Validation Guide. Thermo Scientific HyPerforma Single-Use Bioreactor Systems. Validation Guide. Revision A

Transcription

1 Guide Thermo Scientific HyPerforma Single-Use Bioreactor Systems Guide Revision A DOC0016 December 2015

2 Table of Contents Section 1 Section 2 Section 3 Section 4 Scope Introduction Process Qualification Sterility Assurance Level Endotoxin and Particulate Functionality...6 Sparger Designs...8 Probe Assembly Design...10 Individual Component Evaluation...13 Probe Assembly Evaluation Oxygen Transfer Mixing Studies Additional 2,000L Studies S.U.B. 50 to 1,000L Temperature Mapping Studies Sterility Testing...56 Condenser System 3.1 Functional Overview Peristaltic Pump Chiller (TCU) Condenser Plate Assembly Functional Overview Condenser Testing...72 Quality Control 4.1 Introduction Inspection...74 Incoming Inspection In-Process Inspections and Testing 4.3 BPC Lot Record Release and Certificate of Analysis Sample C of A Traceability Shelf Life Sample Certificate of Irradiation...78 Thermo Scientific Single-Use Bioreactor (S.U.B.) 2

3 Section 5 Section 6 Regulatory 5.1 General BPCs...79 Appendix 6.1 References Abbreviations and Acronyms Legacy Oxygen Transfer Data Test Methods for Comparing Drilled Hole to Open Pipe Spargers...87 Thermo Scientific Single-Use Bioreactor (S.U.B.) 3

4 Section 1 Scope Section 1 Overview 1.1 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. 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 4

5 Section Introduction 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. 2.2 Process Verification 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. testing verifies and confirms that product manufactured according to the Batch Record meets the acceptance criteria set by engineering during the verification phase. 2.3 Sterility Assurance Level 2.4 Endotoxin and Particulate of the gamma irradiation sterility assurance level (SAL) for BPC is performed per ANSI/AAMI/ISO :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 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 5

6 2.5 Functionality Individual Component Designs Hub Assembly Design 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. Figure 2.1 Hub assembly 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. Seal Cup Design 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 6

7 V-Ring Seals Gasket Bearings Dust Cover Hub Design Impeller Design 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 Figure 2.2 Impeller and tubing design Thermo Scientific Single-Use Bioreactor (S.U.B.) 7

8 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. 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 ) Figure 2.3 Open pipe sparger Table 2.1 Open pipe sparger ID 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 2.4 Porous Frit Sparger Thermo Scientific Single-Use Bioreactor (S.U.B.) 8

9 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 Figure 2.6 Drilled hole sparger top view 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 100L Porous Frit and Drilled Hole Spargers 9cm (3.5 ) disk with 570 x 0.178mm holes 250L Porous Frit and Drilled Hole Spargers 12cm (4.8 ) disk with 760 x 0.233mm holes 500L Porous Frit and Drilled Hole Spargers 17cm (6.75 ) disk with 980 x 0.368mm 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 Spargers Table 2.2 Drilled hole sparger configuration by volume 2x 17cm (6.75 ) disks with 690 x 0.582mm holes (1,380 holes) Thermo Scientific Single-Use Bioreactor (S.U.B.) 9

10 Probe Assembly Design 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 Interface Sleeve Fitting Figure 2.7 Model of probe adapter nut Adapter Nut Sleeve Probe Figure 2.8 Model showing the probe, adapter nut and sleeve assembly 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 10

11 strength were obtained using a stainless steel probe and a PC adapter fitting. Average maximum torque strength values (to failure) were in-lbs for pre-irradiated samples and 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. Sleeve Fitting 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. Manufacturer Required Specifications to Fit Probe Assembly Length From O-ring (mm) Length From O-ring (mm) Diameter (mm) Applikon Biotechnology DO Applikon Biotechnology ph Mettler Toledo DO Mettler Toledo ph Broadley James DO Broadley James ph Finesse Solutions DO Finesse Solutions ph Table 2.3 Specifications for resuable probes for use in the probe assembly Figure 2.9 Model of probe sleeve fitting Thermo Scientific Single-Use Bioreactor (S.U.B.) 11

12 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. Kleenpak Connectors Temperature/Sampling Fitting Design Chamber Design 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 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 ( ) ID x 1.59mm ( ) 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 12

13 Individual Component Evaluation Hub Assembly Evaluation (S.U.B. Testing) Seal Cup Figure 2.12 is a cross sectional view of the hub assembly and a threedimensional view of the seal cup. Figure 2.12 Seal cup design Seal Cup Engagement Test 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 13

14 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 preirradiation 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 277.5lb f with a standard deviation of 26.3lb f. 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 198.6lb f is acceptable due to no tensile load being applied to the seal cup during use. Seal Cup Disengagement 1/27/2006 Rate: 12.7cm (5 ) per minute Sample lb f Stats ave stdev 26.3 min max Table 2.4 Seal cup disengagement results 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 14

15 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 15

16 Figure 2.15 Tests #10-5 to 10-7, non-product contact v-seal only 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. Gasket Pressure Test Hub Sub-Assembly Testing 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 16

17 Impeller to Hub Tubing Pull Test Probe Assembly Evaluation Leak/Burst Testing 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. 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 No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material No Leak Sleeve Material Avg SD Table 2.5 Leak burst test results 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 ( ) 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 17

18 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. Temperature Probe Sample Tube Figure 2.16 Model of the temperature/sampling (TS) fitting Thermo Scientific Single-Use Bioreactor (S.U.B.) 18

19 TS Fitting Temperature Profile Temperature ( C) Time (min) Stainless Steel Sheath (1/4) (1/4) Probe Control Silicone Sheath (1/8) 1/8 Probe Control Graph 2.1 Temperature map profile (temperature/sampling fitting) Temperature ( C), 1/4 Probe Temperature ( C), 1/4 Probe Time (min) SS-Sheath Control Delta Silicone Sheath Control Delta Ramp Rate: 2 C/min Table 2.6 Temperature offset summary Assembled BPC Evaluation (S.U.B. Testing) Visual Inspection Dimensional Inspection Leak/Burst Test Unsupported 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 19

20 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 and 1.064psi, respectively. Average maximum pressures were psi for the 50L and 4.07psi for the 250L S.U.B. BPC. Container Burst Strength Test Supported 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. Sample Burst Location Max Failure Type Pressure (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 Film and Port Weld Seam Strengths Peel tests were performed on all welded seams (film-to-film and port-tofilm). 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 20

21 2.6 Oxygen Transfer Introduction The S.U.B. BPC is designed to provide an acceptable range of k L a 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. S.U.B. BPCs Range of Operating Parameters with Open Pipe and Frit Spargers 50L 100L 250L 500L 1,000L 2,000L Temperature ( C) ± 0.1 Operating Volume (L) 25 to to to to to 1,000 1,000 to 2,000 Agitation Rate (rpm) 30 to to to to to to 75 Recommended Max. Gas Flow Rates Open Pipe Porous Frit Overlay Open Pipe Porous Frit Overlay Open Pipe Porous Frit Overlay Open Pipe Porous Frit Overlay Open Pipe Porous Frit Overlay Open Pipe Porous Frit (3) Overlay Air (slpm) O 2 (slpm) CO 2 (slpm) N 2 (slpm) Total (slpm) Exhaust Load (slpm) Table 2.8 Operating parameters using open pipe and porous frit spargers S.U.B. BPCs Range of Operating Parameters with Drilled Hole and Frit Spargers 50L 100L 250L 500L 1,000L 2,000L Temperature ( C) ± 0.1 Operating Volume (L) 25 to to to to to 1,000 1,000 to 2,000 Agitation Rate (rpm) 30 to to to to to to 75 1 Recommended Max. Gas Flow Rates Drilled Hole Porous Frit Overlay Drilled Hole Porous Frit Overlay Drilled Hole Porous Frit Overlay Drilled Hole Porous Frit Overlay Drilled Hole Porous Frit Overlay Drilled Hole Porous Frit Overlay Air (slpm) O 2 (slpm) CO 2 (slpm) N 2 (slpm) Total (slpm) Exhaust Load (slpm) Table 2.9 Operating parameters using drilled hole and porous frit spagers 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 21

22 Results Overview Experiments were performed using 50, 250 and 2,000L vessels to measure the mass transfer of oxygen and CO 2 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 CO 2 stripping, and are presented as k L a for various sparge flow rates for each vessel size. Two dimensional plots are used to show individual sparger results for oxygen delivery and CO 2 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 k L a 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 22

23 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Open Pipe Sparger Graph 2.2 Results for 50L S.U.B. with porous frit sparger Graph 2.3 Results for 50L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger 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 Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.6 Results for 50L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 23

24 100L Results with Porous Frit and Open Pipe Spargers 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Open Pipe Sparger Graph 2.7 Interpolated results for 100L S.U.B. with porous frit sparger Graph 2.8 Interpolated results for 100L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla 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 Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.11 Interpolated results for 100L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 24

25 250L Results with Porous Frit and Open Pipe Spargers Results of experiments with 250L vessels using porous frit and open pipe spargers are shown below Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Open Pipe Sparger Graph 2.12 Results for 250L S.U.B. with porous frit sparger Graph 2.13 Results for 250L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla with Open Pipe Sparger Graph 2.14 Results for 250L S.U.B. with porous frit sparger Graph 2.15 Results for 250L S.U.B. with open pipe sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 30 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.16 Results for 250L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 25

26 500L Results with Porous Frit and Open Pipe Spargers 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. Oxygen Delivery kla for Frit Sparger Oxygen Delivery kla for Frit Sparger Graph 2.17 Interpolated Results for 500L S.U.B. with porous frit sparger Graph 2.18 Interpolated Results for 500L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla with Open Pipe Sparger Graph 2.19 Interpolated Results for 500L S.U.B. with porous frit sparger Graph 2.20 Interpolated Results for 500L S.U.B. with open pipe sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 35 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.21 Interpolated Results for 500L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 26

27 1,000L Results with Porous Frit and Open Pipe Spargers 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Open Pipe Sparger Graph 2.22 Interpolated Results for 1,000L S.U.B. with porous frit sparger Graph 2.23 Interpolated Results for 1,000L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla with Open Pipe Sparger Graph 2.24 Interpolated Results for 1,000L S.U.B. with porous frit sparger Graph 2.25 Interpolated Results for 1,000L S.U.B. with open pipe sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 35 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.26 Interpolated Results for 1,000L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 27

28 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 Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Open Pipe Sparger Graph 2.27 Results for 2,000L S.U.B. with porous frit sparger Graph 2.28 Results for 2,000L S.U.B. with open pipe sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla with Open Pipe Sparger Graph 2.29 Results for 2,000L S.U.B. with porous frit sparger Graph 2.30 Results for 2,000L S.U.B. with open pipe sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.31 Results for 2,000L S.U.B. with both porous frit and open pipe spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 28

29 50L Results for Porous Frit and Drilled Hole Spargers The results of experiments with 50L vessels using porous frit and drilled hole spargers are shown below. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.32 Results for 50L S.U.B. with porous frit sparger Graph 2.33 Results for 50L S.U.B. with drilled hole sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Spripping kla with Drilled Hole Sparger Graph 2.34 Results for 50L S.U.B. with porous frit sparger Graph 2.35 Results for 50L S.U.B. with drilled hole sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.36 Results for 50L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 29

30 100L Results for Porous Frit and Drilled Hole Spargers 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.37 Interpolated Results for 100L S.U.B. with porous frit sparger Carbon Dioxide Stripping kla with Frit Sparger Graph 2.38 Interpolated Results for 100L S.U.B. with drilled hole sparger Carbon Dioxide Stripping kla with Drilled Hole Sparger Graph 2.39 Interpolated Results for 100L S.U.B. with porous frit sparger Graph 2.40 Interpolated Results for 100L S.U.B. with drilled hole sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.41 Interpolated Results for 100L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 30

31 250L Results for Porous Frit and Drilled Hole Spargers Results of experiments with 250L vessels using porous frit and drilled hole spargers are shown below. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.42 Results for 250L S.U.B. with porous frit sparger Carbon Dioxide Stripping kla with Frit Sparger Graph 2.43 Results for 250L S.U.B. with drilled Hole sparger Carbon Dioxide Stripping kla with Drilled Hole Sparger Graph 2.44 Results for 250L S.U.B. with porous frit sparger Graph 2.45 Results for 250L S.U.B. with drilled hole sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 35 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.46 Results for 250L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 31

32 500L Results for Porous Frit and Drilled Hole Spargers 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.47 Interpolated Results for 500L S.U.B. with porous frit sparger Carbon Dioxide Stripping kla with Frit Sparger Graph 2.48 Interpolated Results for 500L S.U.B. with drilled hole sparger Carbon Dioxide Stripping kla with Drilled Hole Sparger Graph 2.49 Interpolated Results for 500L S.U.B. with porous frit sparger Graph 2.50 Interpolated Results for 500L S.U.B. with drilled hole sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 30 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.51 Interpolated Results for 500L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 32

33 1,000L Results for Porous Frit and Drilled Hole Spargers 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. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.52 Interpolated Results for 1,000L S.U.B. with porous frit sparger Carbon Dioxide Stripping kla with Frit Sparger Graph 2.53 Interpolated Results for 1,000L S.U.B. with drilled hole sparger Carbon Dioxide Stripping kla with Drilled Hole Sparger Graph 2.54 Interpolated Results for 1,000L S.U.B. with porous frit sparger Graph 2.55 Interpolated Results for 1,000L S.U.B. with drilled hole sparger Combine kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 30 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.56 Interpolated Results for 1,000L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 33

34 2,000L Results for Frit and Drilled Hole Spargers The results of experiments with 2,000L vessels using porous frit and drilled hole spargers are shown below. Oxygen Delivery kla with Frit Sparger Oxygen Delivery kla with Drilled Hole Sparger Graph 2.57 Results for 2,000L S.U.B. with porous frit sparger Graph 2.58 Results for 2,000L S.U.B. with drilled hole sparger Carbon Dioxide Stripping kla with Frit Sparger Carbon Dioxide Stripping kla with Drilled Hole Sparger Graph 2.59 Results for 2,000L S.U.B. with porous frit sparger Graph 2.60 Results for 2,000L S.U.B. with drilled hole sparger Combined kla Oxygen Delivery At 0.15 HP / 1000gal Agitation 30 kla 1/hrs Macro Sparge slpm Micro Sparge slpm Graph 2.61 Results for 2,000L S.U.B. with both porous frit and drilled hole spargers Thermo Scientific Single-Use Bioreactor (S.U.B.) 34

35 2.7 Mixing Studies The recommended range of mixing rates for the range of S.U.B. systems is as follows: 50L 100L 250L 500L 1,000L 2,000L Operating Volume (L) ,000 1,000-2,000 Agitation Rate (rpm) Table 2.10 Recommended range of mixing rates 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 S.U.B. 100L S.U.B. Half Volume Full Volume Half Volume Full Volume Mixing Agitation Mixing Agitation Mixing Agitation Time Speed Time Speed Time Speed (sec) (rpm) (sec) (rpm) (sec) (rpm) Agitation Speed (rpm) Mixing Time (sec) L S.U.B. 500L S.U.B. Half Volume Full Volume Half Volume Full Volume Mixing Agitation Mixing Agitation Mixing Agitation Time Speed Time Speed Time Speed (sec) (rpm) (sec) (rpm) (sec) (rpm) Agitation Speed (rpm) Mixing Time (sec) ,000L S.U.B. Half Volume Full Volume Mixing Agitation Time Speed (sec) (rpm) Agitation Speed (rpm) Mixing Time (sec) Table 2.11 Summary of mixing study results for the 50 to 1,000L S.U.B. systems Thermo Scientific Single-Use Bioreactor (S.U.B.) 35

36 Mixing Study 50L S.U.B Concentration (%) Time (sec) 50 rpm avg 100 rpm avg 150 rpm avg 200 rpm avg Graph 2.62 Mixing study 50L S.U.B. full volume Mixing Study 50L S.U.B Concentration (%) Time (sec) 50 rpm avg 100 rpm avg 150 rpm avg Graph 2.63 Mixing study 50L S.U.B. half volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 36

37 Mixing Study 100L S.U.B. Concentration (%) Time (sec) 50 rpm avg 100 rpm avg 150 rpm avg 200 rpm avg Graph 2.64 Mixing study 100L S.U.B. full volume Mixing Study 100L S.U.B. 140 Concentration (%) Time (sec) 50 rpm avg 100 rpm avg 150 rpm avg Graph 2.65 Mixing study 100L S.U.B. half volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 37

38 Mixing Study 250L S.U.B concentration (%) Time (sec) avg 60 rpm avg 100 rpm avg 120 rpm avg 140 rpm Graph 2.66 Mixing study 250L S.U.B. full volume Mixing Study 250L S.U.B Concentration (%) Time (sec) 40 rpm avg 60 rpm avg 80 rpm avg Graph 2.67 Mixing study 250L S.U.B. half volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 38

39 Mixing Study 500L S.U.B. (Full Volume) Concentration (%) Time (sec) 30 rpm avg 70 rpm avg 110 rpm avg 150 rpm avg Graph 2.68 Mixing study 500L S.U.B. full volume Mixing Study 500L S.U.B. (Half Volume) Concentration (%) Time (sec) 30 rpm avg 70 rpm avg 110 rpm avg 150 rpm avg Graph 2.69 Mixing study 500L S.U.B. full volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 39

40 10% Mixing Study 1000L S.U.B. (average of 3 sample locations) 8% Concentration 6% 4% 2% % time(s) 60rpm 70rpm 80rpm 90rpm 100rpm 110rpm Graph 2.70 Mixing study 1,000L S.U.B. full volume 100% Mixing Study 1000L S.U.B. (average of 2 sample locations) 80% Concentration 60% 40% 20% 0% time(s) 60 rpm 45 rpm 30 rpm Graph 2.71 Mixing study 1,000L S.U.B. - half volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 40

41 Mixing Study (2,000L) 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 Test #* Tank Volume Impeller Location/Shaft Power/Vol (Hp/1,000 Length gal) RPM M1 Nominal, 2,000L 1 diameter from bottom / M2 Nominal, 2,000L 1 diameter from bottom / M3 Nominal, 2,000L 1 diameter from bottom / M10 ½ Volume, 1,000L 1 diameter from bottom / M11 ½ Volume, 1,000L 1 diameter from bottom / M12 ½ Volume, 1,000L 1 diameter from bottom / Table 2.12 Mixing study test matrix 2,000 L S.U.B. Thermo Scientific Single-Use Bioreactor (S.U.B.) 41

42 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) ,000L S.U.B. (Half Volume Mixing Time) RPM Average Mix Time - Worst Case (sec) Average Mix Time - All Probes (sec) Table 2.13 Mixing study results 2,000L S.U.B. Graph 2.72 Mixing study 2,000L S.U.B. - full volume Graph 2.73 Mixing study 2,000L S.U.B. - half volume Thermo Scientific Single-Use Bioreactor (S.U.B.) 42

43 2.8 Additional 2,000L Studies Drain Time and Hold-up Volume Test Procedure 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 (SV ) 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 (SV ) 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 Results 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. Both tests passed the mandatory four-liter maximum allowable hold-up volume requirement as shown in table 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 43

44 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. Section 2 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 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 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.). Thermo Scientific Single-Use Bioreactor (S.U.B.) 44

45 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 45

46 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 2 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. Thermo Scientific Single-Use Bioreactor (S.U.B.) 46

47 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 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 volume and 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 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 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 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 NOTE: The jacketed S.U.B. systems have an approximate total jacket heat transfer area shown in Table Thermo Scientific Single-Use Bioreactor (S.U.B.) 47

48 Vessel Temp Start ( C) Temp Finish ( C) Min/Max Temp ( C ) Time (Minutes) Time (Hours) Average Slope ( C /min) Watts 50L Resistive / K 100L Resistive / K 250L Resistive / K 500L Resistive / K 1,000L Resistive / K 50L Jacketed / K 100L Jacketed / K 250L Jacketed / K 500L Jacketed / K 1,000L Jacketed / K 50L Jacketed / K 100L Jacketed / K 250L Jacketed / K 500L Jacketed /50 N/A N/A N/A 10K 1,000L Jacketed / K Table 2.15 Heating and cooling statistics Vessel - Jacketed Jacket Area in In 2 Jacket Area in Cm 2 50L L L L ,000L Table 2.16 Jacket surface area Thermo Scientific Single-Use Bioreactor (S.U.B.) 48

49 50L Resistive SUB S.U.B. Heat Heat Up Up Time Time Degrees C Hours 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 100L Resistive SUB S.U.B. Heat Heat Up Up Time Time Degrees C Hours AVERAGE 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 49

50 250L Resistive SUB Heat Up Time 250L Resistive S.U.B. Heat Up Time Degrees 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 500L 500L Resistive SUB S.U.B. Heat Heat Up Up Time Degrees C Hours AVERAGE Graph 2.80 Temperature profile of 500L resistive S.U.B. from 5 to 37 C maximum resistive blanket temperature of 50 C Thermo Scientific Single-Use Bioreactor (S.U.B.) 50

51 1000L Resistive SUB S.U.B. Heat Up Time Degrees C Hours AVERAGE Graph 2.81 Temperature increase in 1,000L resistive S.U.B. from 5 to 37 C maximum resistive blanket temperature of 50 C 50L Jacketed S.U.B. SUB Heat Up Time Degrees 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 51

52 100L 100L Jacketed SUB S.U.B. Heat Up Time Degrees C Hours AVERAGE Graph 2.83 Temperature increase in 100L jacketed S.U.B. from 5 to 37 C with a maximum TCU temperature of 50 C 250L 250L Jacketed SUB S.U.B. Heat Up Time Degrees C Hours AVERAGE Graph 2.84 Temperature increase in 250L jacketed S.U.B. from 5 to 37 C with a maximum TCU temperature of 50 C Thermo Scientific Single-Use Bioreactor (S.U.B.) 52

53 500L 500L Jacketed Jacketed SUB S.U.B. Heat Heat Up Up Time Degrees C Hours AVERAGE Graph 2.85 Temperature increase in 500L jacketed S.U.B. from 5 to 37 C with a maximum TCU temperature of 50 C 1000L 1000L Jacketed Jacketed SUB Heat S.U.B. Up Heat Time Up Time Degrees 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 53

54 50L 50L Jacketed SUB S.U.B. Cool Down Time Degrees C Hours 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. 100L 100L Jacketed SUB S.U.B. Cool Down Time Degrees 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 54

55 250L 250L Jacketed SUB S.U.B. Cool Cool Down Down Time Time Degrees 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. Degrees C 1000L Jacketed S.U.B. SUB Cool Down Time 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 55

56 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 Sterility Testing Initial Evaluation Sterility Testing Secondary Evaluation An initial sterility run was conducted using the hub assembly. A total of three samples were tested. These samples represented a worstcase 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. 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). Thermo Scientific Single-Use Bioreactor (S.U.B.) 56

57 Sterility Testing Final Evaluation 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. Results and Discussion Sterility Testing Initial Evaluation 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) Thermo Scientific Single-Use Bioreactor (S.U.B.) 57

58 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. Sterility Testing Secondary Evaluation 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. Sterility Testing Final Evaluation Conclusions 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 58

59 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 2 Thermo Scientific Single-Use Bioreactor (S.U.B.) 59

60 Section 3 Condenser System Section 3 Condenser System 3.1 Functional Overview 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. Exhaust Line From S.U.B. Filter Straps Filter Bracket Assembly Condenser Post Assembly Exhaust Vent Filters Condenser Plate Assembly Condenser Return Line Back to S.U.B. Dual Headed Peristaltic Pump Post Receivers Condenser Bag Gas Outlet Port Dual Chamber Condenser Bag Condenser Bag Gas Inlet Port Gripping Tabs Closed Bath Recirculating Chiller Cart Assembly Condenser Bag Liquid Drain Ports Alignment Holes Condenser Disposables Condenser Hardware Condenser System Figure 3.1 Condenser system overview Thermo Scientific Single-Use Bioreactor (S.U.B.) 60

61 Section 3 Condenser System Materials Table Bill of materials for single-use condenser components per SH2B2004. Item Part Number Description 1 1 Each 1 Each SH2B Container: Bioreactor Condenser Bag 2 2 Each 2 Each SV Filter: Ultracap, 0.2 um 3/4 HB x 3/4 HB, Meissner 3 2 Each 2 Each SV Fitting: Polypropylene 90 elbow, 5/8 4 1 Each 1 Each SV Fitting: Polypropylene ST. Conn., 3/16 x 1/4 5 1 Each 1 Each SV Fitting: Polypropylene Y. Conn., 3/4 x 5/8 x 5/8 6 3 Each 3 Each SV Fitting: Polypropylene Y. Conn., 1/4 7 2 Each 2 Each SV Fitting: Polypropylene end plug, 1/2 8 2 Each 2 Each SV Fitting: Polypropylene T. Conn., 5/ m (2.67 ) 0.82m (2.67 ) SV Tubing: Silicon braided, 5/8 ID x OD m (7 ) 1.52m (5 ) SV Tubing: C-Flex (R ), 1/4 ID x 3/32 Wall m (3.5 ) 1.07m (3.5 ) SV Tubing: Pharmapure, 3/16 ID x 1/16 Wall m (7 ) 1.32m (4.33 ) SV Tubing: Silicon braided, 3/4 ID x 1.10 OD m (2 ) 0.60m (2 ) SV Tubing: Silicone, 1/2 ID x 1/8 Wall m (1.33 ) 0.80m (1.33 ) SV Tubing: C-Flex (R ), 5/8 ID x 1/8 Wall 15 1 Each 1 Each SV Fastener: Snapper clamp, 1/2-3/ Each 34 Each SV20030 Fastener: Cable tie m (4 ) 1.22m (4 ) SV50072 Packaging Material: Bubble wrap Table 3.1 Parts list Pressure vs. Exhaust Flow Rate 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 L SUB Internal Pressure vs. Sparge Flow with Standard Condenser Bag and Varying Vent Filters Internal Pressure (psi) Sparge Flow (L/min) 2 x 10" Meissner 1 x 10" Meissner 2 x KA3 1 x KA3 Filter 2,000L System Back Pressure per Sparge Flow Rate (psi) Configuration 10Lpm 15Lpm 20Lpm 25Lpm 30Lpm 35Lpm 40Lpm 45Lpm 50Lpm 2x10 Meissner x10 Meissner xKA xKA Table 3.2 Filter performance testing on production intent condenser system Thermo Scientific Single-Use Bioreactor (S.U.B.) 61

62 Section 3 Condenser System 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 # (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 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. Figure 3.3 Spallation comparison of peristaltic pump tubing types Thermo Scientific Single-Use Bioreactor (S.U.B.) 62

63 Section 3 Condenser System 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 63

64 Section 3 Condenser System 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. Door Cover Plate O-ring Seal Coolant Channel Bleed Vent Bag Tension Latch Plate Coolant Inlet Port Coolant Inlet Port Plate Insulation Bag Center Baffle Bar Door Latch Condenser Bag Alignment Buttons Figure 3.5 Condenser plate assembly exploded view Condenser Plate Structural Integrity 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 Thermo Scientific Single-Use Bioreactor (S.U.B.) 64