Affinity chromatography as per PCI

The presentation covers the principle, operation technique and applications of affinity chromatography

1. Affinity Chromatography

2. Introduction

3. Introduction

4. Thank You

5. Operation technique:

Binding of biomolecule to the ligand of

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the solid medium (resin) is packed into a it.

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from the support.

Add clution buffer to column and
retain the flow through

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‘Separated biomolecule Example: Interaction favors at pH 2 : Use for incubation
No interaction at pH 6 : Use for elution

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6. Immoblized
Receptor

Separation Elution

2. Washing away |

non-bound sample
components from
the support.

conditions

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7. Es
€, Preparation of stationary phase

・ The chromatographic resin is packed into a column. 7 à

・ Short or wide columns can be used for separation as per need < 从 后 y

+ The specific ligand is linked by the linker to the chromatographic |” =
matrix. omy, a

+ Various activated reactive chromatographic matrices are also
available for this purpose

Ligand in the Affinity media Separation of biomolecule

Amino Acid Variety of serum proteins, peptides, enzymes

Lectins Carbohydrate, glycoproteins

Glutathione Glutathione S-transferase (GST) tagged recombinant
proteins

Heparin Plasma coagulation proteins, lipases

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8. 1. Preparation of stationary phase

Ligand in the Affinity media Separation of biomolecule
Nucleotide/Coenzyme Dehydrogenases, kinases, and transaminases
Nucleic Acid mRNA, DNA, rRNA
Immunoglobulins Protein A/G
Hormone receptor Hormones
Antigen Antibody
Metal chelate His-tag fusion protein

| Of the molecules present in the sample, only the ones having a “matching” binding site can bind to the
specific ligand molecules. Other molecules can be readily washed off.

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9. A 2 Sample preparation
The sample must be a clear solution free from solid
particles.
・ This can be achieved by centrifugation or filtration.
・ Protein solutions should be centrifuged at least 10000 g.
Cell lysates should be centrifuged at 40-50000 g.
+ A 0.45-um pore size filter can be used for filtration.

It is also necessary to consider how the solubility and

stability of the sample biomolecule can be influenced by

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> Salt concentration

> The presence of any organic solvent

・ These factors affect the interactions between the desired
target protein and the matrix-bound ligand.

・ The composition of the initial binding buffer must be
adjusted accordingly.

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10. rer Elution of the molecules of interest

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3. Sample application and Equilibration with a buffer facilitating the

specific interaction

・ The chromatographic column is washed with 3-4 column volumes of
the starting (binding) buffer.

・ The sample must also be equilibrated with this starting binding buffer.

・ During sample loading. consider the strength of the interaction.

* Incase of high-affinity samples, a high flow rate may be applied.

・ Incase of a weak interaction reduce the rate of sample loading.

* sample application. the column should be further washed with binding
buffer until all unbound components are removed.

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Elution via pH change or ionic strength changes YAA octal
Elution by changing the composition of the mobile phase 7 3
Competitive elution ： y
Use of chaotropic agents i E -

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11. a 4. Elution of the molecules of interest

A. Elution via pH and/or ionic strength changes

One possible and simple means of elution is achieved
through decreasing the interaction strength between
the ligand and the target protein.

Changes in the pH will change the ionization state of
charged groups of the ligand and/or the target protein,
thereby changing the strength of the interaction.

Similarly, increasing the ionic strength (usually by
raising the NaCl concentration) will generally reduce
the interaction strength between ligand and target
protein.

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Medium,

B. Elution by changing the composition of the mobile phase
+ ‘The molecules of interest can be isolated in a pure form by
changing the composition of the mobile phase.

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12. a 4. Elution of the molecules of interest

C. Competitive elution

For competitive elution, materials are applied that
react with the ligand, competing for the pre-existing
interaction.

For instance, different proteins can be readily
displaced from the metal chelate matrix by imidazole
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D. Use of chaotropic agents

+ In cases when the target-ligand interaction is very strong. the
above elution methods may turn out insufficient for eluting the
protein of interest.

+ In these cases, chaotropic agents like urea or guanidine can be used
to wash off the target protein from the column.

+ This naturally will involve the denaturation of the protein, which
can then be renatured in some cases under suitable conditions

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13. Applications

1) Purification of biomolecules

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Affinity chromatographic purification is frequently of great
importance in the case of recombinant proteins.

Recombinant proteins are often produced in a way that they
contain a fused “label” at their N- or C-terminal, resulting
from genetic cnginccring.

This way, if the label gives the protein an ability to enter into
affinity binding, the recombinant protein can be simply “fished
out” of the cell extract via affinity chromatography

of stationary phase (resin)

Purification of recombinant proteins

Label of the protein favors binding with ligand

Cell extract

Recombinant proteins

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Applications
1) Purification of biomolecules

Example:

・ One of the most widely used label which is
fused to protein terminal is tag glutathione
S-transferase (GST), which is fused to N
terminal of recombinant protein

・ GST of recombinant protein binds to a
glutathione-conjugate matrix (Ligand of the
stationary phase)

+ Elution of GST tagged recombinant protein is
then carried out by adding excess of
glutathione in elution buffer as discussed
earlier

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15. Applications

2) Separation of glycoproteins from non-glycosylated proteins, or
separation of one glycoform from another glycoform

・ Lectin affinity chromatography is a form of affinity
chromatography where lectins are used to separate components
within the sample.

・ Lectins are the proteins which can bind to specific carbohydrate
(sugar) molecules.

・ The most common application is to separate glycoproteins from
non-glycosylated proteins, or one glycoform from another
glvcoform

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16. Applications

3) Immunoglobulin purification
(antibody immobilization)

・ Antibodies can be immobilized by both covalent and
adsorption methods

・ Random covalent immobilization methods generally link
antibodies to the solid support via their free amine groups
using cyanogen bromide, tresyl chloride, or tosyl chloride.

・ Once these antibodies are immobilized, selective compounds
with high affinity towards immunoglobulins can be separated
and hence can be purified

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17. Applications
4) Protein A, G, and L purification

Proteins A, G. and L are native or recombinant proteins of microbial origin which bind
specifically to immunoglobulins including immunoglobulin G (IgG, 80% of serum
immunoglobulins.)

+ Native and recombinant protein A can be cloned in Staphylococcus aureus
+ Recombinant protein G (cell surface protein) is cloned in Streptococcus
+ Recombinant protein L is cloned from Peptostreptococcus magnus.

+ Both protein A and G specifically bind the Fe region of IgG
・ Protein L binds to the kappa light chains of IgG

The most popular matrixes or supports for affinity applications which utilize protein A, G.
or L is IgG beaded agarose

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18. REFERENCES

1. PT Mane et al., Basics of Instrumental Analysis, Volume — I, Marvin Press,
2017, New Delhi, India.

2. A.V. Kasture et al., Pharmaceutical Analysis, Volume — IL Nirali Publication,
2018, Pune, India

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