CELL DISRUPTION

EXTRACTING TARGET PRODUCT

LOCATION OF BIOLOGICAL PRODUCTS

In bioprocesses by fermentation or cell culture a wide
varieties of cells containing a diverse range of products in
different locations……..

1
• Intracellular products occur in a soluble form in the cytoplasm or are
produced as inclusion bodies (fine particles deposited within the
cells).
• Examples of intracellular products include recombinant insulin and
recombinant growth factors.
• Certain biological products have to be extracted from tissues, an
example being porcine insulin which is obtained from pig pancreas.
CELL SUSPENSION
DO
CENTRIFUGATION
OR FILTRATION
EITHER OR BOTH
 TYPES OF CELLS
Cells
• Gram positive bacteria
• Gram negative bacteria
• Yeast
• Mould
• Cultured mammalian
• Cultured plant
• Ground tissue
e.g. Lactobacillus and Streptococcus

•Sub-micron from 0.5 to 2 microns () in size

•Have thick cell walls, 0.02-0.04 microns,
peptidoglycan + polysaccharide+ teichoic
acid
•Phospholipid cell membrane present
e.g. E. Coli, Pseudomonas and Salmonella Typhimuriam

•Sub-micron from 0.5 to 1 micron in size

•Cell capsule present
(LPS)
"camouflage" •Peptidoglycan layer is thin

(about 7.5 to 10 nm thick). •Periplasmic space present

•Mechanically less robust than gm+ bacteria
•Chemically more resistant than gm+ bacteria
 YEAST & MOULD CELLS
Yeast: 2-20 microns in size, spherical or ellipsoid

Yeasts are unicellular

Moulds (fungus): Bigger and filamentous are, unicellular

to multi-cellular

Very thick cell walls are present in both

Cell wall is mainly composed of polysaccharides such as

glucans, mannans and chitins

Plasma membranes are mainly made up of phospholipids

and lipoproteins
 Animal cells
•Animal cells do not have cell walls
•Animal cells are very fragile
•Cultured animal cells are several microns in size
•Spherical or ellipsoid

Plant cells
•Plant cells can be bigger
•Plant cells have thick and robust cell walls mainly
composed of cellulose
•Plant cells are difficult to disrupt
•Cultured plant cells are less robust than real plant cells
 EXTENT OF FORCES
FOR CELLS DISRUPTION

1 psi is approximately equal to 6895 N/m2.

12
13
 DRIVING FORCESS FOR
CELL DISRUPTION

Classified broadly into following types :

 Nonmechanical :
1) Physical methods
2) Chemical and enzymatic methods
3) Physicochemical methods
 Mechanical methods
1) Bead mill homogenizer
2) High pressure homogenizer
Cell Disruptions
Some Issues During Cell Breakage
(e.g. Polymyxins disrupt both the outer and inner
membranes of Gram-negative bacteria after binding to
LPS. Penicillin, Cephalosporins, Bacitracin, Vancomycin
are also used to kill the gram +/- bacteria.)
 Physicochemical Methods
(Combination of the following
Physical & Chemical Methods)
•Detergents
•Enzymes
•Organic solvents
•Osmotic shock
•Heat shock or Thermolysis
•Mechanical Cell Disruptions by
physical forces
 Also mannanase, chitinase for yeast/mould cell wall

 Cellulase and pectinase for plant cell walls

 Lysozymes damage bacterial
cell walls
• Lysozymes damage bacterial cell walls by
catalyzing hydrolysis of 1,4-beta-linkages
between N-acetylmuramic acid and N-acetyl-D-
glucosamine residues in a peptidoglycan and
between N-acetyl-D-glucosamine residues in
chitodextrins.
 THERMOLYSIS
• This is relatively an easy and economical process but
can be used only if the product is stable to heat
shock.

• Basically it inactivates the organisms by destabilizing

and then disrupting the cell wall without affecting the
products.

• The effect of heat shock depends on parameters such

as pH, ionic strength, presence of chelating or
sequestering agents such as EDTA which binds
magnesium thereby destabilizing the cell wall and
presence of proteolytic and other hydrolytic enzymes.
 Freeze-Thaw method
• The freeze-thaw method is commonly used to lyse
bacterial and mammalian cells.
• The technique involves freezing a cell suspension in
a dry ice/ethanol bath or freezer and then thawing
the material at room temperature or 370C.
• This method of lysis causes cells to swell and ultimately
break during thawing as ice crystals form during the
freezing process and then contract.
• Multiple cycles are necessary for efficient release
recombinant proteins located in the cytoplasm of
bacteria and are recommended for the lysis of
mammalian cells in some protocols.
The osmotic pressure Π (Pi) of a solution containing n moles of solute
particles in a solution of volume V is given by the van't Hoff equation:
Π = nRT / V= RTC where C=n/V (concentration)
 ULTRASONIC VIBRATIONS

Ultrasound
generator

Ultrasound tip

Cell suspension

•Application: Bacterial and fungal cells

•Mechanism: Cavitation followed by shock waves
•Frequency: 25 kHz
•Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10
minutes
•Used in conjunction with chemical methods
SDS, CTAB, Sodium taurocholate (natural),
CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) , CHAPSO (3-[(3-
cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate), Tween Series,
Deoxycholate, DHPC (1,2-diheptanoyl-sn-glycero-3-phosphocholine) & DOPC (1,2-dioleoyl-sn-
glycero-3-phosphocholine) (Native)
IN CASE OF MAMMALIAN CELLS
A non-ionic detergent such as saponin or steroid
glycoside digitonin or digitin, which binds b-
hydroxysterols and is capable of complexing
membrane cholesterol has been used to release
intracellular proteins by permeabilizing the plasma
membrane alone without affecting the organelle
membranes.
 Alkali Treatment
•This is a cheap and effective method but very harsh.

•Alkali acts on the cell wall in a number of ways including

saponification of the lipids.

•Treatment is carried out with pH 11-12 for about 20-30

mins.

•Proteases are inactivated by this treatment.

•Pyrogen free therapeutics enzymes e.g. l-asperginase.

MECHANICAL CELL DISRUPTION METHODS
This methods are used in the lab scale as
well as industrial scale operation.

In the laboratory, wet grinding with a

Ball Mill or a Waring Blender may be used and
particularly effective for animal cells and
tissues as well as mycelial organism.

In industrial scale, cell disruption is carried

out by using e.g.

(i) Bead Mill or (ii) High Pressure Homogenizer

 MECHANICAL CELL DISRUPTION METHODS
There are several type of homogenizers e.g.

 French press (pressure cell) and high-pressure homogenizers.

 Mostly used in Analytrical and Preparative purposes

 However the cell suspension is drawn through a check valve into a pump
cylinder.

 Then , it is forced under pressure (up to 1500 bar) through a very narrow
annulus (orifice) or discharge valve, over which the pressure drops to
atmospheric range.

Cell disruption is primary achieved by high liquid shear in the orifice

and the sudden pressure drop upon discharge causes explosion of the
cells.

1 bar = 100 kPa = 1,000,000 dynes per square centimeter

Homogenization
1. Hand-held piston/plunger device
animal cells are easily disrupted
2. High-pressure homogenizer
suitable for large scale operation
principles;
sample narrow orifice High pressure
cell crushing cell breaking
pumping

usually, increasing the pressure increase the amount and rate of product release

High-pressure homogenizer Mechanical homogenizer

 A Dounce homogenizer consists of a round glass pestle
that is manually driven into a glass tube.
Liquid based homogenization is the most widely used cell disruption technique for
small and large volumes and cultured cells. Cells are lysed by forcing the cell or
tissue suspension through a narrow space known as clearance space thereby
shearing the cell membranes.
Dounce homogenizer (Hand operated)
Dounce Homogenizer
 Energy used in grinding

 Compression forces are used to fracture friable or crystalline matters

 Combined impact and shearing forces are required for fibrous matters
 Shearing forces are used for fine grinding of softer matters
Product release from disrupted cells

Time
C  t
 1  exp    Single pass
C max  

N
C   t 
 1  exp     Multi pass
C max    
Product Released……..
Problem
Colloidal Meal
 ROTOR/STATOR MILL

A colloid mill or rotor--stator mill device consists of a stationary block with a tapered cavity called
the stator and a truncated cone shaped rotating object called the rotor.
Typical rotation speeds are in the 10,000 to 50,000 rpm range.
The cell suspension is fed into the tiny gap between the rotating rotor and the fixed stator.
The feed is drawn in due to the rotation and expelled through the outlet due to centrifugal action.
The high rate of shear generated in the space between the rotor and the stator as well as the
turbulence thus generated are responsible for cell disruption.
These mills are more commonly used for disruption of plant and animal tissues based
material and are operated in the multi-pass mode, i.e. the disrupted material is sent
back into the device for more complete disruption.
 French press
•Application: Small-scale recovery of
intracellular proteins and DNA from
bacterial and plant cells
•Primary mechanism: High shear rates
within the orifice
•Secondary mechanism: Impingement
•Operating pressure: 10,000 to 50,000 psig

•The device consists of a cylinder fitted with a plunger which is connected

to a hydraulic press.

•The cell suspension is placed within the cylinder and pressurized using the
plunger.

•The cylinder is provided with an orifice through which the suspension

emerges at very high velocity in the form of a fine jet.
Cell disruption using French press
The cell disruption takes place primarily due to the high shear rates and differential
pressure. The internal FRENCH Pressure Cell pressure increases as the pressure
developed by the Laboratory Press increases. The intracellular pressure increases as
well. As the sample is dispensed through the sample outlet tube, the external pressure
on the cell wall drops rapidly toward atmospheric pressure. The pressure within the cell
drops as well but not as quickly as the pressure external to the cell. This pressure
differential causes the cell wall membrane to burst, releasing the intra-cellular contents.
A French press is frequently provided with an impact plate, where the jet impinges
causing further cell disruption. Typical volumes handled by such devices range from a
few milliliters to a few hundred milliliters. Typical operating pressure ranges from 10,000
to 50,000 psi.
Advantages:
•— This technique results in more uniform and complete disruption
•— Easy to use
•— Cells do not require pre-treating.
 Bead Mill Disruption
It may be horizontal or vertical
consists of a grinding cylinder with a
central shaft fitted with a number of
impellers and driven by a motor.
The cylinder is partially filled with
wear resistance grinding elements or
beads made from glass, alumina,
titanium carbide, zirconium oxide or
zirconium silicate.
Horizontal bead mills have the
advantages over vertical type due to
its higher loading capacity of beads
of smaller size, uniform distribution of
beads for good grinding at lower Cell suspension is pumped and cell disruption occurs due to shear forces
speeds and lower energy input. produced between velocity gradients because of he rotary movement of the
cells and beads within the cylinder.
Collision between the cells and bids, grinding of cells between rolling
beads also contribute to the disruptive forces of the cell.
Disruption takes place due to the grinding action of the rolling beads and
the impact resulting from the cascading ones
 Bead mill

Cascading
beads
Rolling
beads Cells being
disrupted

•Bead milling can generate enormous amounts of heat

•Cryogenic bead milling : Liquid nitrogen or glycol cooled unit
•Application: Yeast, animal and plant tissue
•Small scale: Few kilograms of yeast cells per hour
•Large scale: Hundreds of kilograms per hour.
 Cell disruption using bead mill

Bead mill equipment consists of a tubular vessel made of metal or thick

glass within which the cell suspension is placed along with small metal or
glass beads. The tubular vessel is then rotated about its axis and as a result
of this the beads start rolling away from the direction of the vessel rotation.
At higher rotation speeds, some the beads move up along with the curved
wall of the vessel and then cascade back on the mass of beads and cells
below. The cell disruption takes place due to the grinding action of the
rolling beads as well as the impact resulting from the cascading beads.
 Cell disruption using bead mill
Bead milling can generate enormous amounts of heat. While
processing thermolabile material, the milling can be carried out at low
temperatures, i.e. by adding a little liquid nitrogen into the vessel. This
is referred to as cryogenic bead milling. An alternative approach is to
use glycol cooled equipment. A bead mill can be operated in a batch
mode or in a continuous mode and is commonly used for disrupting
yeast cells and for grinding animal tissue. Using a small scale unit
operated in a continuous mode, a few kilograms of yeast cells can be
disrupted per hour. Larger unit can handle hundreds of kilograms of
cells per hour.

Disadvantages:
Occasional problems with foaming and sample heating, especially
for larger samples.
Tough tissue samples such as skin or seeds are difficult to disrupt
unless the sample is very small or has been pre-chopped into small
pieces.
HIGH PRESSURE HOMOGENIZER
 CELL DISRUPTION IN A HIGH PRESSURE HOMOGENIZER

Dynamic pressure,

PS = ½( v2 )

It consists of a high pressure positive displacement pump coupled to an adjustable

discharge valve with a restricted orifice
The cell suspension is in stress and disrupted when pumped at high pressure
Normal and shear stress also present there due to passage of the fluid through the
narrow orifice and pressure rapidly reduces to almost atmospheric pressure as the cell
suspension passes out of the orifice respectively .
 Mortar and Pestle
It is a manual grinding method, most commonly used to
disrupt plant cells. In this method, tissue is frozen in liquid
nitrogen and then crushed using a mortar and pestle.
Because of tensile strength of the cellulose and other
polysaccharides comprising the cell wall, this method is
the fastest and most efficient way to access plant protein
and DNA.

Mortar and Pestle

The rate and degree of cell disruption depend on
several parameters :

1. The nature of microorganisms----its size, cell wall

composition and thickness and concentration of microbial
cells
2. Product location within the cells-----cytoplasm, periplasmic
region or organalle
3. Type of Homogenizer-- type of impeller, its agitation speed
specifically the tip speed of he impeller
4. Bead size, its density and loading
5. Residence time
6. Temperature
7. Type of valve and seat
8. Operating pressure
9. Number of passes of cell suspension through the
homogenizer
Sequential disruption of microbial cells and
differential release of intracellular product
INDUSTRIAL SEQUENTIAL DIASRUPTION