thermo scientific hyperforma single-use bioreactor systems...

Thermo ScientificHyPerforma Single-Use Bioreactor Systems Validation Guide

Validation

Gu

ide

Revision ADOC0016 • December 2015

Thermo Scientific 2 Single-Use Bioreactor (S.U.B.)

Table of Contents Scope ......................................................................................................4

Validation2.1 Introduction ......................................................................................52.2 Process Qualification .........................................................................52.3 Sterility Assurance Level ....................................................................52.4 Endotoxin and Particulate .................................................................52.5 Functionality .....................................................................................6

Sparger Designs ...........................................................................8Probe Assembly Design .............................................................10Individual Component Evaluation ............................................13Probe Assembly Evaluation ........................................................17

2.6 Oxygen Transfer ..............................................................................212.7 Mixing Studies ................................................................................352.8 Additional 2,000L Studies ...............................................................432.9 S.U.B. 50 to 1,000L Temperature Mapping Studies ........................472.10 Sterility Testing..............................................................................56

Condenser System Validation 3.1 Functional Overview .......................................................................603.2 Peristaltic Pump ..............................................................................623.3 Chiller (TCU) .................................................................................633.4 Condenser Plate Assembly Functional Overview .............................643.5 Condenser Testing ...........................................................................72

Quality Control 4.1 Introduction ....................................................................................744.2 Inspection .......................................................................................74 Incoming Inspection In-Process Inspections and Testing4.3 BPC Lot Record Release and Certificate of Analysis .......................754.4 Sample C of A .................................................................................764.5 Traceability ......................................................................................774.6 Shelf Life .........................................................................................774.7 Sample Certificate of Irradiation......................................................78

Section 1

Section 2

Section 3

Section 4


Regulatory 5.1 General ...........................................................................................795.2 BPCs ...............................................................................................79

Appendix 6.1 References .......................................................................................806.2 Abbreviations and Acronyms ...........................................................806.3 Legacy Oxygen Transfer Data ..........................................................826.4 Test Methods for Comparing Drilled Hole to Open Pipe Spargers ....87

Section 6

Section 5


Section 1Validation Overview

Section 1 Scope

This validation guide contains information about standard Thermo Scientific™ HyPerforma™ Single-Use Bioreactors (S.U.B.s) and includes the Thermo Scientific™ HyPerforma™ S.U.B. systems, component lists, test methods and results, product design verification methods, operational qualification methods, quality control, validation and regulatory information.

Validation for the Thermo Scientific™ HyPerforma™ BPCs used in the HyPerforma S.U.B. systems can be found in the Thermo Scientific™ BioProcess Container validation guide. Please contact your local sales representative for more information.

The standard component library maybe updated at any time. As of the date of this publication the data in this guide is current and correct.

1.1 Scope


Section 2Validation

Section 2 Validation

Product and process validations have been developed and implemented as defined in the BPC validation master plan, which is in compliance with the concepts of cGMP for medical devices. Meaningful product validations demonstrate compliance with release criteria and approved product claims.

Irradiation, endotoxin and particulate validation is based on current testing standards. These tests evaluate manufacturing conditions as well as product cleanliness and consistency.

Process verification consists of a production build and validation testing when a new product or change in manufacturing process is introduced. The production build takes place under the defined specification. The acceptance criteria set from the process verification results or design of experiment (DOE) are utilized as the requirements for validation testing. BPCs are tested functionally when applicable. Validation testing verifies and confirms that product manufactured according to the Batch Record meets the acceptance criteria set by engineering during the verification phase.

Validation of the gamma irradiation sterility assurance level (SAL) for BPC is performed per ANSI/AAMI/ISO 11137-2:2006 guideline. The standard outlines the VDmax25 test methods following the use of a “simulated product.” The test kit consists of a 200L chamber and a representative sample of connectors and tubing used on standard BPCs. This BPC is referred to as the “monster bag” and evaluates a worst case configuration for sterility. This method validates a minimum irradiation dose of 25kGy for all products and provides a sterility assurance level (SAL) of 10-6.

Process validations and monitoring are established for endotoxin and particulate for the manufacture of BPCs. Particulate (USP 788) and endotoxin (USP 85) assays in conjunction with bioburden testing on sample flexible containers to demonstrate product consistency are performed. Test samples of BPCs consistently meet or exceed the acceptance criteria.

2.1 Introduction

2.2 Process Verification

2.3 Sterility Assurance Level

2.4 Endotoxin and Particulate


Section 2Validation

Descriptions of the design of individual components of the S.U.B. are provided in the following sections. Highlights of the designs of the bearing port, impeller, sparger and probe assembly are included.

The following section details the individual components comprising the hub assembly. See figure 2.1 for components.

2.5 Functionality

Figure 2.1 Hub assembly

Individual Component Designs

Hub Assembly Design

Highlights of the hub assembly design include the following:• Under-cut lip which allows snap in feature of the seal-cup.• Locating lip to interface with drive motor housing.• Tortuous sterility path when fully assembled with all components.• Polyethylene construction for seal capability with film.

Highlights of the design are the following:• Seven snap legs. These legs take minimal force to snap in seal cup

to hub assembly, but a larger force to remove seal cup.• Three concentric rings for seal and gasket placement.• Animal-derived component-free polycarbonate construction.

Seal Cup Design


Section 2Validation

V-Ring Seals Details of the seal are as follows:• High wear resistance Viton™ o-rings.• Reliable sealing capabilities. • Animal-derived component-free construction.

Silicone gasket provides a seal between bearing port and contact fluids. Details of the gasket:

• Animal-derived component-free construction.• Molded from qualified Silicone.

Hub assembly design makes use of two sealed bearings. This locks the hub to the bearing port and eliminates the failure mode of the hub slipping and causing seal misalignment.

An oil seal is used as a dust cover to enclose the assembly.

Stainless steel construction due to product contact nature. This piece provides the hollow pass through for the drive shaft to couple with the mixing impeller. Provides sealing counter faces for the v-ring seals.

• Stainless steel 316 construction.• Hexagon profile couples with drive shaft. • Sealing counter faces: machined to a high quality polished surface.

The geometry of each impeller was defined by the following guidelines and illustrated in figure 2.2:

• Style: Pitched Blade• Impeller Diameter: 14.6cm (5.75”); 20.0cm (7.875”) or 25.1cm

(9.875”)• Blade Angle: 45°• Number of Blades: 3• Impeller Tubing: C-Flex™ tubing, animal-derived component-free

Gasket

Bearings

Dust Cover

Hub Design

Impeller Design

Figure 2.2 Impeller and tubing design


Section 2Validation

Sparger Designs

Open Pipe Sparger The open pipe sparger design (figure 2.3) is based on a simple open pipe formed from a molded silicone port adapter which extends 3.8cm (1.5”) into the standard S.U.B. BPC. The inner diameter (ID) of the open pipe sparger varies across the S.U.B. product line as indicated in table 2.1.

Porous Frit Sparger The porous frit sparger design is based on a PVDF (polyvinylidene fluoride) frit with 20-40µm pore size. The porous frit sparger is 12mm (0.472”) in diameter and extends 8cm (3”) into the standard S.U.B. BPC. This is the same in all standard S.U.B. BPCs in sizes from 50 to 1,000L (figure 2.4). The 2,000L standard BPC that uses an open pipe sparger includes three porous frit spargers. The 2,000L standard BPC with drilled hole spargers contains two porous frit spargers along with the two drilled hole spargers.

Figure 2.3 Open pipe sparger

Figure 2.4 Porous Frit Sparger

Size ID

50L 3.18mm (0.125”)

100L 3.18mm (0.125”)

250L 4.78mm (0.188”)

500L 6.35mm (0.25”)

1,000L 6.35mm (0.25”)

2,000L 6.35mm (0.25”)

Table 2.1 Open pipe sparger ID


Section 2Validation

Drilled Hole Sparger The drilled hole sparger (figure 2.5 and 2.6) is a film-based sparger disc with drilled pores of specific sizes and quantities, tailored to various S.U.B. sizes as shown in table 2.2. It is intended as a macro sparger, and used in combination with a porous frit sparger (micro) in one style of the standard S.U.B. BPC.

Figure 2.5 Drilled hole sparger cross-section

System Size Standard Sparger Configuration Drilled Hole Sparger Configuration

50L Porous Frit and Drilled Hole Spargers 9cm (3.5”) disk with 360 x 0.178mm holes




1,000L Porous Frit and Drilled Hole Spargers 17cm (6.75”) disk with 1,180 x 0.445mm holes

2,000L Porous Frit and Drilled Hole Spargers2x 17cm (6.75”) disks with 690 x 0.582mm holes

(1,380 holes)

Figure 2.6 Drilled hole sparger top view

Table 2.2 Drilled hole sparger configuration by volume


Section 2Validation

Adapter Nut Fitting The design intent of the adapter is to provide an interface for the dissolved oxygen (DO), pH probes and the sleeve fitting. The probe adapter nut was designed to interface with the probe (threaded section) and sleeve fitting (barb section).

Probe Assembly Design

Figure 2.7 Model of probe adapter nut

The barb feature of the adapter nut provides for an expansion of the sleeve with sufficient area for the cable tie attachment. The part design captures a 3-thread minimum, and allows for a theoretical full compression of the o-ring to the Teflon™ backing ring. Two flats on the threaded section outer diameter (OD) allow the operator to wrench down the probe if required. The threads have torque strength seven times (7x) greater than the manufacturer-supplied probe plastic threads. Tests for maximum torque

Figure 2.8 Model showing the probe, adapter nut and sleeve assembly

Probe Interface

Sleeve Fitting

SleeveAdapter Nut

Probe


Section 2Validation

strength were obtained using a stainless steel probe and a PC adapter fitting. Average maximum torque strength values (to failure) were 233.75 in-lbs for pre-irradiated samples and 234.1 in-lbs in post-irradiated samples.

The flange on the probe interface section is designed as a positive stop for the sleeve, and as a retention feature to secure the position of the probe relative to the S.U.B. hardware. The adapter cavity is designed to allow the Kleenpak™ connector to fit in this area providing an additional 2.54cm (1.0”) of compression on the sleeve to account for manufacturer variation in probe length.

The sleeve fitting (figure 2.9) is designed to connect the Kleenpak connector to the adapter nut fitting. The sleeve functions to displace the probe tip approximately 8.89cm (3.5”) from the Kleenpak connector interface into the chamber at a nominal 2.54cm (1.0”) depth. The sleeve will accommodate the DO/pH probes (figure 2.10 and 2.11) from major probe suppliers such as those listed in table 2.2.

Sleeve Fitting

Manufacturer

Required Specifications to Fit Probe Assembly

Length From O-ring (mm)

Length From O-ring (mm)

Diameter(mm)

Applikon Biotechnology – DO 235 235 12

Applikon Biotechnology – pH 235 235 12

Mettler Toledo – DO 215 215 12

Mettler Toledo – pH 195 219 12

Broadley James – DO 215 214 12

Broadley James – pH 225 219 12

Finesse Solutions – DO 225 220 12

Finesse Solutions – pH 225 220 12

Table 2.3 Specifications for resuable probes for use in the probe assembly

Figure 2.9 Model of probe sleeve fitting


Section 2Validation

Figure 2.10 Model of the probe assembly with a probe installed

Figure 2.11 Probe extended into the chamber

Pressure testing was performed on the sleeve/adapter nut/Kleenpak connector assembly. Test data indicated no leaks up to a yield pressure of 7psig. The pressure rating for the sleeve is defined at 3.5psi (2x safety factor), which corresponds to an 2.44m (8.0’) hydrostatic column of water.

Cycle fatigue testing was performed on five sleeve samples. Each unit was cycled (extended : compressed) 500 times, followed by a leak test at 3psig. No failures, defects or anomalies were observed.

Aseptic connections through the S.U.B. ports are made using the Kleenpak connectors. The design of the connector includes peel away strips which cover both the male and female section. Once the male to female connection is made, the strips are removed which completes the fluid pathway while maintaining the sterility of the system. For further information regarding the Kleenpak connector design and testing, refer to the Pall’s Kleenpak Connector Validation Guide.

The temperature/sampling (TS) fitting is designed from silicone material to provide a sleeve for a temperature probe and a tube for direct sampling. The part was designed with a 4.76mm (0.1875”) ID x 1.59mm (0.0625”) wall thickness silicone tube to accommodate a 3.175mm (0.125”) OD temperature probe. The silicone material type and wall thickness prevent the tube from sealing off when kinked during the packaging of the S.U.B. BPC. A polypropylene luer insert is installed in the temperature line to prevent tube diameter restriction that would prevent the probe from being inserted. This also served for retention of the RTD (resistance temperature detector) when in use.

The chamber design met the design input requirements for geometry and porting requirements.

Kleenpak Connectors

Temperature/Sampling Fitting Design

Chamber Design


Section 2Validation

Individual Component Evaluation

Figure 2.12 is a cross sectional view of the hub assembly and a three-dimensional view of the seal cup.

Hub Assembly Evaluation (S.U.B. Testing)

Seal Cup

Seal Cup Engagement Test

Figure 2.12 Seal cup design

This evaluation was to verify that all snap legs on the seal cup were properly seated in the hub assembly. A total of 46 hub assemblies were evaluated; 24 samples evaluated upon completion of full functional testing post-irradiation and 22 samples upon completion of leak/burst testing pre-irradiation. Results were that all legs were fully seated except those legs that were either partially or fully located in the lifter gap. Those legs did flex out into their relaxed free state and would be captured by the undercut lip if it had existed in that area.

All samples met the requirement of no unseated engagement legs.


Section 2Validation

Seal Cup Disengagement Test

This testing was done to determine the forces required to disengage the fully seated seal cup from the hub assembly. Samples tested were pre-irradiation and consisted only of the bearing port and seal cup. Samples were fixed on the Instron tensile test machine, and tested under tensile load at a crosshead speed of 12.7cm (5”) per minute. No requirements were set for this test and it was conducted for information only.

Table 2.3 results show an average removal force of 277.5lbf with a standard deviation of 26.3lbf. The large standard deviation results from the varying loads due to the random alignment of the seal cup leg relative to the bearing port. The resulting lower 3-σ value of 198.6lbf is acceptable due to no tensile load being applied to the seal cup during use.

Table 2.4 Seal cup disengagement results

Seal Cup Disengagement1/27/2006Rate: 12.7cm (5”) per minute

Sample lbf

1 293.3 2 281.93 267.54 300.15 322.26 242.87 255.08 293.89 278.310 240.3

Stats

ave 277.5stdev 26.3min 240.3max 322.2

V-Seal Wear Evaluation The installation parameters of the v-ring seals (figure 2.13) are critical to the functionality of the system. The critical parameters being the stretch of the inner diameter of the seal and the compression of the lip. This lead to optimization of the seal installation for the S.U.B. application through extensive testing evaluating stretch and lip compression. The resulting seal final assembly performed as intended.


Section 2Validation

Figure 2.13 Hub assembly cross-section

Sterility Testing of Hub Assemblies

A final sterility run was conducted on a total of seven samples. Samples one to four contained the product contact 35mm seal only, samples five to seven contained the upper 50mm seal only (see figures 2.14 and 2.15). This arrangement allowed testing and demonstrating reliability with single seals, thus when multiple seals are used in the final configuration they are redundancies to the system to improve reliability. Hub assemblies were sealed into sample BPCs that were then filled with TSB by the liquid media group. Access holes were drilled into the port bodies to directly challenge the seals with positive growth media. House air was filtered into the BPCs to create a positive pressure environment. Testing was conducted in the validation incubation room.

Figure 2.14 Tests #10-1 to 10-4, product contact v-seal only


Section 2Validation

Samples were tested over a five month period resulting in a total of 28.9 million revolutions. The design requirement is 120rpm for 21 days, which equates to 3.63 million revolutions. All samples met the requirement of no sterility failures.

This testing was conducted to test the square profile gasket. Samples were molded out of silicone. In use, the gasket will experience an approximate pressure range of 0.5 to 1.0psi. Testing was limited to 40psi maximum due to safety concerns. Results show that all samples were tested up to 40psi with no leaks. All samples passed the requirement of >5psi without leakage.

This testing was to evaluate the hub assembly for functionality and durability. A total of eight samples were assembled in the clean room and gamma irradiated. The hub assembly was tested for seven days at 360rpm. This resulted in the required number of revolutions based upon the 21-day run at 120rpm requirement established in the design input. Testing was accomplished on prototype test stands; samples did not house a drive shaft, impeller, and were not sealed into a BPC. Results were that all samples functioned properly with no issues and all engagement legs were fully seated. Samples were taken apart upon testing conclusion and were visually evaluated. Results show that the v-ring seals demonstrated a consistent wear pattern and performed as intended. No abnormalities were noted.

Figure 2.15 Tests #10-5 to 10-7, non-product contact v-seal only

Gasket Pressure Test

Hub Sub-Assembly Testing


Section 2Validation

Impeller to Hub Tubing Pull Test

Testing was performed to evaluate the connection of the impeller tubing to the impeller on one end and the stainless hub on the other end. The tubing tested was 19.05mm (0.75”) ID, 3.175mm (0.125”) wall thickness animal-derived component-free C-Flex™ tubing, supplied from the vendor at a specified pre-cut length. A total of ten samples were scheduled for testing; one sample was not tested. The samples were tested after the supported burst testing. Samples were tested, and results verified that the pull strength of the connection exceeded normal forces during drive shaft insertion into the impeller assembly.

Probe Assembly Evaluation

Leak/Burst Testing Ten probe assemblies were subjected to a leak burst test. The units were pressured to 5psi, held for two minutes, then examined for leaks in the individual components and connection points by pressure decay and water immersion test methods. Test results indicated that the parts maintained integrity and demonstrated average yield strength of 7.7psi (see table 2.5). The maximum pressure of the system during operation is the hydrostatic pressure of 2.5 to 3.0psi. The system allows for a >2x margin of safety.

Sample Hold Pressure(psi)

Yield Pressure(psi)

Burst Pressure(psi)

Leak /No Leak Burst Location

1 6.700 7.660 6.710 No Leak Sleeve Material2 5.330 7.490 6.020 No Leak Sleeve Material3 5.290 7.730 6.160 No Leak Sleeve Material4 5.370 7.620 5.920 No Leak Sleeve Material5 5.410 7.630 5.970 No Leak Sleeve Material6 5.280 7.580 6.120 No Leak Sleeve Material7 5.250 7.630 5.920 No Leak Sleeve Material8 5.370 7.630 5.880 No Leak Sleeve Material9 5.180 7.720 5.900 No Leak Sleeve Material

10 5.220 7.770 6.090 No Leak Sleeve MaterialAvgSD

5.440.45

7.650.08

6.070.25

Tube Port Testing The lip seal is designed to allow the probe/fitting to penetrate through the flange section, while creating a seal. Four samples each were subjected to a 15psi seal integrity test using a standard 12mm diameter probe and the silicone temperature/sampling fitting. The probe(s) and temperature/sampling fitting has a nominal OD of 12mm (0.4725”) and 13mm (0.52”), respectively. The design meets the 1.5psig requirement.

Pressure testing of eight samples indicated that the part met the minimum 15psig seal integrity with no yielding of the tube at a 30psig pressure.

Table 2.5 Leak burst test results


Section 2Validation

Nominal assembly forces were required to install the Kleenpak connector and various fittings. The tubing did not buckle during installation. Application use will not exceed 3.5psi (>9x safety factor).

The column height was designed to allow for a 30mm (1.18”) length section for a tube clamp between the flange and barb-fitting. The heavy duty tubing clamp has approximately 6.35mm (0.25”) side clearance for installation. No issues were observed for operators installing the clamp onto the tube. The TS fitting and tubing port was pressure tested to 20psig. No leaks were observed. Results indicated that the TS fitting and tubing port met the 15psig minimum pressure requirement.

Temperature mapping of the system was performed on TS assemblies. Two drops of glycerol were added to facilitate heat transfer. Normal S.U.B. ramp rates were approximately 0.07 to 0.12°C per minute during heat-up, which indicates that the system differential as 0.05°C. A rapid ramp rate was used to represent a worst case scenario of 2°C per minute which is a much more rapid rate than will be achieved in this S.U.B. system. Testing indicated that the sheathed probes lagged behind the controls during the ramp, but were within 0.05°C after 10 minutes of equilibrium at 37°C. The 3.175mm (0.125”) RTD was tested with and without sheaths.

Twenty-two tubing samples were tested post gamma irradiation (25 to 38kGy) for kinking. Samples of tubing were kinked with a slide clamp and cable tie to simulate a worst case kink in the tube. Data indicated that the silicone material showed no negative results after irradiation.

Figure 2.16 Model of the temperature/sampling (TS) fitting

Temperature Probe

Sample Tube


Section 2Validation

Graph 2.1 Temperature map profile (temperature/sampling fitting)

Tem

pera

ture

(˚C)

Time (min)

TS Fitting Temperature Profile

Stainless Steel Sheath (1/4) (1/4) Probe Control Silicone Sheath (1/8) 1/8 Probe Control

Time(min)

Temperature (˚C), 1/4” Probe Temperature (˚C), 1/4” Probe

SS-Sheath Control Delta SiliconeSheath Control Delta

5 0.485 0.328 0.157 0.598 0.458 0.14017 24.954 25.435 -0.481 24.692 25.700 -1.00834 37.049 37.056 -0.007 37.297 37.250 0.047

Ramp Rate: 2˚C/min

Table 2.6 Temperature offset summary

Eleven 50L and eleven 250L assembled S.U.B./S.U.M. BPCs were visually inspected. All containers met the visual requirements for appearance, quality and workmanship. There were no abnormalities noted.

Dimensional inspection was performed on each of the 50 and 250L finished S.U.B. BPCs. All measurements were within drawing specifications for both S.U.B.s.

Leak and burst testing was performed on the 50 and 250L S.U.B. BPC in an unsupported condition. Testing procedures included filling the S.U.B. BPC to 1psi and holding for two minutes. All units passed the test requirements with no issues. Average leak hold pressures for the 50 and 250L S.U.B. systems were 1.17 and 1.13psi. Average maximum pressures were 3.23 for the 50L and 1.82psi for the 250L S.U.B.

Assembled BPC Evaluation (S.U.B. Testing)

Visual Inspection

Dimensional Inspection

Leak/Burst Test – Unsupported


Section 2Validation

Sample Burst LocationMax

PressureFailure Type

(Material/Seam)Pass/Fail

1 Bearing port 3.21 Material Pass

2 Top seam 3.59 Material Pass

3 Top seam 3.69 Material Pass

Table 2.7 Half volume burst testing – 250L S.U.B. BPC

Leak/burst tests were performed on the 50 and 250L S.U.B. BPCs, supported in the corresponding size S.U.B. tank. Testing procedures included filling the S.U.B. BPC to 1psi and holding for two minutes. All of the S.U.B. BPCs passed the test requirements. Average leak hold pressures for the supported 50 and 250L S.U.B. BPCs were 1.154 and 1.064psi, respectively. Average maximum pressures were 6.758 psi for the 50L and 4.07psi for the 250L S.U.B. BPC.

Three samples of the 250L S.U.B. BPC were burst upon conclusion of a 21 day functional sterility test. S.U.B. BPCs were pressurized in order to generate burst data for liquid filled and supported conditions at an operating temperature of 37°C. Results below show location of burst to be in the headspace of the bag. The three samples passed the burst requirement of no seam failures.

Container Burst Strength Test – Supported

Film and Port Weld Seam Strengths

Peel tests were performed on all welded seams (film-to-film and port-to-film). All weld seam strengths met standard S.U.B. BPC requirements according to our control methods for manufacturing the 50 and 250L S.U.B. systems.


Section 2Validation

Introduction The S.U.B. BPC is designed to provide an acceptable range of kLa values to support rapid growth for an array of common cell platforms using the operating parameters shown in Table 2.7 for BPCs equipped with open pipe and porous frit spargers, and Table 2.8 for BPCs equipped with drilled hole and porous frit spargers.

Study Method Experiments were designed to estimate and model mass transfer of gasses in S.U.B. systems. For more information about methods and procedures, see the test methods detail in section 6.4 in the appendix of this manual.

2.6 Oxygen Transfer

S.U.B. BPCs Range of Operating Parameters with Open Pipe and Frit Spargers

50L 100L 250L 500L 1,000L 2,000LTemperature (°C) 2.0 - 40.0 ± 0.1

Operating Volume (L) 25 to 50 50 to 100 125 to 250 250 to 500 500 to 1,000 1,000 to 2,000

Agitation Rate (rpm) 30 to 200 30 to 200 30 to 150 30 to 150 20 to 110 20 to 75

Recommended Max. Gas Flow Rates

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

(3)

Over

lay

Air (slpm) 1 0.5 5 2 1 10 5 2.5 10 10 5 15 10 8 15 12 16 15

O2 (slpm) - 0.25 - - 0.5 - - 1.25 - - 2.5 - - 4 - - 8 -

CO2 (slpm) - 0.1 - - 0.2 - - 0.5 - - 1 - - 1 - - 1 -

N2 (slpm) - 0.25 - - 0.5 - - 1.25 - - 2.5 - - 2.5 - - 2.5 -

Total (slpm) 1 0.85 5 2 1.7 10 5 4.25 10 10 8.5 15 10 13 15 12 25 15

Exhaust Load (slpm) 20 20 20 40 40 90

S.U.B. BPCs Range of Operating Parameters with Drilled Hole and Frit Spargers50L 100L 250L 500L 1,000L 2,000L

Temperature (°C) 2.0 - 40.0 ± 0.1

Operating Volume (L) 25 to 50 50 to 100 125 to 250 250 to 500 500 to 1,000 1,000 to 2,000


Recommended Max. Gas Flow Rates Dr

illed

Hole

Poro

us F

rit

Over

lay

Drille

d Ho

le

Poro

us F

rit

Over

lay

Drille

d Ho

le

Poro

us F

rit

Over

lay

Drille

d Ho

le

Poro

us F

rit

Over

lay

Drille

d Ho

le

Poro

us F

rit

Over

lay

Drille

d Ho

le

Poro

us F

rit

Over

lay

Air (slpm) 2.5 1 5 5 2 10 12 4 14 25 6 35 100 8 60 200 16 1292

O2 (slpm) - 1 - - 2 - - 4 - - 6 - - 8 - - 16 -

CO2 (slpm) - 0.25 - - 0.5 - - 1 - - 1.5 - - 2 - - 4 -

N2 (slpm) - 1 - - 2 - - 4 - - 6 - - 8 - - 16 -

Total (slpm) 2.5 1.25 5 5 2.5 10 12 5 14 25 7.5 35 100 10 60 200 20 129

Exhaust Load (slpm) 20 20 90 90 180 270

Table 2.8 Operating parameters using open pipe and porous frit spargers

Table 2.9 Operating parameters using drilled hole and porous frit spagers


Section 2Validation

Results Overview Experiments were performed using 50, 250 and 2,000L vessels to measure the mass transfer of oxygen and CO2 stripping and the results for 100, 500 and 1,000L vessel sizes have been interpolated theoretically from those results.

The results in this section show the mass transfer of oxygen and CO2 stripping, and are presented as kLa for various sparge flow rates for each vessel size. Two dimensional plots are used to show individual sparger results for oxygen delivery and CO2 stripping, separately. Three dimensional plots are used to show the combined micro/macro (porous frit/open pipe or porous frit/drilled hole) sparger oxygen delivery behavior in terms of kLa response at different combined flow rates for each vessel size.

Results, unless otherwise specified, are at an agitation power input per volume (PIV) of 0.15 HP/1,000gal (29.6 W/m3).

Results for vessels using porous frit and open pipe spargers are presented first. Results for vessels using porous frit and Drilled Hole Spargers are presented in separate, subsequent sections.


Section 2Validation

0

0.5

1

1.5

22.5

33.5

44.5

5

0

5

10

15

20

25

30

35

40

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM

Combined kLa Oxygen Delivery At 0.15 HP / 1000gal Agitation

35-40

30-35

25-30

20-25

15-20

10-15

5-10

0-5

0.00

1.00

2.00

3.00

4.00

5.00

0 0.2 0.4 0.6 0.8 1 1.2

1/hr

kLa

slpm flow rate

Carbon Dioxide Stripping kLa with Frit Sparger

0.00

0.50

1.00

1.50

2.00

0 0.5 1 1.5 2 2.5 3

1/hr

kLa

slpm flow rate

Carbon Dioxide Stripping kLa with Open Pipe Sparger

Graph 2.4 Results for 50L S.U.B. with porous frit sparger

Graph 2.5 Results for 50L S.U.B. with open pipe sparger

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0 0.2 0.4 0.6 0.8 1 1.2

1/hr

kLa

slpm flow rate

Oxygen Delivery kLa with Frit Sparger

0.00

0.50

1.00

1.50

2.00

0 0.5 1 1.5 2 2.5 3

1/hr

kLa

slpm flow rate

Oxygen Delivery kLa with Open Pipe Sparger



50L Results with Porous Frit and Open Pipe Spargers

The results of experiments with 50L vessels using porous frit and open pipe spargers are shown below.

Graph 2.6 Results for 50L S.U.B. with both porous frit and open pipe spargers


Section 2Validation

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0 0.5 1 1.5 2 2.5

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


0.00

1.00

2.00

3.00

4.00

0 0.5 1 1.5 2 2.5

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


Graph 2.7 Interpolated results for 100L S.U.B. with porous frit sparger

Graph 2.11 Interpolated results for 100L S.U.B. with both porous frit and open pipe spargers

Graph 2.8 Interpolated results for 100L S.U.B. with open pipe sparger

Graph 2.9 Interpolated results for 100L S.U.B. with porous frit sparger

Graph 2.10 Interpolated results for 100L S.U.B. with open pipe sparger

0

1

2

3

45

67

89

10

0

5

10

15

20

25

30

35

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


30-35

25-30

20-25

15-20

10-15

5-10

0-5

The data shown below for 100L vessels is estimated, and has been interpolated from experimentally-derived 50 and 250L S.U.B. data and biased by vessel volume.



Section 2Validation


0.00

5.00

10.00

15.00

20.00

25.00

30.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

2.50

0 2 4 6 8 10 12 14

1/hr

kLa

slpm flow rate



Graph 2.16 Results for 250L S.U.B. with both porous frit and open pipe spargers




0

2.5

5

7.5

1012.5

1517.5

2022.5

25

0

5

10

15

20

25

30

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


25-30

20-25

15-20

10-15

5-10

0-5

0.00

1.00

2.00

3.00

4.00

0 2 4 6 8 10 12 14

1/hr

kLa

slpm flow rate


0.000.501.001.502.002.503.003.50

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


Results of experiments with 250L vessels using porous frit and open pipe spargers are shown below.


Section 2Validation


0.00

5.00

10.00

15.00

20.00

25.00

0 2 4 6 8 10 12

1/hr

kLa

slpm flow rate

Oxygen Delivery kLa for Frit Sparger

0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 5 10 15 20 25 30

1/hr

kLa

slpm flow rate


Graph 2.17 Interpolated Results for 500L S.U.B. with porous frit sparger

Graph 2.21 Interpolated Results for 500L S.U.B. with both porous frit and open pipe spargers

Graph 2.18 Interpolated Results for 500L S.U.B. with open pipe sparger


Graph 2.20 Interpolated Results for 500L S.U.B. with open pipe sparger

0

5

10

15

2025

3035

4045

50

0

5

10

15

20

25

30

35

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


30-35

25-30

20-25

15-20

10-15

5-10

0-5

0.00

5.00

10.00

15.00

20.00

25.00

0 2 4 6 8 10 12

1/hr

kLa

slpm flow rate

Oxygen Delivery kLa for Frit Sparger

0.000.501.001.502.002.503.003.50

0 2 4 6 8 10 12

1/hr

kLa

slpm flow rate


Data shown below for 500L vessels is estimated, and has been interpolated from experimentally-derived 250 and 2,000L vessel data and biased by vessel volume.


Section 2Validation

1,000L Results with Porous Frit and Open Pipe Spargers

0.00

5.00

10.00

15.00

20.00

25.00

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


-0.500.000.501.001.502.002.503.003.50

0 10 20 30 40 50 60

1/hr

kLa

slpm flow rate


Graph 2.22 Interpolated Results for 1,000L S.U.B. with porous frit sparger

Graph 2.26 Interpolated Results for 1,000L S.U.B. with both porous frit and open pipe spargers

Graph 2.23 Interpolated Results for 1,000L S.U.B. with open pipe sparger


Graph 2.25 Interpolated Results for 1,000L S.U.B. with open pipe sparger

0

10

20

30

4050

6070

8090

100

0

5

10

15

20

25

30

35

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


30-35

25-30

20-25

15-20

10-15

5-10

0-5

0.00

2.00

4.00

6.00

8.00

10.00

0 10 20 30 40 50 60

1/hr

kLa

slpm flow rate


-0.500.000.501.001.502.002.503.003.50

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


The data shown below for 1,000L vessels is estimated, and has been interpolated from experimentally-derived 250 and 2,000L vessel data and biased by vessel volume.


Section 2Validation

2,000L Results for Porous Frit and Open Pipe Spargers

The results of experiments with 2,000L vessels using porous frit and open pipe spargers are shown below.

0.00

5.00

10.00

15.00

20.00

25.00

0 10 20 30 40 50

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

2.50

0 10 20 30 40 50 60

1/hr

kLa

slpm flow rate


Graph 2.27 Results for 2,000L S.U.B. with porous frit sparger

Graph 2.31 Results for 2,000L S.U.B. with both porous frit and open pipe spargers

Graph 2.28 Results for 2,000L S.U.B. with open pipe sparger


Graph 2.30 Results for 2,000L S.U.B. with open pipe sparger

0.00

2.00

4.00

6.00

8.00

0 10 20 30 40 50 60

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 10 20 30 40 50

1/hr

kLa

slpm flow rate


0

20

40

60

80100

120140

160180

200

0

5

10

15

20

25

30

35

40

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


35-40

30-35

25-30

20-25

15-20

10-15

5-10

0-5


Section 2Validation

50L Results for Porous Frit and Drilled Hole Spargers

0.005.00

10.0015.0020.0025.0030.0035.0040.00

0 0.2 0.4 0.6 0.8 1 1.2

1/hr

kLa

slpm flow rate


0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 0.5 1 1.5 2 2.5 3

1/hr

kLa

slpm flow rate

Carbon Dioxide Spripping kLa with Drilled Hole Sparger


Graph 2.36 Results for 50L S.U.B. with both porous frit and drilled hole spargers

Graph 2.33 Results for 50L S.U.B. with drilled hole sparger



0

0.5

1

1.5

22.5

33.5

44.5

5

0

5

10

15

20

25

30

35

40

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


35-40

30-35

25-30

20-25

15-20

10-15

5-10

0-5

0.002.004.006.008.00

10.0012.00

0 0.5 1 1.5 2 2.5 3

1/hr

kLa

slpm flow rate

Oxygen Delivery kLa with Drilled Hole Sparger

0.000.501.001.502.002.503.003.504.00

0 0.2 0.4 0.6 0.8 1 1.2

1/hr

kLa

slpm flow rate


The results of experiments with 50L vessels using porous frit and drilled hole spargers are shown below.


Section 2Validation


-5.00

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

0 0.5 1 1.5 2 2.5

1/hr

kLa

slpm flow rate


0.001.002.003.004.005.006.007.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate

Carbon Dioxide Stripping kLa with Drilled Hole Sparger


Graph 2.41 Interpolated Results for 100L S.U.B. with both porous frit and drilled hole spargers

Graph 2.38 Interpolated Results for 100L S.U.B. with drilled hole sparger



0

1

2

3

45

67

89

10

0

5

10

15

20

25

30

35

40

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


35-40

30-35

25-30

20-25

15-20

10-15

5-10

0-5

-2.000.002.004.006.008.00

10.0012.0014.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


0.000.501.001.502.002.503.003.504.00

0 0.5 1 1.5 2 2.5

1/hr

kLa

slpm flow rate


The data shown below for 100L vessels is estimated, and has been interpolated from experimentally-derived 50 and 250L data and biased by vessel volume.


Section 2Validation


0.00

5.00

10.00

15.00

20.00

25.00

30.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


0.001.002.003.004.005.006.007.00

0 2 4 6 8 10 12 14

1/hr

kLa

slpm flow rate



Graph 2.46 Results for 250L S.U.B. with both porous frit and drilled hole spargers

Graph 2.43 Results for 250L S.U.B. with drilled Hole sparger



0

2.5

5

7.5

1012.5

1517.5

2022.5

25

0

5

10

15

20

25

30

35

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


30-35

25-30

20-25

15-20

10-15

5-10

0-5

0.002.004.006.008.00

10.0012.0014.00

0 2 4 6 8 10 12 14

1/hr

kLa

slpm flow rate


0.000.501.001.502.002.503.003.504.00

0 1 2 3 4 5 6

1/hr

kLa

slpm flow rate


Results of experiments with 250L vessels using porous frit and drilled hole spargers are shown below.


Section 2Validation


0.00

5.00

10.00

15.00

20.00

25.00

0 2 4 6 8 10 12

1/hr

kLa

slpm flow rate


0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 5 10 15 20 25 30

1/hr

kLa

slpm flow rate



Graph 2.51 Interpolated Results for 500L S.U.B. with both porous frit and drilled hole spargers




0.002.004.006.008.00

10.0012.00

0 5 10 15 20 25 30

1/hr

kLa

slpm flow rate


0.000.501.001.502.002.503.003.50

0 2 4 6 8 10 12

1/hr

kLa

slpm flow rate


The data shown below for 500L vessels is estimated, and has been interpolated from experimentally-derived 250 and 2,000L data and biased by pore diameter of the drilled hole spargers.

0

5

10

15

2025

3035

4045

50

0

5

10

15

20

25

30

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


25-30

20-25

15-20

10-15

5-10

0-5


Section 2Validation

1,000L Results for Porous Frit and Drilled Hole

Spargers

0.00

5.00

10.00

15.00

20.00

25.00

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


0.001.002.003.004.005.006.007.008.00

0 20 40 60 80 100 120

1/hr

kLa

slpm flow rate



Graph 2.56 Interpolated Results for 1,000L S.U.B. with both porous frit and drilled hole spargers

Graph 2.53 Interpolated Results for 1,000L S.U.B. with drilled hole sparger


Graph 2.55 Interpolated Results for 1,000L S.U.B. with drilled hole sparger

0

10

20

30

4050

6070

8090

100

0

5

10

15

20

25

30

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM

Combine kLa Oxygen Delivery At 0.15 HP / 1000gal Agitation

25-30

20-25

15-20

10-15

5-10

0-5

0.002.004.006.008.00

10.0012.0014.00

0 20 40 60 80 100 120

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

2.50

3.00

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


Data shown below for 1,000L vessels is estimated, and has been interpolated from experimentally-derived 250 and 2,000L data and biased by pore diameter of the drilled hole spargers.


Section 2Validation

2,000L Results for Frit and Drilled Hole Spargers

0.002.004.006.008.00

10.0012.0014.0016.00

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


0.001.002.003.004.005.006.007.008.00

0 50 100 150 200 250

1/hr

kLa

slpm flow rate



Graph 2.61 Results for 2,000L S.U.B. with both porous frit and drilled hole spargers

Graph 2.58 Results for 2,000L S.U.B. with drilled hole sparger


Graph 2.60 Results for 2,000L S.U.B. with drilled hole sparger

0

20

40

60

80100

120140

160180

200

0

5

10

15

20

25

30

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Mac

ro S

parg

e sL

PM

kLa

1/hr

s

Micro Sparge sLPM


25-30

20-25

15-20

10-15

5-10

0-5

0.00

5.00

10.00

15.00

20.00

0 50 100 150 200 250

1/hr

kLa

slpm flow rate


0.00

0.50

1.00

1.50

2.00

0 5 10 15 20 25

1/hr

kLa

slpm flow rate


The results of experiments with 2,000L vessels using porous frit and drilled hole spargers are shown below.


Section 2Validation

2.7 Mixing Studies The recommended range of mixing rates for the range of S.U.B. systems is as follows:

Table 2.11 Summary of mixing study results for the 50 to 1,000L S.U.B. systems

50L S.U.B. 100L S.U.B.

Half Volume Full Volume Half Volume Full VolumeAgitation

Speed(rpm)

Mixing Time (sec)

Agitation Speed(rpm)

Mixing Time (sec)


Mixing Time (sec)


Mixing Time (sec)

50 80 50 150 50 55-60 50 80-85100 20 100 50 100 30-35 100 40-45150 15 150 40 150 20-25 150 35-40

200 10 200 30-35

250L S.U.B. 500L S.U.B.

Half Volume Full Volume Half Volume Full VolumeAgitation

Speed(rpm)

Mixing Time (sec)


Mixing Time (sec)


Mixing Time (sec)


Mixing Time (sec)

40 50 60 60 30 75 30 8060 30 100 40 70 25 70 6080 20 120 30 110 20 110 50

140 20 150 15 150 25

1,000L S.U.B.

Half Volume Full VolumeAgitation

Speed(rpm)

Mixing Time (sec)


Mixing Time (sec)

30 68-89 60 37-4745 37-40 70 30-3960 35-39 80 30-43

90 26-34100 20-29110 19-28

Study Method(50 to 1,000L)

The mixing efficiency was estimated for the range of agitation rates by measuring the conductivity of the liquid contents of the S.U.B. BPC at different locations within the system after the addition of sodium chloride solution. Conductivity was measured with three conductivity probes positioned at the top, middle and bottom. The time to achieve uniform distribution of sodium chloride throughout the BPC was designated as the mixing time. Since multiple sensors were used the average time was determined when concentration readings of all the sample locations achieved a minimum of 95% of the final concentration. The study was conducted at maximum and minimum operating volumes for 50, 100, 250, 500 and 1,000L S.U.B. systems.

50L 100L 250L 500L 1,000L 2,000LOperating Volume (L) 25-50 50-100 125-250 250-500 500-1,000 1,000-2,000Agitation Rate (rpm) 30-200 30-200 30-150 30-150 20-110 20-75

Table 2.10 Recommended range of mixing rates


Section 2Validation

Mixing Study50L S.U.B.

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 50 100 150 200 250 300

Time (sec)

Conc

entra

tion

(%)

50 rpm avg 100 rpm avg 150 rpm avg 200 rpm avg


0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 50 100 150Time (sec)

Conc

entra

tion

(%)

50 rpm avg 100 rpm avg 150 rpm avg

Graph 2.63 Mixing study 50L S.U.B. – half volume

Graph 2.62 Mixing study 50L S.U.B. – full volume


Section 2Validation




020406080

100120140160

0 20 40 60 80 100 120

Time (sec)

Time (sec)


Conc

entra

tion

(%)

Conc

entra

tion

(%)

0

20

40

60

80

100

120

140

0 20 40 60 80 100 120




Section 2Validation


Mixing Study 250L S.U.B.

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 50 100 150Time (sec)

Time (sec)Co

ncen

tratio

n (%

)



0.0

20.0

40.0

60.0

80.0

100.0

120.0

0 20 40 60 80 100 120 140 160 180co

ncen

tratio

n (%

)

avg 60 rpm avg 100 rpm avg 120 rpm avg 140 rpm



Section 2Validation


Mixing Study500L S.U.B. (Full Volume)

0

20

40

60

80

100

120

140

160

0 20 40 60 80 100 120

Time (sec)

Conc

entra

tion

(%)


Mixing Study500L S.U.B. (Half Volume)

0

20

40

60

80

100

120

140

160

180

200

0 20 40 60 80 100 120 140

Time (sec)

Con

cent

ratio

n (%

)




Section 2Validation

Graph 2.71 Mixing study 1,000L S.U.B. - half volume


(average of 3 sample locations)

0.00%

20.00%

40.00%

60.00%

80.00%

100.00%

0 50 100 150 200 250

time(s)

60rpm 70rpm 80rpm 90rpm 100rpm 110rpm


(average of 2 sample locations)

0%

20%

40%

60%

80%

100%

0 100 200time(s)

60 rpm 45 rpm 30 rpm

Conc

entra

tion

Conc

entra

tion

Graph 2.70 Mixing study 1,000L S.U.B. – full volume


Section 2Validation

Theory

Procedure

Mixing performance was evaluated using an electrolyte solution and conductivity sensors. These sensors offer a very fast response time and both stable and repeatable readings. Mixing time is defined as the time elapsed between when stock solution is added and the average sample location reading exceeds 95% of final concentration. These mixing tests represent best case time estimates as the salt is added as a pre-mix solution.

Each bag was filled with DI water to the test volume, heated to 40ºC, and a salt solution was prepared. For the tests a 1 liter volume of solution (300 grams per liter dissolved Sodium Chloride) was introduced at the top of the BPC. Each test consisted of verifying the correct agitation speed and starting values of conductivity, adding the appropriate amount of salt solution, and then recording the readings on the probes utilizing the Kaye Validator™ thermal validation system until the conductivity leveled off. After the data were collected and entered, the percentages compared to the final reading were calculated for each sample taken. The percentage values from the probes were then averaged to approximate the mixing time. All three probes were used for both full and half volume calculations.

Three conductivity sensors from the same model and manufacture were attached to a rod installed into the BPC from the top. These sensors were positioned next to the top mounted mixer drive motor. It is anticipated that the location on this side of the tank represents worst case mixing because they are located the greatest distance from the high shear region of the impeller. The sensor positions represent three column height locations of low, middle, and high. The low and high positions were each located approximately 30.5cm (12.0”) from the respective ends of the fluid column. The mid probe was located at the 1,000L mark (half volumes). In an effort to obtain a representative reading the sensor tips protruded into the tank no less than 2.54cm (1.0”) from the inside of the tank wall. For half volume mix tests, the low and high probes were located approximately 15.2cm (6.0”) from the respective ends of the fluid column and the mid probe was located at the 500L mark.

Mixing Study (2,000L)

Mixing Test #* Tank VolumeImpeller Location/Shaft

LengthPower/Vol (Hp/1,000

gal)RPM

M1 Nominal, 2,000L 1 diameter from bottom / 82.9” 0.05 60



M10 ½ Volume, 1,000L 1 diameter from bottom / 82.9” 0.05 47



Table 2.12 Mixing study test matrix 2,000 L S.U.B.


Section 2Validation

Results All tests conducted were done in duplicate. The first column in results Table 2.13, below, was calculated by determining which probe (top, mid, or bottom) resulted in the slowest mix time for each respective test and averaging those together. The second column was calculated by averaging the all of the probes.

Mixing time performance at full volume is near equivalent at 75 and 95rpm.

Mixing at high impeller speed (>60rpm) at half volume is not recommended and will result in less than desirable performance and accelerated shaft wear (excessive power input, poor power dissipation result in lack of turn-over in a non-baffled tank).

2,000L S.U.B. (Full Volume Mixing Time)

RPM Average Mix Time - Worst Case (sec) Average Mix Time - All Probes (sec)

60 44 31.5

75 31.5 24

95 31 22.8

2,000L S.U.B. (Half Volume Mixing Time)

RPM Average Mix Time - Worst Case (sec) Average Mix Time - All Probes (sec)

47 28 20.8

60 21 16.8

75 53 24.8

Table 2.13 Mixing study results 2,000L S.U.B.

Graph 2.73 Mixing study 2,000L S.U.B. - half volumeGraph 2.72 Mixing study 2,000L S.U.B. - full volume


Section 2Validation

2.8 Additional 2,000L Studies

Drain Time and Hold-up Volume Test

Procedure

Results

Tanks were filled to nominal volume with water and heated to 37ºC. Drain line was opened and drain time tracked with a stopwatch. All testing was done gravity drain (no pump). When flow ceased the drain line was capped and relocated from the floor drain to a catch pan. The line was un-capped and bag manipulated to ensure remaining fluid draining into catch pan. The fluid was weighed and hold-up volume determined.

Test #1 had the following drain line configuration:• Drain Port: Standard 2.54cm (1.0”) BPC port (SV20522.01)• Reduced to 1.9cm (0.75”) ID line, 1.5m (5.0’) long• Tri-clamped to a 2.54cm (1.0”) ID line approximately 6.1m (20’)

long and inserted into floor drain

Test #2 had the following drain line configuration:• Drain Port: Standard 2.54cm (1.0”) BPC port (SV20522.01)• Reduced to 1.9cm (0.75”) line, 1.5m (5.0’) long• Reduced to a 1.27cm (0.50”) ID line approximately 4.6m (15’)

long and inserted into floor drain

Both tests passed the mandatory four-liter maximum allowable hold-up volume requirement as shown in table 2.13. The faster drain time of Test #1 was expected due to the cross-sectional area of the drain line being more than 2x that of Test #2.

Test # Drain Time Hold-Up Volume

1 (1.9cm ID restriction) 1 hr 40 sec 0.34 Liter

2 (1.27cm ID restriction) 2 hr 45 min 0.66 Liter

Table 2.14 Mixing study test matrix 2,000L S.U.B.


Section 2Validation

Temperature Mapping The heat capacity of the thermal jacket was evaluated on the 2,000L S.U.B. hardware. Several tests were performed, as noted below.

• 5 to 37°C at 40°C max for TCU• 5 to 37°C at 50°C max for TCU• 37 to 10°C using in-house cooling

Chilled water was added to the disposable S.U.B. at nominal volume (2,000L), and then cooled to 5°C using dry ice. Using a standard temperature control unit (TCU), (specifications: 34 kW, 480VAC, 3 phase, 2 hp pump motor) the water was heated up to the 37°C.

By interpreting the data from the linear portion of the Graph 2.74, the heat time from 5 to 37°C, with a maximum temperature of 40°C, for the 2,000L unit was approximately four hours. In a similar test, the maximum temperature was set at 50°C and the Graph 2.75 shows a heat time from 5 to 37°C of less than two and a half hours. Using this TCU, the solution warmed up at a rate of 0.245°C/min during full ramp. When testing with a maximum of 40°C for the jacket temperature, the slope tapered off when the jacket temperature neared 40°C and created a longer heat up time. For the 50°C maximum jacket temperature test, the ramp up rate was 0.245°C/min, until the batch temperature was reached.

To find temperature consistency of the full jacket height, three thermocouples were used, media bottom, media mid, and media top and compared in both tests, maximum 40°C and maximum 50°C. The largest difference for both tests was 0.08°C. These values were evaluated in the steady state portion of the tests.

NOTE: The upper jacket has an approximate surface area of 44,081.8 sq. cm (6,832.7 sq. in.) and the lower 5,980 sq. cm (927 sq. in.) for a combined total of 50,062 sq. cm (7,759.7 sq. in.).


Section 2Validation

Graph 2.74 Thermal profile of a 2,000L jacketed S.U.B. from 5 to 37°C with a max water jacket temperature of approximately 40°C.

Graph 2.75 Thermal profile of a 2,000L jacketed S.U.B. from 5 to 37°C with max water jacket temperature of approximately 60°C.


Section 2Validation

Graph 2.76 Thermal profile of a 2,000L jacketed S.U.B. from 37 to 11°C with a chilled water jacket temperature of approximately 10°C.

The cooling capacity of the thermal jacket was also evaluated on the 2,000L S.U.B. hardware. The S.U.B.s were heated to a temperature of 37°C and then cooled using in-house chilled water.


Section 2Validation

2.9 S.U.B. 50 to 1,000L Temperature

Mapping Studies The heating capability of all water jacketed and resistive electric heater blanket S.U.B. units was evaluated. Several tests were performed, as noted below.

• 5-37°C at 50°C maximum for TCU on jacketed S.U.B. systems• 5-37°C at setpoint of 40°C for the resistive electric blanket style

S.U.B. systems• 37-5°C at varying °C minimums for the TCU on jacketed S.U.B.

systems (varying setpoints are explained below for each unit)

Chilled water was added to the disposable S.U.B. at nominal volumeand then cooled to 5°C using dry ice with agitation enabled. Using the TCU on the jacketed S.U.B. and the built-in resistive blanket on the electric heating S.U.B. units, chilled water was heated to a setpoint of 40°C. S.U.B. systems with the resistive electric heating blankets were controlled using a PXG4CRM1-FVY00 Fuji Electric controller. Jacketed S.U.B. temperature was controlled by the thermal control unit (TCU) directly.

To verify temperature consistency, multiple thermo couples were used to measure bulk fluid temperature in the resistive heated vessels. The thermocouples had a 99% confidence of 0.05°C between each other.

Time for given S.U.B. configuration water temperatures to increase from 5°C to within 0.1°C of 37°C, using a maximum heating setting temperature of 50°C, are listed in table 2.15. Graphs 2.77 through 2.86 display plots of the averaged thermo couples used in the resistive and jacketed vessels. The average slope in °C/minute is taken from the linear regression of vessel temperature change in graphs 2.77 through 2.86 and included in table 2.15.

A cooling test was performed for each size of the jacketed S.U.B. systems at nominal volume using a thermal control unit (TCU) with the wattage listed in Table 2.15. The tests started at a temperature of 37°C with a setpoint of 5°C.

The times it took to cool from 37°C down to within 0.5°C of the target low temperature setpoint are listed in Table 2.15 and shown in graphs 2.87 though 2.90.

NOTE: The jacketed S.U.B. systems have an approximate total jacket heat transfer area shown in Table 2.16.


Section 2Validation

VesselTemp Start (°C)

Temp Finish (°C)

Min/Max Temp (°C )

Time (Minutes)

Time (Hours)

Average Slope

(°C /min)Watts

50L Resistive 5 37 5/50 259 4.32 0.124 0.46K

100L Resistive 5 37 5/50 293 4.88 0.109 0.87K

250L Resistive 5 37 5/50 449 7.48 0.071 1.36K

500L Resistive 5 37 5/50 628.8 10.48 0.051 2.6K

1,000L Resistive 5 37 5/50 720 12.00 0.044 5K

50L Jacketed 5 37 5/50 105 1.75 0.305 10K

100L Jacketed 5 37 5/50 111 1.84 0.288 10K

250L Jacketed 5 37 5/50 115 1.92 0.278 10K

500L Jacketed 5 37 5/50 170 2.83 0.188 10K

1,000L Jacketed 5 37 5/50 379 6.3 0.084 10K

50L Jacketed 37 5 5/50 165 2.75 -0.194 10K

100L Jacketed 37 5 5/50 265 4.6 -0.121 10K

250L Jacketed 37 5 5/50 240 4 -0.133 10K

500L Jacketed 37 5 5/50 N/A N/A N/A 10K

1,000L Jacketed 37 5 5/50 1020 17 -0.031 10K

Table 2.15 Heating and cooling statistics

Vessel - Jacketed Jacket Area in In2 Jacket Area in Cm2

50L 551 3554100L 977 6302250L 1947 12558500L 3469 22375

1,000L 5604 36146

Table 2.16 Jacket surface area


Section 2Validation

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50

Deg

rees

C

Hours

100L Resistive SUB Heat Up Time

AVERAGE

Graph 2.77 Temperature increase in 50L resistive S.U.B. from 5 to 37°C with a maximum resistive blanket temperature of 50°C

Graph 2.78 Temperature increase in 100L resistive S.U.B. from 5 to 37°C with a maximum resistive blanket temperature of 50°C

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Deg

rees

C

Hours


AVERAGE

50L Resistive S.U.B. Heat Up Time



Section 2Validation

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Deg

rees

C

Hours


AVERAGE

Graph 2.79 Temperature profile of 250L resistive S.U.B. from 5 to 37°C w maximum resistive blanket temperature of 50°C

Graph 2.80 Temperature profile of 500L resistive S.U.B. from 5 to 37°C maximum resistive blanket temperature of 50°C

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

Deg

rees

C

Hours


AVERAGE




Section 2Validation

Graph 2.81 Temperature increase in 1,000L resistive S.U.B. from 5 to 37°C maximum resistive blanket temperature of 50°C

5.07.09.0

11.013.015.017.019.021.023.025.027.029.031.033.035.037.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Deg

rees

C

Hours

50L Jacketed SUB Heat Up Time

AVERAGE

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00

Deg

rees

C

Hours


AVERAGE

Graph 2.82 Temperature increase in 50L jacketed S.U.B. from 5 to 37°C with a maximum TCU temperature of 50°C


50L Jacketed S.U.B. Heat Up Time


Section 2Validation

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50

Degr

ees C

Hours


AVERAGE


5.07.09.0

11.013.015.017.019.021.023.025.027.029.031.033.035.037.0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00

Deg

rees

C

Hours


AVERAGE





Section 2Validation

5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00

Deg

rees

C

Hours


AVERAGE


5.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Deg

rees

C

Hours


AVERAGE

Graph 2.86 Temperature increase in 1,000L jacketed S.U.B. from 5 to 37°C with a maximum TCU temperature of 50°C




Section 2Validation

5.07.09.0

11.013.015.017.019.021.023.025.027.029.031.033.035.037.0

-0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00

Deg

rees

C

Hours

50L Jacketed SUB Cool Down Time

AVERAGE

Expon. (AVERAGE)

Graph 2.87 Temperature decrease in 50L jacketed S.U.B. from 37 to 5°C with a minimum TCU temperature of 5°C. Due to limitations of the test environment, some data is interpolated using an exponential curve, as shown.

3.005.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Deg

rees

C

Hours


AVERAGE

Graph 2.88 Temperature decrease in 100L jacketed S.U.B. from 5 to 37°C with a minimum TCU temperature of 5°C

50L Jacketed S.U.B. Cool Down Time



Section 2Validation

3.05.07.09.0

11.013.015.017.019.021.023.025.027.029.031.033.035.037.0

-0.50 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

Deg

rees

C

Hours


AVERAGE

Expon. (AVERAGE)

Graph 2.89 Temperature decrease in 250L jacketed S.U.B. from 37 to 5°C with a minimum TCU temperature of 5°C. Due to limitations of the test environment, some data is interpolated using an exponential curve, as shown.

NOTE: The 500L jacketed S.U.B. data was insufficient because of facility limitations during testing. The 500L test will be performed at a later date.

3.005.007.009.00

11.0013.0015.0017.0019.0021.0023.0025.0027.0029.0031.0033.0035.0037.00

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

Deg

rees

C

Hours


AVERAGE

Graph 2.90 Temperature decrease in 1,000L jacketed S.U.B. from 5 to 37°C with a minimum TCU temperature of 5°C




Section 2Validation

2.10 Sterility Testing

Introduction The S.U.B. BPC is expected to retain functionality and sterility for 21 days of continuous operation at normal parameters.

Materials and Methods

Figure 2.15 Hub assembly with secondary and tertiary seals removed (primary, product contact seal only).

Figure 2.16 Hub assembly with primary and secondary seals removed (tertiary, upper seal only).

Sterility TestingInitial Evaluation

An initial sterility run was conducted using the hub assembly. A total of three samples were tested. These samples represented a worst-case scenario in that they were previously used during a mechanical performance evaluation. The mechanical evaluation included connection to a drive motor and spinning the hub at a rate of 360rpm for 15 hours. The nominal agitation rate for the 250L S.U.B. is 120rpm, thus the mechanical performance test was three times the standard operating conditions. Seals were also removed from the assemblies in order to challenge remaining individual seals. Access holes were drilled into the port bodies, providing access to each seal in order to directly challenge them with live bacterial culture. The assemblies were welded into sample BPC, irradiated and then aseptically filled with tryptic soy broth (TSB). Testing was conducted in a 37º C incubation room.

Further evaluation of the hub assembly were performed using a total of seven samples. Samples one to four contained only the primary (product contact) 35mm seal (figure 2.15), while samples five to seven contained only the tertiary (upper) 50mm seal (figure 2.16). This was done in order to determine the reliability of individual seals. The standard arrangement contains three seals as a sterility barrier, thus providing redundancy to improve system reliability.

Sterility Testing Secondary Evaluation


Section 2Validation

The assemblies were sealed into sample BPC, irradiated and then aseptically filled with TSB. Once again, access holes were drilled into the assembly bodies to directly challenge the seals with live bacterial culture. Ambient air was filtered and sparged into the BPC via the direct sparge port to create a slightly positive pressure environment, as typical with a S.U.B. Testing was conducted in a 37°C incubation room.

In order to establish system reliability against a 21 day requirement, 22 fully functional systems were tested (six units each of the 50 and 250L S.U.B.s in stage one; five units each of the 50 and 250L S.U.B. in stage two). Testing was staged due to constraints in space and power availability. All samples were built complete with all ports, tube sets and filters. Shipping tests were conducted to verify packaging maintained product integrity. All samples were packaged and palletized according to specifications, irradiated and tested according to ISTA 1E. This testing included vibration, impact and drop testing.

All samples passed this series of tests. Samples were then unpacked and used in functional sterility testing.

All samples were aseptically filled with TSB. Stage one testing included the insertion of six probes into dry BPC (prior to TSB addition) using the autoclaved probe and probe assembly. Stage two testing included the insertion of three probes into filled BPC (post TSB addition). Stage one testing was performed at half volume (25 or 125L in the 50 or 250L S.U.B. respectively) with an agitation rate scaled to the working volume of the system (92.9 or 59.1rpm respectively). Stage two testing was performed at full working volume with an agitation rate at nominal speed (186 or 120rpm respectively). Agitation rates were controlled using the onboard controller and mixing drive. External air pumps were used to supply filtered ambient air to each system through the direct sparge port at average rates of 250 or 500mL/minute respectively. All systems were operated at a temperature of 37°C and controlled using the onboard temperature controller.

Samples were tested according to the following schedule:

• Day 0 – Initiated test with an agitation rate of 120rpm• Day 2 – Sample BPC filled with TSB• Day 3 – Primary inoculation (20µl of live culture)• Day 20 – Secondary inoculation (20µl of live culture)

Results and Discussion

Sterility TestingInitial Evaluation

Sterility TestingFinal Evaluation


Section 2Validation

• Day 24 – Test criteria achieved, no sterility failures• Day 175 – Test discontinued, no sterility failures

The samples were tested for more than 30.2 million revolutions. The design requirement was 120rpm for 21 days, which equates to 3.63 million revolutions. All samples met the requirement.

Samples were tested according to the following schedule:

• Day 0 – Initiated test with an agitation rate of 260rpm (maximum speed)

• Day 1 – Sample BPC filled with TSB• Day 8 – Agitation rate reduced to 200rpm due to heat

generation from drive motors causing incubation room to overheat

• Day 10 – Agitation rate reduced further to 150rpm due to heat generation

• Day 20 – Primary inoculation (20µl of live culture)• Day 42 – Secondary inoculation (20µl of live culture)• Day 80 – Tertiary inoculation (20µl of live culture)• Day 131 – Test discontinued, no sterility failures

The samples were tested for a total of 28.9 million revolutions. The design requirement was 120rpm for 21 days, which equates to 3.63 million revolutions. All samples met the requirement.

Each stage ran for a period of 21 days and evaluated for any functional sterility issues. All samples functioned properly for the 21 day period with no sterility failures.

The hub assembly are a key component for the successful operation of the S.U.B. They provide the means for a stirred-tank operation while maintaining a sterile environment. Mechanical and sterility barrier properties must be robust in order for the S.U.B. to replace traditional stainless steel systems in bioproduction.

Three separate studies were performed, each testing the capability of the assembly to function as both hub and sterility barrier under normal to extreme conditions for extended periods of time. Each of these evaluations included one or more of the following perturbations:

• Running at intervals of approximately six months• Removing seals within the assembly• Multiple inoculations of live bacterial cultures above

individual seals

Conclusions

Sterility TestingSecondary Evaluation

Sterility TestingFinal Evaluation


Section 2Validation

• Operating at high rotational speeds at up to three times of nominal

• Mechanical performance testing prior to sterility testing• Vibration, impact and drop testing prior to sterility testing

The results of the evaluations show the hub assembly exceeds all requirements as a sterility barrier and help to demonstrate the S.U.B. is a viable alternative to traditional stainless steel systems for biopharmaceutical manufacturing.


Section 3Condenser System Validation

Section 3 Condenser System Validation

The condenser system is intended to be used as an accessory to the Single-Use Bioreactor (S.U.B.) as an alternative to vent filter heaters. It is an integral part of a standard 2,000L S.U.B. system and can be provided as a custom option with other S.U.B. sizes. The condenser’s purpose is to prevent liquids from condensing and collecting inside of the vent filters of the S.U.B. The condenser system cools the exhaust gasses leaving the S.U.B. chamber, and as a result it condenses the moisture out of the saturated gasses coming from the S.U.B. The liquid condensate that is stripped from the exhaust gasses is then pumped back into the S.U.B. chamber, creating a sterile loop. The condenser plate is chilled by a closed-bath recirculating chiller which has sufficient capacity to cool two condenser plates simultaneously if desired. Figure 3.1 is a functional diagram of the condenser system.

3.1 Functional Overview

Figure 3.1 Condenser system overview

CartAssemblyClosed Bath

RecirculatingChiller

Condenser BagGas Inlet Port

Gripping Tabs

Alignment Holes

Condenser ReturnLine Back to S.U.B.

Filter Straps

Dual HeadedPeristaltic Pump

Condenser Plate Assembly

Post Receivers

Condenser Post Assembly

Filter BracketAssembly

Exhaust LineFrom S.U.B.

Exhaust VentFilters

Condenser BagGas Outlet Port

Dual ChamberCondenser Bag

Condenser BagLiquid Drain Ports

Condenser Disposables Condenser Hardware Condenser System



0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0 10 20 30 40 50 60

Inte

rnal

Pre

ssur

e (p

si)

Sparge Flow (L/min)

2000L SUB Internal Pressure vs. Sparge Flow with Standard Condenser Bag and Varying Vent Filters

2 x 10" Meissner 1 x 10" Meissner 2 x KA3 1 x KA3

Table 3.1 Parts list

Table 3.2 Filter performance testing on production intent condenser system

Materials Table

Pressure vs. Exhaust Flow Rate

Bill of materials for single-use condenser components per SH2B2004.

Several exhaust filter configurations were evaluated on the condenser. The system back-pressure was measured with each filter configuration while incrementally increasing the sparge flow rates from 10 to 50Lpm. The results are shown in table 3.2.

Filter Configuration

2,000L System Back Pressure per Sparge Flow Rate (psi)10Lpm 15Lpm 20Lpm 25Lpm 30Lpm 35Lpm 40Lpm 45Lpm 50Lpm

2x10” Meissner 0.01 0.02 0.03 0.03 0.05 0.07 0.08 0.10 0.131x10” Meissner 0.02 0.03 0.04 0.06 0.07 0.08 0.10 0.14 0.17

2xKA3 0.02 0.03 0.04 0.06 0.07 0.09 0.12 0.15 0.181xKA3 0.03 0.05 0.08 0.10 0.14 0.16 0.20 0.24 0.29

Item .01 .02 Part Number Description1 1 Each 1 Each SH2B1008.01 Container: Bioreactor Condenser Bag2 2 Each 2 Each SV21050.01 Filter: Ultracap, 0.2 um 3/4” HB x 3/4” HB, Meissner3 2 Each 2 Each SV20510.06 Fitting: Polypropylene 90° elbow, 5/8”4 1 Each 1 Each SV20505.02 Fitting: Polypropylene ST. Conn., 3/16” x 1/4”5 1 Each 1 Each SV21043.06 Fitting: Polypropylene Y. Conn., 3/4” x 5/8” x 5/8”6 3 Each 3 Each SV20506.03 Fitting: Polypropylene Y. Conn., 1/4”7 2 Each 2 Each SV20503.08 Fitting: Polypropylene end plug, 1/2”8 2 Each 2 Each SV20504.05 Fitting: Polypropylene T. Conn., 5/8”9 0.82m (2.67’) 0.82m (2.67’) SV20448.05 Tubing: Silicon braided, 5/8” ID x 0.965” OD10 2.13m (7’) 1.52m (5’) SV20678.07 Tubing: C-Flex (R70-374-000), 1/4” ID x 3/32” Wall11 1.07m (3.5’) 1.07m (3.5’) SV20706.03 Tubing: Pharmapure, 3/16” ID x 1/16” Wall12 2.13m (7’) 1.32m (4.33’) SV20448.06 Tubing: Silicon braided, 3/4” ID x 1.10” OD13 0.60m (2’) 0.60m (2’) SV20019.10 Tubing: Silicone, 1/2” ID x 1/8” Wall14 0.80m (1.33’) 0.80m (1.33’) SV20678.23 Tubing: C-Flex (R70-374-000), 5/8” ID x 1/8” Wall15 1 Each 1 Each SV20031.03 Fastener: Snapper clamp, 1/2” - 3/4”16 32 Each 34 Each SV20030 Fastener: Cable tie17 1.22m (4’) 1.22m (4’) SV50072 Packaging Material: Bubble wrap



3.2 Peristaltic Pump A peristaltic pump is used to transfer the liquid condensate from the condenser bag back into the batch. A pump is required in order to overcome the pressure inside of the batch. The pump that was chosen was the Masterflex™ L/S™ Digital Drive, 600rpm, 115/230VAC, 50 to 60Hz Thermo Fisher Scientific #07523-80 (See figure 3.2). This model of pump was chosen for its high endurance, low maintenance brushless DC motor. This pump drive also has the option for auto restart, which is recommended for this application. The pump will come standard with two single-track Easy-Load II pump heads. The dual heads facilitate the use of two chill plates, allowing the user to service two S.U.B.s with one condenser cart if desired. The recommended speed for the peristaltic pump is 12 to 30rpm; however, the best indication of adequate pump speed is that there will be no liquid buildup above the drain ports in the condenser bag.

Figure 3.2 Peristaltic pump drive and head

Figure 3.3 Spallation comparison of peristaltic pump tubing types

Peristaltic Pump Tubing The pump tubing chosen for this application is MasterFlex™ PharmaPure™ size L/S 25. This tubing was selected due to its high resistence to particulate generation (spallation) inside of the tubing. The tubing is classified as USP Class VI compliant, as well as gamma stable up to 48kGy. Figure 3.3 shows a spallation comparison of several types of commonly used pump tubing.



3.3 Chiller (TCU) The specified chiller is the Thermo Scientific™ ThermoFlex 900 IPR with a PD1 (Positive Displacement 1) pump (see figure 3.4). This chiller was chosen because it is the smallest closed-bath recirculating refrigerant cooled chiller provided by Thermo Fisher. The requirements for the chiller are a minimum cooling capacity of 400W at 5°C, and a minimum flow of 1Lpm at 15psi head. This is sufficient for cooling two condenser plates. Another requirement for the chiller is that it cannot generate pressures above 30psi inside of the cooling plate. The ThermoFlex 900 IPR has an internal pressure regulation system that can be set to achieve the pressure requirement. The chiller also has an auto restart function which is recommended to be activated for this application.

Figure 3.4 Thermo Scientific Neslab ThermoFlex 900 IPR TCU



3.4 Condenser Plate Assembly Functional

Overview

The single-use condenser bag straddles, and is enclosed by, the condenser plate assembly. The purpose of the condenser plate assembly is to provide a cold heat sink for the condenser bag, and this sink cools the exhaust gasses. The assembly consists of the main cooling plate, insulation, bag alignment and tensioning hardware, transparent doors and baffling hardware. The doors are spaced 6.35mm (0.25”) away from the surface of the main cooling plate to provide a volume for the condenser bag to inflate into while in use. Self-skinning foam is applied to the exterior surfaces of the plate to minimize thermal load on the chiller, mitigate ambient condensation on the plate, and control condensation formation inside of the condenser bag near the outlet port. An exploded view of the condenser plate assembly is shown in Figure 3.5.

The cooling plate assembly was tested to ensure that neither the plate nor the doors would fail under potential pressurization scenarios. The condenser bag will experience the same internal pressure that the S.U.B. chamber experiences. That same pressure will be transmitted directly to the door assemblies. Pressure testing was done on the door assembly up to 15psi without failure of any kind in the door assembly. This demonstrated

Figure 3.5 Condenser plate assembly exploded view

DoorCoverPlate

Bag CenterBaffle Bar

DoorLatch

Condenser BagAlignment Buttons

CoolantInlet Port

CoolantInlet Port

PlateInsulation

LatchPlate

O-ringSeal

CoolantChannel

BleedVent

BagTension

Condenser Plate Structural Integrity



a 2x safety factor over the in-tank burst pressure of the 50L S.U.B. chamber (highest burst pressure for S.U.B. chambers). The coolant also exerts pressure on the condenser plate walls. The plates were tested and showed no permanent deformation, and no coolant leaks at 30psi. The chiller is equipped with an internal pressure regulator which will be factory set at 30psi to ensure that the condenser plate does not over-pressurize and leak.

In a humid environment, the condenser plate and coolant lines will form condensation on their exterior surfaces. In order to minimize the amount of condensation formation, the cold components of the system are insulated. The condenser plate assembly has a self-skinning foam coating on the surfaces that aren’t important for cooling the condenser bag. The coolant lines come with a rubber insulation jacket around the exterior, and sleeves are added to the coolant lines which can be moved up to cover the couplers between the condenser plate and the coolant lines. The system was tested by a third party in North Carolina, USA where the plant ambient conditions were measured to be approximately 55 to 60 percent relative humidity and 23 to 25°C. The amount of condensation which formed on the exterior surfaces of the condenser system was reported to be at an acceptable level.

The true maximum functional capacity of the condenser system depends on the temperature of the cooling plate, the ambient air temperature, batch temperature, and gas flow rate. The true sign that the condenser system has reached its maximum functional capacity is when the tubing between the vent filters and the condenser bag begin to fog up on the interior. This is a sign that the temperatures of the gasses exiting the condenser are higher than room temperature, and gas-to-liquid phase change is happening in the vent lines, which is what the condenser system is designed to prevent. Since the goal is to cool the gasses below room temperature (18-23°C), test scenarios were performed to see what gas flow rates through a 40°C batch would cool to 18°C in the condenser. The temperature of the gasses flowing through the condenser bag was measured at multiple locations along the gas flow path. Figure 3.6 shows the thermocouple locations inside of the condenser bag. The results from the temperature mapping show that the condenser has ample capacity to cool exhaust gasses from the S.U.B. at sparge rates well above the original design requirement of 50L/min. gas. The testing also shows that the condenser plate does not have to be cooled as low as 5°C in order to cool 50L/min. gas rate to an acceptable level. The data from the temperature mapping tests are presented in tables 3.3 through 3.7. Care must be taken when pushing high levels of gas through the S.U.B. At elevated flow rates foam is produced more rapidly, and the pressure inside of the S.U.B. increases. Foam control and pressure monitoring are highly recommended.

Ambient condensation prevention

Condenser Functional Capacity



45

67

8 9

Dot = Thermocouple

32 14

15

16

17

18

19

2021

1 13

Condenser Inlets

Condenser Outlets

Figure 3.6 Thermocouple locations in condenser during temperature mapping



50 L/min 5°C Plate

Position Temp °C Position Temp °C

1 32.4 13 31.4

2 26.9 14 27.9

3 21.7 15 23.0

4 14.9 16 16.7

5 14.3 17 17.1

6 12.2 18 13.0

7 11.7 19 12.6

8 10.7 20 12.6

9 11.5 21 12.8

50 L/min Gas, 5˚C Cooling Plate

Thermocouple Position

Bolted side of plate (Bottom Axis) Solid side of plate (Top Axis)

Gas

Tem

p ˚C

Table 3.3 Condenser temperature profile 40°C batch, 50L/min. gas, 5°C plate



75 L/min 5°C Plate

Position Temp °C Position Temp °C1 34.0 13 32.5

2 29.4 14 29.2

3 25.1 15 25.5

4 21.5 16 23.4

5 18.7 17 20.3

6 15.0 18 15.3

7 14.0 19 14.1

8 13.3 20 14.7

9 13.1 21 13.7




Gas

Tem

p ˚C

Table 3.4 Condenser temperature profile 40°C batch, 75L/min gas, 5°C plate






Gas

Tem

p ˚C

Table 3.5 Condenser bag temperature profile 40°C batch, 100L/min gas, 5°C plate

100 L/min 5°C Plate


2 31.6 14 31.1

3 28.2 15 28.0

4 25.5 16 26.0

5 21.9 17 23.1

6 18.1 18 17.5

7 16.5 19 16.4

8 16.3 20 16.7

9 15.4 21 15.8






Gas

Tem

p ˚C


95 L/min 8°C Plate


2 31.4 14 31.1

3 28.4 15 28.3

4 25.9 16 25.9

5 22.7 17 23.3

6 19.3 18 18.5

7 17.9 19 17.9

8 17.5 20 17.9

9 16.6 21 17.1






Gas

Tem

p ˚C

75 L/min 10°C Plate

Position Temp °C Position Temp °C

1 34.3 13 33.0

2 30.1 14 30.1

3 26.9 15 27.0

4 23.9 16 24.7

5 21.3 17 22.8

6 18.2 18 18.4

7 17.2 19 17.5

8 16.7 20 17.7




Sample Number (50L/min Direct Sparge, 40°C Batch Temp)1 2 3 4 5 6 7 8 9 10 Avg Stdev

Pressure (psi) 0.08 0.08 0.08 0.1 0.08 0.1 0.09 0.09 0.1 0.11 0.09 0.011

Filter #1 Int. Temp. (°C)

22.9 20.5 22.9 24.5 23.1 21.9 22.3 23 20.6 21 22.3 1.28

Filter #1 Int. RH (%)

51 54 53 57 56 58 56 53 54 59 55 3

Filter #2 Int. Temp. (°C)

21.7 22.2 21.9 22.4 23.1 22.1 24.4 23.3 19.4 20.8 22.1 1.37

Filter #2 Int. RH (%)

57 61 58 61 57 56 55 56 63 62 59 3

Amb. Temp. (°C) 24.6 23.9 24.7 25 25.1 24.8 25.95 26.5 22.2 23.2 24.6 1.25

3.5 Condenser Testing

Testing was done to show the repeatability of the condenser system. Six measurements were taken across ten separate condenser disposables sets. The measurements taken were: S.U.B. internal pressure, Filter #1 internal temperature, Filter #1 internal relative humidity, Filter #2 internal temperature, Filter #2 internal relative humidity, ambient temperature. The data is presented in Table 3.8.

During development, six separate tests were performed using growth media inside of the S.U.B. to prove that the condenser system was capable of maintaining sterility. None of the six tests lost sterility. Five of the six sterility runs were also cell culture runs (CHO cells). The cell cultures all grew as expected, however, there was one unforeseen anomaly that has only been observed during the cell runs. As the cell growth accelerates, residue begins to build up inside of the condenser bag outlets. The residue was analyzed and it was found that no cells, whole or partial, were present in the residue.

Several fault conditions were identified during the development of the condenser system. A summary of the conditions along with the resulting hazards and contingencies are shown in Table 3.9.

Repeatability of Condenser System

Sterility/Cell Culture Testing

Fault Conditions

Table 3.8 Condenser repeatability test data



Fault ConditionResulting

HazardContingency Actions

Kink in exhaust tubing between S.U.B. and condenser bag

Excessive pressure in S.U.B.

• Ensure single-use condenser components are properly installed onto condenser hardware

• Inspect tubing regularly for kinks• Continuous pressure monitoring on S.U.B. is strongly recommended

Liquid buildup in condenser bag outlet tubing and/or vent filter


• Check that chiller power is on• Check that auto-restart option on chiller is activated• Check chiller temperature (setpoint and actual)• Check coolant level in chiller• Check that batch flow rates do not exceed recommendations• Check coolant lines between chiller and condenser plate for abnormalities• Temporarily plug off vent filter (one at a time) while manipulating the tubing to

drain liquids back into condenser bag• Continuous pressure monitoring on S.U.B. is strongly recommended

Exhaust tubing between S.U.B. and condenser bags creates a ‘P-trap’


• Organize the tubing or condenser system such that no ‘P-trap’ is created • Continuous pressure monitoring on S.U.B. is strongly recommended

Liquid buildup in condenser bag


• Check that pump power is on• Check that auto-restart option on pump is activated• Check that pump is set to recommended speed (12-30rpm)• Check that pump head is turning at set speed• Check for kinks or blockages in liquid drain line tubing• Check that pump tubing is properly installed in pump head (direction matters)• Continuous pressure monitoring on S.U.B. is strongly recommended

Foam reaches condenser bag


• Reduce gas flow rates in S.U.B.• Add anti-foam agent to S.U.B.• Once foam source has been controlled, foam will naturally dissipate and drain

out of condenser bag• Continuous pressure monitoring on S.U.B. is strongly recommended

Table 3.9 Fault condition descriptions and contingency action plans


Section 4Quality Control

Section 4 Quality Control

Only the highest quality components are used in the manufacture of the S.U.B. Each component is given a unique part number and has a controlled material specification. In addition, we actively promote communication with vendors, conduct audits of vendors and maintain a vendor evaluation program.

Incoming components are quarantined until they have met the approved component specification’s criteria. During the inspection process, lot numbers and part numbers are recorded for traceability purposes. Once components have satisfied the requirements in the specification they are released into inventory by QC personnel.

In-process inspections and testing take place during the manufacturing process to ensure that each production run of BPCs is being manufactured to the approved specifications.

Film Inspection

Ports, fittings, tubing, end treatments

• Contamination• Gels or carbons• Width and gusset dimensions• Film thickness• Tensile strength• Chemical composition using

FT-IR spectrometer

• Appearance and visual inspection

• Dimensional analysis

Flexible container chambers Inspection

Finished flexible containers

• Appearance• Seam and port seal strength• Dimensional analysis• Leak and burst testing

• Correctness• Completeness• Particulate debris• Defects and damage• Correct packaging

4.1 Introduction

4.2 Inspection

Incoming Inspection Component

In-Process Inspections and Testing



Production control process ensures traceability for each lot of BPCs. The process control document becomes the stepwise manufacturing record, which physically accompanies the lot through manufacturing. At the end of the production process quality assurance reviews the lot record for completeness and correctness prior to release of the lot. At this time a Certificate of Analysis (COA) is issued.

Lot record review Certificate of Analysis• Bill of Materials• Certificate of Irradiation• Production quality inspections• Production integrity testing• Labels• Deviations• Certificate of Analysis

• Product name• Catalog number• Lot number• Expiration date• Irradiation dosage

4.3 BPC Lot Record Release

and Certificate of Analysis



Life Technologies Corporation 1726 HyClone Dr, Logan Utah 84321 thermoscientific.com/bioproduction

SAMPLE CERTIFICATE OF ANALYSIS Single-‐Use Bioreactor BioProcess Container Systems

Product: BioProcess Container:

XX L SAMPLE Lot Number: AAANNNNNN Catalog Number: SAMPLE Expiration Date: MMM/YYYY Drawing Number: SAMPLE Drawing Revision: “-‐” Manufacture Date: MMM/YYYY

============================================================================================= Test: Specification: Units: Results:

=============================================================================================

Irradiation Dose: 25 – 40 kGy Pass

� Inspection: This lot of BioProcess Container Systems has been 100% visually inspected per specification.

� Biological Reactivity: All product contact materials have passed USP Class VI testing (USP <88>).

� Cytotoxicity: All Thermo Scientific product contact films have passed Cytotoxicity testing (USP <87> MEM Elution).

� Physicochemical: All Thermo Scientific product contact films have passed USP Physicochemical Tests for Plastics (USP <661>).

� EP Testing: All Thermo Scientific product contact films have passed EP <3.2.2.1> “Plastic Containers for Aqueous Solutions for

Parenteral Infusion”.

� Endotoxin: Samples of representative BioProcess Container Systems are routinely tested in periodic validations for the

presence of endotoxin per the USP Bacterial Endotoxin Test (USP <85>). Aqueous extracts contained < 0.25 EU/ml as

determined by the Limulus Amebocyte Lysate Test (LAL).

� Particulate: Samples of representative BioProcess Container Systems have been routinely tested in periodic validations and

have passed requirements per Particulate Matter in Injections Light Obscuration Particulate Count Test (USP <788>).

� Sterility: Routine sterility testing is performed on representative samples of Thermo Scientific BioProcess Container Systems

following ANSI/AAMI/ISO 11137 guidelines. Periodic validation has determined that an irradiation dose of 25 – 40 kGy provides a minimum Sterility Assurance Level (SAL) of 10⁻⁶ for product contact surfaces.

_______________________________________________ Quality Department / Date Issued

4.4 Sample C of A



Traceability is maintained on BPCs and components.

Shelf life of BPCs is based on real-time stability and accelerated aging tests performed during product development. All Standard BPCs have a three-year expiration date from date of irradiation.

Customization is available on all standard BPC standards. Custom chambers with standard components can have a shelf life equal to a standard BPC, which is based on real-time stability and accelerated aging tests performed on the standard components during product development. All custom chambers with standard components on the BPCs can receive a three-year expiration date from date of manufacture if requested by the customer.

Customization to our standard BPCs to meet the specifications of our customers is available. Custom designs with custom components receive a manufacture date on the Certificate of Analysis. Shelf life data may not be available for some custom items requested by the customer or supplied by the customer.

Standard Chambers

4.6 Shelf Life

Custom Chambers with Standard Components

Custom Chambers with Custom Components

4.5 Traceability



4.7 Sample Certificate of

Irradiation


Section 5Regulatory

Section 5 Regulatory

Thermo Scientific annually registers with the Food and Drug Administration (FDA) as a Medical Device Establishment to be a manufacturer and contract manufacturer of medical devices for sera and media products.

As a manufacturer of medical devices for human use, our facilities are inspected by the FDA for compliance with current Good Manufacturing Practices (cGMP) as listed in 21 CFR Chapter 1 Part 820, Quality System Regulation.

The intended use statements and promotional materials define the regulatory status of Thermo Scientific HyPerforma products and verbal claims associated with them. The medical devices are listed with the FDA using Form FDA 2892. Class I medical devices are not licensed by the FDA. Serum and media for cell culture use are also exempt from pre-market notification requirements (21 CFR 864). They may be used as a component in the manufacture of a device or drug that requires licensing. It is the responsibility of the device or drug manufacturer to determine if serum and/or cell culture media are suitable for their application.

Although we have implemented the same cGMP compliant quality system for BPC manufacture and the BPC facility is covered by our ISO 9001 registration, BPCs are not considered medical devices. The FDA does not regulate flexible containers.

Master files are submitted to the FDA to provide requested proprietary information. The FDA may review a master file in conjunction with the review of a customer’s product after we have authorized the review in a written letter to the FDA. A master file on BPCs has been submitted to the FDA along with subsequent updates to that master file. FDA CBER has given the BPCs master file the designation BB-MF-6657. Master files are not released to customers.

5.1 General

5.2 BPCs


Section 6Appendix

Section 6 Appendices

Validation References:

• U.S. Pharmacopoeia• European Pharmacopoeia• ASTM• ANSI/AAMI/ISO 11137• ANSI/AAMI/ISO 15843: 2000• ANSI/AAMI/ISO TIR27: 2001

%EL Percent elongation

AAMI Association for the Advancement of Medical Instrumentation

ANSI American National Standards Institute

ASTM American Society for Testing and Materials

BPC BioProcess Container

C Celsius

cGMP Current Good Manufacturing Practices

COA Certificate of Analysis

DFMEA Design Failure Mode Effect Analysis

DHS Drilled Hole Sparger

DOE Design of Experiments

DV Design Verification

EP European Pharmacopoeia

CBER Center for Biologics Evaluation and Research

F Fahrenheit

FDA Food and Drug Administration

FEA Finite Element Analysis

g Gram

GPa Giga Pascal

h Hour

in Inch

Appendix 6.1 Validation

References

Appendix 6.2 Abbreviations and

Acronyms


Section 6Appendix

in/min Inches per minute

ISO International Organization for Standardization

kGy Kilo Gray

L Liter

LAL Limulus Amebocyte Lysate

lbf Pound-force

MFC Mass Flow Controller (gas)

MPa Mega Pascal

mL Milliliter

NAO Non-Animal Origin

OQ Operational Qualification

PC Polycarbonate

PCD Process Control Document

PE Polyethylene

PFMEA Process Failure Modes and Effects Analysis

PP Polypropylene

PQ Process Qualification

PS Polysulfone

psi Pounds per square inch

psig Pounds per square inch gauge

PVC Polyvinyl chloride

RH Relative Humidity

RTD Resistance Temperature Dector

Tg Glass Transition Temperature

USP United States Pharmacopoeia

UTS Ultimate Tensile Strength

VIP Validation Information Package

WID Work Instruction Document

WVTR Water Vapor Transmission Rate


Section 6Appendix

Appendix 6.3Legacy Oxygen Transfer

Data

Introduction The S.U.B. BPC is designed to provide an acceptable range of kLa values to support rapid growth for a range of common cell platforms using the following operating parameters.

Table 6.1 Operating conditions for cell culture applications (full and half volume)

Study Method A design of experiments was generated for creation of mass transfer models for the S.U.B. systems. Thirty five randomized combinations (105 total) over the anticipated range of agitation and sparge rates (porous frit and open pipe) were tested for each volume S.U.B. The following table shows the testing media and ranges of experimental conditions used across all volumes used to generate these models. Please note the ranges shown are not applicable to all volumes.

Mathematical models were generated for the different S.U.B. sizes based on the results of these experiments. The ranges represented can be considered typical for the operation of the S.U.B.

Operating Condition Experimental Setup (50 to 1,000L)

Vessel fluidDeionized water containing1 g/L poloxamer 188 and 0.2 mL/L 10% antifoam

Temperature (C) 37

Porous frit gas flow rate range (lpm) 0 to 20

Open pipe flow rate range (lpm) 0 to 40

Overlay gas flow rate (lpm) 0

Agitation Speed (RPM) 20 to 230

Table 6.2 DOE for Mass Transfer Models

S.U.B. Range of Operating Parameters

50L 100L 250L 500L 1,000L 2,000LOperating Volume (L) 25 to 50 50 to 100 125 to 250 250 to 500 500 to 1,000 1,000 to 2,000


Temperature (°C) 2.0-45.0 ± 0.1

Recommended Max. Gas Flow Rates

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

Over

lay

Open

Pip

e

Poro

us F

rit

(3)

Over

lay

Air (lpm) 1 0.5 5 2 1 10 5 2.5 10 10 5 15 10 8 15 12 24 15

O2 (lpm) - 0.25 - - 0.5 - - 1.25 - - 2.5 - - 4 - - 8 -

CO2 (lpm) - 0.1 - - 0.2 - - 0.5 - - 1 - - 1 - - 1 -

N2 (lpm) - 0.25 - - 0.5 - - 1.25 - - 2.5 - - 1 - - 2.5 -

IMPORTANT NOTE: The data in this appendix was published in earlier versions of this Validation Guide. Since that time, test methodology and test procedures have changed. This legacy data is provided for reference.


Section 6Appendix

0.00

2.00

4.00

6.00

8.00

10.00

12.00

0 50 100 150 200 250

Agitation speed (rpm)

kLa (1/hr)

0.2 LPM

0.4 LPM

0.6 LPM

0.8 LPM

1.0 LPM

0.2 LPM + OP

0.4 LPM + OP

0.6 LPM + OP

0.8 LPM + OP

1.0 LPM + OP

k La (1

/hr)

Effect of Agitation Speed on kLa

50L S.U.B.

Porous Frit Sparger OnlyPorous Frit Sparger Plus 0.02 VVM (1 LPM)

Through Pipe

RPM Frit Flow (LPM)Open Pipe

(LPM)kLa RPM Frit Flow (lpm)

Open Pipe (lpm)

kLa

80 0.2 0 1.45 80 0.2 1 2.3480 0.4 0 2.60 80 0.4 1 3.5080 0.6 0 3.58 80 0.6 1 4.4980 0.8 0 4.39 80 0.8 1 5.3080 1 0 5.02 80 1 1 5.94115 0.2 0 1.35 115 0.2 1 2.32115 0.4 0 2.69 115 0.4 1 3.67115 0.6 0 3.86 115 0.6 1 4.85115 0.8 0 4.86 115 0.8 1 5.85115 1 0 5.68 115 1 1 6.68150 0.2 0 1.54 150 0.2 1 2.60150 0.4 0 3.08 150 0.4 1 4.14150 0.6 0 4.44 150 0.6 1 5.51150 0.8 0 5.63 150 0.8 1 6.70150 1 0 6.64 150 1 1 7.72185 0.2 0 2.04 185 0.2 1 3.17185 0.4 0 3.76 185 0.4 1 4.90185 0.6 0 5.32 185 0.6 1 6.46185 0.8 0 6.69 185 0.8 1 7.85185 1 0 7.89 185 1 1 9.06220 0.2 0 2.82 220 0.2 1 4.04220 0.4 0 4.74 220 0.4 1 5.96220 0.6 0 6.48 220 0.6 1 7.71220 0.8 0 8.05 220 0.8 1 9.29220 1 0 9.44 220 1 1 10.69

Table 6.3 Results for 50L S.U.B.

Graph 6.1 Results for 50L S.U.B.

Results


Section 6Appendix


0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

0 20 40 60 80 100 120 140 160

Agitation Speed (RPM)

k La (1

/hr)

kLa (1/hr)

1 LPM2 LPM3 LPM4 LPM5 LPM1 LPM + OP2 LPM + OP3 LPM + OP4 LPM + OP5 LPM + OP

250L S.U.B.


Through Pipe

RPMFrit Flow

(lpm)Open Pipe

(lpm)kLa RPM

Frit Flow (lpm)

Open Pipe (lpm)

kLa

30 1 0 2.13 30 1 5 3.4230 2 0 4.57 30 2 5 5.7230 3 0 6.49 30 3 5 7.4930 4 0 7.86 30 4 5 8.7230 5 0 8.71 30 5 5 9.4160 1 0 2.36 60 1 5 3.5260 2 0 5.07 60 2 5 6.0860 3 0 7.23 60 3 5 8.1060 4 0 8.87 60 4 5 9.5960 5 0 9.96 60 5 5 10.5490 1 0 3.26 90 1 5 4.2990 2 0 6.21 90 2 5 7.1090 3 0 8.64 90 3 5 9.3790 4 0 10.52 90 4 5 11.1190 5 0 11.87 90 5 5 12.32

120 1 0 4.81 120 1 5 5.70120 2 0 8.02 120 2 5 8.77120 3 0 10.69 120 3 5 11.30120 4 0 12.83 120 4 5 13.29120 5 0 14.44 120 5 5 14.75150 1 0 7.01 150 1 5 7.77150 2 0 10.47 150 2 5 11.09150 3 0 13.41 150 3 5 13.88150 4 0 15.80 150 4 5 16.13150 5 0 17.66 150 5 5 17.84

Table 6.4 Results for 250L S.U.B.

Graph 6.2 Results for 250L S.U.B.


Section 6Appendix

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

0 20 40 60 80 100 120 140


kLa (1/hr)

4 LPM8 LPM12 LPM16 LPM20 LPM4 LPM + OP8 LPM + OP12 LPM + OP16 LPM + OP20 LPM + OP

k La (1

/hr)


1,000L S.U.B.


Through Pipe

RPMFrit Flow

(lpm)Open Pipe

(lpm)kLa RPM

Frit Flow (lpm)

Open Pipe (lpm)

kLa

20 4 0 0.77 20 4 10 1.8375294720 8 0 1.87 20 8 10 2.8399345320 12 0 2.70 20 12 10 3.569252720 16 0 3.25 20 16 10 4.0254839720 20 0 3.53 20 20 10 4.2086283440 4 0 0.86 40 4 10 2.0497670940 8 0 2.18 40 8 10 3.2717966540 12 0 3.23 40 12 10 4.220739340 16 0 4.00 40 16 10 4.8965950540 20 0 4.50 40 20 10 5.299363960 4 0 1.40 60 4 10 2.7081767960 8 0 2.94 60 8 10 4.1498308360 12 0 4.20 60 12 10 5.3183979760 16 0 5.20 60 16 10 6.2138782160 20 0 5.92 60 20 10 6.8362715480 4 0 2.38 80 4 10 3.8127585780 8 0 4.14 80 8 10 5.4740370980 12 0 5.62 80 12 10 6.8622287280 16 0 6.84 80 16 10 7.9773334480 20 0 7.78 80 20 10 8.81935126100 4 0 3.81 100 4 10 5.36351242100 8 0 5.79 100 8 10 7.24441543100 12 0 7.49 100 12 10 8.85223154100 16 0 8.92 100 16 10 10.1869607100 20 0 10.08 100 20 10 11.2486031120 4 0 5.68 120 4 10 7.36043835120 8 0 7.88 120 8 10 9.46096584120 12 0 9.81 120 12 10 11.2884064120 16 0 11.46 120 16 10 12.8427601120 20 0 12.84 120 20 10 14.1240269

Table 6.5 Results for 1,000L S.U.B.

Graph 6.3 Results for 1,000L S.U.B.


Section 6Appendix


k La (1

/hr)


2,000L S.U.B.


Through Pipe

RPMFrit Flow

(lpm)Open Pipe

(lpm)kLa RPM

Frit Flow (lpm)

Open Pipe (lpm)

kLa

20 8 0 3.77 20 8 12 5.9020 12 0 6.11 20 12 12 8.2220 16 0 7.92 20 16 12 10.0120 20 0 9.19 20 20 12 11.2620 24 0 9.92 20 24 12 11.9735 8 0 3.94 35 8 12 6.2335 12 0 6.41 35 12 12 8.6635 16 0 8.33 35 16 12 10.5735 20 0 9.72 35 20 12 11.9435 24 0 10.58 35 24 12 12.7755 8 0 5.61 55 8 12 8.0855 12 0 8.23 55 12 12 10.6855 16 0 10.31 55 16 12 12.7455 20 0 11.86 55 20 12 14.2755 24 0 12.87 55 24 12 15.2675 8 0 5.98 75 8 12 8.6575 12 0 8.76 75 12 12 11.4175 16 0 11.01 75 16 12 13.6375 20 0 12.71 75 20 12 15.3175 24 0 13.88 75 24 12 16.4695* 8 0 6.77 95* 8 12 9.6395* 12 0 9.71 95* 12 12 12.5595* 16 0 12.11 95* 16 12 14.9395* 20 0 13.98 95* 20 12 16.7795* 24 0 15.31 95* 24 12 18.08

Table 6.6 Results for 2,000L S.U.B.

Graph 6.4 Results for 2,000 L S.U.B.

*NOTE: Standard operating range is 20 to 75rpm. Custom operating limit up to 95rpm at 90 to 110% working volume only.


Section 6Appendix

Appendix 6.4 Test Methods Used to Compare Drilled Hole

Sparger to Legacy Open Pipe Sparger

This appendix contains a detailed description of the test methods used to compare drilled hole sparger performance to the performance of the legacy open pipe sparger. The actual test results appear in the Oxygen Transfer topic in Section 2 of this guide.

Manufacturing Equipment Standard drilled hole sparger disc and BPC, component welding tools and ancillary items

Test Equipment • Druck DPI 705 pressure measurement instrument• 50, 250, and 2,000L S.U.B. plus Finesse TK and Delta V

controllers paired with Lauda T 1200 and Advantage Sentra SK 1035 and SK 3475 TCU units

• Sufficient air flow to operate the DHS at up to 0.1 VVM flow rates and the porous frit sparger up to 0.02VVM flow rates via the S.U.B. controller and stand-alone Alicat MFCs (mass flow controllers)

• Extech 461825 optical tachometer to verify agitation rates• Hobo U30 weather station with barometric pressure logging in lab

to compensate for ambient pressure shifts post data collection• Finesse stand alone and built in "blade" TruFluor DO transmitters

and associated cables with reader heads• Proprietary testing equipment for evaluating frit sparger behavior

MaterialsMaterials included prototypes of the following• Pre-tested-porous frits• Standard drilled hole sparger discs incorporated into the BPCs

noted in the following line. Drilled hole sprager test BPCs outfitted with new drilled hole sparger standard parts plus a pre-tested porous frit (including a spare BPC for each vessel size)

• Open pipe sparger test BPCs outfitted with an open pipe sparger plus a porous frit sparger (including a spare BPC for each vessel size)

• Mettler Toledo disposable pH electrodes• Finesse TruFluor™ Dissolved Oxygen (DO) sensors• Source for Air, N2, CO2, gassses at 30psig• NIST 7, 4.01, and 10.01 pH calibration buffer set

Test Protocol


Section 6Appendix

Standard Setup• BPCs loaded and filled with test solution (DI H2O + lg/leter

poloxamer 188 + 3.5g/liter HEPES buffer)• pH balanced to 7.25 at 100% air saturation using minimal naOH or

HCI• Temperature control stabilized at 37°C• Agitation at 0.15 HP/1k gal. PIV

Baseline Testing1. In previous testing, the open pipe data was not tested at actual

macro sparge flow rates. As such, it was important to run enough data points to generate representative kLa behavior and stacking efficiency when run in tandem with the porous frit sparger and by itself. Additionally, since the kLa testing procedure had changed since the original kLa was taken, it was important that general porous frit sparger and open pipe sparger performance behavior was re-evaluated (see next section for kLa analysis procedure).

- The BPCs with open pipe and porous frit spargers had O2 delivery and CO2 stripping kLa parameters run at a PIV of 0.15 electric hp/1,000 U.S. liquid gallons (29.56 Watt/m3). The closest match rpm values at 50, 250, and 2,000L S.U.B. vessels were 218, 139 and 87 respectively.- O2 delivery and CO2 stripping kLa testing varied micro and macro sparge flow rates according to the test map shown below.

2. Due to time constraints, the drilled hole sparger was characterized at 50, 250, and 2,000L vessel volumes. Vessel volumes in between these were estimated via scaled interpolation (which assumes representative general behavior patterns). As such, it was important to run enough data points to generate representative behavior and stacking efficiency when run in tandem with the porous frit sparger and by itself. The drilled hole sparger and frit testing repeated the conditions and varied micro and macro sparge rates used for frit and open pipe testing.

Test Map

0 0.0025 0.005 0.01 0.02 Micro VVM0 X X X X

0.125 X X X X

0.025 X X X

0.05 X X X X

0.1 X X X X X

0.15 X

Macro VVM


Section 6Appendix

kLa Analysis Process Test Name Standard O2 Delivery and CO2 Stripping kLa Performance Test

Test Stage Overview1. Setup2. Calibration3. kLa DO Delivery and CO2 Stripping Characterization4. kLa Data Processsing5. Step Response Testing (Optional)6. Step Response Data Processing (Optional)

Stage 1 - Setup

1. The BPC was filled to rated vessel volume with DI H2O loaded with 3.5 grams per liter HEPES buffer and one gram per liter poloxamer 188, or an alternative specified test solution.

2. Agitation value was set to the target for test (agitation was given at least 5 minutes to stabilize pattern before running a new test).

3. Temperature was set to 37°C, or an alternative specified test temperature.

4. Single-use Finesse TruFluor DO sensors were connected.5. Mettler Toledo single-use pH electrodes to be used were connected

and calibrated in NIST 4.01, 7, and 10.01 calibration buffers. 6. Calibrated Mettler Toledo pH sensors were inserted.7. Ensured that data was logging correctly to Finesse TK and DeltaV

control systems.8. Set agitation to target test value.9. Set system temperature to 37°C.10. Connected air lines to MFCs to be used for testing (controller and

stand-alone).11. Set macro sparge/open pipe sparge rates to 0.02VVM (example: in a

50L reactor this would be one liter per minute) air flow.12. Set micro sparge/porous frit sparge rates to 0.01VVM air flow.13. Waited for the system temperature to stabilize for at least 30 minutes.

Air flow was enabled for at least 60 minutes before proceeding.

Stage 2 - Calibration

1. Took one point offset on pH sensors. 2. Adjusted measured pH using minimal amounts of NaOH and/or HCl

to reach 7.25 pH. Documented amount of acid or base used. Allowed 10 minutes to stabilize before proceeding.

3. Stopped air flow and sparged N2 instead, at previous air flow rates.4. Performed the first of a two point calibration on all DO sensors after

N2 had been sparging into the system for at least one hour.


Section 6Appendix

5. Took low/0% air saturated calibration value. Documented ambient lab pressure at this time.

6. Stopped all air sparging and started air flow at 0.02VVM through the micro sparger/porous frit sparger. Did not proceed until DO measurement had not changed or was “bouncing” between a final value range, and the value range had not further increased for at least five minutes.

7. Took high/100% air saturated calibration value.8. Allowed air flow to continue for at least 45 minutes after

callibration. Verified that measured DO values remained at approximately 100% of high calibration result.

Stage 3 - kLa DO Delivery and CO2 Stripping Characterization

1. Ensured that airflow had stabilized and measured DO values for at least 60 minutes to establish initial 100% air saturation value.

2. Ensured that agitation was set to target for kLa test for at least five minutes prior to starting the test.

3. Stopped all air flow to vessel.4. Set micro sparge to 0.02VVM N2 gas flow (example: in a 50L

reactor this would be one liter per minute).5. Set macro sparge to 0.05VVM N2 delivery.6. Let system run until measured DO was below five percent, and

documented system clock time just before starting the next steps.7. Stopped N2 flow for five minutes to allow holdup gas to leave

vessel.8. Set macro and micro sparge directly and immediately to kLa test

values. 9. Documented approximate start time of introducing air to system for

kLa test.10. Let system run until measured DO is above 90% air saturation.11. Repeated steps 2 through 10 for each set of kLa test parameters in the

test map.12. At least once every 24 hours, swapped step 1 with step 10 to re-

establish 100% air saturation measurment (note that air flow rates were set to 0.01VVM and 0.02VVM, micro and macro air flow rates respectively).

13. Repeated step 12 (re-establish 100% air saturation for at least 60 minutes) at the end of testing regardless of time since last re-establishment of 100% air saturation.

14. When performing CO2 stripping, replaced all N2 sparge steps with CO2 and documented process for CO2 stripping (note that CO2 stripping response is significantly slower than O2 delivery response).

NOTE: CO2 stripping tests were always performed after O2 delivery


Section 6Appendix

tests. CO2 has a strong affinity for water and is difficult to completely remove. When more O2 delivery tests were performed, air was sparged into the system per step 4 for at least six hours to ensure purge of excess CO2 from the system.

Stage 4 - kLa Data Processing

1. Collected data for vessel temperature, sparge air, primary N2 and CO2 flow rates, agitation rpm and ambient lab pressure during that time.

2. DO data sets were synchronized with ambient lab pressure data. 3. Normalized data to ambient pressure. a. The average ambient lab pressure that existed when kLa experiments were first run was considered the “baseline pressure.” b. Multipled all measured DO values by the “baseline pressure.” c. Divided all measured DO values by the actual ambient lab pressure, to normalize % DO values to their relative correct partial pressure and keep kLa data consistent with respect to changing lab pressure.4. Normalized data to “true” 100% air saturation. a. The average of the last five minutes of each 60 minute 100% air saturation reestablishment segment was used to re-calibrate data after the experiment was completed in order to compensate for drift. Calculated the average %air saturated DO for the last five minutes of each of these segments; these are the “true” 100% air saturation DO values. b. Performed linear interpolations between the first two “true” 100% air saturation DO values (called “true”1 and “true”2) by using the equation: interpolated “true” 100% air sat. = “true”1 + (“true”2 – “true”1)*(currentime – time1)/(time2 – time1). c. Repeated steps 4.a and 4.b to interpolate between any other “true” 100% air saturation DO values.This produced a relative scaled “true” 100% air saturated DO value to re-calibrate all measured DO values with, and removed any standard sensor drift. d. Divided all measured DO values by the calculated interpolated “true” 100% air saturation values, making values close to 100% equal to 1. e. Multiplied all measured DO values by 100% to return to a % air saturation measurement now normalized based on usable post calibration points.5. In order to process the test data into kLa values, took the linear

regression of the log adjusted DO data from 20% to 80% air saturation with respect to change in time. Log adjustment of DO data is as follows:


Section 6Appendix

In C* – C0C* – Ct

( ) where C* is the DO saturation value and C0 and Ct are the initial DO concentration and the current concentration at time t. If the data has been correctly normalized as outlined previously, C* should always be 100%.6. Ensured that all calculated linear regression adjusted R2 (correlation

coefficient) values were 0.97 or better (1 is ideal). a. If the R2 value was less than 0.97, checked the corresponding plot to determine the cause and re-ran test to ensure accuracy, if necessary.7. Copied resulting values to target report and generated performance

plots and 3D behavior maps from the data.

Stage 5 - Step Response Testing

1. Set up two 1 Liter or larger vessels filled with DI H2O and temperature controlled to 37°C.

2. Enabled micro sparging of air to vessel 1, and N2 to vessel 2 at 0.1 VVM.

3. Covered each vessel with an opening just large enough to insert DO sensors used in kLa testing. Let gasses sparge for at least 30 minutes.

4. Inserted a DO sensor into vessel 1 and allowed it to equilibrate for 20 minutes. Ensured that no bubbles were attached to sensing chemistry or membrane. Note: If the probe head can be set just below the bubble path of the sparger it should will avoid bubbles building up on sensing head.

5. Rapidly moved the sensor to vessel 2 such that insertion into vessel 2 liquid occured at the exact documented time. Note: This was always accomplished in under 5 seconds.

6. Checked for bubbles interfering with sensing membrane or chemistry. Documented if bubbles were present, and shook them off.

7. Allowed to equilibrate in vessel 2 for at least 20 minutes.8. Rapidly moved the sensor to vessel 1 such that insertion into vessel

1 liquid occured at an exact marked time (accomplish in under 5 seconds).

9. Checked for bubbles on sensing membrane or chemistry, shook them off, and documented this if necessary.

10. Repeated steps 5 through 8.11. Repeated full procedure for each DO sensor that needed its response

time checked.


Section 6Appendix

Stage 6 - Step Response Data Processing

1. Measured the time required from insertion into the new vessel liquid until the sensor reached 63% and 90% of the new steady state for all sensor transfers that were carried out between vessels 1 and 2.

2. Multiplied the 63% time by 5. This approximates a 99% of steady state response time estimate.

3. Averaged the 63% response time estimates, then calculated the 99% confidence and documented both values.

4. Repeated step 3 for 90% and 99% estimate. These are the measured T63, T90, and T99 response times for the tested DO sensor.

Note: It has been suggested by Van’t Riet and others that a response time (T63) that is too close to 1/kLa values will skew measured kLa using the dynamic method outlined in this document. Generally speaking, a response time value that is 20% or less of 1/kLa should provide acceptable results. T63 response times that are greater than 30% of 1/kLa being measured will require mathematical response time compensation of the O2 sensor data being used prior to estimating kLa. The sensors tested were found to have adequate response times for the testing performed.

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