Development

 of  Single-­‐Use  Pneuma5c  Bioreactor  System TM  

Brian  Lee1  ,  Yas  Hashimura1,  Daniel  Giroux1,  Yoo-­‐Chang  Park2,  Yong-­‐Jae  Kim2  ,  Sang-­‐Hoon  Paik2,  Jae-­‐Choon  Kim3,  Duk-­‐Jae  Oh3,  Baron  Lumani4,  Dieter  Eibl4  
1  PBS  Biotech  Inc.,  Camarillo,  California,  U.S.A.,    2  Green  Cross  Corp.  Central  Research  Center,  KyungGi-­‐Do,  Korea,    3  SeJong  University,  BioScience  and  Biotechnology  Dept.,  Seoul,  Korea  
4  University  of  Zurich,  Department  of  Applied  Life  Sciences,  Wadenswil,  Switzerland  

PBS-­‐80L   PBS-­‐3L  

Abstract   Turbulent  Energy  Dissipa5on  Rate   Biological  Test  in  PBS  Bioreactors   Yield     Polymer     HCP     Host  DNA  
(%)   (%)   (ppm)   (pg/mg)  
Stainless-­‐steel   bioreactors   have   tradi4onally   been   used   in          
cell   culture   processes   for   recombinant   protein   expression.         Cell  culture  performance  was  evaluated  in  250L  PBS  bioreactor  against  the    250L  PBS   47.2   1.15   7.22   0.80  
Recently  the  benefits  of  single-­‐use  bioreactors  have  become    following  bioreactor  systems:  25L  WAVETM,  300L  stainless-­‐steel,  5L  glass              
more  evident,  resul4ng    in  their  rapid  adop4on  over  the     s4rred-­‐tank  (not  shown),  and  1L  shake  flask  (not  shown).        Evalua4on        300L  stainless-­‐steel   44.0   0.92   4.81   9.52  
stainless-­‐steel  systems.    A  novel  single-­‐use  bioreactor  system   Against  WAVE,  glass  s4rred-­‐tank,  and  shake  flask  was  performed  using    
has   been   developed   using   a   pneuma4c   mixing   mechanism         CHO-­‐S  cell  line  in  chemically-­‐defined  medium  (CD-­‐CHO  AGTTM,  Invitrogen).      
to   improve   limita4ons   of   the   current   single-­‐use   bioreactor          
system.    Pneuma4c  Bioreactor  System  (PBS)  offers  fast,  low     Comparable  product  yield  and  impurity  concentra4ons  were  achieved  in    
shear,  and  homogeneous  liquid  mixing  without  any  external   the  250L  PBS  bioreactor  and  the  300L  stainless-­‐steel  bioreactor.      
mechanical  devices,  yet  scalable,  easy-­‐to-­‐use,  and  more    
compact   than   any   other   single   use   bioreactors   currently                
available  in  the  market.      

Key  Factors  for  Ideal  Bioreactors        CFD  modeling  was  used  to  predict  the  distribu4on  of  turbulent     250L  PBS  
300L  stainless-­‐steel   250L  PBS  
         energy  dissipate  rate  in  10L  PBS  at  20  RPM,  1000L  PBS  at  10     300L  stainless-­‐steel  
1)  Low  shear  stress            RPM,  and  typical  2000L  s4rred-­‐tank  bioreactor  at  20  RPM.        
 To  allow  comparable  cell  growth  and  produc4vity              
     Narrower  energy  dissipa4on  rate  distribu4on  was  demonstrated    
when  scaling  up  process  
         in  the  PBS  system  compared  to  s4rred-­‐tank,  which  indicate                    
2)  High  mass  transfer  rate  (kLa)            more  uniform  and  homogeneous  mixing    in  the  PBS  bioreactors.      
 To  provide  sufficient  oxygen  to  and  remove  carbon            
dioxide  from  cell  culture  
High  Mass  Transfer  Rate  (kLa)        Comparable  peak  viable  cell  density  (1.0x107  cell/mL)  and  viability    
3)  Rapid,  homogeneous  mixing            profile  (>95%)  were  achieved  in  both  the  250L  PBS  bioreactor  and  the    
   To  distribute  medium  nutrients,  heat,  metabolites                         10L            25L  WAVE  (graph  on  le+).  
                     quickly  and  uniformly  
   Gas  Flow  Rate          Comparable  final-­‐day  product  4ter  (600  mg/L),  cell  growth  rate  (20-­‐hr    
0.125   0.135   0.145   0.155   Comparable  in-­‐vitro  bioac4vity  was  achieved  in  the  250L  PBS  bioreactor  and  
(VVM)            doubling  4me),  peak  viable  cell  density  (1.0x107  cell/mL),  and  viability     the  300L  stainless-­‐steel  bioreactor,  as  demonstrated  by  the  ELISA  assay                    
         profile  were  achieved  in  the  250L  PBS  bioreactor  and  the  300L  stainless-­‐          
Low  Shear  Stress      kLa  (hr-­‐1)   1.60   12.00   17.20   19.12  
         steel  bioreactor  (graph  on  right).    
(graph  on  le+)  and  the  MTS  assay  u4lizing  L929  mouse  myeloma  cells    
(graph  on  right).      

High  gas-­‐liquid  mass  transfer  rate  (up  to  20  hr-­‐1)  was  achieved  in        
the  10L  PBS  system  using  a  dual  sparger  system:    1.25  SLPM  gas           M 1 2 3 M 1 2 3 M 1 2
flow  rate  through  the  open-­‐tube  sparger  and  0.30  SLPM  flow  rate  
through  the  microsparger.      
Conclusions  and  Future  Direc5ons  
6.9
200 150
116 100 6.0
75
97   A  simple,  user-­‐friendly,  single-­‐use  Pneuma4c  Bioreactor  System  (PBS)  with    
Short  Fluid  Mixing  Time   66
55
50
5.3
5.2          novel,  pneuma4c  Air-­‐Wheel  mixing  technology  has  been  developed      
37
4.5
2L   10L   50L   250L   1,000L   5,000L   37 25 4.2   Pneuma4c  mixing  provides  sufficient  power  for  homogeneous  fluid  mixing,    
20
31            high  gas-­‐liquid  mass  transfer,  and  low  shear  environment  needed  for  op4mal
   Gas  Flow   3.5
0.07   0.06   0.05   0.04   0.03   0.02   22  cell  culture  process,  at  gas  flow  rate  input  comparable  to  tradi4onal  s4rred-­‐  
Rate  (VVM)  
14            tank  systems      
   Wheel  Speed  
20   18   16   14   10   8   1:    250L  PBS   1:    250L  PBS   1:    250L  PBS  
(RPM)     The   Pneuma4c   Bioreactor   System   has   been   successfully   scaled   up   from   2L   to  
2:    300L  stainless-­‐steel   2:    300L  stainless-­‐steel   2:    300L  stainless-­‐steel  
     Shear  stress  was  calculated  from  CFD  modeling  on  FLUENT                                Mixing  Time     250L   in   biological   tests,   and   proof   of   concept   has   been   demonstrated   at                  
10   17   25   30   36   42   3:    Reference 3:    Reference
           soaware  (v.  6.2)   (sec)   5,000L  working  volume      
SDS-­‐PAGE     SDS-­‐PAGE     IEF
     Lower  maximum  shear  stress  was  demonstrated  in  the  PBS     (Non-­‐reducing) (Reducing)   The  broad  scalability  of  the  Pneuma4c  Mixing  System  will  allow  PBS  Biotech    
         system  compared  to  a  conven4onal,  s4rred-­‐tank  bioreactor     Fluid   mixing   4me   in   PBS   bioreactors   was   quan4fied   using   visual       to   offer   a   product   line   with   a   wide   range   of   working   volumes   for   upstream        
         operated  at  typical  impeller  speed   observa4on   upon   dye   addi4on,   pH   changes   with   acid/base                         bioprocessing  needs      
addi4on,   and   CFD   modeling     at   various   wheel   rota4on   speeds                 Comparable   product   purity   profiles   were   achieved   in   the   250L   PBS                                
(RPM)   for   different   bioreactor   scales.     Significantly   shorter                         bioreactor  and  the  300L  stainless-­‐steel  bioreactor,  per  SDS-­‐PAGE  gel    (both  
     Consistent  maximum  shear  stress  in  PBS  system  from  10L          
         to  1,000L  working  volumes  confirmed  its  broad  range  of       mixing   4mes   were   observed   in   PBS   systems   with   2L   to   5,000L             non-­‐reducing  and  reducing)  demonstra4ng  similar  size  distribu4on  and  IEF                                 TM
working  volumes  compared  to  conven4onal  s4rred  tanks.       showing  similar  electric  charge  distribu4on.           PBS  Biotech  thanks  Life  Technologies  for  generous  contribu4on  of  
         scalability     culture  medium  for  these  studies.  

QMSF-4000 C Product Data Sheet