See American Water Works Association

See also Virus Separation

Definitions

Detergents: Detergents are amphipathic molecules, meaning they contain both a nonpolar “tail” having aliphatic or aromatic character and a polar “head”. Ionic charadter of the polar head group forms the basis for braod classification of detergents; they may be ionic (charged, either anionic or cationic), nonionic (uncharged), or zwitterionic (having both positively and negatively charged groups but with a net charge of zero). Detergents are a class of molecules whose unique proterties enable manipulation (disruption or formation) of hydrophobic – hydrophilic interactions among molecules in biological samples. Detergents are used to lyse cells (release soluble proteins), solubilize membrane proteins and lipids, control protein crystalization, prevent nonspecific binding in affinity purificaiton and immunoassay procedures and are used as additives in electrophoresis. Detergents can be denaturing or non-denaturing with respect to protein structure. Denaturing detergents can be anionic such as sodium dodecyl sulfate (SDS) or cationic such as ethyl trimethyl ammonium bromide. These detergetns totally disrupt membrane and denature proteins by breaking protien-protein interactions. Non-denaturing detergents can be divided into nonionic detergents such as Triton X-100, bile salts such as cholate and zwitterionic detergents such as CHAPS. Generally, moderate concentrations of mild (i.e., nonionic) detergents compromise the interity of cell membranes, thereby facilitating lysis of cells and extraction of soluble protein, often in native form. Non-denaturing detergents such as Triton X-100 have rigid and bulky nonpolar heads that do not penetrate into water-soluble proteins. consequently, they generaly do not disrupt native interations and structures of water-soluble proteins and do not have cooperative binding proterties. (ThermoFisher Scientific, “Detergents for cell lysis and protein extraction”). 

Particular Non-Antibody Proteins Purified

Allergens: Goda (US4,902,783) discloses a purification method for removing allergen from gene expression products using a sequence of steps of adsorption treatment with silica gel, activated carbon at least two density gradient centrifugation  and two equilibrium density gradients steps. 

Annexins: Lipocortins:

Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. They are found in a wide range of tissues, with not well understood, but apparently very diverse phyjsiological effects. Their amino acid sequences show a high degree of similarity, with a segment of about 70 amino acids being repeated four times (eight times in annexin VI). (Lewit-Bentley, European J of Biochemistry 210:1 (1992), 73-77. 

Lipocortins are a family of proteins that display homology and include PP4, PP4-X, PAP III, p68 and liporcortins I and II. These proteins have antiinflammatory and anicoagulant effects. Rumisch (US 5,136,026). 

–Annexin A5: represents a protein of high therapeutic interest. It has been used in the prevention of atherothrombosis and/or plaque rupture, and the treatment of vacular dysfunction. (Moks, US 15/760,641, published as US 20200262866).

—-Anion Exchange (AEX):

Juergen (EP0441274) discloses a process for the purifcation of lipocortins by treatment of a solution of a lipocortin such as PP4 with an AEX in the presence of a chelating agent and detergent. Accordingly to the method, centrifuged lystates of E. coli in Tris/HCL, pH 6.8, containing rPP4-X is mixed with Triton X-100 to a final concentration of 1.0%, incubated for 10 min with shaking and then centrifuged. 

Mitterer (US 2012/0108513) discloses a method for the purificaition of a divalent cation binding protein such as Annexin V which includes the steps of loading an AEX with the protein in a loading buffer in the absence of divalent cations and optionally washing the laoded ANEX with a washing buffer in the absence of divalent cations and then eltuing the divalent cation binding protein with an eluant that includes at least one divalent cation (examiles include Ca2+, B32+, Mg2+, Cu2+) to form an eluate containing the divalent cation binding protein wherein the eluant has a pH higher than the pH of the washing buffer or in case no washing step is used of the loading buffer. In certain emobdiments the AEX has a positively charged group such as DEAE, DMAE, TMAE, PEI, QAE and quaternary ammonium (Q). 

Rumisch (US 5,136,026) discloses a method for removing toxins from protein solutions containg PP4 which includes AEX in the presence of a chelating agent adn an ionic detergent. After addition of EDTA tto PP4 containing solution, the solution was brought into contact with DEAE Sepharose, washed and the absorbed prtoeins were eluted with an NaCL gradient increasing linearly. 

Wang ((Protein expression and purificaiton 45(2006) 8-87) disclsoes a single step IEX with DEAE Sepharose for purifying annexin A5. Annexin A5 was eluted at abuot 35 mM NaCl. Single protein band exhibiting a moleccle mass of 36 kDa present in SDS-PAGE gell. The purity of rh-annexin A5 was tested by HPLC showing greater than 98% purity as estimated form teh percentage of total peak area. 

——Homogenisation (non-ionic detergent)-AEX-Affinity Chromatography:

Moks, (US 15/760,641, published as US 20200262866; see as US Patent Application 17/332,968, published as US 2021/0347819) discloses a process for the purification of Annexin A5 (ANxA5) from an enotoxin producing host cell with a cell wall that includes releasing the intracellular AnxA5 protien in the presence of a homogenisation buffer that includes a non-ionic detergent such as Tween 20 or Tween 80. The non-ionic detergent (e.g., Tween 20/80) can be included in the homogenisation buffer which is then added to the cells or the cells may be suspended in the homogenisation buffer without the non-ionic detergent, and then the non-ionic detergent can be added. The non-ionic detergent prevents binding between Annexin A5 and endotoxin. In one embodiment the homogenisation buffer may also include a calcium metal ion chelotor such as ethylene glycol tetraacetic acid (EGTA) that does not strongly bind Mg2+. In one example, the buffer includes 50 mM Tris, pH 7.4, 1 mM MgCL2 and 1% Tween 80. Then the process can include the step of clarification to produce a clarified product that includes the released AnxA5 protein. The process can next include an anion exchange capture (AEX) step. It may be beneficial to include one or more types of additional selection metal ions (such as Mg2+) where the metal ions are selected such that the calcium metal ion chelator has a binding affintiy for the selected metal ions that is greater than its binding affinity for the AEX but less than its binding affinity for cacium ions.   This is important becasue EDTA can impact negatively on the efficacy of AEX steps. Free EDTA can bind directly to the AEX function groups and thereby reduce the capacity and the separation acheived by an AEX. Theparin affinity chmatography, it may be preferred to sue an elution buffer that includs a cacium metal ion chelator such as EDTA. The chelation reaction specifically elutes Annexin A5 which can only bind to Heparin in the presence of cacium. Next, the AnxA5 product can then also be further polished with another AEX step. Because the eluted AnxA5 product of the heparin affinity chromatography steps contains high levels of cacium ion chelator, it is again desirble additional selected metal ions such as Mg2+ are added prior to the AEX step. 

Jurgen (EP 0441274A2) dsiclsoes a process for the purification of lipocortins such as PP4 by treating a solution with the lipocortin with an AEX in the presence of a chelating agent and a detergent. 

Park (Molecular Medicine 22: 424-436, 2016) disloses transfer of annexin A5 plasmids into E coli, havesting and purifying the lysate with Ni+ affinity chromatography. The eluted protein was concentrated and analyzed using 12% gradient SDS-PAGE cells, Coomassie Brilliant blue staining, and anti-annexin A5 antibody (abcam). Endotoxin was removed from purified proteins by using Triton X-114 Surfact-Amps detergent (thermo Scientific). Endotoxin level (less than 0.1 EU/mg protein) and the level of bacterial DNA (0.1 ng/mg prtoein) were assessed using Limulus amoebocyte lysate (LAL) QCL-1000 (LONZA Ltd). 

—-Expanded Bed Chromatography:

Frej (Biotech & Bioengineering 44 (1994) 922-929) discloses using expanded bed adsorption with a novel IEX absorbent for recovery of recombinant human placental annexin V from E coli homogenate. 

—-Immunoaffinity:

Han (EP 0452849) disclsoes purificaiton of PP4 from human placenta by an affinity gel coupled to a mAb against PP4). 

–Immobilized Metal Chelate Affinity Chromatography (IMAC):

Ni-NTA (Nicel NTA) refers to a nickel2- ion that has been coupled to Nitrilotriacetic acid (NTA). Ni-NTA can be coupled to agarose resin or magnetic beads for IMAC. This is a purificaiton method to obtain functional His-tagged protein. 

Adenoassociated virus (AAV): See Virus Separation

Amino acids: Zou (CN 1515543) discloses separating amino aicds diluting protein hydrolyzate and flowing it through active carbon adsoprtion column, then flowing through AEX, mixsing the eluent with arginine and using flow through CEX. 

Exosomes (extracellular vesicles, EVs):

Exosomes are membrane-bound nanovesicles with a diameter of 30-150 nm and a phospholipid bilayer structure on the surface that are actively secreted out of a cell by endosomal membrane budding. Due to the secretion and release from different types of cells, exosomes usually carry cell-specific components such as various proteins, mRNAs and miRNAs. They participate in regulating a variety of signaling pathways by transmitting signal molecules and can direclty fuse iwth cells by endocytosis an dother means. (Tang US 17/628,754, published as 17/628,754)

EVs are loaded with a cargo of proteins, lipids and RNA, and they are tagged with surface markers which favor uptake by target cells. Thus, they are a key mode of cell-to-cell communcation. Naive EVs isolated from sources such as platelets and stem cells are also of great interest in regeneative medicine. Traditional prufication of EVs is based on ultracentrifugation (UC). 

Existing commonly used methods for separation and extraction of exosomes mainly include ultracentrifugation, density gradeint centrifugation, membrane filtraiton and size exclusion chromatography. However, most of them have low recvoeyr rates and are time-consuming and the exosomes are easy to rupture to produce a large amount of protiens and sufffer form lipid contamination. Tang (US 17/628,754, published as 17/628,754)

–Ligand-based exosome affinity purification (LEAP): has proven to be an efficient, scalable, low-cost and highly reproducible EV purification platform suited to the manufacture of EV. LEAP is based on selective capture (binding) and release (elution) of EVs from media. Previous research into EV IEX primarily has explored AEX for EV purificaiton. By contrast LEAP technology is based on cation exchange. Although EVs have a net negative charge, local areas of positive charge exist on the outer surface of their lipid bilayer structure. LEAP technoloy uses shcarged R groups spaced about 5 A apart along the LEAP resin “backbone” to selectively cpture EVs by interacting with those positive charges. (Law et al. “Ligand-based exosome affinity purfication” BioProcess International, June 2021, number 6, pp. 28-35). 

–Immunomagnetic microsphere with antibody to Bio-markers for isolation of nerve-tissue exosomes:

Mitsuhashi (US 2018/0340945) disclsoe a method of capturing exosomes by contacting a sample with a solid support such as a magnetic beac that include capturing agents such as an antibody which selectively bind to a biomarker on the exosomes. 

Tang (US 17/628,754, published as 17/628,754) discloses a silver iron oxide (Ag-Fe3O4) immunomagnetic microsphere that includes coupling an S100beta or MBP antibody via a poly-D-lysine to the microsphere. The microspheres can be used for extracting nerve tissue-derived exosomes.  S100beta protein is expressed and secreted by neuroectoderm cells. Myeline basic protein (MBP) accounts for 30% of the total myelin protein and is located on the serosal surface of myelin. MBP is mainly synthesized and secreted by oligodendrocytes in the central nervous system and Schwann cells in the periopheral nervous system, and plays a vital role in the differentation of nerve cells, myelination and the maintenance of the stability of the nervous system. 

Zubo (CN109576210(A)) discloses a method for rpaidly separating exosomes whcih includes an ultrafiltraiton pretreatment step followed by using superparamagentic Fe3O4 nano-particlesn which have polyethylene glycol (PEG) as a psacer and a ligand such CD63 which can separate the exosome under.a magnetic field.  

Complement Proteins:

Bansal (US 13/063301, published as US 2011/0160636) discloses an extracoporeal device for inhibiting AP pathway acitvation that includes a support with an anti-complement antibody. In one embodiment, the antibody is bound to a protein-G matrix.

Lambris (US 2011/0269113) discloses a method for reducing or eliminating biomaterial-induced procagulant activity in blood subject to extracorporeal treatment which includes treating the blood with a complement inhibitor in am amount effective to reduce or prevent C5a/C5aR mediated tissue factor formation. Any inhibitor of the complement cascade leading to the formation or activity of C5a or the C5aR can be used in the method. In one embodiemnt, an extracorporeal treatment device includes a complement inhibitor treated biomaterial and the device is a hemodialysis unit. 

Storr (US 15/895551, published as US 2019/0247560) disclsoes a blood treatment device for remvoing a complement factor form blood wehre the device includes a matrix configued to immobilie said compement factor. 

Human Serum Albumin: Ohmura (US5294699) discloses culturing clls in the presence of an absorbent such as an anion exchange or active carbon which binds coloring contaminants so as to separate said coloring contaminants from the albumin. The method solves the problem that HSA has a dark yellow or dark yellowish brown color when HSA is produced by recombinant DNA technology as opposed to the yellow color of serum derived HSA. 

Erythropoietin: Schmalz (WO2010/034442A1) discloses a method for purifying erythropoietin comprising at least one chromatography step of which hydroxyapatite is used. The method comprises binding erythropoietin in a solution containing calicum ions and then a further solution containing less than 0.5 mM clacium ions is applied whereby erythropoietin is detached from the HA.

Factor VIII protein: Factor VIII concentrates for the treatment of hemoophilia have been prepared by fractionation of plasma. Methdos are also available for production of Factor VIII in cell culture using recombinant DNA techniques. Hemophilia is an inherited disease with Hemophilia A the most frequent form. It affects only males with an incidence of 1-2 persons per 10k liveborn males. The diesease is caused by a strongly decreased level or absence of biologically active coagulation Factor VII (also known as antihemophilic factor, AHF). Ljungqvist (WO 00/63243) disclsoes using modified B or Z domains from staphylococcal protein A (SpA) for the purificaiton of human Factor VIII. Preferably at least 4 amino acids of the SPA domain have been substituted. 

Hemoglobuilin: Pliura (US5,439,591 and 5,545,328) discloses a process for purifying hemoglobuilinwhere in the first stage the sample is applied to an anion exchange such that Hb and contaminants having lower affinity for the column are displaced such that certain contaminics which are more acidic than the Hb species are absorbed onto the solid phase and are thereby separated form the Hb which appears in the eluent along with the more acidic contaminants. Then, in a second stage, the eluent is applied to a CEX until saturation loading is execeeded so as to create an overload condition in the column so that at the overload condition, the Hb species is eluted from the column by displacement by the contaminants of greater affinity under the chosen conditions and the hb is thereby collected in the eluent at a very high state of purity.  See overload chromatography

Insulin: is produced in the B cells of the pancreas in response to hyperglycemia. Diabetes mellius, caused by a deficienty in insulin production, is the largest cause of human death in industrialized nations. Traditionally, insulin is purified form animal sources by extraction procedues followed by suitable chromatographic operations. Frozen bovine or porcine pancreases are cut up and extracted with ethanol. They are then acidified to pH 2. This helps remove (or inactive) the trypsin, which can degrade insulin. The extracted and acidic insulin solution is neutralized with calcium carbonate. Vacuum extraction is used to concentrate the insulin. Salt addition thenn precipitates the insulin. Reprecipitation of the insulin is done by redissolving the insulin followed by adjusting the ph to the isoelectric point of insulin. Further purification is done by redissolving the insulin followed by adjusting the pH to the isoelectric point of insurin. Further purification is done by gel filtration chromatography and/on IEX. Standard methods for large scale purificaiton of recombinant insulin include IEX, gel permeation and reverse phase chromatography. (Sadan, Biopharm Manuf. 7(3), pp. 34-43, 1994).

Latex: from plants is a complex emulsion of proteins, alkaloids, starches, sugards, oils, tannins, resins and gums. In most of the plants, the latex is white, but some have yellow, organe or scarlet latex due to the presence of some organic compouns. Some melanin-like compounds present in the latex of the genus Ficus impart brown color to the latex and also bind to the proteins and prevent the adsorption of the proteins to the matrix of an ion exchanger, thus posing problems during the purificaiton process. (Kumari, J. Agric. Food Chem. 2010, 58, 8027-8034) teaches that activated charcoal is one of the most widely used adsorbent for organic compounds and has many applications in isolation and purificaiton of biomolecules from curde fermentation broths. Kumari teaches a simple and inexpensive process for the decolorization of crude altex of Ficus religiosa using activated charcoal. 

mRNA: 

–Affinity chromatography:

Affinity-based chromatographic isolation of mRNA is a robust technique that is useful as an industrial platform. An mRNA construct contains a 3′ polyadenylic acid )polyA) tail to increase in vivo stability. That enables the use of affinity purificaiton with oligo-deoxythymidinic acid (oligo(dT) probes that are covalently coupled to solid supports. (Aviv, PNAS 69(6), 1972: 1408-1412. ). 

For example, the POROS Oligo(dT)25 affinity resin includes a 50 um porous poly(styrene-co-divinylbenzene) base bead with a polydeoxythymidine (poly-T) 25-mer (dT-25) conjugated to the surface using a proprietary linker. The poly-T ligand on the surface allows capture of mRNA molecules with a poly-A tail that are manufactured using an in vitro transcription process. (Sikka “Advancing Vaccine development with novel chromatogrpahy solutions” BioProcess international, 21(9), 2023)

Neonatal Fc receptor (FcRn): is a heterodimer with pH-dependent IgG affinity, structurally similar to MHC Class I molecule. FcRn is a heterodimer of beta2-microglobulin (14 kDa) and an alpha-chain (46 kDa), structurally homologous to the alpha-chain of teh MHC class I molecule. 

–IgG Affinity chromatography: 

Sedmak (“isolation form human placenta of the IgG transporter, FcRn, and localization to the syncytiotrophoblast” J Immunology, 1996, 157: 3317-3322) discloses IgG affinity purificaiton of hFcRn, one of 46 kDa and the other of 14 kDa, form a detergent solubilized human placenta. The two proteins bound to immobilized IgG at pH 6 but not at pH 8. The 14-kDa protein was identified as beta2-microglobulin.  

Tissue plasminogen activator (tPA) is produced in teh tissue of a higher animal, and is a protein which activates plasminogen, a precursor of plasmin which is a proteolytic enzyme specific to fibrin. 

Mori (EP 0256836) discloses brining a crude tPA preparation into contact with hydroxyapatite to absorbe the various tPA species to teh HA. The pHs and/or salt concentraitons of eluents are cahgned according to a tPA species of a desired MW to be eluted. A salt is used to separate tPA from HA. The salt has no chelating ability. For example, a salt containing a kaotropic ion such as thiocyanate or perchlorate, a chloride, flluoride, phosphate, corabonate, sulfate, nitrate, borate, acetate or the like mabye be used. Specific examples include sodium phosphate, potassium phosphate, dosium chloride, potassium chloride, ammonium thiocyanate, sodium sulfate, sodium borate, sodium acetate, etc. 

Bacteria/Microorganisms/Parasites/Toxins

–Bordetella pertussi: causes whooping caugh (Pertussi) which is a highly contageious respiratory infection. Whooping cough commonly affects infactns and young children, but can be prevented by immunization. Commonly purified and isolated antigenic components include pertussis toxin (PT), filamentous haemagglutinin (FHA), Fimbriae (FIM) and 69 kD outer membrane proteins also known as Pertactin (PRN).

Mago (WO2009/016651) discloses a method for purifying PRN from Bordetella pertussis by centrifuging a harversted culture of Bordetella pertussis to segregate the cell biomass from the supernatant, DF, precipitated with ammonium sulphate followed by hydroxyapatie and AEX. 

–Diphteria toxin:  Diphteria toxin is a proteinaceous toxin which is synthesized and secreted by toxigenic strans of Corynebacterium deptheriae. DP and its mutatn forms have found applications in both vaccines, as a carrier protein and anticancer drugs. (Goerke US 2014/0193876). 

—-DF -AEX -HIC: 

Goerke (US2014/0193876) discloses purification of diphteria toxin clarifcation of the broth, diafiltration to reduce ionic strenght by removing salts and other ions smaller than the MWCO of the DM against a low ionic strenght buffer such as phosphates, AEX such as Q sepharose and DEAE which has been equilibrated with a buffer such as Bis-Tris and elution with a buffer such as Bis-Tris

Wolfe (US 6,689,871) discloses a method of purifying diphteria toxin from a culture of toxin producing bacterial by DF against a low ionic strenght buffer such as Tris, Bis-Tris and pohsphate , then AEX such as DEAE which may first be equilibrated with a buffer such as Bis-Tris and elution of the toxin followed by a hydrophobic matrix such as HIC. 

Particular Contaminants Removed:

Endotoxins:

–Using Detergents:

(Pabst “Removal of endotoxin from protein solutions by phase separation using Triton X-114) disclose reuction of endotoxin contamination of protein solutions by using the detergent, Triton X-114. Protein solutions containing endotoxin were treated with Triton X-114 on ice, the solution was then warmed to 37C whereupon two phases forms, a triton X-114 phase, containing the endotoxin, was precipitated by centrifugation. Teh samll amount of detergent that persisted in protein solution could be removed by gel filtration or absorption. 

Nucleic Acid (RNA (mRNA), genomic DNA (gDNA) and plasmid DNA (pDNA): 

Nucleic acid is usually produced by amplifying replicable plasmid DNA in a gram negative bacteria such as E. coli. After lysis, it is centrifuged and the supernatant is shaken out with phenol. Subsequently an ultracentrifugation on a caesium chloride gradient is carried out. However, such preparations contain endotoxins, phenol, caesium chloride and/or eitidium bromide as a dye. Kuhne (US 6,750,333)

Most processes available for pDNA purification are conducted on a small laboratory scale. Theyy mainly involve cell lysis using enzymes like RNAs or lysozyme, extraction with organic solvents and ultracentrifucation in density gradients. Different chromatography such as AEX has been used for manufacturing scale processes (Urthaler US2004/0002081)

A further process is described in the QIAGEN Plasmid Handlbook (Qiagen Inc., Chatsworth, USA) and EP-B0268946 where cell lysate obtained after the conventional lysis is chromatographed on QI (Kuhne (US 6,750,333).

–Chromatography methods

DNA is the second common biological polymer molecule besides proteins. Increasing amounts of pure plasmid DNA are for example needed in gene therapy, while fast and reliable analysis of vairous DNA samples remains a challenge in biotechnology. Chromatography is increasingly discussed as a powerful tehnique for DNA preparations. From a chemical point of view, DNA molecules are polyelectrolytes and, more specifically, polyanions. (UNO Q and UNO S respectively). (Ruth, “Novel approaches to the chomatography of proteins” Biotechnoly and Bioprocessing/Biotechnol. Bioprocess. 27, 2003 455-502)

—-Affinity chromatography: WO95/21177discloses a process for the islation and purificaiton of nucliec acids for use in gene therapy by centrifugation, filtration, affinity chromatography with subsequent chromatography on an ion exchanger.

—-Hydroxylapatite: 

Kuhne (US 6,750,333) discloses a process for the production of plasmid DNA which involves hydroxylapatite chromatography. Preferably, ion excahnge is carried out before the HA.

Yamamoto (US 5,843,731) discloses a method for isolating plasmid DNA by suspending hydroxyapatite particles in a buffer solution haveing a pH of about 6-9, about 100 mM water soluble calcium salt and about 1-100 tris(hydroxymethl) aminomethane, adding a solution containing RNA and plasmid DNA obtained by bacgeriolysis thereby absorbing the RNA onto the HA and recovering the plasmid DNA froma supernatant of the suspension. 

Prions: Gilljam (US2010/0210821) disclsoes a process for isolation and purificaiton of a target protein from a mixture containing prions by mixed (multi modal) mode or hydrophobic charged induction resin. using different types of wash buffers which include detergents, alcohols and amino acids such that smaller amount of the prions that bind the resin can be washed out prior to eluting the products. 

Soy protein extracts: How (J. Food Science, 47 1982) teaches subjcting soy protein extracts to activated carbon and ion exchange process treatments to remove phenolic compounds which are responisble for adverse color and flavor characteristics of soy protein products. 

Particular Methods Used for Purificaiton of Non-antibody Proteins

Immunoaffinity chromatography:

Suarez (Applied and encironmental microbiology, 1997, 4990-4992) discloses generation of anti-nisin A specific monosclonal antibodies and the developmetn ofnisn immunoassays for teh detection and quatification of this bacteriocin. Monocllaon antibodies of the IgG1 isotype were produced by a hybridoma cell line and coupled to a column. The column was then used for immunoaffinity of nisn A. 

Yang (US 16/604,123, published as US 2020/0377560) discloses immunoaffinity of native royalactin using a minoclonal antiody that binds to the native royalactin.