles in Test 6. This shows that thaw rate was controlled independent of load.
9
Figure 12.
-100.00
-80.00
-60.00
-40.00
-20.00
0.00
20.00
40.00
0.0 200.0 400.0 600.0 800.0 1000.0 1200.0 1400.0 1600.0 1800.0 2000.0
Tem
per
atu
re (
oC
)
Time (Minutes)
Controlled Rate Thaw Test 8
1 9-L Bottle
Bottle 1 Control
Conclusions
A method was successfully developed to control the desired freezing and thawing rate using a
Farrar Scientific Model 4002 Controlled Rate Freeze/Thaw Chamber. It was validated with
different loads (1, 6, and 12 bottles) and product positions in the chamber. A program was
developed that a client can use to reliably and uniformly bulk freeze and thaw their biological
formulation.
References:
1. King J, Freezing and Thawing Rate Controlled Validation 16-L Bag Study. BioProcess
Int. May 2016: 1-4.
2. Puri M, et al. Evaluating Freeze-Thaw Processes in Biopharmaceutical Development –
Small-Scale Study Designs. BioProcess Int. 13(1) 2015: 34-45.
3. Cao E, et al. Effect of Freezing and Thawing Rates on Denaturation of Proteins in
Aqueous Solutions. Biotechnol. Bioeng. 82(6) 2003: 684-690.
Technical Article for BioProcess International Magazine
March 7, 2016
Author: Jerry King, Ph. D.
Senior Scientist
Farrar Scientific
Office: 740-374-8300
Email: [email protected]
