See also graft chains and ionic functional groups attached to membranes (under “chromatography” and “graft chains”)

Types of Materials Used

Hollow fiber: In ultrafiltration applications, one very common membrane form for industrial and laboratory uses is the hollow fiber. Hollow fiber membranes are mounted, usually in cartridges, with the open ends of the fibers potted at each end in an adhesive plug sealed to the cylindrical cartife walls at either end thereof. The membrane barrier layer or “skin” is usually on the inside of the hollow fiber or “lumen”. The process feed usually enters one end of the hollow fiber and a concentrated solution exists at the other end (US 4,990,252).

Mircroporous:

–ceramic: Couto (US 20050260672) teaches that membranes which which separate a desired species from undersirable species without substantial clogging problems and at a rate sufficient for continuous operation of the system include microporous membranes with pore sizes typically from 0.1 to 10 micrometers and are prefeably ceramic. 

–Positively Charged Microporous membranes

Wu (US 7,223,341) discloses a positively charged microporous membrane ahving a protien binding capacity of aobut 25 mg/ml or greater that includes a hydrophilic porous substrate and a crosslinked coating that provides a fixed positive charge to the membrane. The coating can be prepared by crosslinking a composition that includes a diallylamine copolymer haivng epoxy grounds and pendant cationic groups, a polyamine such as a polyakyleneamine, and an amin reactive compoudn having a cationic group. In some bodiments, the psotively charged microporous membrane includes a hydrophilic porous substrate and a coating preapred by crosslinking a composition that includes diallylamine, a diallyldiakylammonium halide, an acrylic monomer having a quaternary ammonium group and a crosslinking agetn. 

Polysulfone type: polymers have been exensively used in the manufacture of ultrafiltration membranes becuase of excellent hydrolytic stability and high temperautre properties. Commercially avilalbe polysulfone polymers include UDEL polysulfone manufactured by Union Carbide Corp and Victrex polyethersulfone manufactured by Victrex (ICI) (US 4,990,252). Couto (US 20050260672) teaches that UF membranes which have smaller pores and are characterized by the size of the protein that will be retained are available in increments from 1000 to 1 million Dalton NMW and are commonly made of polysulfone.

Hydrophilic microporous membranes:

It is well known that membranes with hydrophilic surface are usually less prone to fouling and cake formation. Numerous attempts have been undertaken to hydrophilize UF membrane surface by blending orignal polymers with hydrophilic polymers, by sorption of hydrophilic macromolecules, by pretreatment of polysulfone (PS) membranes with poly(N-vinylpyrrolidone) (PVP), poly(vinyl alchol) or methylcellulose and by introducing ionogenic groupons onto the membrane surface. Surface charge of the membrane has also an influence on membrane fouling. If functional group swith the same charge sign as the seaprated components are introduced on the surface of polymer membranes, membrane foulind decreases. (Tischchenko “Purificaiton of polymer nanoparticles by diafiltration with polysulfone/hydrophilic polymer blend membranes” Separation and purification technology, 22(23), 2001)

–cellulose:

Cellulose is the most hydrophilic filter material in use, and this natural property of cellulose maximizes the yield of protein recovery by minimizing protein adsorption . In addition, cellulose maintains high output over long filtration times and is minimally affected by detergent. (see AsahiKasei, “PLANOVA filters” “Virus removal for biotherapeutic products).

The virus removal filter PLANOVA is composed of the hollow fibre membrane BMM made of a cuprammonium regenerated cellulose, is able to remove particles effectively, mianly based on their size. (Manabe, “Removal of virus through novel membrane filtration method” pp. 81-90).  PLANOVA filters are commercially available from Asahi Chemical Ind. Co. Ltd. 

WO01/14047 describes a filtration membrane for physiologically active substances wherein the logarithmic removing ratio for parovivus is 3 or more. The main membrane comprises hollow fibers made of cellulose. It has been described as disadvantageous in that when it is wet with water, the mechanical strenght is low, filtration pressure cannot be made higher and thus it is difficult to achieve a high permeation rate.

—-Cuprammonium Regnerated Cellulose

AsahiKasei sells Planova filters which utilize a hollow-fiber microporous membranemposed of naturally hydrophilic cuprammonium-regenerated cellulose inside a polycarbonate housing. The filters are sold under the tradenames Planova 15N, 20N, 35N and 75N. The 75N filter is designed to remove impurities or aggregated proteins prior to final virus filtration.

–Synthetic Polymers

—-Polyvinylidene fluoride (PVDF):

Koguma (US2006/0016748 and EP1552878A1) discloses a hydrophilic microporous membrane comprising a thermoplastic resin such as polyolefin and fluoride resins (e.g., polyvinylidene fluoride). The vinyl group is graft polymerized to impart the surface of the micropores with hydrophilicity thereby reducing adsorption of physiologically active substances such as protein.

Yanagida (12/993512) discloses that many methods for hydrophilizing a membrane formed of a synthetic polymer, such as a method of graft polymerizing a hydrophilic vinyl monomer with one vinyl group onto the pore surface of a microporous membrane at a graft ratio of 3 to 50% (WO2004/035180), a method of bonding a polymer formed of a polyfunctional acrylate or methacrylate to a membrane via a crosslinking agent (JP-A-2000-1548), or a method of impregnating a membrane with a solution incluidng a polymerization initiator and a hydrophilic monomer, and polymerizing the monomer inside the pores of the membrane (WO91/16968). The purpose is to imrorve filterability of the membrane formed of a hydrophboic synthetic polymer with respect to the intermediate protein product.

ASAHI KASEI BIOPROCESS sells hydrophilic modified polyvinylidene fluoride (PVDF) hollow fiber membranes under the trademark Plnova BioEX.

–Polyether sulfone (PES): is reported by the manufacturers (Millipore, Bedformd MA) to filter large volumes of sample without protein binding. PES membranes are cheaper than those made of PVDF and larger volume of sewage sample was reportedly filtered with the same PES filter unit. PES membranes are also cheaper than PVDF membranes (Moce-Llivina (J. Virological Methods, 109, 2003, 99-101).

Companies: Asahi kasei Bioprocess, Inc.; Asahi Kasei Medical; Millipore

See also Virus filtration during antibody purification under “antibodies” 

Types of Membranes  See outline    Conditions/Parameters See “operating conditions”

Since infection with an infectious virus among pathogenic agents may cause serious diseases, removal of contaminating viruses is highly required (EP 1552878A1). Regulatory guidelines require that recombinant DNA dervied protein products for human use meet a criterion of less than 1 virus particle per million doses. There is also a requirement to demonstrate that virus inactivation and clearance are accomplished using at least threee different mechanbisms. Common methds for removal of viruses include sized based filtration, affinity and anion exchange chromatography. Methods for inactviation often include low pH, heat, and use of solvents and detergents. (Van Reis, “Bioprocess membrane technology, Science 297 (2007) 16-50) 

Shukla, (J. Chromatogr. B 848 (2007) 28-39) discloses employing viral filtration in antibody purification scheme to complement a low pH viral inactivation step. 

Current virus retentive filters are ultrafilters or microfilters with very small pores. Virus filtration membranes are made from hydrophilic polyethersulfone (PES), hydrophilic polyvinylidene (PVDF) adn regenerated cellulose. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)

Nanofiltration for Removing Viruses: 

Nanofiltraiton is a very effective method for removing viruses. In this respect, the pore size of the filter has to be smaller than the effective diameter of the virus which is to be removed. In addition, the temperature, the properties of the materials and the buffering conditions are of importance. Parvovirus can be reliably removed using filters having a pore diameter of 15 nm and nanofiltraiton has been used for separating hepatits A virus and parvovirus from factor IX preparations with filters such as Virsolve 70, Planova 15N and Pall Ultipor DV20. In contrast, blood coagulation factor IX has a low MW of 56kDa and is thus not retained by membranes employed for nanofiltration, Large proteins such as fibronogen, von Willebrand factor and factor VII are alos too big to be freed from viruses by filtering them through a nanofilter haivng a pore size of from 15-35 nm. (Lengsfeld US 2003/0232969). 

Size of Viruses Determine Diameter of Filter to Use

Types of viruses include smallest viruses, such as parvovirus, with a diameter of about 18-24 nm, medium-sized viruses, such as Japanese encephalitis virus, with a diameter of about 40-45 and relatively large viruses, such as HIV, with a diameter of about 80-100 nm. In order to remove these virus groups physically by the membrane filtration method, a microporous membrane having a pore size of about 10-100 nm is required.

As to antibody purification using filtration see “antibody purification”

Typical components which pass through and are retained by membranes (WO 2005/091801)

Cell culture supernatant generally has an ionic strenght between 100 and 150 mM, pH 7.5 and is likely to contain anionic or hydrophobic species other than DNA such as RNA, host cell protein and surfactants (Charlton, Bioseparation, 8, 1999, 281-291). 

DNA: 

The structure of DNA is such that electrostatic interactions are possible along the polyphosphate backbone of the molecule and hydrophobic interactions are possible within the grooves of the helix. Such a combination of both electrostatic and hydrophobic interactions for absorption of DNA using various depth filters are reported by (Charlton, Bioseparation, 8, 1999, 281-291). 

Plasma Proteins:

Fibrinogen/fibronencting, coagulation factor V, coagulation factor VII, von Willebrand Factor: 

Fibrinogen is a 340 kDa hexameric glyoprotein. It is a large protein and thus far been regarded as being too big to be freed from viruses by filtering them through a nonfilter having a pore size of from 15-35 nm. Thus far fitration methods fo fibringone have been described for a filter pore size of 35 nm. A problem here is that at a pore size of 35 nm, relatively small, noenveloped virsues such as hepatits A and parvovirus cannot be removed. However, Lengsfeld (US 2003/0232969) discloses separating off viruses from a protein solution by nanofiltration by adding a chaotropic substances such as arginine, guanidine, cirtulline, urea and derivatives thereof using filters with a pore size ranging from 15-25 nm.

Ristol Debart (EP 1,457,497) discloses remvoing viruses in fibrinogen solutions by freezing an adjusted purified solution (a cryoprecipitate, the first fraction of Cohn method or equivalent fibrinogen containing fraction is precipitated preferably with glycine) and then thawing at a temperature between 5-20C. The undissolved materials associated with the fibrinogen are subsequently separated, the temperature is adjusted and the resultant solution is finally subjected to nanofiltration using filters smaller than 35 nm. The freezing, thawing and anofiltration are carried out in the presence of at least one amino acid.  With the controlled freezing and thawing, insoluble, aggregated or partially denatured materia is precipitated that would, in pracvtice, prevent the filtration of the solution through pore sizes smaller than 35 nm. Separation of the precipitated material allows nanofiltration to a pore size smaller than 35 nm.   

Takahashi (US6,867,285) also discloses that contaminant viruses can be efficiently removed by subjected a plama protein such as fibrinogen, fibronectin, coagulation factor V, coagulation factor VII, von Willebrand Factor, coagulation factor XIII, retinol binding protein, alpha-globulin, beta-globulin and gamma-globulin to a porous membrane having a pore size greater than a single particle size of the virus which permits passage of the virus. During filtration through a porous membrane, chaotrropic amiono acid may be added. This allows enhanced expression of the effect that contaminant viruses can be removed efficiently by the porous membrane treatment without losing the activity of the protein.  

–Dual/successive filters (larger pores to smaller pores): 

Burton (US 8,198.407) disclsoes affinity capture of von Wiilbrand Factor/Factor VIII on an affinity adsorbent developed for the capture of vWF/FVIII Filtered plasma was applied on the column and the flow-through effluent was collected when the adsorbance at 280 nm reached 5%. The column was washed with  to collect the column effluent until adsrobance dropped won to 5% of the adsorbance full scale. The solution was mixed and then filtered through a 3/0.8 um SartoCelanCA followed by a 0.45/0.22 um Sartobran P filter. 

Viruses:

Viruses to be used in validation studies for plasma products should include HIV-1 , a model for HPC (e.g., Sindbis or bovine viral diarrhoea virus (BVDV); non-enveloped viruses (animal parvovirus, hepatitie A) and an enveloped DNA virus (e.g., a herpesvirus). (Brandwein, “membrane filtration for virus removal” Dev Biol. 1999, 102, pp. 157-163).  

Hepatitis E Virus (HEV): The sensitivity of HEV to heat has been shown to vary greatly depending on the heating conditions whereas HEV particles were completely removed using 20-nm nanofilters (unoki, “extent of hepatitis E virus elimination is affected by stabilizers present in plasma products and pore size of nanofilters” Vox Sanguinis 2008, 95, 94-100).

Murine Leukaemia Virus: Hazel Aranha-Creado (Biological (1998 26, 167-172) report using a hydrophilic polyvinylidenefluoride (PVDF) microporus membrane (Ultipor VF grad DV50 virus removal filter) was effective to remove endogenous retrovius like contaminants such as MLV from monoclonal IgG rpoducts.  

 Porcine parvovirus (PPV): Hongo-Hirasaki (J. Membrane Science 278 (2006) 3-9) discloses using Planova20N was effective in the removal of small viruses such as parvovius from IgG solution. Shile proteins such as IgG pass through the capillary void pore structure, viruses are exlcuded from passing through the capillary ports. Thus even in the case wehre the size of filter object (i.e., parvovirus) is very close to that of IgG, Flanova 20N shows high virus removaility less than 1/10 rejection of the original solution without damaing the IgG permeability).  For purification of viruses from antibody, see “antibody purification”.

Other Components:

Outer Membrane Vescies (OMV) from bacteria:

Olivieri (US 2007/0087017) discloses ultrafiltration for preparing bacterial OMVs. The process incldues post-ultrafiltraiton steps where the OMVs may be sterilised by passing the OMVs thorugh a standard 0.22 um filters. Because these filters can become clogged it is rpeferred to perfrom sequential steps through a series of filters of decreasing pore size, finishing with a standard sterilistation 0.22 um filter. Examples of preceeding filters include those with a pore size of 0.8 and 0.45 um. 

Bar: 1 Bar=14.5037738 psi.  1 Bar= 100 kilopascals  1 kilopascal = 0.001 Megapascals

Crossflow Velocity:  refers to the velocity of the fluid across the top of the membrane surface. A “flow” is usually given in terms of volume per unit membrane area per unit time as with liters/m2/h (LMH). CF=Pi-Po where Pi is pressure at the inlet and Po is pressure at the outlet and is related to the retentate flow rate .It provides the force that sweeps away larger molecules that can clog the membrane .

Delta p: is pressure drop from retentate inlet to outlet. It is (pi-p0) where pi is the inlet pressure and p0 is the outlet pressure. 

Depth filtration: see outline

Direct flow filtration device (DFF): is a filtration device that has one inlet and one outline. DFF devices are tpyically single use devices. Depending upon the selected pore size, molecules smaller than the aerage membrane pore size will pass through the filter. In contrast, tangential flow filtraiton (tff) devices have one inlet, one retentate outlet and at least one permeate outlet (see outline for TFF). 

Filtrate Flux (J): is the rate at which a portion of the sample has passed through the membrane.

Flow: is typically given in terms of volumn per unit membrane area per unit time as in liters/m2/h (LMH). A cross flow, for example, might be given as ml/min for a membrane area of xm2.

Flux (permeate flow): the flow of fluid across the membrane (i.e., through the pores of the membrane).

Log reduction value (LRV):  LRV is obtained by calculating the cahnge in viral concentration in say an antibody solution between before and after filtration with a virus removing membrane. LRV=Log10(C0/CF) where C0=viral concentration in an antibody solution before filtration with the virus removing removing membrane and CF=viral concentration in the antibody solution after filtration with the virus removing membrane.

nanofiltration: is a special kinda of ultrafiltration (Bachacher “Purification of intravenous immunoglobulin G from human plasma – aspects of yeild and virus safety” Biotechnol. J. 2006, 1, 148-163).

Nominal Molecular Weight Cut Off (NMWCO): is the size (kilodaltons) designation for the ultrafiltration membranes. The MWCO is defined as the molecular weight of the globular protein that is 90% retained by the membrane. 

Percent Solids by Weight (% w/v): is defined as the weight of dry solids in a given volume of a susension divided by the total weight of that volume of the suspension, multipled by 100.  (Menyawi, US 17/054,018, published as US 2021/0246162)

Retentate: is the portion of the sample that does not pass through the membrane, also known as the concentrate. Retentate is typically re-circulated during TFF. 

Surface (Zeta) potential: When the zeta potential of monoclonal antibodies under given solution conditions is designated as Eil (mV) and the zeta potential of a virus removing membrane under given solutios conditions is designated as Em (mV), the two desirably have the following relationship: 0 mV≤Eil-Em≤20mV. When the result of Eil-Em is within the range that allows interaction between the antibodies and the membrane to decrease, it is thought to have an effect of improving the filtration rate of the membrane. A value for Eil-Em of more than 20 mV causes electrostatic interaction between antibodies and the membrane to increase, having an adverse effect on filtration. Also, when the virus removing membrane is negatively charged within a certain pH range and the antibodies are positively charge, the relationship between the zeta potential Eil of antibodies and the zeta potential Em of a membrane is desirably represented by -4% x Em ≤Eil1≤-550% x Em. Hongo (US 13/260419). 

Transmembrane pressure (TMP): refers to the pressure which is applied to drive the solvent and components smaller than the cut-off value of the filtration membrane through the pores of the filtration membrane. In tangential flow systems, highest TMP’s are at the inlet (beginning of flow channel) and lowest at the outlet (end of the flow channel). TMP is calculated as an average pressure of the inlet, outlet, and filtrate prots. The TMP is the force that actually pushes molecules through pores of the filter. TMP can be calculated as TMP [bar]=(PF + PR)/2-Pf where PF is the feed pressure, PR is the retentate pressure and Pf is the filtrate pressure. In other words, the driving force (transmembrane pressure or TMP) is the difference between the average of the membrane feed pressure (P1) and the retentate pressure (P2) minus the permeate pressure (P3); TMP=(P1+P2)/2-P3). Schilog (WO 02/00331)

Volume concentration factor (VCF): refers to the ratio of the initial feed volume to the retentate volume. For examplke, if 20 L of feedstock are processed until 18 L have passed through to the filtrate and 2 L are left in the retentate, a 10 fold concentration has been performed so the VCF is 10. 

See also “purification fo antibodies” using filtration, “Definitions” and  graft chains and ionic functional groups attached to membranes (under “chromatography” and “graft chains”)

Companies/Products:  3 M purificaiton .  Pall Life Sciences Mini Kleenpak  

Sartoclean CA mini cartidges (available with 3.0/0.8 um and 0.8/0.65 um cellulose acetate double membranes for the retention of partciles and larger microorganisms by means of fractionated membrane filtraiton, as well as simple membrane mini cartidge with 0.2 and 0.45 um pore size. Main applicaiton is prefiltraiton in combination with subsequent Sartogran P mini cartridge. ) 

Sartogran Sartorius 0.45/0.2 um filtration unit. 

Introduction:

Filtration is a commonly used technique for separating proteins. The filters used during filtration are often classified by retained particle size. For example, membrane microfilters generally retain partciles 0.1-10 microns in diameters.

Ultrafiltration (see outline)

Ultrafiltration membranes have pore sizes between 1 and 20 nm and are designed to provide high retention of proteins and other macromolecules. They can also be used for HPTFF. Microfiltration membrane have pore size between 0.05 and 10 um and are designed to retain cells and cell debris while allowing proteins and smaller solutes to pass into the filtrate. Virus filtration membranes are sometimes (but incorrectly) referred to as nonofiltration membranes based on their 20-70 namometer pore size. Nanofiltration is properly defined as a process that spearates solvent, monovalent salts and small organics from divalent ions and larger species. Depth filters are not typically considered as membranes since they retain key components throught the porous structure. (Van Reis, “Bioprocess membrane technology, Science 297 (2007) 16-50) 

The majority of established applications are still performed on conventional ion exchangers such as Sepharose Fast Flow Q (GE Healthcare) but charged membrane filtration (Pall Corporation, Sartorius) is becoming increasingly popular (Gagnon, p. 492).

Diafiltration

Diafiltration is a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular weight material and/or to cause the rapid change of ionic and/or pH environments. Diafiltration is performed with the same membranes as ultrafiltration and is a tangential flow filtration. During diafiltration, buffer is introduced into the recycle tank while filtrate is removed from the unit operation. In processes where the product is in the retentate (for example IgG), diafiltration washes components out of the product pool into the filtrate, thereby exchanging buffers and reducing the concentration of undersiable species. Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate of about equal to the ultrafiltration rate.

Diafiltration is thus a convenient and efficient technique for removing or exchanging salts, removing detergents, spearating free from bound molecules, removing low MW materials, or rapidly changing the ionic or pH enviornment (WO 2005/091801).

Shaban (US 14/809,211) discloses a method of purifying an antibody from a  biologic composition by diafiltering the composition with phosphate buffered saline (PBS) such as sodium phosphate and NaCL. The method is conveniiently used with anion exchange since the method removes (Bis-tris) and particularly with the mixed mode resin Capto adhere affinity chromatography which is a mixed mode of anion exchange and hydrophobic interaction since Bis-tris is a buffer used in a Capto adhere purificaiton step and is an comtaminate. 

Hemodiafiltration: combines both standard dialysis and hemofiltration into one process, whereby a dialyzer cartridge containing a high flux membrane is used to remove substances from the blood both by diffusion and by convection. The removal of substances by diffusion is accomplished by establishing a concentraiton gadient across a semipermeable membrane by flowing a dialysate solution on one side of the membrane while simultaneously flowing blood on the opposite side of the membrane. Collins (US 6,406,631) discloses a blood dialysis system which includes a hemodiafiltraiton sytem comprising a first dialyzer and second dialysate compartment.

Depth Filtration (see outline)

Membrane Chromatography

Membrane chromatography: function similarly to packed chromatography columns, but in the format of conventional filtration modules. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)

Microfiltration

Microfiltration: is a pressure driven separation process that uses membranes of a given pore size to separate components in a solution or suspension on the basis of their size differences. It is not fundamentally different from ultrafiltration or nanofiltration except in terms of the size of the molecules it retains. (Yigzaw, Biotechnol. Prog. 2006, 22, 288-296).

Type of Membranes  see outline

Typical components which pass through and are retained by membranes (WO 2005/091801)

Modes of Filtration (see outline)

Methods of Extracting Proteins from starting precipitate

By Repeated Dilution of precipitate to a final dilution factor:

(Menyawi, US 17/054,018, published as US 2021/0246162) discloses a continuous extraction/separation process for maximizing the recovery of a protein of interest from a starting precipitate/material using filtration. The method also allows liquid or diluent to be re-circulated in a closed system and thus the quantity of the liquid is maintained through the process while footprints such as large tank volume can be reduced. The method recovers at least 95% of the protein of interest in the protein comprising precipitate. According to the method, the precipitate is first mixed with a liquid in a first tank to form a suspension having a first diltuion factor (DF) and the suspension is then fed into a first filtration unit to product a first retentate and a first permeate enriched with the protein of interest. The suspension can then be diluted by adding liquid or by streaming the first retentate into the first tank to a second dilution factor. These steps are repeated until either a final dilution factor has been acheived in the first tank or a protein concentraiton of the supension in the first tanke of 0.001-0.1 g?L has been acheived. 

Types of Filter Units

Ribault (US2009/0181450A1) discloses a filter body vaing a first and second filter membrane where the second membrane has a pore diameter smaller than the pore diameter of the first membrane. A method of using the filter body where a sample is passed through the first and second membranes is also disclosed.

See also capillary electrophoresis under Diagnostic Techniques and Common Analytical Techniques

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells) to investigate subunit compositions and to purify the protein for subsequent applications. In polyacrylamide gel electrophoresis, proteins migrate due to an electrical field through pores in the gel matrix. Pore size decreases with higher acrylamide concentrations. The migrations of the protein is determined by the gel pore size and protein charges, size and shape.

Isolectric Focusing:

The basis principale behind isoelectric focusing in a pH gradeint is that a charged molecule will become immobilzied in an electric field when it migrates to a position in the pH gradient that is equal to its isoelectric point (zero net charge). This process occurs independently of the initial location of a specific prtoein in the solution. It is the result of the disappearance of the effective electrical charge of the protein when migrating to the region where pH is equal to pH. (Zilberstein, US 7914656)

One dimensional gel (1-D) electrophoresis

1-D electrophoresis under denaturing conditions (i.e., in the presence of 0.1% SDS) separates proteins based on molecular size. Most proteins bind SDS in a constant weight ratio, leading to identical charge densities for the denatured proteins. Thus the SDS protein complexes migrate in the polyacrylamide gel according to size, not charge. Most proteins are resolved on polyacrylamide gels containing from 5% to 15% acrylamide. The relationship between the relative mobility and log molecular weight is linear over these ranges. After the proteins are solubilized by boiling in the presence of SDS, an aliquot of the protein solution is applied to a gel lane, and the individual proteins are separated electrophoretically.  2-mercaptoethanol is added during solubilization to reduce disulfide bonds. Comparison of reducing and nonreducing gels can provide valuable information about the number of disulfide cross linked subunits in a protein complex. If the subunits are held together by disulfide linkages, the protein will separate in denaturing gels as smaller sized subunits as compared to nonreducing conditions where the protein will separate as a complex.

The polyacrylamide gel is cast as a separating gel topped by a stacking gel and secured in an electrophoresis apparatus. After leaving the stacking gel, the protein enters the separating gel which has a smaller pore size, a higher salt concentration and higher pH compared to the stacking gel. The proteins are separated according to either molecular size in a denaturing gel (containing SDS) or molecular shape, size, and charge in a nondenaturing gel.

Two-dimensional  (2-D) gel electrophoresis

2-D gel electrophoresis separates proteins in the first dimension by isoelectric focusing and in the second dimension by electrophoresis in the presence of SDS. Thus information is obtained not only about size as in one-dimensional gels but also about the charge of the protein. In isoelectric focusing a pH gradient is established using ampholines. Proteins migrate to their isoelectric point (pH) at which there is no net charge.

The protein spots can be visualized by staining. An imaging system is used to record an image of the stained gel to provide a record of the protein distribution in the sample. These images can be analyzed, compared, and archived with software packages (e.g., MELANIE, PDQuest, Z3, Progenesis Workstation, ProteomeWeaver, ProteinMine, Delta2D, DeCyder).

Challenges: A concern with 2D gels is that despite their resolving power, 2D gels do not completely resolve all proteins into single spots. Many spots contain 2-5 proteins. Another concern is that even with the most sensitive stains, there is a limited dynamic range for protein detection. Cellular expression levels of different proteins can differ by as much as a million-fold whereas the dynamic range for protein staining is about 100-2000 fold. Thus, 2D gels typically detect only the most abundantly expressed proteins.

2-D in Combination with other Techniques

2D-SDS-PAGE combined with a high-throuput MS analytical method (MALDI-TOF MS). Gel digestion is used to cleave the proteins to peptides which are then analyzed by MALDI-TOF MS. The MS data is then analyzed with a peptide mass fingerprinting algorithm and software which identifies the proteins present. A second approach is to use peptide sequence identification by tandem MS. Here, one digests the proteins in the mixture to peptides which are then resolved (at least partially) by chromatography and then electrospray tandem MS. These spectra are mapped to protein sequences from databases with the aid of Sequest or similar search tools. The 2 distingushing characteristics of this approach are 1) the analysis primarily involves working with peptides rather than with proteins and the protein identification is based on the MS-MS fragmentation spectra, rather than on peptide mass fingerprinting.

Fractionation of Proteins by pI value and then 2-D: Hagner-mcwhirter (WO 2007/058584) discloses a method of loading a sample containing a complex mixture of proteins onto media which can be chromatography matrix and eluting by centrigual force with at least three buffers having different pH in a stepwise manner to obtain at least three fractions separated accordingl to pI value and then subjecting each fraction to further separation by 2D electrophoresis.

Alpha-1 proteinase inhibitor (a1PI), also named alpha-1-antitrypsin (AAT) is a glycopeptide inhibitor of proteases, and is found in human serum and other fluids. Isolation of a1PI from human plasma is presently the most efficient practical method of obtaining a1PI in quantity and human plasma is the only FDA approved source Matthiessen (US 2007/0037270). 

AAT is an essential protease inhibitor found minly in the blood. AAT normally protects connective tissue, such as the elastic tissues of the lungs, from degradation by elastase, an enzyme released by neutrophils at sites of inflammation. (Kumpalum US 8,580,931).

By Alcohol/Ethanol Precipitation steps

Most published processes for AAT isolation begin with one or more fractions of human plasma known as Cohn fraction IV precipitates such as Cohn fraction IV, or more specifically fraction IV1 and fraction IV1-4 as well as precipitates of Kistler-Nitschmann supernatant A or A-I which are obtained from plasma as a paste after a series of ethanol precipitations and pH adjustments. Brinkman (WO2009/025754).  Cohn IV-1 precipitate is usually derived after ethanol fractionating of plasma by precipitation with 20% ETOH at pH 5.2 (Matthiessen (US 2007/0274976). 

In the Cohn-Oncley method 6 which consists of 3 precipitation steps, the first precipitation step, referred to as Fraction I precipitation, is performed at high pH (7.2) and low ethanol concentration (8-10%) to precipitate proteins such as fibrinogen and Factor XIII away from IgG and A1PI, which remain in the supernatant. IgG is then precipitated from the Fraction I supernatant in a second precipitation step, referred to as a Fraction II+III precipitation, performed at moderate pH (6.8) and high ethanol concentration (20-25). The bulk of the A1PI remains in the supernatant of the Fraction II+III precipitation, and is subseqeuntly separated from albumin in a third precipitation step, referred to as a Fraction I-IV-1 precipitation, performed at low pH (5.2) and moderate ethanol concentration (18%). 

Starting with Cryosupernatant

Bruckschwaiger (US13/776,448, published as US 2013/0224183) teaches that a single initial precipitation step that captures all of the proteins normally precipitated in the Fraction I, Fraction II+III and Fraction IV-1 precipitates combined using an initial low pH, high alcohol precipitation and that IgG and ApI could be efficiently extracted from this precipitate wihtout the use of subsequent protein precipitations. Bruckshwaiger teach that this is accomplished using a high inital ETOH precipitation step (20-30% ETOH at pH 5-6) without any prior alcohol precipitation steps resulting in co-precipitation of the immunoglobulins with A1PI. This precipitate can then be solubized, forming a suspension having an soluble porition where the immunoglobulins are located and an insoluble porition which A1PI is located for further processing.  

Starting with Effluent/Supernatant II+III

Bastek “Purification of alpha-1-proteinase inhibitor using ligands from combinatorial peptide libraries” dissertation, North Carolina State University, 2000) disclsoes that the effluent II+III in the Cohn fraction contains about 30 mg/ml totoal protein 3% of which is A1PI. Eff. II+III  main component is hSA, in addition to anti-thrombin III, IgG, IgA, Apo A-1, transferrin, haptoglobin and alpha-1 acid glycoprotein. Bastek teaches that Eff+III contains 87% of the total A1PI in plasma compared to only 49% in redissolved IV-1 paste (see starting with Fraction I-IV precipitate below) and thus has a distinct advantage over IV-1 paste for A1PI recovery.  

Kupalume (US2009/p292114) also teaches that Cohn fraction II+III supernatant (also known as supernatant A in the modified Cohn fractionation method described by Kistler and Nitschmann, 1962, Vox Sang, 7. p. 414 to 424) have been used as a source of AAT and that Cohn fraction II+III supernatant contains 2-3 times more active AAT than Cohn fraction Iv-1. 

Starting with Fraction I-IV1 Precipitate: 

Bastek “Pufication of alpha-1-proteinase inhibitor using ligands from combinatorial peptide libraries” dissertation, North Carolina State University, 2000) disclsoes that A1Pi has been purifed from redissolved Cohn fraction IV-1 paste as starting material. The Cohn process has 6 major precipitation steps starting with the centrifual harvest of cryoprecipitate from freshly thawed plasma. The effluent is brought to 8% ETOH at pH 7.2, -2.5C and the resulting fraction I precipitate is discarded. The supernatant I composition is modifed to 25% ETOH at pH 6.6, -6C. A second precipitation, Paste II+III, is collected by centrifugation and used as the intermediate for immunoglboulin renrichment. The II+III eflluent pH is decreased to 5 to yield fraction Iv-1. IV-1 paste frequently serves as the intermediate for isolation of A1pI and anti-thrombin III, while further fractionation of the eflluent yeidls albumuin (Fraction V). The IV-1 paste is redissolved at pH 7-8, 20C and contains 5-10% A1PI. The major contaminants are anti-thrombin III, albumin, lipoproteins, cerulpolasmin and trqansferrin. 

Brinkman (WO2009/025754) discloses separating alpha-a-antitrypsin (AAT) from apolipoprotein A-I (ApoA-1) from Fraction IV1 (obtained by a first pricipitation using 8%ETOH, pH 7.2, a second precipitation using 20% ETOH, pH6.9, taking the supernatant (the precipitate FRaction II+III is set aside for other purposes) and adjusting the ph of the superntant to about 5 and ETOH to 21%. The precipitate that forms (Fraction IV1) in this 3rd precipitate contains AAT, ApoA-I and other contaminintng proteins/lipids. APOA-I and AAT can then be seaprated from each other  by suspending the fraction IV1 at pH 9.6, coooling and adding ETOH 12%, pH 5.4. The soluble AAT filtrate is then searpated form the insoluble ApoA-I material by filtration. 

Kee (WO2004/060528) discloses preparation of a Cohn Fraction IV1-4 fraction by 1st cooling human plasma to -2-2C, adjust pH 69-7.5, cold ETOH 6-10%, lower temp to -4-0C, the precipitate that forms (Fraction I) is removed by centrifugation or filtration, 2nd, the filtrate/sueprnatant adjusted to pH 6.77.1, ETOH added 18-22%, temp -7-03C, the precipitate that forms (Fraction II+III) is set aside for other purposes, 3rd, the filtrate/sueprnatant is adjsuted to pH 4.9-5.3, ETOH 16-20%, temp -7-03, after the supension settles, pH adjusted 40-44% and ETOH added 40-44%, the precipitate that forms (Fraction IV1-4) is removed. It contains AAT as well as contaminating proteins. The Fraction IV1-4 paste is resupended in buffer, pH 7.5-9.5 and treated with dithiothreitol (DTT) and fumed silica. The solutlbe AAT product is separated from the precipitated fumed silica using a filter press. The ATT final filtrate is then applied to an IEX, the eluate to HIC, it is stablized by pasteurization, then NF. 

By chromatography

Affinity Chromatography:

Buettner (US 15/770,111, published as US 2018/0305401) discloses a method for purifying a first protein of interest and second protein of interest which can be IgG and A1PI starting from a fibringoen depleted plasma solution (see plasma proteins) using one affinity chromatography such as CAPTURESECT taht binds to a CH3 domain of IgG  and purifying the AqPI in the flow-through fraction using a second affinity chromastography. The IgG is eluted from teh 1st affinity chromatography and polished by IEX and viral filtration. The A1PI is also eluted from the 2nd affinity chromatography. A flwoth-through fraction of the A1Pi purificaiton process can e further processed to isolate addtional proteins such as prothrombin complex followed by albumin. 

AEX: 

Coan (US4,697,003) teaches a method for separating alpha-1-proteinase inhibitor (AKA alpha-1 antitrypsin) starting with either Cohn Effluent II_III, Cohn Effluent I and cryosupernatant solution. According to the method, the aqueous solution is treated to lower the concentraiton of salts such as by DF, contacting the solution with an AEX in bind and elute mode. 

Glaser (Preparative Biochemistry, 5: 333-348 (1975) discloses isolating AAT from Cohn fraction IV1 paste. The paste is stirred in phosphate buffer at pH 8.5 in order to reactivate AAT which is largely inactivated by the pH of 5.2 employed in Cohn fractionation. After dialysis and centrifugation, the supernatant is subjected to two rounds of AEX. 

Schulz (US 2006/0194300) discloses a process for preparing A1AT from a solution containing A1AT such as a reconstituted plasma fraction IV1 (Cohn) which inlcues subjecting the solution to AEX (DEAE) , eluting the A1AT foloowed by solvent/Detergent treatment. 

AEX-CEX: 

Lebing and Chen (US5,610,285) describe an initial AEX followed by CEX at low pH and low ionic strenght to purify human AAT from plasma. 

Wood (CA2432641) discloses purification of alpha-1 proteinase inhibitor (alpha-1 PI) from aqueous solutions such as human plasma by precipitation of contaminating protens by adding PEG followed by AEX in bind and elute mode. Further purificaiton may be done by CEX which takes advantage of the fact that alpha-1-PI does not bind to the CEX under certain conditions. 

AEX-HA: Mattes (Vox Sanguinis, 81: 29-36 (2001) and US 6.974,792) discloses a method for isolating AAT from Cohn fraction IV that involves ethanol precipitation, AEX and adsorption on HA. 

Schulz (US2006/0194300) discloses a process for preparing A1AT by subjecting an A1At containing solution to IEX, adding detergents and optionally a solvent for inactivating lipid enveloped viruses followed by increasing the salt concentraiton to salt out the detergent. 

See also IgA separation under antibody purification 

IgA on mucosal surfaces is produced locally and not dervied from circulating IgA. IgA is one of the gamma globulins on the basis of its electrophoretic mobility. It is composed of two H chains and 2 L chians. it may be monomeric, dimeric or trimeric. IgA mononomers are joined together as dimers at the constant regions of thier H chians by a J chain. IgA is secreted as one of two subclasses, IgA1 and IgA2. IgA1 predominates in the circulating blood wehrein most of it occurs as a monomer. Most IgA on mucosal surfaces, sucha s the surfaces of the trachea, bronchi, and bronchioles in the lungs, occurs as dimers or trimers joined by J chains. IgA dimers and timers have an increased ability to bind to and agglutinate target molcuels. (Simon, US 6,932,967). 

Starting Preparations/Samples

IgA can be frationated from plasma or other biological fluid using various combinations of precipitations and chromatographic techniques. (Menyawi US 14/377,535).

IgA is a byproduct in the cold ethanol fractionation process to prepare immunologlobulin G. In the fractionation method, pooled human plasma is first treated to produce a cryoprecipitate and cryo-supernatant. The cryo-supernatant is subjected to a first ethanol fractionation to yeild a supernatant I which is subjected to a second ethanol fractional to yield fraction II+III. Fraction II+III is subjected to a third ethanol fractionation procedure to yield a supernatant III and fraction III precipitation. The fraction III precipitate enriched in IgA is generally discarded as an unwanted byproduct. Simon (US 15/205, 359, published as US 2016/0319039). 

Fraction III precipitate:

Fraction III precipitate, contains the majority of serum IgA, but also a variety of other proteins as well as lipidic material which is difficult to process. Further purification procedures have thus been developed to obtain IgA (Leibl, J. Chromatography B, 678 (1996) 173-180).

Fraction III is obtained as by-product of large scale fractionation of human plasma. This fraction corresonds to the insoluble proteins after extraction of fraction II+III. (Pejaudier, Vox Sang, 23: 165-175 (1972).

The fraction III precipitate enriched in IgA is generally discarded as an unwanted by-product. (Simon, US 6,932,967). 

Particular Purification Schemes

Affinity Chromatography:

Corthesy (US 2015/0056180) disclosses preparation of human plasma IgA by affinity chromatography using CaptureSelect Human IgA resin using 3 different sources of plasma IgA; cryo-depleted plasma, resolubilised cold ethanol fractionation paste or a strip fraction from ACX. Secretory IgA was latter obtained by combining in vitro IgA with recombinant human secretory component. 

Jacalin: Roque-Barreira (J Immunology, 134(3), 1985) disclose that jacalin can precipitate IgA and describe an affinity chromatography procedure using jacalin.

Anion Exchance (AEX): 

Chtourou (US2004/0132979) discloses a prepurification step (e.g., a fraction I+II+III precipitate obtained from plasma is resuspended in acetate buffer and octanoic acid is added, precipitate is separated , subjected tto virus inactivation) and a single AEX step carried out at alkaline pH (e.g., TMAE-Fractogen quilibrated with a glycine-NaCl mixture pH 9), washed with the equilibrating buffer and then a sequenctial elution is performed using a first phosphate buffer at pH 6.2 to elute the IgGs, then a second elution with the same buffer to which has been added 150 mM NaCl which elutes an IgA and IgG4 enriched fraction. 

Leung, (Kindey International, 59, 2001, pp. 277-285) discloses separation of polymerica nd monomeric IgA1 pooled fractions by AEX which include eluting with 12.5 mL of elution buffer (a linear salt gradient from 0-1 mol/L NaCL in 20 mmol/L Tris-HCL, pH8.0. Subfractions of 250 ul were collected throughout the elution. 

Menyawi (US 14/377,535, published as US 2015/0005476) discloses a process for enriching IgA from a composition, such as one that has been obtained from plasma, by loading the composition on an AEX, either strong or weak,  under conditions that allow the IgA to bind, optionally applying a pre-elution step by applying a low conductivity soltuion, preferably at a weakly acidic to neutral pH and then applying an alkaline first elution solution with a substance having at east 2 acid groups such as a multivalent carboxylic acid wherein the protein eluted is enriched for monomeric IgA, but essentially devoid of IgM. An additional optional step of applying an acidic elution solution that comprises a strong competitor for the AEX is then performed so as to enrich dimeric IgA. 

Moller (US5,410,025) discloses precipitation of a Cohn Fraction II/III or III in a buffer, elimination of impurities by precipitation with octanoic acid at pH 4-6, then treating at a low conductivity with an AEX, attaching most of the IgA and IgM Since the anticomplementary activity of the IgG fractio nnot attached to the AEX is low, the fraction can be employed in conjunction with the fractions taht contain IgA and/or IgM to prepare mixture that can be converted into IVIG preparations.

Pejaudier, (Vox Sang, 23: 165-175 (1972) disclsoes various schemes of preparation of IgA from a Fraction III recipitate  by resuspending the fraction III precipitate with acetate buffer, pH 5.7, stirring for 30 min and keeping overnight at 4C, the precipitate was discarded and the supernatant passed on DEAE cellulose equilibrated at pH 5.7 with acetate buffer. Elution was done with acetate, pH 5.7. Then ethanol 32%, pH 5.7, superntant revmoed and precipitate dissolved in 0.9% NaCl, pH 5 with caprylic acid, recipitate remove (alpha2M) and supernatnt contains IgA.

Uemura (US 5,258,177) discloses a method of prepararing an IgA rich preparation froma  plasma fraction which incorporates DEAE Sepahdex condition with 0.005 mM naCl, pH 7.0, washing several times and then eluting the IgA using 100 mM NaCl which was substqeuntly concentrated by UF. 

AEX-HA:

Leibl (US6,646,108 and WO97/25352) discloses a method for the separation of IgG and IgA form an immunoglublin containing starting material by mxing the sample with Fractogen EMD TMAE 650 suspended in 50 mM sodium acetateaceitic acid buffer, pH 5.0 (non-bound material separated over a suction filter), washing twice with 50 mM sodium acetate/acetic acid buffer, pH 5.0, then stirring the gel for 2 h at 4C with 50 mM sodium acetate/acetic acid buffer +0.5 M NaCL, pH 5.5 (elution carrier out twice). The eluted material was dialysed against buffer pH 7.5. 3% of buffer B (0.5 M NaH2PO4/Na2HPO4 +150 mM NaCl, pH 6.8) was added to the retained material and this material was mixed with hydroxylapatite (ceramic) which had been equilibrated with 975% buffer A (PBS, pH 7.4 and 3% buffer B (0.5 M NaH2PO4/Na2HPO4 + 150 mM NaCl, pH 6.8) and suspended in the same buffer mixture. The sueprnatant was suction filtered and the HA washed which was combined with the first sueprnatant. The eluates of HA were mixed by stirring with ammmonium sulfate, the precipitate resuspended. analysis shoed that IEX brought about a partial enrichment of IgA but still contained HMWPs and/or aggregates. The HA treatmetn increased the ration of IgA to IgG. 

AEX-thiophilic resin-Metal chelate chromatography:

disclose that IgA can be separated by other proteins such as alpha anti-trypsin, IgG and IgM contained in a fluid such as plasma or serum or mucosal secretions (milk, colostrum, tears and saliva) by metal chelate chromatography. The IgA is absorbed to the metal ion matrix (ions include zinc, copper, nickel, iron, manganese, chromium, cadmium, calcium, magnesium) and then selectively eluted. Alternatively, the fluid containing IgA is applied to the metal ion matrix under conditions such that at least one of the others proteins is absorbed while the IgA is not substantially absorbed. According to the disclosed scheme, ethanol (8%) was added to plasma, the suerpnatant removed by UF, then delipidated with aerosil, the delipidated solution loaded onto a DEAE Sepharose Fast Flow (euqilibrated with 10 mM sodium acetate at pH 5.2), IgG and transferrin were removed in the void volumen and albumin eluted with 25 mM sodium acetate pH 4.5. IgA, haptoglobin, ceruloplasmin, alpha antitrypsin, IgM complement (C3) and small amounts of IgG and albumin co-eluted with 0.5 M sodium chloride pH 5.2, a solution of 1.7M ammonium sulfate, 0.1M sodium acetate was added to the eluate from the DEAE column with 0.5 M sodium chloride at pH 5.2, the solution loaded onto a thiophilic resin, IgA rich material eluted with sodium acetate, ammonium sulfated and dialyzed solution loaed onto a chemalte agarose column after Zn2+ had been immobilized. 

Gel permeation

(Leibl, J. Chromatography B, 678 (1996) 173-180) discloses a quick separation method  of IgG and IgA by starting with a saline extract of fraction III precipitate treated with 2 M ammonium sulfate, dissolving the precipitate and running directly on a Superdex gel permeation column. Six peaks were obtained and the amterial fractionated in the maximum of peak 4 consisted of rather pure IgA.

 Hydroxyapatite:

–HA-AEX:

(Leibl, J. Chromatography B, 678 (1996) 173-180) disscloses a method of purifying IgA by extraction of a fraction III precipitate, removal of coagulation proteins and lipoprotines by heparin-Sepharose and precipitation with dextran sulfate (Heparin Sepharose also adsorbed two thirs of the alpha macroglobulin), removal of remaining alpha macroglobulin by HA, Fractogel TMAE to further increase the IgA content and then gel permeation.

Purification of dimeric/polymeric IgA containing Secretory Component (SC)

Simon (US 15/205, 359, published as US 2016/0319039) discloses a method of purifying IgA with recombinant secretory component. According to the procedure a fraction III precipitate that is produced as a byproduct from production of IgG by ethanol fractionation of pooled human plasma is further purified by ion exchange adsorption purificaiton follwed by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. From 4-22% of plasma IgA is dimeric and polymeric IgA. The resulting dimeric IgA is further coupled to recombinant secretory comonent that is produced by recombinant techniques. the coupling is accomplished by forming disulfide bonds under mildly oxidizing conditions. Dimeric IgA cotnaining both J chain and SC is again purified by ion-exchange and size exclusion chromatography and/or UF. The purified dimeric and polymeric IgA containing SC is iptionally stablized for example by the addition of human serum albumin to a final concentraiton of 5%.