Biotechnology manufacturing

Biopharmaceutical Manufacturing

Deanna Scott, B.S., RACMIP 480

Lab Basics for the Biotech Industry

Downstream Processing

Deanna Scott, M.S., RACMIP480

Lab Basics for the Biotech Industry

General Steps in Downstream Protein Purification

Remember in Upstream Processing…

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

Upstream

Proteins are targeted to different compartments of the cells and subcellular localization of recombinant protein affects:Post-translation modificationsAggregation of proteinRefoldingEnzymatic activitySecretion

Upstream, Fermentation, Downstream Integration

E. Coli

Most important organism in bioprocessing.Gram Negative – inner membrane – cell wall – external membraneSecretion 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

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

FiltrationCentrifugationSedimentationFlocculation a process where a solute comes out of solution in the form of floc or flakes. Gravity settling

Product Isolation

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

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

ChromatographySeparation of mixturesPassing 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

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

silica layer in thin layer chromatography

Chromatography Terms:Mobile Phase

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

The substance that is to be separated during chromatographyExamples:

E. coli culture filtrateYeast cell extrations

Chromatography Terms:Chromatograph

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

Chromatography Terms:Chromatogram

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

Chromatography Terms:Preparative vs. Analytical

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

Column ChromatographyPlanar ChromatographyPaper ChromatographyThin layer Chromatography

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 columnopen tubular column

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

Column Chromatography-Expanded Bed AdsorptionFluidized bed vs. solid phase This allows omission of initial clearing steps:

Centrifugation Filtration, for culture broths Slurries of broken cells

The principle of EBA is to allow the chromatography beads to fluidize in the feed stream which is pumped at low pressure. 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.

Planar Chromatography

The stationary phase is present as or on a plane.

Paper, serving as such or impregnated by a substance as the stationary bed

Paper chromatographyLayer of solid particles spread on a support such as a glass plate

Thin layer chromatography

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

Is a polar moleculeCompounds 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.

Thin Layer ChromatographySimilar to paper chromatographyInstead 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 fasterBetter separationsChoice between different adsorbents.

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

Techniques by physical state of mobile phase

Gas chromatographyLiquid chromatography

Gas ChromatographyMobile phase is a gas. Carried out in a columnBased on a partition equilibrium of analyte between a solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often Helium). 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). This causes a difference in retention time

Gas Chromatography

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

Liquid Chromatography

Mobile phase is a liquid. Carried out either in a column or a plane.HPLCIn 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

Techniques by separation mechanism

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

Definition: Ion

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)

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

"polishing" Tertiary/Quaternary structure (native)

Size Exclusion Chromatography (SEC)

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.

Definitions: Polarity

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

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)

When the analyte, is passed over a HIC column in a highly salty buffer (Binding Buffer), the hydrophobic regionsStick 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

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


Binding bufferAn 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.


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


Elution bufferA 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.

Affinity Chromatography

Immunoadsorbent Antibody bindingElution Dialysis

Affinity ChromatographyImmunosorbant

Column ChromatographySolid matrix:

AgaroseSephadexDerivatives of cellulose, or other polymers

Affinity ChromatographyAntibody 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.

Affinity Chromatography Elution

Disruption of non covalent interactionBuffers

High [NaCl] and/or Low pH

Denaturing agents: 8 M ureaSoluble 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 ChromatographyDialysis

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

Product Polishing

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

CrystallizationDesiccationLyophilizationSpray drying May include:

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

viruses or depyrogenation

Green Fluorescent Purification




Engineering

Biotechnology manufacturing