BTE4481 (Separation Processes II) Sem.

1, 2015-2016

Materials related to Chp1

Todays meeting will cover:

Bioseparation: what is it?

Overview of bioproducts: primary & secondary
metabolites, macromolecules, particulates
Introduction to Principles of Engineering Analysis in
Bioseparation

Introduction to Bioseparation:

Bioproduct of interest

upstream processes
e.g. fermentation

downstream processes
purification or bioseparation

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.

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.

BIOSEPARATION Processes:
Isolation, Separation, Purification & polishing of bioproducts
extracted, purified
and market ready
bioproduct

Cells in bioreactor
BIOSEPARATION
PROCESSES

Plant tissues
Animal tissues
Immobilized
biocatalysts

bioproducts

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

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
Slide credit to: Professor Ian Marison, Head of School of Biotechnology, Dublin City University

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!

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

Broad Categories of Bioproducts & their Sizes

Table 1.2 (Harrison et al, 2003)

Bioproduct

Small molecules
{can not be sedimented,
but can be separated by
extraction}

Large molecules
{highly adsorptive}

Particles
{can be collected by
sedimentation or filtration}

Examples

Molecular Weight
(Da)

Typical radius

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

Proteins
Polysaccharides
Nucleic acids

103-106
104-107
103-1010

3-10 nm
4-20 nm
2-1,000 nm

Ribosomes
Viruses
Bacteria
Organelles
Yeast cells
Animal Cells

25 nm
100 nm
1 m
1 m
4 m
10 m

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

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

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

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

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

PRIMARY Metabolites
formed during the primary growth phase of the organism
key metabolic intermediates in catabolism & anabolism

Some primary products of microbial metabolism

and their commercial significant
Primary
Metabolites

Commercial Significance

Citric acid
Glutamic acid
Lysine
Nucleotides
Phenylalanine
Polysaccharides

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

Vitamins
Lactic acid
Formic acid
Fructose
Acetic acid

(Standbury et al, 2000 and Table 1.4)

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
Penicillin core structure, where
"R" is the variable group.
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.

MACROMOLECULE bioproducts
Proteins
Nucleic Acids
Polysccharides

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

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

Classification of Proteins according to prosthetic group

Classification of Proteins according to function

Nucleic acids: RNA & DNA

polynucleotides: repeating units of nucleotides
phosphodiester linkage between 3OH & 5OH of ribose or deoxyribose

DNA (DeoxyriboNucleic Acid)

* DNA - chemically more robust than
proteins
* 2o structures are highly stable, being
based on base-pairing hydrogen bond

RNA (RiboNucleic Acid)

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

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

* 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

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

Allahu akbar

Recall:
Introduction to Bioseparation

Bioproduct of interest

upstream processes
e.g. fermentation

downstream processes
purification or bioseparation

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.

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.

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.)

Introduction to Bioseparations: Engineering Analysis

Stages of Downstream Processing

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

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.)

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)

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
Specific activity=

Units of biological activity

mass

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
Yield =

Amount of product produced

Amount of product in feed

PURIFICATION TABLE example

PURIFICATION TABLE example

Specific
activity
(U/mg)

https://novoprotein.wordpress.com/2012/09/29/the-purification-table/

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
https://novoprotein.wordpress.com/2012/09/29/the-purification-table/

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

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

Recall,
BIOSEPARATIONS:
a sequence of recovery and
separation steps
maximize the purity of the
bioproduct
minimizing the processing
time, yield losses and costs
(*Pyrogen: any substance that produces a fever)

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)

PURITY of BioProducts and DSP

Slide credit to: Professor Ian Marison, Head of

School of Biotechnology, Dublin City University

Bioproducts Production Level and Price

(Inverse r/ship betw. selling price & prod. level)
(continuous improvements required to remain competitive!)

BIOPHARMACEUTICAL ROUTE TO MARKET

Sale of bioproducts (e.g. biopharmaceuticals) must recover

cost of development, production, marketing & clinical trials (for
human therapeutics)

Bioproducts: Sales & Future Production

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

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)

Integrated USP & DSP Design criteria

Slide adapted from Professor Ian Marison, Head of School of Biotechnology, Dublin City University

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