CHAPTER 11: INTRO TO BIOSEPARATION ENGINEERINGportal.unimap.edu.my/portal/page/portal30/Lecturer...
Transcript of CHAPTER 11: INTRO TO BIOSEPARATION ENGINEERINGportal.unimap.edu.my/portal/page/portal30/Lecturer...
CHAPTER 11:
INTRO TO BIOSEPARATION
ENGINEERING
Sem 2 2011/2012 ERT 313 BIOSEPARATION ENGINEERING
MOHAMAD FAHRURRAZI TOMPANG
Course details
Credit hours/Units : 4
Contact hours : 3 hr (L), 3 hr (P) and 1 hr (T) per week
Evaluations
Final Exam – 50%
Midterm Tests – 20%
Course works – 30%
Laboratories – 10%
Assignments – 10%
PBL – 10%
CARRY MARKS – 50%
Course details
Course Outcome (COs) will be covered:
CO3 – Ability to apply principles, analyze mechanical-
physical separation process and develop design of
membrane unit (C4, P3, A3)
Course works (Overall evaluations)
Assignments – 2 (Uploaded in portal)
Quizzes – Friday, 18 May 2012
Midterm test – Monday, 21 May 2012
Class participations – Max. of 3 points
Important reminder
Attendance should not less than 80%, or else you will be barred from taking final examination.
Plagiarism and copying other students’ work is strictly prohibited especially in doing assignments and lab reports, or else both parties will get zero.
Cheating in quizzes and examinations is also prohibited, or else both parties will get zero.
Therefore, study hard and smart. Take note of the important chapters or things that will be highlighted throughout lectures.
BIOPROD UCT?
Chemical substances / combination of chemical substances
that are made by LIVING THING range from methanol to
whole cells.
Derived by EXTRACTION from whole plants and animals
By synthesis in bioreactors containing cells / enzymes
Biological products - chemical classification
Solvents, e.g. ethanol, acetone, butanol
Cells, e.g. bakers yeast, brewers yeast, freeze dried lactobacillus
Crude cellular extracts, e.g. yeast extract, soy extracts
Organics acids, e.g. citric acid, lactic acid, butyric acid
Vitamins, e.g. ascorbic acid, vitamin B12
Amino acids e.g. lysine, phenylalanine, glycine
Gums and polymers, e.g. xanthan, gellan, dextran
Antibiotics, e.g. penicillins, rifanpicin, streptomycin
Proteins, e.g. industrial enzymes, egg proteins, milk proteins, whey protein
therapeutic enzymes, monoclonal antibodies, plasma proteins
Sugars and carbohydrates, e.g. glucose, fructose, starch, dextran
Lipids, e.g. glycerol, fatty acids, steroids
Nucleic acids, e.g. plasmids, therapeutic DNA
BIOLOGICAL PRODUCTS (with
different classification)
Biological products - applications
Industrial chemicals, e.g. solvents, organic acids, industrial enzymes
Agrochemicals, e.g. biofertilizers, biopesticides
Pharmaceuticals, e.g. antibiotics, hormones, monoclonal antibodies,
plasma proteins, vaccines
Food and food additives, e.g. whey proteins, milk proteins, egg proteins,
soy proteins
Nutraceuticals, e.g. vitamins, amino acids, purified whey proteins
Diagnostic products, e.g. glucose oxidase, peroxidase
Commodity chemicals, e.g. detergent enzymes, insecticides
Laboratory reagents, e.g. bovine serum albumin, ovalbumin, lysozyme
Cosmetic products, e.g. plant extracts, animal tissue extracts
BIOLOGICAL PROD UCTS (with different
bioseparation process)
Product Nature of bioseparation required
Alcoholic beverages:
Beer, wine, spirits
Clarification, distillation
Organic acids:
Acetic acid, citric acid
Precipitation, filtration, adsorption,
solvent extraction
Vitamins:
Vitamin C, vitamin B12, riboflavin
Precipitation, filtration, adsorption,
solvent extraction
Amino acids:
Lysine, glycine, phenylalanine
Precipitation, filtration, adsorption,
solvent extraction
Antibiotics:
Penicillins, neomycin, bacitracin
Precipitation, filtration, adsorption,
solvent extraction
Carbohydrates:
Starch, sugars, dextrans
Precipitation, filtration, adsorption
Lipids:
Glycerol, fats, fatty acids
Precipitation, filtration, adsorption,
solvent extraction
Proteins:
Food and food additives
Nutraceuticals
Industrial enzymes
Hormones
Pharmaceutical enzymes
Plasma derived products
Monoclonal antibodies
Growth factors
Clotting factors
Thrombolytics
r-DNA derived proteins
Diagnostic proteins
Vaccines
Filtration, precipitation, centrifugation,
adsorption, chromatography, membrane
based separations
DNA based products:
DNA probes, plasmids, nucleotides,
oligonucleotides
Filtration, precipitation, centrifugation,
adsorption, chromatography, membrane
based separations
OVERVIEW OF BIOPROCESSING OF BIOCHEMICAL &
PHARMACE UTICAL PRODUCTS
One of the major segments within biotechnology where
R&D is bioprocessing which deals with the manufacture of
biochemicals, food, neutraceuticals and agrochemicals.
New biologically derived product have been developed,
approved and licensed.
Eg: Monoclonal antibodies (used for treatment of canser)
OVERVIEW OF BIOPROCESSING OF
BIOCHEMICAL & PHARMACE UTICAL PROD
UCTS
all biochemicals & pharmaceutical
product MUST be extensively P U RIFIED
before used in respective application.
Bioprocessing / downstream processing of
biochemical & pharmaceuticals products refer
to the SYSTEMATIC study of the scientific
and engineering principle utilized for the large
scale purification of biological products.
OVERVIEW OF BIOPROCESSING OF
BIOCHEMICAL & PHARMACEUTICAL
PRODUCTS
2 categories of bioprocessing :
i) reactive bioprocessing
-bio-separation process
follows some form of
biological reactions
ii)extractive bioprocessing
-almost entirely involves
bioseparation
REACTIVE BIOPROCESSING
Bioseparation follow some form of
BIOLOGICAL REACTION
Eg: Antibiotic production (separation &
purification following microbial fermentation)
Upstream
processing
Biological
Reaction Bioseparation
Biological
products
Biocatalyst
Screening
Formulation
Media
optimization
Fermentation
Cell culture
Enzymatic reaction
EXTRACTIVE BIOPROCESSING
Almost entirely involved bioseparation
(With extractive bioseparation, upstream
processing involves raw material acquisition &
pre-treatment)
Eg: Manufacture of plasma proteins from
blood Upstream
processing Bioseparation
Biological
products
Synthesis in
VIVO in their
respective
natural source
Bioseparations engineering
Definition: Recovery, isolation, purification
and polishing of products synthesized by
biotechnological processes
Extended definition: Final polishing steps of
processes such as biotechnology based effluent
treatment and water purification
Upstream
processing Bioreaction
Downstream
processing
Bioproduct/s
Impurities
Why do we need bioseparation?
Enrichment of target product
Reduction in bulk
Removal of specific impurities
Enhancement of product stability
Achievement of product specifications
Prevention of product degradation
Prevention of catalysis other than the type desired
Prevention of catalyst poisoning
Challenges in Bioseparations Engineering
•Low product concentration
•Large number of impurities,
•Thermolabile bioproducts.
•Narrow operating pH and ionic strength window
•Shear sensitivity of bioproducts
•Low solubility of bioproducts in organic solvents
•Instability of bioproducts in organic solvents
•Stringent quality requirements •Percentage purity
•Absence of specific impurities
An ideal bioseparation process should combine high throughput
with high selectivity, and should ensure stability of product.
A Good Bioseparation Process:
Ensures desired purity of product
Ensures stability of product
Keeps cost low
Is reproducible
Is scalable
Meets regulatory guidelines
Economic Importance of Bioseparation
Engineering
The purification of biological product from their
respective starting material. Eg: cell culture media
: technically difficult and expensive
The critical limiting factor in the
commercialization of biological product
Many cases, bioseparation cost can be a
substantial component of the total cost of
bioprocessing
ERT 313/4 BIOSEPARATION ENGINEERING
SEM 2 (2010/2011)
Economic importance of bioseparation
engineering (cost of bioseparation)
Product Bioseparation
cost (%)
Solvents e.g. ethanol, acetone 15-20
Cells, e.g. bakers yeast, brewers yeast 20-25
Crude cellular extracts, e.g. yeast extract 20-25
Organics acids, e.g. citric acid, lactic acid 30-40
Vitamins and amino acids e.g. lysine, ascorbic acid 30-40
Gums and polymers, e.g. xanthan, gellan 40-50
Antibiotics, e.g. penicillins, rifanpicin 20-60
Industrial enzymes, e.g. Amyloglucosidase, glucose isomerase 40-65
Non-recombinant therapeutic proteins, e.g. pancreatin, papain 50-70
r-DNA products, e.g. recombinant insulin, recombinant streptokinase 60-80
Monoclonal antibodies 50-70
Nucleic acid based products 60-80
Plasma proteins, human albumin, human immunoglobulins 70-80
Strategies for Bioseparation
A large number of bioseparation methods are available
The strategy is based on how best these can be utilized for a given separation
The following need to be taken into account:
The volume of process stream
The relative abundance of the product in this process stream
The intended use of the product, i.e. purity requirements
The cost of the product
Stability requirements
Conventional strategy:
The RIPP scheme
Recovery, isolation, purification and polishing
Based on a logical arrangement of bioseparation
methods
Low-resolution (less selectivity), high-throughput
(product) techniques (e.g. precipitation, filtration,
centrifugation, crystallization) are first used for recovery
and isolation
High-resolution techniques (e.g. adsorption,
chromatography, electrophoresis) are then used for
purification and polishing
Conventional strategy:
The RIPP scheme
Biotechnological processes usually yield products at very low
concentrations in the product stream.
The product of interest also needs to be separated from a large
number of impurities, some of which have physical and
chemical properties not too different from the product.
If such a product stream is sent directly to a high-resolution
separation device, it will soon be overwhelmed and fail to
function properly.
Therefore it makes sense to use high throughput-low resolution
techniques first to significantly reduce the
volume/concentration of process stream.
Conventional strategy:
The RIPP scheme These high throughput-low resolution steps are
referred to as recovery and isolation steps.
The processed product stream is then further
processed by high resolution-low throughput steps to
get pure finished products.
With the advent of membrane separation processes it
is now possible to replace the conventional RIPP
scheme. Membrane processes give high throughput
and can be fine-tuned/optimized to give very high
resolution. The use of membrane technology reduces
the number of bioseparation steps and hence
contributes towards high product recovery.
The RIPP Scheme
• Multi-technique separation
• Process design should take into consideration the
following:
• The nature of starting material
• The initial location of the target product
• The volume of process stream
• The relative abundance of the product in the starting
material
• The susceptibility to degradation of the product
• The desired physical form of the final product
• The quality requirements
• Costing
Commonly Used Bioseparation Processes
Low resolution-high throughput Cell disruption
Precipitation
Centrifugation
Liquid-liquid extraction
Leaching
Filtration
Supercritical fluid extraction
Microfiltration
Dialysis
High resolution-low throughput Ultracentrifugation
Adsorption
Packed bed chromatography
Affinity separation
Electrophoresis
High resolution-high throughput
•Ultrafiltration
•Fluidized bed chromatography
•Membrane chromatography
Objective & Typical Unit Operations of
The 4 Stages in Bioseparation
Stage Objectives Typical unit
operation
Recovery
(separation of
insoluble)
Remove/ collect cells, cells debris /other
particulate.
Reduce volume
Filtration,
sedimentation,
extraction,
adsorption
Isolation of
product
Remove material have properties widely
different from those desired in product.
Reduce volume
Extraction,
adsorption,
ultrafiltration,
precipitation
Purification Remove remaining impurities which
typically similar to desired product in
chemical functionality & physical
properties.
Chromatography,
affinity method,
functional
precipitation
Polishing Remove liquid.
Convert product to crystallized form
Drying,
crystallization
• Largely based on chemical separation
techniques
• Chemical separation techniques are modified
based on specific requirements
• Novel separations may be necessary in some
cases
• High throughput/productivity
• High selectivity
• Need to satisfy stringent quality requirements
• Need to take into account degradable material
• Low temperature operations
• Multi-technique separation
Nature of Bioseparation
• Dilute biological products
• Impurities & by-product also present
• Stringent require for product (quality
requirement)
• Susceptible to denaturation &
degradation
• Thermo bile
• Multi technique separation
Different Attributes of Bioseparation
Basis of Separation
Biological products are separated based on one or several of
the following in combination:
• Size, e.g. filtration, membrane separation, gel-
filtration, centrifugation
• Density, e.g. centrifugation, sedimentation, flotation
• Diffusivity, e.g. membrane separation, supercritical
fluid extraction
• Shape, e.g. centrifugation, filtration, sedimentation
• Polarity, e.g. extraction, chromatography, adsorption
• Solubility, e.g. extraction, membrane separation,
precipitation, crystallization
• Electrostatic charge, e.g. adsorption, membrane
separation
• Mobility, e.g. electrophoresis, membrane separation
• Volatility e.g. distillation, membrane distillation,
pervaporation
Cell culture
supernatant Microfiltration
Ammonium
sulfate
Precipitation Filtrate
Precipitate
Solution
Buffer
Ultrafiltration Buffer
Ion-exchange
chromatography Buffer
Ultrafiltration Gel
filtration
Monoclonal
antibody
Monoclonal antibody
purification
1. What is the product?
2. What is the value of product?
3. What is the acceptable product quality for the
proposed end use?
4. Where is the product in the complex mixture?
5. What are the physical and chemical properties
of the product and the impurities?
6. Is the product stable?
7. What are the economic of the various isolation
procedure?
8. Are they any contamination / health risk?
9. Can the isolation procedure be scaled up?
HOW TO CHOOSE
SEPARATION METHOD
1. Keep the number of step to a
minimum.
2. Select the component that is easiest
to remove first
3. Leave the most difficulty isolation
step for last
RULES OF THUMB FOR
BIOSEPARATIONS
Replacement of the conventional RIPP scheme
by using new techniques which can
significantly cut down the number of steps
needed to bioseparation
Some of these new and emerging techniques
are:
• Membrane chromatography
• Expanded-bed chromatography
• High-resolution ultrafiltration
Current Paradigm in The Bioseparation