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
(50-60C) 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 (75C)
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

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
=
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?