CELL DISRUPTION

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Introduction

u Cell disruption is an essential part of biotechnology and the downstream processes related to the manufacturing of biological products.

u It is necessary for the extraction and retrieval of the desired products, as cell disruption significantly enhances the recovery of biological products.

u It affects the physical properties of the cell slurry, thus indirectly influencing further downstream processes.

u Several types of cell disruption methods exist, as biological products may be extracellular, intracellular or periplasmic.

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Methods of microbial cell disruption

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u Cell disruption methods can be categorized into mechanical methods and non- mechanical methods.

u Different cells have different structures; hence they require different methods for disruption.

Ideal technology characterization

Maximum release of the product of

interest.

No mechanical or

thermal denaturation of the product during

disruption.

Minimal release of proteases which may degrade the product.

Minimal release of particulates or

soluble contaminants that may influence

downstream

processing.

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Contd. Ease of extraction from the cell debris.

Speed of the method.

Cost of the method.

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Susceptibility of the cells to disruption.

Product stability.

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Mechanical Methods

u Principle - cells are being subjected to high stress via pressure, abrasion with rapid agitation with beads, or ultrasound.

u Methods of disruption include cavitation, shearing, impingement, or combination of those.

u Intensive cooling of the suspension after the treatment is required in order to remove the heat generated by the dissipation of the mechanical energy.

u Some high-pressure methods can only be applied in laboratory scale, such as French press and Hughes press.

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Bead mills

u Consists of a jacketed grinding chamber with a rotating shaft, running in its centre.

u Agitators are fitted with the shaft, and provide kinetic energy to the small beads that are present in the chamber which makes the beads collide with each other.

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u Glass Ballotini or stainless steel balls are used, size range being selected for most effective release of the enzyme required.

u Increased number of beads increases the degree of disruption, due to the increased bead-to-bead interaction.

u However, it also affects the heating and power consumption.

Drawbacks

systems are required.

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The high temperature

rises with increase of

bead volume and so

additional cooling

2

Poor scale-up.

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High chance of

contamination.

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Ultrasound 15

u Ultrasonic disruption is caused by ultrasonic vibrators that produce a high frequency sound with a wave density of about 20 kHz/s.

u A transducer then converts the waves into mechanical oscillations through a titanium probe, which is immersed into the cell suspension.

u Very effective in small scale work.

Drawbacks

01 Upscaling is very poor.

02 Has high energy requirements.

03 High health and safety issues, due to noise.

04 It is not continuous.

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French press and high pressure homogeniser

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u In a French press, or high pressure homogenization, the cell suspension is drawn through a valve into a pump cylinder.

u Then it is forced under pressure of up to 1500 bar, through a narrow annular gap and discharge valve, where the pressure drops to atmospheric.

u Cell disruption is achieved due to the sudden drop in pressure upon the discharge, causing the cells to explode.

High pressure homogenizers

u Consists of a displacement pump which draws the cell suspension through a check valve into the pump cylinder.

u High pressures of upto 150 Mpa and flow rate of 10,000 L/hr.

u Main disruptive factor – pressure applied and pressure drop across the valve.

21M

anton - Gaulin hom

ogenizer

Non-mechanical physical methods1. Thermolysis

u Periplasmic proteins in G(-) bacteria are released when the cells are heated up to 50ºC.

u Cytoplasmic proteins can be released from E.coli within 10min at 90 ºC.

u Improved protein release has been obtained after short high temperature shocks, than when at longer temperature exposures at lower values.

u The results are highly unreliable, as the protein solubility changes with temperature fluctuations.

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2. Decompression

u The cell suspension is mixed with pressurized subcritical gas for a specified time, depending on the cell type.

u The gas enters the cell and expands on release, causing the cell to burst.

u Advantages - supercritical CO2 is able to extract off-flavours that are caused by lipid components.

u Gentle on the cells, resulting in large debris that are easier to remove in order to obtain the desired product.

u Disadvantages - low efficiency, high dependency on pressure release and time of contact between the cell suspension and the gas.

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3. Osmotic shock

u Caused by a sudden change in salt concentration.

u Cells are first exposed to either high or low salt concentration.

u Conditions are quickly changed to opposite conditions which leads to osmotic pressure and cell lysis.

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Disadvantages

01

Low efficiency.

02

Require enzymatic pre-treatment to weaken the cells.

03

Requires addition of high amounts of salts.

04

Water usage is high.

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Product may be diluted which increases downstream processing costs.

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Non-mechanical chemical methods1. Detergents

u Detergents damage the lipoproteins of the microbial cell membrane and lead to release of intracellular components.

u Detergents that are used for disrupting cells are divided into anionic, cationic and non-ionic detergents.

u Commonly used anionic detergent is sodium dodecyl sulfate (SDS) which reorganizes the cell membrane by disturbing protein-protein interactions.

u Triton X100, a non-ionic detergent solubilize membrane proteins.

u Cationic detergent, ethyl trimethyl ammonium bromide acts on cell membrane lipopolysaccharides and phospholipids.

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Cell disruption with detergents28

• Detergents interact with cell membrane compounds which will lead to disassembly of cell membrane.

DisadvantagesProteins will

be

denatured in

lysis process.

Detergents

may also

disturbsubsequent

downstream

processing

steps.

Additional

purification

step may berequired

after cell

lysis.

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2. Solvents

Solvents which can be used for cell lysis include

alcohols, dimethyl sulfoxide, methyl ethyl

ketone or toluene.

Extract cell wall’s lipid components

Leads to release of

intracellular components

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3.Alkali treatment

Used for hydrolysis of microbial cell wall material provided that the desired

enzyme will tolerate a pH of 10.5 to 12.5

for 20 to 30 minutes.

Disadvantages chemical costs for neutralization

of alkali are high.

Product may not be stable in alkali conditions.

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Non-mechanical enzymatic methods

u Use of digestive enzyme decomposes the microbial cell wall.

u Used enzyme depends on microbe.

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Lysozyme is commonly used

enzyme to digest cell wall of gram positive

bacteria.

It hydrolyzes β- 1-4-glucosidic bonds in the

peptidoglycan

Contd.

Enzymes commonly

used for degradation

of cell wall of yeast

and fungi include different cellulases,

pectinases, xylanases and chitinases.

The enzyme’s high

price and limited availability limits their

utilization in large scale processes.

In addition, the added enzyme may

complicate downstream

processing (e.g. purification).

Drawbacks could be

minimized by immobilization of

enzymes

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Summary

u Certain mechanical methods are only viable at a laboratory scale, due to their cost- effectiveness and scale-up difficulty.

u Mechanical methods are well suited for industrial scale, and are the most popular disruption methods in use.

u High energy requirements and high pressure requirements are disadvantages of mechanical methods.

u Many variables ranging from required apparatus to optimal materials affect the efficacy of mechanical methods.

u Methods like ultrasound may offer significant energy savings when compared to solid shear mechanical methods.

u The difficulty of sterilization and cleaning procedures makes mechanical methods susceptibleto contamination though.

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u Mechanical methods, like sonication, have severe health and safety issues, resulting fromnoise. u Mechanical and physical methods have specific condition requirements, including pressure

requirements and temperature requirements.

u These conditions need to be strictly monitored as they may affect protein release, protein solubility and cause undesirable effects in the products.

u Cwhitahntghienfgortm

ematpioenraotfucreryss, tuaslse.d in thermolysis, may cause cells to burst or may damage cells

u Temperature variation also affects the activity of enzymes and may alter three-dimensional structures.

u A major problem with physical methods is their high cost.

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u Chemical methods are risky to use for the disruption of sensitive cells, as the used solvents anddetergents can cause protein denaturation, damaging the final product.

u A significant issue is the removal and recovery of the chemical disrupter, making chemical methods highly applicable at a laboratory scale.

u Chemical methods also have low efficacy, making them more expensive and less useful asdisruption methods.

u The high consumption of solvents and water makes chemical methods environmentally unfriendly.

u Enzymatic methods are gentle, with fewer side effects, yet high costs make them impractical.

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References

u Cell disruption methods. Natalia Kakko, Nicoletta Ivanova and Anssi Rantasalo.

u Stanbury, P., Whitaker, A. & Hall, S. (2016) Principles of Fermentation Technology (Third Edition), Chapter 10.

u Abhilasha S. Mathuriya. (2009) Industrial biotechnology, Ane Books Pvt Ltd. Chapter 13.

u Elliott Goldberg. (1997). Handbook of downstream processing. Blackie Academic and Professional, London. Chapter 1.

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