CHEE 450: Insulin Design Project

CRYSTALLIZATION
UNIT
Rachel Adams
Jana Dengler
Megan MacLeod
Kyla Sask
CRYSTALLIZATION UNIT
Outline
• Purpose of Crystallizer
• Methods of Crystallization
• Design Specifications
• Engineering Drawing
• Alternative Cost and Suppliers
• Alternative Processes
• Questions
CRYSTALLIZATION UNIT
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 the
purification and concentration of insulin

• 98% of the insulin must be crystallized
CRYSTALLIZATION UNIT
Mechanism of Crystallization
• Crystal nucleation and amorphous precipitates
are in competition during supersaturation
conditions

• Nucleation favored by slowly exceeding the
equilibrium point of saturation
– permits time for the protein structure
to orient in a crystalline lattice
CRYSTALLIZATION UNIT
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, high
viscosity or many impurities
– Can produce high quality crystal

CRYSTALLIZATION UNIT
Methods of Crystallization
• Supersaturation: liquid (solvent) contains more
dissolved solids (solute) than can ordinarily be
accommodated at that temperature

• Can be achieved by several methods:
– Cooling
– Evaporation
– Solvent addition
– Precipitant Addition
CRYSTALLIZATION UNIT
Cooling Method
• Concentrated solution
gradually cooled below
saturation temperature
(50-60°C) to generate a
supersaturated state
• Yields well defined
micron-sized crystals
• Shell and tube heat
exchanger is used to
cool solution

CRYSTALLIZATION UNIT
Cooling Method
• Advantages:
– High purity downstream
• Disadvantages:
– Temperature change does not always have a positive
effect on supersaturation in proteins
– Protein stability may be at risk
– Solubility can be relatively insensitive to temperature
at high salt concentrations
– Cooling will only help reach supersaturation in
systems where solubility and temperature are directly
related


CRYSTALLIZATION UNIT
Evaporation Method
• Solute dissolves in solution when heated to a
certain temperature (75°C)
• Slowly cooled until crystals precipitate
• Shell and tube heat exchanger is used to heat
and cool solution
CRYSTALLIZATION UNIT
Evaporation Method
• Advantages:
– high purity levels downstream
• Disadvantages:
– Vaporization chamber requires high pressures
– Protein viability very sensitive to high
temperatures

CRYSTALLIZATION UNIT
Solvent Method
• Solvents are generally good protein
precipitants
• Their low dielectric constants lower the
solvating power of their aqueous solutions
• Requires acidic solvent
– For crystallization, an insulin protein falls
out of solution at isoelectric point
pH 5.4-5.7
CRYSTALLIZATION UNIT
Solvent Method
• Advantages:
– Proteins viability not at risk due to
temperature change
• Disadvantages:
– Possible protein contamination due to
insufficient downstream solvent recovery

CRYSTALLIZATION UNIT
Addition of Zinc Ions
• In the presence of zinc ions, insulin proteins
orient to form hexamer structures

• Zinc ions render insulin insoluble which results
in micro-crystallization and precipitation

Human Insulin Hexamer with Zinc ion

CRYSTALLIZATION UNIT
Seeding Techniques
• Primary nucleation is the first step in
crystallization - growth of a new crystal
– Can bypass primary nucleation (creation of
new crystals) by "seeding" the solution

• Secondary nucleation is crystal growth
initiated by contact
– Accelerated by "seeding" adding existing
insulin crystals to perpetuate crystal growth
CRYSTALLIZATION UNIT
Progression of Crystallization
http://www.cheresources.com/cryst.shtml
CRYSTALLIZATION UNIT
Crystal Size and Growth Rate
• Crystal size distribution is important for the production
process; affects:
– downstream processing
– solids transport
– caking and storage properties of the material
• Correct crystal size vital for economic production
• Crystals produced in commercial crystallization
processes are usually small
– 30 to 100 um in diameter
CRYSTALLIZATION UNIT
Crystal Size and Growth Rate
• Assumptions:
– Continuous
– Constant-volume
– Isothermal
– Well-mixed

• Relates population density
and crystal size
( ) t
µ
G L
L k
L k
v
a
/ exp n n
M : Mass Crystal
A : Area Crystal
o
3
c
2
c
÷ =
=
=
• Mechanism of crystal growth
to determine crystal growth
( )
s
v
a s
c c
3 dt
dL
÷ =
µ k
k k
CRYSTALLIZATION UNIT
Crystallizer Design
• Addition of acidic solvent to decrease pH to
achieve supersaturation
• Addition of Zinc ions to initiate Insulin
precipitation
• Implementing of “seeding” technique
• Minimize heat variation to maintain protein
stability
• Washing and extensive solvent recovery
downstream
CRYSTALLIZATION UNIT
Design Equations
( ) ( )
( ) ( )
( )
2.5D H
H
4
D
V
slurry of gravity specific 24 . 6
slurry of quality
Volume
crystals of sg 75 . 0 solutio of sg 25 . 0
crystals of sg solution of sg
slurry of gravity Specific
.25 quantity/0 crystal quality Slurry
flowrate time retention quantity Crystal
2
=
· =
·
=
· + ·
·
=
=
· =
t
CRYSTALLIZATION UNIT
Proposed Design
Temperature 25 °C
Pressure 1.013 bar
Flowrate 111.842 kg/batch
Volume 0.29 m
3
Diameter 0.529 m
Height 1.325 m
Residence Time 23.98 h
CRYSTALLIZATION UNIT
Engineering Drawing
http://sundoc.bibliothek.uni-halle.de/diss-online/04/04H181/prom.pdf
CRYSTALLIZATION UNIT
Costing Estimates
• Three costs involved:
– Crystallizer unit
– Zinc Chloride Solution and Water
– Power Requirements
CRYSTALLIZATION UNIT
Costing Estimates
• Crystallizer Unit www.matche.com

CRYSTALLIZATION UNIT
Costing Estimates
• Crystallizer Unit
Batch, Atmospheric Crystallizer
0
10000
20000
30000
40000
50000
60000
70000
80000
0
.
0
0
0
.
1
5
0
.
3
0
0
.
4
5
0
.
6
1
0
.
7
6
0
.
9
1
1
.
0
6
1
.
2
1
1
.
3
6
1
.
5
2
1
.
6
7
1
.
8
2
1
.
9
7
Size (m
3
)
C
o
s
t

(
U
S

$
)
Carbon Steel Stainless Steel
CRYSTALLIZATION UNIT
Costing Estimates
• Zinc Chloride Solution
– Many suppliers
– $15.00 - $27.00 for 500g




• Power Requirements
– Canadian Hydro: 8.99 cents/kWh (April, 2006)
CRYSTALLIZATION UNIT
Crystallizer Suppliers
• GEA Niro Inc.
– Companies in over 50 countries
– Copenhagen, Columbia, Germany, USA

GEA Kestner Evaporator/Crystallizer

• Swenson Technology Inc.
– Illinois, USA

• HPD Inc.
– Illinois, USA


CRYSTALLIZATION UNIT
Alternative Processes
• For special drug purposes and when a
zinc-free product is needed
• Alternative processes that can be used
include:
– Isoelectric Precipitation
– Gel Chromatography
– Ultrafiltration
CRYSTALLIZATION UNIT
Isoelectric Precipitation
• Protein purification
procedure that can be
used with crystallization
or on its own



• The pH of a mixture is adjusted to the pI of the
protein to be isolated to selectively minimize its
solubility
CRYSTALLIZATION UNIT
Gel Filtration Chromatography
• Molecules are separated
according to their size and
shape
• Filtration column is filled with
porous beads
• Solution passes through
column
• Elution through the gel occurs
in order of decreasing
molecular masses
CRYSTALLIZATION UNIT
Ultrafiltration
• Ultrafiltration used to concentrate
macromolecular solutions
• Forced under pressure or by centrifugation
through a semipermeable membranous disk
• Solvent and small solutes pass
through the membrane, leaving
behind a more concentrated
macromolecular solution
CRYSTALLIZATION UNIT
QUESTIONS?

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