Expression  system  

Complexity  

Single    Enzyme   Bacteria   Yeast   Mammalian  Cells  

2  

Bioreactor  opera1on  

Batch   Fed-­‐Batch  

Feed  

Perfusion  

Filtra;on/Sedimenta;on  Chromatography  

Size/Weight  charge/affinity  

Downstream  

Balasundaram,  B.,    Trends  in  Biotechnology  Vol.27  No.8,  2009  

-­‐  Quality  -­‐  Market  -­‐  Economy  of  the  process  (produc;vity)    -­‐  Legisla;on  (CO2  emission,  renewable  resources)  

-­‐  Technical  knowledge  -­‐  Risk  factors    

Bioprocesses  versus  chemical  industry  

1990,  Roche:  Vitamin  B2  from  chemical  synthesis  to  biological  process  based  on  Bacillus  sub;lis  

-­‐  Quality  -­‐  Market  -­‐  Economy  of  the  process  -­‐  Legisla;on  

Small  chemicals  

 -­‐  75%  less  non-­‐renewable  resources    -­‐  emissions  are  heavily  reduced  -­‐  decrease  in  the  product  cost      

 à  Roche  becomes  world-­‐wide  leader  producer      

Fermenta1on  

2001,  Cargill-­‐Dow  Chemical:  Lac;c  acid  from  corn  (starch),    140,000  tons  of  LA  per  year,  around  400,000  tons  of  corn  (maize)  processed    Possibility  to  produce  PLA  at  around  1$  per  kilo  

PLA  

-­‐  Quality  (only  L(+)  form)  -­‐  Market  -­‐  Economy  of  the  process  

Lactobacillus  Bulgaricus  

Lac1c  Acid  

Corn,  Apples,  Potatoes,  Cassava,  …  

Lac1c  Acid  

Y.-­‐J.  WEE  et  al:  Biotechnological  Produc;on  of  Lac;c  Acid,  Food  Technol.  Biotechnol.  44  (2)  163–172  (2006)      

Ethanol  

-­‐  Quality  -­‐  Market  -­‐  Economy  of  the  process  

Sugar    Brazil  à  sugar  cane;      US  à  corn  starch  

Source:  Renewable  Fuels  Associa;on  (RFA)    

Synthe1c:    Cracking  oil  à  Ethylene,  +  Steam  +  catalyst  àEthanol  +  toxic  by-­‐products!  

Bioprocess  

Yeast  

Ethanol  

Sanchez,  O.J.  and  Cardona,  C.A.,  Bioresource  Technology  99  (2008)  5270–5295      

Therapeu1c  proteins  

Biologics  sector  Biotechnology  firms  

Source:  “Organisa;on  for  Economic    Co-­‐opera;on  and  Development”  

hkp://www.oecd.org/science/keybiotechnologyindicators.htm  

Monoclonal  An1body  

Strategies  and  challenges  for  the  next  genera;on  of  therapeu;c  an;bodies  Alain  Beck,  Thierry  Wurch,  Chris1an  Bailly  &  Nathalie  Corvaia  Nature  Reviews  Immunology  10,  345-­‐352  (May  2010)    

Monoclonal  An1body:  Discovery  Phage  display   Ra;onal  design  

Muta;ons  

Target  

Ab  

Iden;fica;on  from  human  samples  

Therapeu1c  proteins  

Host: enzymes mammalian cells microorganism

Products:

Aspirin 21 atoms Hormon

~3000 atoms Antibody ~25 000 atoms

Cost of development / complexity

Plane Bike

Car

Biopharmaceutical Industry Industrial (white) Biotechnology

-­‐  Quality  -­‐  Market  -­‐  Economy  of  the  process  

15  

Produc1on  and  Development  

10  years   (successrate:  1/10)  

Behme  S.  Manufacturing  of  Pharmaceu;cal  Proteins  –  From  Technology  to  Economy  

Therapeu1c  proteins  

Complexity  

Single    Enzyme   Bacteria   Yeast   Mammalian  Cells  

Animal  cells  e.g.  Chinese  hamster  ovary  (CHO)    

à  dominant  host:    robust,  protein  folding,    

non-­‐immunogenic  glycosyla;on  pakern  

1  µm,  simple,  fast  growth  (1/min)  

10  µm,  comples,  slow  growth  (1/day),  

glycosila;on  

17  

Post-­‐transla1on  modifica1ons  

•  Glycosyla1on    •  Heterogeneous  product  

•  Protein  folding    

•  Therapeu;c  efficacy  (ADCC,  CDC,  life-­‐;me,  ..)  

•  immunogenicity  

•  Charge  Isoforms  •  Heterogeneous  product  

•  Chemical  or  enzyma;c  modifica;on    

•  Protein  charge  distribu;on    •  Therapeu;c  efficacy  and  safety  unknown  

•  Structural  Changes    •  Fragmenta;on  and  aggrega;on  

•  Comprises  drug  efficacy  and  safety  

IgG  1  

Highly  variable  product  

“Microheterogeneity”  

N-­‐linked    glycosyla;on  

C-­‐terminal  lysine  cleavage  

Disulphide  scrambling  misfolds  

N-­‐terminal  glutamine  cycliza;on  An;body-­‐Dependent  Cell-­‐Mediated  Cytotoxicity  (ADCC),  and  Complement  Dependent  Cytotoxicity  (CDC)  

Hercep1n  charge  isoforms  

•  Hercep;n  (Trastuzumab)  •  IgG1,  pI  =  8.45  •  Final  drug  substance  contains    •  mul;ple  isoforms    

with  different  ac;vi;es  

18  

Analytical weak cation exchange chromatogram

W1 W2 W3 W4

P (140% activity)

www.drugbank.ca

S1 (12% activity)

Batch   Fed-­‐Batch  

Feed  

Perfusion  

0 2 4 6 8 10 12 14 16 180.0

0.2

0.4

0.6

0.8

1.0

Probability for protein modification

Perfusion Fed-batch

Prot

ein

frac

tion

/ (-)

Time / (days)

Perfusion:    τmean  =  1  day  

Fed-­‐Batch:    τmean  =  9  days  

Residence  1me  distribu1on:    

Bioreactor  opera1on  

1.  Separate  product  from    i.  Process  related  impuri;es  (DNA,  HCP,  cell  depries)  ii.  Product  related  impuri;es  (aggregates,  fragments)  

2.  Make  sure  the  target  protein  is  stable  and  pure:  Separa;on  task  is  a  mixture  (charge  isoforms)  from  a  mixture  (DNA,  HCP,  Aggregates,…)  à  challenging!!!  

3.  Product  needs  to  be  in  correct  buffer  system  

20  

Objec;ves  in  Downstream  process  development