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One  and  Done  –  Ge+ng  Biopharmaceu5cal  Produc5on  Processes  Right  the  First  Time  

Ying  Huang,  Ph.D.  Associate  Director,  Process  Development  

KBI  Biopharma,  Durham  NC  

Presented  at:  World  Orphan  Drug  Congress,  Washington  DC,  April  25,  2013  

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Pre-Clinical Phase I Phase II Phase III

Process Development Process

Characterization Process

Validation Process Monitoring &

Improvement

FIH Process •  Deliver clinical process

quickly •  Platform process •  Clinical Supply

Submission & Approval

Lifecycle management

BLA Prep & PAI

Commercial Process •  Deliver manufacturing process for

registrational trials and market •  Design keeping large-scale manufacturing

in mind •  Improve productivity, efficiency, robustness,

manufacturability, COGs •  Analytical characterization and method

development

Process Characterization and Validation •  Develop IPC strategy through understanding of process inputs and

outputs (design space) •  Scale-down characterization and validation studies •  Large-scale process validation to demonstrate process consistency •  BLA preparation •  Supporting documents for licensure inspections •  Post-commercial process improvements (CI) •  Post-commercial process monitoring

FIH process Commercial process

Gottschalk U., Brorson K., Shukla A. Nature Biotechnology, 30(6), 489-491, 2012

Biologics  Commercializa5on  

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Approaches  to  Get  Every  Step  Right  at  the  1st  Time?  •  Screen  the  best  protein  candidate  construct  by  transient  transfec3on.  •  Iden3fy  the  stable  high  producing  clones  within  a  short  3meline    •  Pool  enrichment  with  FCAS  to  achieve  a  good  heterogeneous  pool  •  Single  cell  cloning  with  ClonePix  to  improve  selec3vity  

•  Define  the  op3mized  cell  culture  process  by  using  high  throughput  microbioreactor  with    rapid,  accurate  3ter  and  product  quality  assessment.  •  Develop  an  efficient  downstream  purifica3on  processes  using  selec3ve  column  washes.  

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Cell  Line  Development  –  Ini5a5on  of  Biologic  Produc5on  

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Vector  Construc5on  

Transfec5on  &  Selec5on  (Amplifica5on)  

Pool  Enrichment  

Clone  Isola5on  &  Screening  

Stability  Assessment    

Strong  vector  w/  enhance  element  

Transient  Transfec5on  

Gene  

Stable  Pool  

FACS  

High  throughput  cloning  (FACS  or  ClonePix)  

Earlier  material  generaHon  

Stable  Cell  Line  GeneraHon  

Gene  codon  op5miza5on  

Valid  Clone  Screening  Process  

CSI  

Process  Development  

Process  Development  

Cell  Bank  

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Decide  the  Right  Molecule  with  Transient  Gene  Expression  (TGE)  

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7  to  14  days  

Large  Amount  of  Star5ng  Cells  (K-­‐Sep)  

Large  Scale  FB  Culture  for  Produc5on  (Wave)  

Benefit  of  TGE  Ø   Screening  candidate  molecules  –  lock  the  best  construct  Ø   Fast  material  genera3on  –  analy3c  method  development  

0   1   2   3   4   5   6   7   8  

Titer  

Culture  Days  

GOI_1  GOI_2  GOI_3  

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Pool  Enrichment  with  FACS  

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#3.  2nd  Round  of  Enrichment  Sorting  

#2.  1st  Round  of  Enrichment  Sorting  

#1.  Original  Population   Gate1  

Using  the  FACS  Jazz  instrument  to  sort  the  top  5%  of  the  popula5on  

0.0  

0.5  

1.0  

1.5  

2.0  

2.5  

3.0  

Original   Round  1  Enriched  

Round  2  Enriched  

Fold  In

crease  

Pools  

Pool  Produc5vity  Assessment  (  7-­‐day  batch  culture)  

Time:  ~  2  weeks  

Ø FACS  allows  sor5ng  of  high  producing  cells  to  an  enriched  pool,  which  provides  a  be_er  pool  for  earlier  material  genera5on  and/or  cloning.  

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•  Automated  Colony  Picking  Robot  •  High  throughput  •  Asep3c  

•  Fluorescent  Technology  for  Selec3ve  Cloning  •  Ranks  and  selects  only  the  highest  

producing  clones  

0

5000

10000

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1G7

1E10

1C12

1F10 2B6

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1G5

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2B1

2B9

2A1

2A6

1G4

1G12 1B5

2A2

2A4

1B8

1H7

2G8

2A9

1F1

2B5

1B9

2G11 1A2

1A5

1F3

2D2

1C4

2F12 1F9

2B2

2E11

2G10 1F2

2A10

1E5

2E10

1H5

1E8

1F8

1C2

1B11

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2F10 2E4

2C12

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1F4

1H8

2F1

1F5

2G12 2F5

2G7

2G2

1F12 1B4

2A12

2A11

2E3

1E6

2C2

1C10 2F9

2F4

1G8

2F3

1E9

1E2

1C5

1H9

2F2

2B10

2E5

2E12

1G2

2E2

1E1

1E4

2E1

1H4

2F8

2E9

2B8

2F11 1A9

2A3

2H8

1C11

Colony

Tite

r (PD

U/m

LL)

Semi-­‐Solid  Matrix  with  Conjugated  An3body  

Colony    (White  light)  

Halo  (Florescent  light)  

Image  Plane  

High  throughput  Cloning  with  ClonePixTM  

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100%  is  the  1ter  of  best  clone  from  ClonePixTM    ClonePix vs LDC

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20

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Clone

Rel

ativ

e R

anki

ng (%

)

ClonePix Top 30

LDC Top 30

ClonePixTM    Can  Isolate  Be_er  Clones  with  Less  Effort  than  LDC  

Clones  

Rel

ativ

e Titer (

%)

 ClonePixTM  Top  30  Clones  

 LDC  Top  30  Clones  

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Run  specific  assays  for  protein  glycan  and  protein  charge  variant  for  harvest  samples  with  turnaround  of  1-­‐2  days.    

§  Use  24  miniaturized  Bioreactors  (10-­‐15mL  working  volume).  

§  DOE  design  of  factors    (pH,  DO,  temperature,  feeds  volume  and  frequency).  

§  Perform  daily  analysis  to  monitor  cell  growth  and  key  metabolites.  

ambrTM  

ProA-­‐based  3ter  analysis  of  in  process  cell  culture  samples  with  quick  turnaround  of  same  day.  

Ø  Cell  growth,  cell  viabili3es,  metabolite  profiles    

For  each  tested  condi5on  

Ø  Titers  and  Product  Quality  

+  

Selec3on   of   best   process  condi3on(s)   with   respect   to   cell  growth,  produc3vity  and  product  quality.  

Integrated  High  throughput  Process  Development  and  Analy5cal  Support  

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forteBIO  Octet   Caliper  LabChip  GX  II  

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Cell  Culture  Process  Op5miza5on  with  ambrTM  –  Growth  Profile  

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Ø Real  5me  monitor  of  cell  growth  profiles  by  tes5ng  mul5ple  culture  condi5ons  like  pH,  temperature,  DO  level  and  feeds.  

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Cell  Culture  Process  Op5miza5on  with  ambrTM  –  Produc5vity  

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Ø   Produc5vity  and  protein  quality    (data  not  shown)  reported  by  high  throughput  assays  with  a  quick  turnaround  5me.  

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A  CHO  cell  line  producing  a  recombinant  glycoprotein  

Cell Growth

Titers Product Quality Attributes

Scalable  Model  Micro-­‐bioreactor  :  ambrTM  

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Ø The  process  decisions  and  results  from  ambrTM  were  reproducible  to  other  tradi5onal  bioreactor  scales  (10  L  and  200  L).  

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Taking  Advantage  of  Modulators  in  Downstream  Purifica5on  •  Incorpora3on  of  modulators  into  process  can  help  increase  selec3vity  and  purity  of  product  

•  Combina3ons  of  modulators  can  further  enhance  process  step  

•  Goal:  U3lize  mobile  phase  modulators  to  decrease  HCP  levels  during  Capto  MMC  process  step  for  an3body  purifica3on  

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Incorpora5ng  Modulators  into  Wash  steps  

•  Case  Study:    »  Target  molecule:  E.  coli  derived  

recombinant  protein  »  Process  step:  Phenyl  Sepharose  FF  »  Ini1al  product  yield:  88.8%    

»  Result:    –  Individual  modulators  showed  some  

selec3vity  enhancement  but  also  product  loss  

–  A  combina3on  of  urea,  sodium  thiocyanate  and  glycerol  in  the  wash  step  increased  product  purity  to  >95%  

Shukla AA, et al., 2002. Biotechnol Prog 18: 556–564.

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0.50  

0.70  

0.90  

1.10  

1.30  

1.50  

0.0%   10.0%   20.0%   30.0%   40.0%   50.0%   60.0%   70.0%   80.0%   90.0%   100.0%  

Normalized

 HCP

 

Recovery  

HCP  vs.  Recovery  aier  Intermediate  Wash  for  Capto  MMC  Capture  

baseline

During  Capture  step  

Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.

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0.40  

0.50  

0.60  

0.70  

0.80  

0.90  

1.00  

1.10  

0.0%   10.0%   20.0%   30.0%   40.0%   50.0%   60.0%   70.0%   80.0%   90.0%   100.0%  

Normalized

 HCP

 

Recovery  

HCP  vs.  Recovery  aier  Intermediate  Wash  for  Capto  MMC  Polishing  

baseline

During  Polishing  step  

Wolfe LS, et al., 2014. J. Chromatogr. A 1340: 151-156.

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Process  Impact  on  HCP  Clearance  of  Using  Selec5ve  Column  Washes    •  Inclusion  of  an  intermediate  wash  using  Tris,  0.1M  NaCl,  50mM  arginine,  5%  ethylene  glycol,  pH  7.0  resulted  in  2-­‐fold  lower  HCP  levels  when  compared  to  process  where  a  modulator  was  not  u3lized.  

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Conclusions  

•  Orphan  biopharmaceu3cal  development  needs  par3cular  emphasis  on    •  Genera3ng  a  high  producing  cell  line  with  short  3meline  

»  Earlier  material  genera1on  »  Iden1fying  high  producer  clones  with  an  integrated  approach  

•  Developing  a  process  with  the  end  in  mind  (licensure  filing)  to  avoid  mul3ple  changes  along  the  way  »  Op1mizing  cell  culture  process  with  high  throughput  technologies  

•  U3lizing  mixed  mode  chromatography  to  improve  product  purity  and  maintain  process  step  yield  

•  A  dedicated  CDMO  with  the  right  knowledge  and  capabili3es  can  help  smooth  the  development  pathway  

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Acknowledgments  

•  Abhinav  Shukla,  Ph.D.    Vice  President,  Process  Development  &  Manufacturing  

•  Michael  Cavanaugh  Vice  President,  Business  Development  

•  Sigma  Mostafa,  Ph.D.    Director,  Process  Development    

•  Shahid  Rameez,  Ph.D.  Process  Development  Scien3st  II,  Upstream  Process  Development    

•  Leslie  Wolfe,  Ph.D.    Process  Development  Scien3st  II,  Downstream  Process  Development    

•  Rich  Harper,  B.S.    Process  Development  Scien3st  I,  Cell  Line  Development