OVERVIEW OF

DOWNSTREAM
PROCESSING
CENTRIFUGATION, FILTRATION AND
CHROMATOGRAPHIC TECHNIQUES

1
INTRODUCTION
• Purification and recovery of the molecule of interest from harvested cultures.
• Steps include:
 Separation
 Cell disruption (for intracellular products)
 Extraction
 Isolation
 Purification
 Polishing
 Viral clearance/filtration/inactivation (in case of mammalian system)
 Concentration (crystallization/precipitation)
 Lyophilization

2
INTRODUCTION
Milestones in the history of
protein purification

3
SEPARATION OF PRECIPITATES AND PARTICULATE MATERIAL: FILTRATION
• One of the core unit operations across biopharmaceutical downstream processing.
• Separates components of a fluid mixture according to size during passage through a porous medium.
• If liquid on one side of a porous material is exposed to pressure, it will start to move through the porous
material, provided the pressure on the opposite side is lower.
 Feed: The starting fluid.
 Permeate or filtrate: Solution that passes through.
 Flux: The volume passing through the membrane per unit time and per membrane area (L/m2/h), also
abbreviated to LMH.

Figure 1: General principles of flow through a porous material. When Pfeed > Ppermeate, flow through the porous material will occur
4
FILTRATION: Darcy’s Law
• Foundation for simulations and evaluations of filtration.
• Developed by Henry Darcy in the 19th century.
• Forms the basis for analysing filtration behaviour by relating flow to permeability, membrane area, pressure
drop, viscosity, and path length.

∆
𝑄=− ……………………………..1

Q (m3/s) The liquid flow

κ (m2) The intrinsic permeability of the medium
A (m2) The cross-sectional area for flow
ΔP (Pa) The total pressure drop
μ (Pa s) The liquid viscosity
L (m) The length over which the pressure drop is taking place

• Higher pressure and permeability increase flow, while higher viscosity and resistance reduce it.
5
FILTRATION
A

Filtration set up
• In bioprocess filtration, different pressure sources are
used.
• The most common are a liquid pump or pressurised air. B
• Typical filtration equipment includes a pump, the
pressure sensor, and the filter, all connected in a series
(A), in which vessels for feed and filtrate are also
required.
• Some systems (B) do not include a pump, but instead,
the liquid is pushed through the filter using a
pressurised gas (e.g., air). Here, the feed vessel is
typically a pressure chamber connected to a pressurised Figure 2: Filtration process set-up with pressure source
gas source. (A) pump and (B) pressurised air, pressure gauge, and
filtration media
6
FILTRATION
• Modes of filtration: 2 main modes
1. Normal flow filtration (NFF), also called dead-end filtration.
 All flow is passed directly through the filter.
 Material filtered out (retained) is accumulated on the membrane surface or inside the membrane’s pore
structure.
2. Cross-flow filtration(CFF), also called tangential flow filtration(TFF).
 The majority of the flow is directed across, or tangential to, the filter membrane (cross flow or retentate flow).
 A fraction of the cross flow, that is permeating through the membrane, is called a permeate flow.
 The sweeping action of the cross-flow prevents the build-up of foulants on the membrane surface.

Figure 3: The two most common filtration modes in bioprocessing: (A) normal flow filtration and (B) cross-flow filtration 7
FILTRATION
• Each filtration process is characterised by a unique relationship between the membrane flux and the pressure drop
across the membrane.
• The process can be operated either under
 Constant flux (pressure drop across the membrane will be increasing) or
 Constant-pressure-drop (flux through the membrane will decrease).
• In both cases, the non-constant parameter is being monitored, and the recorded changes are used to assess the filter
performance (i.e., the extent of filter fouling or clogging).

• For NFF, a pressure gauge is placed on the filter inlet, and a flow
meter is placed on the filter inlet or outlet.
• For CFF, pressure gauges are placed on the feed, retentate, and
permeate sides of the filter, and flow meters can be placed on the
feed or retentate side, and on the permeate side.
• Two different pressures are usually controlled or monitored during a
CFF process:
Figure 4: Cross-flow filtration set-up.
I. Pressure drop, ΔP
II. The transmembrane pressure, TMP
8
FILTRATION
∆𝑃 = 𝑃feed − 𝑃retentate ………………………………………......…………..2

𝑇𝑀𝑃 = − 𝑃permeate………………………………………….3
where Pfeed, Ppermeate and Pretentate are pressures measured at the inlet and outlet of retentate channels
• Pressure drop, ΔP:
 ΔP helps track or infer the retentate-side circulation flow.
 It reflects the pressure loss along the retentate channel from inlet to outlet.
 When the feed pump drives the retentate flow, this pressure drop indicates how strongly liquid is being
pushed through the channel.
 It helps monitor the retentate flow rate or estimate it when no flow meter is installed.
• TMP
 Average pressure acting across the membrane wall
 It is the actual driving force for liquid passing across the membrane into the permeate side.

9
FILTRATION: Types of filters and materials of construction
• Filters are broadly divided into 2 types
I. Membrane-based filter.
II. Non-membrane porous media

Membrane-based filter Non-membrane porous media

Application For defined separations For coarse separations, prefiltration, and cell removal

Material type Inert polymer material that does not Woven media, such glass fiber or polypropylene fibers
interact with the material that is
being filtered

• Materials of construction for filters and membranes are diverse.

• Some examples include sintered metal, cellulose, or polymers such as polyvinylfluorodyne (PVDF), polyethersulfone
(PES), polytetrafluoroethylene (PTFE), or Nylon.
• Material choice affects hydrophilicity, strength, chemical compatibility, solvent resistance, pH tolerance, and
adsorption behaviour.
• Hydrophilic membranes are preferred for aqueous streams, while hydrophobic membranes are preferred for gas
filtration.
10
 FILTRATION: Membrane structure and morphology
• Membranes are characterised by pore size, pore-size distribution, pore connectivity, pore shape, and
porosity.
• Symmetric membranes have similar pore structures throughout the thickness.
• Asymmetric membranes have more open pores on one side and tighter pores on the other. They often
provide lower back pressure and higher particulate capacity.

A B

Figure 5: Schematic view of (A) a symmetric membrane, (B) an asymmetric (also called an anisotropic membrane)
11
FILTRATION
• Microfiltration versus ultrafiltration
 Microfiltration is generally used for particulate removal and
is rated in microns, typically 0.1 to 20 µ.
 Ultrafiltration is used for macromolecular separations, such
as proteins, and is rated by NMWCO in Daltons, from about
1000 Da to 1 M Da.
 MF membranes are often more symmetric, while UF Figure 6: Effect of retention on different-
membranes typically have an asymmetric structure with a
shaped particles
selective skin layer.

• Retention:
 Retention refers to the extent to which a filter allows passage of a certain substance or particle in relation to a
specific membrane.
 The level of retention is expressed as the ratio of the concentration of the penetrating solute in the permeate
to its concentration in the feed and is called the sieving coefficient, S (value varies between 1 and 0).

𝑆= ………………………………………………………….4

where S is the sieving coefficient (–), CPermeate is the solute concentration in permeate, and CFeed is the solute
feed concentration. 12
FILTRATION: Fouling
• Occurs when material, typically solids or proteins, is captured on and/or inside the porous matrix, restricting
flow.
• Under constant pressure, fouling lowers flow; under constant flow, it increases pressure drop.
• Fouling reduces effective pore volume or membrane area and limits membrane capacity.
• Fouling mechanisms: Three major fouling mechanisms
 Particulates trapped on top of a membrane surface due to their size or shape (cake formation).
 Particulates trapped inside the matrix due to their size or shape.
 Adsorption of particulates on the pores of the membrane.
• In practice, more than one fouling mechanism usually occurs during a filtration run.
• Filter aids (e.g. celite) can reduce dense cake formation by generating voids around the large particles,
preventing them from building up a dense filter cake.

Figure 7:Different fouling mechanisms: (A) Cake filtration, (B) pore blocking, and (C) adsorption 13
FILTRATION: Fouling
• Fouling is, in general, not reversible during a
process.
• In normal flow filtration, filters are commonly
discarded after use.
• For cross-flow filtration, fouling is remedied by a
clean-in-place (CIP) step, which is performed after
the unit operation is complete.
• CIP is typically performed with caustic agents.
• Membrane polarization:
 Occurs when the cross-flow force is not strong
enough to sweep away concentrated material
from the membrane surface within CFF.
 Polarization can be reduced or removed by
increasing the sweeping force (i.e., increasing Figure 8: Polarization
the cross-flow rate or stopping or reducing
the flux).

14
SEPARATION OF PRECIPITATES AND PARTICULATE MATERIAL: CENTRIFUGATION
• Relies on the sedimentation of particles in an increased gravitational field.
• Takes advantage of density differences between the solid and liquid phases present in the suspension.
• In bioprocessing, centrifugation operations are applied for the clarification of fermentation broths and cell lysates,
recovery of inclusion bodies and density gradient separations.
• The G-force or relative centrifugal force is defined as
𝐺= …………………………………………..........................4
ω is the angular velocity
r is the radius of the centrifuge
g is the acceleration due to gravity.
• The settling velocity in a centrifugal field is given by(valid for Re < 1)

𝑣𝑤 = (ρ𝑠 − ρ0)ω2𝑟 …………………………………………...5

vw is the terminal settling velocity,

d is the diameter of the spherical particle,
ρs is the solid density and
ρ0 is the density of the fluid
η is the viscosity of the fluid
15
SEPARATION OF PRECIPITATES AND PARTICULATE MATERIAL: CENTRIFUGATION
• For rigid spherical particles, the time required for a particle to
sediment in a given medium from the meniscus to the bottom of
the tube is given by,

𝑡= 𝑙𝑛 ………………………6
( )

where Xt = radial distance of meniscus

Xb = radial distance of the bottom of the tube
ρs = Density of particle
ρ0 = Density of solution
Figure 9: Forces acting on a particle in a
ω = Angular velocity of rotor centrifuge rotor
η = Viscosity of the fluid
rs = Stokes's radius of particle

10
𝑅𝐶𝐹 = 𝑅𝑃𝑀2 ∗ 1.118 ∗ ∗ r…..……7

16
CENTRIFUGATION: ROTOR TYPES
1. Fixed-angle rotors
• Hold the sample tubes at an angle of 45° in relation to the axis of the
rotor
1
• The particles strike the opposite side of the tube, where they finally slide
down and are collected at the bottom
• faster than other types of rotors as the path length of the tubes increases
• However, as the direction of the force is different from the position of the
tube, some particles might remain at the sides of the tubes
2. Swinging bucket rotors/ Horizontal rotors
• Hold the tubes at 90° as the rotor swings during process start-up.
2
• The tubes are suspended in racks that allow them to be moved enough to
reach the horizontal position.
• The particles are present along the direction of the force, which allows
them to be moved away from the rotor towards the bottom of the tubes.
3. Vertical rotors
• Provide the shortest path length, fastest run time, and the highest
resolution of all the rotors.
• The tubes are vertical during the operation of the centrifuge 3
• Instead of settling down, particles tend to spread towards the outer wall
of the tubes.
• commonly used in isopycnic and density gradient centrifugation.
Beckman coulter 17
CENTRIFUGATION: TYPES
1. Benchtop centrifuge: Driven by an electric motor, where the tubes are rotated about a fixed axis.

2. Continuous flow centrifuge: Allows the centrifugation of large volumes of samples without affecting the sedimentation
rates.

3. Gas centrifuge: Explicitly used for the separation of gases based on their isotopes (e.g. uranium -235 and uranium-238).

4. Hematocrit centrifuge (11,000 rpm and RCFs of up to 15,000 g): For the determination of the volume fraction of
erythrocytes (RBCs) in a given blood sample.

5. High-speed centrifuge: 15,000 to 30,000 rpm.

6. Low-speed centrifuge: 4000-5000 rpm.

7. Microcentrifuge: 12,000-13,000 rpm, for smaller volumes ranging from 0.5 to 2 µl.

8. Refrigerated centrifuges: temperature control ranging from -20°C to -30°C, RCF of up to 60,000 xg.

9. Ultracentrifuges: as high as 150,000 rpm, separate molecules in large batches and in a continuous flow system.

10. Vacuum centrifuge/Concentrators: Utilise centrifugal force, vacuum, and heat to accelerate evaporation of laboratory
samples.
18
SEPARATION OF PRECIPITATES AND PARTICULATE MATERIAL: CENTRIFUGATION
• Process centrifuges include tubular centrifuges and disc centrifuges.
• For large-scale bioprocessing and for continuous operations, the disc centrifuge is commonly
used.
• A disc centrifuge consists of a series of inverted cones stacked with minimal spacing between
them.
• As the feed stream moves upwards, rotation of the discs causes solid particles to move along
the radial direction towards the walls.
• The accumulated solids then move along the wall and can be continuously separated from the
liquid.
• Depending on the centrifugal rotational speed, microbial cells and debris can be easily
separated from the fermentation broth.
• The clear supernatant is withdrawn as the effluent stream.

Figure 10: Schematics

of a disc centrifuge

19
COLUMN CHROMATOGRAPHY
• The name literally means “colour drawing" and was used to describe the
separation of natural pigments on filter papers by differential retardation
(one-dimensional).
• Column of matrix material equivalent to layering together large numbers of
strips of paper (two-dimensional).
• As the solvent moves through the column, solutes present in the original
sample can do one of three things.
I. Completely partitioned into the solvent, they will run with the solvent
front (Rf = 1) and be washed out quickly.
II. Totally adsorbed to the matrix (Rf = 0), they will remain in their starting
position.
III. 0 < Rf < 1, where partial adsorption retards the solute, but eventually it
is eluted.
• Two solutes with closely similar retardation constants (Rf ) would be eluted
with midpoints at different times; if the column is long enough and
longitudinal diffusion is not too great, a complete separation can be
achieved.
• The wide variety of adsorbents suitable for proteins has made column
chromatography the most important basic procedure for protein Figure 11: Principles of chromatography.
separation.
20
SEPARATION BY ADSORPTION
• True chromatography describes the separation of solutes according
to their different partitioning between two phases, stationary and
mobile (in most cases, solid and liquid), resulting in differing
mobilities down a column (or sheet, layer) of solid particles in the
presence of a flowing phase.
• Partition coefficient: The fraction of the solute that is adsorbed on
the stationary phase (mostly solid in column) or the ratio of the
solute's concentration in the stationary phase (Cs)to its
concentration in the mobile phase(Cm).

𝑘𝑑 = ……………………….8

• Components with a higher affinity for the stationary phase have

higher (Kd) values and move slower, while those preferring the
mobile phase move faster.
• Dissociation constant (Kp):
.
𝐾𝑝 = ……………………9
p = concentration of protein in free solution at equilibrium
with the adsorbent
Figure 12: Distribution of solute molecules in an
q = concentration of matrix-bound protein adsorbent column consisting of packed beads
m =concentration of free effective binding sites
21
TYPES OF ADSORBENTS USED IN PROTEIN CHROMATOGRAPHY
• 2 essential features:
I. The nature of the matrix
II. The type of functional group
• Types:
I. Ion exchangers (ion exchange chromatography)
a) Anion exchangers (positively charged matrix), e.g.
diethylaminoethyl- (DEAE-) and quaternary amino
ethyl- (TEAE-, QAE-) substituents attached directly
to hydroxyl groups on the matrix.
b) Cation exchangers (negatively charged matrix): 3
chemical categories.
 "Weak" carboxymethyl substitute (CM)(above
pH 4.5) Figure 13: Structures of the commonly used charged
 “Strong" sulfonate groups (S- and SP)(Below substituents on anion exchangers.
pH 4) (a) diethyl aminoethyl-, DEAE-;
(b) hydroxypropyl diethyl aminoethyl-,(quaternary) Q-;
 Phosphates (out of use)
(c) trimethyl aminoethyl-, TMAE- or Q-;
(d) carboxymethyl-,CM-;
(e) sulfopropyl-, SP- or S-. 22
TYPES OF ADSORBENTS USED IN PROTEIN CHROMATOGRAPHY
II. Hydrophobic and Reverse Phase Adsorbents (hydrophobic
interaction chromatography, HIC)
• Hydrophobic absorbents exploit variability in external
hydrophobic amino acid residues on different proteins.
• Proteins interact with hydrophobic adsorbents because, in
aqueous solvents, hydrophobic patches on proteins seek
out other hydrophobic surfaces preferentially.
• Hydrophobic interactions are strengthened by high salt
concentrations and higher temperatures and are
weakened by the presence of detergents or miscible
organic solvents.
• The most common hydrophobic adsorbents used in
protein chromatography consist of short aliphatic chains
(C4 to C10), or a benzyl (phenyl) group attached through
simple chemistry to hydroxyls on the matrix.
• The extent of binding depends on Figure 14: Structures of some commonly used
a) The type and density of substitution of the matrix hydrophobic adsorbents
b) The nature of the buffer conditions
23
TYPES OF ADSORBENTS USED IN PROTEIN CHROMATOGRAPHY
II. Hydrophobic and Reverse Phase Adsorbents (hydrophobic interaction chromatography, HIC)
• Reverse-phase adsorbents are essentially similar to the hydrophobic, containing aliphatic chains between 8
and 18 carbons long.
• The matrix is hydrophobic(nonpolar), and the solvent, at least in the starting conditions, is polar, mainly water
(opposite of normal phase).
• Highly successful when dealing with small, structurally sturdy proteins, up to about 30,000 Da in size.
• Operates by denaturing the polypeptide chains and exposing the internal hydrophobic residues to the
adsorbent, followed by elution by weakening the interaction forces with organic solvents.
III. Inorganic adsorbents
IV. Affinity Adsorbents: Mimic a natural protein-ligand interaction
a) Highly specific
b) Group adsorbents
• Pseudo-affinity adsorbents: biomimetic, in which the binding site interacts relatively nonspecific
"multifunctional" adsorbents that operate by a variety of types of interactions.
V. Immunoadsorbents and Protein A/G:
• Columns that specifically bind antibodies are in great demand for antibody purification.
• Mostly based on the proteins isolated from Streptococcus sp.
24
ION EXCHANGERS
• Proteins bind to ion exchangers by electrostatic forces
between the proteins' surface charges (mainly) and the dense
clusters of charged groups on the exchangers.
• The charges are balanced by counterions such as metal ions,
chloride ions, and sometimes buffer ions.
• A protein must displace the counterions and become
attached.
• 2 forms of interaction:
Figure 15: Illustration of the "ion exchange" occurring when a
a) Irreversible negatively charged protein adsorbs to an anion exchanger

b) Reversible
• The energy of interaction per mole Table: Change in the energies of interaction of enzymes with cm-
cellulose for each positive charge on the enzyme
𝛥𝐺0 = −2.3𝑅𝑇𝑙𝑜𝑔𝐾𝑝……………………………………10
• The rate of change of energy of interaction per mole per
charge z

= −2.3𝑅𝑇 = −2.3𝑅𝑇𝑠…………...………11

• s =slope of the plot of log Kp against the number of charges on

the protein molecule. 25
ION EXCHANGERS:
• Adsorptive Capacities of Ion Exchangers
 Depends greatly on the size of the protein molecule.
 The smaller molecules adsorb to a greater extent.
 Molecules with molecular weights greater than 106 are likely to be excluded from most cellulose-based ion
exchangers.
• Decisions for choosing an adsorbent
 The charge ( plus or minus) and the nature of the group responsible for that charge.
 The nature of the matrix particles, in terms of bead size, flow rate required under pressure, capacity, and often,
cost per unit volume.
• pH and Donnan Effects
 The pH in the microenvironment of an ion exchanger is not
exactly the same as that of the applied or eluting buffer.
 Donnan effects can repel or attract protons within the
adsorbent matrix and limit the operational pH range of ion
exchangers.
 In general, the pH in the matrix is up to 1 unit higher than
that in the surrounding buffer in anion exchangers, and 1
unit lower in cation exchangers. Figure 16: The Donnan effect on pH in the
microenvironment of ion exchangers 26
ION EXCHANGERS:
• Elution of adsorbed protein: 2 general methods in theory
a) Change the buffer pH to a value where binding is weakened---lower pH for an anion exchanger, or higher pH
for a cation exchanger.
b) Increase the ionic strength, thereby weakening the electrostatic interaction between the protein and the
adsorbent.
 Salt gradients are most commonly used, usually either potassium or sodium chloride.
 The action of the salts can be considered in one of two ways.
a) The salt can directly displace the protein.
b) The system can be regarded as an equilibrium in which even strongly bound proteins spend some time not
adsorbed; the presence of the salt ions between the unattached protein and the adsorbent greatly weakens
the attraction between the two.
• Choice of ion exchanger

Table: Choice of ion exchanger for purification of a protein with a known isoelectric point

27
INORGANIC ADSORBENTS:
• Include oxides, insoluble hydroxides, and phosphates.
• The most important example is calcium hydroxyphosphate (hydroxyapatite); its gelatinous form, calcium phosphate gel (suitable for
batch adsorption)
• Advantage: low cost (especially in large-scale industrial applications), so reuse is often unnecessary.
• Mode of action: not as clearly explained.
• Protein adsorption likely involves electrostatic interactions and possibly polar dipole-dipole bonding on the crystal surface.
• Adsorption pH range: most likely 6–9, where proteins often contain adjacent positive and negative groups.
• In practice, adsorption is more complex because buffer
ions, especially phosphate, also adsorb to the inorganic
surface and can alter its charge.
• Low salt concentration favours adsorption, while higher
salt concentration promotes elution; in columns,
• Elution can be achieved using a salt gradient.
• These adsorbents may be used either to bind the target
protein or to remove unwanted contaminants while
leaving the desired protein in solution.
• Though considered crude and old-fashioned, they Figure 17: Dipolar interactions of proteins on inorganic salt surface:
remain useful and economical for commercial-scale (a) Simple interaction, (b) with the intervention of buffer ions.
protein purification
28
HYDROPHOBIC ADSORBENTS:
• Discovery: Spacer arms (e.g. hexamethylene chains) without ligands unexpectedly bound proteins via hydrophobic
interactions between aliphatic chains and hydrophobic protein regions.
• Mechanism: Hydrophobic patches on proteins bind immobilised alkyl chains.
• Common adsorbents: C4-, C6-, C8-, C10-alkyl chains, phenyl-Sepharose, and terminal amino-modified chains
(multifunctional).
• Conditions: Adsorption at high salt for hydrophilic proteins, and at low salt for hydrophobic proteins.
• Advantages: High capacity (10-100 mg/cm³), excellent recovery due to salt stabilisation, works with crude samples.
• Limitations: Less sharp separations than ion exchange due to slow association-dissociation, protein-protein interactions
cause peak overlap.
• Applications: Complements ion exchange; useful when other methods fail; ideal for proteins stable at high salt.
• Elution of Protein from Hydrophobic Columns:

Highly hydrophobic proteins (Low salt sample application) Highly hydrophobic proteins (high salt sample application)
Lowering of temperature Lowering salt concentration
Inclusion of organic solvent
Inclusion of polyols, especially ethylene glycol
Inclusion of detergent
By increasing the pH 29
HYDROPHOBIC ADSORBENTS: REVERSE PHASE CHROMATOGRAPHY
• Uses hydrophobic adsorbents (C8-C18 chains on silica beads); sample applied in aqueous solvent (often dilute acid),
eluted with organic solvent gradient (methanol/acetonitrile).
• Protein behavior: Smaller proteins resolve earlier; proteins >40 kDa may not elute readily.
• Mechanism: Proposed denaturation on adsorption, renaturation on elution.
• Suitable for: Sturdy proteins that withstand chromatography conditions, or where inactive proteins are acceptable.
• Strength: Very high resolution; widely used for peptide separations (enzymic digests).
• Applications: Small, acid-stable therapeutic proteins/peptides that resist denaturation.
• Limitations: Not generally suitable for sensitive intracellular proteins and enzymes.

30
IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)
• Technique relies on weak coordinate bonds between
immobilized metal ions and protein basic groups (mainly
histidine residues).
• Adsorbent: Matrix + spacer arm + chelator (IDA or TED); binds
divalent transition metals (Fe, Co, Ni, Cu, Zn) or trivalent (Fe, Al).
• Mechanism: Chelator leaves metal with spare coordination sites
(occupied by water/buffer); these are displaced by protein
groups.
• Protein binding: Histidine is most attracted; also tryptophan,
cysteine (neutral pH); residues must be surface-located. Figure 18: IMAC working
• Selectivity: A single histidine in small proteins is sufficient (kp
~10⁻⁶ M); most crude extract proteins don't bind strongly
• Examples:
i. Zn-TED (Tris-carboxymethyl ethylenediamine) column:
Specifically binds α₂-macroglobulins from serum (others
pass through).
ii. Fe(III)-IDA: Binds phosphorylase and lactate dehydrogenase Figure 19: Structure
from muscle extract. of three types of
• Affinity-like behaviour: Not biospecific (no natural ligand immobilized metal ion
relationship) but selective for specific proteins. chelating ligands
31
AFFINITY CHROMATOGRAPHY
• Principle
 Developed during the 1960s and 1970s,
 Referred originally to the use of an immobilized natural ligand, which
specifically interacts with the desired protein (biospecific interaction).
 The ligand is immobilized on suitable particles which can be packed
into a column.
 A sample containing the protein is passed into the column.
 The specific interaction holds back the desired protein, while others
pass through.
• The main requirements for a successful affinity adsorbent
 The ligand should be attached to the matrix in such a way that the
ligand's binding to the protein concerned is not seriously disturbed.
 A "spacer arm" setting the ligand away from the matrix should be used Figure 20: Basic principle of affinity
to make it more accessible to the protein. adsorption chromatography.
 Nonspecific interactions should not be so great that many other
proteins are adsorbed in addition to the one required.
 The linkages should be stable under the likely conditions to be used
during the chromatography, including "cleaning-up" procedures before
reuse. 32
IMMUNOADSORBENTS
• Ultimate adsorbent: Immobilized antibody raised specifically against the target protein for reuse.
• Specificity: Binds a single surface feature of a protein (not necessarily a ligand binding site).
• Binding strength: Recognizes exact amino acid sequence + 3D organization; very high binding constants (low kp)
• Common challenge: Too-tight binding is often problematic for elution.
• Antibody types:
 Polyclonal: Multiple antibodies
 Monoclonal: Single antibody species
• Reproducibility limitation: Depends on the availability of the same antibody preparation between labs.

33
IMMUNOADSORBENTS
• Considerations for the choice of type of antibody:
1. The availability of facilities and expertise for monoclonal work.
2. The ultimate aim of the exercise (e.g., is this a one-off purification for research purposes, or is there a
foreseeable continuous requirement for the product?)
3. The quantities of the end product needed.
4. The overall costs of the procedure (particularly important for a long-term commercial operation).
5. Does the simplest method give the best product?
• Relative Advantages of Polyclonal and Monoclonal Antibodies

34
SEPARATION IN SOLUTION: GEL FILTRATION
• Other names: gel exclusion and molecular sieving.
• Gel permeation: Sometimes used interchangeably, but uses an organic solvent.
• Rapid separation of macromolecules based on size.
• The gel consists of an open, cross-linked, three-dimensional molecular network, cast in bead
form for easy column packing and optimum flow characteristics.
• The pores within the beads are of such sizes that some are not accessible by large
molecules, but smaller molecules can penetrate all pores. Figure 21: Two-dimensional
representation of pores of gel
• Under optimum flow conditions, all accessible pores can be filled as the proteins pass by. filtration material and
Therefore, equilibrium rather than kinetic effects are involved. accessibility of various areas by
molecules of different sizes.
• Examples: Sephadex (dextran based), Sepharose(agarose based), Superdex(composite resin
of dextran and agarose.

Figure 23: Scale diagram to illustrate the relative sizes of molecules and beads used in gel filtration
Figure 22: Principles of gel filtration to separate
different-sized molecules in a column 35
SEPARATION IN SOLUTION: GEL FILTRATION

Figure 24: Prepacked column from Tofflon Life

sciences

Figure 26: Low-pressure preparative

chromatography columns

Figure 27: Novasep LC 60 high

pressure column
Figure 25: XK empty column from Cytiva 36
REFERENCES
• [Link]
processing.
• Chapter 14, Filtration Principles, Jakob Liderfelt, Jonathan Royce, GE Healthcare Life Sciences, Uppsala,
Sweden.
• Protein purification. Principles and Practice. Robert K. Scopes.
• [Link]
innovator/how-separation-works/how-a-disc-stack-centrifuge-works/
• [Link]
42/[Link]
• [Link]

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