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imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

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imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Executive Summary

The purpose of this mixing study is to characterize the performance of ASI’s imPULSE Single-Use Mixing System in a range
of sizes and solution viscosity. Conductivity, pH and optical density, where applicable, were monitored to indicate the time
required to achieve a uniform solution. In addition, the heat transfer capability of jacketed systems was also evaluated by
recording the temperature at various points over time while mixing.

The solutions used in the tests were characterized as:

• Buffer Salts in Water, Low Viscosity 1 cP (Phosphate Buffered Saline – PBS)
• Buffer Salt in Corn Syrup / Water, Medium Viscosity 170 cP (Sodium Chloride)
• Buffer Salts in Corn Syrup / Water, High Viscosity 790 cP (Sodium Chloride)
• Media Powder in Water (Bovine Serum Albumin – BSA)
• Temperature Mapping Study (Water)



Background

ASI is a leading global provider of advanced single-use systems for the Healthcare and Life Sciences Industries. ASI
contracted with Foster Wheeler Biokinetics, an industry leader in the full service engineering, design, construction and
validation of biopharmaceutical facilities worldwide to perform mixing studies on the imPULSE Single-Use Mixing System.

The imPULSE Mixing System uses irradiated disposable Class VI compliant process contact surfaces; each mixing bag
contains a patented bottom mounted mixing device. The product is available in volumes from 30 to 5,000 Liters.

The mixing device is an integrated part of the disposable bag and its shape is engineered with multiple slots and film flaps.
The flaps open and close as the mixing device moves up and down throughout the mixing bag. On the downstroke, the
flaps close and energy is directed toward the bottom of the mixing bag and up the sidewalls. On the upstroke the flaps
open allowing the fluid to flow through the slots, thus producing one way flow and very effective mixing.

Upstroke Downstroke

2 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Introduction

This study presents the overall mixing performances of the imPULSE Single-Use Mixing Systems for large scale
preparation of media. The sizes selected for this study are the 250 (Jacketed), 750 and 1,500 Liters. This range of sizes
represent the imPULSE System’s mixing performance in small, medium and large vessels.

This summary has been developed from evaluation tests that were performed in ASI’s Test Center in Millersburg, PA on
July 18, 2012.

The following tests were performed:

• Dissolving of Phosphate Buffered Saline (PBS) in Water – A common buffer salt, which settles to the bottom of liquids
• Dissolving of Sodium Chloride in a Corn Syrup / Water Solution – Two different concentrations were used to simulate

medium and high viscosity liquid
• Mixing Bovine Serum Albumin – A media powder which floats on the surface and can be difficult to hydrate with water
• Temperature Mapping in a Jacketed imPULSE Mixing System – Mixing Water

Calibrated Instruments were used and the measuring parameter(s) were recorded over time.

Materials and Methods

The list of Materials and Equipment Used for this Application:

• Standard Single-Use Mixing Bag for imPULSE (Sizes 250 L, 750 L, 1,500 L)
• imPULSE Mixing System Equipment (Sizes 250 L, 750 L, 1,500 L)
• Mixing Devices
- 250 L: Diameter (in.) 12.0 and Stroke (in.) 8
- 750 L: Diameter (in.) 17.0 and Stroke (in.) 8
- 1,500 L: Diameter (in.) 21.0 and Stroke (in.) 8
• Instruments, Meters and Testers Used in This Study
- pH: Oakton, Multi-parameter PCS Tester 35
- Conductivity: YSI Model Pro Plus with 6051030 Cable and conductivity probe
- Optical Density: Ocean Optics USB 4000 Spectrophotometer Serial Number USB4A00240
- Temperature Recorder: Monarch Instruments Data Chart 6000
- Viscometer: Cannon-Fenske Routine Viscometer 100 to 600 cSt range
• Chemical Supplies Used in This Study
- Sodium Chloride
- Potassium Chloride
- Sodium Phosphate Dibasic
- Potassium Phosphate Monobasic
- Bovine Serum Albumin
- Corn Syrup

3 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Methods Used
Mixing of Buffer Salts (PBS) in Water (Low Viscosity)
The objective of this test is to characterize the time to mix common buffer salts in a low viscosity solution (water) using
ASI’s imPULSE Single-Use Mixing Systems. Phosphate Buffered Saline (PBS), a common buffer salt, which sinks to the
bottom of vessel is used as the test ingredient. Tests were performed on the 250 L, 750 L and 1,500 L mixing systems with
PBS as the test ingredient.
250 L Mixing Results
Conductivity was recorded every 30 seconds. Temperature and pH measurements were also recorded at various intervals.

Conductivity with Time

After two minutes of mixing, the conductivity measurements level off and remain constant for the remainder of the study.
These results show the buffer salts are dissolved in the 250 Liters of water within two minutes. This was confirmed visually.
750 L Mixing Results
Conductivity was recorded every 30 seconds. Temperature and pH measurements were also recorded at various intervals.

Conductivity with Time

After one and one-half minutes of mixing, the conductivity measurements level off and remain constant for the remainder
of the study. These results show the buffer salts are dissolved in the 750 Liters of water within one and one half minutes.
This was confirmed visually.

4 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

1,500 L Mixing Results
Conductivity was recorded every 30 seconds. Temperature and pH measurements were also recorded at various intervals.

Conductivity with Time

After thirty seconds of mixing, the conductivity measurements level off and remain constant for the remainder of the
study. These results show the buffer salts are dissolved in the 1,500 Liters of water within thirty seconds. This was
confirmed visually.

Mixing of Sodium Chloride in corn syrup / water (Medium and High Viscosity)
The objective of this test is to characterize the time to mix a common buffer salt in medium and high viscosity solutions
(corn syrup / water) using ASI’s imPULSE Single-Use Mixing Systems. Tests were performed on the 250 L Single-Use Mixing
System with Sodium Chloride as the test ingredient. Medium and high viscosity solutions to test dissolution of the sodium
chloride were achieved by mixing corn syrup with water. 75% by volume corn syrup presents a medium viscosity solution,
while an 82% by volume concentration is high viscosity.

Medium Viscosity Results
Conductivity was recorded every 30 seconds. Temperature and pH measurements were also recorded at various intervals.
• Initial Temperature: 25.5 oC
• Viscosity as measured: 131.7 centiStokes (cSt)
• Density of mix contents was determined by weighing 50 ml of the corn syrup solution.
• Weight of syringe with 50 ml of corn syrup: 64.33g
• Density : 64.33g / 50.0 ml or 1.287 g/ml
• Viscosity of solution in cP (density x cSt): 170 cP

5 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

170 cP, Conductivity with Time

After four and one-half minutes of mixing, the conductivity measurements level off and remain constant for the remainder
of the study. These results show the buffer salts are dissolved in the 250 Liters of corn syrup solution, 170 cP within four
and one half minutes. This was confirmed visually.
High Viscosity Results
Conductivity was recorded every 30 seconds. Temperature and pH measurements were also recorded at various intervals.
• Initial Temperature: 28.4 oC
• Viscosity as measured: 587 centiStokes (cSt)
• Density of mixer contents was determined by weighing 50 ml of corn syrup solution.
- Weight of syringe with 50 ml of corn syrup: 67.2 g
- Density: 67.2 g / 50.0 ml or 1.344 g/ml
• Viscosity of solution in cP (density x cSt): 790 cP

790 cP, Conductivity with Time

The conductivity continued to rise with time about 1% per minute after 20 minutes of mixing. The test was concluded after
41 minutes of mixing.

6 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Mixing of Media Powder in Water
The objective of this test is to characterize the time to mix media powders in water using ASI’s imPULSE Single-Use
Mixing Systems.
Bovine Serum Albumin (BSA) was used to simulate media powder components resembling amino acids and supplements.
BSA is a hydroscopic powder that lies on top of water until dissolved. BSA dissolution progress is measured using optical
density measurements at 280nm (OD280).
Optical density measurements at 280nm were recorded with time.

OD 280 with Time

After four minutes of mixing, the optical density measurements level off and remain constant for the remainder of the
study. These results show the powder is dissolved in 250 Liters of water within four minutes. This was confirmed visually.

Temperature Mapping (Water)
The objective of this test is to characterize the temperature uniformity within the mixing bag of ASI’s imPULSE
Single-Use Mixing System.
Tests were performed on the 250 L Single-Use Mixing System with water as the test ingredient. Four thermocouples
equally spaced vertically in the bag recorded temperature of the process fluid over time during heat up and cool
down cycles.

7 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Heating Cycle
Analysis of heating thermocouple data indicates all data is well within success criteria of +/- 1 oC variation.

60
50

40

Heating Cycle
30

Variance

20

10

0

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170
Time

Cooling Cycle
Analysis of cooling thermocouple data indicates all data is well within success criteria of +/- 1o C variation.

40 Cooling Cycle
35 Variance
30
25
20
15
10

5
0

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110
Time

8 | imPULSE Mixing Study Summary

imPULSE Mixing Study Summary

Study of the imPULSE Single-Use Mixing Systems Performance:
Rapid Preparation of Media Powders in Low and High Viscosity Solutions.

Conclusion
Mixing studies were performed on the imPULSE Mixing Systems in various sizes (250, 750 and 1,500 Liters).
The purpose of the tests was to challenge the mixing with different criteria and record the results.
The challenges included:
• Mixing Buffer Salts which settle to the bottom of solutions in water at various volumes
• Mixing Sodium Chloride Salt in 250 Liters of a medium and a high viscosity corn syrup solution
• Mixing a Media Powder, which floats on the surface of the liquid and is difficult to hydrate in 250 Liters of water
• Heating and cooling 250 Liters of water while measuring the temperature at various locations throughout the mixing
• bag to determine uniformity.
The results indicate that ASI’s unique mixing device mixes powders and solutions efficiently in small, medium and
large vessels.
• Buffer Salts are dissolved in water in less than four (4) minutes
• Buffer Salts dissolve in a medium viscosity solution within five (5) minutes
• The ASI Mixing System proved effective at mixing Buffer Salts in high viscosity solutions, close to 1000 cP. The results
• indicate that the conductivity of the solution was changing by 1% every minute after 20 minutes of mixing,
• Media Powders which tend to float and resist hydration were dissolved within 4 minutes
• The vessel jacket designs of the imPULSE Mixing System proved effective at maintaining temperature uniformity
• throughout the process fluid during heat up and cool down cycles.

9 | imPULSE Mixing Study Summary