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Deanna Scott, B.S., RAC
MIP 480
Lab Basics for the Biotech Industry

Deanna Scott, M.S., RAC
Lab Basics for the Biotech

General Steps in Downstream
Protein Purification

Remember in Upstream
z Biological activity of a protein is based on
hydrophobicity and charge.
z Post translational modifications play a key
role in activity.
z Therefore, the expression system chosen has
a profound affect on protein localization, and
hence the down stream purification needs.

Use of Protein Production


Upstream z Proteins are targeted to different compartments of the cells and subcellular localization of recombinant protein affects: z Post-translation modifications z Aggregation of protein z Refolding z Enzymatic activity z Secretion .

Upstream. Downstream Integration . Fermentation.

z Gram Negative – inner membrane – cell wall – external membrane z Secretion mechanisms typically direct an accumulation of the recombinant protein between the two membranes. z This creates the need for a milder treatment to release the product. .E. Coli z Most important organism in bioprocessing.

Stages in Downstream Processing z Removal of Insolubles z Product Isolation z Product Purification z Product Polishing z A few product recovery methods may be considered to combine two or more stages. . z For example. Affinity chromatography often isolates and purifies in a single step. expanded bed adsorption accomplishes removal of insolubles and product isolation in a single step.

Removal of Insolubles z Separation of cells. z Gravity settling . cell debris or other particulate matter z Typical operations to achieve this: z Filtration z Centrifugation z Sedimentation z Flocculation a process where a solute comes out of solution in the form of floc or flakes.

Product Isolation z Removal of those components whose properties vary markedly from that of the desired product. ultrafiltration. z Water is the chief impurity z Isolation steps are designed to remove it (i. z Solvent extraction.e. dialysis) z Reducing the volume z Concentrating the product. and precipitation are some of the unit operations involved. adsorption. .

. crystallization and fractional precipitation. reversed phase chromatography. z Expensive to carry out z Require sensitive and sophisticated equipment z Significant fraction of the entire downstream processing expenditure. size exclusion. z Examples of operations include affinity.Product Purification z Done to separate those contaminants that resemble the product very closely in physical and chemical properties.

which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated.Chromatography z Separation of mixtures z Passing a mixture dissolved in a "mobile phase" through a stationary phase. .

z Examples: z silica layer in thin layer chromatography .Chromatography Terms: Stationary Phase z The substance which is fixed in place for the chromatography procedure.

. z Liquid (LC and CEC) z Gas (GC) z Supercritical fluid (supercritical-fluid chromatography. SFC). z The mobile phase consists of the sample being separated/analyzed and the solvent that moves the sample through the column.Chromatography Terms: Mobile Phase z The phase which moves in a definite direction.

coli culture filtrate z Yeast cell extrations .Chromatography Terms: Analyte z The substance that is to be separated during chromatography z Examples: z E.

g. gas chromatographic or liquid chromatographic separation. .Chromatography Terms: Chromatograph z A chromatograph is equipment that enables a sophisticated separation e.

.Chromatography Terms: Chromatogram z The visual output of the chromatograph z In the case of an optimal separation. different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

Analytical z Preparative chromatography z Separate the components of a mixture for further use (and is thus a form of purification).Chromatography Terms: Preparative vs. z Analytical chromatography z Operates with smaller amounts of material z Seeks to measure the relative proportions of analytes in a mixture. z The two are not mutually exclusive .

Techniques by chromatographic bed shape z Column Chromatography z Planar Chromatography z Paper Chromatography z Thin layer Chromatography .

z The particles of the solid stationary phase or the support coated with a liquid stationary phase may fill the whole inside volume of the tube: z packed column z open tubular column z Differences in rates of movement through the medium are calculated to different retention times of the sample.Column Chromatography z Column chromatography is a separation technique in which the stationary bed is within a tube. .

Column Chromatography- Expanded Bed Adsorption z Fluidized bed vs. for culture broths z Slurries of broken cells z The principle of EBA is to allow the chromatography beads to fluidize in the feed stream which is pumped at low pressure. . z The expanded bed allows particulate impurities in the feed stream to pass freely – and at very high flow rates – through the system without any clogging or pressure building up throughout the process. solid phase z This allows omission of initial clearing steps: z Centrifugation z Filtration.

Planar Chromatography z The stationary phase is present as or on a plane. z Paper. serving as such or impregnated by a substance as the stationary bed z Paper chromatography z Layer of solid particles spread on a support such as a glass plate z Thin layer chromatography .

z Cellulose (aka “paper’): z Is a polar molecule z Compounds within the mixture travel farther if they are non- polar. and therefore do not travel as far. .Paper Chromatography z Compounds in the sample mixture travel different distances according to how strongly they interact with the paper. z More polar substances bond with the cellulose paper more quickly.

or cellulose on a flat. alumina. . it involves a stationary phase of a thin layer of adsorbent: z Silica gel.Thin Layer Chromatography z Similar to paper chromatography z Instead of using a stationary phase of paper. z Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. inert substrate. z Compared to paper: z Runs faster z Better separations z Choice between different adsorbents.

Techniques by physical state of mobile phase z Gas chromatography z Liquid chromatography .

z Carried out in a column z Based on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone- based material) and a mobile gas (most often Helium). z This causes a difference in retention time . Gas Chromatography z Mobile phase is a gas. z The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary column) or a solid matrix inside a larger metal tube (a packed column).

Gas Chromatography z It is widely used in analytical chemistry z High temperatures used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature them) .

z HPLC z In the HPLC technique. .Liquid Chromatography z Mobile phase is a liquid. z Carried out either in a column or a plane. the sample is forced through a column that is packed with irregularly or spherically shaped particles or a porous monolithic layer (stationary phase) by a liquid (mobile phase) at high pressure.

HPLC Configuration .

Techniques by separation mechanism z Ion exchange chromatography z Size exclusion chromatography (SEC) z Reversed-phase chromatography (RP-HPLC) z Two-dimensional chromatography (2D) z Hydrophobic Interaction chromatography (HIC) .

z FPLC .Ion Exchange Chromatography z Used charged stationary phase to separate charged compounds z Resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained.

giving it a positive or negative electrical charge. formed when an atom loses electrons in a reaction. z Anions are negatively charged ions.Definition: Ion z Ion is an atom or molecule which has lost or gained one or more valence electrons. Anions are negatively charged because there are more electrons associated with them than there are protons in their nuclei. formed when an atom gains electrons in a reaction. . z Cations are positively charged ions. forming an 'electron hole'.

Size Exclusion Chromatography (SEC) z Gel permeation/filtration chromatography (GPC) z Separates molecules according to their size z Low resolution z "polishing" z Tertiary/Quaternary structure (native) .

Size Exclusion Chromatography (SEC) .

Reverse-Phase Chromatography z Reversed-phase chromatography is an elution procedure used in liquid chromatography in which the mobile phase is significantly more polar than the stationary phase. .

z Molecular polarity is dependent on the difference in electronegativity between atoms in a compound and the asymmetry of the compound's structure.Definitions: Polarity z The dipole-dipole intermolecular forces between the slightly positively-charged end of one molecule to the negative end of another or the same molecule. .

it can be possible to separate compounds that are indistinguishable by one-dimensional chromatography.Two-dimensional chromatography z Insufficient separation of some analytes. z It is possible to direct a series of unresolved peaks onto a second column with different physico-chemical z Since the mechanism of retention on this new solid support is different from the first dimensional separation. .

the hydrophobic regions z Stick to the HIC beads. z Other proteins which are less hydrophobic (or more hydrophilic) pass right through the column.Hydrophobic Interaction Chromatography (HIC) z When the analyte. is passed over a HIC column in a highly salty buffer (Binding Buffer). z This procedure allows the purification of one protein from a complex mixture of bacterial proteins. .

Green Fluorescent Protein Purification Using HIC z Equilibration buffer z A medium salt buffer (2 M (NH4)2SO4) which is used to "equilibrate" or "prime" the column .

z When in a high salt solution. .Green Fluorescent Protein Purification Using HIC z Binding buffer z An equal volume of high salt Binding Buffer (4 M (NH4)2SO4) is added to the bacterial lysate. the hydrophobic regions of proteins are more exposed and are able to interact with and bind the hydrophobic regions of the column. z Supernatant containing GFP has the same salt concentration as the equilibrated column.

3 M (NH4)2SO4) is used to wash weakly associated proteins from the column z Proteins which are strongly hydrophobic (GFP) remain bound to the column. .Green Fluorescent Protein Purification Using HIC z Wash buffer z A medium salt Wash Buffer (1.

z In low salt buffers (which have a higher concentration of water molecules). causing the GFP to have a higher affinity for the buffer than for the column. the conformation of GFP changes so that the hydrophilic residues of GFP are more exposed to the surface. thereby allowing the GFP to wash off the column. 10 mM Tris/EDTA) is used to wash GFP from the column. .Green Fluorescent Protein Purification Using HIC z Elution buffer z A low salt buffer (TE Solution.

Affinity Chromatography z Immunoadsorbent z Antibody binding z Elution z Dialysis .

or other polymers .Affinity Chromatography Immunosorbant z Column Chromatography z Solid matrix: z Agarose z Sephadex z Derivatives of cellulose.

z Antibodies bind noncovalently to matrix z Antibodies of other specificities (green) and other serum proteins (yellow) will pass through unimpeded. .Affinity Chromatography Antibody Absorption z The serum is passed over the immunoadsorbent.

. z These compete with the immunoadsorbent for the antigen-binding sites of the antibodies and release the antibodies to the fluid phase.Affinity Chromatography Elution z Disruption of non covalent interaction z Buffers z High [NaCl] and/or z Low pH z Denaturing agents: 8 M urea z Soluble form of the antigen.

. for example.Affinity Chromatography Dialysis z The eluate is then dialyzed against. buffered saline in order to remove the reagent used for elution.

z Crystallization z Desiccation z Lyophilization z Spray drying z May include: z Sterilization of the product z Remove or deactivate trace contaminants which might compromise product safety ƒ viruses or depyrogenation .Product Polishing z End with packaging of the product in a form that is stable. easily transportable and convenient.

Green Fluorescent Purification .

Green Fluorescent Purification .

Green Fluorescent Purification .

Green Fluorescent Purification .