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

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

Existing Sampling Methods Hot plate to maintain temperature Sampling syringe ethanol DOsparger Temperature probe pH probe Agitator

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

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

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

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

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

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

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

Assessment of Design Idea #2 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) Advantages:Disadvantages:

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

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

Assessment of Design Idea #3 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 Advantages: Disadvantages:

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

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

Assessment of Design Idea #4 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 Advantages:Disadvantages:

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

AdvantagesDisadvantages Final Design 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 the hood Reservoir of new syringes is briefly exposed to outside environment each time a sample is transferred to a collecting tube

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

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

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)

Parts Information

Cost of Resources Natural Gas Supply –$1.186/therm* Electricity –$ /kWhr (first 2000 kWhr/month)** –$ /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,

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

AutoCAD Drawing

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

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

References ABEC Website, B. Braun Biotech Website, Bailey, James E., and Ollis, David F. Biochemical Engineering Fundamentals. McGraw-Hill Inc.: St. Louis, Balcarcel, R. Robert. Associate Professor of Chemical Engineering, Vanderbilt University. New Brunswick Scientific Website, Todar, Kenneth. “The Control of Microbial Growth.” 21 September 2000