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)

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PENICILLIN

Biological activity: anti-microbial

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STREPTOKINASE

Biological activity: anti-coagulant

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CHYMOSIN

Biological activity: enzimatic

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ANTI-hCG

Biological activity: molecular recognition

•Human chorionic gonadotropin

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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

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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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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?

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SOME CELLS USED TO PRODUCE BIOLOGICAL PRODUCTS

Saccharomyces cerevisiae

Escherichia coli

Aspergillus niger

Chinese Hamster Ovary (CHO) Dunaliella salina Baby Hamster Kidney (BHK)

?

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THE CASE OF INFLUENZA VACCINES

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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

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Nordic Innovation Centre

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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

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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

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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)

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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 –

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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

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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

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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)

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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.

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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

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PRIMARYISOLATION

INTERMEDIATEPURIFICATION

DOWNSTREAMPROCESSING

FINALPURIFICATION

FiltrationCentrifugationCell lysisLiquid-liquid extractionAdsorption

DOWNSTREAM PROCESSING - Overview

Liquid-liquid extractionPrecipitationAdsorptionUltrafiltration

ChromatographyCrystallizationDryingLyophilisation

Incoming material

Bulk product

Unit operation

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DOWNSTREAM PROCESSING - Overview

Volume of process streams decreases towards the end of the process: a lot of water is removed

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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

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Properties of biological products

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Properties of biological products

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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

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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

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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

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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

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4. Market size/annual production

1. What is the size of the market?

2. What is the market share?

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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

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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

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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?

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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?

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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

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5. Establish process diagram

iv. Environmental impact

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5. Establish process diagram

v. Safety

Biohazards?

Dangerous materials ?

end users

community

workers

product

process

factory

raw materials

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5. Establish process diagram

vi. Scale-up

Lab scale Process scale

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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

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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

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Measures of downstream processing performance

yield

number of operations

purity

100

cost

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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

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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

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Q17. TB vaccine

5 × 1013 AdV particles/L

Upstream

Yield = 95%

Calculate the annual volume of culture required:

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1011 AdV35 particles/dose

180 x106 doses/year

Downstream

mammalian cell culture