Lecture 4: Industrial Purification

What did the upstream process include?

• Fermentation product

• Protein product (in cells or in media), in addition to contaminants:

  • Host proteins, DNA, membrane, metabolites (lipids, sugars), endotoxins from G-negative bacterial hosts, viruses, bacteria

  • Fermentation media components, purification reagents, metals, column/reservoir/tubes leachables

  • Product variants (fragments, aggregates, isomers, etc)

The downstream process includes all steps required to do what?

• steps required to purify a biological product from cell culture broth to a final purified product, it involves multiple purification steps

Each step in the downstream process might include what to purify?

• Include 1 or more technique to purify

Specific downstream steps and their number depend on what?

• Depend on the product and production system, cost (economically scalable) and the challenges you have with every system

• We can pick and choose one or more based on case by case

What are the downstream steps?

• Initial purification (target capture)

• Intermediate purification

• Final purification

• Sterilization and formulation

  • *Can have multiple steps in each process depending

What is the goal of initial purification?

• Concentrate the protein in a fast/quick way (rough purification)

What does initial purification include?

• Includes removal of cells and cell debris

• It can include 1 or more steps

What are the most common initial purification steps?

• Centrifugation

• Cell lysis

• Filtration

• Dialysis

• Precipitation (salting out)

• Expanded Bed Adsorption (EBA)

What is centrifugation?

• Uses centrifugal force to separate particles from solution according to their size, shape, density, and medium viscosity

What is cell lysis?

• Uses physical or chemical force to breakdown the cell membrane which leads to release of the cell contents

What is filtration (depth filtration)?

• Uses variety of filters with different sizes that involves a porous filtration medium to retain particles throughout the medium, rather than just on the surface of the medium

What is dialysis?

• Separation of particles in a liquid on the basis of differences in their ability to pass through a membrane

What is precipitation (salting out)?

• Addition of large conc of salts causes precipitation of total proteins

  • MORE SALT INCREASES SOLUBILITY OF PROTEIN, the ionic strength also increases and repulsion between proteins are less

  • Too much salt causes them to aggregate and precipitate out (this is a way of initial purificiation)

In Expanded bed adsorption (EBA), what is used from the fermenter? What does this enable?

• Lysates/cells are used directly from fermenter

• Enables the clarification, concentration, and semi-purification to be achieved in a single step

In EBA, what are the steps?

• Pack the column with coated beads

• Apply mobile phase upwards → causes bed expansion

• Apply sample upwards causes protein of interest to be adsorbed on beads while cell debris, particulates and containments are removed through the top of the column

• Apply pressure downward to get rid of extra liquid and concentrate the protein-bound beads; adding buffer can be used to elute the (semi)purified protein from the beads

What does intermediate and final purification include? What does choice of purification depend on?

• Multiple steps of purification

• Choice of purification depends on the type of other proteins

What does intermediate and final purification rely on? What are examples?

• Rely on Chromatography: based on the differences in the competition affinity between a stationary phase and a fluid mobile phase

• Ex:

  • Ion exchange chromatography

  • Adsorption (Normal Phase) Chromatography

  • Hydrophobic Interaction Chromatography (HIC)

  • Affinity chromatography

  • Size exclusion (gel permeation) chromatography

What is ion exchange chromatography?

• Separation based on binding with opposite charged binding sites

In ion exchange chromatography, if your protein of choice is positive, what do you use?

• Cation exchange chromatography

  • Positively charged protein (cation) binds to negative charged bead

  • Negatively charged protein (anion) AND neutral flows through

  • THEN increase salt concentration, increase pH and then collect the eluted positive charged protein

  • pH one unit BELOW (net positive charge)

In ion exchange chromatography, if your protein of choice is negative, what do you use?

• Anion exchange chromatography

  • Negative charged protein (anion) binds to positive charged bead

  • Negatively charged protein (anion) AND neutral flows through

  • THEN decrease salt concentration, decrease pH and then collect the eluted negative charged protein

  • pH one unit ABOVE (net negative charge)

What are the phases in adsorption (normal phase) chromatography? What happens to sample molecules here?

• Stationary phase is relatively polar

• Mobile phase is relative non-polar

• Sample molecules that have high affinity to stationary phase (polar) will stay longer on the column and elute later

What does the adsorption chromatography enable?

• Enables high ratios of product load, thus is economically scalable

For hydrophobic interaction chromotography, what are proteins in physiological conditions?

• Proteins are hydrated: hydrophilic AA attract water molecules and are exposed to the surface while most hydrophobic amino acid residues are located inside the protein core

In HIC, high salt causes what?

• High salt will attract the water from the protein causing exposing of hydrophobic residues

  • Alanine, Valine, Leucine, Isoleucine, Methionine, Tyrosine, Tryptophan, Phenylalanine

• Mild technique: suitable for proteins

What is affinity chromatography based on? What are some examples?

• Based on strong interaction between protein of interest and another substance

• Ex:

  • Glycoproteins bind to lectin

  • Serine proteases bind to lysine

  • Protein A or G bind to Fc region of immunoglobulins

In affinity chromatography, what are tagged proteins?

• Have protein of interest, and engineer the tag to have affinity to a chromatography medium

  • Get rid of contaminant, and purify the protein

What is immuno-affinity chromatography? What is the affinity based on?

• A type of affinity chromatography

• Based on affinity of antibodies with their epitopes

What are advantages of immuno-affinity chromatography?

• High specificity combination of concentration purification in one step

What are the disadvantages of immuno-affinity chromatography?

• Occasional very strong antibody-antigen binding requiring harsh conditions for elution

• Disruption of the covalent bond linking the “receptor” to the matrix

  • Used for purification of Urokinase, factor VIII factor X, erythropoetin

What is Size Exclusion Chromatography known as? What is separation based on and the characteristics?

• aka Gel permeation

• Separation based on their shape and size

• Slow and thus is commonly used late in the purification step when protein is more concentrated

In summary, what are the purification techniques separation based on?

• Centrifutation → density

• Dialysis → size

• Depth filtration → size

• Precipitation → change in solubility

• Ion exchange chromatography → charge

• Adsorption chromatography → hydrophilicity (covalent/non-covalent interactions)

• Hydrophobic interaction chromatography -. hydrophobicity

• Affinity chromatography → affinity (specific ligand-substrate interaction)

• Gel permeation chromatography → size

How to get rid of viral contaminants?

• Heat tx (pasteurization)

• Nanofiltration

• Ion exchange chromatography

• Immuno affinity chromatography

How to get rid of bacterial contamination?

• Filter sterilization: 0.2 um filters

• Sterilization of raw material at 121 C (for 15 min)

• Strict aseptic conditions (clean rooms, filtered air)

• antibiotics (not penicillin: antibiotic free operations are preferable)

How to get rid of pyrogen contamination?

• Ion (anion) exchange chromatography of the product

• Heating materials (glass and metal used in the process) in dry heat ovens (>180 C)

How to get rid of cellular DNA contamination?

• Detection using dye-binding fluorescence or PCR

Proteins need to adopt what structure to be functional?

• Adopt a distinct tertiary structure where they expose certain region for recognition by other receptors or substrates and hence become functional

What is occasionally produced as insoluble?

• Recombinant proteins are occasionally produced as insoluble in the form of inclusion bodies

To confirm proper protein folding, how do we detect protein folding?

• Circular Dichroism (CD)

• Fluorescence

• Fourier Transform Infrared (FTIR)

What is Circular Dichroism?

• The difference between the absorption of left and right handed circularly polarized light

What is fluorescence?

• The visible or invisible radiation emitted by certain substances as a result of incident radiation of a shorter wavelength such as X-rays or UV light

What is Fourier Transform Infrared?

• Measures the IR spectrum absorption or emission of a protein

How can the alpha-helices be estimated by?

• CD in the far UV region (180-260 nm), however occasional overlap from beta sheets (weak signals)

• FTIR, however occasional overlap from loop structures

How can beta sheets be estimated by?

• CD: weak variable signals due to twists of interacting beta strands

• FTIR: EFFICIENTLY estimates B-structure and differentiates between parallel and antiparallel forms

How can aromatic amino acids be identified?

• CD in the near UV region (250-340 nm); can also provide info on disulfide structures

• Fluorescence

What is characteristic of CD, FTIR, and fluorescence spectroscopy?

• Require less protein conc

• Fast

• Give no info on the full protein structure or the exact location of each AA

What are some other methods to confirm proper protein folding?

• Differential Scanning Calorimetry: measures the heat required to unfold a protein

• Chromatography

• Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

• Static and Dynamic Light Scattering techniques

• Analytical Ultracentrifuge: permits the measurement of the conc of a sample versus position within a spinning centrifuge cell. It analyzes the sedimentation velocity or sedimentation equilibrium

What are some techniques that can allow proper folding of misfolded proteins?

• Dilution

  • Easy

  • Requires huge vessels

  • Requires reconcentration

• Dialysis

  • Easy

  • Time consuming

• Chromatography

  • Achieve purification and refolding in 1 step

  • Time saving

  • High refolding efficiency

• High hydrostatic pressure (high pressure to solubilize aggregates)

  • Rapid

  • Achieve solubilization and refolding in 1 step