cell disruption chapter (1)

8/20/2019 Cell Disruption Chapter (1)

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Cell disruption

Introduction

Biological products synthesized by fermentation or cell culture are either intracellular

or extracellular. Intracellular products either 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. A large number of recombinant products form inclusion bodies in

order to accumulate in larger quantities within the cells. In order to obtain intracellular

products the cells first have to be disrupted to release these into a liquid medium

before further separation can be carried out. Certain biological products have to be

extracted from tissues, an example being porcine insulin which is obtained from pig

pancreas. In order to obtain such a tissue-derived substance, the source tissue first

needs to be homogenized or ground into a cellular suspension and the cells are then

subjected to cell disruption to release the product into a solution. In the manufacturing

process for intracellular products, the cells are usually first separated from the culture

liquid medium. This is done in order to reduce the amount of impurity: particularlysecreted extracellular substances and unutilized media components. In many cases the

cell suspensions are thickened or concentrated by microfiltration or centrifugation in

order to reduce the process volume.

CellsDifferent types of cell need to be disrupted in the bio-industry:

• Gram positive bacterial cells

• Gram negative bacterial cells

• Yeast cell

•

Mould cells• Cultured mammalian cells

• Cultured plant cells

• Ground tissue

Fig. 4.1 shows the barriers present in a gram positive bacteria. The main barrier is the

cell wall which is composed of peptidoglycan, teichoic acid and polysaccharides and

is about 0.02 to 0.04 microns thick.

The plasma or cell membrane which is made up of phospholipids and proteins isrelatively fragile. In certain cases polysaccharide capsules may be present outside the


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cell wall. The cell wall of gram positive bacteria is particularly susceptible to lysis by

the antibacterial enzyme lysozyme.

Fig. 4.2 shows the barriers present in a gram negative bacteria. Unlike gram positive

bacteria these do not have distinct cell walls but instead have multi-layered envelops.

The peptidoglycan layer is significantly thinner than in gram positive bacteria.

An external layer composed of lipopolysaccharides and proteins is usually present.

Another difference with gram positive bacteria is the presence of the periplasm layers

which are two liquid filled gaps, one between the plasma membrane and the

peptidoglycan layer and the other between the peptidoglycan layer and the external

lipopolysaccharides. Periplasmic layers also exits in gram positive bacteria but these

are significantly thinner than those in gram negative bacteria. The periplasm is

important in bioprocessing since a large number of proteins, particularly recombinant proteins are secreted into it. An elegant way to recover the periplasmic proteins is by

the use of osmotic shock. This technique is discussed at the end of the chapter.

Yeasts which are unicellular have thick cell walls, typically 0.1 to

0.2 microns in thickness. These are mainly composed of polysaccharides such as

glucans, mannans and chitins. The plasma membrane in a yeast cell is composed of

phospholipids and lipoproteins. Mould cells are largely similar to yeast cells in terms

of cell wall and plasma membrane composition but are multicellular and filamentous.

Mammalian cells do not possess the cell wall and are hence quite fragile i.e. easy to

disrupt. Plant cells on the other hand have very thick cell walls mainly composed of

cellulose and other polysaccharides.

Cell wall wherever present is the main barrier which needs to be disrupted to recoverintracellular products. A range of mechanical methods can be used to disrupt the cell

wall. Chemical methods when used for cell disruption are based on specific targeting

of key cell wall components. For instance, lysozyme is used to disrupt the cell wall of

gram positive bacteria since it degrades peptidoglycan which is a key cell wall

constituent. In gram negative bacteria, the peptidoglycan layer is less susceptible to

lysis by lysozyme since it is shielded by a layer composed of lipopolysaccharides and

proteins.

Cell membranes or plasma membranes are composed of phospholipids arranged in the

form of a bi-layer with the hydrophilic groups of the phospholipids molecules facing

outside (see Fig. 4.3). The hydrophobic residues remain inside the cell membranewhere they are shielded from the aqueous environment present both within and


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outside the cell. The plasma membrane can be easily destabilized by detergents, acid,

alkali and organic solvents. The plasma membrane is also quite fragile when

compared to the cell wall and can easily be disrupted using osmotic shock i.e. by

suddenly changing the osmotic pressure across the membrane. This can be achieved

simply by transferring the cell from isotonic medium to distilled water.

\

Cell disruption methods can be classified into two categories: physical methods and

chemical methods.

Physical methods

•

Disruption in bead mill

• Disruption using a rotor-stator mill

• Disruption using French press

• Disruption using ultrasonic vibrations

Chemical and physicochemical methods

• Disruption using detergents

• Disruption using enzymes e.g. lysozyme

• Disruption using solvents

• Disruption using osmotic shock

The physical methods are targeted more towards cell wall disruption while the

chemical and physicochemical methods are mainly used for destabilizing the cell

membrane.

I. Physical methods for cell disruption

1. Cell disruption using bead mill

Fig. 4.4 illustrates the principle of cell disruption using a bead mill. This 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


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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.

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.

Cell disruption primarily involves breaking the barriers around the cells followed by release

of soluble and particulate sub-cellular components into the external liquid medium. This is arandom process and hence incredibly hard to model. Empirical models are therefore moreoften used for cell disruption:


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Where

C = concentration of released product (kg/m3)

C max = maximum concentration of released material (kg/m3)

t = time (s)

θ = time constant (s)

The time constant θ depends on the processing conditions, equipment and the properties of the cells being disrupted.

For multiple passes, the following relation can be used:

Where N is the number of passes.

ExampleA batch of yeast cells was disrupted using ultrasonic vibrations to release an

intracellular product. The concentration of released product in the solution was

measured during the process (see table below):

Solution

2. Cell disruption using rotor-stator millFig. 4.6 shows the principle of cell disruption using a rotor-stator mill. This 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 thespace between the rotor and the stator as well as the turbulence thus generated are


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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. The

cell disruption caused within the rotor-stator mill can be described using the equations

discussed for a bead mill.

3. Cell disruption using French pressFig. 4.8 shows the working principle of a French press which is a device commonly

used for small-scale recovery of intracellular proteins and DNA from bacterial and

plant cells. 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. The cell disruption

takes place primarily due to the high shear rates influence by the cells within the

orifice. 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 millilitres to a few hundred millilitres. Typical operating pressureranges from 10,000 to 50,000 psig.


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4. Cell disruption using ultrasonic vibrationsUltrasonic vibrations (i.e. having frequency greater than 18 kHz) can be used to

disrupt cells. The cells are subjected to ultrasonic vibrations by introducing an

ultrasonic vibration emitting tip into the cell suspension (Fig. 4.9). Ultrasound

emitting tips of various sizes are available and these are selected based on the volume

of sample being processed. The ultrasonic vibration could be emitted continuously or

in the form of short pulses. A frequency of 25 kHz is commonly used for cell

disruption. The duration of ultrasound needed depends on the cell type, the sample

size and the cell concentration. These high frequency vibrations cause cavitations, i.e.

the formation of tiny bubbles within the liquid medium (see Fig. 4.10). When these bubbles reach resonance size, they collapse releasing mechanical energy in the form


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of shock waves equivalent to several thousand atmospheres of pressure. The shock

waves disrupts cells present in suspension. For bacterial cells such as E. coli, 30 to 60

seconds may be sufficient for small samples. For yeast cells, this duration could be

anything from 2 to 10 minutes. Fig. 4.11 shows a laboratory scale ultrasonic cell

disrupter.

Ultrasonic vibration is frequently used in conjunction with chemical cell disruption

methods. In such cases the barriers around the cells are first weakened by exposing

them to small amounts of enzymes or detergents. Using this approach, the amount of

energy needed for cell disruption is significantly reduced.

II. Chemical and physicochemical methods of cell disruption

1. Cell disruption using detergents


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Detergents disrupt the structure of cell membranes by solubilizing their phospholipids.

These chemicals are mainly used to rupture mammalian cells. For disrupting bacterial

cells, detergents have to be used in conjunction with lysozyme. With fungal cells (i.e.

yeast and mould) the cell walls have to be similarly weakened before detergents can

act. Detergents are classified into three categories: cationic, anionic and non-ionic.

Non-ionic detergents are preferred in bioprocessing since they cause the least amountof damage to sensitive biological molecules such as proteins and DNA. Commonly

used non-ionic detergents include the Triton-X series and the Tween series. However,

it must be noted that a large number of proteins denature or precipitate in presence of

detergents. Also, the detergent needs to be subsequently removed from the product

and this usually involves an additional purification/polishing step in the process.

Hence the use of detergents is avoided where possible.

2. Cell disruption using enzymesLysozyme (an egg based enzyme) lyses bacterial cell walls, mainly those of the gram

positive type. Lysozyme on its own cannot disrupt bacterial cells since it does not lyse

the cell membrane. The combination of lysozyme and a detergent is frequently usedsince this takes care of both the barriers. Lysozyme is also used in combination with

osmotic shock or mechanical cell disruption methods. The main limitation of using

lysozyme is its high cost. Other problems include the need for removing lysozyme

from the product and the presence of other enzymes such as proteases in lysozyme

samples.

3. Cell disruption using organic solventsOrganic solvents like acetone mainly act on the cell membrane by solubilizing its

phospholipids and by denaturing its proteins. Some solvents like toluene are known to

disrupt fungal cell walls. The limitations of using organic solvents are similar to those

with detergents, i.e. the need to remove these from products and the denaturation of

proteins. However, organic solvents on account of their volatility are easier to remove

than detergents.

4. Cell disruption by osmotic shockAs discussed early in this chapter, osmotic pressure results from a difference in solute

concentration across a semi permeable membrane. Cell membranes are semi

permeable and suddenly transferring a cell from an isotonic medium to distilled water

(which is hypotonic) would result is a rapid influx of water into the cell. This would

then result in the rapid expansion in cell volume followed by its rupture, e.g. if red

blood cells are suddenly introduced into water, these hemolyse, i.e. disrupt therebyreleasing hemoglobin. Osmotic shock is mainly used to lyse mammalian cells. With

bacterial and fungal cells, the cell walls need to be weakened before the application of

an osmotic shock.

cell disruption chapter (1)

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