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

Biopharmaceutical

Manufacturing

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

Downstream Processing Deanna Scott, M.S., RAC MIP480 Lab Basics for the Biotech Industry
Downstream
Processing
Deanna Scott, M.S., RAC
MIP480
Lab Basics for the Biotech
Industry

General Steps in Downstream Protein Purification

General Steps in Downstream Protein Purification
General Steps in Downstream Protein Purification

Remember in Upstream Processing…

Remember in Upstream Processing… Biological activity of a protein is based on hydrophobicity and charge. Post

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

Use of Protein Production Platforms

Use of Protein Production Platforms
Use of Protein Production Platforms
Use of Protein Production Platforms

Upstream

Upstream Proteins are targeted to different compartments of the cells and subcellular localization of recombinant

Proteins are targeted to different compartments of the cells and subcellular localization of recombinant protein affects:

Post-translation modifications Aggregation of protein Refolding Enzymatic activity Secretion

Upstream, Fermentation, Downstream Integration

Upstream, Fermentation, Downstream Integration
Upstream, Fermentation, Downstream Integration
E. Coli Most important organism in bioprocessing. Gram Negative – inner membrane – cell wall

E. Coli

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

Stages in Downstream Processing

Stages in Downstream Processing Removal of Insolubles Product Isolation Product Purification Product Polishing A

Removal of Insolubles Product Isolation Product Purification Product Polishing A few product recovery methods may be considered to combine two or more stages.

For example, expanded bed adsorption accomplishes removal of insolubles and product isolation in a single step. Affinity chromatography often isolates and purifies in a single step.

Removal of Insolubles Separation of cells, cell debris or other particulate matter Typical operations to

Removal of Insolubles

Separation of cells, cell debris or other particulate matter Typical operations to achieve this:

particulate matter Typical operations to achieve this: Filtration Centrifugation Sedimentation Flocculation

Filtration Centrifugation Sedimentation Flocculation a process where a solute comes out of solution in the form of floc or flakes. Gravity settling

Product Isolation

Product Isolation Removal of those components whose properties vary markedly from that of the desired product.

Removal of those components whose properties vary markedly from that of the desired product. Water is the chief impurity

Isolation steps are designed to remove it (i.e. dialysis) Reducing the volume Concentrating the product. Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations involved.

Product Purification

Product Purification Done to separate those contaminants that resemble the product very closely in physical and

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

Chromatography

Chromatography Separation of mixtures Passing a mixture dissolved in a "mobile phase" through a stationary

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

Chromatography Terms:

Stationary Phase

Chromatography Terms: Stationary Phase The substance which is fixed in place for the chromatography procedure.

The substance which is fixed in place for the chromatography procedure. Examples:

silica layer in thin layer chromatography

Chromatography Terms:

Mobile Phase

Chromatography Terms: Mobile Phase The phase which moves in a definite direction. Liquid (LC and CEC)

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

Chromatography Terms:

Analyte

Chromatography Terms: Analyte The substance that is to be separated during chromatography Examples: E. coli culture

The substance that is to be separated during chromatography Examples:

E. coli culture filtrate Yeast cell extrations

Chromatography Terms:

Chromatograph

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

A chromatograph is equipment that enables a sophisticated separation e.g. gas chromatographic or liquid chromatographic separation.

Chromatography Terms:

Chromatogram

Chromatography Terms: Chromatogram The visual output of the chromatograph In the case of an optimal separation,

The visual output of the chromatograph In the case of an optimal separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

separation, different peaks or patterns on the chromatogram correspond to different components of the separated mixture.

Chromatography Terms:

Preparative vs. Analytical

Chromatography Terms: Preparative vs. Analytical Preparative chromatography Separate the components of a mixture for

Preparative chromatography

Separate the components of a mixture for further use (and is thus a form of purification).

Analytical chromatography

Operates with smaller amounts of material Seeks to measure the relative proportions of analytes in a mixture.

The two are not mutually exclusive

Techniques by chromatographic bed shape

Techniques by chromatographic bed shape Column Chromatography Planar Chromatography Paper Chromatography Thin

Column Chromatography Planar Chromatography Paper Chromatography Thin layer Chromatography

Column Chromatography Column chromatography is a separation technique in which the stationary bed is within

Column Chromatography

Column chromatography is a separation technique in which the stationary bed is within a tube. 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:

packed column open tubular column Differences in rates of movement through the medium are calculated to different retention times of the sample.

Column Chromatography- Expanded Bed Adsorption

Column Chromatography- Expanded Bed Adsorption        

 

 
 

 
Planar Chromatography The stationary phase is present as or on a plane.   Paper chromatography

Planar Chromatography

The stationary phase is present as or on a plane.

 

Paper chromatography

Layer of solid particles spread on a support such as a glass plate

Thin layer chromatography

Paper Chromatography

Compounds in the sample mixture travel different distances according to how strongly they interact with the paper. Cellulose (aka “paper’):

Is a polar molecule Compounds within the mixture travel farther if they are non- polar. More polar substances bond with the cellulose paper more quickly, and therefore do not travel as far.

non- polar. More polar substances bond with the cellulose paper more quickly, and therefore do not
non- polar. More polar substances bond with the cellulose paper more quickly, and therefore do not
Thin Layer Chromatography Similar to paper chromatography Instead of using a stationary phase of paper,

Thin Layer Chromatography

Similar to paper chromatography Instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent:

Silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper:

Runs faster Better separations Choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent.

compounds in the sample mixture travel different distances according to how strongly they interact with the

Techniques by physical state of mobile phase

Techniques by physical state of mobile phase Gas chromatography Liquid chromatography

Gas chromatography Liquid chromatography

Techniques by physical state of mobile phase Gas chromatography Liquid chromatography

Gas Chromatography

Gas Chromatography  

 

Gas Chromatography

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

used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature
used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature
used in GC make it unsuitable for high molecular weight biopolymers or proteins (heat will denature
Liquid Chromatography Mobile phase is a liquid. Carried out either in a column or a

Liquid Chromatography

Mobile phase is a liquid. Carried out either in a column or a plane. HPLC In the HPLC technique, 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

HPLC Configuration

HPLC Configuration
HPLC Configuration

Techniques by separation mechanism

Techniques by separation mechanism Ion exchange chromatography Size exclusion chromatography (SEC) Reversed-phase

Ion exchange chromatography Size exclusion chromatography (SEC) Reversed-phase chromatography (RP-HPLC) Two-dimensional chromatography (2D) Hydrophobic Interaction chromatography (HIC)

Ion Exchange Chromatography Used charged stationary phase to separate charged compounds Resin that carries charged

Ion Exchange Chromatography

Used charged stationary phase to separate charged compounds

Resin that carries charged functional groups which interact with oppositely charged groups of the compound to be retained. FPLC

charged functional groups which interact with oppositely charged groups of the compound to be retained. FPLC

Definition: Ion

Definition: Ion Ion is an atom or molecule which has lost or gained one or more

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

Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC) Gel permeation/filtration chromatography (GPC) Separates molecules according to

Gel permeation/filtration chromatography (GPC) Separates molecules according to their size Low resolution

"polishing" Tertiary/Quaternary structure (native)

molecules according to their size Low resolution "polishing" Tertiary/Quaternary structure (native)

Size Exclusion Chromatography (SEC)

Size Exclusion Chromatography (SEC)
Size Exclusion Chromatography (SEC)
Reverse-Phase Chromatography Reversed-phase chromatography is an elution procedure used in liquid chromatography in

Reverse-Phase Chromatography

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

used in liquid chromatography in which the mobile phase is significantly more polar than the stationary

Definitions: Polarity

Definitions: Polarity The dipole-dipole intermolecular forces between the slightly positively-charged end of one

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

Two-dimensional

chromatography

Two-dimensional chromatography Insufficient separation of some analytes. It is possible to direct a series of

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

Hydrophobic Interaction Chromatography (HIC)

Hydrophobic Interaction Chromatography (HIC) When the analyte, is passed over a HIC column in a highly

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

Green Fluorescent Protein Purification Using HIC

Green Fluorescent Protein Purification Using HIC Equilibration buffer A medium salt buffer (2 M (NH4)2SO4) which

Equilibration buffer A medium salt buffer (2 M (NH4)2SO4) which is used to "equilibrate" or "prime" the column

Green Fluorescent Protein Purification Using HIC

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

regions of proteins are more exposed and are able to interact with and bind the hydrophobic
regions of proteins are more exposed and are able to interact with and bind the hydrophobic

Green Fluorescent Protein Purification Using HIC

Wash buffer A medium salt Wash Buffer (1.3 M (NH4)2SO4) is used to wash weakly associated proteins from the column Proteins which are strongly hydrophobic (GFP) remain bound to the column.

weakly associated proteins from the column Proteins which are strongly hydrophobic (GFP) remain bound to the
weakly associated proteins from the column Proteins which are strongly hydrophobic (GFP) remain bound to the

Green Fluorescent Protein Purification Using HIC

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

the GFP to have a higher affinity for the buffer than for the column, thereby allowing
the GFP to have a higher affinity for the buffer than for the column, thereby allowing
Affinity Chromatography Immunoadsorbent Antibody binding Elution Dialysis

Affinity Chromatography

Immunoadsorbent Antibody binding Elution Dialysis

Affinity Chromatography Immunoadsorbent Antibody binding Elution Dialysis

Affinity Chromatography Immunosorbant

Column Chromatography Solid matrix:

Agarose Sephadex Derivatives of cellulose, or other polymers

Immunosorbant Column Chromatography Solid matrix: Agarose Sephadex Derivatives of cellulose, or other polymers
Immunosorbant Column Chromatography Solid matrix: Agarose Sephadex Derivatives of cellulose, or other polymers
Affinity Chromatography Antibody Absorption The serum is passed over the immunoadsorbent. Antibodies bind

Affinity Chromatography Antibody Absorption

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

to matrix Antibodies of other specificities (green) and other serum proteins (yellow) will pass through unimpeded.

Affinity Chromatography Elution

Affinity Chromatography Elution Disruption of non covalent interaction Buffers High [NaCl] and/or Low pH Denaturing

Disruption of non covalent interaction Buffers

High [NaCl] and/or Low pH

Denaturing agents: 8 M urea Soluble form of the antigen.

These compete with the immunoadsorbent for the antigen-binding sites of the antibodies and release the antibodies to the fluid phase.

Affinity Chromatography Dialysis

Affinity Chromatography Dialysis The eluate is then dialyzed against, for example, buffered saline in order to

The eluate is then dialyzed against, for example, buffered saline in order to remove the reagent used for elution.

The eluate is then dialyzed against, for example, buffered saline in order to remove the reagent

Product Polishing

Product Polishing End with packaging of the product in a form that is stable, easily transportable

End with packaging of the product in a form that is stable, easily transportable and convenient.

Crystallization Desiccation Lyophilization Spray drying May include:

Sterilization of the product Remove or deactivate trace contaminants which might compromise product safety

viruses or depyrogenation

Green Fluorescent Purification

Green Fluorescent Purification

Green Fluorescent Purification
Green Fluorescent Purification

Green Fluorescent Purification

Green Fluorescent Purification
Green Fluorescent Purification

Green Fluorescent Purification

Green Fluorescent Purification
Green Fluorescent Purification

Green Fluorescent Purification

Green Fluorescent Purification