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INSTITUTO SUPERIOR TINSTITUTO SUPERIOR TÉÉCNICOCNICO
Departamento de Engenharia Departamento de Engenharia QuQuíímica emica e BiolBiol óógicagica
SeparationSeparation andand PurificationPurification ofof BiologicalBiological ProductsProducts
INTRODUCTIONINTRODUCTION
Miguel Prazeres
2009
PACLITAXEL (TAXOLTM)
Biological activity: anti-cancer
•Pacific yew (teixo)
Miguel Prazeres
PENICILLIN
Biological activity: anti-microbial
Miguel Prazeres
STREPTOKINASE
Biological activity: anti-coagulant
Miguel Prazeres
CHYMOSIN
Biological activity: enzimatic
Miguel Prazeres
ANTI-hCG
Biological activity: molecular recognition
•Human chorionic gonadotropin
Miguel Prazeres
GARDASILTM
•$270-$360 /3 doses
•Protection against cervical cancer
Biological activity: anti-viral (papilloma)
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INFLUENZA VACCINE
Biological activity: anti-viral
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YELLOW FEVER VACCINE
Biological activity: anti-viral
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Bacillus thuringiensis
Biological activity: insecticide
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Q1.
Rank the following biological products on the basis of their size:
1. ethanol2. Escherichia coli
3. red blood cells
4. Saccharomyces cerevisiae5. pUC 18
6. insulin
7. glucose
8. erythropoietin
ethanol < glucose < insulin < erythropoietin < pUC 18 < E. coli < S. cerevisiae < red blood cells
Miguel Prazeres
Q2. Consider:
1. erythropoietin for injection (rEPO)
2. vitamin C
3. phospholipase C for detergents
The annual production in kg increases in the order:
a) phospholipase C, vitamina C, rEPO
b) rEPO, phospholipase C, vitamin Cc) vitamin C, phospholipase C, rEPO
The approximate cost/kg increases in the order:
a) phospholipase C, vitamin C, rEPOb) rEPO, phospholipase C, vitamin C
c) vitamin C, phospholipase C, rEPO ����
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Miguel Prazeres
1E+0
1E+1
1E+2
1E+3
1E+4
1E+5
1E+6
1E+7
1E+8
1E+9
1E+10
1E-4 1E-3 1E-2 1E-1 1E+0 1E+1 1E+2 1E+3 1E+4 1E+5 1E+6 1E+7
Log10 produção anual (T/ano)
Log1
0 pr
eço
($U
S/k
g)eritropoietina
α-interferão
taxol
ácido cítrico
penicilinaenzimas p/ detergentes
DNase
tPA
HSA
pectinasedigitalis
vinblastina mAbs
insulina
WORLD PRODUCTION AND PRICE OF BIOLOGICAL PRODUCTS
Miguel Prazeres
Q3. Consider:
1. mass: 100 mg2. purity: 98.0 %3. biological activity: 100 U/mg
1. mass: 100 mg2. purity: 99.9 %3. biological activity: 50 U/mg
What is preferable?
Miguel Prazeres
Miguel Prazeres
SOME CELLS USED TO PRODUCE BIOLOGICAL PRODUCTS
Saccharomyces cerevisiae
Escherichia coli
Aspergillus niger
Chinese Hamster Ovary (CHO) Dunaliella salina Baby Hamster Kidney (BHK)
?
Miguel Prazeres
THE CASE OF INFLUENZA VACCINES
Miguel Prazeres
Platform Vaccine type
Embryonated eggs inactivated whole virion, inactivated split virus, inactivated antigens,
live attenuated
Mammalian cells inactivated whole virion, inactivated split virus, inactivated antigens,
live attenuated, viral vector-based
Insect cells virus-like particles (VLPs), recombinant antigen
Yeast recombinant antigen
Bacteria recombinant antigen, plasmid DNA
Which vaccine type? Which platform?
SOME CELLS USED TO PRODUCE BIOLOGICAL PRODUCTS
Escherichia coli
Miguel Prazeres
Miguel Prazeres
Nordic Innovation Centre
Miguel Prazeres
NATURAL SOURCES OF BIOLOGICAL PRODUCTS
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RAWMATERIALS
UPSTREAMPROCESSING
DOWNSTREAMPROCESSING
FILL AND FINISH
bulk product
final product
MANUFACTURING OF BIOLOGICAL PRODUCTS - Overview
Extraction
Cell cultureSynthesis
GOAL OF DSP : purify the biological product from the starting material (tissues, biological fluids, cell culture, reaction media) by removing impurities until the pre-specified purity/biological activity is met
NOTE: impurity ≠ contaminant
Miguel Prazeres
RAWMATERIALS
UPSTREAMPROCESSING
DOWNSTREAMPROCESSING
FILL AND FINISH
bulk product
final product
RAW MATERIALS
Extraction
Cell cultureSynthesis
The selection of raw materials is very important:
Dirty raw materials – cheaper but have a higher impact in the DSP
Cleaner (pure) raw materials – more expensive but you pay in advance for the purification
Miguel Prazeres
Q4. Consider the production of:
1. penicillin (molasses, 5 unit operations)
2. yogurte (leite, 1 unit operation)
3. insulin (refined sugar, 30 unit operations)
In which case is the following ratio higher?:
Cost of raw materials in Upstream processing
Total cost of raw materials (Upstream + Downstream processing)
Miguel Prazeres
RAWMATERIALS
UPSTREAMPROCESSING
DOWNSTREAMPROCESSING
FILL AND FINISH
bulk product
final product
UPSTREAM PROCESSING
Extraction
Cell cultureSynthesis
The selection of the production technology may impact the whole DSP:
Intracellular vs extracellular product –
Starting concentration –
Miguel Prazeres
CELLS DISRUPTION SOLID-LIQUIDSEPARATION
VOLUMEREDUCTION
UPSTREAM PROCESSING: intracellular vs extracelullar product
CELL CULTURE SOLID-LIQUIDSEPARATION
intracellular
extracellular
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Escherichia coli
inclusion bodies
UPSTREAM PROCESSING: intracellular vs extracelullar product
Miguel Prazeres
UPSTREAM PROCESSING: starting concentration
Ethanol 100 g/L
Antibiotics 10-30 g/L
Proteases 2-5 g/L
mAbs 0.1-5 g/L
Plasmids 0.1-1 g/L
Adenovirus 0.01-0.1 g/L
Price
Concentration
Miguel Prazeres
Q5. Consider:
1. 2000 children w/ growth hormone deficiency (hGH)2. 15 mg/week for 1 year
Compare production from:
i) human pituitary glands (≈ 4,3 mg hGH/gland)
ii) genetically modified E. coli (≈ 1 g hGH/L culture medium)
iii) transgenic goats (2 g hGH/L milk, 800 L milk/year.goat)
Miguel Prazeres
Q6. Consider:
1. 1.6 billion (25% world coverage) influenza vaccine2. 1 shot per patient (15 µg HA antigen)
Compare production from:
i) Eggs (≈ 15 µg HA/egg)
ii) MDCK (Madin-Darby canine kidney) cells (≈ 12 µg HA/mL culture medium)
iii) Insect cells (70 µg HA/mL culture medium)
Assume 50% purification yield in all cases.
Miguel Prazeres
DOWNSTREAMPROCESSING
Remove solidsReduce volumeRelease product
DOWNSTREAM PROCESSING - Overview
Reduce volumeRemove impurities with properties very different
from the product
Remove remaining impuritiesRemove liquids
Bulk product
PRIMARYISOLATION
INTERMEDIATEPURIFICATION
FINALPURIFICATION
Incoming material
Miguel Prazeres
PRIMARYISOLATION
INTERMEDIATEPURIFICATION
DOWNSTREAMPROCESSING
FINALPURIFICATION
FiltrationCentrifugationCell lysisLiquid-liquid extractionAdsorption
DOWNSTREAM PROCESSING - Overview
Liquid-liquid extractionPrecipitationAdsorptionUltrafiltration
ChromatographyCrystallizationDryingLyophilisation
Incoming material
Bulk product
Unit operation
Miguel Prazeres
DOWNSTREAM PROCESSING - Overview
Volume of process streams decreases towards the end of the process: a lot of water is removed
Miguel Prazeres
start
Volume
endmid
dilution
process I process II
process III
downstream processing
Volume of equipment decreases towards the end of the process
SYNTHESIS OF DOWNSTREAM PROCESSES
1. Know the product and associated impurities
2. Product specifications
3. Analytical techniques
4. Market size/annual production5. Establish process diagram
i) literature
ii) experienceiii) rules of thumb (heuristics)
iv) environmental impact
v) safety
vi) scale-upvii) GMPs and regulations
Miguel Prazeres
1. Know the product and associated impurities
1. Molecular weight/size/shape
2. Isoelectric point3. Tridimensional structure and composition
4. Relevant functional groups (e.g. –COOH, -NH2, etc)
5. Diffusivity, solubility, etc…6. Stability (T, pH, shear stress, …)
Clues for the selection of unit operations
Miguel Prazeres
Properties of biological products
Miguel Prazeres
Miguel Prazeres
Properties of biological products
Miguel Prazeres
Properties of biological products
Q7. Erythromycin
Solubility:
i) in water: 2 mg/mlii) in ethyl acetate: >>100 mg/ml
Suggest a unit operation to remove erythromycin from the medium
Saccharopolyspora erythraea
Miguel Prazeres
erythromycin crystals
2. Product specifications
1. Purity2. Specific activity (potency)
3. Acceptable impurity/contaminant levels
4. Appearance (color, viscosity, particle size, ….5. pH
6. Identity, structure
7. …….
Clues for the selection of unit operations
Tells you where to go
Miguel Prazeres
Start with the end in mind
Specification Analytical method Acceptance criteria
Plasmid
1. Appearance Visual inspection Clear, colourless solution
2. Identity Restriction mapping, sequencing, PCR
According to expected map Homology
3. Homogeneity Agarose gel + densitometry > 90 % supercoiled
4. Concentration A260, HPLC, fluorescence According to the application
5. Potency Cell transfection According to the application
Impurities
1. Protein Bicinchoninic acid assay (BCA) SDS-PAGE
Not detected< 0.01 µg /dose
2. RNA Agarose gel 0.8% Not detected
3. genomic DNA Hybridization, PCR, fluorescence < 0.05 µg /µg plasmid< 0.01 µg /dose
4. Endotoxins LAL test (Limulus AmeobocyteLysate)
< 0.1 EU/µg plasmid< 5 EU/kg body mass
Ex: plasmids for gene therap or DNA vaccines
2. Product specifications
Miguel Prazeres
3. Analytical techniques
1. HPLC/mass spectrometry
2. Electrophoresis3. Spectroscopy (absorption, fluorescence, Circular Dichroism, …
4. ELISA (enzyme linked immuno-sorbent assay)
5. Hibridization6. Protein tests
7. Activity (potency) tests
8. Microbiological tests (sterility, microbiologic load, endotoxins, viruses, phages….
Process monitoring (know were you are)
Product Quality (know if you have arrived)
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3. Analytical Techniques
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Prod1
-20
0
20
40
60
80
100
120
140
0 5 10 15 20 25
Time (min)
mA
U
Prod1
3. Analytical Techniques
Miguel Prazeres
Q8. Associate the parameter to the technique(s)
f. Absorbancy6. RNA impurity
e. Northern hibridization5. Bacterial contamination
d. HPLC4. DNA impurity
1. Molecular weight a. Southern hibridization
2. Purity b. Cultures in agar plates
3. Concentration c. LAL test
7. Bacterial endotoxins g. electrophoresis
Miguel Prazeres
4. Market size/annual production
1. What is the size of the market?
2. What is the market share?
Miguel Prazeres
Q9. Flu vaccines
Worldwide annual production: ~10 million doses
Suppose:
10% market share
1 dose = 3 different virus strains (3 x 15 µg each virus)
50 µg virus/embryonated eggPurification yield = 60 %
How many eggs are required per year?
5. Establish process diagram
i. Literature
ii. Experienceiii. Rules of thumb (heuristics)
iv. Environmental impact
v. Safetyvi. Scale-up
vii. Other aspects
viii. GMPs and regulations
Miguel Prazeres
5. Establish process diagram
i. Literature
ii. Experience
Miguel Prazeres
Patents
Scientific articles
ReportsThesis
Books
http://www.google.com/patents
http://ep.espacenet.com/
5. Establish process diagram
i. Literature
ii. Experience
Miguel Prazeres
Patents
Scientific articles
http://ep.espacenet.com/, http://www.google.com/patents,
http://apps.isiknowledge.com/, http://www.sciencedirect.com/,
http://www3.interscience.wiley.com/cgi-bin/home
http://www.springerlink.com/home/main.mpx
http://www.ncbi.nlm.nih.gov/pubmed/
5. Establish process diagram
iii. Rules of thumb (heuristics)
Miguel Prazeres
1. Remove the most plentiful impurities first
2. Remove the easiest to remove impurities first
3. Make the most difficult and most expensive separations last
4. Select processes that make use of the greatest differences in the properties of the product and impurities
5. Select and sequence processes that exploit different separation driving forces
6. Just because it works in the lab doesn’t mean it’s right for the factory
5. Establish process diagram
iii. Rules of thumb (heuristics)
Miguel Prazeres
1. Remove the most plentiful impurities first
2. Remove the easiest to remove impurities first
Cell brothUnit
operation
cells
Unitoperation
water
water
productextracellularproduct
Cell broth Unitoperation
water (most abundant ‘impurity’, > 90%)
cells
intracellularproduct
Miguel Prazeres
3. Make the most difficult and most expensive separations last
5. Establish process diagram
iii. Rules of thumb (heuristics)
Chromatography is many time used to separated product isoforms or variantsThis high resolution and high cost operation is usually performed towards the end
Q10.
product: pI = 8,0, MW = 50 000
Impurity 1: pI = 4,2, MW = 10 000Impurity 2: pI = 7,8, MW = 35 000
Which impurity should be removed last?
0
20
40
60
80
100
120
0 5 10
Time (min)
mA
U
IgG
IgG dimersIgGaggregates
Miguel Prazeres
4. Select unit operations that make use of the greatest differences in the properties of the product and impurities
5. Establish process diagram
iii. Rules of thumb (heuristics)
Q11.
product: pI = 7,0, MW = 170 000
Impurity 1: pI = 6,7, MW = 20 000
What property should be explored first to perfom this separation?
Miguel Prazeres
5. Select and sequence processes that exploit different separation driving forces
6. Just because it works in the lab doesn’t mean it’s right for the factory
7. Keep It Simple
5. Establish process diagram
iii. Rules of thumb (heuristics)
UO1 UO2 UO3
UO1 UO2 UO3
≠ charge ≠ solubility ≠ molecular weight
≠ charge ≠ charge ≠ charge
option1
option2
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Q12. Extracelullar protein
a )
b )
chromatography precipitation
chromatographyprecipitation
supernatant
supernatant
What is the best option to recover the protein from the medium?
Miguel Prazeres
5. Establish process diagram
i. Literature:
ii. Experienceiii. Rules of thumb (heuristics)
iv. Environmental impact
v. Safetyvi. Scale-up
vii. Other aspects
viii. GMPs and regulations
Miguel Prazeres
pDNA
Culture
Recovery
Lysis
Chromatography
Ammonium sulfate
pp
Isopropanol pp
0
500
1000
1500
2000
2500
processopadrão
70%reciclagem
semisopropanol
Impa
cto
ambi
enta
l (po
ntos
/kg) Outros
Sulfato de amónio
Isopropanol
Miguel Prazeres
5. Establish process diagram
iv. Environmental impact
Miguel Prazeres
5. Establish process diagram
v. Safety
Biohazards?
Dangerous materials ?
end users
community
workers
product
process
factory
raw materials
Miguel Prazeres
5. Establish process diagram
vi. Scale-up
Lab scale Process scale
Miguel Prazeres
5. Establish process diagram
vii. GMPs and regulations
viii. Other aspects
Robustness – ability to accommodate small variations in materials/operating conditions
Consistency – lot-to-lot quality should be maintained
Costs –
LawsRegulationsGuidelines
Miguel Prazeres
Measures of downstream processing performance
C, Concentration (mg/ml) – mass/volume
P, Purity (%) – product mass/total mass of solutes
A, Specific activity (U/mg) – biologic activity/solute mass
Vi-1
Ci-1
Pi-1
Ai-1
Vi
Ci
Pi
Ai
1i1i
iiCVCV
100−−
=η
1i
iCC
CF−
=
1i
iAA
PF−
=Purification factor, PF
Yield, η
Concentration factor, CF >1 – concentration
< 1 - dilution
>1 – purification
< 1 – no purification
UO i
Miguel Prazeres
Measures of downstream processing performance
yield
number of operations
purity
100
cost
Miguel Prazeres
Measures of downstream processing performance
N1 2 i-1 i
CFVV
CVCV
CVCV
CVCV
CVCV
CVCV
1
N
11
NN
1N1N
NN
ii
1i1i
1i1i
ii
11
22i
N
1total ==×××××=∏=
−−
++
−−LLηη
i
N
1total CFCF ∏=
i
N
1total PFPF ∏=
Q13. Solid phase synthesis of peptides
NH2
HOOC
matrix
a.a
peptide
1. Yield per addition/purification of 1 aa = 97%
What is the expected yield in the synthesis of a 50 aa polipeptide?
a) 50 x 0.97
b) 500.97
c) 0.9750
d) 0.97/50
Miguel Prazeres
Q14. Purification of a protein
volume = 100 Ltotal protein = 0,1 mg/mLactivity = 1 U/mL
OU
volume = 2 Ltotal protein = 1 mg/mLactivity = 40 U/mL
Activity of the pure protein = 100 U/mg
Calculate:
a) Yield
b) Concentration factor
c) Purification factord) Purity
c) Specific activity
Miguel Prazeres
Q15. Viral load reduction
volume = 100 mLviral load = 109 vp SV40
OU
volume = 10 mL
viral load = 2 × 103 vp SV40
Calculate the reduction in the viral load in log10
Miguel Prazeres
waste
Q16. Purification of IgG
purity = 25% (IgG/total protein)IgG mass = 100 g
OU
Yield = 95%95% removal of impurities
Calculate:
a) final purityb) final IgG mass
c) mass of impurities in and out
Miguel Prazeres
Q17. TB vaccine
5 × 1013 AdV particles/L
Upstream
Yield = 95%
Calculate the annual volume of culture required:
Miguel Prazeres
1011 AdV35 particles/dose
180 x106 doses/year
Downstream
mammalian cell culture
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