For antibody purification using partitioning see “antibody purification”

Many thermoseparating polymers contain ethylene oxide groups. PEG is a thermospearating polymer but its cloud point is too high (above 100C) for use in separating bioaterials. Ethylene oxide (EO-propylene oxide (PO) random copolymers have lower cloud points and have been used in two stage separation system for protein purification. In the first stage, the target protein is partitioned to the EOPO rich phase. In the seocnd stage, the EOPO rich phase is thermoseparated. A new two phase system is formed with a EOPO-rich bottom phase and a water rich top phase. In this system, proteins are partitioned to the water phase and the copolyer can be recycled. (Johansson, “Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification” Biotechnology and bioengineering, 1999, 66(4), 247-257). 

Examples of polymers which display thermoseparation in water are poly(vinylcaprolactam), poly(N–isopropylacrylamide), EHEC (ethyl-hydroxyethyl-cellulose) and random or block copolymers of ehtylene oxide (EO) and propylene oxide (PO). The random copolymers of EO and PO segments EOPO polyers) are seaprated as a liquid phase pon temperature increase. Most studies of biomolecule partitioning in thermoseparated systems have been done in EOPO-polymer containing systems. In these systems the concentraiton of EOPO-polymer in the bottom phase is usually 40-60 %, while the top phase contains almost 100% water. Thus these systems are called water/EOPO systems. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

EOPO/Salts –

A thermoseparating random copolymer (Ucon 50-HB-5100) composed of  50% ethylene oxide and 50% propylene oxide has been used to form an aqueous two-phase system by heating the polymer water solution above the cloud point of the copolymer. In the formed two phase system a water rich top phase is in equilibrium with an aqueous polymer rich bottom phase. Hydrophobic peptides (containing aromatic amino acids) were strongly partitioned to the polymer rich phase, while hydrophilic peptides were enriched in the water rich phase. A decreased partitioning to the polymer rich phase was obtained upon temperature increase. With the salt NaCLO4, the polypeptide was partitioned to the polymer rich phase while with Na2SO4 the polypeptide was partitioned to the water rich phase. (Hans-Olof Johansson, Biochimica et Biophysica Acta 1335 (1997), 315-325.

Hallgren (US2008/0293926) disclose a method of separating a target from a liquid by providing a polymer in an aqueous liquid such as water which the polymer comprises at least one hydrophobic portion, contacting the polymer-containing liquid with the liquid comprising the target, applying a stimulus such as temperature or pH change to the resulting mixture and maintaining it until a reversible phase separation is obtained. One phase is polymer rich and contains target(s) and another phase is polymer poor. In an advantageous embodiment, the polymer present has a predominating hydrophobic character but also comprises one or more hydrophilic porition. Useful polymers include a block copolymer cased on ethylene oxide and proylene oxide. In one embodiment, Hallgren discloses isolating an antibody from an unclarified CHO brother by combining in a containiner the broth with ethylene oxide propylene oxide (EOPO) copolymer and at least one added salt selected from NaCl, Na2PO4, KPO4, NaSO4, potassium citrate, (NH4) SO4, sodium citrate, sodium acetate and ammonium acetate in a concentraiton of 100-300 mM and gently mixing the liuqid under conditions where the polymer is above its cloud point so that it forms a one thermoresponsive polymer, two phase system wehre the antibody partitiontions into the phase not enriched in the thermoresponsive polymer while nontarget compounds paritition to varying degrees to the phase interface or the thermoresponsive polymer enriched phase. 

EOPO/Dextran- WO2004/020629 discloss EOPO polyers to further facilitate the separation of plasmids which have already partitioned in a two polymer phase system. At room temperature the two-polymer, two phase system formed with EOPO and dextran polyers forms in same manner as PEG and dextran system. The less dense EOPO enriched upper phase is isolated from the EOPO and dextran polymer aqueous two phase system. The temperature of the EOPO enriched phase is then raised so that the upper phase undergoes further phase separation into a water enriched phase and a self associated EOPO polymer enriched phase. Advantageously, the water enriched phase should contain the desired target. 

Van Alstine (WO 2010/080062) disclose enriching one target protein from a liquid by differentially partitioning between two aqeuous phases. The phases are formed by adding a single type of responsive, self associating hydrophilic polymer and optionally use additional salts to an aqeuous biological solution (such as a fermentation sample) under thermal conditions where the solution separates into a one polymer, two phase system with one phase enriched in the polymer. The polymers used are aqueous in the sense that they form aqueous phases when combined with water. In one embodiment, the hydrophilic poly(ether) is a syntehtic polyer comprising ethylene oxide units such as poley(ethylene)glycol (PEG), ethylene oxide propylene oxide (EOPO) in either random copolymer form (e.g., Breox® or UCON polymers) or block polymers (e.g., Pluronic® polymers) ethoxy-containing polysaccharides and isopropylacrylamide modified polyers. EOPO for example separates into two phases when above its cloud temperature (Tc) and is consequently regarded a thermoseparating polymer. 

Tjerneld (US6,454,950) disclose a method for partitioning a compound in a two phase system in which one of the phases is rich and the other poor in a  thermoseparating polymer, collecting one of the phases and if desired further working up said compound from the collected phase. In one embodiment the polymer is HM-EOPO and in the other Breox copolymer (random copolymer with equal amount of ethylene oxide and propylene oxide. Partitioning of various proteins are illustration including BSA, lysozyme and apolipoprotein A-1. The technique of back extraction is also illustration from the HM_EOPO polymer phase to the water phase (first protein is partitioned in a 4% HM-EOPO polymer water system wehre the apolipoportein A1 is partitioned to the HM-EOPO polymer, the top water phase removed and exchange for buffer and the system mixed to allow for separation again (protein now back extracted into the new water phase). 

—EOPO (UCON) – Dextran – triethylene glycol-diglutaric acid (TEG-COOH): Ferreira (J. Chromatography A, 1195 (2008) 94-100 disclose using a thermosenstitive polymer of UCON (ethylene oxide/propylene oxide 50:50) with Dextran and TEG-COOH for the purificaiton of IgG from a CHO cell supernatant. Without TEG-COOH, IgG preferentally partitioned to the less hydrophobic dextran-rich phase (the recvoery yield of IgG in the top UCON-rich pahse was lower than 50%).  However, with the adding a free ligand containing carboxylic functionalities (TEG-COOH), a significant increase in the partitione of IgG from the bottom phase to the top phase was observed. 

–Poly(acrylic acid) (PAA) + EOPO + Salt: Hjorth (WO2008/156409 and US2010/0174052) dicloses partitioning in a multiphase system with a  1) first polymer which is synthetic poly(acid) such as poly(acrylic acid) (PAA) or polyacrylate, 2) a second synthetic polymer which ispoly(ether) such as poly(ethylene)glycol (PEG)  or ethylene oxide properylene oxide (EOPO) and 3. at least one salt. 

Johansson (US8,268,915) also teaches using a first polymer which is a poly(acid) such as poly(acrylic acid) (PAA) or poly(methacrylic acid) and a second polymer is is a poly(ether) such as a synthetic polymer comprising ethylene oxide units which includes poly(ethylene)glycol )PEG), ethylene oxide proylene oxide (EOPO) in either random copolymer form (e.g., Breox polymers) or block copolymers 9e.g., Puronic polymers) and at least one salt for the separation of biomolcules 

As to partitioning of antibodies see “purification of antibodies”

Extraction is the prcoess of moving one or mroe components of a mixture form one phase to another (compare to precipitation which is the process of removing one or mroe components from solution to form a solid phase, the precipitate). Liquid-liquid extraction using organic and aqueous extraction media is a traditional separation operation that can be applied to the purificaiton of biopharmaceuticals. In three-phase partitioning, proteins can be purified directly form cell homoenates by partitioning between a layer of butanol and a strong aqueous salt solution. Under these conditions, cell debris tends to separate into the organic phase and nucleic acids preciptiate at the interphase while proteins remain in solution. Aqueous to-phase extraction systems are the most widely used extraction operation, empoloying a mixture of aqueous polymers and/or salts. One phase generally contains polyethylene glycol (PEG) and the other contains a different polymer, such as dextran, or the salt potassium phospahte. Under ideal conditions, the desired protein can be separated into the PeG phase while the majority of contaminating proteins as well as other contaminants are trapped in the second phase, o in the interphase., and can be removed by centrifugation. (Uwe Gottschalk, Sartorius Biotech GmbH, “Downstream Processing” Chapter 18 in Filtration and Purificaiton in the Biopharmaceutical Industry, Second Edition. Informa healthcare 2008). 

Two-polymer-Two Phase systems: Generally

Aqueous two-phase systems (ATPS) are formed when aqueous solutions of two mutually incompatible components separate into two phases of different densities under the force of gravity. This technique allows clarification, concentration and partial purificaion to be integrated in one step. (Ferreira, J. Chromatography A, 1195 (2008) 94-100).

ATPS formed by mixing two water soluble incompatible polymers have been used for separation of sensitive biological materials. The most well known are the PEG/dextran system (PEG=poly(ethyleneglycol) and PEG/phosphate system). It is the possibility to form two phase systems with high water concentration in both phasses that makes the PEG/dextran and PEG/phosphate useful for sensitive biological materials. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

The traditional aqueous two phase systems have been the PEG/dextran which dervies from polymer-polymer incompatability and PEG/salt systems which derives from salting out of the polymer with a salt. The PEG/dextran systems are used for small scale separations of macromolecules, membranes, cell particles and cells. The PEG/salt systems are mainly used in large scale enzyme extractions. (Tjerneld (US 6,454,950). 

More novel two-phase systems have utlized polymers that have a solubility in water that decreases upon increasing the temperature (thermoseparating polymers). In these case two macroscopic phases (one polymer enriched bottom phase and one water rich top phase) can be obtained upon heating a solution of the polymer a few degrees above the cloud point (the temperature at which the phases start to searate out). The cloud point for an aqueous solution of a given polymer depends on polymer concentration and amount and type of other components added. The lowest cloud point is called the lowest critical solution temperature (LCST). (Tjerneld, US 6,454,950). 

Aqueous polymer two phase-systems: 

Polymers used in two phase systems are aqueous in the sense that they form aqueous phases when combined with water. Aqueous polymer two phase-systems are more useful for biologicals. One reason for this is the high water concentration in both phases. They are formed by mixing certain hydrophilic and typically neutral polyers in aqueous solution. These include dextran (polyclucose) and poly(ethylene glycol) (PEG) as well as polysucrose (such as Ficoll™) and PEG or linear polyacrylamid and PEG. Typically concentrations of each polymer are 50-10% w/w. At such concentrations, entropic and other forces tend to drive the formation of two phases both of which are typically greater than 90% (ww) water. The phases are typically enriched in one polymer and have low interfacial tension. Phase density differences drive the phases to separate by gravity or centrifugation. One advantage of the PEG and Dextran type of two-phase syste is that target proteins may partition in favour of the PEG enriched, less dense, upper phase while cell debris may partition (or sediment) to the interface or complementary lower phase. Independent of the challenge of adding and then removing two polymers from the bioprocess stream is the cost of polymers such as dextran and PEG (WO2010/080062).

In the biotechnical field, aqueous polymer two phase systems formed with two polymers or with one polymer in presence of significant added salt are of general interest. Many undesired components such as cell debris will tend to appreciably partition to the lower (dextran-rich or salt richs, respectively) phase in a PEG and dextran, or a PEG and salt two phase system. Drawbacks include phase component cost, phase component removal and effect of phase components on other downstream operations.

The application of an ATPS at process-scale has been hampered by the complexity of the system combined with the fact that partition mechanisms are poorly understood and method development is fairly empirical. The partitioning of biological compounds in an ATPS depends not only on the physico-chemical properties of the biomolecule, such as surface hydrophobicity, charge and size, but also on the system composition.  (Azevedo, “Chromatography-free recovery of biopharmaceutical through aqueous two-phase processing” Trends in Biotechnology, (2009), 27(4):240-247.

Conditions: PEG characteristics, including weight, size and concentration are very important factors in the properties of the phase forming system. Higher molecular weight of PEG has less coefficient factor and then lower polymer concentraiotn needed for high separation. Increases in salt concentraiton also result in an increase in partition cefficients of bioproducts in upper phase or interface due to salting out. Partitioning of proteins and enzymes to the phases in the ATPE system also depends on their isoelectric points. The pH of the system, however, affects the cahrge of target protein and ion composition as well as introduces differential partitioning into the tow phases. A pH value above 7 is suitable fot eh PEG/phosphate system and a pH below 6.5 is compatible with the PEG/sulphate sustem. Most fo the biomolecuels, especially proteins and enzymes, are stable at neutral pH that is favorable condition to conduct ATP partitioning. The selection of salts for ATPS depends on their ability to promote hydrophobic interactions between biomolecuels. The PEG/phosphate system is widely used for recovery of bioproducts. Other salts having similar properties to phosphate, such as sulphate and citrate, have also been used. (Goja J. Bioproces Biotechniq 2013, 4:1)

PEG/Salt Systems

The impact of salt concentration has been widely studies. Increases in salt concentraiton result in an increase in partition coefficients of bioproducts in upper pahse or interface due to salting out. In general, proteins with the negative charge tend to partition to the top phase in PEG/Salt systems while those with the positive charge usually go to the bottom phase. (Goja J. Bioproces Biotechniq 2013, 4:1) 

PEG/dextran system: The PEG/dextran system is a particulalry mild system and is thus used for more sensitive biological materiasl such as cells and cell organells. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

PEG/phosphate system: is used for large scale purification of proteins. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

Fusion Proteins which direct Product Protein to Particular Phase

Hydrophobin-Protein fusions:

Hydrophobins are bipolar and small proteins, consisting of about 100-150 amino acids, of which 8 are cysteine residues. They are expressed by filamentous fungi to help the organisms adapt to the environment. Hydrophobins and hydrophobin fusion proteins can be purified and concentrated with a surfactant. (Joensuu (US 14/902851, published as US 2016-0159854)

Collen (Biochimica et Biophysica Acta (2002) 1569, pp. 139-150) discloses partitioning and purification of the fusion protein endoglucanase – hydrophobin I (EGI-HFBI) from a culture filtrate originating from Trichoderma reesei fermnetnation. The micelle extraction system was formed by mixxing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Rppal PES100. The detergent/polymer aqueous two-phase sytems resultsted in better separation compared to cloud point extraction in a Tris X-114/water system. After the primary recovery step, EGI-HFBI was back extracted into a water phase by adding (EOPO) copolymers to the micell rich pahse and a temerpature induced phase separation at 55C. 

Penttila (US7,335,492 and US 7,060,669) disclose the separation of molecules by fusing them with a targetting protein having the capability to carry the molecule to a desired phase in ATPS. Examples of molecuels suited as tareting proteins are hydrophobin like small proteins (hydrophobins). The hydrophoin like protein is bound to the product molecule to be separated. Phase forming materials and possibly also additional salts are added to a water solution containing the fusion moecule. The two phases are formed either by gravity settling or centrifugation. The targeting protein drives the product to for instance the detergent rich phase which can be the top or bottom phase. 

Insertion of Trp rich peptides into C terminus of Product protein:

Hassien (J Chromatography A, 668 (1994) 121-128) iclsoes insertion of AlaTrpTrpPro near the C temrinus of ZZT0. The Trip rich peptide strongly increasedthe partitioning of ZZTO into the PEG rich pahse in a PEG-potassium phosphate aqueous twp phase system. 

One polymer-Two Phase systems: Thermoseparating polymer/water two phase systems

Thermoresponsive hydrophilic polymers exhibit inverse thermal solubility such that as temperature is raised above a certain cloud temperature (Tc) which is related to the polymer’s lower critical solubility temperature (LCST), they self associate and start to form a unique polymer rich phase. Such polymers include copolymer or block copolymers frored with mixture of ethyelene oxide (EO) and propylene oxide (PO) monomeric groups, so called EOPO polymers, polysaccharides modified with EO, PO or similar groups (e.g., ethylhydroxyethycellulose or EHEC) or polyers formed using N-isopropylacrylamide (NIPAAM). 

The system is referred to as a “thermoseparated aqueous two phase system”. Two phases are formed by heating an aqueous solution of a thermoseparating polymer. Above a cricial solution teemperature, the cloud point, the solution will separate into two macroscopic phases. One of the phases (often the bottom phase) is enriched with polymer, the other is depleted. The lowest cloud point of the system (LCST) is the lower critical solution temperature. (Hans-Olof Johansson, Bioseparation 7: 259-267, 1999).

In general thermoseparating phases have normally been used together with dextran or similar polysaccharid in a two step process. Thus selectivity over target and contaminant protein occurs in the first partition step followed by use of temperature induced phase separation (of typically EOPO polymer rich phase) to isolate target and polymer into target containing aqueous phase floating on top of a self associated polymer rich denser phase. 

Modes of Operation

Continuous ATPS:

Palomares (MX2009013602) discloses a device for the continous recovery of bioparticles by means of aqueous two-phase systems which is perforemd in a continous mannter where the partition, separation and/or recvoery of proteins in continous systems. The singe device performs continous bioparticle priamry recvoery whithout requiring centrifugation, agitation and decantation, allowing biopartciles recovery more efficiently than recvoeyr processes sysing other systems and can be implemented onan industrial scale. The recovery of each phase is carried out in separate containers. 

Villegas (J. Sep. Wol. 2013, 36, 391-399) discloses the application of a continous aqueous two-phase system for the recovery of biomlecules. Compared with batch systems, continous operation increased partition coefficient with higher recovery efficiencies. Befiefly, an equilibrated 3 kg system phases were separated into two different containers and fed independently at 10 mL/min while spiked SPE sample was injected by a third inlet at 1 mL/min. Mixingagitation was accomplisehd with a static mixer. Phases were continously harvested at the end of the system, as well as the interphase by a coupled three way outlet connected. 

Companies:   Apec Water    AquaNu  Evoqua Water Technologies  ESP Water Products

Halosource  Hosptial Supply Corporation    Proctor & Gambel   (water floculation agent used to provide clean water) BioSciences Inc  Siemens   Water Research Center  

Winter Water

Water/Waistewater Training: University of Florida 

Miami Dade Water Quality Reports

Safe Aquatic Herbicides:

Solitude Lake Management

Contaminants in Tap Water

Chlorine and organic substances: As Tap or city water is subjected to chlorination with chlorine gas or sodium hypochlorite, it contains residual chlorine dissolved in the form of hypochlorous ion (CLO-_ or hypochlorous acid (HCLO). Such residual chlorine gives rise to a smell or bleaching powder odor. Tap water also contains a small amount of organic chlorine compounds, including trihalomethane compounds such as chloroform CHCl3 an bromodichloromethane (CHCl2Br, which are prooduced by reaction of chlorine with organic substances. The presence of trihalomethane compounds in tap water is drawing increasing public attention as they are carcinogenic, harmful subjstances. In addition, phytoplanktons tend to increase and propagate in water sources due to water pollution so that smelly or malodorous organic substances which presumably are metabolite or secreta of phytoplanktons are present in a small content. These substances generally referred to as “musty-smelling” substances. (Hiasa (US 5,607,595). 

Chloramines: Increasingly drinking water utilities are gradually switching over to the use of choramines – in particular, monochloramine – disinfection of drinking water. In general, chlroamines are safe in drinking water in low dosages. 

Microbiological contaminants

Total coliform Bacteria should be tested.

Inorganic contaminants:

Include antiomny, arsenic, barlum, chromium, copper, fluoride, lead nitrate, nitrite, selenium and sodium. 

Radiactive contaminants:

includ alpha emitters, combined radium and uranium. 

Methods/Substances used to Purify Water

Activated Carbon

Activated carbon refers to carbonaceous substances that are characterized primarily by their surface area, pore size distribution and sorptive and catalytic properties. Activated carbon can be made from a variety of carbonaceous materials and processed to enhance its adsorptive properties. Some common materials that are used to make activated carbon are bituminous coal, bones, coconut shells, lignit, peat, pecan shells, petroleum based residues, pulp mill black ash, surgar, wastwater treatment sludge and wood. Carbon and other adsorbents in various forms have been used for the treatment of water and as detoxifying pharmaceutical agents in medicine for many centuries. Graculated activated carbon (GAC) and powdered activated carbon (PAC) have been used in the US to control taste and odors in drinking water. Today, there are GAC beds in US water treatment plants.

Activated carbon has been used in water purification processes (Cehn, J. Biol. Chem, 242: 173-181 (1967); Nakano, Anal Biochem, 129, 64-71 (1983); Nikolaev Int. J. Art. Rog, 14: 179-185 (1991). Typical activation processes involve subjecting a carbon source such as resin wastes, coal, coal coke, petroleum coke, lignites, polymeric materials and lignocellulosic materials including pulp and paper, wood, nut shell to a thermal process (e.g., with an oxidizing gas) or a chemical process (e.g., with phosphoric acid or metal sals, such as zinc chloride). An exemplary chemical activaiton of wood based carbon with phosphoric acid (H3PO4) is disclosed in US Re. 31,093 and US 5,162,286 teaches phosphoric acid activaiton of wood based material. 

There are several differnt types of activated carobn filters commercially avilable, including service carbon, backwashable carbon and steam or hot water sanitizable carbon (Siemens Water Treatment Technologies).

–For purification of Tap Water: 

A conventional process for producing drinking water involves screening which separates out the coarse impurities, pretreatment by introducing reagents followed by flocculation and settling so as to eliminate materials in suspension, sand filtering during which ammonia is biologically  nitriified and impurities in suspension are eliminated, injetion of ozone to kill bacterai and viruses, filtering by gracular activated carbon to eliminate organic materials and chlorination (Montagnon US5,037,550). 

Conventionally, water purifying devices have been used for domestic purposes in order to remove harfmful and smelly substances. In early water purifiers, it has been customary to use granular activated charcoal which is capable of removing resiude chlorine as well. It is believed that residual chlorine is removed by chemical adsorption at the active sites located at the surface of activated charcoal and thus adsorption capability of granula activated charcoal is dependent on the specific surface area of activated charcoal. In constrast, trihalemthanes and smelly organic substances are believed to be physically adsorbed by activated charcoal, with the hydrated molecules being trapped in the micropores of activated charcoal. In recent water purifiers, activated carbon fibers manufactured by carbonizing acrylic or phenolic fibers follwoed by activation have been increaseingly used because of the iproved adsorption speed as compared with granular activated charcoal and due to the advantage of having a narrower pore diameter distribution. In this instance, typical water purifiers markedted today are designed such that carttridges of activated carbon fibers are replaced each 6 months or a year. (Hiasa (US 5,607,595). 

Mouri 9US2005/0181931) discloses a removing agent for heavy metal such as chromium, manganese, cadmium, lead and mercury, particularly lead in water containing synthetic zeolite in which 10% or more of the total amount of exchangeable cation is substituted with magnesium ion and 60% or more is substituted with magnesium and calcium ion and activated carbon at a ratio of 2:98 to 50:50. In addition, the concurrent sue of this calcium and magnesium type zeolite with activated carbon at a specific ratio allows a superior adsorbent, which does not remove calciu and magensium ion and can efficiently remove residual chlorine and trihalomethane. 

–Filters with Activated Carbon:

Farrelly (US2009/0188854) discloses a filter device comrpising a housing for securing to a source and having an upstream filter elelment of coarse particle size and downstream filter element of fine filter size. Granulated activated carbon materials dervied form charcoal is provided in the filter chambers for removing and elimating bacteria such as E coli as well as chemical and metals including iron form water.

Mitchel (US2003/0217963) discloses a filter for providing potable water. The filter includes a housing having an inlet and outlet and a filter materials in the housing formed at least in part from activated carbon filter particles.

Air Stripping: is a process of moving air through contaminated groundwater or surface water in an above group treatment system. Air stripping removes chemicals called “volatile organic compounds” or “VOCs”. VOC easily evaporate, which means they can change from a liquid to a gas. The air passing through contaminated water helps evaporate VOCs faster. After treating the water, the air and chemical vapors are collected and either removed or vented outside if VOC levels are low enough. Air tripping is commonly used at public water treatment plants, particularly where the water contains organic materials. 

–Particular Schemes using Activated Carbon

—-AC-CEX–AEX

Hall (US 4,474,620) disclsoes a water purificaiton aprratus which has an untreated water reservoir at the top. The untreated water flows by gravity through a series of three water treatment beds containing AC, CEX and AEX. The media in each chamber is in cartridge form and may be easily removed. Treated water from the final chamber is collected in a reservoir. 

Flocculant/Disinfectant Powders

–Ferric sulfate –calcium hypochlorite

P&G has developed a pwodered derric sulfate (a flocculant) and calcium hypochlorite (a disnfectant) for the purification of water. One opens the sachet, adds the contents to an open bucket containing 10 liters of sater, stirs for 5 minutes, let the solids settle to the bottom of the bucket, strains the water through a cotton cloth into a second containing and waits 20 mninutes for the hypochlorite to inactivate the microorganisms.

Ozonation    see Water Research Center

Reverse Osmosis: 

Reverse osmosis (RO) membranes provide a cost effective water purificaiton solution for wastewater reclamation facilities and are an integral part of many public water treatment plants. Advantages of RO systems are that (1) they are simple to design and operate, have low mintenance requirements and are modular in nature, making expansion easier, (2) both inorganic and organic contaminatns can be removed, (3) there is no effect on the material being recvoered and RO prcoesses can reduce the volume of waste streams so that these can be treated more efficiently and cost effectively by other processes such as incineration. In addition, RO systems can replace or be used in conjunction with other treatment processes such as oxidation, adsorption, stripping or biological treatment to produce high quality water. 

Water Filtration:

Products:  LifeSaver Bottle

Filtration is a physical process that occurs when liquids, gases, dissolved or suspended matter adhere to the surface of, or in the pores of, an absorbent medium. Filtration of contaminants depends highly on the amount of contaminant, size of the contaminant particle, and the charge of the contaminant particle. Depending on the household’s water needs, pretreatment before filtration may include the addition of coagulants and powdered activated carbon, adjustments in pH or chlorine concentration levels, and other pretreatment processes in order to protect the filter’s membrane surface. See CDC

Physical removal of waterborne Crytosporidium oocysts and Giardia cysts is ultimately achieved by properly functioning conventional filters, providing that effective pretreatment of the water is applied. Disinfection by chemical or physical methods is finally required to inactivate/remove the infectious life stages of these organisms. See Bentancourt

Membrane filtration systems have long been used for water and waste-water treatment, with applications primarily in reverse osmosis plants in water-scarce regions. Of the membrane filters, ultrafiltration membranes with a typical pore size between 0.002 and 0.1 μm have shown higher removal of pathogens such as Cryptosporidium, Giardia, and bacteria, viruses, and parasites. See Francis

Katouli evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. They also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n
53 15.4 per 10 liters) and cysts (n
59 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 8, P
0.015; cysts, 49.8 12.2, P
0.4260), and SMF (oocysts, 16.2 2.8, P
0.0079; cysts, 35.2 3, P
0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.

Absorption: In Tata Swach, adsorption is through rice husk ash (activated silica and activated carbon) which is impregnated with silver nanoparticles to target microbes.

Coagulation and filtration is one of the most common water treatment techniques used by larger water systems, used for removing particulates and turbidity from surface water. A coagulant (typically either iron or aluminum salts with polymeric materials) is added and mixed with the influent water. The larger particles formed by coagulation are then removed from the water by filtration (typically sand, anthracite coal, or a combination of the two). See EPA

Microfiltration: A microfiltration filter has a pore size of approximately 0.1 micron (pore size ranges vary by filter from 0.05 micron to 5 micron). Microfiltration has a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia). Microfiltration has a moderate effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Microfiltration is not effective in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Microfiltration is also not effective for removing chemicals. See CDC

Ultrafiltration:  An ultrafiltration filter has a pore size of approximately 0.01 micron (pore size ranges vary by filter from 0.001 micron to 0.05 micron; Molecular Weight Cut Off (MWCO) of 13,000 to 200,000 Daltons). Ultrafiltration filters remove particles based on size, weight, and charge; Ultrafiltration has a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia); Ultrafiltration has a very high effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Ultrafiltration has a moderate effectiveness in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Ultrafiltration has a low effectiveness in removing chemicals. See CDC

There are modular variants of U. F purifiers which are suitable for community scale like SkyHydrant, Lifestraw Family and also mobile variants like Jaldoot and Perferctor E. Several stationary household UF purifiers are available like Moselle, Jaltara and Waterife Little Star Gold. See Kedare

There is a unique experiment with plant xylem-based ultrafiltration. Bacteria up to 3 LRV can get filtered out with sapwood (predominantly xylem) of trees like pine which is easily available, inexpensive, biodegradable and suitable for resource-constrained environments. See Kedare

Nanofiltration:  A nanofiltration filter has a pore size of approximately 0.001 micron (pore size ranges vary by filter from 0.008 micron to 0.01 micron; Molecular Weight Cut Off (MWCO) of 200 to 2000 Daltons); Nanofiltration filters remove particles based on size, weight, and charge; Nanofiltration has a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia); Nanofiltration has a very high effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Nanofiltration has a very high effectiveness in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Nanofiltration has a moderate effectiveness in removing chemicals.

Reverse Osmosis: Links of interest: Elsevier. EPA.   Companies: See AquaSana

Reverse Osmosis Systems use a process that reverses the flow of water in a natural process of osmosis so that water passes from a more concentrated solution to a more dilute solution through a semi-permeable membrane. Pre- and post-filters are often incorporated along with the reverse osmosis membrane itself. A reverse osmosis filter has a pore size of approximately 0.0001 micron. Reverse Osmosis Systems have a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia);  Reverse Osmosis Systems have a very high effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Reverse Osmosis Systems have a very high effectiveness in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Reverse Osmosis Systems will remove common chemical contaminants (metal ions, aqueous salts), including sodium, chloride, copper, chromium, and lead; may reduce arsenic, fluoride, radium, sulfate, calcium, magnesium, potassium, nitrate, and phosphorous, See CDC

Unlike activated carbon filtration technology that uses certain materials to attract contaminants that bind like magnets, reverse osmosis uses a semi-permeable membrane for filtration. Think of it like a mosquito net. Reverse osmosis forces water through a semipermeable membrane, leaving all particles larger than the net behind.  It uses a considerable amount of water pressure, making it the most effective water purification technique on the market. See AquaSana

Solar Water Disinfection

Companies: GoSun

Products: GoSun Flow. Solvaten.

Solar water disinfection is a sort of portable water purification that cleans water through solar energy in order to remove contaminants such as bacteria, viruses, and protozoa. It does so through a mixture of electricity generated by solar PV panels, solar heating, or solar ultraviolet light collection. Solar disinfection, which combines thermal and UV radiation, has been repeatedly shown to be effective for eliminating microbial pathogens and reduce diarrhoeal morbidity (Hobbins 2004) including epidemic cholera (Conroy 2001). Among the most practical and economical is the “SODIS” system, developed and promoted by the Swiss Federal Institute for Environmental Science and Technology.

It is well documented that solar energy can be an effective means of cleaning contaminated water. This is because ultraviolet (UV) light destroys the formation of DNA linkages in microorganisms, thereby preventing them from reproducing and thus rendering them harmless. The World Health Organization  lists solar disinfection in clear bottles by the combined action of UV radiation, as well as thermal disinfection (pasteurization) in opaque vessels with sunlight from solar cookers or reflectors and combination systems employing chemical coagulation-flocculation as some of the most promising and accessible technologies for household water treatment. See Climate Technology Center

SODIS: 

Popularly abbreviated as SODIS, solar water disinfection is a simple, safe, economical and effective method of disinfecting contaminated water. The method simply involves the use of sunlight and plastic PET bottles for purifying or improving the quality of drinking water.  See Water Treatment Plants

Solar disinfection (SODIS) was developed in the 1980s to inexpensively disinfect water used for oral rehydration solutions. In 1991, the Swiss Federal Institute for Environmental Science and Technology began to investigate and implement SODIS as a household water treatment option to prevent diarrhea in developing countries. Users of SODIS fill 0.3-2.0 liter plastic soda bottles with low-turbidity water, shake them to oxygenate, and place the bottles on a roof or rack for 6 hours (if sunny) or 2 days (if cloudy). The combined effects of ultra-violet light (UV)-induced DNA damage, thermal inactivation, and photo-oxidative destruction inactivate disease-causing organisms. See CDC

Fontan discloses water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvumoocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) than the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times). See Fontan

Solar radiation and heat produced by the sun have a synergistic effect in killing cysts of Giardia duodenalis and Entamoeba histolytica/dispar when temperatures rise above 50°C, with complete death at 56°C, using painted 2‐l PET containers. See Mduluza

However, despite an extensive SODIS promotion campaign Coliford found only moderate compliance with the intervention and no strong evidence for a substantive reduction in diarrhoea among children. These results suggest that there is a need for better evidence of how the well-established laboratory efficacy of this home-based water treatment method translates into field effectiveness under various cultural settings and intervention intensities. Further global promotion of SODIS for general use should be undertaken with care until such evidence is available. See Coliford 

Ion Exchange

During ion exchange treatment, water is passed through a resin containing exchangeable ions. Stronger binding ions displace weaker binding ions and are removed from the water. There are two types of ion exchange—anion exchange and cation exchange. Anion exchange resins generally exchange chloride for anionic contaminants, like uranium. Cation exchange resins generally exchange sodium or potassium for cationic contaminants, such as radium. Mixed bed resins with cation and anion exchange media in two layers are available for systems that need to remove both radium and uranium.  Ion exchange is also effective for the removal of beta particles and photon emitters. See EPA

Chemical Treatment 

Chlorination is a simple, affordable and scalable method of water disinfection through the use of sodium hypochlorite NaOCl (liquid) (Fig. 6), NaDCC (solid) and calcium hypochlorite (Ca(OCl)2) (solid). It gives residual protection due to the availability of free chlorine; however, there may not be any improvement in terms of turbidity. With a dosage of 2 mg/L for about 0.5 h, chlorination can offer about 3 LRV of enteric bacteria. For a reduction in turbidity as well as microbial disinfection, combined methods such as coagulant/flocculant as well as chemical disinfectant powders/tablets are used. See Kaderi.

Chlorine in one form or another is by far the most commonly used chemical for the disinfection of water supplies. It is also active for other purposes assocIated with water treatment and supply, such as prevention of algal, bacterial and general slime growths in treatment plants and pipeworks, control of tastes and odours, and removal of ¡ron, manganese and colour See WHO

Other Water Purification Techniques:

Distillation Systems use a process of heating water to the boiling point and then collecting the water vapor as it condenses, leaving many of the contaminants behind. Distillation Systems have a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia); Distillation Systems have a very high effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Distillation Systems have a very high effectiveness in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Distillation Systems will remove common chemical contaminants, including arsenic, barium, cadmium, chromium, lead, nitrate, sodium, sulfate, and many organic chemicals.  See CDC

Ultraviolet Treatment with pre-filtration is a treatment process that uses ultraviolet light to disinfect water or reduce the amount of bacteria present.Ultraviolet Treatment Systems have a very high effectiveness in removing protozoa (for example, Cryptosporidium, Giardia); Ultraviolet Treatment Systems have a very high effectiveness in removing bacteria (for example, Campylobacter, Salmonella, Shigella, E. coli); Ultraviolet Treatment Systems have a high effectiveness in removing viruses (for example, Enteric, Hepatitis A, Norovirus, Rotavirus); Ultraviolet Treatment Systems are not effective in removing chemicals. See CDC

Aquaguard Compact is an example of UV-based purifier. See Kedare

Water Softeners use ion exchange technology for chemical or ion removal to reduce the amount of hardness (calcium, magnesium) in the water; they can also be designed to remove iron and manganese, heavy metals, some radioactivity, nitrates, arsenic, chromium, selenium, and sulfate. They do not protect against protozoa, bacteria, and viruses. See CDC

Herbal Remedies: 

–Tulsi leaves (Ocmium sanctum) & Neem leaves (Azadirachta indica) have the capacity to purify water. See Tanushree

–Moringa oleifera Lam belongs to the family Moringaceae and is a valuable plant, found in many countries of the tropics and subtropics. Its leaves, fruit, flowers and immature pods are used as a highly nutritive vegetable in many countries, particularly in India, Pakistan, Philippines, Hawaii and many parts of Africa. Seed extract is observed to have a protective effect by decreasing liver lipid peroxides and is antihypertensive.  M. oleifera roots, leaves, seed, fruit, flowers, bark and immature pods are used as cardiac and circulatory stimulants, contain antipyretic, antiepileptic, antitumor, antiinflammatory, antiulcer, diuretic, antihypertensive, cholesterol lowering, antispasmodic, antidiabetic, hepatoprotective, antioxidant, antibacterial and antifungal activities, and are being used for the treatment of various ailments in the indigenous system of medicine.  Moringa oleifera seed powder has shown a significant reduction of turbidity and coliform count when it was used at smaller concentrations without altering the pH of the water. Moreover, the extracts of the seed with different solvents showed antibacterial activity to all the four test organisms, i.e., Escherichia coli (ATCC2592), E. coli (clinical isolate), Salmonella typhii (clinical isolate) and Shigella dysenteriae(clinical isolate). The acetone extract is the most effective in inhibiting and killing the test organisms at a very low concentration (MIC and MBC = 6.25 mg/mL) for Salmonella typhii (clinical isolate). The present study has suggested that the acetone extracts of M. oleifera seeds have potential as antibacterial compounds against pathogens and their ability to either block or circumvent resistance mechanisms could improve the treatment and eradication of microbial strains. Thus, plant seed extracts could be used in the treatment of infectious diseases caused by microbes. See Husen

Crushed Moringa seeds clarify and purify water to suit domestic use and lower the bacterial concentration in the water making it safe for drinking. By using Moringa seeds people will no longer be depending on expensive means originating from the West. Using Moringa to purify water replaces chemicals such as aluminum sulfate, which are dangerous to people and the environment, and are expensive. Moringa oleifera seeds treat water on two levels, acting both as a coagulant and an antimicrobial agent. It is generally accepted that Moringa works as a coagulant due to positively charged, water-soluble proteins, which bind with negatively charged particles (silt, clay, bacteria, toxins, etc) allowing the resulting “flocs” to settle to the bottom or be removed by filtration. The antimicrobial aspects of Moringa continue to be researched. Findings support recombinant proteins both removing microorganisms by coagulation as well as acting directly as growth inhibitors of the microorganisms. While there is ongoing research being conducted on the nature and characteristics of these components, it is accepted that treatments with Moringa solutions will remove 90-99.9% of the impurities in water. See Miricle Trees

Emergency Disinfection of Water  See EPA

Detection and Monitoring of Water

Cabelli discuses a membrane filter procedure for enumerating Escherichia coli was developed and evaluated. The method quantifies E. coli within 24 h without requiring subculture and identification of isolates. It incorporates a primary selective-differential medium for gram-negative, lactose-fermenting bacteria; resuscitation of weakened organisms by incubation for 2 h at 35 degrees C before incubation at 44.5 degrees C for 18 to 22 h; and an in situ urease test to differentiate E. coli from other thermotolerant, lactose-positive organisms.  See Cabelli

Particular Type of Chromatography

Anion exchange:

–for adenovirus purification: it is usually preferred to use at least one AEX step. (Weggeman (US2009/0123989). 

Hydroxyapatite chromatograpy: has been studied for viral clearance for about 45 years. The mechanism of binding can be due to either synergistic effects of multi-site binding, strong interactions of clustered surface phosphates or both. The original eluant used almost exclusively was phosphate but sodium chloride has also been sued. A variety of other additives such as polyethylene glycol (PEG) has been employed as elution modifiers. PEG has been shown to improve aggregate separation by differentially enhancing retention of larger solutes. The tight binding of viruses to CHT can be explained at least in part by the multi-modal nature of hydroxyapatite interactions. For enveloped viruses, the significant number of phosphate groups on the lipd envelope interact strongly with the calcium atoms via a chelation mechaism as has been previously shown for both proteins and nucleic acids. In general, the much larger surface area provided by viruses would be expected to result in ighter binding as compared to much smaller bio molecules such as antibodies (US 13/178970). 

Particular Types of Viruses 

Adeno-associated virus (AAV):

AAV has become a vector of choice becasue of its safety profile. It can not replicate on its own and is not integrated directly into the host genome. The process begins by inserting a desired genetic payload sequence into a virus and producing vectors using transfected host cells. Host cells are lysed, viral particles extracted and purified to eliminate host cell contaminant and nimimiz AAV virons that are missing the therapeutic sequence. AAV manufacturing using human embryonic kidney cells in either adherent or suspension mode includes several tyipcal processing steps: cell expansion, plasmid transfection, viral vector production, cell lysis, puficaiton and fill and finish. The purificaiton process usually invlves clarifciaiton, capture through affintiy chromatography, polishing with AEX, TFF concentration/diafilgration and final filtration. Traditional chromatographic approaces used for separation of empty and full capsids are based on monoliths or columns packed with resin. A promising alternative is the use of membrane adsorbers. Used with small conductivity step changes (rather than linear gradients), Pall Mustang Q membranes can yield distinct elution peaks for both DNA-free and DNA-containing capsides. (Cameau “Overcoming obstacles in AAV viral vector manufacturing” BioProcess International, June 2021, 19(6); pp. 68-69)

AAV is a helper-dependent DNA paravovirus that belongs to the genus Dependovirus. AAV requires co-infection with an unrelated helper virus such as adenovirus, herpes virus or vaccinia in order for a productive infection to occur. In the basence of a helper virus, AAV establishes a latent state by inserting its genome into a host cell chromosome. Subsequent infetion by a helper virus rescues the integrated viral genome, which can then replicate to product infectious viral progeny. AAV has a wide host range and is able to replicate in cells from any species in the presence of the suitable helpter virus. AAAV has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. Wright et al. (US 2013/0072548). 

An important objective in the design of rAAV production and purificaiton systems is to implement stategies to minimize/control the generation of production related impurities such as proteins, nucleic acids, and vector related impurities, including wild-type/pseudo WT AAV species and AAV encapsidated residual DNA impurities. Removal of impurities in AAV vectors is complicated due to the wayrAAV vectors are produced. In one production process, rAAV vectors are produced by a transient transfection proces using three plasmids. Significant amounts of plasmid DNA are introduced into the ells to produce rAAV vectors. In addition, when rAAV vectors are released from the producing ells, cellular proteins and nucleic acids are co-released. Considering that the rAAV vector represents only aobut 1% of the biomass, it is very challenging to purify rAAV vectors to a level of purity which can be used as a clinical human gene therapy product. Wright et al. (US 2013/0072548).

–AEX-CEX or CEX-AEX:

Adenovirus is usually preferred to use at least one AEX step. (Weggeman (US2009/0123989).

Qu (US 16/088743, published as US 2020/0299650) discloses a method for purifying an AAV vector that includes harvesting cells which include the AAV vector, lysing the cells, treating the lysate with a nuclease to reduce nucleic acid, filtering the nucleic acid reduced lystate and subsecting the lysate to AEX followed by CEX or vice versa, followed by filtration.

Wright et al. (US 2013/0072548) discloses a method for puriyfing an AAV vector that includes a transgene encoding a therpaeutic protein which includes harvesting cells transduced with AAV, concentration the cells via TF, lysing the cells by micofluidization to form a lysate, clarificaiton of the AAV particles and then ion exchange column chromatography which can include various combinations of AEX and CEX.

—AEX/CEX – SEC –CEX/AEX:

 Wright (US Patent Application No: 16/088,743, published as US 2020/0299650) discloses that including a size exclusion column chromatography (SEC)in betweenthe CEX/AEX steps is particularly effective in removing impurities. 

–Apatite Chromatography:

Thorne (US 2015/0024467) disclsoes a process for isolating rAAV by capturing the rAAV aprticles on an aptite chromatogrpahy medium in the presence of polyethylene glycol (PEG). The method also includes upstream processing steps such as for example centrifugation, AEX and doenstream processing steps such as HIC, size exclusion chromatography and/or AEX. 

–Hydrophobic interaction chromatogrpahy (HIC): Thorne (US 2015/0024467) discloses a method for isoalting rAAV particles which includes contacting a feedstream with rAAV particles with a HIC medium in a high salt buffer, wherein the rAAV particles bind the HIC and eluting the rAAV partciels with a medium salt buffer. 

Hepatitis B surface antigen: Infection with HBV leads to the production of large virus particles. Vaccination of several million individuals world wide with HBsAg purified form the plasma of asymptomatic human carriers was used for almost two decades as an effective means of prevenitng HB associated health problems such as liver failure. However, the manufacturing process was tedious and time consuming requiring procuedures to inactivate infectious HBV that might be present in plasma. By the early 1980s advances in genetic engineering and biotechnology allowed the first HBV to be obtained by formulation of HBsAg produced in recombinant straints of yeast Sacharamyces cerevisiae followed by cell disruption and purified by several extraction and chromatographic steps. Following disuprtion of the cell, cell debris was diluted with potassium thiocyanate containing disruption buffer and the pH adjusted from 8 to an optimun vlaue of 4.5 so as to precipitate non-protein contaminants. Acid precipitation was followed by centrifugation to remove the precipitated proteins, carbohydrates and lipids. The the clarified HBsAg prepration is subjected to bach adsorption at acid pH onto diatomaceous earth matrix followed by elution form the matrix at low ionic strnght and basic pH (Hardy Journal of Biotechnology 77, 2000, 157-167). 

The HBV has a spherical shape with a lipoprotein coating mostly of HBV surface antigen (HBsAg). Recombinant HBV vaccines have been created based on teh HBsAg syntehsized in yeast foor ammalian cells. Transformed cells are cultures in indstrial scale fermentors, the antigen collected, purified and formulated into spherical particles that epxose the highly immunogenic “a” antigenic determinant region. The main purificaion step is immunoaffinity chromatography (IAC). (BioProcess International, March 2023, volue 21, number 3, Quintana “Purificaiton of hepatitis B virus surface antigen for vaccine products”. 

Filtration

Weggeman (US2009/0123989) discloses a method for the purifiction of a virus comprising a step of ultrafiltration wherein the retentate contains the virus, wherein back pressure of at least 5 kPa is applied on the permeate side. This results in an improvement over processes wherein no back pressure is applied on the permeate side. 

Filtration in combination with CEX: Mheta (US2011/0034674) discloses method of subjecting a protein/antibody mixture to CEX and an endotoxin removal step prior to passing through a virus filter. The method improves the capacity of the virus filter during protein purification. 

Whey can be characterized as milk from which 90-95% of the casein and the fat have been removed. Whey comprises a variety of proteins such as alpha-lactablumin, beta-lactalbumin, immunoglobulins, albumin, enzymes, growth factors and hormones.

Specific Proteins in Whey

Alpha-lactalbumin: is a protein found in the milk of mammals. However, beta-lactoglobulin does not occur in human milk but is found in bovine milk. Some infants show different degrees of intolerance to bovine milk. Alpha-lactalbumin is largely used both in preparation of humanized milk and compositions of non-allergenic milk products for infants who are allergic to beta-lactoglobulin. Accordingly, there is interest to fractionate bovine whey proteins resulting in a whey protein isolate suitable for infant formulas.

Caseinomacropeptide (CMP also known as GMP (glycomacropeptide) and CDP (casein derived peptide): is a complex mixutre of macropeptides found in sweet wheys (rennet and chesse wheys) derived from kappa-casein by the action of the enzymes chymosin and/or pepsin. CMP is rich in sialic acid and has a number of potential therpaeutic uses. Ayers (WO02/28194)

Human alpha-lactalbuin (HAMLET) has been shown to induce cell death in tumor cells by interacting with specific histone proteins and with nucleosomes (Svanborg, US 2006/0233807A1). 

Purification Schemes Used

–Ion Exchange:

Ion-Exchange: chromatographic methods, particularly IEX, for whey protein separation has been developed and used successfully on a commerical scale. 

(i) Cation Exchange

Ahmed (US5756680) discloses a method for the sequential separation of why proteins. In one embodiment, when is passed through a chromatographic column prepacked with a strong S cation exchange resin, equilibrated with acetate buffer at pH 3.8 where all the whey proteins bind, washing and then sequentially eluting immunoglobulin and beta-lactoglobulin at pH4, followed by alpha lactalbumin at pH 5, followed by BSA at pH 7 and then lactoferrin at pH 7.5.

Etzel (US 5,986,063) discloses a process to yield a stream enriched in beta-lactoglobulin and a second stream enriched in alpha-lactalbumin from a solution containing whey proteins by contacting the solution with a cation exchanger and selectively eluting the bound beta-lactogobulin fraction and the bound alpha-lactalbumin protein fraction by changes in the eluting pH. More specifically, the pH of the solution containing why proteins is adjusted to a pH of less than about 4.5, then the pH of the bound fraction is adjusted to about 4.9, elution of the beta-lactoglobulin fraction and adjusting the pH to about 6.5 and eluting the alpha-lacalbumin fraction.  

Mozaffar (US 6096870) discloses separation of whey proteins through the use of chromatography such as cation and anion exchange. Included in the procedure is pH adjustment to 3.8 prior to loading onto cationic exchanger washing and two sequential elutions the first elution for beta-Lactoglobulin and the second for alpha-Lactalbumin.

Hansen (US 13/063579) discloses a method for isolation of a b-lactoglobulin product and an alpha-enriched whey prtoein isolate from whey which provides the steps of (i) providing whey obtain from an animal, (ii) adjusting the pH value of the whey to pH 4.5 or below, (iii) loading the whey to a chromatographic support, (iv) optionally washing the chromatographic support, (v) eluting the beta-lactoglobulin from the support using a first elution buffer having a pH of 4 or less and a salt concentration of at least 0.1M and (vi) eluting the alpha renriched whey protein isolate with a second eltuion buffer. 

Hansen (US 2007/0092960) also discloses that the combination of high operating temperature and high flow rate applied upon loading biomolecule containing fluids onto a chromatographic column significantly improves the adsorptive capactiy and the productivity of the adsorbent of the column. In one embodiment sweet whey is pumped through a heat exchanger to reach 50C before being loaded onto a chromatography column and the pH adjusted to 4.7. Non bound material was washed out and the bound proteins eluted in two separate steps. In elution 1, all the bound beta-lactoglobulin is recovered and in elution 2 all the bound immunoglobulin G, bovin serum albumin and alpha lactalbumin is recovered.

–Overload Mode:

Ayers (US6,592,905 and WO/1997/027757) discloses a process for producing an immunoglobulin enriched whey protein fraction from a whey protein solution using CEX.  Accordingly to the invention, the CEX is contacted with an excess of adsorabel protein at a pH level below about 4.5 which causes the CEX to adsorb preferentially whey proteins with the immunoglobulin being displcaed. 

(ii) Anion Exchange (AEX):

Ayers (WO02/28194) discloses a process for producing a CMP isolate and an acid and heat stable beta-lactoglobuilin enriched WPI from a feedstock containing whey proteins including CMP and beta-lactoglublin by coacting the feestock with an AEX under conditions in which CMP and beta-lactoglobuilin are both adsorbed, eluting the beta-lactoglobuilin and the CMP from the AEX and contacting the eluate with a second AEX under conditions whereby CMP is selectively adsorbed, collecting the beta-lactoglobulin continaing flow through from the second anion exchanger and eluting CMP from the second AEx.

Mozaffar (US 6096870) discloses subjecting whey to an anionic exchange resin at about pH 7.5 wherein the beta-lactoglobulin adsorbs, collecting the flow through which is alpha-lactalbumin, immunoglobulins, bovine serum albumin and lactoferrin and eluting the adsorbed beta-lactoglobulin with a buffer at a pH of about 7.5.

Outinen (WO 95/19714) discloses fractionating alpha-lactalbumin and beta-lactoglobulin by passing a whey solution through an anion exchange resin, washing the column with deinonized water to obtain alpha-lactalbumin and eluting with a weak acqueous NaCl solution to recover beta-lactoglobulin.

–AEX-CEX: 

Gerberding & Byers, “Preparative ion-exchange chromatography of proteins from dairy whey” J. Chromatography A, 1998(808): 141-151). discloses separating proteins such as alpha-lactalbumin, beta-lactoglobulin, BSA and IgG and lactose from a sweet dairy whey mixture using AEX and then CEX. The AEX was most effective in separating beta-lactoglobuilin. CEX was used to further recover the IgG. 

(iii) Filtration

Ultrafilitration: UF is the most widely used commercial method of whey fractionation. It is employed after an initial chromatographic prcoessing step to further separate the whey proteins. However, it suffers form the drawback of high capital and operating costs, membrane fouling, incomplete removal of low Molecular mass solutes thereby requiring in place cleaning and sanitation of the membrane to minimize microbial problems. (Gerberding, J. Chromatography A 808 (1998) 141-151)

(Cheang J. Membrane Science 231 (2004) 159-167) discloses use of a two stage tangential flow filtration system for the purificaiton of alpha LA and beta LG from whey protein isolate. Separation was aheived using 100 and 30 kDa membranes in series in both the order of 100kDa followed by 30 kDa and 30 kDa followed by 100 kDa. In the second strategy, the 30kDa membrane was used to obtain purified alpha LA in the permeate solution while retaining the beta LG and BSA. The collected retentate was then searpated in stage 2 using a 100 kDa membrane.

(iv) Membrane Separation

EP-A 0 311 283 teaches the use of UF membrane for achieving an alpha-La/beta-Lg ratio of at the most 3/1 in the permeate.

(iv) Precipitation

–Ammonium Sulfate-IEX: (Lozano, , International Dairy J. 18(1), 2008, p. 55-63) discloses differential precipitation with ammonium sulfate to isolate beta-lactoglobulin from other whey proteins using 50% ammonium sulfate. The precipitate was dissolved and separated again using 70% ammonium sulfate, leaving a supernatant liquid enriched in beta-lactoglobulin. After dialysis and lyophilization, isolation of the protein was performed by IEX. 

For purification of antibodies from Milk see Purification of antibodies

Separation of Casein from Whey (for separation of specific proteins in Whey see outline)

The supernantant fluid generated during cheese manufacturing is commonly referred to as whey. Proteins contained in whey, which are soluble proteins including lactoferrin, lactoperoxidase, immunoglobulins, albumin, alpha-lactalbumin and beta-lactoglobulin are referred to as “whey proteins”. Whey has long been the predominant source of milk proteins and significant effecrts have been devloted to separation and isolation of various whey proteins.

Although whey is discarded during cheesemaking (the “curd” which is formed when milk is acidified (becomes sour) is used to make cheese) it has significant value as a source of nutrition. It is used in andies and special cheese products. In addition large quantities of dry and concentrated forms are mixed with food and feeds.

The separation of Casein from Whey involves separation of casein, an insoluble protein contained in whole milk, from other components by precipitation. The two predominant precipitation techniques are rennet precipitation and acid precipitation, which are ulternatively utilized, depending on the specific type of cheese to be produced. 

Rennet precipitation (Sweet Whey): is produced when the caseins are enzymatically coagulated using rennet type enzymes at a pH of 5.9 to 6.3. rennin is added to warm milke caseins which are precipitated leaving the whey proteins in solution. Whey produced by this method is referred to as “sweet whey”. By far of the whey produced as a cheese manufacturing by product, nearly all of it was sweet whey. (Gerberding, J. Chromatography A 808 (1998) 141-151)

Acid precipitaiton (Acid Whey): is carried out at the isoelectric point of milk (i.e., 4.7) through the use of acid to precipitate out casein and leave the whey in solution. Whey produced by this method is referred to as “acid whey”. (US 6096870).

Filtration: 

Multi-filters

Couto (US2004/0167320) teaches a process that employess three filtration unit operations that clarify, concentrate and fractionate a product such as an antibody from a transgenic milk volume contianing the molcule of interest. 

Koph (US 2003/0059512) teaches a method of separation of a casein-rich fraction and a casein-depleted fraction of milk by (a) providing a source of milk, (b) optionally flowing the milk through a cream separator to remove all or at least a portion of the fatty component of the milk, (c) optionally pastuerizing the milk, (d) flowing the milk through a cross-flow filtration module to separate the milk into a casein rich retentate fraction and a casein depleted permeate fraction and (e) recovering both the casen rich fraction and the casein depleted fraction. 

Microfiltration

Milk serium protein can be made by MF of milt to remove the caseins. Etzel (WO 2012/012237)

Charged Ultrafiltration

–AEX Membranes (positively charged membranes):

Etzel (WO 2012/012237) discloses a method for fractionating a protein mixture that includes multiple protein species such as milk by (a) adjusting the pH of the mixture to an isoelectric point just below the pI of the protein of interest, thereby rendering a net charge of about zero on the protein of interest and also adjusting the conductivity of the mixture such that shielding of the multiple species other than the protein of itnerest is limited to the extent that the other species are rejected by the charged UF. The charged UF may have a pore size rating 150-500kDa and the protein of interest includes glycomacropeptide (GMP*, alpha-lactalbumin (ALA), IgG and/or beta-lactoglobulin (BLG). For example, milk serum proteins can be made by MF of milk to remove the caseins. Alpha-lactalbumin is smaller and more acidic  (14.4 kDa; pI 4.4) than beta-lactoglobulin (18.67 kDa pI 5.1). By adjusting the milk serum protein to about pH 4.0-4.5, the alpha lactalbumin has little to no net charge while the beta lactoglobulin has a net positive charge. The alrge beta lactoglobulin will be subject to electrostatic repulsion by a positivley charged UF while the smaller alpha-lactalbumin that has little to no net charge can permeat the charged UF. Howev, increasing the conducitvity of the protein mixture increases shielding of the charges on the proteins. As conductivity increases to above about 100 mS/cm, charge shielding is great enough to negate the effect of electrostatic repulsion. Thus there is a blance between pH and conductivity. 

Natural interferon production has traditionally involved ammonium chloride treatment of buffy coats to lyse the red blood cells and to isolate the leukocytes, followed by viral stimulation of leukocytes with subsequent large scale harvesting of culture medium. The interferons are then isolated by various precipitation, adsorption, or immuno-affinity techniques.

Concentration of Interferon from Luekocyte Culture:

Traditionally, luekocytes have been collected as whole blood and stored in impermeable plastic bags at about 4 C-25C until processed into plasma and red blood cells. The white blood cell layer (buffy coat) is generally discarded as a side product which can be collected and treated with ammonium chloride to lyse the contaminating red blood cells. The remaining leukocytes are then cultured in media containing a serum and activated with a viral inducer to produce interferon. Morris (US6,350,589) disclosed a process where whole blood is collected and centrifuged to produce a buffy coat. Collection of the buffy coat is done with a peristaltic pump and a specially designed manifold that is much gentler than a vacuum pump. An initial wash with PBS serves to remove both platelets and some serum contaminants. After centrifugation, the supernatant is removed with a peristaltic pump and specially designed aspirators. The cells are gradually brought to isotnoic osmolarity with PBS washes. 

Subsequent Purification Techniques

Affinity Purification Techniques:

–Antibody affinity – RPHPLC: US 4,551,271 discloses passing a partically purified preparation through an antibody affinity column and a reversed phase high performance liquid chromatographic column. 

Controlled Pore Glass (CPG):

Billiau (Antimicrobial Agents and Chemotherapy, July 1979, p. 49-55) disclsoes purification of human fibroblast interferon by absorption of F-inteferon on CPG beads at neutral pH and more or less selective relase at pH about 2.0. 

–CPG – Zinc-Chelate Chromatography:

Heine (Methods in Enzymology, 78, pp. 448-456, 1981) discloses purificaiton of crude human fiblast interferon by CPG adsorption and then a stecond step of zinc-chelate chromatographic purificaiton. 

Hydroxyapatite (HA) chromatography: Morris (US6,350,589) discloses taking a crude interferon (e.g., such as a cation exchange capture) and applying it to a HAC column. 

Particular types of Interferons

INF-alpha: Bodo (US 5196323) discloses a process for purification of rrecombinant  IFN-alpha by tandem chromatography comprising separation on a cellulose column followed by an anti-alpha-interferon monoclonal antibody affinity column, then subjecting the purifed material to isolectric precipitation of impurities at about pH 4.0 to about pH 4,8 and then chromatography on a high performance cation exchange solumn using a volatile buffer. 

Glutamic-oxalacetic transaminase: Cammarata “Fractionation and properties of glutamic-oxalacetic transaminase” (1951) teaches that low temperature alcohol fractionation, so successful in plasma protein fractionation, offers advantages over ammonium sulfate in the purification of gluatmic-oxalacetic transaminase. Care was taken to add the 95% alcohol dropwise or in a fine spray to avoid excess local concentration.

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.