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Biosepartaion engineering
Bioproducts: chemical substances or combination of chemical substances that are made by living
things from methanol to whole cells
Must be extracted and purified to some degree before being suitable for market
Choice of separation method: the nature of the product
Removal of solids
• Filtration, Centrifugation, Sedimentation
Purification
• Chromatography. electrophoresis, precipitation
• Chromatography over half of the purification costs of biopharmaceuticals
Polishing
• Crystallization, Drying
Erythropoietin or EPO is a glycoprotein hormone that controls red blood cell production. It is a cytokine for
erythrocyte (red blood cell) precursors in the bone marrow. It is produced by the kidney in response to low oxygen
pressure in the blood. (Amgen & Genentech $800,000/g)
Interferons (IFNs) are natural cell-signaling proteins produced by the cells of the immune system of most
vertebrates in response to challenges such as viruses, parasites and tumor cells. Interferons assist the immune
response by inhibiting viral replication within host cells, activating natural killer cells and macrophages, increasing
antigen presentation to lymphocytes, and inducing the resistance of host cells to viral infection. The protein
interferon, produced by animal cells when they are invaded by viruses, is released into the bloodstream or
intercellular fluid to induce healthy cells to manufacture an enzyme that counters the infection. Interferon is
therefore considered a potential medical resource as a BIOPHARMACEUTICAL. For many years the supply of
human interferon for research was limited by costly extraction techniques. In 1980, however, the protein became
available in greater quantities through GENETIC ENGINEERING. -interferon has been approved for therapeutic
use against hairy-cell LEUKEMIA and Hepatitis C.
Vinblastine is an anti-mitotic drug used to treat certain kinds of cancer, including Hodgkin's lymphoma, non-
small cell lung cancer, breast cancer, head and neck cancer, and testicular cancer. Vinblastin: Vinblastin/vincristin
from Catharanthus roses acts as cell-cycle inhibitor and thus can be used as anticancer drugs.
Digitalis is also known as digoxin and digitoxin. It's a drug that strengthens the contraction of the heart
muscle, slows the heart rate and helps eliminate fluid from body tissues. It's often used to treat congestive heart
failure and is also used to treat certain arrhythmias. Digitalis has been described in medical literature for over 200
years. It's derived from the foxglove plant.
Digoxigenin (DIG) is a steroid found exclusively in the flowers and leaves of the plants Digitalis purpurea and
Digitalis lanata. It is used as a molecular probe to detect DNA or RNA. It can easily be attached to nucleotides by
chemical modifications. DIG molecules are often linked to uridine nucleotides; DIG labeled uridine (DIG-U) can
then be incorporated into RNA probes via in vitro transcription. Once hybridisation occurs in situ, RNA probes
with the incorporated DIG-U can be detected with anti-DIG antibodies that are conjugated to alkaline phosphatase.
To reveal the hybridised transcripts, alkaline phosphatase can be reacted with a chromogen to produce a colour
precipitate.
Pectinase is a general term for enzymes that break down pectin, a polysaccharide substrate that is found in the
cell walls of plants. One of the most studied and widely used commercial pectinases is polygalacturonase. It is
useful because pectin is the jelly-like matrix which helps cement plant cells together and in which other cell wall
components, such as cellulose fibrils, are embedded. Therefore pectinase enzymes are commonly used in processes
involving the degradation of plant materials, such as speeding up the extraction of fruit juice from fruit, including
apples and sapota. Pectinases have also been used in wine production since the 1960s. They can be extracted from
fungi such as Aspergillus niger. The fungus produces these enzymes to break down the middle lamella in plants so
that it can extract nutrients from the plant tissues and insert fungal hyphae. If pectinase is boiled it is denatured
(distorted) making it harder to connect with the pectin at the active site, and produce as much juice.
SOLID Enzymatic
Small molecules: amino acids, sugar, fine chemicals, antibiotics, hormones, vitamins
can not be sedimented, but can be separated by extraction
Lipids
Particulate products
can be collected by sedimentation or filtration
1 cm
Insects
1 mm
Hair
Bacteria
10 m
1 m
100 nm
Virus
Ribosome
10 nm Antibodies
Amino Acids
1 nm
Sucrose
Water
4.5 nm
2.4 nm 14 nm
Naturally occurring compounds and metabolites such as sugar, citric acid, vitamins, amino
acids, lipid, and antibiotics
Catabolism: the process by which microorganisms obtain energy from organic compounds.
Catabolism is the metabolic process that breaks down molecules into smaller units. It is made up
of degradative chemical reactions in the living cell. Large polymeric molecules (polysaccharides,
nucleic acids and proteins) are processed into their constituent monomeric units (i.e.
monosaccharides, nucleotides and amino acids, respectively).
Anabolism (biosynthesis) is the metabolic process that builds larger molecules from smaller
ones. Anabolic processes tend toward "building up" organs and tissues. These processes produce
growth and differentiation of cells and increase in body size, a process that involves synthesis of
complex molecules. Examples of anabolic processes include growth and mineralization of bone
and increase of muscle mass.
CH2OH CH2OH
O OH H O H CH2OH H
H O
H H
H OH H H HO
OH
HO H HO O CH2OH
OH H
H OH H OH
Glucose Fructose
Sucrose
Lipids can be broadly defined as any fat-soluble (hydrophobic) naturally-occurring molecules, that
are insoluble in water but soluble in nonpolar organic solvents such as ether, benzene, chloroform.
Highly extractable into nonpolar solvents
Although the term lipid is sometimes used as a synonym for fat, it is in fact a subgroup of lipids
called triglycerides.
The term is more-specifically used to refer to fatty-acids and their derivatives (including tri-, di-, and
monoglycerides and phospholipids) as well as other fat-soluble sterol-containing metabolites such as
cholesterol.
Fatty acid are usually esterified to glycerol to form di- & triglycerides
Diglycerides are usually esterified to a phosphate group (phospholipids), which may in turn be
esterified to ethanolamine or choline
Fatty acids and phospholipids are amphiphilic with a strongly polar “head” & a strongly nonpolar
“tail”
OH
OH
O O
OH
OH
NaOH, H2O
heat
OH + RCO2- Na+
OH + R'CO2- Na+
OH + R"CO2- Na+
-
O O
O O
-
-
Free soap Hydrophobic tail O O
O O
O
-
-
- O O
O - O O O - O
O O O O
-
-
-
O
O O Micelle
dirt
Glycerol esterified with two fatty acids and one phosphate group
Second most abundant group of natural lipids
Fatty acids and phospholipids are amphiphilic with a strongly polar “head” & a strongly nonpolar “tail”
H3C
C D H3C H
A B H H
HO
Vitamin A (retinol)
• Essential to vision OH
Vitamin E
• Mixture of isomers; α-tocopherol most important
• Protects against oxidative damage to cells from radicals
CH3
HO
CH3 CH3
CH3 O
CH3
CH3
a-Tocopherol
Hydrophobic antioxidant vitamin
Ch. 1: Bioseparation & biological materials
KAIST 1.3 Small molecules Bioseparation Eng.
Penicillin binds at the active site of the transpeptidase enzyme that cross-links the peptidoglycan
strands. The labile β-lactam ring in penicillin reacts with a serine residue in the transpeptidase as
shown below. This reaction is irreversible and so the growth of the bacterial cell wall is inhibited.
The resulting complex is stable to water and remains attached to the polypeptide chain.
Proteins
Polysaccharides
Nucleic acids
Peptide bond
Alpha helix
Coil held by hydrogen bonding every 4th AA
5.44Å
2.3Å
Bio.kaist.ac.kr/~hskim/ppt/nano_lecture.ppt & http://myhome.hanafos.com/~s9euno/ch3-1.htm
sheet: side-by-side polypeptide chains are cross linked by interchain hydrogen bonds
Typical of fibrous proteins such as silk
CD (circular dichroism): detect changes in the 2dary structure by measuring the difference in the abs.
of left circularly polarized light & right circularly polarized light estimate the fraction of a protein
in helix, sheet, and random coil configurations
7.0
Å
Ser Val
Cys
Asp
Asp
Lys
Lys
OH
HS
SH
OH
☞ DTT causes the disulfide bond in the protein to be broken. The breaking of the disulfide bonds
causes the protein to unfold and the 3D structure to become less compact move more slowly
Reducing agent
2-mercaptothanol: a mild reducing agent that is ideal for cleaving disulfide bonds to thiols.
DTT: a water-soluble reagent that reduces disulfide bonds
OH
HS
SH
-mercaptoethanol OH
HSCH2CH(OH)CH(OH)CH2SH
Dithiothreitol
Folded peptide chains interact with one another in the native conformation of an oligomeric protein
Hemoglobin molecule: 2 -globin and 2- globin peptide chains
Bacterial RNA polymerase: several subunits
Quaternary structure is maintained by intermolecular bonds: ionic & disulfide bond
Structure of Heme B
Heterocyclic organic ring, porphyrin
Prosthetic group
The majority of proteins are conjugated proteins that contain not only AAs, but other organic and
inorganic compounds Prosthetic group: non-amino acid portion of a conjugated protein
Posttranslational modification: any changes to protein molecules other than the addition of AAs via
peptide bond formation
Glycosylation: oligosaccharides are added to active side chains of specific AAs (Ser, Asn, Thr)
Side chain oligosaccharide serve extremely important functions such as stability, catalytic function,
binding specificity, solubility, targeting, and transport
Human EPO, which is normally made in cells of the kidney and transported via the blood to the bone
marrow recombinant protein by animal or yeast cell culture (not by bacteriaglycosylation)
Antibody structure
Globular proteins synthesized by B lymphocytes in animals
Highest association constant with antigen
Y-shaped protein molecules with an invariant stem
Each pair of N-terminal residues constitutes an antigen binding site
CH2O denotes carbohydrate
C: constant region
V: variable region
H: heavy chain
L: light chain
Fc of heavy chain: [-chain: IgA] [-chain: IgD] [-chain: IgG] [-chain: IgM] [-chain: IgE]
WBC: leukocytes
1. Lymphocyte
B cell: synthesize antibody proteins
T cell: Inflammatory T cell, cytotoxic T cell, helper T cell ( activate B cell, macrophage, etc.)
2. Monocyte: become macrophage
3. Granulocyte
Epitope: a portion of a molecule to which an antibody binds. Epitopes can be composed of sugars,
lipids or amino acids. In most cases, epitope tags are constructed of amino acids. (antigenic
determinant)
How to create a cell line that could be grown in culture & would produce monoclonal antibody: a trick
has been devised for producing them in bioreactors
B lymphocytes do not reproduce in culture
Normal B lymphocytes sometimes become cancel cells (myelomas) that acquire the ability to grow in
culture while retaining many of the attributes of B cells
*Hybridoma hybridizing one B lymphocyte with a lymphoma (blood cell tumor) cell
Hybridoma, which grows in culture and secretes a single type of antibody molecules, is the source of a
monoclonal Ab.
Hybridomas produce progeny indefinitely in vitro which make monoclonal antibodies.
Ribonuclease
Trypsin
Enzymes Urokinase
DNA polymerase
Cellulase
Hemoglobin
Serum albumin
Transport proteins
Myoglobin
β1-Lipoprotein
Ovalbumin
Nutrient, storage protein
Casein
Actin
Contractile or motile Myosin
Tubulin
Keratin (skin)
Fibroin (silk)
Structural Collagen (tendons, ligaments)
Elastin (joints)
Proteoglycans (cell walls)
Immunoglobulin (antibodies)
Venom proteins
Defense
Ricin
Interferon
Insulin
Growth hormone
Regulatory Lymphokines/cytokines
Protein kinases (signal transduction)
DNA binding repressors and activators
Soybean trypsin inhibitor
Inhibitors Plasminogen activator inhibitor
HIV protease inhibitor
• High specificity
Regio-specificity
Stereo-specificity
• High turnover rate
• Instability
Heat
Organic solvents
Denaturants
Lysozyme-substrate complex
Lysozyme: The enzyme functions by attacking peptidoglycans (found in the cell walls of bacteria, especially Gram-positive
bacteria) and hydrolyzing the glycosidic bond.
Since the active function of proteins is dependent on tertiary and quaternary structures, the 3D
configuration of each molecule must be maintained
Protein degradation
deamidation of asparagine and glutamine
Since the active function of proteins is dependent on tertiary and quaternary structures, the 3D
configuration of each molecule must be maintained
Protein degradation
deamidation of asparagine and glutamine
oxidation of methionine to methionine sulfoxide form
oligomerization
aggregation
fragmentation (depolymerization)
cross-linking
inter- and intramolecular rearrangements involving cysteine disulfide exchanges
denaturation: breakdown of H-bonds and ionic bonds that determine tertiary structure
Temperature
Thermal denaturation for many proteins begins to occur at 45 to 50 oC
An example of the thermal denaturation of the protein ribonuclease (Fig. 1.17)
High temperature break H-bond & -pleated sheet structure is disrupted
Still leaves the disulfide bonds intact
Some enzymes from thermophilic bacteria inhabiting hot springs
active at high temperature above 85oC
GSTglutathione
MBPamylose: affinity tags
Genetic engineering to avoid the expression of insoluble inclusion bodies fusion protein
Expressed as C-terminal fusion with another protein called a carrier protein (higher solubility)
Carrier proteins: GST (glutathione-S-transferase), MBP (maltose binding protein), NusA protein,
thioredoxin
Buffer flow
Target protein His tag Ni
Non-His tag
fused proteins
Resin : Ni-matrix
Ni ion interact with Histidine hexamer
His-tag fused protein retains in the column (Ni-resin)
The other non-his tag fused proteins are washed away
Finally, His-tag fused target proteins are eluted from the column purely
Buffer flow
Target protein His tag Ni
Elution of His-tag
fused proteins
Resin : Ni-matrix
Ni ion interact with Histidine hexamer
His-tag fused protein retains in the column (Ni-resin)
the other non-his tag fused proteins are washed away
Finally, His-tag fused target proteins are eluted from the column purely
☞ Imidazole (the side chain of histidine)
For protein with cysteine, add a reducing agent such as -mercaptoethanol, dithiothreitol (DTT),
dithioerythritol, or cysteine to allow reduction of the interchain disulfide bonds by thiol-disulfide
exchange
NH2C(:NH)NH2·HCl
NH2C(O)NH2
Guanidine hydrochloride
Urea
Structure of DNA
James Watson & Francis Crick at 1953
Native DNA consists of two long chains (strands) that form a
double-stranded helix
Coiled polynucleotide chains of DNA: hydrogen bonds between
the bases
Base pairing rule: A-T(U) & G-C
A-T: two hydrogen bonds & G-C: three hydrogen bonds
- Wallace rule (less than 15mer): Tm = 2oC (A+T) + 4oC (G+C)
Nucleotide: nitrogenous base, ribose (deoxyribose) sugar, one or more phosphate groups
Polysaccharides
Large molecules
• Proteins (3-10 nm)
• Polysaccharides (4-20 nm)
• Nucleic acids (2-1,000 nm)
Particulate products
• Virus (100 nm)
• Bacteria (1um), yeast cells (4 um), animal cells (10 um)
• Liposomes or natural vesicles (100 nm)
• Bacterial inclusion bodies (100-1,000 nm)
• Subcellular particles or organelles Ribosomes (25 nm)
• Natural hormone granules (200 nm)
Subcellular components
• Early bioseparation steps include flocculation, sedimentation, filtration
Material balance
accumulation = inflow - outflow + amount produced - amount consumed
Equilibria
equilibrium constant for chemical reaction
Keq=[C]/([A][B]) for A + B C
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
CS: conc. in the adsorbent phase
C: conc. in the liquid phase (C: chemical species & S:adsorption site)
Keq=[CS]/[C] linear adsorption equilibrium (valid at low conc.)
JD = - D(dc/dx)
D = kT/6πμa (k: Boltzmann constant, T: absolute temp., μ: viscosity, a: particle radius)
http://pathmicro.med.sc.edu/fox/lps.jpg
Extremely heat stable – recommended conditions for inactivation are 180 0 C for 3 hours.
New drug discovery: several years for the completion of the route (Fig. 1.24)
Preclinical trial: animal test, toxicity test
Phage I: safety for usually healthy adults
Phage II: efficacy for 20-80 patients
Phage III: efficacy for hundreds – thousands patients