Automating The Cell Culture Sampling Process

Automating The Cell Culture Sampling Process Michael Phipps Tara Ryan Advisors: Dr Rick Haselton & Dr Paul King BME 273 April 23, 2002 Abstract Research and Development portions of pharmaceutical companies contain cell culture labs in which cells are grown in various-sized bioreactors Each of these cultures must be sampled at least once daily Methods of manually withdrawing a sample from the bioreactor can be reliable but still come with risks of contaminating the culture Lab workers who take the samples from the reactors must be trained and experienced in sterile technique Cell culture lab workers often must go into work on weekends, holidays and during vacations if they are unable to find someone they can trust to sample their cultures during that time Thus, the development of an automated cell-culture sampling device would prove to be a worthwhile endeavor While reducing the risk of contamination that occurs due to sampling, an automatic sampling system will also reduce the time it takes a lab worker to draw a sample from a culture and reduce the skill and training required by that lab worker Using cell culture experts and literary sources, background research was conducted, design ideas were brainstormed and evaluated, and then a final design idea was selected The components of the final design were identified and researched, and an AutoCAD drawing as well as an economic analysis of the proposed system was generated The design idea that was selected consists of a compartmentalized hood environment that contains the bioreactors, a reservoir of syringes, a syringe disposal bag, wires serving as a heat sterilization source, collecting tubes, and a mechanical arm that moves along a track from station to station throughout the hood as it carries out the sampling process Although initial equipment costs for the proposed system are high, once labor nd the probability of contamination occurring are factored into the cost of the current system, the proposed device’s cost does not end up being much more expensive The profitability of the proposed system increases as the number of bioreactors contained in it increases, and the proposed system’s advantages also become more evident as the number of contaminations occurring in the current system increases The cost of maintaining one bioreactor for a year under the current sampling system is $35,597, while the cost of maintaining one bioreactor under the proposed sampling system is $102,633 However, when the system encloses eight bioreactors and four contaminations per year are assumed to occur in each reactor in the current sampling system, the yearly operating cost under the current sampling system rises to $367,603, while the similar cost using the proposed sampling system (assuming no contaminations) is only $348,901 Introduction Research and Development Departments of pharmaceutical companies contain fermentation and cell culture labs in which genetically engineered cell lines are grown and maintained in bioreactors of various sizes These reactors range from about 500mL for research purposes to greater than 40L for the production of material to be used in small-scale studies (Even larger fermenters (of working volumes in the hundred thousand Liters range) are used at manufacturing facilities Such enormous reactors are used to produce the large quantities of the protein that will be dispensed to the public after the drug has received FDA approval.) At least once daily, lab workers, using sterile technique, must pull samples (usually about 10-15mL for a 1.5L reactor) from the bioreactors and measure levels of carbon dioxide, glucose, phosphorous, lactate, and ammonia The researchers also perform cell counts to determine the concentration of cells in the bioreactor as well as the viability of the culture Methods of manually withdrawing a sample from the bioreactor can be reliable but still come with risks of contaminating the culture Lab workers who withdraw the samples from the reactors must be trained and experienced in sterile technique Also, cell culture lab workers often must come into the lab on weekends, holidays, and during vacations if they cannot find someone they can trust to sample their cultures during that time Thus, the development of an automated cell-culture sampling device would prove to be a significant and useful advance in pharmaceutical technology While reducing the risk of contamination that occurs due to sampling, an automatic sampling system will also reduce the time it takes a lab worker to draw a sample from a culture and reduce the skill and training required by that lab worker Similar general processes and procedures are followed by the Research and Development Departments of most pharmaceutical companies (Lee) Molecular biologists genetically engineer a particular type of cell, generating a number of different cell lines, all of which have had their DNA altered so that they will produce the desired biopharmaceutical protein At Regeneron Pharmaceuticals, Inc., a smaller pharmaceutical company, eight 1.5L bioreactors were the reactors were commonly used for research (rather than production) purposes One common application of these small-scale reactors is to study and compare different cell lines Each cell line that produces the particular protein of interest is grown in one of these reactors for several weeks, and samples taken periodically from these cultures are analyzed in order to determine which line has the most favorable growth rate, death rate, specific productivity, and yields the exact protein that is desired This preliminary research comparing different cell lines is then used to select which line should be scaled up into the larger bioreactors and used to produce large amounts of the protein material At Regeneron Pharmaceuticals, Inc., the 1.5L bioreactors manufactured by B.Braun Biotech, Inc are sampled at least once daily (twice on days when the culture is split, i.e when the medium is exchanged and the protein material is harvested, which comes out to approximately every three days, depending on the cell line and protein being produced.) Each time a bioreactor is to be sampled, the laboratory worker puts on rubber gloves and cleans them with an ethanol solution The 30mL disposable syringe connected through the tubing line through the sampling port of the bioreactor is used to draw up 20-30mL of medium solution from the bioreactor A fresh 30mL, disposable syringe is unwrapped and its cap is removed, the worker handling the fresh syringe carefully so that nothing touches its exposed tip The syringe attached to the tubing line through the sampling port, now containing 20-30mL of medium, is unscrewed from the Lüer lock on the end of the tubing and is quickly replaced with the newly unwrapped, fresh syringe The medium withdrawn into the syringe that has just been removed from the tubing is waste; its purpose was to flush the sampling port tubing line so that no residual medium in the tubing that is uncharacteristic of the bioreactor’s contents is present in the sample The new syringe that has just been attached to the tube that runs through the reactor’s sampling port is used to pull another 15-20mL of medium out of the bioreactor A second fresh, disposable 30mL syringe is unwrapped, the cap is removed from its tip, and it is quickly screwed onto the sampling port tubing, replacing the syringe that had just drawn the 15-20mL of sampling fluid from the reactor At the end of these two syringe switches, a new, empty 30mL syringe is connected to the tube that runs through the sampling port of the reactor, and two used, unconnected syringes now lying on the benchtop contain medium from the culture (the first one drawn being waste and the second one taken being the actual sample.) Thus, each sampling process uses two disposable 30mL syringes and removes about 50mL of fluid from the reactor This sampling method used in the pharmaceutical industry is also commonly used in academic research, as Dr Robert Balcarcel, now an Assistant Professor of Chemical Engineering at Vanderbilt University, used similar sampling methods while conducting his graduate research at the Massachusetts Institute of Technology The automatic cell culture sampling system envisioned from the onset of the project was desired to operate in the following manner: Activate machine Ensure sterility of syringe tip Obtain new syringe Make sure tip is okay to enter culture Draw sample from culture Insert syringe tip into culture Dispose of used syringe Move collecting tube to analysis machines Pull sample into syringe tube Deposit sample into collecting tube Move syringe to collecting tube Remove syringe tip from culture According to Angela Younger, a former Research Associate at Regeneron Pharmaceuticals, Inc., in Tarrytown, New York, contamination of 1.5L working-volume bioreactors, in her experience, was not an everyday occurrence, but it did happen quite regularly If a reactor of 1.5L were to be left running continuously for an indefinite period of time, Younger estimated that the culture would probably become contaminated, on average, after about three months Following Younger’s estimations, a 1.5L working-volume bioreactor, if maintained yearround, would become contaminated an average of four times over the course of a year In addition to affirming Younger’s estimate, Frank Lee, Ph.D., a former Cell Culture Scientist at Regeneron Pharmaceuticals, Inc., cited inadequate laboratory ventilation and/or erroneous sampling technique as the two main sources of culture contamination Different types of microorganisms are able to survive in a wide range of environments Characteristics that are responsible for such flexibility are microorganisms’ small size and easy dispersal, their occupancy of very little space, their need for small amounts of nutrients, their wide variety of nutritional requirements, and their ability to adapt to changes in environmental conditions (Todar “Nutrition” 1) However, growth of microorganisms is influenced by many abiotic factors, including the pH, temperature, oxygen availability, and moisture of the environment (Todar “Nutrition” 1) Most species of bacteria grow best in a medium of a relatively warm temperature and with a pH of around 7.0 (Todar “Nutrition” 1) Unfortunately for cell culture researchers, cultures used in biopharmaceutical production are usually maintained at warm temperatures and at pHs of 7.2-7.4, depending on the cell type being grown (Todar “Control” 1) Thus, average culture conditions create an environment that is conducive to rapid bacterial growth Methods of sterilization used in laboratory practices diminish the activity of microorganisms by destruction (such as heat, radiation, or chemical or mechanical means), removal (meaning filtration or centrifugation), or inhibition (via refrigeration, dessication, water depletion, and chemicals) (Bailey 441) Heat is the most widely used method of sterilization in the laboratory (Since bacterial endospores are considered to be the most heat-resistant of all cells, when sterilizing by heat, destruction of these endospores guarantees sterility (Todar “Control” 1).) Autoclaving is a technique usually used in the laboratory to sterilize glassware Autoclaving kills all forms of life, including bacterial endospores (Todar “Control” 1) However, autoclaving obviously cannot be used for substances that will denature at high temperatures, and autoclave machines are usually not useful in sterilizing large pieces of equipment, since big, bulky equipment often cannot be moved to the location of the autoclave and/or the autoclave is not large enough to hold such equipment Another, very simple and crude, heat sterilization method sometimes used in the lab is the use of a Bunsen burner flame Antimicrobial agents are chemicals that kill microorganisms or inhibit their growth (Todar “Control” 1) Such agents include antiseptics and disinfectants Antiseptics are harmless enough to be applied to the skin and mucous membranes; silver nitrate, iodine solution, alcohols, and detergents are all antiseptics (Todar “Control” 1) Disinfectants include chlorine, formaldehyde, hypochlorites, chlorine compounds, copper sulfate, and quaternary ammonium compounds Disinfectants are not as useful as antiseptics since the former are not safe enough to apply to living tissues, and because they kill microorganisms but not necessarily kill their spores too (Todar “Control” 1) Irradiation techniques are most often used to sterilize the surfaces of objects, and these methods act by destroying or distorting the nucleic acids of the microorganisms (Todar “Control”) Ultraviolet light, x-rays, and microwaves are all potential irradiation agents Filtration methods consist of physically removing cells from a liquid or gas, and they are especially important in sterilizing solutions of antibiotics, amino acids, and vitamins, since the components of such solutions would be denatured if they were to be heat-sterilized (Todar “Control” 1) Methodology In beginning the design of an automated cell culture sampling mechanism, several people with experience in pharmaceuticals and bioreactor maintenance in particular were contacted Such experts included: Dr Robert Balcarcel, an Assistant Professor of Chemical Engineering at Vanderbilt University; Dr Todd Giorgio, an Associate Professor of Biomedical Engineering at Vanderbilt University; Dr Ken Brigham at Medical Center North in the Vanderbilt University Medical Center; Dr Xiao-Ping Dai, formerly a Protein Purification Scientist at Regeneron Pharmaceuticals, Inc.; Dr Frank Lee, formerly a Cell Culture Scientist at Regeneron Pharmaceuticals, Inc., and Angela Younger, M.S., formerly a Research Associate at Regeneron Pharmaceuticals, Inc In addition to speaking with various researchers, a literature search was also conducted in order to gather information regarding bacterial growth, instances of contamination in pharmaceutical settings, and sterilization techniques that may be employed to destroy microorganisms After researching the problem of cell culture contamination, several ideas were generated during a brainstorming session (see Results section) Each design idea was evaluated, and during the assessment process, other possible designs evolved and were later explored as well After determining the advantages and disadvantages of each idea, a final design was selected for the device A list of the components of the final design was generated, and such parts needed to build the proposed system were researched Laboratory equipment manufacturer websites were accessed and searched for parts specifications and pricing information Sales representatives of such manufacturers were often contacted via email and/or telephone in order to obtain accurate and up-to-date price quotes, and catalogs, price lists, pamphlets, and other product literature from various companies were requested and received in the mail Electricity costs and labor costs figures were also researched Once the dimensions of each component of the system were specified, a to-scale, three-dimensional diagram of the device was drawn using the AutoCAD software The prices obtained from the manufacturers of the components were used to generate an economic analysis of the proposed system In this analysis, the costs of the current and proposed systems were calculated and compared For each system, the cost for the system to encompass one through eight bioreactors was calculated To more accurately project the costs of the current system, contamination was assumed to occur, once, twice, three, or four times (an average of two weeks into the culture) per bioreactor per year In addition, labor costs were included in the current system, since currently lab workers are responsible for taking the time to manually sample the bioreactors The general equations used in the economic analysis are as follows: Eqn (1) Cost of Proposed System (1 bioreactor) = sum of all component parts Eqn (2) Cost of Proposed System (n bioreactors) = Eqn (1) + n*(Eqn (1) - cost of arm - cost of track - cost of hood - cost of dividing doors) Eqn (3) Cost of Current System (1 bioreactor) = sum of all component parts + cost of labor to sample/yr Where cost of labor to sample/yr = (#samples/yr)*(seconds/sample)*(wage/year)*(1yr/365days)*(1day/24hr)*(1hr/3600sec) Eqn (4) Cost of Current System (n bioreactors) = Eqn (3) + n*(Eqn(3)) Eqn (5) Cost of Current System (1 bioreactor, Contamination/yr)* = Eqn (3) + ((2/52)*cost of labor to sample/yr)*((2/52)*cost of syringes/yr)*((2/52)*cost of collecting tubes/yr) Eqn (6) Cost of Current System (2 bioreactors, Contaminations/yr)* = (Eqn(4), n=2) + 2((4/52)*cost of labor to sample/yr)*((4/52)*cost of syringes/yr)*((2/52)*cost of collecting tubes/yr) +Note: This assumes the contamination occurs weeks into the bioreactor run; hence, for each contamination in each reactor, two weeks worth of labor and two weeks worth of some of the materials are lost Results and Discussion Below are the design ideas that were generated (in sequential order): Continuous Circulating Bioreactor Loop (Figure 1) Both ends of a tube are immersed in the bioreactor fluid, and somewhere in the tubing loop there is a three-way valve that connects the loop to another tubing line This third tubing branch leads to a syringe or other type of sample collecting tube Fluid from the bioreactor continuously circulates throughout the tubing loop, and when it is time to sample, the three way-valve is turned so that the flow Figure Continuous Circulating Bioreactor Loop through the loop is directed through the third branch and fluid is collected in the syringe/sampling tube Once the sample is collected, the branch tubing is clamped, and the valve is returned to its original position As the bioreactor fluid begins to circulate through the tubing loop once again, the currently used practice of sampling via two syringe switches (as described in the Introduction section) is used to retrieve the sample from the syringe connected in the apparatus Advantages of this first idea are that it is simple, inexpensive, and easy to set up After rejecting this design idea, it was learned that cell culture workers at Pfizer, Inc in Groton, Connecticut, actually relied on this setup and method to sample their bioreactors However, a significant disadvantage of this method is that it does not avoid having to switch the syringes twice, and thus the tubing end is still exposed to the outside environment twice each time a sample is taken In addition, this idea also fails to reduce the time or labor that is needed to sample Thus, this setup and method of sampling does not meet most of our project’s objectives Enclosed septum Track Environment Mechanical arm Syringe disposal container septum Wash EtOH Reservoir of new syringes Using Alcohol for Sterility (Figure 2) The bioreactor(s) Autoclavable, contained environment are enclosed in an autoclavable, enclosed environment along with a mechanical arm, a tube filled with alcohol, a Figure Enclosed Environment Using Alcohol for Sterility tube filled with a sterilized washing fluid, a reservoir or new syringes, and a syringe disposal container The mechanical arm, stationed towards the back of the enclosed environment and operating along a track that extends along the length of the enclosed space, obtains a new needle syringe, dips the syringe in alcohol, dips the syringe in the wash fluid, and then proceeds to insert the needle into the bioreactor top (through a septum that covers the sampling port) and retrieve a sample from the bioreactor The mechanical arm then sticks the needle through a septum in the bottom surface of the enclosed environment, with this septum leading to a collecting tube located outside of the enclosed space The sample is released from the syringe needle, through the septum, and into the collecting tube The arm ejects its used needle syringe into a waste disposal container that is located on one side of the enclosed environment The mechanical arm then obtains a new syringe needle and the process is repeated for another bioreactor that needs to be sampled Advantages of this second idea are that there is very little risk of contamination, the device can enclose many reactors, and it will reduce the time and labor needed to sample However, in this design, the wash will probably have to be changed frequently, possibly even while a culture is being grown in the bioreactor There is also a high cost associated with the track and mechanical arm parts that are included in this design In addition, there is a chance of alcohol residue being left on the syringe needle tip after it is sterilized If this happens and the alcohol enters the reactor, the composition and viability of the culture will be influenced Enclosed septum Track Environment Using Mechanical arm Heat for Sterility Syringe disposal container Flame Reservoir of new syringes septum (Figure 3) The bioreactor(s) are contained in an Autoclavable, contained environment autoclavable, enclosed environment along Figure Enclosed Environment Using Heat for Sterility with a mechanical arm, a heat source (such as a Bunsen burner), a reservoir of new syringes, and a syringe disposal container The mechanical arm, stationed towards the middle of the enclosed space and operating along a track that extends along the length of the enclosed environment, obtains a new syringe needle, brings the needle portion to the open flame and holds it there for a sufficient length of time, lets the needle cool completely, and then inserts the needle into the bioreactor top (through a septum that covers the sampling port) to retrieve a sample from the bioreactor The mechanical arm then sticks the needle through a septum in the bottom surface of the enclosed environment, the septum leading to a collecting tube located outside of the enclosed space The sample is released from the syringe needle, through the septum and into the collecting tube The mechanical arm ejects its used syringe needle into a waste disposal container that is located on one side of the enclosed environment The arm then obtains a new syringe needle and the process is repeated for another bioreactor that needs to be sampled Advantages of this idea are that the risk of contamination is greatly reduced, the system can enclose many bioreactors, and that the time and labor needed to sample is also reduced In addition, this design idea is more reliable than the previous one When the needle was washed with ethanol in the previous design, some of the alcohol could have gone undetected and remained on the needle when the needle entered the culture However, in this design, the time required for the needle tip to cool after sterilization can be calculated and accounted for in the system so that the needle is guaranteed to be safe to enter the culture Disadvantages of this design are the high cost associated with the mechanical arm and track system, the possibility that 10 heat from flame may influence the temperature of the enclosed environment, and/or the cultures, and the precautionary measure that no flammable materials should be present in the enclosed space Hood-like Environment with Removable Dividing Door (Figure 4) The bioreactor(s) are enclosed in a hoodTrack Syringe disposal container Mechanical arm septum is divided into two Pure air inlet Reservoir of new syringes Test tube rack with collecting tubes like environment that Autoclavable, contained environment Figure Hood-like Environment with Removable Dividing Door compartments Contained in the hood are: a mechanical arm, a reservoir of new syringes, a test tube rack containing sample collecting tubes, a syringe disposal container, and a heat source (such as a Bunsen burner.) The mechanical arm, stationed towards the middle of the hood and operating along a track that extends along the length of the hood, obtains a new syringe needle, holds it in the open flame for a sufficient amount of time, lets it cool, and then inserts it into the bioreactor top (through a septum that covers the sampling port) to retrieve a sample from the bioreactor An airflow inlet source at one end of the hood turns on, and a movable door (located towards the opposite end of the hood) slides out of the hood While the opening of this door exposes the bioreactors to the outside environment of the laboratory, the continuous air stream ensures the maintenance of a sterile environment by blowing air out of the hood, and thus preventing any foreign matter from the surrounding area from entering the hood The mechanical arm transports the sample through the door opening and to a collecting tube The sample is deposited into the collecting tube that is resting in a test tube rack (A laboratory worker later analyzes the samples that are collected.) The mechanical arm passes back through where the door had moved, then the door closes, the airflow inlet turns off, and the arm continues to move back towards its starting point in the hood, ejecting its used syringe needle into a waste disposal container located on one side of the hood The arm obtains a new syringe needle and the process is repeated for another bioreactor that needs to be sampled Advantages of this design include the reduced risk of culture contamination, the flexibility to enclose multiple bioreactors, the reduced time and labor needed to sample, and the fact that the cooled syringe needle tip can be guaranteed to be safe to enter the culture 11 Some drawbacks of this device are its expensiveness and the fact that the reservoir of syringes is briefly exposed to the outside environment each time a sample is taken when the divider door opens Also, again there is the possibility of heat from the flame influencing the temperature of the enclosed environment, and as a precautionary measure, no flammable materials should be present in the environment Track Syringe disposal container Mechanical arm septum Movable dividing door Hot wires Pure air inlet Reservoir of new syringes Test tube rack with collecting tubes Movable insulating door Autoclavable, contained environment Figure Final Design Idea Final Design Idea (Figure 5) The bioreactor(s) are enclosed in a hood-like environment that is divided into three compartments In the first compartment is the reservoir of new syringes as well as voltage-producing wires that act as a heat source The second compartment contains the bioreactor(s), and the third compartment is exposed to the outside environment and contains a syringe disposal container and a test tube rack with collecting tubes into which the samples will be deposited The mechanical arm, stationed towards the middle of the hood and operating along a track that extends along the length of the hood, obtains a new needle syringe, touches its tip to the hot wire ends, lets it cool, and then the insulating door located between the wires and the bioreactors opens up The arm inserts the syringe needle into the bioreactor top (through a septum that covers the sampling port) to retrieve a sample from the bioreactor An airflow inlet, stationed at one end of the hood, turns on, and a second movable door slides out of the hood, opening the bioreactors up to the outside environment While the opening of this door exposes the bioreactor environment to the surrounding area in the lab, the constant airflow outwards protects the sterility of the enclosed environment, continuously blowing air out of the hood and thus preventing any foreign matter from the surrounding area from entering the hood The mechanical arm then transports the sample through the second door opening and to a collecting tube The sample is released from the syringe needle into a collecting tube (A laboratory worker will later analyze the samples in the rack.) The mechanical arm ejects the used needle 12 syringe into a waste disposal container, and the arm begins retreating back into the hood, the second door closes, the air stream turns off, the arm continues moving deeper into the contained environment, the insulating door closes, and the mechanical arm returns to its original starting position, obtaining a new syringe needle and then repeating the process when another bioreactor needs to be sampled This final design idea includes all of the advantages seen previously in the other designs: it meets all of the project’s goals, reducing the time and labor needed to sample, and reducing the risk of contamination; the addition of the door between the bioreactors and the syringe reservoir maintains the bioreactors in a sterile environment should the reservoir of syringes need to be replaced or the mechanical arm need to be repaired; the wires providing the heat sterilization source eliminates the need to turn a flame burner on and off each time a sample is drawn; the air source creates a hood that prevents foreign particles from flowing into the sterile confines of the enclosed environment; and the heat provided by the wires for the purpose of sterilizing the syringe needle will not influence the temperature of the surrounding area nearly as much as the flame would have A major drawback of the final design idea is its high cost, although the economic analysis (in the pages to follow) suggests that the system is worth the extra cost since it drastically reduces the chances of contaminating the culture Information regarding the components of the proposed device is listed in the table on the next page 13 Manufacturer Test Tubes Globe Scientific Inc 500 for $138 polypropylene with attached screw cap Test Tube Rack Globe Scientific Inc $7.60 high density polypropylene Disposable Syringes With Needles MedPlus Corporation $65/1000 pieces plastic Syringe Disposal Bag GRP Medical Services: Biohazard Disposal Supplies Enclosed Environment and Air Inlet Mechanical Arm Allometrics, Inc Intelitek Price Material Composition Dimensions Reference Part $8 (or 12 for $90) $23,274.66 $20,825 Track Wires Intelitek N/A $23,352 negligible Metal Dividers N/A negligible N/A Other Notes http://www.gl obescientific.c om 15mL 25/bag, sterile http://www.gl autoclavable; obescientific.c nonfloating in om/cpage40.h water bath; holds 16mm diameter tml 60 tubes http://www.m sterilized with edpluscorp.co ethylene oxide, m/pricenon toxic, pyrogen20mL volume list.htm free gallon http://sharpss upply.com/mc art/ price estimate is based on determining the cost per additional m^3 of volume, and scaling up the 10m^3 (10m http://www.al cost from an includes motor and long x 1m wide lometrics.com existing product blower (115V) x 1m tall) /hoods.htm quote N/A L=479 mm, W=490 mm, H=206 mm (L=18.8" W=19.3", H=8.11") overall travel = 1800mm, overall length = extruded 2100mm, travel aluminum beam velocity = construction 500mm/sec N/A N/A 1m X 1m (2 of N/A these) http://www.in telitek.com/pr oducts/roboti cs/systems/sc ora-er-14s.html http://www.in telitek.com/pr oducts/roboti cs/accessories /slidebases/sli debase-914s.html N/A See website for more detailed specifications; price estimate includes gripper price estimate is based on determining the cost per additional m of length, and scaling up the cost from an existing product quote N/A *Bioreactors used in the system are New Brunswick Scientific’s BioFlo 3000 Universal Fermentor See the Appendix section for its specification 14 Cost Analysis for the Proposed System: (see Methodology section for equations used, and see Appendix for entire spreadsheets) # Reactors bioreactor bioreactors bioreactors bioreactors bioreactors bioreactors Total Cost/year of production ($) 102657.798 137838.937 173020.075 208201.214 243382.352 278563.491 bioreactors bioreactors 313744.629 348925.768 Cost Analysis for the Current System*: (see Methodology section for equations used, and see Appendix for entire spreadsheets) Total Cost/year of production reactor reactors reactors reactors reactors reactors reactors reactors contamination 35596.97221 71193.94443 106790.9166 142387.8889 177984.8611 213581.8333 249178.8055 284775.7777 contamination contaminations contaminations contaminations 38185.3306 40773.689 43362.04739 45950.40578 76370.66121 81547.37799 86724.09478 91900.81156 114555.9918 122321.067 130086.1422 137851.2173 152741.3224 163094.756 173448.1896 183801.6231 190926.653 203868.445 216810.2369 229752.0289 229111.9836 244642.134 260172.2843 275702.4347 267297.3142 285415.823 303534.3317 321652.8405 305482.6448 326189.512 346896.3791 367603.2462 *Each contamination case assumes that each reactor in that case experiences that number of contaminations per year Also, each contamination incidence assumes the culture was contaminated two weeks into the run Based on the results of the economic analysis, the extra initial equipment costs associated with the proposed system prove to be worth it when the system is expanded to include multiple bioreactors and is assumed to have prevented several contaminations during the year In addition, the proposed system also has significantly lower labor resource costs since the proposed device is virtually self-sustaining Figure shows the yearly production costs versus the number of bioreactors incorporated for the proposed system, the current system assuming no 15 contaminations occur throughout the year, the current system assuming two contaminations per bioreactor per year occur, and the current system assuming four contaminations per bioreactor per year occur As Figure shows, if there are about seven bioreactors to be sampled and each one becomes contaminated approximately once every three months, the proposed device becomes the more cost-efficient sampling system 4.00E+05 Yearly Operation Costs ($/yr) 3.50E+05 3.00E+05 Figure Yearly Operation Costs versus Number of Bioreactors in the System for both the Proposed and Current Systems 2.50E+05 no 2.00E+05 Proposed System (No Contamination) 1.50E+05 Current System - No Contamination 1.00E+05 Current System - Contaminations/yr While exact Current System - Contaminations/yr 5.00E+04 0.00E+00 Number of Bioreactors Linear (Proposed Sys tem (No Contamination)) Linear (Current System - Contaminations/yr) 10 Linear (Current System - No Contamination) Linear (Current System Contaminations/yr) number of existing pharmaceutical companies that contain cell culture- 2labs could be obtained, there is sufficient reason to believe that there will be a significant market potential for the proposed system While Regeneron Pharmaceuticals, Inc is one of the smaller existing pharmaceutical companies with 200-300 employees, an investment in the proposed system would be of definite value to the company Thus, it is logical to assume that the majority of pharmaceutical companies, being larger than Regeneron, would also have an interest in such proposed automated technology In addition, when this project was recently presented as part of a job interview at Pfizer, Inc., the cell culture lab workers there showed tremendous interest in the proposed design system, and even touted it as being a “great idea.” After completing a safety analysis of the proposed system using the Design Safe program, no major safety concerns were found to be associated with the device’s implementation Conclusions 16 The final design of the automatic sampling system meets all of the preliminary project goals: while reducing the incidence of contamination of cell cultures, it will also reduce the time and effort laboratory workers will have to spend on sampling the cultures Although the equipment cost for the proposed system is larger than the equipment cost for the current system, it will be more than compensated for once the labor costs and costs due to contaminations are accounted for in the current system model In addition, as more bioreactors are added into the system, the proposed device’s cost per bioreactor decreases, whereas the current device’s cost per bioreactor remains the same With the cost of the system not being a significant issue, there appears to be a sufficient demand among the pharmaceutical industry for the automated technology this proposed system will provide Recommendations Several suggestions regarding improvement and implementation of the proposed system were made towards the end of the project Cell culture workers at Pfizer, Inc., at the Global Research Headquarters in Groton, Connecticut, suggested putting the system on a timer so that no employee would have to come in to work on weekends and holidays in order to take samples The people at Pfizer, Inc also suggested examining the use of the septum in the lid of the bioreactor as the sampling port, since they have had a few problems with contamination of their septa in the past Dr Robert Galloway, Professor of Biomedical Engineering at Vanderbilt University, pointed out that the wires acting as the heat sterilization source could become corroded over time Galloway suggested dipping the syringe needle into a radioactive solution before sampling the culture Another suggestion made was to have the movable dividing doors fold downward in an accordion-like fashion rather than rolling up or out to the side and out of the hood environment If the doors are made so that they fold downward, the door material will remain in the enclosed environment throughout the sampling process Thus, collapsible doors will not introduce any foreign, potentially-contaminating particles from the outside environment into the hood when the doors reseal off the compartments of the system at the end of the sampling process Before the design of this device continues any farther, some sort of interest survey among all existing pharmaceutical companies might be helpful, even if only to provide a rough estimate of what the market for the proposed device may look like 17 References Associated Bioengineers and Consultants, Inc (ABEC) Company Website 25 January 2002 Allometrics, Inc., Specialty Laboratory Services Website 20 March 2002 B Braun Biotech Website 25 January 2002 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 28 January 2002 Charton, R “Simplifying Sterile Access in Bioreactor Operations.” American Biotechnology Laboratory December 1990 Globe Scientific, Inc Website 17 March 2002 Intelitek Website 22 March 2002 Lee, Dr Frank Cell Culture Scientist, Regeneron Pharmaceuticals, Inc Summer 2000 MedPlusCorporation Website 20 March 2002 New Brunswick Scientific Website 28 January 2002 Todar, Kenneth “The Control of Microbial Growth.” 21 September 2000 16 January 2002 Todar, Kenneth “Nutrition and Growth of Bacteria.” 16 January 2002 Younger, Angela Research Associate III, Regeneron Pharmaceuticals, Inc Summer 2000 18 ... from the outside environment into the hood when the doors reseal off the compartments of the system at the end of the sampling process Before the design of this device continues any farther,... that covers the sampling port) to retrieve a sample from the bioreactor The mechanical arm then sticks the needle through a septum in the bottom surface of the enclosed environment, the septum... towards the opposite end of the hood) slides out of the hood While the opening of this door exposes the bioreactors to the outside environment of the laboratory, the continuous air stream ensures the