1. Purification of Enzymes
Basic science:
Its specificity for substrates
Kinetic parameters
Means of regulation
Structure
Mechanism of catalysis
Understand the role of enzymes in more complex systems
Use in medical and industrial applications

2. Initial Recovery of Protein
Intracellular or Extracellular?
Cell Disruption
Animal cells (no CW):
Potter homogenizer
Osmotic shock
Freeze-thaw cycles
Plant cells (CW):
The Waring blender

3. Microbial cells (CW):

4. Removal of Whole Cells and Debris-1
Centrifugation; batch vs. continuous-flow
5000 g for 15 min for cells
10 000g for 45 min for cell debris
High capital and running costs
Filtration; depth vs. membrane filters ( μm)
Separation of whole cells from fermantation media
Removal of whole cells and cell debris after cell disruption
Elimination of microbial species from product

5. Removal of Whole Cells and Debris-2
Aqueous two-phase partitioning
Gentle
Stabilization of
proteins
Yield of protein
activity high
Easy scale-up
Empirical

6. Removal of Whole Cells and Debris-3
Removal of nucleic acids
Liberation of large amounts of nucleic acids increases viscosity of cellular homogenate
 difficult to process
Nucleic acid removal is especially important in the preparation of therapeutic proteins
Methods: precipitation (by polyethylenimine) or treatment with nucleases
Removal of lipids
It is a contaminant and can interfere with subsequent purification steps
Removal: Glass wool or a cloth of very fine mesh size

7. Concentration and Primary Purification
Large volumes of  Manageable
dilute solution amount
In laboratory scale:
Ultrafiltration
Precipitation
Ion-exchange
Dialysis
Freeze drying
Addition of dry Sephadex G-25

8. Concentration by precipitation-1

9. Concentration by precipitation-2
One of the oldest methods
Straight forward to perform
Uncomplicated equipment
High recovery of biological activity
Many precipitants are highly corrosive
Inefficient if initial protein concentration is low
Some precipitants are highy inflammable, some are expensive
Many precipitants must be disposed carefully
In many cases, precipitant must be removed totally

10. Concentration by Ion-Exchange-1
Isoelectronic point of proteins are different
(+)ly charged proteins  cation exchanger (CM)
(-)ly charged proteins  anion exchanger(DEAE)
Elution with a high ionic strength solution

11. Concentration by Ion-Exchange-2
Batchwise:
Extracellular proteins from fermentation broths or cell culture media
Cell debris from cell homogenates
Effective and relatively inexpensive
Easily regenerated
Considerable clarification of solution
Limited amount of protein purification

12. Concentration by ultrafiltration-1
Most widely applied method both in laboratory and industrial scale
Ultrafiltration membranes (pore diameters: 1 – 20 nm)
Molecular mass cut-off: 1 – 300 kDa (globular proteins)
Traditional materials: cellulose acetate and cellulose nitrate
Nowadays: PVC and polycarbonate
Concentration polarization can be a problem...

13. Concentration by ultrafiltration-2
Gentle
High recovery rates (even > 99 %)
Quick
Little ancillary equipment is needed
Some degree of protein purification
Susceptibility to rapid membrane clogging

14. Column Chromatography
Separation of different protein types from each other according to their differential partitioning between two phases:
A solid stationary phase
A liquid mobile phase
Separation based on size and shape, overall charge, presence of surface hydrophobic groups, and ability to bind various ligands

15. Different Chromatographic Techniques

16. Gel Filtration Chromatography-1
Also named as Size Exclusion Chromatography
Separation based on size and shape
Porous gel matrix in bead form is used:
e.g. xlinked dextran, agarose, acrylamide
Large proteins come first....

17. Gel Filtration Chromatography-2

18. Gel Filtration Chromatography-3
EXAMPLES
Sephadex: dextran based, G-25 to G-200
Sephacryl: allyl dextran based, more rigid and physically stable so suitable for large scale
Sepharose: agarose based, lack of physical stability
Bio-Gel P: acrylamide based
A: agarose based
Fractogel: A copolymer, very high degree of mechanical stability

19. Gel Filtration Chromatography-4
Long chromatographic columns are needed (length/width = 25-40)
Rarely employed during the initial stages
Protein solution is significantly diluted
Column flowrates are often considerably lower

20. Ion-Exchange Chromatography-1
FACTS
Proteins possess both (+) and (-) charges
At pH=7:
Aspartic and glutamic acid have negatively charged side groups
Lysine, arginine, histidine have positively charged side groups
pH of medium vs. pI of protein

21. Ion-Exchange Chromatography-2
PRINCIPLE
Reversible electrostatic attraction of a charged molecule to a solid matrix possessing opposite charge
Elution is done by increasing salt concentration or changing pH

22. Ion-Exchange Chromatography-3
Single most popular chromatographic technique...
High level of resolution
Easy scale-up
Ease of usage
Easy column regeneration
One of the least expensive

23. Ion-Exchange Chromatography-4
Inert, rigid and porous matrix materials are desirable
EXAMPLES
Cellulose-based
Improved cellulose-based, e.g. diethylaminoethyl (DEAE) Sephacel
Sephadex; charged groups attached to Sephadex G-25 or G-50
Based on polymers: Agarose and Sepharose
Alternative one: tentacle type

24. Hydrophobic Interaction Chromatography-1
8 out 20 commonly found a.acids in proteins are classified as hydrophobic
In most proteins the majority of hydrophobic residues are buried inside the protein
Different proteins have different degree of hydrophobic surface

25. Hydrophobic Interaction Chromatography-2
EXAMPLES
Most popular resins are hydrophobic group attached xlinked agarose gels
e.g. octyl- and phenyl-Sepharose gels

26. Affinity Chromatography-1
Described as the most powerful highly selective method
It relies on the ability of most proteins to bind specifically and reversibly to their ligands
Generally used in late purification steps

27. Affinity Chromatography-2
Biospecific affinity chromatography
General ligand approach: Cofactors (NAD+) or lectins
Specific ligand approach: enzyme substrates, substrate analogues or inhibitors, antibodies
Pseudoaffinity chromatography: e.g. Dye affinity chromatography

28. Affinity Chromatography-3
Biospecific Affinity Chromatography
Choice of affinity ligand
Specificity, reversible binding, stability
Choice of support matrix
Stability, rigidity, inertness, porosity, derivatizable, inexpensive, reusable
e.g. agarose, cellulose, silica and various organic polymers
Choice of chemical coupling technology
nonhazardous, inexpensive, rapid. Spacer arm?

29. Affinity Chromatography-4
Increase in purity of over 1000-fold, with almost 100 % yields are reported (at least in lab scale)
Drastically reduce number of subsequent steps
Ligands are extremely expensive and often exhibit poor stability
Ligand coupling techniques are chemically complex, hazardous, time-consuming and costly
Leaching of ligand causes:
The reduction of system effectiveness
The presence of undesirable contaminant in product

30. Affinity Chromatography-4
Immunoaffinity purification
Polyclonal antibodies: low binding capacity, some other proteins can also bind
Monoclonal antibodies: monospecific
Relatively high cost technique
Antibody leakage may occur
Elution is difficult (e.g. glycine-HCl buffer with pH )

31. Affinity Chromatography-5
Lectin affinity chromatography
Lectins are a group of proteins synthesized by plants, vertebrates and some invertabrates (e.g. concanavalin A, soybean lectin)
In glycoprotein purification
Many lectins are expensive
Co-purification of glycoproteins
Little track record

32. Affinity Chromatography-7
Dye affinity chromatography
Triazine dyes (e.g. cibacron blue F3G-A) are used
Dyes are available in bulk and relatively inexpensive
Chemical coupling is easy
Dye-matrix linkage is relatively resistant
The protein binding capacity is high
Elution is relatively easy
Textile dyes contain varying amount of impurities
Highly empirical

33. Affinity Chromatography-8
Metal chelate affinity chromatography
Iminodiacetic acid (IDA)
e.g. Ni, Cu, Zn, Fe
Basic groups, mostly
side chain of His
Mostly used in recombinant protein purification

34. Chromatofocussing Separation based on isoelectic point of proteins
Pre-equilibrate a ion-exchange column at a pH
Pour slowly a buffer of different pH
Due to natural buffering capacity of exchanger, pH gradient will occur along the column
Usually a weak anion exchanger is used
Pre-equilibrated with high pH value
Pass a low pH value buffer

35. High Pressure Liquid Chromatography-1 (HPLC)
Microparticulate stationary phase media of narrow diameter is used
Time for diffusion is reduced
Sample fractionation time is reduced
BUT pressure increases
Ideal support material
Mechanically & chemically stable
Low degree of non-specific adsorption
Reusable and inexpensive
Available in small size with narrow distribution
High degree of porosity
Silica gel, xlinked polystyrene are generally used

36. High Pressure Liquid Chromatography-2
Preparative HPLC
Length: up to 80 cm, wider diameter
Analytical HPLC
Length: cm, diameter: mm
Many small molecules can be purified by HPLC
In industrial scale, preparative HPLC is used in purification of insulin, interleukin-2

37. High Pressure Liquid Chromatography-3
Superiour resolution due to small particle size
Fast
High degree of automation
Cost
Capacity
Generally used for high value proteins intended for therapeutic use

38. Fast Protein Liquid Chromatography (FPLC)
Operating pressure is significantly lower
Glass or inert plastic columns in stead of stainless steel
Economically more attractive than HPLC
Pharmacia’s BioPilot and BioProcess systems are commercial FPLC systems designed for pilot and industrial scale use
Flowrates up to 400 L/h are achievable in BioProcess system

39. Expanded bed chromatography-1
Particulate matter in protein sample should be removed before conventional purification procedures
Expanded bed chromatography aims to overcome this requirement
Duration and cost decrease
Design considerations:
Bead density
Flow rate of mobile phase
Bead size distribution

40. Expanded bed chromatography-2
The use of beads with an appropriate diameter range is important for the generation of a stable expanded bed ( μm)

41. Purification of recombinant proteins
Same techniques but generally more straight forward because of high expression of recombinant protein
Specific peptide or protein tags can be incorporated for rapid purification
Polyarginine or polylysine tag: cation exchange chromatography
Polyhistidine tag: metal chelate chromatography
Flag (a synthetic peptide) tag: immunoaffinity chromatography
Removal of the tag is generally desirable afterwards

42. Protein Inactivation and Stabilization

43. Approaches to protein stabilization
Buffered solution
Temperature control
Minimization of processing time
Avoid vigorous agitation or addition of denaturing chemicals
Add substances inactivating known inactivators
Include stabilizing agents
Glycerol, sugars and PEG: they decrease free water activity
BSA: as “bulking” protein

44. Storage
Optimization of storage conditions is a trial and error process...
Optimum T and pH for maximum stability
In liquid format: add stabilizing agents, filter-sterilization is advised
In frozen format: quickly freeze the solution, preferably in liquid nitrogen, then store in -70OC
In dry format: protein may be more stable

45. Lyophilization-1
Lyophilization involves the drying of protein directly from frozen state
Freeze the sample
Apply vacuum
Increase the temperature  sublimation
Many commercial proteins (e.g. vaccines, hormones, antibodies) are marketed in freeze-dried form

46. Lyophilization-2 One of the least harsh method for protein drying
Lightweight product  distribution easier
Can be rapidly rehydrated
Accepted by regulatory authorities
Equipment is extremely expensive
Running cost high
Long processing times
Some proteins exhibit an irreversible decrease in biological activity

47. Characterization-1

48. Characterization-2 Functional Studies
Determination of specific activity
Determination of substrate range and specifity
Kinetic characteristics
Effect of various influences on activity

49. Characterization-3
Evidence of purity
1-D SDS-PAGE: The most common method used is 1-D polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS)
Purpose:
Determination of purity
Determination of molecular mass

50. Characterization-4

51. Characterization-5
1-D PAGE: proteins are under non-denaturing conditions.
Some sort of activity stain can be used
Isoelectric focussing: in stead of SDS, a mixture of low molecular mass organic acids and bases are used
A pH gradient forms in the gel
Protein will stop moving when it comes to the pH equals its pI value

52. Characterization-6
2-D Electrophoresis: combines SDS-PAGE with isoelectric focussing

53. Characterization-7
Capillary Electrophoresis: not in polyacrylamide gel but along a narrow capillary tube packed with a fused silica matrix, generally for low Mw substances. HPLC: superior peak resolution and fast At least 2 different HPLC column types are used

54. Characterization-8 Molecular Mass Determination Mass Spectroscopy
Gel filtration analysis
Non-denaturing electrophoresis (Ferguson plot)
Analytical ultracentrifuge:
Specially designed sample cells are used
Svedberg equation is used to find molecular mass from sedimentation coefficient

55. overview Cell Disruption Removal of Whole Cells and Debris
Concentration and Primary Purification
Purification (column chromatography)
Characterization