pharmaceutical biotechnology lesson one -...
TRANSCRIPT
By Yuqiong Xia
3/27/2017
药物制造过程:终产品的制造The drug manufacturing process: Production of
final product
Pharmaceutical Biotechnology西安电子科技大学 夏玉琼
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Production of final product
1. cell harvesting and recovery of crude product
2. concentration and initial purification
3. main purification(chromatography)
4. final product formulation
5. product filling, freeze-drying and sealing
6. labeling and packaging
1. working cell bank vial removed from storage
2. propagation of working bank cells, generating starter cultures
3. production-scale cell culture
Upstream processing
Downstream processing
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The drug manufacturing process
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Upstream processing: cultivation
Downstream processing: purification
Downstream processing: formulation
http://www.zet.or.at/spool/gfx/1201165054-SerumfreeDB.jpg
http://images.medscape.com/pi/features/drugdirectory/octupdate/AMG01440.jpg
Target protein Impurities
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Outline
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Upstream processing: cultivation
Downstream processing: purification
Downstream processing: formulation
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The master cell bank/working cell bank system
Culture of the newly constructed
production cell line
Aliquoted into ampoules
Master
cell bank
Working cell bank number 1
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Commonly used E.coli growth media
http://en.wikipedia.org/wiki/File:Anthrax_culture.jpg
http://www.exptec.com/Bacterial%20E.coli%20Growth%20Media.htm
• LB (Luria Bertani) Miller broth (1%NaCl): 1% peptone(蛋白胨), 0.5%
yeast extract, and 1% NaCl
• LB (Luria Bertani) Lennox Broth (0.5% NaCl): 1% peptone, 0.5%
yeast extract, and 0.5% NaCl
• SOB medium (Super Optimal Broth): 2% peptone, 0.5% Yeast
extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl2 , 10mM MgSO4
• SOC medium (Super Optimal broth with Catabolic repressor): SOB
+ 20mM glucose
• 2x YT broth (2x Yeast extract and Tryptone): 1.6% peptone, 1%
yeast extract, and 0.5% NaCl
• TB (Terrific Broth) medium: 1.2% peptone, 2.4% yeast extract, 72
mM K2HPO4, 17 mM KH2PO4 and 0.4% glycerol
• SB (Super Broth) medium: 3.2% peptone, 2% yeast extract, and
0.5% NaCl
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Animal Cell Cultures
http://www.zet.or.at/spool/gfx/1201165054-SerumfreeDB.jpg
http://ibidi.com/uploads/pics/coatings2.jpg
https://encrypted-tbn2.gstatic.com
Cell subculture: PBS, trypsin, culture medium (MEM, DMEM, RPMI 1640)
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Cultivation Medium for animal cells西安电子科技大学 夏玉琼
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Formulation for DMEM ATCC® 30-2002西安电子科技大学 夏玉琼
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Stirred-tank bioreactor
起泡器
挡板
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Outline
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Upstream processing: cultivation
Downstream processing: purification
Downstream processing: formulation
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Basic operations required for the purification西安电子科技大学 夏玉琼
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Contaminants of biopharmaceuticals
and
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Methods of purification
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Chromatography (层析/色谱)
Filtration and centrifugation
Precipitation
https://chem.libretexts.org/
http://www.spectrumlabs.com/filtration/Edge.html
http://chemwiki.ucdavis.edu/@api/deki/files/1325/precipitation_reactions.jpg?revision=1
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Column Chromatography
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Separate sample by
separation column
Separation column is
packed with stationary
phase
With the addition of mobile
phase (solvent), different
samples are eluted at
different timehttps://chem.libretexts.org/Textbook_Maps/Analytical_Chemistry_Textbook_Maps/Map%3A_Analytical_Chemistry_2.
0_(Harvey)/12_Chromatographic_and_Electrophoretic_Methods/12.2%3A_General_Theory_of_Column_Chromatograp
hy
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Chromatography methods
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Mode/principle Separation based on
Ion-exchange Charge
Gel permeation Size
AffinitySpecific ligand-substrate
interaction
Adsorption Covalent/noncovalent binding
Hydrophobic
interactionHydrophobicity
https://chem.libretexts.org/
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Cation ion exchange chromatography (IEC) and
anion IEC
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IEC is always the
first purification
method
IEC removes
proteins and other
impurities with
different charge
First adsorption
and then elution
https://www.labome.com/method/Protein-Purification.html
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Hydrophobic interaction chromatography
Hydrophobic
Hydrophilic
Hydrophobic aggregates
salt
High saltLow salt
http://upload.wikimedia.org/wikibooks/en/thumb/0/00/Hicsalt.png/500px-Hicsalt.png
• high recoveries, folded correctly and
are separated from contaminants that
are structurally related
• Ideally after ion-exchange
chromatography
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Adsorption chromatography
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“normal
chromatography”
Protein of interest
binds to stationary
phase
High-ratio adsorption
Economically scalable
http://ts2.mm.bing.net/th?id=H.4677323748673621&pid=1.7
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Immunoaffinity chromatography
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Based on
Antibody-antigen binding
Problems
Hard removal of ligand
Limited scale-up
Protein
of inerestBinding
material
Unbounded
protein
http://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Purification/Affinity_chromatography
http://media.opencurriculum.org/articles_manual/ck12_biology/immune-response/4.png
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Gel permeation chromatography
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Commonly used as the final step in purification
Also called size exclusion chromatography
Separate proteins according to their shape and size
http://www.tissuegroup.chem.vt.edu/chem-ed/sep/lc/graphics/size-exc.gif
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Filtration and centrifugation
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Filtration
Cross flow (tangential flow filtration)
Ultrafiltration
Centrifugation
Buoyant density centrifugation
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Cross flow (Tangential flow filtration)
http://www.spectrumlabs.com/filtration/Edge.html
http://www.bbc.co.uk/bitesize/ks3/science/images/filtration2.gif
Common filtration Common filtration
Leads to dead-end
in industry
Tangential flow
filtration
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Ultrafiltration
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Separate molecules of different sizes
Concentrate macromolecules
Change the aqueous phasehttp://www.toltec.biz/how_hemodialysis_works.htm
http://blog.labplanet.com/wp-content/uploads/2012/09/Centrifugal.jpg
https://www.emdmillipore.com/images/large/Amicon05_ReverseSpin-71[83660-ALL].jpg
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Centrifugation
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Buoyant density centrifugation
viscous fluid with different densities in a tube
http://www.freewebs.com/ltaing/centrifugation.htm
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Precipitation
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Scalable, simple, economical
Limited purity, too many precipitates are hard to
handle
http://chemwiki.ucdavis.edu/@api/deki/files/1325/precipitation_reactions.jpg?revision=1
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Use salt to precipitate
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Chaotropic series with increasing “salting out”
effects
Ammonium sulfate
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Use water-miscible organic solvents to precipitate
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water-miscible organic solvents
Change dielectric constant ε
Examples
Polyethylene glycol –(CH2CH2O)n
Trichloracetic acid CCl3COOH
Under certain conditions, chitosan and non-ionic
polyoxyethylene detergents
F = 𝑞1𝑞2/(4𝜋𝜀𝑑)
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Processing of glycosylated recombinant
interferon (~28 kDa): 1/3
UF, ultrafiltration; DF, diafiltration
F, filtration
TFF, tangential flow filtration
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Processing of glycosylated recombinant
interferon (~28 kDa): 2/3
A, adsorption
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Processing of glycosylated recombinant
interferon (~28 kDa): 3/3 西安电子科技大学 夏玉琼
Outline
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Upstream processing: cultivation
Downstream processing: purification
Downstream processing: formulation
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Components in marketed formulations西安电子科技大学 夏玉琼
Solubility enhancers
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Amino acids such as lysine and arginine
used to solubilize tissue plasminogen activator, t-PA
Surfactants such as SDS
to solubilize non-glucosylated IL-2
Solubility increase with higher
arginine concentration
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Protein adsorption and aggregation
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Protein can be adsorbed to the interface like
water/air, water/container wall and water/needle
Adsorbed proteins form aggregates and precipitate
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Anti-adsorption and anti-aggregation agents
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1% Albumin (白蛋白)
Adsorb to surfaces instead of drugs
Surfactant
Adsorb to the hydrophobic interfaces and turn the
interface hydrophilic
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Buffer components
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Short, temporary pH change can cause
aggregation
Phosphate (pH 5.8-8.0, 11.0-11.9), citrate (pH 3.0-
6.2) and acetate buffer (pH 3.7-5.6)
http://delloyd.50megs.com/moreinfo/buffers2.html
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Preservatives (防腐剂) and antioxidants
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Antioxidants belong to preservatives
Easily oxidized amino acids
Methionine, cysteine, tryptophan, tyrosine, histidine
Inert gas in the vials can reduce oxidation
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Antioxidants
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Ascorbic acid (抗坏血酸,维生素C)
Acetylcysteine (乙酰半胱氨酸)
http://www.medicalook.com/molecular/ascorbic_acid.gif
http://ts1.mm.bing.net/th?id=H.4941541510548252&pid=1.7
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Lyoprotectants help to keep structures of protein
during freeze-drying
Glycol
Lyoprotectants
http://www.food-manufacturer.com/faqs/freeze-dried-knowledge/freeze-drying-process.html
Blue circles: Water molecules
dry dry rehydrate
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Osmotic agents
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Adjust the osmotic pressure
Saline and mono- or disaccharide solutions
http://chemistry2.csudh.edu/rpendarvis/monosacch.html
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Summary
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Upstream processing: cultivation
industry-scale bioreactor
Downstream processing: purification
IEC, GPC, hydrophobic interaction chromatography,
adsorption chromatography, affinity chromatography,
tangential flow filtration, ultrafiltration, buoyant density
centrifugation, precipitation
Downstream processing: formulation
Solubility enhancers, anti-adsorption, buffer, preservative,
lyoprotectant, osmotic agents
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