Biological products, Applications, types of bioseparation engineering,

1. ANISHA
INTEGRATED M.TECH
8th semester

2.  BIOLOGICAL PRODUCTS
 NATURE OF BIO SEPARATION
 CELL DISRUPTION & ITS TYPES
 PRECIPITATION METHODS
 MEMBRANE BASED BIO SEPARATION

3.  DEFINITION

4.  Types of Biologically derived products based on chemical nature
1. Solvents
2. Organics acids
3. Vitamins
4. Amino acids
5. Antibiotics
6. Sugars and carbohydrates
7. Lipids
8. Nucleic acids
9. Semi-purified proteins
10. Purified proteins
11. Cells
12. Crude cellular extracts
13. Hydrolysates
Ethanol, butamol. Acetone
Citric acid, lactic acid, butyric acid
Ascorbic acid, vitamin B12
Lysine, phenylalanine, glycine
Penicillins, rifanpicin, framycetin,
Glucose, fructose, starch, dextran, xanthan, gellan
Glycerol, fatty acids, corticosteroids, prostaglandins
Plasmids, therapeutic DNA,
Industrial enzymes, egg proteins, milk proteins,
Therapeutic enzymes, cytokines, hormones,
Bakers yeast, brewers yeast, freeze dried lactobacillus
Yeast extract, soy extracts, animal tissue extract
Soy hydrolysates, whey hydrolysates, animal tissue

5.  Types of biological products based on Applications
1. Industrial chemicals
2. Agrochemicals
3. Biopharmaceuticals
4. Food and food additives
5. Nutraceuticals
6. Diagnostic products
7. Commodity chemicals
8. Laboratory reagents
9. Cosmetic products
Solvents, organic acids, industrial enzymes
Biofertilizers, biopesticides
Antibiotics, hormones, monoclonal antibodies,
proteins, vaccines, hormones, cytokines,
Whey proteins, milk proteins, egg proteins, soy
proteins, protein hydrolysates
Vitamins, enzymes, coenzymes, cofactors,
amino
Glucose oxidase, peroxidase, HCG
Detergent enzymes, insecticides
Bovine serum albumin, ovalbumin, lysozyme
plant extracts, animal tissue extracts

6. 1. Separation on chemical processes.
2. Low molecular weight compound such as amino acid , vitamins are purified
by conventional processes. These are:
 liquid-liquid extraction,
 packed bed adsorption,
 evaporation
 drying with practically no necessary modificationss.
NATURE OF BIO SEPARATION

7. Bases of separtion would be.,
Density, size, porosity, shape , polarity, solubility, diffusivity, volatality,
charges, molecular weight, partition coeeficent,l ight absorption
DIFFERENCES BETWEEN BIOLOGICAL SEPARATION AND
SYNTHETIC SEPARTTION
I. Large volume of dilute streams are needed even for low
concentration compound separation.,ex : For monoclonal
antibodies in 0.1mg/ml in mammalian cell culture supernatants.
II. Bioseparation should be Selective in nature due to similiarty
between impurities and product of interest.
III. Potential to avoid extreme physiochemical conditions., pH,
temperature etc due to degradtion of biological products.
IV. Sometimes sub – ambient temperature required for heat labile.
V. Solutions are prepared inside bioprocess for inectables
therapeutics and endotoxin free compound

8. CELL DISRUPTION
 The plasma membrane of different oragnism can be destroyed or disrupted by
using solvents,detergents,osmoticshock etc.It can be physical or chemical.
 Disrupted cells can be gram negative, gram positive, mould cells,yeast
cells,mammalian cells,cultured plant cells and ground cells etc.
 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.
• Gram negative have multilayered envelops and thinner peptodoglycan.
• Yeasts which are unicellular have thick cell walls, typically 0.1 to
0.2 microns in thickness (such as glucans, mannans and chitins.)
 They are composed of phospholipids arranged in the form of a bi-layer with the
hydrophilic groups of the phospholipids molecules facing outside .
 The hydrophobic residues remain inside the cell membrane where they are
shielded from the aqueous environment present both within and outside
the cell.

10. PHYSICAL METHOD
 Disruption in bead mill
 Disruption using a
rotor-stator mill
 Disruption using French
press
 Disruption using
ultrasonic vibrations
 Disruption using
detergents
 Disruption using
enzymes e.g. lysozyme
 Disruption using
solvents
 Disruption using
osmotic shock
CHEMICAL METHOD

11. ULTRASOUND DISRUPTION
HAEMOLYSIS BY OSMOTICSHOCK
DISRUPTION
Diagrams showing cell disruption
method:

12. Detergent disruption French press disruption

13. BEAD MILL METHOD
 This device consists of a stationary
block with a tapered cavity 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.
 High rate shear generated in the space
between the rotor and the stator as
well as the turbulence thus generated
are responsible for cell disruption.
ROTOR-STATOR MILL

14. FRENCH PRESS METHOD
 It consist of cylinder fitted
with plunger connencted
to hydraulic press.
 Cells placed inside cylinder
and pressurized (10k to 50k
psi) using plunger ,
through an orfice
suspension cells emerges at
high velocity due to
primary shear stress cell
starts disrupting.
 Ultrasonic vibrations of
frequency greater than
18kHz are used to disrupt
cells.
 Vibrations create
cavitation i.e., formation of
bubbles and they reach
resonance size where they
collapse releasing
mechanical energy in form
of shock, and disrupts the
cell membrane.
ULTRASONIC VIBRATION

15. DETERGENT DISRUPTION
 They disrupts the cell
membranes and phosholipids
of mammalian cells mainly.
 Bacterial celle needs
conjunction with lysozyme to
disrupts by weaken its walls.
 Non ionic for bioprocessing
havinf least damage effect
such as tween series
detergents.
 Need for removal.
 Enzymes are used to
destroy the cell membrane.
 Such as lysozymes,
pectinase used for
disruption and breaking
the bonds between the
membranes.
 But they are costly.
 Need for removal after
action.
ENZYMES DISRUPTION

16. OSMOTIC SHOCK
 Semi premeable cell
membrane are transferred
into hypotonic solution
from isotonic that results
into rapid expansion of
cell & ruptures called
osmotic shock.
 Used to lyse mammalian
cells,remove periplasmic
substances by expelation.
 Acetone act on cell
membrane solubilizing
its phosholipids and
denature the protein.
 Toluene for fungalcells.
 Important to remove by
volatility.
ORGANIC SOLVENT

17.  Precipitation based bioseparation essentially involves selcective conversion of a specific
dissolved component of a complex mixture to an insoluble form using appropriate physical
or physicochemical means.
 The insoluble form which is obtained as a precipitate is sepatated from the dissolved
components by appropriate solid-liquid separation techniques such as centrifugation.
Biological macromolecules can be precipitated by:
1. Cooling
2. pH adjustment
3. Addition of solvents such as acetone and ethanol
4. Addition of anti-chaotropic salts such as ammonium
sulphate and sodium sulfate
5. Addition of chaotropic salts such as urea
and guanidine hydrochloride
6. Addition of biospecific reagents as in immunoprecipitation
PRECIPITATION

18. USING ORGANIC
SOLVENTS
• By reducing dielectric constant
USING ANTI-CHAOTOPIC
SALTS
• By decreasing solubility of proteins
PRECIPITATION METHODS
A.
B
.

19.  Solvent based precipitation method
reduced the dielctric constant of
medium in which they are present.
 ln {S/Sw}={[A/RT ][1/ew]- [1/e]}
 S= solubility of protein, Sw =
solubility of protein in water, A=
constant, e = dielectric constant of
medium, ew = dielectric constant of
water.
 Protein have lower solubiltiy in
medium[ethanol] than in water.
 lower concentration of organic
solvent are used in precipitation
processes at low temperature
 Ex; human plasma protein
purification by cohn fractionation.
 Reducing protein solubility by
increasing salt concentration results
in increase in protein protein
hydrophobic interaction.
 ln(S)=B – Ks Cs i.e., Cohnequation
 B= Constant, Ks= salting out
constant, Cs= salt concentration
 At low temp. 4degree celsius, &
constant depend on salt, pH and
protein solution.
 More protein precipiated out &
stability increases at low temp. by
synergetic rxn.
 Salt used = Ammonium salts, NaCl
 Can be dirct addition or saturated
 Ex; 30% -50% Ammoniumsalt cut.
A. B.

20. STAGES OF PRECIPITATION:
1. Mixing
2. Nucleation
3. Diffusion limited growth
4. Convection limited growth
• Formation of homogenous mixture and mix it .Time needed for mixiing can
be determined by: Tm= (l)(l)/4D ; l= avg eddy length, D= diffusivity.
• At supernaturation minute particles form called nucleation .
• Diffusion limited growth increases collision between particel hence increses
rate of fomation of bigger particles in microns.
• Mixing directly proportional to frequency of collision.
• Aging process takes place i.e., time given to precipitate & supernatant
formation( 6h-12hrs at 4 degree celsius)
Mechanism

21. Particle-liquid separation
Particle-solute separation
Solute-solvent separation
Solute-solute separation
MEMBRANE BASED SEPARATION

22. Membranes are divided into:
1. Symmetric- similar compostition and morphology
2. 2.Asymmetric- non-identical composition
Basically membranes are:
1. Flat sheet membrane
2. Tubular membrane
3. Hollow fibre membrane

23. 1. MICROFILTRATION
This method is used for separation of fine particles from
solutions. The transmembrane pressure ranges from 1 to 50 psig. Most
microfiltration membranes capture particles by surface filtration, i.e. on
the surface of the membrane. In some cases depth filtration is also used.
Uses: for clarification, sterilization and slurry concentration.
2. ULTRAFILTRATION
Membranes retain macromolecules such as proteins while allowing
smaller molecules to pass through.
(a) separate large molecules from solvents,
(b) separate large molecules from smaller molecules,
(c) separate large molecules from one another.
The primary separation mechanism is size exclusion, but
physicochemical interactions between the solutes and the membrane, and
operating conditions can influence the process quite significantly.
 Pressure ranges from 10 to 100 psig.
 Most UF membranes are asymmetric.

24. 3. NANOFILTRATION
 Allow salts and other small molecules To pass through but retain
larger molecules such as peptides, hormones and sugars.
 Pressure in NF ranges from 40 to 200 psig.
 MostNF membranes are composite i.e. asymmetric.
4.Liquid membrane processes
TRansport of solutes across a thin layer of a third liquid interposed
between two miscible liquids.
liquid membranes:
1. Emulsion liquid membranes (ELM)
2. Supported liquid membranes (SLM)

25. 5. MEMBRANE CHROMATOGRAPHY
 Adsorption and chromatographic separations are
traditionally carried out using packed beds.
 Separation mechanisms These include; 1.
Affinity binding 2. Ion-exchange interaction
3.Hydrophoboic

26.  Diagram showing dialysis
 Solute separation occurs
primarily because
smaller solutes partition
into the membrane
better than bigger
solutes because the
degree to which the
membrane restricts the
entry of solutes into it
increases with solute
size.
 Smaller solutes also
diffuse more rapidly than
larger ones.
6.DIALYSIS

27. REFERENCES:
 Research gate
 NCBI
 WIKIPEDIA
 BOOK- PRINCIPLES OF BIO- SEPARATION ENGINEERING BY
RAJA GHOSH
 BIOCHEMISTRY TECHNIQUES BOOK BY STRYER
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