Transcript Document

Capillary- pore and Tortuous- pore membranes
• Capillary pore- straight through
cylindrical capillaries
• Tortuous pore-sponge with network
of interconnecting tortuous pores
Pore-size measurement techniques
1. Bubble –point method
2. Scanning elctron microscopy
3. Mercury porisimetry
Organisms retained in various pore sizes
Polytetra fluoroethylene (PTFE)
membrane
Polypropylene membrane
CRYOGELS
Doublet and triplet pores in capillary pore membranes
Pore size distribution in 0.45 µm membranes (by
mercury porisimetry)
Plate (Sheet) and frame format
membranes
Hollofibre – Tubular format membranes
Enhanced Cross-Flow Filtration Systems
Hollow-fibre filtration system
Manual benchtop tangential flow filtration (TFF) system.
Typical Applications
- Concentration and filtration
- Desalting and buffer exchange
- Cell harvesting/clarification
- Virus harvesting/clarification
Pellicon XL 50 Ultrafiltration Device
Applications
- Concentration, desalting, and
buffer exchange of proteins,
polysaccharides, lipid solutions,
viruses, colloids, cell suspensions,
and mammalian cells
- Sample preparation
- Preparation of material for
clinical trials
- Small volume manufacturing
Labscale TFF System
Centrifugation
A centrifuge is a device for separating particles from a solution
according to their size, shape, density, viscosity of the medium
and rotor speed. In biology, the particles are usually cells, sub
cellular organelles, viruses, large molecules such as proteins and
nucleic acids.
Analytical centrifugation
Analytical centrifugation involves measuring the physical properties
of the sedimenting particles such as sedimentation coefficient or
molecular weight.
Preparative centrifugation
In preparative centrifugation objective is to isolate specific particles
which can be reused. There are many type of preparative centrifugation
such as rate zonal, differential, isopycnic centrifugation.
Centrifugation classification based on speed
Another system of classification is the rate or speed at which the
centrifuge is turning.
Ultracentrifugation is carried out at speed faster than 20,000 rpm.
Super speed centrifugation is at speeds between 10,000 and
20,000 rpm.
Low speed centrifugation is at speed below 10,000 rpm.
Differential centrifugation
A third method of defining centrifugation is by the way the samples
are applied to the centrifuge tube. In moving boundary (or differential
centrifugation), the entire tube is filled with sample and centrifuged.
Through centrifugation, one obtains a separation of two particles but
any particle in the mixture may end up in the supernatant or in the pellet
or it may be distributed in both fractions, depending upon its size, shape,
density, and conditions of centrifugation.
Denisty gradient centrifugation
It allows separation of many components in a mixture
by creating density gradient during centrifugation
There are two forms of density gradient centrifugation:
rate zonal and
isopycnic
Rate Zonal Centrifugation (also termed sedimentation velocity,
zone centrifugation)
In rate zonal centrifugation, the sample is applied in a thin zone
at the top of the centrifuge tube on a density gradient. Under centrifugal
force, the particles will begin sedimenting through the gradient in
separate zones according to their size shape and density. The run must
be terminated before any of the separated particles reach the bottom
of the tube.
Swing bucket rotor
Isopycnic centrifugation (also termed sedimentation
equilibrium centrifugation)
During the centrifugation, the CsCl generates a gradient (“self-generating
gradient”), and the molecules move to the position in the gadient where
their density is the same as the gradient material. Isopycnic means
“same density,” so the molecules move to their “isopycnic position.”
Fixed angle or swing bucket rotor
Gradients
Sucrose
Cscl2
Glycerol
Dextran
Centrifugation
The performance of a centrifuge is characterised by:
Vc = dp2 . (ds - dl) . W2 r / 18 n
where Vc = centrifugal sedimentation rate (gs-1),
dp = particle diameter, ds = density of solid, dl =
density of liquid,
w= angular speed, r = distance of the particle to the
axis of rotation
and n = viscosity of medium.
Major forces acting on solid particle during settlingGravitational force (FG)
Drag force (FD)
Buoyant force (FB)
When the particles reach a terminal settling velocity, forces
acting on a particle balance each other, resulting in zero net
force. That is
FG = FD + FB
Nomogram for converting
maximum relative centrifugal force (RCF, i.e., g-force) to RPM
RCF to RPM: Determine centrifuge 's radius of rotation (in mm) by measuring
distance from center of centrifuge spindle to bottom of device when inserted into
rotor. Lay a ruler or draw a line from radius value in right-hand column value that
corresponds to the device 's maximum rated g-force. Then read the maximum value
from column at left.
R
I
P
P
Isolation/Extraction
Release of protein from biological host
• To gain access to the product
• Access to the product is simple and inexpensive when the
protein is produced extracellularly
• Microbial sources are preferred
• Mammlian cell hosts are preferred when posttranslational
modification is essential for the function of eukaryotic
proteins
• Bulk enzymes are invariably produced extracellularly by
Bacillus species & fungi, as are the proteins produced
by mammalian cell culture
Cell envelops of bacteria and yeast
Lipopolysaccharide
membrane
Mannan partially crosslinked by phosphodiester
bridges
Peptidoglycon
layer
Glucan layer
with proteins
Cytoplasmic
membrane
Gram-positive
bacteria
Gram-negative
bacteria
Animal cells: no cell wall, thus fragile in breaking
Plant cells: composed of cellulose and other polysaccharides
Yeasts
Cell Disintegration Techniques
Technique
Example
Principle
Gentle
Cell lysis
Erythrocytes
Enzyme digestion
Lysozyme treatment
of bacteria
Chemical solubilization/ Toluene extraction
autolysis
of yeast
Hand homogenizer
Liver tissue
Minicing (grinding)
Muscle etc.
Osmotic disruption of cell
membrane
Cell wall digested, leading
to osmotic disruption
Cell wall (membrane) partially solubilized chemically;
lytic enzymes released
complete the process
Cells forced through
narrow gap, disrupts cell
membrane
Cells disrupted during
minicing process by shear
force
Cell Disintegration Techniques
Technique
Example
Moderate
Blade homogenizer
(waring type)
Muscle tissue, most
animal tissues, plant
tissues
Grinding with abrasive Plant tissues, bacteria
(sand, alumina)
Vigorous
French press
Bacteria, plant cells
Ultrasonication
Cell suspensions
Principle
Chopping action breaks up
large cells, shears apart
smaller ones
Microroughness rips off
cell walls
Cells forced through small
orfice at very high pressure; shear forces disrupt
cells
Micro-scale high-pressure
sound waves cause disruption by shear forces
and cavitation
Cell Disintegration Techniques
Technique
Bead mill
Manton-Gaulin
homogenizer
Example
Cell suspension
Cell suspension
Principle
Rapid vibration with glass
beads rips cell walls off
As for French press, but on
a larger scale
Mechanical Methods
• Mechanical methods can be applied to a liquid or solid medium
• Most common mode, despite higher capital and operating costs
• Disruption is based primarily on liquid or solid shear forces
• Liquid shear cell disruption is associated with cavitation phenomenon
that involves formation of vapor cavities in liquid due to local reduction
in pressure that could be affected by ultrasonic vibrations, local increase
in velocity, etc. Collapse and rebound of the cavities will occur until an
incresae in pressure causes their destruction
• On the collapse of the cavitation bubble, a large amount of energy is released
as mechanical energy in the form of elastic waves that disintegarte into
eddies which impart motions of diferent intensities to the cell, creating
pressure difference across the cell.
When the kinetic energy content of the cell exceeds the cell wall strength,
the cell disintegrates.
Mechanical Methods
Ultrasonic vibrators (sonicators) are used to disrupt the cell wall and membrane
of bacterial cells. An electronic generator is used to generate ultrasonic waves,
and a transducer converts these waves into mechanical oscillations by a
titanium probe immersed in a cell suspension. Wave density is usually around
20 kc/s.
• Rods are broken more readily than cocci, and gram negative cells more easily
than gram positive cells
• The technique is not used at industrial scale primarily because the ultrasonic
energy absorbed into suspension ultimately apears as heat, and good
temperature control is necessary
• In some cases results in denaturation of sensitive enzymes and fragmentation
of cell debries
Mechanical Methods
High-Pressure homogenization:
French Press: The french press is a hollow cylinder in a stainless-steel
block that is filled with cell paste and subjected to high pressure. The
cylinder has a needle value at the base, and the cells disrupt as they are
extended through the value to atmospheric pressure. The flow restriction
in the value assembly drives up pressure (in the range of 50 and 120 MPa)
• Disruption follows first-order process at a given pressure in a highpressure homogenizer. The extent of protein release is represented
by
Rm
= kNP
ln Rm – R
Where Rm and R are the maximal amount of protein available for
release and the protein amount released at a certain time, respectively
(kg protein/kg cells), k is the first-order rate constant, N the number
of passages, P the operating pressure.
Mechanical Methods
High-Pressure homogenization:
Manton-Gaulin homogenizer: Traditional form of high-pressure
homogenizer, works as french press but on a larger scale.
Bead Mill Disruption: Stirring a cell suspension with glass beads
is an effective method of disruption of organisms. The process
is normally performed in a bead mill, such as Dyno-Mill
The principle of operation is to pump the cell suspension through
a horizontal grinding chamber filled with about 80% beads.
Within the grinding chamber is a shaft with specially designed
discs. When rotated at high speeds, high shearing and impact
forces from millions of beads break cell walls.
•
•
•
•
Can be used effectively at large scale
Available in sizes upto 275 l and can process 2000 kg/h of a cell
suspension or about 340 kg dw/h of yeast
Can work with algae, bacteria and fungi
Better temperature control
Mechanical Methods
Limitations
• High risk of damage to the product
• Heat denaturation a major problem
• The release of proteases from cellular compartments can
lead to enzymatic degradation of the product
• Bead mill have comparatively long residance times, products
released early may be damaged
• Products released encounter an oxidizing environment, that
can cause denaturation and aggregation
Non-Mechanical Methods
Physical Rupture of Microbial Cells
Desiccation: by slow drying in air, drum drying, etc followed
by extraction of the microbial powder
Osmotic shock: Changes in the osmotic pressure of the medium
may result in the release of certain enzymes, particularly
periplasmic proteins in gram negative cells. Suspending
a cell suspension in a solution with high salt concentration
High temperature: Exposure to high temperature can be an
effective approach to cell disruption but is limited to
heat-stable products. Heating to 50 – 55 ºC disrupts outer
membrane, releases periplasmic proteins. Heating at 90 ˚C
for 10 min can be used for releasing cytoplasmic proteins
Non-Mechanical Methods
Physical Rupture of Microbial Cells
Freeze-thawing: Rupture with ice crystals is commonly used
method. By slowly freezing and then thawing a cell paste,
the cell wall and membrane may be broken, releasing enzymes
into the media
Nebulization: In nebulization gas is blown over a surface of liquid
through a neck. Because of the differential flow within the neck,
the cells are sheared
Decompression: When pressurized, the microbial cells are gradually
penetrated and filled with gas. After saturation by the gas, the
applied pressure is suddenly released when the absorbed gas rapidly
expands within the cells leading to rupture
Note: Methods produce low protein yields and require long process time