CRYSTALLIZATION UNIT

Insulin Design Project

Outline

Purpose of Crystallizer
Methods of Crystallization
Design Specifications
Engineering Drawing
Alternative Cost and Suppliers
Alternative Processes
Questions

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

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

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

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

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

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

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

Evaporation Method
Advantages:
high purity levels downstream

Disadvantages:
Vaporization chamber requires high
pressures
Protein viability very sensitive to high
temperatures

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

Solvent Method
Advantages:
Proteins viability not at risk due to
temperature change

Disadvantages:
Possible protein contamination due to
insufficient downstream solvent
recovery

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

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

Progression of Crystallization

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

Crystal Size and Growth Rate

Assumptions:
Continuous
Constant-volume
Isothermal
Well-mixed

Relates population density and crystal

size
Mechanism of crystal growth to
determine crystal growth

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

Design Equations

Proposed Design

Engineering Drawing

Costing Estimates
Three costs involved:
Crystallizer unit
Zinc Chloride Solution and Water
Power Requirements

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

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

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

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