ERT320 BIOSEPARATION ENGINEERING

PTT 302 DOWNSTREAM
PROCESSING TECHNOLOGY
SEMESTER 1 2013/2014
Lecture 1:
Introduction to Bioproducts and Bioseparation
Definitions
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Bioseparations
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A sequence of recovery and separation steps – maximize the purity
of the bioproduct while minimizing the processing time, yield losses
and costs
Bioproducts
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Chemical substances or combinations of chemical substances that are
made by living things
Can be broadly classified into three categories of sources:
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Whole cells: single-cell protein, baker’s yeast and animal feed supplements
derived from yeast fermentation
Intracellular macromolecules: 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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The choice of separation method depends on the
nature of the product – purity, yield and activity are
the goals
Bioproducts are sold for their chemical activity,
example:
 Methanol for solvent activity
 Ethanol for its neurological activity or as fuel
 Penicillin for its antibacterial activity
 Streptokinase (an enzyme) for its blood clot
dissolving activity
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The design of a large-scale purification process
requires three main considerations :
 Clearly
defining the final product objective (therapeutic for
human or animals, industrial enzymes)
 Characterizing the starting material (from bacteria, yeast,
mammalian cell, knows the physicochemical properties of
the product such as surface hydrophobicity, pI, stability etc.)
 Defining possible separation steps and constraints
regarding the protein product, operations and conditions to
be used
Rules of Thumb
In selection of separation sequence, five main heuristics or rules of thumb provide a
good basis for process selection which are as follows:
Rule 1: Choose separation processes based on different physical, chemical or
biochemical properties
Rule 2: Separate the most plentiful impurities first
Rule 3: Choose those processes that will exploit the differences in the physicochemical properties of the product and impurities in the most efficient
manner
Rule 4: Use a high-resolution step as soon as possible
Rule 5: Do the most harduous step last
Objectives and Typical Unit Operations of
the Four Stages in Bioseparations
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Individual recovery operations can be grouped into different categories, depending on
their general purpose.
The sequence of recovery operations that typically employed in practice is as follows:
 Separation of insolubles. Insoluble materials include whole cells, cell debris, pellets of
aggregated protein, and undissolved nutrients. Common operations for this purpose
are sedimentation, centrifugation, and filtration.
 Isolation and Concentration. Generally refers to the isolation of the desired product
from unrelated impurities. Significant concentration is achieved in the early stages, but
concentration accompanies purification as well. This category includes extraction,
ultrafiltration, precipitation, and ion exchange.
 Primary Purification. More selective than isolation; some purification steps can
distinguish between species having very similar chemical and physical properties.
Primary purification techniques include chromatography, affinity methods, and
fractional precipitation.
 Final Purification (Polishing). Necessitated by the extremely high purity required of
many bioproducts, particularly pharmaceuticals and therapeutics. After primary
purification the product is nearly pure but may not be in the proper form. Partially
pure solids may still contain discolored material or solvent. Crystallization and/or
drying are typically employed to achieve final purity.
Categories of Bioproducts and Their
Sizes
Bioproduct
Examples
Molecular Weight
(Da)
Typical radius
Small molecules
Sugars
Amino acids
Vitamins
Organic acids
200-600
60-200
300-600
30-300
0.5nm
0.5nm
1-2nm
0.5nm
Large molecules
Proteins
Polysaccharides
Nucleic acids
103-106
104-107
103-1010
3-10nm
4-20nm
2-1,000nm
Particles
Ribosomes
Viruses
Bacteria
Organelles
Yeast cells
Animal Cells
25nm
100nm
1µm
1µm
4µm
10µm
Characterization of Biomolecules
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Because biomolecules differ greatly in nature, different
separation principles are required for their recovery and
purification.
Their relative molecular masses vary generally,
biomolecules are rather unstable and their stability
depends on many different factors such as:
pH
 Temperature
 Ionic strength
 Type of solvent used
 Presence of surfactant
 Metal ions
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Small Biomolecules
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can be divided into two categories
primary metabolites and secondary metabolites
Primary Metabolites
formed during the primary growth phase of the
organism
Sugar – monosaccharides, disaccharides or
polysaccarides (Figure 1.1)
Organic alcohols, acids, and ketones – can be
produced by anaerobic fermentation of
microorganism ex. Ethanol, isopropanol, acetone,
acetic acid (Figure 1.2)
Vitamins – vitamin A (carotenoid), B, C (ascorbic
acid), D (hormone D, steroid)
Figure 1.1: Sucrose or table sugar is a disacharide composed
of the monosaccharides glucose and fructose
Small Biomolecules (cont’d)
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Amino acids – building blocks of proteins
specific properties of amino acid side groups can be
exploited in purification methods. A protein rich in
acidic @ basic amino acids on its surface can be
adsorbed by ion exchange or separated by
electrophoresis (Figure 1.3)
Lipids – natural fats consist of fatty acids, lipids,
steroids this family of bioproducts- highly extractable
into nonpolar solvents (Figure 1.4)
Figure 1.3: a-Amino acid structure showing
‘zwitterionic equilibrium at neutral pH.
Figure 1.4: Typical structures of three classes of compounds:
a fatty acid (stearic acid), a steroid (estradiol) and
prostaglandin (PGE2).
Some primary products of microbial metabolism and their
commercial significant (Standbury et al, 2000)
Primary Metabolites
Commercial Significance
Ethanol
Citric acid
Glutamic acid
Lysine
Nucleotides
Phenylalanine
Polysaccharides
‘Active ingredient’ in alcoholic beverages
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
Vitamins
Secondary Metabolites
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Not produced during the primary growth phase of a
microorganisms but at or near the beginning of the
stationary phase
Antibiotics – best known most extensively studied
Antibiotics: erythromycin, tetracycline and streptomycin
(Figure 1.5)
Figure 1.5:Structures of three well-known antibiotics: erythromycin, tetracycline,
and streptomycin. The choice of purification method is influenced by the profound
differences in the chemical structure of these compounds.
Macromolecules: Protein
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Primary structure (the covalent amino acid sequence) (Figure
1.6)
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established by the sequence of nucleotide codons of the
messenger RNA for that protein
amino acid- held together in strictly linear sequence and all
backbone bonds are covalent
no branched peptide, despite the existence of amino and
carboxyl side chains capable of forming amine linkages
Secondary structure (the hydrogen-bonded structures)
(Figure 1.7)
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two main forms – the formation of hydrogen bonds between
oxygen and nitrogen atoms in the peptide backbone
two structures that form are the α helix and the βsheet
(or‘pleated sheet’)
Amino acid sequence of bovine insulin.The two polypeptide chains are held
together by disulfide bridges, but they originated from single chain, a
portion of which was excised by the cell.
A peptide chain in an α-helix
configuration, showing hydrogen
bonding (dashed lines) that stabilizes
the structure
Representation of three
parallel peptide chains
in a β-sheet structure.
Figure 1.7: Secondary Structure
Protein (cont’d)
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Tertiary structure (the folding pattern of hydrogen-bonded
and disulfide-bonded structures) (Figure 1.8) the three
dimensional folding of coiled or pleated polypeptide chain
establishes the following: surface properties, catalytic
activity, stability, mechanical strength and shape
Quaternary structure (the formation of multimeric complexes
by individual protein molecules)
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the folded peptide chains interact with one another in the native
conformation of an oligomeric protein ex. A complete hemoglobin
molecule contains two α-globin and two β-globin peptide chains
maintained by intermolecular bonds, including ionic and covalent
linkages
Figure 1.8: Three-dimensional
structure of ribonuclease
determined by x-ray diffraction
analysis
Protein (cont’d)
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prosthethic group and hybrid molecules
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conjugated proteins- contain not only amino acids but other
organic and inorganic compounds
the non-amino acid portion of a conjugated protein- called
prosthetic group
conjugated proteins- classified on the basis of the chemical nature
of their prosthetic group
proteins containing lipids or sugar moieties – called hybrid
compounds
antibodies – hybrid molecules – become a major product of
biotechnology companies (Figure 1.9)
antibodies – globular proteins synthesized by B lymphocytes in
animals
Classification of proteins According to
Prosthetic Group
Figure 1.9: Structural
features of an antibody
(immunoglobulin G. or
lgG) Each pair of Nterminal residues
constitutes an antigen
binding site.
Classification of Proteins According to
Function
Classification of Proteins According to
Function
Commercial Uses of Proteins
Commercial Uses of Proteins
Application
Commercial Enzymes
Animal Feed: Enzyme supplementation
Bio-Feed Pro, Bio-Feed Wheat, BioFeed Beta
Baking: Dough Improvement
Fungamyl Super, Pentopan,
Lipopan™
Dairy: Milk protein modification
Alcalase®, Pancreatic Trypsin Novo
(PTN), Flavourzyme
Detergent: Removal of fatty stains
Lipolase®, Lipolase Ultra,
LipoPrime™
Starch: Starch liquefaction
Termamyl, BAN, Fungamyl
Textiles: Wool finishing
Esperase, Savinase
Stability of Protein
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Stability of protein – temperature, pH, shear
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Protein degrade via several pathways: deamination of
asparagine and glutamine, oxidation of methionine,
oligomerization, aggregation, denaturation (breakdown of
hydrogen bonds and ionic bonds)
Temperature
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as it is increased, denaturation of a protein will start to occur at
some point
Thermal denaturation for many proteins begins to occur at 45 to
500C
Refer to Fig. 1.10 & 1.11; when the temp is high enough, the hydrogen
bonds are broken (dotted line in fig. 1.10 and the b-pleated sheet
structure of the protein is disrupted)
Some proteins – still active at temp much higher than 45 to 500C ; ex.
Enzymes isolated from thermophilic bacteria inhabiting hot springs –
active at temp above 850C
Figure 1.10: Schematic drawing showing the conformational change that occurs when
ribonuclease is heated above its thermal denaturation temperature. Dotted lines indicate
hydrogen bonds, and numbers indicate the positions of the amino acid cysteine. which forms
disulfide bonds between chains.
Figure 1.11: Thermal denaturation of ribonuclease as measured by
∆A 287 , the change in absorbance at 287 nm compared with a
reference solution at zero absorbance.
Protein (cont’d)
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pH
often varied during the d/stream processing of proteins
 Fig 1.12 illustrates the phenomenon for four enzymes for
which effect of pH on the relative activity of the enzymes
has been determined
 In these cases the pH changes- leading to a change in the
ionization state and structure of the active site
 At extreme of pH, proteins are not only denatured but can
be completely hydrolysed to their constituent amino acids
 At extreme of pH, proteins are not only denatured but can
be completely hydrolysed to their constituent amino acids
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Figure 1.12: The effect of pH on the relative activity of the enzymes
trypsin, cholinestrase, pepsin, and papain
Protein (cont’d)
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Shear
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Not significant except when gas-liquid interfaces are present
Ex. In a rotating disk reactor that had a gas phase caused by incomplete
filling of the reactor
This loss of activity – attributed to the proteins being adsorbed to the
gas-liquid interfaces in an unfolded partially active and inactive state
If the turbulence in the solution – sufficiently high, the interface with
protein adsorbed breaks, causing unfolded protein to be inactivated
Therefore, it is important – gas-liquid interfaces be avoided/ minimized
Macromolecules: Nucleic acids and
Oligonucleotides
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Nucleic acids- polynucleotides whose primary structure consists of
repeating units of nucleotides (Figure 1.13)
A nucleotide consists of a nitrogeneous base, a ribose or deoxyribose
sugar and one or more phosphate groups
Ribonucleotide acid (RNA) – polynucleotide containing only ribose sugar
while deoxyribose (DNA) contains only deoxyriboses (DNA) (lacking a
hydroxyl group at the 2’ position)
The use of DNA in tiny quantities; large-scale purification – never been
necessary
However, a new family of nucleic acid products – currently arising
Commercial potential of oligo- and polynucleotides are antisense
sequences, ribozymes and aptamers (combinatorial products)
These three categories of nucleic acid products- targeted for the
therapeutics and diagnostics field
Figure 1.13: The basic structures present in nucleic acids:
(a) pyrimidine bases, (b) purine bases,
(c) the nucleotide adenosine monophospate (AMP),
(d) a section of a DNA
chain
Macromolecules: Polysaccharides
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Of all bioproducts in use; the highest MW and the longest
end-to-end polymer length (Figure 1.14)
Most familiar- starch, glycogen and cellulose
Most of them- extracted from plants, several are produced
microbially; ex. Dextran, alginate
Highly purified- seldom sold, sources and uses of selected
p/saccharides are listed in Table 1.6
Figure 1.14: Two polysaccharides. (a) Amylose, (b) Chitin
Some Polysaccharides, Their Sources and
Uses
Particulate Products
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The form of sub-cellular particles, bacterial inclusion
bodies, whole cells and insoluble macromolecular
aggregates
Purifying of suspended animal and plant cells- to
obtain pure populations for biochem. study and for
food/ feed applications, starting material for pure
bioproducts
Introduction to Bioseparations:
Engineering Analysis
a)
Stages of Downstream Processing
Introduction to Bioseparations:
Basic Engineering Analysis
1. Material balance:
Accumulation = inflow –outflow + amount produced –
amount consumed
2. Equilibria:
Introduction to Bioseparations:
Basic Engineering Analysis
2. Equilibria:
Example: in extraction process that had gone to
equilibrium:
Introduction to Bioseparations:
Basic Engineering Analysis
2. Equilibria:
Example: in adsorption process that had gone to equilibrium:
Introduction to Bioseparations:
Basic Engineering Analysis
2. Flux (Transport Phenomena):
Example: Fick’s Law:
Introduction to Bioseparations:
Basic Engineering Analysis
2. Flux (Transport Phenomena):
Example: Darcy’s Law:
Introduction to Bioseparations:
Engineering Analysis (cont’d)
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Criteria should be used in evaluating and developing 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