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
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