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Protein Concentration and Sample Clarification

Membrane Filtration

Ultrafiltration

Ultrafiltration (UF) is the process of separating extremely small particles and dissolved molecules from fluids. The primary basis for separation is molecular size, although in all filtration applications, the permeability of a filter medium can be affected by the chemical, molecular or electrostatic properties of the sample.

Materials ranging in size from 1K to 1000K molecular weight (MW) are retained by certain ultrafiltration membranes, while salts and water will pass through. Colloidal and particulate matter can also be retained. Ultrafiltration membranes can be used both to purify material passing through the filter and also to collect material retained by the filter. Materials significantly smaller than the pore size rating pass through the filter and can be depyrogenated, clarified and separated from high molecular weight contaminants. Materials larger than the pore size rating are retained by the filter and can be concentrated or separated from low molecular weight contaminants.

Ultrafiltration is typically used to separate proteins from buffer components for buffer exchange, desalting, or concentration. Ultrafilters are also ideal for removal or exchange of sugars, non-aqueous solvents, the separation of free from protein-bound ligands, the removal of materials of low molecular weight, or the rapid change of ionic and/or pH environment. Depending on the protein to be retained, the most frequently used membranes have a nominal molecular weight limit (NMWL) of 3 kDa to 100 kDa.

Ultrafiltration is far gentler to solutes than processes such as precipitation. UF is more efficient because it can simultaneously concentrate and desalt solutes. It does not require a phase change, which often denatures labile species, and UF can be performed either at room temperature or in a cold room.

Comparison of ultrafiltration with other commonly used membrane separation techniques.
Comparison of ultrafiltration with other commonly used membrane separation techniques.

Microfiltration

Microfiltration (MF) is the process of removing particles or biological entities in the 0.025 :m to 10.0 :m range from fluids by passage through a microporous medium such as a membrane filter. Although micron-sized particles can be removed by use of non-membrane or depth materials such as those found in fibrous media, only a membrane filter having a precisely defined pore size can ensure quantitative retention. Membrane filters can be used for final filtration or prefiltration, whereas a depth filter is generally used in clarifying applications where quantitative retention is not required or as a prefilter to prolong the life of a downstream membrane. Membrane and depth filters offer certain advantages and limitations. They can complement each other when used together in a micro-filtration process system or fabricated device.

The retention boundary defined by a membrane filter can also be used as an analytical tool to validate the integrity and efficiency of a system. For example, in addition to clarifying or sterilizing filtration, fluids containing bacteria can be filtered to trap the microorganisms on the membrane surface for subsequent culture and analysis. Microfiltration can also be used in sample preparation to remove intact cells and some cell debris from the lysate. Membrane pore size cut-offs used for this type of separation are typically in the range of 0.05 :m to 1.0 :m.

Recovery

The ultimate aim of ultrafiltration is to maximize recovery of solutes of interest, but there are many membrane characteristics that affect that goal.

Factors Affecting Recovery Include:

  • Nominal molecular weight limit (NMWL)/nucleotide cut-off (NCO)
  • Retention
  • Concentration polarization
  • Flux

Nominal Molecular Weight Limit

A microfiltration membrane’s pore size rating, typically given as a micron value, indicates that particles larger than the rating will be retained. Ultrafiltration membranes are rated according to the nominal molecular weight limit (NMWL), also sometimes referred to as molecular weight cut-off (MWCO). The NMWL indicates that most dissolved macromolecules with molecular weights higher than the NMWL will be retained.

An ultrafiltration membrane with a stated NMWL should retain (reject) at least 90% of a globular solute of that molecular weight in daltons. However, for a wider safety margin, the selected cut-off should be well below the molecular weight of the solute to be retained. When solutes are to be exchanged, the cut-off should be substantially above that of the passing solute. A lower NMWL increases rejection but decreases the filtration rate for the same membrane material.

Retention and product recovery are a function of a variety of other factors, including the molecular shape and size of the molecule; electrical characteristics; sample concentration and composition; operating conditions; and device or system configuration. Two membranes may have the same NMWL but will exhibit different retention of molecules within a relatively narrow range of sizes. In addition, slender, linear molecules (e.g., nucleic acids) may find their way through pores that will retain a globular species of the same weight. Retention can also be affected by hydration with counter-ions. Nevertheless, NMWL has proven to be an effective general indicator of membrane performance for globular proteins.

When using membrane ultrafiltration for sample concentration or desalting, care must be taken to select a membrane (or device) with a NMWL appropriate for the application. Because there are several considerations in determining whether a given solute will or will not be retained by a membrane of a specific cut-off, it is best to choose a device with cut-off at about one half of the molecular weight of the protein to be concentrated. This maximizes protein recovery and minimizes filtration time.

Nucleotide Cut-off (NCO)

For most membranes, the NMWL is determined experimentally under a standard set of operating conditions. These analyses typically employ purified globular proteins to serve as markers or indicators of the retention characteristics of an ultrafiltration membrane. Although this approach is useful for choosing the appropriate NMWL for most protein research applications, selection of a membrane with an appropriate NMWL membrane for nucleic acid or polysaccharide purification is considerably more complex. By virtue of the rod-like three-dimensional structures of these molecules, these types of molecules require a tighter membrane (with a smaller cutoff) than do globular proteins of the same molecular weight. It is therefore convenient to consider the membrane retention characteristics of nucleic acids as being related to their length (in nucleotides) rather than their molecular weight.

Membrane Selection

Types of Membranes

Ultracel Ultrafiltration Membrane

To concentrate or desalt dilute solutions, use Ultracel series regenerated cellulose ultrafiltration membranes. The hydrophilic, tight microstructure of Ultracel membranes assures the highest possible retention with the lowest possible adsorption of protein, DNA or other macromolecules.

Durapore® Microporous Membrane

To clarify biological samples, recover DNA from agarose gels, retain chromatography resins or suspended solid media, use Durapore hydrophilic PVDF microporous membranes. Durapore membranes allow all soluble protein and nucleic acids to pass, retaining sub-cellular fragments, whole cell and particulate materials. Durapore membranes are extremely hydrophilic, and they provide the lowest binding of proteins and other biologicals of all commercially available microporous membranes.

Membranes Organized by Typical Recoveries

For globular proteins, there is a good correlation between molecular weight and Stoke’s radius. This usually allows one to predict the recovery of a protein based on its molecular weight if a membrane with the same nominal molecular weight rating is used (see typical recovery of a panel of protein solutes in the table below, “Membrane selection by recovery”). However, in order to accommodate the wide range of potential protein solutes with different tertiary structures, we suggest initially using the “rule of two” to ensure optimal recovery.

Rule of Two

For Ultracel (regenerated cellulosic) membranes, Millipore recommends using a membrane with a NMWL at least two times smaller than the molecular weight of the protein solute that one intends to concentrate.

Ultrafiltration Membranes vs. Traditional Microporous Membranes

Ultrafiltration Membranes
Cross-section of ultrafiltration membrane with skin and porous substructure.
Ultrafiltration membranes generally have two distinct layers: a thin (0.1- 1.5 μm), dense skin with a pore diameter of 10-400 A and a more porous substructure. Any species capable of passing through the pores of the skin (whose size is precisely controlled in manufacture) can therefore freely pass the membrane.
Traditional Microporous Membranes
Cross-section of traditional membrane with uniform pore structure from top to bottom.
Microporous membranes are generally rigid, continuous meshes of polymeric material with defined pore sizes. They are used to retain bacteria, colloids and particulates. Species are either retained on the membrane surface or trapped in its substructure.

Membrane Selection by Recovery
Membrane Selection by Recovery

Applications by Membrane Type
Applications by Membrane Type

출처: http://www.millipore.com