Advanced Methods of Adenovirus Vector Production for Human

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2868B Domstc 11 14 03 1 50 PM Page 76, Materials and Methods and characterization of the ARM tion Two adenoviral vectors Ad5 Luc. is available at The Williamsburg and Ad5 GFP encoding either luciferase. Cells and Viruses BioProcessing Foundation website or green fluorescent protein respective. The human embryonic kidney 293 www wilbio com The cells were main ly were used in the virus production. HEK 293 cell line was used to propa tained in DMEM Ham s F 12 50 50 optimization experiments. gate adenovirus type 5 Ad5 based mix medium supplemented with 10. recombinant vectors Five vials of mas fetal bovine serum FBS and 4 mM L Cell Counting. ter cell bank MCB HEK 293 cells Glutamine Confluent cultures of HEK Infected cells were harvested from. Magenta Corporation Rockville MD 293 cells were infected with the virus at the culture vessel such as a flask or. were used to generate the Working Cell a multiplicity of infection MOI of 100 roller bottle and counted in a hemocy. Bank WCB Upon characterization viral particles per cell at which point tometer Cell viability was determined. the cells were accepted by the the growth medium was replaced with by trypan blue exclusion In the micro. Adenovirus Reference Material Working the medium containing 2 5 FBS carrier experiments a simpler way of. Group as a cell bank for the Adenovirus Infected cells were incubated until a monitoring cell growth was to count the. Reference Material ARM project 1 All cytopathic effect CPE was observed released nuclei as originally described. information regarding the development typically 48 to 72 hours post infec by Sanford et al with modifications by. van Wezel 2 7 Specifically cells grown,on microcarriers were incubated in a. hypotonic solution of citric acid and the,nuclei released by lysis were stained. with 0 1 w v crystal violet followed by,enumeration in a hemocytometer At. least 100 cells or nuclei were counted,for each time point with these counts.
being performed in quadruplicate for,each cell density data point. Cell Lysis Virus Purification and,Once CPE developed the cells were. harvested and resuspended in the con,ditioned medium in a volume repre. senting 5 to 10 of the volume of cul,Microcarrier concentration mg mL. tivation medium The cells were then,freeze thawed three times in dry.
Figure 1 Effect of microcarrier concentration on final cell density. ice ethanol bath cell debris was,removed by centrifugation and the. clarified lysate was treated with sterile,Benzonase enzyme at 50 U ml for 30. minutes at room temperature to digest,cellular DNA The supernatant was lay. ered onto a preformed step gradient of,CsCl with a density range of 1 4 to 1 25. g ml and centrifuged at 23 000 rpm for,90 minutes at 4 C in a Beckman SW28.
rotor Beckman Coulter Inc Fullerton,CA Banded virus was collected dilut. ed twice with 10mM Tris HCl pH 8 0,2mM MgCl2 and purified by equilibri. um centrifugation in the same CsCl gra,dient once more The virus was dialyzed. against 10mM Tris HCl pH8 0 2mM,MgCl2 10 glycerol and stored at 80. C The virus physical and infectious,titers were determined according to.
Figure 2 Effect of starting cell bead ratio on final cell density of HEK 293 cells in microcarrier standard operating procedures SOPs. culture established at the University of Alabama,76 BioProcessing Journal September October 2003. 2868B Domstc 11 14 03 1 50 PM Page 77,at Birmingham UAB vector and vaccine. production facility The concentration,of adenoviral particles in purified. preparation of virus was measured by,UV spectrophotometry at 260 nm The. infectious titer method used CPE that,adenoviruses have on cells as the read.
out to detect infection in HEK 293 cells,10 days post infection with an adenovirus. sample tissue culture infectious dose,TCID 50 assay Both optical density at. 260 nm and the TCID 50 protocols were,adapted from ARM SOPs. Virus Propagation,The cells were propagated and aden. ovirus vectors were produced in 500,cm2 triple flasks Nalge Nunc.
International Rochester NY that were,placed in a regular CO2 incubator 850. cm2 roller bottles Corning Incorporated Figure 3 3 liter Cell Optimizer System Wheaton with microcarriers. Acton MA at 0 25 rpm and in two, condition controlled systems One of filtration rate thereby improving cell the most economical in terms of media. these was the 3 L Cell Optimizer System viability This design allows the cells to costs. Wheaton Science Products Millville grow on and around the fibers to At this point in the study we. NJ that was employed with Cytodex 3 extremely high densities or more than employed roller bottles as a standard. microcarriers Amersham Biosciences 108 cells ml A typical system consists technique for vector production with. Piscataway NJ Cell growth and virus of a microprocessor controlled pump a good results While scaling up the pro. propagation in the Cell Optimizer medium reservoir and a cartridge duction process our concerns were. System took place in a spinner flask The microprocessor controlled pump improved control of culture parameters. with four side arms The system s assembly allows control of pressure pH and gas tensions for example. Control Tower and Support Unit auto wave shape and provides flexible control reduced requirements for labor and. matically controlled pH via addition of options The system s positive pressure lower risk of contamination all benefits. sodium bicarbonate and CO2 whereas displacement pumping system ensures of microcarrier culture Although. the dissolved oxygen was adjusted with long cartridge life and facilitates nutri microcarrier culture is an advanced. airflow The volumetric airflow rate was ent and waste exchange across the fiber technique it is based on prior knowl. from 4 to 10 liters per liquid liter per edge of a cell type s growth characteris. hour throughout the production cycle tics Information about cell morpholo. Temperature was maintained with a gy plating efficiency and other growth. heating pad which eliminated the need Before proceeding to virus produc properties in traditional monolayer cul. for an incubator The system s BIOPRO tion preliminary experiments to deter ture is invaluable when optimizing cell. software provided the data acquisition mine the yield of the HEK 293 cells in propagation on microcarriers In this. capability for the controller and records each propagation system were per regard the most efficient technique. storage formed The cells were maintained as ensures maximum attachment of the. The other system used was the described in the Materials and Methods cell inoculum to the microcarriers and. FiberCell Hollow Fiber Cell Culture section Table 1 shows the average total results in a rapid homogeneous growth. System Bellco Glass Inc Vineland yield in each system type in million of cells to the highest possible density. NJ Hollow fibers are relatively new cells after 72 hours of cultivation The To determine such a technique sev. tools in cell cultivation that allow data was calculated based on eight inde eral optimization experiments were. designing compact systems 3 Potted pendent measurements after harvesting performed Figure 1 shows the effect of. at both ends with medical grade cells from each cell propagation system microcarrier concentration on the final. polyurethane these hollow fiber filters These results indicate that the same cell density In most situations micro. create a semipermeable barrier between number of cells can be grown in one 3 carriers are used in stirred cultures at a. the extracapillary area where the cells liter microcarrier culture twenty one concentration from 1 to 5 mg ml In. grow and the intracapillary space where roller bottles thirty nine triple flasks or our initial experiments the optimal. the medium flows The naturally ninety T 175 single flasks In addition bead concentration was between 2 5 and. hydrophilic polymer provided a high the microcarrier culture seemed to be 5 mg ml and all further studies were. www bioprocessingjournal com September October 2003 77. 2868B Domstc 11 14 03 1 50 PM Page 78, performed at 3 mg ml Optimization of stage and the total cell yield Figure 2 complete small volume cell culture. this parameter ensured a balance shows that 20 to 40 cells per bead are spinner This system provides quick. between sufficient surface area for cell required for maximum utilization of accurate determination of optimal. growth and minimized microcarrier these microcarriers All the experi parameters for cell growth The Cell. costs ments for the production of adenovirus Optimizer System s vessels are consistent. The other parameter to optimize was vectors were subsequently accom and similar in their design 1 to 45 L. the cell density at inoculation Fig 2 plished at starting cell densities of volumes which along with its modu. Cells survival and growth rates depend 300 000 to 500 000 cells ml which were lar construction makes the system con. on the inoculation density and condi calculated based on the finding above venient versatile and easy to scale up. tioning effects At low plating efficiency and the known number of beads per mg Using this cultivation system in com. the cells tend to be very sensitive to cul of microcarriers bination with microcarrier based cul. ture under low density conditions The The microcarrier bioreactor system ture allows easy aseptic sampling Fig. starting cell number or inoculation we used for cell culture process opti 3 at any time making it possible to. density affects both the proportion of mization and viral production studies monitor both cell expansion and virus. beads bearing the cells at the plateau was the 3 L Cell Optimizer System a production Because the system is in a. biosafety cabinet the culture was under,required containment conditions thereby. allowing easy removal of the samples,for microscopic examination cell.
counting titration and other assays,Figure 4 shows a typical growth. curve of HEK 293 cells in the microcar,rier culture system demonstrating that. there was no drop in cell density below,the starting cell number during the lag. phase first 24 hours in culture which,often occurs when culture initiation. conditions are not optimal In addition,the final cell density was generally at.
least 1 x 106 cells ml after 72 hours in,culture infection point The virus. titer accumulation curve is shown in,Figure 5 Vector harvesting was typical. ly between 48 and 72 hours post infection, Figure 4 Growth Curve of HEK 293 Cells in Microcarrier Culture. Microcarrier culture facilitates micro,scopic observation of the virus produc. ing cells due to transparency of the,beads Examining cells by microscopy is.
a vital part of the technique in order to,assess the state of the culture confluen. cy cell counting and CPE development,Fig 6A B and C Counting cells after. trypsinization can be used to quantify,cells but more a rapid method is to. count the released nuclei In this,method cells grown on microcarriers. are incubated in a hypotonic solution,and nuclei released by lysis are stained.
then counted with a hemocytometer,Overall in contrast to roller bottles. the microcarrier based system intro,duced new options and required less. hands on time This production system,facilitated the high yield production of. HEK 293 cells and vectors Although, Figure 5 Ad5 Luc Production in Microcarrier Culture TCID 50 ml the results we obtained with microcarri. 78 BioProcessing Journal September October 2003,2868B Domstc 11 14 03 1 50 PM Page 79.
Figure 6 Monitoring of microcarrier culture, er culture were very promising cGMP are adapted to suspension culture large contained inside the closed environ. regulations and the need to validate quantities of adenovirus can be pro ment of the hollow fiber cartridge. cleaning sterilization and preparation duced in a single FiberCell hollow fiber extracapillary space Adenovirus har. of reusable equipment for example cartridge Under these conditions large vesting took only a few minutes and. spinner flasks encouraged us to test the numbers of cells up to 3 x 109 were resulted in highly concentrated samples. recently introduced FiberCell system grown in a small volume 15 ml extra of the virus At least 1013 virus particles. which comes presterilized certified and capillary space Efficient viral infection could be produced per preparation in. ready to use 8 occurs due to the high cell densities. Testing of the FiberCell system began Because direct control over cell den. with adapting cells to serum free condi sity in the FiberCell system is somewhat. tions using the original HyClone proto difficult two alternative ways to moni. col 4 Briefly the procedure was to sub tor cell growth were used based on. culture cells in decreasing concentra either glucose consumption or lactate. tions of serum until a concentration of production or both In our Ad vector. 2 5 serum was reached Once cells production experiments we established. were in 2 5 serum and a passage was the correlation of the lactate production. completed as normal cells were incu rate with the cell number by taking. bated to form a monolayer the serum small samples from the extracapillary. containing medium was then replaced space and counting the cells Figure 7. with HyQ protein free PF 293 medium demonstrates very good correlation. ing associated with production of recombinant adenoviruses make it diffi cult for many researchers to utilize these vectors This is particularly true with respect to cell culture optimization and the virus propagation protocols employed in vector production In this regard the development of innovative cell culture techniques has become vital

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