Materials Related to Chp1
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Transcript of Materials Related to Chp1
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Todays meeting will cover: Bioseparation: what is it?
Overview of bioproducts: primary & secondary
metabolites, macromolecules, particulates
Introduction to Principles of Engineering Analysis in
Bioseparation
Materials related to Chp1
BTE4481 (Separation Processes II) Sem. 1, 2015-2016
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where the cell mass from the upstream are processed to meet purity and quality requirements. Unit operation in downstream processing is
usually divided into several stages such as cell /harvest/disruption, product isolation, purification and polishing.
Introduction to Bioseparation:
Bioproduct of interest
upstream processes
e.g. fermentation Cell isolation and cultivation, cell banking and culture expansion of the cells until final harvest; also concerns with inoculum development, media development, improvement of inoculum by genetic engineering process, optimization of growth kinetics.
downstream processes
purification or bioseparation
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BIOSEPARATION Processes:
Isolation, Separation, Purification & polishing of bioproducts
Plant tissues
Animal tissues
Immobilized
biocatalysts
Cells in bioreactor
bioproducts
BIOSEPARATION PROCESSES
extracted, purified and market ready bioproduct
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Bioseparation challenge: to remove process & product contaminants & impurities from desired bioproduct i.e. to efficiently & economically recover a high purity bioproduct from a complex mixture of
related & functional molecules, impurities & contaminants which may have similar physical &
chemical properties
Slide credit to: Professor Ian Marison, Head of School of Biotechnology, Dublin City University
IMPURITIES components required for production and/or purification, but found in purification step
host cells, host cell proteins, nucleic acids, lipids
separation components (extractants; salts; urea; antifoam; etc..)
degraded products (misfolded; aggregated)
CONTAMINANTS components which may enter production process
foreign cells (bacteria, viruses) endotoxin (bacteria lipo-polysaccharides) extractables from membranes/filters/resins cleaning chemicals
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Bioseparation: separation and purification of bioproducts A sequence of recovery and separation steps to: maximize the purity of the bioproduct minimize the processing time minimize yield losses minimize costs Continuous improvements required to remain competitive!
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BIOPRODUCTS
Produced by Living Organisms
enormous range of physical & chemical properties enormous range of product values & production levels
3 CATEGORIES OF SOURCES:
Whole cells: single-cell protein, bakers yeast and animal feed supplements derived from yeast fermentation
Intracellular macromolecules: protein in cytoplasm, protein in
inclusion bodies from recombinant bacterial fermentations,
starch in inclusion bodies found in plant cells and intracellular
proteins
Extracellular products: proteins, antibiotics, organic acids, and
alcohols secreted during microbial fermentations or cell culture
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Bioproduct Examples Molecular Weight
(Da)
Typical radius
Small molecules {can not be sedimented,
but can be separated by
extraction}
Sugars
Amino acids
Vitamins
Organic acids
200-600
60-200
300-600
30-300
0.5 nm
0.5 nm
1-2 nm
0.5 nm
Large molecules {highly adsorptive}
Proteins
Polysaccharides
Nucleic acids
103-106
104-107
103-1010
3-10 nm
4-20 nm
2-1,000 nm
Particles {can be collected by
sedimentation or filtration}
Ribosomes
Viruses
Bacteria
Organelles
Yeast cells
Animal Cells
25 nm
100 nm
1 m
1 m
4 m
10 m
Table 1.2 (Harrison et al, 2003)
Broad Categories of Bioproducts & their Sizes
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
(A) Sugars: monosaccharides, disaccharides & polysaccharides
as products in bioprocess, examples:
- mannitol to fructose (partial oxidation by acetic acid bacteria)
- glucose to fructose (glucose isomerase)
where fructose corn syrup: sweeteners in soft drinks
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
(B) Organic alcohols, acids, and ketone
- can be produced by the anaerobic fermentation of microbes
- Examples: ethanol, isopropanol, acetone, acetic acid, lactic
acid, propionic acid
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
(C) Vitamins: e.g. Vitamin C
(ascorbic acid) Most vitamins can be synthesized in organic chemical reaction (may have harmful impurities) Extraction from plants and fermentation produces vitamins naturally (may incur at higher cost )
example of a present-day market tension: natural vs synthetic
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
(D) Amino acids
* Building blocks of proteins
* Specific properties of amino acid side group (review)
Acidic or basic AAs: adsorption by ion exchange or separation by electrophoresis
Many aliphatic side chains: preferential adsorption onto or extraction into nonpolar media
Free SH (sulfhydryl) Histidine
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
(E) Lipids
any fat-soluble (hydrophobic), naturally-occurring molecules, that are insoluble in water but soluble in nonpolar organic solvents. - highly extractable into nonpolar solvents
Triglycerides Phospholipids Steroids Waxes
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PRIMARY Metabolites formed during the primary growth phase of the organism key metabolic intermediates in catabolism & anabolism
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Primary Metabolites
Commercial Significance
Citric acid
Glutamic acid
Lysine
Nucleotides
Phenylalanine
Polysaccharides
Vitamins
Lactic acid
Formic acid
Fructose
Acetic acid
Various uses in the food industry
Flavour enhancer
Feed supplement
Flavour enhancers
Precursor of aspartame, sweetener
Applications in the food industry
Enhanced oil recovery
Feed supplements
Biodegradable polymers
Fine chemicals
Food, fermentation
Food (vinegar) and fine chemicals
(Standbury et al, 2000 and Table 1.4)
Some primary products of microbial metabolism
and their commercial significant
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SECONDARY Metabolites not formed during the primary growth phase of the organism formed near the beginning of the stationary phase
not associated with primary growth cycle
generally more complex with multiple functional groups
Example:
- penicillin, produced by Penicillium species
- used as antibiotic
- 3 x 107 kg/yr ; US$120/kg ; US$4 billion/yr
Produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin.
-ketoglutarate + AcCoA homocitrate L--aminoadipic acid L-lysine + -lactam L-lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be avoided in penicillin production.
Penicillin core structure, where "R" is the variable group.
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MACROMOLECULE bioproducts Proteins Nucleic Acids Polysccharides
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PROTEIN bioproducts
includes enzymes, hormones, clotting factors, antibodies natural or by recombinant gene technology
Example: - erythropoietin (EPOGEN), 30000 g/mol - 2 kg/yr ; US$80 million/g ; US$2 billion/yr
Recombinant protein expression
to get more protein production & also improve protein purification by designing
fusion protein (inserting removable tags at C- or N-terminus of polypeptide)
His6x tag ; Glutathione S-transferase tag (GST); Maltose binding protein (MBP)
Sometimes recombinant proteins produced as improperly folded entities known
as inclusion bodies
Must be solubilized
Must allow for proper refolding
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Classification of Proteins according to prosthetic group
PROSTHETIC GROUPS IN PROTEINS & HYBRID MOLECULES
Prosthetic group: non-amino acid portion (organic & inorganic compounds)
of a conjugated protein
e.g. glycosylation: oligosaccharides conjugated post-translationally to specific
AAs (Ser, Asn, Thr)
help stability, catalytic function, binding specificity, solubility, targeting, & transport
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Classification of Proteins according to function
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Nucleic acids: RNA & DNA
polynucleotides: repeating units of nucleotides phosphodiester linkage between 3OH & 5OH of ribose or deoxyribose
DNA (DeoxyriboNucleic Acid) RNA (RiboNucleic Acid)
* DNA - chemically more robust than proteins * 2o structures are highly stable, being based on base-pairing hydrogen bond
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Oligo-or polynucleotides with commercial potentials
1. Antisense:
* bind to mRNA and inactivate it
* Antisense therapy: in vivo treatment of a genetic disease by blocking
translation of a protein with antisense DNA or RNA
2. Ribozyme:
* can react with RNA and catalyze its cleavage at sequence-specific site
* Ribozyme specific binding for cleavage of viral or bacterial genes
3. Aptamer:
Aptamers are nucleic acid species that have been evolutionary engineered
through in vitro selection or equivalently, SELEX (systematic evolution of
ligands by exponential enrichment) to bind with high affinity to various
molecular targets such as small molecules, proteins, nucleic acids, and even
cells, tissues and organisms.
Aptamers offer the utility for biotechnological and therapeutic applications as
they offer molecular recognition properties
In addition to their discriminate recognition, aptamers offer advantages over
antibodies as they can be engineered completely in a test tube
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POLYSACCHARIDES
Highest Mwt of all bioproducts in use
* Not necessarily linear like peptides & nucleic acids
* Numerous OH : possible branching via typical glycosidic linkage
* Most familiar polysaccharides: starch, glycogen, cellulose
Starch:
* Major storage polysaccharide in higher plants.
* Mixture of amylose (-1,4, 2025%) & amylopectin (-1,4 & -1,6 per 24-30 residues, 7580%) * Provides 80% of dietary calories in humans worldwide
Glycogen
* Major storage polysaccharide in animals.
* Long straight glucose chains (-1,4) & branched (-1,6) * More branched than starch
Cellulose (-1,4): most abundant in nature & linear unbranched structural homopolysaccharides
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* Starch and glycogen are stored fuels
* Bacterial polysaccharides: component of a number of vaccines
* Microbial polysaccharide: xanthan gum, dextran, alginate,
pullulan
* Cellulose, agarose, dextran are used in media for bioseparation
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PARTICULATES Subcellular particles, bacterial inclusion bodies, whole cells, insoluble
macromolecular aggregates
Generally purified by centrifuge
Some very small particles ultracentrifugation
SCP (single-cell protein): microbial cells grown for food or feed applications
(algae, bacteria, yeast, filamentous fungi)
Subcellular components Bacterial inclusion bodies (100-1,000 nm)
Ribosomes (25 nm)
Liposomes or natural vesicles (100 nm)
Natural hormone granules (200 nm)
Early bioseparation steps include flocculation, sedimentation, filtration
CELLS yeasts, bacteria, mammalian (RBC), polio virus cells
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Allahu akbar
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where the cell mass from the upstream are processed to meet purity and quality requirements. Unit operation in downstream processing is
usually divided into several stages such as cell /harvest/disruption, product isolation, purification and polishing.
Recall:
Introduction to Bioseparation
Bioproduct of interest
upstream processes
e.g. fermentation Cell isolation and cultivation, cell banking and culture expansion of the cells until final harvest; also concerns with inoculum development, media development, improvement of inoculum by genetic engineering process, optimization of growth kinetics.
downstream processes
purification or bioseparation
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Fermentation parameter factors affecting the DSP
properties of MOs (safety, classification, morphology, thermal stability, tendency to flocculate, size, cell wall rigidity, ....) influence the filterability, sedimentation & homogenisation performance
location of the product (intracellular, deposited in vacuoles or inclusion bodies or excreted into the broth - or biomass itself) will define the initial separation steps and purification strategy.
stability of the product defines the need & kind of pretreatment for inactivation or stabilisation.
product, by-products and impurities as well any additions to the broth (e.g. antifoam) may form an interfacial layer in extraction steps, give peaks in chromatography, block membranes in ultrafiltration and analytical equipment; also salts and trace elements often have to be removed prior to pharmaceutical use.
nutrient medium residues (pesticides, herbicides, etc.)
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Introduction to Bioseparations: Engineering Analysis
Stages of Downstream Processing
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SLECTED PHYSICAL BASIS FOR BIOSEPARATION
density: centrifugation, settling
size: membranes, filtration, size exclusion chromatography
charge: IEC, electrophoresis
solubility: precipitation, crystallization
affinity: affinity chromatography
liquid partitioning: extraction
solid partitioning: adsorption, chromatography
hydrophobicity: HIC
Other considerations:
thermal stability, diffusivities, reaction rate constants, separation
thermodynamics
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BASIC PRINCIPLES OF ENGINEERING ANALYSIS
Governing equations:
material balance, equilibria, flux (or transport phenomena)
Material balance
Accumulation = inflow outflow + amt produced amt consumed
Equilibria extraction process: partition coeff. K=y/x y: conc. of a separand in the extract phase x: conc. of the same separand in the raffinate (usually heavy) phase
adsorption process
equilibrium constant for chemical reaction: Keq=[C]/([A][B]) for A + B C CS: conc. in the adsorbent phase
C: conc. in the liquid phase (C: chemical species & S:adsorption site)
[CS]=Keq[C] linear adsorption equilibrium (valid at low conc.)
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BASIC PRINCIPLES OF ENGINEERING ANALYSIS Flux relationships (transport phenomena)
Flux = coefficient driving force flux: flowing per unit area per unit time
driving force: gradient down which units flow
coefficient: permeability or the inverse of a resistance (properties of medium)
Ohms law: Je= CE Je: current density
C: electrical conductivity
E: electrical potential gradient
Ficks first law for diffusive flux due to a concentration gradient dc/dx in one dimension
D: diffusion coefficient property of the medium In some cases, calculable from the Stokes-Einstein equation for spheres
JD= -D(dc/dx)
D = kT/6a (k: Boltzman constant, T: absolute temp., : viscosity, a: particle radius)
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PROCESS & PRODUCT QUALITY
Purity = Amount of product Amount of product + amount of total impurities
Fold purification = the ratio of the purity at any stage in the process to
the purity at the start of the purification process
Another measure of purity is
where units of biological activity are assayed by means of a
biological test, such as moles of substrate converted per second
per liter or fraction of bacterial cells killed
Specific activity= Units of biological activity
mass
Yield = Amount of product produced Amount of product in feed
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PURIFICATION TABLE example
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PURIFICATION TABLE example
https://novoprotein.wordpress.com/2012/09/29/the-purification-table/
Specific activity
(U/mg)
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PURIFICATION TABLE
https://novoprotein.wordpress.com/2012/09/29/the-purification-table/
Table 1 from previous slide:- Volume (ml) this refers to the measured total solution volume at the particular stage in the isolation.
Total protein (mg) the primary measurement is of protein concentration, i.e. mgml-1, which is obtained using a protein assay. Multiplying the protein concentration by the total volume gives the total protein (i.e. mg/ml x ml = mg).
Total activity (units) the activity, in units ml-1, is obtained from an activity assay. Multiplying the activity by the total volume gives the total activity (i.e. units/ml x ml = units).
Specific activity (units/mg) the specific activity is obtained by dividing the total activity by the total protein. Alternatively, the activity (units/ml) can be divided by the protein concentration (mg/ml), in which case the ml cancels out, leaving units/mg.
Purification (fold) Fold refers to the number of multiples of a starting value. In this case it refers to the increase in the specific activity, i.e. the purification is obtained by dividing the specific activity at any stage by the specific activity of the original homogenate. The purification per step can also be obtained by dividing the specific activity after that step by the specific activity of the material before that step.
Yield (%) the yield is based on the recovery of the activity after each step. The activity of the original homogenate is arbitrarily set at 100%. The yield (%) is calculated from the total activity (units) at each step divided by the total activity (units) in the homogenate, multiplied by 100. The yield can also be calculated on a per step basis by dividing the total activity after that step by the total activity before that step and multiplying by 100. The efficiency of a step is calculated as:- Purification (for that step) x % yield (for that step) /100
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CRITERIA USED FOR DEVELOPING AND
EVALUATING A BIOSEPARATION PROCESS
product purity cost of production as related to yield scalability reproducibility and ease of implementation robustness with respect to process stream variables
The integration of unit operations for the efficient synthesis
of bioseparation processes will be discussed in
Bioseparation Design
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CRITERIA USED FOR DEVELOPING AND
EVALUATING A BIOSEPARATION PROCESS
A therapeutic protein can be 99.99% pure but still unacceptable if any pyrogen* is present
Industrial enzyme: enzyme product with practically any impurities that do not inhibit the activity of the product or endanger the user are allowed
(*Pyrogen: any substance that produces a fever)
Recall,
BIOSEPARATIONS: a sequence of recovery and
separation steps maximize the purity of the bioproduct
minimizing the processing time, yield losses and costs
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PURITY REQUIREMENTS
depends critically on application/use
therapeutic proteins: regulated (in USA by FDA) - DNA levels < 100 picograms (1 x 10-10 g) per dose - virus < 1 per million doses - bacteria need to be undetectable on agar plate
citric acid: regulated by FDA if food grade - Cl- < 50 mg/kg - oxalic acid < 100 mg/kg - calcium < 200 mg/kg - water < 0.5%
Industrial Enzymes/chemicals gluco-amylase, subtilisin, cellulase (30 80% Pure)
Diagnostic Enzymes and Food Grade Chemicals (90 95% Pure)
Therapeutic biopharmaceuticals (>97% Pure)
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PURITY of BioProducts and DSP
Slide credit to: Professor Ian Marison, Head of
School of Biotechnology, Dublin City University
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Bioproducts Production Level and Price (Inverse r/ship betw. selling price & prod. level) (continuous improvements required to remain competitive!)
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Sale of bioproducts (e.g. biopharmaceuticals) must recover cost of development, production, marketing & clinical trials (for human therapeutics)
BIOPHARMACEUTICAL ROUTE TO MARKET
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Bioproducts: Sales & Future Production
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Documentation of Pharmaceutical Bioproducts
The official documentation of all compounds sold for medication must appear in the U.S. Pharmacopeia
For each substance the following data are given: structural formula empirical formula potency packaging and storage reference standards labelling chemical identification methods and assays for identity and purity
Validations of these properties- responsibility of the manufacturer
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Bioproduct Production Regulation
cGMP: current good manufacturing practices What are these?
Set of guidelines used for all areas of producing, purifying, packaging, etc. any bioproducts (e.g. drug & food) to ensure safety and quality of the product. Everything is controlled and documented
Materials release and use (logs), equipment operation and cleaning (SOPs, logs), batch production records (PBRs) Personnel are held individually responsible
Mandated by the US Food and Drug Act of 1976
FDA Code of Federal Regulations, Title 21, Parts 210&211 (21 CFR Parts 210 and 211)
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Slide adapted from Professor Ian Marison, Head of School of Biotechnology, Dublin City University
Integrated USP & DSP Design criteria
Concentration Productivity (volumetric, specific) Yield/ conversion Quality Purity Sequence Glycosylation Activity (in vitro, in vivo)
Design criteria: pharmaceutical product Order of importance Quality Concentration Productivity Yield/ Conversion High added value products
Design criteria: bulk product Order of importance Concentration Productivity Yield/ Conversion Quality Low added value products