Bioprocessing separation methods

Ali AhmadpourChemical Eng. Dept.Ferdowsi University of Mashhad

Contents Introduction Separation processes

Liquid-liquid extraction Aqueous two-phase separation Reverse micelle technique Chromatographic separations Dialysis Liquid membrane separation Adsorption separation Electrically enhanced separations

Protein stabilization & denaturation٢

Introduction Proteins are produced from animal tissues (insulin, human

growth hormone (hGH), erythropoietin, interferon), plant tissues, blood, microorganisms or cell cultures.

They have to be separated from a very large number of contaminants, other proteins, nucleic acids, polysaccharides and many other components present in the cell culture used to manufacture these proteins.

For human use, the levels of purity should be of the order of 99.9% or 99.98% or even higher.

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Cont. Competitive advantage in production depends not only on

innovations in molecular biology and other areas of basic biological sciences, but also on innovation and optimization of separation and downstream processes.

The main issues important for the development of novel separation techniques are: Improved resolution Simplicity Speed Ease of scale-up Possibly continuous operation

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Cont.

In separations, the physicochemical properties of proteins should be considered. They are: PI (point of isoelectric) Surface charge as a function of pH Biological affinity (towards certain ligands e.g.

metal ion and dyes) Surface hydrophobicity Size Stability

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Physicochemical basis for separationprocesses

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Separation processes

Different classes of separation processes: Equilibration (LLE, IE chromatography, affinity chrom.,

membrane chrom., electrofiltration, adsorption)

Rate-governed (electrophoresis, membrane processes)

Mechanical (ultrasonic, press, mixers)

Most separation techniques for proteins are rate-governed processes.

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Liquid-liquid extraction LLE has been used in the antibiotics industry for several

decades.

It is now considered as a potentially useful separation step in protein recovery and separation, particularly because it can readily be scaled-up and can be operated on a continuous basis.

LLE advantages: High capacity

Good selectivity

LLE limitations: Poor solubility of the large protein

molecules in typical organic solvents restricts the range of solvents used

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Cont.

Two classes of solvents used for protein recovery or separations are: Aqueous polymer/salt (or polymer/polymer) systems

Reverse micellar solutions (water in oil emulsion)

In both cases, two phases are formed and the separation exploits the difference in partitioning of the proteins in the feed and extraction phases.

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Cont. In the aqueous polymer/salt separation systems the partitioning

of the protein occurs between two immiscible aqueous phases; one rich in a polymer (usually PEG) and the other in a salt (e.g. phosphate or sulphate).

Reverse micelles utilize the solubilizing properties of surfactants that can aggregate in organic solvents to form so-called inverted or reverse micelles. These aggregates consist of a polar core of water and the solubilized protein stabilized by a surfactant shell layer.

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Aqueous two-phase separation Partitioning in two aqueous phases (in a single step or

as a multistage process) can be used for the separation of proteins from cell debris as well as for purification from other proteins.

Most soluble and particulate material partitions to the lower, more polar (e.g. salt) phase and the protein of interest partitions to the top less polar phase, usually PEG.

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Cont. Separation of actual proteins in such systems is based

on the partition coefficient (K).

Manipulating of K can be done by altering parameters such as: Average molecular weight of the polymer Type of phase forming salt used for the heavy phase Types of ions included in the system Ionic strength of added salts (e.g. NaCl)

phase aqeousin ion concentratprotein Pure phase organicin ion concentratprotein PureΚ =

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Reverse micelle technique In RM technique, surface active agents (surfactants)

are used to dissolve proteins in organic solvents. Surfactants create micelles that separate proteins

from the aqueous phase. Micelle: A colloidal aggregate of surfactant

molecules, which occurs at a well-defined concentration known as CMC. The typical number of aggregate molecules (aggregation No.) is 50 to 100.

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Cont. CMC: The characteristic concentration of surfactants

in solution above which the appearance and development of micelles brings about sudden variation in the relation between the concentration and certain physico-chemical properties of the solution (such as surface tension).

Above the CMC ,the concentration of singly dispersing surfactant molecules is virtually constant and the surfactant is at essentially its optimum of activity for many applications.

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Micelles

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Micelles

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Micelle separation

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Reverse micelle

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Reverse micelle extraction After RM extraction, there are two techniques for

transferring proteins into the micellar phase. The most widely used method involves extraction of the

protein with liquid-liquid extraction system. One phase is the aqueous solution of the protein, and the other the organic micellar solution, usually in equal volume. By gently shaking the two phases, the protein partitions from the aqueous into the micellar phase.

The second method is solid state extraction of the protein, in which the protein powder is suspended in the micellar phase and gently stirred.

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Cont. The protein in the RM solution can be transferred back into an

aqueous solution, by contacting it with an aqueous solution containing a high concentration of a particular salt (KCl, CaCl2), which has the capability to exchange with the protein in the micelles.

Protein extraction by RM can be tailored to a specific protein by controlling the micellar parameters such as: The water content, The type and concentration of surfactant, The type and concentration of salt, The pH.

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Protein partition into reverse micelles

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Chromatographic separations

Chromatography techniques for protein separations are: Ion-exchange chromatography Gel filtration (molecular sieve or size exclusion)

chromatography Affinity chromatography Hydrophobic interaction chromatography Reversed chromatography

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Cont.

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Cont.

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Ion exchange chromatographyElution of a sample mixture from a chromatographic column.

a) A sample containing 3 different molecules is loaded onto a column. As the mobile phase carries the sample components through the column, their rate of movement is affected by the relative tendency to adsorb to the stationary phase, slowing their elution. b) The three molecules are graphed according to their elution times

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Ion-exchange chromatography

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Gel filtration chromatography

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Molecular sieve or size exclusion

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Affinity chromatography

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Cont.

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Cont.

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Dialysis separation

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Dialysis bag

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Cont.

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Examples

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Removing ammonium sulphate by dialysis

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Removing ammonium sulphate by gel filtration

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Liquid membrane separation Liquid membrane extraction is a relatively new and

selective separation technology for concentration of low MW chemicals produced by fermentation and food-processing industries.

Separation is achieved by the transport of the solute from a feed phase across a film of organic solvent into a stripping phase (known as facilitated transport).

Examples include organic acids such as citric acid, lactic acid and amino acids.

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Schematic diagram of liquid membrane processes

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Cont. The liquid membrane consists of the organic solvent which

separates the two aqueous phases (the feed and stripping phases), and contains a carrier species to enhance both selectivity and rates of extraction.

Aliphatic diluents are generally preferred as the solvent because of their lower solubility in water. In an ideal situation, the solvent should have no solubility in water to ensure that there is no aqueous phase contamination by trace organics.

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Cont. There are two main configurations for liquid membrane

extraction: Supported liquid membrane (SLM): It is achieved by impregnating a

porous solid film with an organic solvent, which is held in place by capillary forces that exist within the pores in polypropylene, polysulphone, or other hydrophobic materials. Typical dimensions are a membrane thickness of 25-50 µm, with pore sizes of 0.02-1.0 µm.

Emulsion liquid membrane (ELM): It is formed by creating, under high shear, a dispersion of the stripping phase within the organic solvent which forms a nonporous film around the stripping phase droplets. The emulsion thus formed is dispersed into the feed phase containing the solute, which is then transported into the stripping phase. Depending on the dispersion conditions the globule diameter is 1-2 mm and the internal phase droplets are micron sized.

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Liquid membrane separation ELM has very fast extraction kinetics and allow the use

of conventional LLE equipment; but they are prone to emulsion swelling, which gives rise to dilution and instability problems. Also, it is necessity to make and break an emulsion.

SLM system has slower kinetics and loss of the membrane phase.

Liquid membrane processes offer high separation factors, low capital and operating costs, a lower solvent inventory than solvent extraction, ease of scale-up and the possibility of continuous operation.

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Schematic diagram of supported liquid membrane (SLM)

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Schematic diagram of emulsion liquid membrane (ELM)

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Adsorption separation Adsorption systems:

Expanded bed adsorption or fluidized bed adsorption is a new technique that in one step accomplishes removal of whole cells and cell debris, concentration and initial purification of the target protein.

Continuous adsorption recycle extraction is adapted as a downstream process for efficient separation of proteins from crude feed stocks continuously.

The system's performance is determined by the nature of adsorbent an feed material, flow rates, reactor volume and conditions of the adsorption and desorption stages.

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Continuous adsorption recycle extraction (CARE) process

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Electrically enhanced separations(Polyacrylamide gel electrophoresis)

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Electrophoresis

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Protein stabilization

When proteins are extracted, they may irreversibly be damaged. Some factors should be considered for protein stabilization i.e.: pH Temperature Degradation Enzymes Microbial growth

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Protein denaturation Some physical and/or chemical changes in protein structure

can result in denaturation or aggregation of proteins. Denaturation may be reversible or irreversible. Important

factors are: Heating Freezing Extreme of pH Contact with organic chemicals Presence of denturated materials such as urea

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