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CHEE 450: Insulin Design Project
CRYSTALLIZATION
UNIT
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Outline
Purpose of Crystallizer
Methods of Crystallization
Design Specifications
Engineering Drawing
Alternative Cost and Suppliers
Alternative Processes
Questions
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Purpose of Crystallizer
Used to recover pure solids from solution
Highly desirable end product because of:
Exceptional purity Ease of handling
Long shelf life
One of the final treatment steps in thepurification and concentration of insulin
98% of the insulin must be crystallized
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Mechanism of Crystallization
Crystal nucleation and amorphous precipitatesare in competition during supersaturationconditions
Nucleation favored by slowly exceeding theequilibrium point of saturation
permits time for the protein structure
to orient in a crystalline lattice
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Continuous or Batch Design
Benefits of Continuous
Can maintain solution in supersaturated state
Large fluidized bed for crystallization
Minimizes operation costs Minimize down time (startup and shutdown)
Benefits of Batch
Good when have low concentration of product, highviscosity or many impurities
Can produce high quality crystal
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Methods of Crystallization
Supersaturation: liquid (solvent) contains moredissolved solids (solute) than can ordinarily beaccommodated at that temperature
Can be achieved by several methods:
Cooling
Evaporation Solvent addition
Precipitant Addition
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Cooling Method
Concentrated solutiongradually cooled belowsaturation temperature(50-60C) to generatea supersaturated state
Yields well definedmicron-sized crystals
Shell and tube heatexchanger is used tocool solution
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Cooling Method
Advantages: High purity downstream
Disadvantages:
Temperature change does not always have a positiveeffect on supersaturation in proteins
Protein stability may be at risk
Solubility can be relatively insensitive to temperatureat high salt concentrations
Cooling will only help reach supersaturation insystems where solubility and temperature are directlyrelated
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Evaporation Method
Solute dissolves in solution when heated to acertain temperature (75C)
Slowly cooled until crystals precipitate
Shell and tube heat exchanger is used to heatand cool solution
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Evaporation Method
Advantages:
high purity levels downstream
Disadvantages:
Vaporization chamber requires high pressures
Protein viability very sensitive to hightemperatures
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Solvent Method
Solvents are generally good proteinprecipitants
Their low dielectric constants lower the
solvating power of their aqueous solutions Requires acidic solvent
For crystallization, an insulin protein fallsout of solution at isoelectric point
pH 5.4-5.7
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Solvent Method
Advantages:
Proteins viability not at risk due totemperature change
Disadvantages:
Possible protein contamination due toinsufficient downstream solvent recovery
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Addition of Zinc Ions
In the presence of zinc ions, insulin proteinsorient to form hexamer structures
Zinc ions render insulin insoluble which resultsin micro-crystallization and precipitation
Human Insulin Hexamer with Zinc ion
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Seeding Techniques
Primary nucleation is the first step incrystallization - growth of a new crystal Can bypass primary nucleation (creation of
new crystals) by "seeding" the solution
Secondary nucleation is crystal growth
initiated by contactAccelerated by "seeding" adding existing
insulin crystals to perpetuate crystal growth
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Progression of Crystallization
http://www.cheresources.com/cryst.shtml
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Crystal Size and Growth Rate
Crystal size distribution is important for the productionprocess; affects: downstream processing
solids transport
caking and storage properties of the material
Correct crystal size vital for economic production
Crystals produced in commercial crystallizationprocesses are usually small 30 to 100 um in diameter
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Crystal Size and Growth Rate
Assumptions: Continuous Constant-volume Isothermal Well-mixed
Relates population densityand crystal size
GL
Lk
Lk
v
a
/expnn
M:MassCrystal
A:AreaCrystal
o
3
c
2
c
Mechanism of crystal growthto determine crystal growth
s
v
as cc3dt
dLk
kk
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Crystallizer Design
Addition of acidic solvent to decrease pH toachieve supersaturation
Addition of Zinc ions to initiate Insulin
precipitation Implementing ofseeding technique
Minimize heat variation to maintain protein
stability Washing and extensive solvent recovery
downstream
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Design Equations
2.5DH
H4DV
slurryofgravityspecific24.6
slurryofqualityVolume
crystalsofsg75.0solutioofsg25.0
crystalsofsgsolutionofsgslurryofgravitySpecific
.25quantity/0crystalqualitySlurry
flowratetimeretentionquantityCrystal
2
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Proposed Design
Temperature 25C
Pressure 1.013 bar
Flowrate 111.842 kg/batch
Volume 0.29 m3
Diameter 0.529 m
Height 1.325 m
Residence Time 23.98 h
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Engineering Drawing
http://sundoc.bibliothek.uni-halle.de/diss-online/04/04H181/prom.pdf
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Costing Estimates
Three costs involved:
Crystallizer unit
Zinc Chloride Solution and Water
Power Requirements
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Costing Estimates
Crystallizer Unit www.matche.com
http://www.matche.com/http://www.matche.com/7/30/2019 Crystallizer Sc
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Costing Estimates
Crystallizer Unit
Batch, Atmospheric Crystallizer
0
10000
20000
30000
40000
50000
60000
70000
80000
0.00
0.15
0.30
0.45
0.61
0.76
0.91
1.06
1.21
1.36
1.52
1.67
1.82
1.97
Size (m3)
Cost(US$
)
Carbon Steel Stainless Steel
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Costing Estimates
Zinc Chloride Solution
Many suppliers
$15.00 - $27.00 for 500g
Power Requirements
Canadian Hydro: 8.99 cents/kWh (April, 2006)
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Crystallizer Suppliers
GEA Niro Inc. Companies in over 50 countries
Copenhagen, Columbia, Germany, USA
GEA Kestner Evapo rator/Crystal l izer
Swenson Technology Inc. Illinois, USA
HPD Inc. Illinois, USA
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Alternative Processes
For special drug purposes and when azinc-free product is needed
Alternative processes that can be usedinclude:
Isoelectric Precipitation
Gel Chromatography
Ultrafiltration
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Isoelectric Precipitation
Protein purificationprocedure that can beused with crystallizationor on its own
The pH of a mixture is adjusted to the pI of theprotein to be isolated to selectively minimize itssolubility
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Gel Filtration Chromatography
Molecules are separatedaccording to their size andshape
Filtration column is filled withporous beads
Solution passes throughcolumn
Elution through the gel occursin order of decreasingmolecular masses
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Ultrafiltration
Ultrafiltration used to concentratemacromolecular solutions
Forced under pressure or by centrifugation
through a semipermeable membranous disk Solvent and small solutes pass
through the membrane, leaving
behind a more concentratedmacromolecular solution
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QUESTIONS?