Automating the Cell Culture Sampling Process

1. Automating the Cell Culture Sampling Process Mike Phipps Tara Ryan BME 273 April 5, 2002

2. Problem/Background • Cell cultures maintained in bioreactors for Research and Development purposes in pharmaceutical companies must be sampled regularly • Samples (10-15mL) are typically taken once most days, and twice every three days or so when the culture is split • methods of manually withdrawing a sample from the bioreactor can be reliable but still come with risks of culture contamination • lab workers must be trained and experienced in sterile technique • lab workers must come into the lab on weekends or during vacations if they cannot find someone they can trust to sample their cultures

3. Temperature DO sparger probe Agitator pH probe Sampling syringe ethanol Hot plate to maintain temperature Existing Sampling Methods

4. Existing Sampling Methods DO sparger Temperature probe pH probe agitator Water out Water in Sampling syringe Sampling port 3-way valve ethanol Water gasket for temperature control

5. Obtain new syringe Ensure sterility of syringe tip Make sure tip is okay to enter culture Remove syringe tip from culture Move collecting tube to analysis machines Dispose of used syringe Deposit sample into collecting tube Move syringe to collecting tube Pull sample into syringe tube Flowchart of the Sampling Process Activate sampling system Draw sample from culture Insert syringe tip into culture

6. Project Goals • reduce the risk of contamination that occurs due to sampling • reduce the time it takes a lab worker to draw a sample from a culture • reduce the skill and training required by a lab worker

7. Design Ideas Idea #1 • Continuous flow of medium and cells through tubing loop • switch 3-way valve to the sampling line in order to draw a sample

8. does not avoid the “syringe switch” does not reduce the time or labor needed to sample Advantages: • simple • inexpensive • easy setup Assessment of Design Idea #1 Disadvantages:

9. Design Ideas Idea #2 • Ethanol and wash sterilize the syringe tip (needle) • Use of septum • Expand to multiple bioreactors

10. Track septum Mechanical arm Syringe disposal container septum Reservoir of new syringes Wash Ethanol Autoclavable, contained environment Design Ideas Idea #2

11. Very little risk of contamination Can enclose/sample many bioreactors Reduces the labor/time needed to sample Ethanol and wash supplies must be changed frequently Expensive Chance of alcohol residue on syringe tip (can kill cells and influence viability counts) Assessment of Design Idea #2 Advantages: Disadvantages:

12. Design Ideas Idea #3 • Open flame sterilizes the syringe tip (needle) • Use of septum • Water-gasket bioreactor system for better maintenance of the culture’s temperature • Expand to a multiple bioreactors

13. Design Ideas Idea #3 Track septum Mechanical arm Syringe disposal container Flame septum Reservoir of new syringes Autoclavable, contained environment

14. Very little risk of contamination Can enclose/sample many bioreactors Reduces the labor/time needed to sample Once cooled, syringe tip is safe to enter the culture (you can calculate how long the tip needs to cool off after submergence in the flame, but in #2, there is no easy way of making sure all the alcohol wash is gone) Expensive Heat from flame may influence temperature of hood environment or of culture No flammable materials/chemicals should be in the hood Assessment of Design Idea #3 Advantages: Disadvantages:

15. Design Ideas Idea #4 • Simpler (fewer steps for mechanical arm) • Reliance on hood to provide sterility • Expand to multiple bioreactors

16. Idea #4 Track septum Mechanical arm Syringe disposal container Pure air inlet Reservoir of new syringes Test tube rack with collecting tubes Autoclavable, contained environment Design Ideas

17. Reduces the risk of contamination Can enclose/sample many bioreactors Reduce the labor/time needed to sample The hood air source only blowing when the door flap is open Expensive Reservoir of new syringes is briefly exposed to outside environment each time a sample is transferred to a collecting tube Assessment of Design Idea #4 Advantages: Disadvantages:

18. septum Final Design Combination of Design Ideas #3 and #4 (uses flame sterilization with the movable door feature) Track Mechanical arm Syringe disposal container Pure air inlet Reservoir of new syringes Test tube rack with collecting tubes Autoclavable, contained environment Movable dividing door

19. Reduces the risk of contamination Can enclose/sample many bioreactors Reduces the labor/time needed to sample Once cooled, syringe tip is safe to enter the culture Heat from flame may influence temperature of hood environment or of culture No flammable materials/chemicals should be in thehood Reservoir of new syringes is briefly exposed to outside environment each time a sample is transferred to a collecting tube Final Design Advantages Disadvantages

20. Conveyor Belt Idea • Conveyor belt would transport multiple bioreactors to a stationary mechanical arm so that arm will not require a track along which it can move • Cost of a 12-feet long conveyor belt with a diameter/width of 30 inches is estimated to be $6000* • Air inlets (nitrogen, oxygen, etc.) come from pipes running down from the ceiling; can’t easily move these with the bioreactor * according to http://www.matche.com/EquipCost/Conveyor.htm

21. septum Modification of Final Design Mechanical arm Sequence: • Addition of a second door Arm gets new syringe Syringe disposal container Track Syringe is sterilized in flame Insulator door opens; flame is extinguished Pure air inlet Sample is drawn from reactor Reservoir of new syringes Air source turns on Second door opens Test tube rack with collecting tubes flame Autoclavable, contained environment Sample is deposited in tube Arm disposes of syringe Arm moves back; air source turns off Arm moves back; second door closes Arm moves back; insulator door closes

22. Parts Information • Bioreactor type used • New Brunswick Scientific - BioFlo3000 Universal Fermentor • glass tube reactor with stainless-steel dished jacketed bottom, stainless-steel head plate with 11 penetrations including septum port for inoculation, harvest tube, sampling system, (2) addition tubes, multiorifice ring sparger, exhaust condenser, thermowell and (2) 6-blade Rushton impellers • 1.25L working volume, 1.6L total capacity, and working minimum volume is 0.6L vessel dimensions: height=19" (48cm), diameter=9.5" (24cm); overall dimensions: height=30" (76cm), width=25.5" (65cm), front-to-back=24.75" (63cm) • price: call for specific quote (~$30,000 per bioreactor)

23. Parts Information

24. Parts Information

25. Cost of Resources • Natural Gas Supply • $1.186/therm* • Electricity • $0.06178/kWhr (first 2000 kWhr/month)** • $0.06817/kWhr (over 2000 kWhr/month)** • Labor • mean hourly wage for ChE is $32.29*** • mean hourly wage for Chemical Technicians is $17.83*** * Based on Nashville Gas personal charges; 1 therm = 100,000 Btu ** Based on Nashville Electric Service personal charges *** Based on Occupational Employment Statistics, http://www.bls.gov/oes/home.htm

26. Economic Analysis • Current system vs. Proposed System • Equipment costs • Production costs/year • Labor costs/year • Single vs. Multiple (4 or 8) Bioreactors • Effects of Contamination on Cost of Current Systems

27. AutoCAD Drawing

28. Future Work • More specifics regarding the system’s design and operation • Complete economic analysis • Complete AutoCAD drawing

29. Suggestions • Run the sampling machine on a timer • Investigate the reliability of the use of septa

30. References • ABEC Website, <http://www.abec.com> • B. Braun Biotech Website, <http://www.bbraunbiotech.com> • Bailey, James E., and Ollis, David F. Biochemical Engineering Fundamentals. McGraw-Hill Inc.: St. Louis, 1986. • Balcarcel, R. Robert. Associate Professor of Chemical Engineering, Vanderbilt University. • New Brunswick Scientific Website, <http://www.nbsc.com> • Todar, Kenneth. “The Control of Microbial Growth.” 21 September 2000 <http://www.bact.wisc.edu/microtextbook/ControlGrowth/sterilization.html>