PurificatioPurificatio n strategie n strategie

ss

By : Miss Thida Chan By : Miss Thida Chanyachukulyachukul

AIDs Production group AIDs Production group

OutlineOutline General concepts in Purification strateg

ies Principle of purification operations Special considerations in our biopharm

aceutical products Ritonavir (Protease inhibitor) Vaccine -2IL

ConclusionsReferences

General concepts General concepts

Purification strategies

important prerequisite in pharmaceutical manufacture

more predictable and controllablenot all problems in purification system are solved

by the acquisition of sophisticated laboratory equipment and column packing

difficult to find optimum conditions of choosing suitable methods

General concepts General concepts

base the purification of biopharmaceutical product on knowledge on

structure / function / particular structural

details

Conversely, results from the application of a particular purification method can often be interpreted in terms of molecular properties

General concepts General concepts Production can be divided into Production can be divided into

upstream processing the initial fermentation process ,

which results in the initial generation of product .

downstream processing the actual purification of the product

and generation of finished product format followed by sealing of the final product contain

ers

Categories of Categories of unwantedunwanted

Components present due to process conditions :

Host-cell-derived components Process-derived components

Components present due to contamination :

Adventitious agent

- In Plant scale wor - In Plant scale workingking

volumes of between 2-5 liters

the most useful methods of purification recrystallization for solids distillation or steam distillation for liquids

chromatography should be avoided but if it is necessary, then medium

pressure liquid chromatography (mplc) is the method of choice

Principle of purifi Principle of purifi cation operations cation operations

FiltrationChromatography

Distillation

Crystallization or Recrystallization

Basic concept of Basic concept of FiltrationFiltrationTechnique : pass the solution, cold or

hot,through a fluted filter paper in a conical

funnel

removes particulate impurities from liquidsor collect insoluble or crystalline solids

from solution

the solid particles are too fine centrifugation should be used

Basic concept of Basic concept of Gel filtrationGel filtrationdifferent amount of time different

solute stay within the liquid phase that is entrapped by the matrix pore dimension gel structure solute size

uncomplicated / straightforward technique

Industrial filtratio Industrial filtratio n devices n devices

Basic concept of Basic concept of chromatographychromatography= a group of separation techniques, which are

characterized by a distribution of the molecules to be separated between two phases, one stationary and the other mobile phase

molecules with a high tendency to stay in the stationary phase will move through the system at a lower velocity than will those which favor the mobile phase.

the shape, rigidity and particle size distribution profile of the gel matrix are important parameters

Basis chromatogr Basis chromatographyaphy Ion exchange

differences in protein surface charge at a given pHGel filtration

differences in size/shape of different proteinHydrophobic interaction chromatography

differences in the size and extent of hydrophobic patches on the surface of

proteinsAffinity chromatography

ability of a protein to bind in abio- specific manner to a Chosen immobilized ligand

mplcmplcsimplifiedcheapereasy to predict which

fractions will contain each component

silica / alumina / ion exchange resin,

the appropriate size of column and the correct solvent

linked to a fraction collector / An UV / refractive index detector / TLC

Basic concept of Basic concept of Ion exchange Ion exchange

chromatography chromatography (IEC)(IEC)

the more highly charged a protein

the more strongly it will bind to a given, oppositely

charged ion exchanger

the more highly charged ion exchangers

bind proteins more effectively than weakly charged

Advantages of Advantages of Ion exchange Ion exchange

chromatography chromatography (IEC)(IEC)

versatilityhigh resolving powerhigh loading capacity

multiple inlets column with large diameter

straight forward basic principle.

Basic concept of Basic concept of affinity affinity chromatographychromatography

= the exploitation of various biological affinities for adsorption to a solid phase

the ligand immobilized on the solid phase

the counterligand passing the chromatographic column

stages : adsorption, washing, elution and column regeneration

Biological Biological interactions interactions

used in affinity used in affinity chromatographychromatography

Ligand CounterligandAntibody antigen, virus, cellEnzyme substrate analogue, inhibitor, co-

factorLectin polysaccharide, glycoprotein, cell

surface receptor, cellNucleic acid nucleic acid-binding protein

(enzyme or histone)Hormone, vitamin receptor, carrier proteinSugar lectin, enzyme or other sugar

binding protein

Special consideration Special consideration on Affinity chromatog on Affinity chromatog

raphyraphyassociation strength, between ligand and

counterligandif it is too weak no adsorptionif it is too strong difficult to elute the protein adsorbed

pHsalt concentrationdissociation substancesappropriate specificity

affecting the protein to be isolated

Basic concept of Basic concept of distillationdistillationThe distillation process

involves boiling a liquid condensing the vapors the resulting liquid

suitable for all organic liquids and most of the low-melting organic solids

the efficiency depends on

the difference in boiling points of the pure material and its impurities

Steps of Steps of crystallization or crystallization or recrystallizationrecrystallization

The impure material dissolved at or near the boiling point to form a

near-saturated solution. the hot solution is filtered to remove any

insoluble particles allowed to cool

fast cooling generate many nuclei of small crystals

the dissolved substance crystallizes out centrifuge or filter crystals from mother liquor washed free of mother liquor with a little fresh

cold solvent dried

Basic concept of Basic concept of crystallization or crystallization or recrystallizationrecrystallization

only as a last stepyield of about 90% at

best10% yield loss is quite

high

purified with additional processing which are much more quantitative such as extraction or chromatography

Special considera Special considera tions in our Bioph tions in our Bioph

armaceutical pro armaceutical productsducts

- 2Vaccine / ILPPPPPPPRitonavirPPPPPPP PPPPPPPP

- Plant scale proce- Plant scale proce ss of protein ss of protein

Working cell bank vial removed from storage pr opagation of working bank cells,

generating starting cultures

cell harvesting and recovery of crude protein - production scale cell culture

concentration (if necessary) and initial purification

main purification (chromatography)

product filling, freezing and sealing final product formulation labeling and

packaging

Upstream purificat Upstream purificat ion of Ritonavir ion of Ritonavir

+ chromatography - 2 aminoaldehyde diols (white solid)

ssssss()

pricipitation filtration

chromatography filtration ssssss() ssssss ssssss () epoxide (white solid)

distillation

chromatography resin compound compound XXXIIIa

Ritonavir

Downstream purific Downstream purific ation of Ritonavir ation of Ritonavir

Hydrophobic interactionAffinity

chromatographychromatography

dryer

final product formulation

ConclusionConclusionpurity is a matter of degreesufficient pure for some intended purposeabsolute purity is an ideal which can never be

shown to be attainedthe starting material should be of the grade

commercially available In general, at least two different methods, such

as recrystallization and distillation, should be used in order to ensure maximum purification

the decision to market the product in liquid or po wder form must be determined experimentally,

as there is no way to predict the outcome for any particular material

ReferencesReferences Berthold, W. and Walter, J. Protein purification: aspects of

processes for pharmaceutical products. Biologicals 1994;22:135-150.

Janson JC. And Ryden L. Protein purification; principles, high-resolution methods, and application: VCH publishers, Inc. 1989.

Hesse F. and Wagner R. Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. Trends in Biotechnology. 2000 ;18(4):173-180

WHO study group. Acceptability of cell substrates for production of biologicals. WHO technical report series; 1987: 747, 1-29.

Walter, J and Allgaier, H. Validation of downstream processes. In: Mammalian Cell Biotechnology in Protein Product.1997; 453-48.

Perrin DD., Armarego WLF. and Perrin DR. Purification of laboratory Chemicals. 2nd ed. : Pergamon Press. 1980.

White HL. Introduction to industrial chemistry : John Wiley & Sons, Inc. 1986

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