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Introduction to protein purification and analytical science

Talk at Teesside University: 16 April 12

mark carlile

on 1 November 2013

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Transcript of Introduction to protein purification and analytical science

Introduction to Protein Purification
Thank you for your attention!
Recap on protein therapeutics
Purification science:
Primary separations
Capture chromatography
Product formulation
Dr Mark Carlile
Undergraduate degree: Applied Biochemistry
Postgraduate degree: Molecular genetics
The biology of endogenous antisense transcripts in developmental patterning

Fermentation scientist - Avecia Biologics
Purification Scientist - Avecia, Merck, FujiFilm

Currently: Principal Scientist FujiFilm
Chemical properies
Physical properties
Size and shape
Centrifugation and filtration are used to give a more clarified sample to take forward for purification

What you want:
To remove cells (and cell debris) from growth media
Primary Separations
What is your expression host cell?
E.coli, yeast, mammalian cell, plant cell
What is your expression format?
Soluble or insoluble
Intracellular or extracellular
Are you using an expression fusion or co-expression partner protein
The next steps
Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.
Washed inclusion bodies - still unfolded

Controllable refolding
Refolding of proteins
Once you have a purified product you need to:
protect it from degradation
get it into an injectable/formulated buffer
get it to the correct concentration

Use methods that can remove any remaining contaminants and allow extra components to be added to the final material
Protein formulation
Dr Mark Carlile
Fujifilm Diosynth Biotechnologies
The story so far ...
Molecular Biology
Fermentation (expression)
Fermentation Broth
(What now?)
Differential centrifugation can be utilised as a means to isolate cellular components for further downstream purification
Differential Centrifugation
Expanded fluidised bed
Principal based on a movable adaptor
Loading in upflow = resin expansion
Post loading in downflow = resin compression
Normal Chromatography thereafter
Expanded Bed Chromatography
Sucrose Density
Equilibrium Centrifugation
Membrane isolation
Subcellular fractionation
Resin Bed
Keep this in mind for later
Breaking cells
The use of high pressures to break cells - mechanical disruption
Use of high frequency sound waves - mechanical disruption
Cell Lysis Agents
Use of cell permeabilising agents (Lysozyme) - Chemical disruption
Intracellular expression:
Insoluble protein aggregates
Mass of unfolded protein - expression rate stops correct folding

The physical nature of the protein mass enables a quick and easy purification:

Cell lysis
Inclusion body harvest - centrifugation
Inclusion body washing (water, detergents)
Inclusion bodies
Intracellular soluble proteins.
Water soluble
Made inside and stay inside the cell – cytoplasmic or periplasmic
Cytoplasmic insoluble proteins
Inclusion bodies - protein aggregates
High concentrations of target
High initial purity
Periplasmic proteins
Made inside the cytoplasm, transported to the periplasm
Oxidising environment which allows disulphide bridges to form
Extracellular proteins
Made in the cytoplasm
Secreted out of the cell
Expression Format
Each format directs a slightly different initial purification strategy
Relatively pure protein mass
+ Chaotropic agents (Urea, Gunadine HCl)
+ Reducing agents (DTT, Mecapto-EtOH)
Protein Conc., pH
Removal of chaotropic agent
+ Oxidising agent
Protein Conc., pH
Rate of addition
Protein Folding
The driving force in protein folding is the hydrophobic residues coming together to generate a hydrophobic core
The multiple states of the unfolded protein located at the top fall into a folding funnel consisting of an almost infinite number of local minima, each of which describes possible folding arrangements in the protein. Most of these states represent transient folding intermediates in the process of attaining the correct native fold. Some of these intermediates retain a more stable structure such as the molten globule, whereas other local minima act as folding traps irreversibly capturing the protein in a misfolded state
The levinthal paradox
A polypeptide of 100 residues will have 99 peptide bonds, and therefore 198 different peptide backbone bond angles.

If protein folding take place on a "try-and-see" basis then a lot of conformations would need to be tried.
If a protein were to attain its correctly folded configuration by sequentially sampling all the possible conformations, it would require a time longer than the age of the universe to arrive at its correct native conformation.
For protein separation - usually use packed-bed chromatography
Control Unit
(Packed resin)
Chromatography Applications
Affinity chromatography (AC)

Ion Exchange Chromatography (IEX)

Hydrophobic Interaction Chromatography (HIC)
Affinity chromatography is a method of separating biochemical mixtures and based on a highly specific interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand. Examples: Immunoaffinity or Immobilized Metal Ion
Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their charge.
Charge can be controlled via pH
Both Anion (negative charge) and cation (positive charge) applications
Hydrophobic interaction chromatography is a method that uses a stationary phase that contains hydrophobic ligands bound to the resin. These hydrophobic ligands (-phenyl, -butyl, -octyl) bind to hydrophobic regions on the protein.
The charge on the protein affects its behavior in ion exchange chromatography. Proteins contain many ionizable groups on the side chains of their amino acids including their amino - and carboxyl - termini. These include basic groups on the side chains of lysine, arginine and histidine and acidic groups on the side chains or glutamate, aspartate, cysteine and tyrosine. The pH of the solution, the pK of the side chain and the side chain’s environment influence the charge on each side chain.
IEX Chromatography
HIC Chromatography
R-NH3+ --> R-NH2
Protein pI
Negative charge (-)

Positive Charge (+)
Q Sepharose HP
Strong Anion Exchanger (-ve)

Separation of refolded protein
1 - correctly refolded product
2 - soluble aggregates
3 - misfolded material
4 - High MW aggregates
IEX Chromatography
Gradient elution - controllable separation of different protein forms

Higher charge = Tighter Binding
HIC Chromatography
Based upon an enzyme-substrate-type interaction
Affinity Chromatography
Effect of pH on HIC
As pH increases above pI HIC binding decreases
But as pH decreases below pI binding seems to increase for some proteins

There are some elements of HIC chromatography that are still not fully understood
Binding of protein is based on the selective interaction between the Glutathione-S-Transferase tag of the recombinant protein and the immobilized glutathione of the affinity resin. The Glutathione-S-Transferase tag is a large affinity tag consisting of 220 amino acids (26 kDa) forming dimer structures.
Elution: via addition of free glutathione
Highly specific = high purification efficiency
High cost
Charging of the resin - metal ions
Low [imidazole] in binding/wash phases - 20 mM
High [imidazole] in eleution buffer - 200 mM

Low pH can be used also for elution
Separation based upon size - gel filtration
Larger molecules elute first
Not easy to use at large scale - good for buffer exchange operations
Size Exclusion Chromatoraphy
Ultrafiltration (UF) is a variety of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molecular weight are retained, while water and low molecular weight solutes pass through the membrane.

Concentration: volume of sample decreases whilst level of protein remains constant

Buffer exchange: feed in new buffer at same rate as permeate exits
Sample volume constant
Keep adding new buffer until entire buffer volume has been changed 5-10 times
Purified product needs to be maintained over time
Buffer components:
- stable pH (usually physiological pH)
- cryo-preservation agents
- stabilising agents
- antibacterial agents

Filtration of final material
Final formulation
40 mL
4000 mL
20 cm
Use of same bed height but increased column diameter
Use of linear flow rate (cm/h)
Increase the filtration membrane area
Keep the same flux L/m2/h
Primary separations

Primary Capture Chromatography

Polishing Chromatography (2-5)

Final Formulation
Generic purification strategy
Product Amount

1 - 10 g/L of sample

0.8 - 4 g/L of sample

0.4 - 2 g/L of sample

0.2 - 1 g/L of sample
The best way to describe protein purification is sophisticated fishing!!

But you have a very large set of tools to help you out

You have to use the physics and biochemistry knowledge you have available before casting your line!!
Cell Harvest Homogenate Washed Inclusion Bodies
Inclusion Body Harvest
Uncontrollably dry your sample
Evaporation under vacuum
Very good for long term stable storage
Freeze drying
Above 100L-scale you move to a continuous system
No pellets - thick "slurry"
Difficult to purify proteins
Membrane proteins
Membrane receptors/transporters generated as recombinant therapeutics are quite scarce
Hydrophobic membrane spanning domains.
Full transcript