Purification by chromatography and formulation

1. Purification by Chromatography
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR

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3. Introduction:
 The biological products of fermentation (proteins, pharmaceuticals,
diagnostic compounds and research materials) are very effectively
purified by chromatography.
 Chromatography usually consists of a stationary phase and
mobile phase.

4.  The stationary phase is the porous solid matrix packed in a column
(equilibrated with a suitable solvent) on to which the mixture of
compounds to be separated is loaded.
 The compounds are eluted by a mobile phase.

5. The different types of chromatography techniques used for
separation of desired product(mainly proteins):
Chromatography Principle
Ion exchange chromatography Net charge
Gel filtration chromatography Size and shape
Affinity chromatography Net charge or
Biological affinity
and Molecular
recognition

6. Ion-exchange chromatography:
Ion-exchange chromatography depends on the ionic
bonding of proteins to an inert matrix material.

7. Ion-exchange chromatography:
 Two of the most commonly employed ion-exchange resins (inert
matrix material) are:
 Diethylaminoethyl cellulose: (+) (binds to negative charged
molecules: Anion exchanger)(DEAE) and
 Carboxymethyl cellulose (-) (CM): Cation exchanger.

8. Column
pH: 11 buffer

9.  It involves the separation of molecules based on their surface
charges.
 Ion-exchangers are of two types (cation- exchangers which have
negatively charged groups like carboxymethyl and sulfonate, and
anion- exchangers with positively charged groups like
diethylaminoethyl (DEAE).

10.  In ion-exchange chromatography, the pH of the medium is very
crucial.
 The ionic bound molecules can be eluted from the matrix by
changing the pH of the elutant buffer.
 Ion-exchange chromatography is useful for the purification of
antibiotics, besides the purification of proteins.

11.  The resin is packed into a column, and the protein solution is
allowed through the column in a buffer whose composition
promotes the binding of some or all of the proteins to the resin.
 Proteins are bound to the resin reversibly and can be displaced
by increasing or changing the ionic strength (or pH) of the
buffer. (which adds small ions to compete with the charged
groups of the macromolecules for sites on the resin).
 Proteins are eluted from the column in order from the least
strongly bound to the most strongly bound.

12. Gel Filtration Chromatography
or
Size exclusion chromatography :

13. Gel Filtration Chromatography:
Agarose
or
Sephadex
G-150
beads are
generally
used in
chromato
grphy
column

14. For example if a solution consists of two different proteins such
as 75 kD and 120 kDa loaded on top of the colum.

15.  Gel filtration separates proteins (or nucleic acids) primarily on the
basis of their effective size.
 Like ion-exchange chromatography, the separation material consists
of gel beads that are packed into a column through which the
protein solution slowly passes.
 The materials used in gel filtration are composed of cross-linked
polysaccharides (agarose or Sephadex G-150 beads) of different
porosity, which allow proteins to diffuse in and out of the beads.

16.  For example if a solution consists of three different proteins such as
120 kDa, 75 kDa and 25 kDa.
 To purify 120 kDa protein form mixture, the sample pass through a
column of Sephadex G-150 beads.
 When the protein mixture passes through the column bed, the 120
kDa protein is unable to enter the beads and remains dissolved in the
moving solvent phase.
 The gel beads allows only the entry of proteins that are less than
about 100 kDa size.

17.  As a result, the 120 kDa protein is eluted as soon as the preexisting
solvent in the column (the bed volume) has dripped out.
 In contrast, the other two proteins can diffuse into the interstices
within the beads and are retarded in their passage through the
column.
 As more and more solvent moves through the column, these proteins
move down its length and out the bottom, but they do so at different
rates.
 Among those proteins that enter the beads, smaller species are
retarded to a greater extent than larger ones.
 Consequently, the 120-kDa protein is eluted in a purified state, while
the 75-kDa and 25 kDa protein remains in the column.

18. Affinity chromatography:

19. Affinity chromatography:

20. Affinity chromatography:
 Affinity chromatography is based on an interaction of a protein with an
immobilized ligand.
 Such interactions including hydrogen bonding, ionic interaction,
disulfide bridges, hydrophobic interaction,
 The ligand can be a specific antibody, substrate, or an inhibitor or
antigen or enzyme.
 The protein bound to the ligand can be eluted by reducing their
interaction. This can be achieved by changing the pH of the buffer.

21. Formulation:
Formulation broadly refers to the maintenance of activity and
stability of a biological products during storage and
distribution.

22. Formulation:
 For certain small molecules like (antibiotics, citric acid),
formulation can be done by crystallization by adding salts.
 The formulation of low molecular weight products can be achieved
by concentrating them with removal of the water

23.  Proteins may be formulated in the form of solutions, or dry
powders.
 The sugars (sucrose, lactose), salts (sodium chloride, ammonium
sulfate), glycerol used as stabilizers for protein formulation.
 Vaccines are formulated by mixing with fluids (such as saline).

24. Drying (Formulation):
 Drying is an essential component of product formulation.
 It basically involves the transfer of heat to a wet product for
removal of moisture.

25.  These two types of dryers are commercially available.
 Spray drying:
 Freeze-drying:

26. Spray drying:
 Spray drying is a method of producing a dry powder from a liquid.
 This method preferred method of drying of many thermally-
sensitive materials such as foods and pharmaceuticals.
stream of hot gas
Spray dryer

27.  Spray drying is used for drying large volumes of liquids.
 In spray drying, liquid containing the product are passed through a
nozzle directing it over a stream of hot gas. (Liquid pumps in the
form of droplets).
 The water evaporates and the solid particles are left behind.
 This method used for formation of milk powder.
 Formulation of vitamins, enzymes, amino acids, antibiotics.

28. Freeze-drying:
 Freeze-drying or lyophilization is the most preferred method for
formulation of a wide-range of products.
 Antibiotics, bacteria, viruses are formulated by using these process.
 In this method, the product is rapidly frozen at a very low
temperature (-70°C) and then dehydrated by vacuum.

29. • Under these conditions, the microbial cells are dehydrated and
their metabolic activities are stopped;
• as a result, the microbes go into dormant state and retain viability
for years.

32. THANK Y OU: