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Cell Lysis

Lecture 4 Spring 2017


Lecture 4

Assignments:
1. HW #3:

2. Read:
Chapter 3
Class notes

Copyright Biological and Agricultural Engineering Department, Texas A&M University


Lecture 4. Objectives

Recognize that cell have to be lyzed for release of


intracellular product

Understand choices of lysis method depends on cell wall


structure/strength and product
Fermenter
Whole broth
Product is the cells Product inside the cells Product outside the cells

Cell Removal Liquid for Cell Removal Cell Removal Cells for
Liquid for Filtration Filtration
disposal Filtration disposal disposal
or recycle Microfiltration or recycle Microfiltration Microfiltration or recycle
Centrifugation Centrifugation Centrifugation

Concentrated cell Cell-free liquor


suspension
Product
Cell disruption Primary isolation
Homogenization Solvent extraction
Aqueous two-phase liquid extraction
Cell Homogenate Waste
Adsorption
Precipitation
Ultrafiltration
Cell debris removal
Cell debris for disposal Filtration
Microfiltration
Centrifugation
Product enrichment Waste
Chromatography
Final Isolation
Crystallization
Product Precipitation
Ultrafiltration
Drying
Introduction
Lysis braking cells and tissues to release intracellular
products

Used intra-cellular products produced in animal / plant /


bacterial cultures

Product isolation is a two step process: disruption of the cell


(cell lysis) followed by separation of the proteins from the
liquid material (filtration and/or sedimentation)

Cell type used for bioprocess dictates choice of disruption


method
Methods of cell lysis

Physical
Chemical
Mechanical
Cell Structure

Prokaryotic
Gram positive peptidoglycan and plasma membrane
Gram negative outer (lipopolysaccharide) membrane,
peptidoglycan layer, and plasma membrane

Gram negative
Eukaryotic Cells
1. Animal cells thin cell coat, plasma membrane, and membrane
enclosed organelles
2. Plant cells complex with strong cell wall

3. Yeast stronger cell wall than bacteria


Cell-wall structures

Gramnegative Grampositive Yeast


Mannan network
Glucan network

Membrane
Chemical Methods

1. Enzyme digestion (lysozyme) expensive but specific

Lysozyme hydrolyzes peptidoglycan of the cell wall

Cellulases used for plant cell wall

2. Solubilization

Detergents disrupting (solubilizing) cell membrane

Detergents: Cationic, anionic or nonionic molecules

Lipid dissolution solubilization of cell-wall lipids


using organic solvents: toluene, benzene, octanol
Physical Methods
Osmotic shock (physical force)

Cell swelling and bursting caused by osmotic flow of


water into the cell; useful for animal cells only.
Submerge cells in high solute concentration (20%
sucrose, 0.5 M NaCl, 20 % glycerol) to release water
and then switch to water

= RT (ci co ) the vant Hoff law

= osmotic (trans-membrane) pressure


c = concentration difference of solutes inside and outside
the cell
Where C is osmolarity = molarity of all solutes (all ions) in
water.
Mechanical Methods
1. Homogenization (Valve type)
Current industrial, pilot, or lab scale high pressure homogenizers are equipped with plunger-type
pumps and valves or nozzles made from abrasive resistant ceramics or hard gemstones .

Stability in the delivered pressure is achieved through an attenuation volume between the pump and
the valve or the use of two or more reciprocating plungers and an overlapping algorithm control.
In a typical valve setup a zirconium or tungsten carbide needle-seat valve or ball-seat valve is used,
with homogenization pressure being controlled by the force (F) exerted over the needle blocking
the fluid flow (Figure B-C).

Common high pressure homogenization valves: (B) ceramic needle and seat; (C) ceramic ball
and seat; (D) diamond, sapphire, or ruby nozzle;

The nozzle orifice is usually <0.35 mm and the specific gemstone depends on the maximum
pressure and required nozzle life-span, with diamond being the most resistant and expensive option.
In the nozzle setup, homogenization pressure is determined by the pump pressure and/or a
diversion to flow.
Kinetics of cell disruption 1st order reaction

= k (Rm R )
dR
dt
k -- 1st order rate const.
Rm -- max releasable protein
R -- amt. of soluble protein released at time t

After integration with limits of R = 0 at t = 0 and R = Rt at t = t



ln 1 =

Industry typically uses multiple passes (N) over time and replaces t with N

Eq. (3.3.2) is a semi-empirical modification of the previous equation


for Manton-Gaulin valve - type homogenizer:

ln (1 R ) = k N P a (3.3.2)

where,

R = (Rt/Rm) -- extent of disruption


k -- kinetic parameter which depends on the T
P -- homogenization pressure
a -- parameter depending on the cell species and culture conditions (a = 0.9-2.9)
N -- number of passes
1. Manton-Gaulin High Pressure
Homogenizer (valve-type)
Product

Feed

(A) Hand wheel for pressure control


(B) Spring-loaded valve rod
(C) Valve (homogenizer)
(D) Valve seat
(E) Impact ring
Protein release from yeast cells at 30oC as a function
of operating pressure

Pressure
(PSI
2. Jet-type microfluidics high-pressure
homogenizer (jet-type)
In the microfluidics system (Figure A), the flow stream is split in two or more channels
that are redirected over the same plane but in right angles and propelled into a single
flow stream. The pressure driving pump (up to 300 MPa) promotes a high speed at
crossover of the two flows which results in high shear, turbulence, and cavitation over
the single outbound flow stream.

Both (valve and jet) designs rely on the conversion of pressure to kinetic energy in a convergent flow
region, yielding a high - velocity liquid stream that ultimately undergoes an approximate 90
change in flow direction
Microfluidics High Pressure Homogenizer
Eq. (3.3.3) is a modification of eq. (3.3.2) describing shear and collision effect on cell breakage
in Microfluidics high-pressure homogenizer :

where,
ln(1 R) = k N b P a (3.3.3)
k & a -- parameters that depend on type of cells

b -- parameter values (0.3-0.9) depend on cell type (native vs. recombinant),


concentration, and spec. growth rate

4. Crushing in ball mill (glass and steel balls)

Eqns. (3.3.4) and (3.3.5) are the governing correlations for batch and
continuous operation

5. Ultrasonication (used on bench scale)


Uses cavitation (gas bubble nucleation, growth, and bubble collapse due to
resonant sound wave
Applied to less resistant cell walls (bacterial)
3. Bead/Ball Mills
Bead Mill (Dyno Mill - ECM type)

New Separation System of the Dyno-Mill ECM-AP


The new separation system of the DYNO-MILL ECM-AP shows a unique forced
circulation of the beads ( 1 ).
The circulation of the beads is caused by the newly developed invert
wall ( 2 ), which directs the beads into an axial flow over the screen ( 3 ).
Afterwards, the DYNO-DSE-Accelerator ( 4 ) directly conveys the
beads back to the invert wall .
Effect of homogenization on enzyme
activity
Note:
Particle size reduction w/ time
Release of fumarase (enzymes) activity increases with time
Another enzyme (alcohol dehydrogenase) is inactivated by shear with
time
Comments on Cell Disruption Methods
Enzymatic release:
Advantages
Gentle, little damage of proteins
Scalable

Disadvantages
Not effective against all organisms
Expensive reagents
Enzyme must be approved for usage (FDA)
Subsequent process steps are needed
Comments on Cell Disruption Methods
Solvents
Advantages
Effective to permeabilize cell membrane
Used often in cell free systems

Disadvantages
Solvent toxicity
Not effective for all organisms
Potential damage to proteins
EPA and OSHA considerations
Comments on Cell Disruption Methods
Osmotic Shock
Advantages
Relatively simple
No equipment involved
Operates at low T
Does not effect protein activity / structure

Disadvantages
Not effective for all organisms (good for animal and red blood cells)
Requires subsequent removal of salts
Comments on Cell Disruption Methods
Sonication
Advantages
Efficient 95% disruption)
Rapid (sec to min)
Effective for many organisms
Simple
Small volumes

Disadvantages
Large amts. of heat
No scalable
Use with low cell concentration
Comments on Cell Disruption Methods
Homogenization
Advantages
Good efficiency with multiple passes
Effective for variety of microorganisms
Tolerates moderate cell concentrations
Scalable (scale up parameters available)
Continuous
Large throughputs

Disadvantages
High pressures
Multiple passages
Possible containment problems: seal leaks
High viscosity fluid due to DNA release
Needs often valve replacement (due to valve erosion)
Comments on Cell Disruption Methods
Ball / Bead Mills
Advantages
High disruption efficiency
Effective for variety of microorganisms
Low pressures: easy containment
High cell concentrations (as high as 500 g/L)
Scalable (scale up parameters available)
Continuous and batch
Low maintenance: low P, few moving parts

Disadvantages
Attrition of beads
Possible product contamination
Example: Homogenization of
Chlorella sp.
Assessment of Chlorella Cell Disruption
100.00
90.00
80.00
Turbidity (%) 70.00
60.00
50.00 pH 6.8
40.00 pH 11.75
30.00
20.00
10.00
0.00
Initial samplePass 1 Pass 2 Pass 3 Pass 4 Pass 5

100.00
Cell count (%)

80.00
60.00
pH 6.8
40.00
pH 11.75
20.00
0.00
Initial samplePass 1 Pass 2 Pass 3 Pass 4 Pass 5
Original Pass 1 Pass 2
sample

Pass 5 Pass 4 Pass 3


Protein Extraction as Function of
Homogenization Time and pH

TSP: pH 6.8 vs 11.75


70.00
TSP (mg/g biomass)

60.00
50.00
40.00
30.00 pH 6.8
20.00 pH 11.75
10.00
0.00
Initial Pass 1 Pass 2 Pass 3 Pass 4 Pass 5
sample
Problem for HW # 3.
Find coefficients a and k for Chlorella sp. homogenized by a valve-type homogenizer.
Experimental data are given below. Assume that total soluble protein (TSP) released using
16,000 psi and N = 5 is the maximum extractable protein (Rm =2520 g/ml). Wet biomass
concentration in this experiment was 10% w/w.

P (psi) N TSP (g/ml) Volume (ml) R

16000 5 2520 59.5 1.0

3500 1 380.6 50 0.151

4000 1 521.7 50 0.207

5000 1 532.7 50 0.211

10000 1 871.0 50 0.346

11000 1 1272.0 50 0.505

17500 1 1553.7 50 0.616