pharmaceutical biotechnology lesson one -...

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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