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)

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 dV f

A dt

p
M
f [ ( c ) rm ]
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 M c cV f , where c: mass of solids deposited per volume of filtrate. Substitute this into
the above equation

1 dV f

A dt

f [ (

p
cV f
A

) rm ]

Assume p is constant (most commonly carried out) and take the reciprocal
A

Vf
f rm
dt
f c

dV f
Ap
p

Integration yields

At (

f c
2 Ap

)V f2 (

f rm
p

)V f

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.

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.

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.
2

rpm
Typical range of centrifugal force: 13000-16000g (g: r (mm)
1.12 )
1000

Disc-stack bowl centrifuge:

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 with continuous

discharge of solids

Disc-Stack bowl centrifuge

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.

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.

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

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

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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.

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PEG phase containing

soluble proteins

Dextran phase containing

cellular debris

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

12

Ultrafiltration: separates polymeric particles of

Reverse osmosis (hyperfiltration): separates particles of

13

Reverse Osmosis

0.0001 m

Ultrafiltration

Microfiltration

0.001 m

0.01 m

0.1 m

0.2 kDa

200 kDa

20,000 kDa

1 m

Clarification

10 m

100 m

Proteins
Salts

Mammalian virus

Yeast

Adapted from MS thesis, G.Y. Chen, NTHU.

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.

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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.

Source: Protein concentration and diafiltration by tangential flow filtration. Millipore Corporation.

Tangential flow filters can serve two purposes:

Membrane adsorbers:

Membranes with ion exchange groups or affinity ligands which bind proteins from
the clarified feed.

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Desorption is carried out by appropriate buffers.

A stack of membranes provides large surface area for adsorption (rending it similar
to chromatography gels).

Solubility of proteins changes with salt

concentration which can be expressed in terms of
ionic strength:
1
I Ci Z i2
2

Protein

solubility

Precipitation

Salt in

Salt out

Salt
concen.

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

16

Addition of
neutral salt

(NH4)2SO4

Addition of
organic
solvent

Acetone,

ethanol

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
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)

Addition of
non-ionic
polymer

PEG

Addition of
charged
polymer

Polyethylen
eimine
Polyacrylic
acid

Change in
pH

Low temperature required for operation (for safety)

Reduction in the effective quantity of water

available for protein solvation
Polymer often has stabilizing effect on proteins

Complex formation between oppositely charged

molecules leads to charge neutralization and
precipitation

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.

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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)

1.

Purification usually accounts for 50-70% of production cost mostly on .

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.

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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.

3.

Popular:

Hydrophobic Interaction Chromatography (HIC)

HI results from waters propensity to repel hydrophobic groups. HI is relatively

weak compared to H-bonds and lacks directionality.

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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.
OH

OH

Sepharose-O-CH2-CH-CH2-O-

Sepharose-O-CH2-CH-CH2-O-(CH2)7-CH3
Octyl group

Phenyl group

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:

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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 Hbonds.

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).

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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).

d.

Binding:

Elution:

Drawback:

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.

e.

Elution is performed by the competition of imidazole (an analogue of histidine).

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.

22

Two important factors to consider:

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)

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

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Chemical

Physical

Detergent (unfold the natural conformation), e.g. SDS

Urea

Guanidine hydrochloride

Solvents (interact with hydrophobic a.a.)

Heavy metals (interact with SH groups)

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)

Biological

Vigorous agitation

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

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2.
3.

York.
Doran, PM (2003) Bioprocess Engineering Principles. Academic Press. San Diego.
Blanch, HW, Clark, DS. (1997) Biochemical Engineering. Marcel Dekker. New York.

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