chap 9 downstream processing - national tsing hua …biohu/download/handouts/2015 introduction...
TRANSCRIPT
1
Chap 9 Downstream Processing
Introduction
Definition: the isolation and purification of biotechnological product to a form suitable
for its intended use.
Culture is harvested at the optimal timing cells are separated from the
mediumdownstream processing.
Biotechnological products include: whole cells, organic acids, amino acids, solvents,
antibiotics, industrial enzymes, therapeutic proteins, vaccines, etc.
The complexity of the processing steps is determined by the required purity, which in
turn, is determined by its application.
Separation principles also depend on the size and nature of products.
For intracellular products==> disrupting cells ==> following purification steps
For extracellular products==>concentrating the medium ==> purification
Imperative to minimize the number of steps and maintain high yields in the different
stages.
Many steps are unit operation
processes, usually divided into four
stages:
Solid-liquid separation or
clarification
Concentration
Purification
Formulation
I. Solid-liquid separation
Refers to the separation of cells from
the culture broth, removal of cell debris, collection of protein precipitate, etc. The unit
operations involved are normally centrifugation and filtration. (note: cell debris: broken cells,
could be generated by cell disruption)
2
Filtration
The mixture goes through a filter which retains the particles according to size while
allows the passage of fluid through the filter.
In cake filtration, the particles are retained as a cake on the filter, resulting in the
resistance. In many cases, the cakes are compressible and the changing effective
pressure difference influences the flow through the filter.
Filtration theory
Rate of filtration is often measured as the rate at which liquid filtrate is collected.
])([
1
m
c
f
f
rA
M
p
dt
dV
A
Vf: filtrate volume; t: filtration time; dVf/dt: volumetric rate of filtration;
A: filter area;
p: pressure drop across the filter; f: filtrate viscosity
Mc: total mass of solids in the cake; : average specific cake resistance [LM-1], a measure of the
resistance of the filter cake to flow, dependent on shape and size of the particles.
rm: resistance from the filter [L-1], includes the effect of filter material and any particles
wedged in it during the initial stages of filtration
Let fc cVM , where c: mass of solids deposited per volume of filtrate. Substitute this into
the above equation
3
])([
1
m
f
f
f
rA
cV
p
dt
dV
A
Assume p is constant (most commonly carried out) and take the reciprocal
p
r
pA
Vc
dV
dtA
mff
f
f
Integration yields
f
mf
f
fV
p
rV
pA
cAt )()
2( 2
Vf or t can be calculated at constant pressure, provided all the constants are known.
Examples of filter are perforated sintered metal, cloth, synthetic fibers, cellulose, etc.
Vacuum filters are also often used due to the simplicity of operation and low
costs==>Rotary drum filtration (often used for bacteria, filamentous fungi and yeast cells)
Pretreatment of broth
Pretreatment by changing the biomass particle size, the broth
viscosity and the interactions between the biomass particles
can facilitate clarification (e.g. filtration).
Filter aid: incompressible discrete particles of high
permeability with size ranging from 2-20 m increase the
porosity of the cake and facilitate the passage of the liquid. It
should be inert to the broth, the most frequently used is
Diatomite (skeletal remains of aquatic plants) or inactive
carbon.
Flocculating agents: can help the agglomeration of
individual cells or cell-particles into large flocs which can be
easily separated at low centrifugal forces. These agents include inorganic salts or
polycations, either cellulosic or synthetic polymers.
4
Flotation:
Particles are adsorbed on gas bubbles, get trapped in a foam layer and can be collected.
The gas may either be sparged into the particulate feed or very fine bubbles can be
generated from dissolved gases by releasing the overpressure.
Formation of stable foam is supported by the presence of long chain fatty acids.
Centrifugation
Separation by means of the accelerated gravitational force achieved by a rapid rotation.
Relies on the density difference between the particles and the surrounding medium, most
effective when the particles to be separated are large, the liquid viscosity is low and the
density difference between particles and fluid is great.
Batch centrifuge is common in the labs but the low processing capacity limits its use in
large scale.
5
Continuous centrifuges are common in large-scale processing in which the deposited
solids are removed continuously or intermittently.
Tubular bowl centrifuge
Very commonly used in food and pharmaceutical industries.
Feed enters under pressure through a nozzle at the bottom, and moves upwards through
the cylindrical bowl.
As the bowl rotates, particles traveling upward are spun out and collide with the walls of
the bowl. Solids hitting the wall can form the cake.
As the feed rate increases, the liquid layer moving up the wall becomes thicker thus
reducing the performance of the centrifuge by increasing the distance a particle must
travel to reach the bowl.
This system lacks a provision of solids rejection, the solids can only be removed by
stopping the machine, dismantling it and scraping or flushing the solids out manually.
Typical range of centrifugal force: 13000-16000g (g: 12.11000
)(
2
rpmmmr )
Disc-stack bowl centrifuge:
6
Contain conical sheets of metal (discs) which are stacked with clearances as small as 0.3
mm. The discs rotate with the bowl to split the liquid into thin layers. Feed is released
near the bottom of the centrifuge and travels upwards through matching holes in the
discs.
Between the discs, heavy components of the feed are thrown outward under the influence
of centrifugal forces as lighter liquid is displaced towards the center of the bowl. As they
are flung out, the solids strike the undersides of the discs and slide down to the bottom
edge of the bowl. At the same time, the lighter liquid flows in and over the upper
surfaces of the discs to be discharged from the top of the bowl.
Disc-Stack bowl centrifuge
Disc-stack bowl centrifuge with continuous
discharge of solids
7
Heavier liquid containing solids can be discharged either at the top of the centrifuge or
through nozzles around the periphery of the bowl.
Typical range of centrifugal force: 5000-15000g
Note: the supernatant obtained by centrifugation is not free of cells (103 to 105 cells/ml)
and costs of maintenance and power consumption are both high. Separation of
particulate debris is inefficient by centrifugation.
II. Release of intracellular components
To release maximum amount of the product in an active state.
Factors to consider: inactivating effects such as shear, temperature and proteases, choice
of disruption methods, subsequent processing steps.
Disruption of microbial cells
Mechanical disruption is the most common means to release intracellular products in
laboratory and in industry.
Ultrasonication is common in the lab-scale but the removal of heat is difficult on a larger
scale.
Two common industrial processes:
High pressure homogenization: the cell suspension is forced at high pressure
through an orifice to emerge at atmospheric pressure. The sudden release of
pressure creates high shear.
8
Vigorous agitation with abrasives: agitation with glass in bead mills ruptures the
cells by high shear and impact with the cells. Size of beads: 0.2-0.5 mm for bacteria;
0.4-0.7 mm for yeasts.
Non-mechanical disruption
freeze/thaw: freeze at -80C and rapid thaw at 37C; disrupt the cells by causing
changes in the structure of the cell wall and membrane.
Organic solvents and detergents: render cells more permeable and soluble.
Enzyme: is mild and has selectivity during the product release.
Lysozyme is often used because it damages the peptidoglycan layer and
therefore the internal osmotic pressure bursts the periplasmic membrane.
Gram+ (no outer membrane) bacteria are more susceptible while the lysis of
Gram- (with outer membrane) bacteria requires the passage of lysozyme
through the outer membrane, which can be aided by the addition of EDTA.
Glucanase and mannase, in combination with proteases, are used for the
degradation of yeast cell walls.
Availability and costs limit the use of enzymatic methods. A combination of
enzymatic/chemical lysis with mechanical methods could be used.
Homogenization of animal/plant tissue:
Animal tissues: cut into small pieces, suspend in ice-cold homogenization buffer and
grind in a blender. Animal cells are usually easier to break because of the absence of cell
walls.
Plant cells have tough cell walls and are more resistant.
Non-fibrous plant tissue: more easily to be macerated (to become soft by putting or being
left in water), rapid homogenization of the material in the buffer using a blender.
Fibrous material: difficult to macerate==>freeze and grind the tissues to dry
powder==>add buffer==>homogenization.
Buffers with pH around 6.5-7.2 are used to neutralize the acidic materials including
the phenols. Buffers also contain reducing agents such as ascorbate and thiols to
prevent the accumulation of quiones and hence the inactivation of enzymes during
extraction.
9
Use of cellulase and pectinase (pectin: gel-like stuff in plants, derived from galacturonic acid)
for digestion of plant cell walls is an attractive alternative for achieving selective
release of the protoplasmic material (but not the vacuole contents).
III. Concentration of biological products
After initial separation, the filtrate contains 85-90% of water. Removal of water can be
done in different ways:
Evaporation
Simple but energy-consuming, normally using steam as the heat source in a large scale.
Applicable for food proteins and other stable biologics, but seldom suitable for
processing of biologically active proteins.
Falling film evaporators: the liquid to be concentrated flows down long tubes/plates and
distributes uniformly over the heating surface as a thin film. Part of the liquid is
evaporized and exits as the vapor. Suited for viscous products and is often used in
fermentation industry.
Liquid-liquid extraction
Applied on a large scale in biotechnology both for concentration and for purification.
Efficiency depends on the
distribution of substances
10
between two phases, defined by:
Extraction of low-MW products (vitamins, antibiotics, 2-propanol, butanol,
caffeine.)
Small lipophillic molecules can be extracted by organic solvents (but it may be
more difficult to design an extraction process for hydrophilic molecules).
Physical extraction: distribution is based on the physical preference. This applies to
nonionizing compounds and the extraction is optimized by screening for the
solvents that have a high K value.
Dissociative extraction: distribution is based on the difference in the dissociation
constant of the ionizable components, e.g. extraction of penicillin.
Reactive extraction: for compounds with high solubility in aqueous solution.
Carriers (e.g. phosphorous compound) are added to the organic solvent to form
selective solvation bonds or complexes that are insoluble in the aqueous phase=>
the compound is carried from the aqueous phase to the organic phase.
After extraction, the product can be recovered from the solvent by distillation. If
the product is heat sensitive back-extraction into a new aqueous phase.
Ex: penicillin is extracted into butyl acetate from the fermentation medium at pH
2.5-3, and back-extracted into aqueous phosphate buffer at pH 5-7.5.
Supercritical fluid (SCF) extraction:
SCF are fluids above their critical temperature and pressure, with
11
Usually, the compressed SCF is contacted with the feedstock to be extracted in
an extraction column, which is then transferred via an expansion valve to a
separator. On lowering the pressure, the fluid turns into gas and releases the
product as a precipitate.
Extraction of proteins
Aqueous two-phase systems (ATPS)
Some polymer solutions are immiscible with each other or with salt solution of high
ionic strength two phase form in the container.
For industrial processes, polyethylene glycol (PEG)/salt system is often used for
their low cost.
12
Dextran phase containing
cellular debris
PEG phase containing
soluble proteins
Partitioning of a component is based on its surface characteristics, nature of phase
components and the ionic composition.
Phase separation is slow (from minutes to hours) but can be speeded up by
centrifugation.
Advantages:
Membrane filtration
Classification:
Microfiltration: separates particles of
13
Ultrafiltration: separates polymeric particles of
Reverse osmosis (hyperfiltration): separates particles of
14
Reverse Osmosis Ultrafiltration Microfiltration Clarification
0.001 μm 0.0001 μm 0.01 μm 0.1 μm 1 μm 10 μm 100 μm
0.2 kDa 200 kDa 20,000 kDa
Adapted from MS thesis, G.Y. Chen, NTHU.
Proteins
Yeast Salts Mammalian virus
Selectivity of membranes, expressed as molecular weight cut-off (MWCO) for
ultrafiltration membranes, is mainly determined by the
Example: the actual molecular mass of albumin is 64 kD, the apparent size of
albumin can increase due to a large “ionic cloud” forming around the molecule in a
low ionic strength soln’, so the protein can behave like a 300 kD molecule and be
subject to full retention when processed with a 100 kD rated membrane.
The MWCO is a nominal size, thus usually we select a membrane cut-off rating
which is 0.2-0.3 times the size of the MW target for retention. e.g. to ensure
retention of a 50 kD molecule, a 10-15 kD membrane should be used.
Dead-end filtration: The direction of flow is perpendicular to the membrane surface.
Deposition of particles and precipitation of small solutes, etc. on the surface can
clog the membrane, hence reducing the flow rate.
15
Cross-flow (tangential flow) filtration: the feed stream is maintained parallel to
the filter surface to provide shear force and prevent cake formation.
1. Stirred Cell (Millipore):
Stir to avoid membrane clogging.
Water, salts and lower MW molecules pass through, while larger
molecules are retained concentration.
Because the pore size is small, pressure can be applied to speed up the
concentration.
2. Hollow-fiber: comprises a bundle of hollow capillaries packed in the tube.
The liquid to be filtered is pumped through the capillary walls and can be
drained as permeate from one end of the cartridge while the concentrated
retentate emerges from the other end.
Tangential flow filters can serve two purposes:
Membrane adsorbers:
Membranes with ion exchange groups or affinity ligands which bind proteins from
the clarified feed.
Source: “Protein concentration and diafiltration by tangential flow filtration”. Millipore Corporation.
16
Desorption is carried out by appropriate buffers.
A stack of membranes provides large surface area for adsorption (rending it similar
to chromatography gels).
Precipitation
Solubility of proteins changes with salt
concentration which can be expressed in terms of
ionic strength:
2
2
1ii ZCI
Zi: ionic charge
Ci: molar concentration of the ionic species (can be neutral salts, organic solvents or
high MW polymers).
salt in: when ion concentrations increase, the additional counter ions
salt out: at high salt concentration,
Table 9.4 Modes of protein precipitation
Mode Example Comments
Pro
tein
solu
bility
Salt
concen.
Salt in Salt out
17
Addition of
neutral salt
(NH4)2SO4 Increased hydrophobic interactions between neutral
protein molecules
salt is removed prior to next purification step (except
for hydrophobic interaction chromatography) by dialysis, UF or
gel filtration
Addition of
organic
solvent
Acetone,
ethanol
Reduced dielectric constant enhances electrostatic
interactions between protein molecules (low dielectric
const. increases the charge-dipole and dipole-dipole attraction between
proteins and increases the precipitation)
Low temperature required for operation (for safety)
Addition of
non-ionic
polymer
PEG Reduction in the effective quantity of water
available for protein solvation
Polymer often has stabilizing effect on proteins
Addition of
charged
polymer
Polyethylen
eimine
Polyacrylic
acid
Complex formation between oppositely charged
molecules leads to charge neutralization and
precipitation
Change in
pH
Low solubility of protein at isoelectric point (the
pH at which the protein has no net charge)
Extremes of pH denature and precipitate sensitive
proteins
All the precipitates can be re-dissolved in small volume of buffer suitable for the next
step.
Adsorption to chromatographic resins (see Chromatography)
IV. Purification by Chromatography
Intro:
The degree of purification in previous steps is
limited, usually need several chromatography
steps to yield high purity. Which
chromatography to use depends on the
characteristics of the proteins, such as size and
shape, overall charge, surface hydrophobic
groups, and ability to bind various ligands.
18
Gels (resins) are usually made of cross-linked polymers:
Agarose: polysaccharide made up of D-galactose and 3,6-anhydro-1-galactose
units
Cellulose: polysaccharide of -1-4 linked glucose units
Dextran: a polysaccharide of -1-6-linked glucose
Polyacrylamide: polymer of acrylamide and bis-acrylamide
e.g. Sephadex (Amersham Pharmacia, now part of GE Healthcare)
Purification usually accounts for 50-70% of production cost mostly on .
1. Size Exclusion Chromatography (SEC) ( )
Protein content monitored by
2. Ion Exchange Chromatography (very often used)
a.a. exhibit different charges. The net charges of proteins depend on the
The pH at which a protein has no net charge is called isoelectric point (pI)
At pI, the proteins do not repel one another and thus can precipitate.
19
ion exchange chromatography is based on the
Charged groups are immobilized to solid matrix (gel)
Positively (Negatively) charged proteins bind to negatively (positively) charged
groups by displacing the H+ (OH-) which is initially bound to the resin.
e.g. + groups: diethylaminoethyl -O-(CH2)2N+H(CH2CH3)2 (also known as
DEAE)
trimethylamino methyl CH2N+(CH3)3 (also known as Q)
– groups: carboxymethyl (CH2COO-), sulphomethyl (CH2SO3-)
After binding, the column is washed several times with
wash buffer to remove non-specifically bound proteins.
After wash, the bound proteins are eluted using the elution
buffer. For elution, a salt containing buffer (often NaCl) of
increasing ionic strength in turn displaces the protein from
the matrix.
Summary:
General procedures: sample loadingwasheselution. In
each step, the samples are collected and can be analyzed for
purity and recovery.
Popular:
3. Hydrophobic Interaction Chromatography (HIC)
HI results from water’s propensity to repel hydrophobic groups. HI is relatively
weak compared to H-bonds and lacks directionality.
20
8 a.a. are hydrophobic (non-polar):
Proteins are folded partly by hydrophobic interaction for which the hydrophobic
residues are buried inside (shielded from aqueous environment), and stabilize the
protein conformation.
However, a minority of hydrophobic a.a. are present on the surface and they tend
to cluster to form a group. These hydrophobic groups are masked by an ordered
film of water molecules.
HIC (also known as reverse phase chromatography) uses the different degrees of
surface hydrophobicity and achieves resolution by thousands of interactions of
solute molecules with the resin. HIC has high resolving power and is a widely used
analytical chromatography.
Hydrophobic groups such as phenyl group or octyl group are immobilized to the
gel.
Samples are loaded into the column and proteins bind to the gel, the more
hydrophobic the protein is, the tighter the protein binds.
Salt (e.g. NaCl, or ammonium sulfate) is added in the sample to increase the ionic
strength,
Elution:
Octyl group
OH
Sepharose-O-CH2-CH-CH2-O-(CH2)7-CH3
OH
Sepharose-O-CH2-CH-CH2-O-
Phenyl group
21
4. Affinity Chromatography
Utilize the affinity of the protein toward the ligands. The binding can be achieved
via the affinity between the protein and the ligand immobilized on the resin.
Most powerful and highly selective.
Categories of affinity interactions
a. Protein A-IgG1 for the purification of monoclonal antibodies (MAb)
b. Immunoaffinity:
Exploits the affinity interactions between Ab and Ag.
The interactions include
Ab is immobilized to the resin so as to bind the Ag (the target protein) in the
sample. This process can achieve
Drawbacks:
One popular method uses a glycine-HCl buffer at pH 2.2-2.8 (resulting in
partial denaturation) for elution. High salt concentration or extremes of pH
disrupt Ag-Ab interactions by decreasing electrostatic interactions and/or H-
bonds.
c. Lectin-glycoprotein (for the purification of glycoproteins)
glycoproteins: proteins with carbohydrate side chains (e.g. hormones, growth
factors)
Hundreds of new protein products are currently under clinical investigation or are awaiting the FDA
approval. MAb constitutes the single largest category (>200 MAb).
22
lectins: a group of proteins that bind carbohydrate molecules, e.g. concanavalin A
(Con A, binds -D-mannose and -D-glucose); wheat germ agglutinin (WGA;
binds N-acetyl--D-glucosamine).
Binding:
Elution:
Drawback:
d. Ni-Histidine (popular in recent years)
Relies on genetically added 6 histidine residues on proteins either at the N- or
C- terminus.
Divalent cations (e.g. Ni2+, Cu2+ or Zn2+) are immobilized on resins and bind
the proteins with His6 tag.
Elution is performed by the competition of imidazole (an analogue of histidine).
e. Others
Summary for protein purification by chromatography:
General procedures (except size exclusion): sample loadingwasheselution. In
each step, the samples are collected and can be analyzed for purity and recovery.
The wash and elution buffers have different compositions. Initially, the desired
proteins can be washed and eluted using linear gradient of eluting agents. After
identifying the optimal eluting agent concentrations, stepwise wash/elution can be
carried out.
23
Two important factors to consider:
High flow rates are desired in order to save time (several hours)
Fast-flow Protein Liquid Chromatography (FPLC)
using rigid gels and stainless steel column to withstand high
pressurehigh flow rates
fast (10 min to 1 hr)
V. Protein Stabilization on Finished Product
Denaturation of proteins and loss of biological activity are problems.
Factors resulting in denaturation and loss of activity
Top: process block
diagram for the
purification of
bovine growth
hormone
(somatotrophin)
produced in E. coli.
(intracellular
product) Bottom: purification
summary for
processing 260 Kg
of inclusion bodies. (Adapted from Blanch,
HW, Clark, DS,
Biochemical
Engineering, 1997)
24
Chemical Detergent (unfold the natural conformation), e.g. SDS
Urea
Guanidine hydrochloride
Solvents (interact with hydrophobic a.a.)
Heavy metals (interact with –SH groups)
Physical Extremes of pH
High temperature (exceptions: proteins in bacteria in hot
spring)
Freeze and thaw (freezing causes changes in
microenvironment and local pH damage can
minimized by rapid freezing)
Vigorous agitation
Biological Protease (can add protease inhibitors, such as aprotinin,
PMSF…)
Stabilization:
High protein purity may decrease the stability
Proteins still lose activity during storage add agents to prolong the shelf life
glycols: glycerol, polyethylene glycol
sugars: sucrose
neutral salts: ammonium sulfate, NaCl
Proper storage:
Long-term: spray drying or lyophilization.
In spraying, the liquid input stream is sprayed through a nozzle into a hot vapor
stream and vaporized. The solvent in the small liquid droplets is quickly
vaporized.
Lyophilization
(1)
(2)
Reference:
1. Walsh, G. (2002) Proteins: biochemistry and biotechnology. John Wiley & Sons. New
25
York.
2. Doran, PM (2003) Bioprocess Engineering Principles. Academic Press. San Diego.
3. Blanch, HW, Clark, DS. (1997) Biochemical Engineering. Marcel Dekker. New York.