Commercial Kits:  Pierce Chicken IgY Purification Kit   Gallus Immunotech  Promega Eggstract®

Ammonium sulfate or sodium sulfate precipitation: has widely been used as an initial purification step in the isolation of immunoglobulin isotypes from serum bile and egg yolk. A high concentration of ammonium sulfate competes with the proteins in a solution for water molecules, reducing the solubility of the proteins and thus causing precipitation (Beetham, Avian Diseases 37: 1026-1031, 1993).

Particular Schemes

 Water dilution – AMS: 

Akita (J food science, 57(3), pp. 629634, 1992) discloses isolation of IgY by first a simple dilution of egg yolk with water in low pH follwoed by ammonium sulfate with 60% saturation.

Larsen (US 12/299670 and WO2007/079755) discloses a classic protocol for isolating antibody from egg yolk with Ammonium Sulphate:   First, separate egg white and yolk, wash the yolk with water, puncture the yolk and dissolve it in  1:10 weight volumn water, reeze and dry the solution, add solid ammonium sulphate to 25% saturation, incubate at room temp for 20 min, centrifuge, transfer supernatant to a new glass and add solid ammonium sulphate to 40% saturation and incubate at room temeprature for 20 min, centrifuge. The pellet is redisosolved.

Henderson (US14/020469) discloses a method for purifying IgY antibodies by (a) contacting a sample with a precipitating agent such as ammonium sulfate and (b) differentially precipitating the IgY antibodies obtained in (a) using the same or different precipitating agent to separate IgY(Fc) and IgY(deltaFc) antibody isoforms. In one embodiment, the method comprises a first precipitation by (a) contacting the sample containing the IgY antibodies with a precipitating agent at a first concentraiton to obtain a first precipitate containing the IgY antibodies (b) resuspending this first precipitate and contacting it with the same or different precipitating agent at a second concentration to obtain a second precipitate containing a majority of one IgY isoform and a supernatant containing a majority of the other IgY isoform (c) contacting the supernatant obtained in (b) with the same or different precipitating agent at a third concentration to obtain a third precipitate containing a majority of the other IgY isoform. The precipitated IgY(Fc) and IgY(deltaFc) antibodies can be further purified ushing other chromatographic techniques. In one embodiment and prior to the differential precipitation above eggs laid from immunized geese are collected, after gently making an opening in the egg shell, egg shite is removed and the yolk rinsed with several columnes of ice cold deionzied water, the washing egg yold is then transferred to an absorbent paper towel to remove residue egg white, the yold sac is then punctured and the yolk contents transferred to a 1L beaker and ice cold deionized water is added 90x the yolk weight. The diluted yold is then gently stirred at 250-300 rpm for 15 min at room temperature and the yolk suspension is acidified by adding 1N HCl until pH 5.0 and then further mixed for 5-10 min and frozen at around -20C. The frozen yolk suspension can be thawed, centrifuged and filtered prior to precipitation above with ammonium sulfat at concentraiton of about 50%.

Water dilution- AMS -Affinity purification: Cook, (J. Bioscience and Bioengineering, 91(3), 305-310, 2001) discloses obtaining IgY from egg yolks by mixxing six fold volume of chilled water and allowing the yolks to settle overnight. The speurnatant was decanted and AMS was added in the amount of 200 g/l supernatant. After one half hour stirring at 4C, the preciptiate containing IgY was recovered by centrifugation and resolubilized in phosphate buffered saline for the final AMS preparation. For a more specific antibody preparation, the AMS precipitate can be passed over an affinity column to give the final affinity purified product. 

Water dilution -AMS – alcohol precipitation: Akita (“Immunoglobuilins form egg yolk: isolation and purification” 57(3), Journal of Food science, 1992, pp. 629) discloses separating egg yolk from the white, washing with distilled water to remove albumen, rolling on paper towels to remove adhering egg white, puncturing the membrane and flowing the yolk into a cylinder without the membrane, deluting with distilled water (acidified with 0.1 N HCL) and held for at least 2  hr before centrifugation for 1 hr or filtration through filter paper. The resulting immunoglobuilin containing filtrate is further purified by salt precipatation (AMS). Then either UF followed by DEAE-Sephacel/gel filtration or alcohol precipitation followed by DEAE-Sephacel/gel filtration or salt precipitation (sodium sulfate) can be used to future purify. 

Bizhanov (Scand. J. Lab. Anim. Sci. 3(31), 2004) discloses replacing the sodium sulfate with lithium sulfate in the ater dilution of Akita above. In the method, yolk diluted 1:9 with distilled water, acidified with HCL to pH 5.0 is incubated overnight at 4C. After futher centrifugation, the precipitate containing Igy was resupended in TBS, precipitated twice in either sodium citrate, lithium sulfate or ammonium sulfate (comparison of all 3 methods) and dialyzed against TBS.

Caprylic acid -ammonium sulfate –DF:

Bhanushall discloses purifying IgG from chicken serum by diluting chicken serum with acetate buffer and adding caprylic acid, removing the precipitate, collecting the supernatant and adding ammonium sulfate (NH4)SO4. The pellet was resupsended in PBX and dialyzed against PBS.

Sodium acetate buffer – PEG – AMS precipitation: Duan (CN101787080, published 7/28/2010) discloses a three step method of preparing egg yolk IgY by (1) removing yolk and stirring in acetic acid-sodium acetate buffer followed by sufficient mixting and then (2) stirring in 40% PEG and centrifuging for 30 min. The supernatant is decanted and the precipitate contacts antibody IgY crude extract was subjected to staturation of 33% ammonium sulfate precipitation and purified by SDS-PAGE analysis with a purity of 90-95%. 

Sodium acetate – ammonium sulfate: Wang, (US 13/956511) teaches extraction of IgY from yolk by diluting the yolk with a buffer solution with sodium acetate at pH from 4.6 to 5.4 stirring the mixture, centrifuging and then adding an inorganic salt such as ammonium sulfate solution to the sueprnatant to salt out the IgY. 

Wu (CN101955532A, published 1/26/2011) teaches a method for rxtracting chicekn egg yolk antibodies mixing in a sodium acetate buffer solution to the yolk at pH of 4.8-5.2 and leeting the mixture stand. To the supernatant, ammonium sulfate is added at 25-30%, then centrifuging. The precipitate is resuspended in PBS and ammonium sulfate solution volume of 0.7 times the begging is stirred in, let stand and then the precipitate was resupended in PBX to give a solution of yolk antibodies. 

Natural Gums – Ammonium suophate precipitation: 

Natural gum zlpah-carrageenan has been used to isolate IgY (IgG) fraction from the yolk of chicken eggs (Hatta, Agric. biol. Chem, 54: 2531-2535 1990). The mechanism is not clear but Hatta showed that the natural gums that isolated the highest percentage immunoglobulin fraction were acidic and that the method is due to the ionic binding between the negative charge of the alpha-carrageenan and the net positive charge of the immunoglobulin fraction.

Tan (J Immunological Methods, “A novel, cost-effective and efficient chicken egg IgY purificaiton procedure” 2012) disclose a method of IgY purification from chicken eggs yolks using the common plant gums pectin and kappa-carrageenan in the presence of calcium chloride to delipidate egg yolk mixtures while maintaining IgY in solution and then ammonium sulphate to subsequently precipitate the resulting IgY antibodies to higher purity. 

PEG-AMS

Rajic (“protein depletion using IgY from chickens immunised with human protein cocktails” Prep Biochem Biotechnol 2009, 39(3) 221-47) discloses IgY extraction procedure using water dilution to deplet lipids and lipoproteins followed by sequential precipitation with 31% AMS and 12% pooy ethylene glycol (PEG).

 Stationary phase (silicate, carbon, cellulose, synthetic fiber)- Ammonium sulfate (first concentration 15-24% -AMS (second concentration 25-40%): 

Chiou ( EP1371665B1; see also US20060223986, US 6,680,376 and US2003/0009014) discloses a method of obtaining IgY(deltaFc) antibody isoform absorbing yolk antibodies in a water miscible fration obtained from egg yold with a water insoluble non-charged active constituent in the stationary phase such as silicate, carbon, cellulose and synthetic fiber, flowing the stationary phase with a buffer to obtain an aqueous fraction containing the yolk antibodies and then salting out this aqueous fraction with (NH4)SO4 at a first concentraiton ranging from about 15-24 (w/v) and then salting out the aqueous fraction with (NH4)2SO4 at a second concentration of 25-40% (w/v). 

Chloroform

–Water dilution Chloroform: Larsen (US 12/299670 and WO2007/079755) discloses an alternative protocol for IgY isolation from egg yolk. First separate egg white and yolk, add 100 mM sodium phosphate buffer pH 7.6 to volume 60 ml and mix, add 40 ml chloroform and mis to a semi solid phase, centrifuge, isolate the supernatant. PEG-6000 is added to a final concentration of 12% w/v, mix until PEG dissolved, centrifuge for 10 min, dissolve pell in 100 mM sodium phosphate buffer pH 7.6. sodium azide is added to 0.05%. 

Salting Out Agents

Ammonium sulfate precipitation: See outline

Sodium Sulfate precipitation:

Jensenius “J Immunol Methods, 46, 63-8, 1981) disclsoes two methods of separation of IgG from egg yolk. In the first methods, egg yolk is combined with TBS, then the supernatant from this mixed with dextran sulphate and CaCl2, the sediment from this discarded and then the supertants mixed with Na2SO4. In the second method, yolk is mixed with water and NaoH ot pH 7.0 and then the IgG precipitated with Na2SO4. Ammonium sulfate may be used instead of sodium sulphate.

–Water dilution – CEX: Aqueous extract-Cation exchange-sodium sulfate preciptation: 

Charter, “A new process for the separation and purification of egg yolk antibodies” Thesis 1987) disclose a three stage process for the separation of an antibody (IgY) form chicken egg yolk which invloves aqueous extraction of the water soluble fraction (WSF) from the yolk by dilution with distilled water and pH adjustment, spearation of IgY from the WSF using a cation exchange column and then precipitation with sodium sulfphate.

Sodium Chloride

–Water dilution – NaCL: Larsen (US 12/299670 and WO2007/079755) discloses an alternative protocol for IgY isolation from egg yolk. First separate egg white and yolk, wash the yolkd with 2 volumes water, puncture the yolk and dissolve it in 1:10 weight/volume water, freeze and dry the solution, add 133.25 g solid NaCl to 25% saturation, incubate at room temp for 20 min, centrifuge for 30 min, transfer supernatant to new glass and add solid NaCl to 40% saturation, incubate at room temp for 20 min, centrifuge. The pellet is redissolved. 

Lithium Sulfate

Bizhanov (Scand. J. Lab. Anim. Sci. 3(31), 2004) discloses replacing the sodium sulfate with lithium sulfate in the ater dilution of Akita above. In the method, yolk diluted 1:9 with distilled water, acidified with HCL to pH 5.0 is incubated overnight at 4C. After futher centrifugation, the precipitate containing Igy was resupended in TBS, precipitated twice in either sodium citrate, lithium sulfate or ammonium sulfate (comparison of all 3 methods) and dialyzed against TBS.

PEG:

–PEG-PEG:

Larsen (US 12/299670 and WO2007/079755) discloses a protocl for islation of IgY from egg yolk using PEG.

Polson, A., M.B. von Wechmar and M.H. van Regenmortel, “Isolation of Viral IgY antibodies from yolks of immunized hens,” Immunological Communicaitons 9:475-493 (1980) disclose isolation of antibodies from the yolks of hens by PEG at 3.5% to casue the lipids and vitellin to separate and then precipitation of IgY with 12% PEG.  

Polyethylene glycol (PEG): by a 25% cold ethanol precipitation to displace the immunoglobulin fraction from chicken egg yolks (Polson “Improvements in the isolation of IgY from the yolks of eggs laid by immunized hens. Immunol. Invest. 14: 323-327 (1985). The mechanism is not clear but Poson speculated that the immunoglobulin fraction in the PEG extraction precipitates by means of displacement.

Polson (US4,550,019) discloses a method of separation of IgY which comprises collecting yolks form a number of eggs, washing them with distilled water to remove all the albumen, dropping them into a large glass funnel  and adding buffer equivalent to two volumes of yolk and adding PEG to a final concentraiton of 3.5% by weight of polymer to volume of diluted yolk. The mixutre was centrigued, causing the seapration of three phases in the centrifuge tubes; a yellow fatty layer on the surface, a clear supernatnat layer occupying the largest volume and a semi solid pliable layer of the builk of the yolk and caseinous protein “pellet” representing about 1/3 of the total volume. The supernatant fluid with the fatty layer was decanted into a funnel contianing an adsorbent cotton plug in the neck of the funnel. This plug filtered off the lipid layer that was decanted with the supernatant fluid. The volume of the clear fitlrate was measured and pulverised PEG was added which caused complete displacement of the IgY.

Mostov (US6,340,743) also discoses extracting chicen antibody IgY from egg yolks by a series of PEG precipitations followed by a series of ammonium sulfate precipitations accoridng to the method of Poison. 

Other Agents

Caprylic (octanoic) acid:

–caprylic acid –ammonium sulfate

Mclaren (J Immunol Methods, 1994, 177(1-2): 175-84) discloses collegcting eggs from immunised hens, harvesting the egg yolk, diluting them with phosphate-buffered saline (PBS), pH 7.5, adjusting pH to 4.6 with acetic acid and caprylic acid, stirring at room temeprature for 2 hour, centrifuging, the resulting supernatant, contining the immunoglobulin fraction, was then collected, the pH adjusted to 7.5 with Tris and centrifuged. The prepration was thenn cooled to 4C and ammonium sulfate was added, stirred for 1 hr and centfiuged for 20. The sueprnatnat discard, the plellet washed by resuspending in ammonium sulphate and recentrifuged. The washed pellet was then dissolved in PBS pH 7.5.

–Caprylic acid -HCIC

Tong (“A new purificaiton process for goose immunoglobuilin IgY (dltaFc) wit6h hydrophobic charge-induction chromatography) Biochemical Engineering J. 56 (2011) 205-211) discloses pretreating goose plasma with caprylic acid to precipitate some impurities and loading the supernatant onto a HCIC column for IgY(deltaFc) separation.

See also extraction of IgY by precipitation

Initial Extraction

All IgY purification methods require an initial delipidation step to extract insoluble lipids and lipoproteins. Among the accepted commercial and laboratory delipidation techniques, the most commonly deployed involve either the use of water dilution, chloroform-PEG, polyanionic polysaccharides such as xanthan, dextran sulpate or other natural gums such as pectin. Tan (J Immunological Methods, “A novel, cost-effective and efficient chicken egg IgY purificaiton procedure” 2012).

UF/DV

Hernanez-Campos (J Agric Food Chem, 2010, 58 187-193) discloses purification of IgY from hen egg yolk water soluble protein fraction by UF/DF. The best results were obtained in the absence of salt at pH values 5.7 and 6.7 using poliethersulfone (PES) and modified PES.

Companies working with Avian Antibodies: Amicus Biotech 

Differences and Advantages of Avian Antibodies

It is known that birds (e.g., laying hens) transfer their immunity to the yolk of their eggs and thereby to their offspring (Polson, US4,550,019). A laying hen can produce a 50-60 gm egg (more antibodies than a typical blood sample) every 24 hours. Chicken secrete all three classes of antibodies into their eggs. IgG (aka IgY) is found in high concentration in the yolk whereas IgA and IgM antibodies are present in the white portion  (US 2007/0238097 A1).

There exist substantial differences between the gamma-globulins which occur in the egg yolk of fowl eggs and mammalic *including human) gammaglobulins. First, the source materials are different. Conventional mammalic immunoglobulin preparations have been mostly recovered form blood serum. This is obtained form whole blood after removal of fibrinogen by clotting. The serum is a clear liquid containing about 6.5-7% by volumen of dissolved proteins represented by about 0.8-1% gammaglobulin, 3.5% albumin, 2.5% miscellaneous proteins. There is no caseinaceous matter in mammalic serum and little or no free lipid matter. By contrast, egg yolk contains about 25% by volume yolk granules and another 25% of caseinaceous proteins and free lipids. Second, the immunoglobulins themselves in egg yolk are chemically and physically significantly different from immunoglobulins in human or other mammalian serum. Differences exist in amino acid compositions and electrophoretic mobilities (polson, US4,550,019). 

Unlike in mammals, there are only three classes of antibodies identified in chicken, IgM, IgA and IgY. Among them, IgY is the predominant form which is continually synthesized, secreted into the blood and transferred to the egg yolk, where it accumulates to a concentration even higher than that in the blood. It has been reported that a single egg contains as much Abs as an average bleed from a rabbit (Wang, 13/956511). 

IGY Structure and Isoforms

Ducks and geese produce three kinds of serum immunoglobulins; IgM and two isoforms of IgY antibodies, a full lenght isoform (IgY(Fc) and a truncated isoform (IgY(deltaFc)) (Y-Neng, EP1371665). 

The intact IgY(Fc) antibody possessed two heavy and two light cains, with the heavy chain having one variable and four constant domains. The molecular weight of IgY(Fc) is about 180 kDa and it has a sedimentation coefficeint of 7.8 Svedberg units (S). The truncated form of IgY(Fc) lacks two Fc domains (CH3 and CH4) of the heavy chain and is similar to a F(ab’)2 fragment of an IgG antibody. IgY(deltaFc) has a molecular weight of about 120 kDa and a sedimentation coefficient of c.7. Henderson (US14/020469) 

Advantages to Using Avian Antibodidies

Avian immunogloublins isolated form egg yolk (so-called IgY) have several important advantages over conventional IgG antibodies due to their higher surface stability, lower corss-reactivity, stronger avidity and higher binding capacity (Fang, Frontiers of Pharmaceuticals and Biotechnology, 4, 2003). 

IgY Isolation: (See outline)

IgA and IgM Isolation

Fackrell (US 13/177114) discloses a method of isolating IgA and IgM antibodies from the egg white by fractionating the white of said egg by raising the volume of the egg white using water, lowering the pH, filtering out the insoluble IgM fraction. The IgA solution is then salting out with amonium sulphate, dialyzing the IgA precipitate and drying the IgA. 

Purification Schemes Following Avian Antibody Isolation

Affinity Purification: Williams (US2004/0115215) discloses using affinity columns containing recombinant toxin A protein from 6 defined intervals to isolate region specific IgY antibodies pools. The 6 intervals were slected because they spanned the entire protein and thus are part of an epitope mapping of the Toxin A gene. IgY was applied to the affinity column so as to isolate region specific antibodies specific to each of the six subregions. The columns were washed and Abs eluted. The antibodies can subsequently be tested for nuetralization ability of toxin A protein. The procedure is designed toxin A subregions that are essential for producing neutralizing antibodies. 

Larsen (US 12/299670 and WO2007/079755) also discloses obtaining Igy antibodies having specificity for secondary antigens to be tested. Accordingly to the proecdure, a library of peptide is used to immunize avian species (e.g., fowls). The antibodies can thereafter be purified using a peptide/protein or chemical which the customer wants antibodies against by coupling the peptide/protein to a resin. The antibodies which is also a library binds the antigens on the resin and antigen specific antibodies are eluted from the column. For the related concept of “epitope mapping” of antibodies see “immunology”. 

Applications

Passive immunization by oral administration of IgY: The method of Polson, A., M.B. von Wechmar and M.H. van Regenmortel, “Isolation of Viral IgY antibodies from yolks of immunized hens,” Immunological Communicaitons 9:475-493 (1980  can be used to produce a preparation of egg-yolk antibodies. In brief, laying hens are inoculated with antigen-adjuvant which causes the hens to produce anti-antigen antibodies which are passively transferred into the egg yold of eggs laid by the hens. Egg yolks or whole eggs containing the antibody can be collected and homogenized to form an emulsion which can be dried to form a powder containing the antibody. This powder can be formulated for oral administration and then adminsitered orally to a human or non-human animal subject (US2010/0233184A1). Because IgY is susceptible to degradation in the gastrointestinal tract, enteric pH sensitive coatings can be used.

Affinity chromatography (Protein A/G)

Affinity – IEX: 

Herrmann (WO 2005/100394A2) discloses a process for purifying IL-15/Fc fusion protein by applying the composition to an affinity chromatography column and applying the eluate to an ion exchange chromatography column. Additionally, the eluate from the ion exchange can be applied to a gel filtration column or to a hydrophobic interaction chromatography column.

Herrman (WO 2005/100394) discloses a process for purifying a fusion protein by applying an affinity chromatography, applying the eluate to an ion exchange chromatography.

Protein A -AEX:

Treyo (US 14/775,992, published as US 2016/0024144; see aslo US 16/042,965, published as US 2019/0085021) discloses a method for purifying a recombinant prtoein such as atumor necrosis factor receptor Fc fusion protein.  such as etanercept. 

–Protein A- AEX –HIC

Kulkarni (US2014/0128577) discloses a method for purificaiton of TNFR:Fc fusion protein compirising Protein A chromatography, HIC in bind and elute mode and AEX which is performed in bind elute mode. In one embodiment AEX is performed prior to the HIC.  

Majumder (WO 2014/102814) disclsoes purification of a TNFR-Fc fusion protein which includes the scheme of Protein A chromatography, AEX  (Q Sepharose FF), HIC (the elute of Q Sepharose FF is added to 0.8 M of ammonium sulphage and the sample after filtraiton is laoded on phenyl sepharose resin preequilibrated with 20 mM Phosphate buffer containing 0.8 M ammonium sulphage. After washing with the equilibraiton buffer the protein is eluted wither with 20 mM Phosphate buffer contnaining 0.3 M ammonium sulphage buffer in step gradient mode or with 20 mM Phosphate buffer in linear gradien mode) HIC output is then diafiltrated and concentration against a suitable buffer followed by nano filtration. . 

–—-Protein A -AEX -HIC -CEX

karur (US 16/083891, published as US 2020/0283472; see also wO 2017/168296) disclsoes a method of purifying an Fc fusion protein such as etanercept (TNF fusion with Fc of IgG1) which includes the steps of AEX in bind and elute mode, HIC using polypropylene glycol (PPG) chromatography in bind and elute mode, followed by CEX in bind and elute mode. 

Protein A – CEX: 

Eon-Duval (US 12/377122) discloses a process for reducing the content of free Fc moeties in a fluid comprising an Fc containing protein (exemplifies a TACI-Fc fusion protein) comprising an affinity A step followed by a strong cation step on Fractogel EMD SO3- which includes a wash step with a first buffer having a conductivity of 8.2 to 9.2 mS/cm and a pH of 6.0 to 7.0 to remove free Fc-moieites. 

A packed bed CEX following Protein a chromatography typicaly has yields of greater than 805 and aggregate levels of less than 0.5%. By contract, an expanded bed chromatography reduced aggregates in yields of between 50-60% and only at the lwoest condutivities with each pH was the level of aggreggates reduced to below 1%. Blank (Bioseparation 10: 65-71, 2001)

Protein A -CEX -AEX:

Nti-Gyabaah (US 2014/0187751) discloses methods for purifying a Fc fusion protein proudced in a eukaryotic system and in particular TNFR-Fc (e.g., Etanercept) which includes the steps of Protein A affinity chromaotgoraphy followed by two ion exchange chromatography steps of CEX followed by AEX both in bind-and-elute modes. 

Protein A/G – CEX – AEX – HA: 

Eon-Duval (WO2008/025747) also discloses a process for the purification of an Fc-fusion protein comprising the steps (1) Protein A/G affinity chromatography, carrying out the elution with a pH gradient, preferably from pH 4.5 to 2.8 and then subjecting the eluate to CEX, subjecting the eluate to AEX and subjecting the flow through to hydroxyapatite chromatography. 

Protein A — HA:

Vedantham (US 7,476,722) discloses purification of a protein that may comprise an Fc porition of an antibody such as TNFR:Fc and a second protein such as Protein A /G using affinity chromatography using the second protein affixed to the solid support as an adsorbent followed by hydroxyapatite in flow through mode. 

Protein A- HIC

Kulkarni (WO 2012/176158) discloses a method of purifying a TNFR:Fc fusion protein that includes the steps of Protein A followed by hydrophobic interaction chromatography in bind and elute mode where the buffer solution does not contain any additives and is at a conductivity of 25-40 mS/cm followed by AEX.

–Protein A -HIC-AEX-Mixed Mode

Banerjee (US 2017/0152298 disclsoes purificaiton of TNFR:Fc fusion prtein by Protein A in bind-elute mode followed by HIC in bind and elute mode followed by AEX in bind -elute mode and then mixed-mode chromatogrpahy in flow through mode.

Protein A -Mixed Mode -HIC:

–Multimodal Anion Exchange:

Keller (WO 2016/009049) discloses a method for purifying a TNFR-Fc fusion protein which includes the steps of subjecting a solution that includes TNFR-Fc to affinity chromatography, optionally subjecting the eluate to AEX, subjecting the eluate to multimodal anion exchange chromatography and then subjecting the flow-through to HIC and collecting the eluate to botain purified TNFR-Fc. 

Hydrophobic Interaction Chromatography (HIC):

Ghil (US 2018/0079796) disclsoes a method of prearing human 75 kDa (p75) TNFR fused to an Fc region of human IgG1 using HIC such that the anti-TNF-alpha polypetpide binds to the HIC regin and eluting the anti-TNF alpha polypetpide. Etanercep is the recombinant human tumor necrossis factor receptor p75Fc fusion protein. Etanercept is well establisehd and widely used in clinical practice. 

Park (US 2017/0369553) discloses a method for preparing a TNFR-Fc fusion proteins which includes the steps of laoding a sample containing a TNFR-Fc fusion protein mixture produced form mammalian cells into a column filled with HIC comprsing an aromatic functional group, which is pre-equilibration with an equilibraiton buffer comprising sodium chloride or ammonium sulfate and collecting an eluate with an elution buffer comprising soidum chloride or ammonium sulfate at the same concentraiton as that of the equilibraiton buffer. 

Won (US 2014/0316114) discloses a method for preparting a TNFR-Fc fusion proteins which includes loading a sample which includes the TNFR-Fc produced in mammalian cells to the HIC column pre-equilibrated with an equilibration buffer that includes one or mroe salts selected form sodium citrate, sodium sulfate and sodium phosphate in an amount of 10-14 gL bed per volume of chromatography resin, washing the column with the same salt as the equilibraiton buffer to remove clipped forms of TNFR-Fc fusion protein and eluting the active TNFr-Fc fuison proteins from the column with an elution buffer have salt concentraiton lower than the equilibration buffer. 

HIC-Pre-filter-Virus filter:

Sharma (US Patent Application 16/708,987, published as US 202010190137) discloses purification of the TNFR-Fc fusion prtoein etanercept which includes HIC (e.g., Toyopearl Butyl-650 M column) and subjecting the elution pool to virus filtration (e.g., VireSolve Pro viral filter). In one embodiment, a prefileter (Viresolve Prefilter A1HC) placed upstream of the viral filtration membrane reduced flux decay.

Hydroxyapatite

For purifying Antibody drug conjugate (ADCs);

Lin (US 216/0051695) discloses purification of an anti-HER2-IgG1 conjugated to maleiminocapyroyl drug using HA with a wash and elution buffer that includes sodium phosphate buffer. 

Nadkarni (US 15/778, 290 published as 15/778,290) discloses purifcation of an ADC in monomer form using HA that employs a low sodium phosphate buffer wash and a high or tradient increasing sodium phosphate buffer. 

Ion Exchange

Cation-Exchange (CEX): CEX removed antibody aggregates. Antibody aggregates are more tightly bound than the antibody monomer and elute after the main peak. Elution conditions must be optimized to elute all the antibody monomer but not the aggregated antibody. Blank (Bioseparation 10: 65-71, 2001)

Mixed Mode Chromatography: 

Mazzola (US 2005/0031627) discloses purification of an antibody-DM1 conjugate with ceramic hydroyapatite column as well as ion exchange chromatography. Dai (US2007/0048314A1) discloses purification of an antibody-agent conjugate to an mixed mode ion exchange chromatography. Snyder US 12/769460) discloses a method of purifying an antibody-agent conjugate wherein the agent comprises the label phycoerythrin using a contacting step comprising an increased concentration of salt and elutuing step comprising increasing the concentration of the buffer. 

TNFR-Fc Fusion:

Banerjee (US 2017/0152298) discloses TNFR-Fc fusion prtoein purificaiton using mixed-mode chromatogrdaphy in flow thorugh mode. In another embodiment, Banerjee disclsoes purifying TNFR-Fc fusion protein which includes the steps of affinity chromatography, mixed mode chromatography in bind and elute mode. In a separate emboidment, the process for purifying TNFR-Fc fusion protein includes the steps of affinity chromatography, HIC in bind an delute mode and then mixed mode chromatogrpahy in bind and elute mode. 

Filtration (See also filtration for antibody purification)

Yamaguchi (US2010/0190965) discloses a method of separating immunoglobulin monomers from aggregates using cross-flow filtration with an ultafiltration membrane. The immungolobulin concnetration is 1-150 g/L and cross flow filtration is performed using an UF membrane having a MWCO of 100k or more and less than 500k so that immunoglobulin monomers pass through the UF membrane with a permeability of 80% or more while achieving a factionation performance in which the permeability ratio of immunoglobulin dimers to monomers that pass throguh the UF membrane is 0.20 or less. 

Tangential flow filtration: Dai (WO/2007/024536) discloses a process for preparing a conjugate comprising a cell binding agent chemically coupled to a drug by contacting the cell binding agent with a bifunctional crosslinking reagent to covalently attach a linker to the cell binding agent, subjecting the first mixture to TFF to prepare a first mixture of cell bindin agents haivng linkers bound thereto and conjugating a drug to the cell binding agents having linkers by reacting them with a drug  in a solution having a pH of about 4-9 to preapre a second mixture comprising a cell binding agent chemically coupled to the linker to the drug, free drug and reaction by products and subjecting hte second mixture to TF to purify the cell binding agents chemically coupled through the linkers to the drug.

Isotypes as Particular Types of Antibodies Purified and Techniques Used:

Antibody Fragments: (for Single Domain Binding Polypeptides and Nanobodies see “domain antibodies” in outline)

–Advantages of Using Antibody Fragments: 

Small genetically engineered immuoglobical constructs are being developed industry wide for a growing range of in vivo applications. Examples include Fab, F(ab’)2, single-chain (sc) Fv, bis-scFV, diabodies, minibodies and single domain antibodies. Their small size potentially tives them access to tissues that are poorly accessible by intact antibodies; rpaid clearance form blood and neotargeted tissues, lower immunogenic response and eye-drop inhalent or oral administration. (Gagnon, “Minibodies and Multimodal chromatography methods” BioProcess Interational Feb 2010).

–Purification by pH gradient

Slough (US Patent Application 15/566231, published as US 2018/010007) discloses the isolation of antibodies that lack an Fc region using a pH gradient, such as pH 6-2.1. As binding to Protein A by antibody fragments that lack an Fc region is known due to binding to the human VH3 variable framework subclass of the V regions, multimeric spcies will have increased avidity for Protein A because they have mroe VH3 regions, requiring a lower pH for elution. In one partciular embodiment a dsFv antibody was purified via a pH gradient elution. 

–Purification using CEX-AEX:

Spitali (US 2013/0184439) discloses purification of an antibody fragemtn from a periplasmic cell extract using a first CEX and a second AEX step. 

—-with Reducing agent (glutathione) See also purification of particular contaminants and the section there entied “disulfide bond variants”

Slough (US 15/538282, published as US 20170342105) discloses purifying of a Fab by expressing the Fab in E coli into the periplasmic space, clarification, addition of glutathione, loading onto CEX with loading, washing and eluting in the presence of glutathione followed by AEX equilibrated with buffer containing glutathione, washing with Tris buffer containing glutathione to recover the Fab in the flow thorugh of the loading and washing steps. 

Immunoglobulin homodimers:

(Christei (US 16/497,788, published as US 20200033363) discloses a method of producing/screening/purifying immunoglobulin homodimers which include identical light chain variable regions (VL) monomers which includes expressing each light chain variable domain monomer in a bacteriophage, performing an affinity screening step and a dimerization screening step. In one example the light chain variable domain (VL) maby be a VL from a kappa light chain. In another example, the VL may be a VL from a lambda light chain. The affinity screening step includes contacting the immunoglobulin homodimers with an antigen and selecting those immunoglobulin homodimers that bind specifically to the antigen. The dimerization screening step includes determining the stochiometric ratio (relative numbers of VL monomers and antigen molecuels in the immunoglobulin-antigen complex) of immunoglobulin monomer to antigen. The stoichimoetric ratio may be determind using for example measuring the molecular weight of proteins such as by size exclusion chromatography coupled with multi-angle laser light scattering (SEC MALLS). The MWs of the complex, immunoglobulin alone and antigen alone can then be compared. Variants of the light chain variable domain (VL) monomers can be geenrated for example by modification of V and J gene segments through general mutagenesis. Sequence of VLs for the V and J gene segments can be obtainable for example from public databases. The VL monomers may be capable of dimerizing to form immunoglobulin homodimers. In one example, the method includes the use of a phage display technique which includes expressing multiple copies of a VL monomer joined to a filamentous pahge protin via a linker protein. The linker protein can include a sufficient number of amino acids to allow sufficient confomational flexibility to enable VL monomer dimiriziation. 

Bispecific Antibodies (see outline)

Catalytic Antibodies (Abzymes)

Natural Abzymes: from the sera of patients are usually polyclonal in origin and are products of different immuno-competent cells. Their purification is one of the most complicated aspects of their study. Affinity chromatography on immobilized hapten usually produces homogeneous monoclonal Abzs, but polyclonal Abs from patients are very heterogeneous and their affinity for an immobilized substrate analogs can differ by several orders of magnitude. The first step of purificaiton should spearate Abs efficiently from other proteins so that a fraction with high affinity for potential substrates is obtained. In the first step, affinity chromatogrpahy on adsorbents bearing anti-IgG, anti-IgM or anti-IgA Abs is often used in conditions to remove non-specificaly bound proteins. However, absdorbents bearing immobilzied Abs are often not convenient for Abx purificaiton and protein A-Sepharose is an optimal resin for the first step because it has a higher affinity for Abzs than for Abs without catalytic activity. (Nevinsky, “Natural Catalytic Antibodies – Abzymes, Chapter 19 from Catalytic Antibodies, 2005, pp. 505-569). 

IgA (see outline)

IGM

–Chromatography methods:

Gagnon (WO2009/149067) discloses that IgMs have characteristics that can limit the application of standard antibody purification tools. IgMs tend to be less soluble than IgGs and more susceptible to denaturation (precipitation, including aggregate formation) at extremes of pH, and under conditions of low conductivity. IgMs are generally tolerant of high salt concentrations, which can be useful for IEX, but are susceptible to denaturation from exposure to strongly hydrophobic surfaces, which can limit the usefulness of HIC. Although IgMs can be eluted from modernately hydrophobic supports for HIC in a well definited peak at reasonably low salt concentraiton, IgMs will precipitate at the higher salt concentraitons that are preferred to support good capacity on moderately hydrophobic mediation. Because IgMs are typically more charged than IgGs, IgMs bind more strongly than IgGs to IEX and hydroxyapatite and often bind much more strongly than most contaminants. The large size of IgMs can be a challenge for purificaiton, due to slow diffusion constants, which can be a problem for porous particle based chromatogrpahy media dependent on diffusion for mass transport. Purification of clinical grade IgM can generally be acheived with 3 bind elute chromatography steps on hydroxyapatite, AEX and CEX. Omitting an affinity step is a positive contribution as well as avoiding intermediate diafiltration by using in-line dilution to load samples. 

Gagnon (WO2009/149067) discloses using a first chromatography step such as HA or IEX with the use of a nonionic polymer for removal of the aggregates of the protein product and then a step of combining a solubility enhacning additive such as a switterion, a urea compound and an alkylene glycose and a second chromatography step comprising the use of IEX. The method is particularly useful for purificaiton of IgM from a mixture. Buffers containing nonionic polymers to enhance aggregate removal can be introduced direclty into the zqitterion contianing compositions that enhance protein solubility and substantially avoid aggregate formation, thereby avoiding additional manipulations such as desalting, polymer removal or buffer exchange that could affect the yield and or quality of the purified protein product. 

–Affinity chromatography

—-Secretory component as the ligand:

Secretory component has high affinity for IgM (Bouvet, Scand. J. Immunol. 31, 437-441, 1990).

Jones (J Immunol Methods 1987 104(1-2): 237-43) discloses human secretory component bound covalently to sepharose 4B used as an affinity adsorbetn to isolate and purify polymeric immunoglobulin M from cell culture supernatants.

——–Isolation of secretory component itself:

Corthesy (US2015/0056180) discloses that secretory component itself can be isolated by attaching a hexa-histidine tag to the SC which can further include a cleavable linker so that the tag may be cleaved off prior to use.

Kraehenbuhl (WO98/20141) discloses purification of (SC(IpaB)6xHis by Ni2+ chelate affinity chromatography).

Underdown (Immunochemistry, 1977, 14, pp. 11-118) discloses that SC can be isoalted from human whey by affinity chromatography on IgM sepharose adsorbents.

IgG

–IgG2:

(Dillon, US14/363735) discloses a method of produing an IgG2 antibody enriched for at least one of the several IgG2 structural variants which differ by disulfide connectivity in the hinge region, comprising contacting a solution containing a recombinantly produced IgG2 antibody with either a strong cation exchange (SCX) matrix, an IgG2 affinity matrix or a Protein L matrix and eluting two or more first elution fractions from the matrix where the IgG2 antibody solution elutes off the matrix as two or ore separate forms corresponding to two or more IgG2 structural variantswhich differ by disulfide connectivity in the hinge region. 

IGE

Procedures for different Isotopes: It has been shown theat the constant domain fragment (Fc) of IgE is more sensitive to low pH/high salt conditions that the IgG1 Fc. These results indicate that purification and formulation strategies commonly used for IgG antibodies are not amendable to IgE. “GE HealthcareLife Sciences, Application note 28-9870-45AA “Better guidance in antibody therapeutics process development using differential scanning calorimetry” 2011, pp. 1-4.

Anti-amyloid beta and anti-infectious disease IGs

–By CEX:

Hofbauer (US14/213,585 and US2014/0271669) (see also Gnauer (US 2017/0218051) discloses using CEX for purification of immunoglobulin enriched in anti-infectious disease immunoglobulins uisng CEX by providing a plasma composition comprising IgG immunoglobulins, binding the IgG to a CEX, eluting a majority of the bound IgG immunoglobulins form the CEX in a first elution step and then eluting the remaining IgG bound to the CEX using a second cation elution buffer having a higher pH or conductivity from the first elution step. Hofbauer discloses that anti-brain disease related protein IgG antibodies present in human plasma such as anti-amyloid beta bind to CEX with higher affinity than do most IgG antibodies present in the plasma. By using an extreme ionic strengh conditions for the second elution (e.g., with a solution containing 2 M sodium chloride), the targetted IgG is obtained.

–By Thiophilic Affinity Chromatography

Relkin (US 2012/0183527) discloses using theiophilic chromatography to detect and isolate antibodies that are specific for various forms of amylid, including monomeric, oligomeric and fibrillar amyloid proteins. The resulting population of amyloid beta specific antibodies is hereogenous but binds to amyloid beta with high affinity. The thiophilic chromaotgraphy is used to enrich the antiboedies prior to detection. Under the proecdure a biological sample such as plasma is contacted with a thiophilic resin to bind an anti-amyloid antibody present in the sample, the antibody is eluted from the resin at a non-denaturing pH to form an eluate (an advantage over say Protein A), and the eluate is contacted with an amyloid anitgen to form a complex between the amyloid anitgen and the anti-amyloid antibody and this complex is detected as by ELISA. In an advantageous embodiment, a chaotropic wash step is performed to reduce the signal assocaited with non-specific and low-avidity anti-amyloid antigen binding. 

For purification of other proteins from milk (which may involve same or similar steps) see Non-Antibody purification

For purificaiton of antibodies and other proteins from Inclusion bodies see outline

Separation of Immunoglobulins from Milk:

Particular Schemes:

–Filtration: 

—-MF-UF:

Doring (US14168586) discloses  a process for obtaining immunoglobuilins by subjecting colostral milk from days 0-7 to thermal treatment, skimming the cream and then subjecting the skimmed milk to microfiltration to obtain a first retentate and first permeate. The first permeate is then subjected to ultrafiltration to produce a seocnd permeate which contains lactose and minerals and a second retentate which contains the immunogloubilons, IgG1, IgA and IgM. The separation of the immunoglobuilins form the second retentate may then be performed according to processes known from the art such as through AEX. 

Hensgens (US 12/992701 and WO2009/139624) teaches a process for the preparation of an S-IgA enriched milk fraction by (a) separating fat from the milk at a temperature of below 55C, (b) separating the casein rich and S-IgA fraction by subjecting the low fat milk having a pH exceeding 5.5 to microfiltration using a porous membrane having an average pore size within a range of 0.1-0.45 micrometers where the casein is in the retentate and the S-IgA is contained in the permeate and (c) further concentration of the S-IgA by concentration as by UF.

Hobman (WO01/03515) discloses a method of purifying immunoglobuilin from colostrum (mammary secretion from a cow during the first 4 days postpartum) by taking a skim colsotrum stream and subjecteing it to MF to produce a permeate rich in colostral serum proteins and a serum deplete retentate enhacned with casein. In a preferred embodiment the permeate may be futher processed by US and/or DF the skim milk may be pasteurised for microbail hygiene prior to the MF. 

—-Cross-flow filtration:

Kothe (US 4644056) teaches a method of preparing immunoglobulins including secretory IgA by processing milk accompanied by preciptiation of the caseins characterized in that milk are acidified to a pH of 4.0-5.5, subjected to cross-flow filtration with a mean pore sieze of 0.1-1.2 um and the low molecular components removed by means of further cross-flow filtration.

Separation of Antibodies from Plants

While mammalian cell culture is the predominant expression system fo mAb production, yeilding mAbs with proper folding and glycosylation, the substantial resource requirements and viral clearance present challenges for this production system. Plant on the other hand offer a unique production platform for mAbs and have shown in recent years that they are able to express high levels of antibody. (Fulton, J Chromatogr A, 1389 128-132 (2015)

Particular Schemes:

DF- Charged polyelectrolyte preciption (+ charged to remove impurities) -Protein A/HIC:

(Fulton, J Chromatogr A, 1389 128-132 (2015) discloses a process of purification of a mAb against Ebola GP1 expressed in the plant N. benthmiana which includes a DF step and a charged polyelectrolyte precipitation. Polyethlenimine (PEI, a positively charged polyelectrolyte was used to precipitate acidic proteins and leave IgG in the supernatant. 

Conditions, such as solution pH, ionic strenght, solvent compoisition, termpature, light and electric field. These polymers are referred to as stimuli response, enivornmentally sensitive, “intelligent” or “smart” polymers. a large number of biologically active molecuels may be combined with intelligent polymer systems. The biomolecules may be conjugated to pendant groups along a polymer backbone or to one or both terminal ends of the polymer. In etiehr case, the samrt polymer may be a soluble polymer a graft or block copolymer, aphysically adsorbed polymer on a solid substrate, or a polymer change segment within a hydrogel. Hoffman, A.S., “Intelligent Polymers in Medicine and Biotechnology”, Macromol. Symp., 98, 645-664 (1995).

pH-sensitive affinity macroligands (AML): 

Eudragit S-100: is a commercially available high molar mass copolymer of methcrylic acid and methyl metharylate which precipitates sharply below pH 4.5 and has been used for the purfication of monoclonal antibodies. A drawback is that the pH of precipitation is outside the stability range of many proteins (Hilbrig, J. Chromatography B, 790 (2003), pp. 79-90).  

N-acryloyl-p-aminobenzoic acid: This AML is water soluble above a pH of 4. Below a pH of 4 the acidic residues on the polymer backbone become neutralized (protonation) and hydrophobic interactions between the uncharged polymer backbones enforce aggregation which causes the AMl to precipitate reversibly when the pH is reduced below this value.

For a polyelectrolytes see outline. 

Hoffman (US 5,998,588) discloses stimuli responsive polymers coupled to recognition biomolecules such that once a ligand is bound, it may also be ejected form the binding site by stimulating one or more conjugated polymers to cause ejection of the ligand. In one embodiment, a first stimulus precipitates the polymer while a second stimulus is ued to release the ligand. Hoffman further teaches that stimuli include pH.

Thermosensitive affinity macroligands:

Thermosenstivie polymers in aqueous solutions become soluble (hydrophoilic) at low temperatures but are insoluble (hydrophobic) at high temperatures. This hydrophilic/hydrophobic transition is reversible and occurs at a transition temperature, which is determiend by a primary structure of polymer.

Temperature sensitive smart polymers have been studied extensively. Many polymers exhibit a cloud point (CP) or lower critical solution temperature (LCST) in aqueous solutions. Examples of thermally sensitive polymers howing LCST bheavrio in aqueous solutions that have alchol groups are hydroxpropyl acrylate, hydroxpropyl methlcellulose, hydroxpropyl cellulose, hydroxyethyl cellulose, methycellulose and poly(vinyl alcohol) derivatives. Examples of thermally sensitive polyers showing LCST behavior in aqueous solutions that have N-substituted amide groups include poly(N-substiuted acrylamides), ply(N-acryloyl pyrrolidine), poly(N-acryloyl piperidein), poly(acryl-L-amino acid amides), and poly(ethyl oxazoline). Hoffman, A.S., “Intelligent Polymers in Medicine and Biotechnology”, Macromol. Symp., 98, 645-664 (1995).

poly-N-alkylacrylamides: such as poly-N-isopropylacrylamide (NIPAM) precipitate if a certain critical solution temperature (CST) is reached. The polymer exhibits thermally reversilbe soluble-insoluble changes in response to teemperature changes across lower critical solution temperature (LCST), typically at 32 C in an aqueous solution. It is present as a clear homogeneous solution at 4C and changes to compact form rapidly above the LCST and precipitates within a few seconds. The precipitate can be easily separated and collected by centrifugation.  

Conjugation of Protein A to Theremosensitive Polymers

–poly(NIPAAm)-protein A conjugates:

Chen (“polymer-protein conjugates” 6055 Biomaterials 11 (1990)) discloses a conjugate of protein A with poly(N-isopropylacrylamide) (polyNIPAAm) and used in the separation of human immunogammaglobulin. In the separation prcocess, the poly(N-isopropylacrylamide) protein A conjugates binds to the immunoglobulin and the complex can tehn be separated by precipitation upon heating aove the lower critical solution temperature of the complex. 

Hoffman, A.S., “Intelligent Polymers in Medicine and Biotechnology”, Macromol. Symp., 98, 645-664 (1995) discloses a thermally induced affinity precipitation process to recvoer IgG form oslution by using a poly(NIPAAm)-protein A conjugate.

–NIPAM

Kamihira (Biotech. & Bioengin. 75(5), 2001) pp. 570-580) dicslose immobilzing antibodies as ligands to NIPAM.

Co-precipitation of non-IgG contaminants with positvely chartged polymers parallels the selectivity of AEX. These reagents selectively co-precipitate acidic host cell protins, DNA and varous cell culture additivies. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

Positively charged soluble polymers (polyallylamine, polyarginine) as well as certain divalent cation (ethacridine, metal ions) have been employed to co-precipitate negatively charged contaminants form antibody preparations. (Gagnon, US 14/555060)

Cationic polyelectrolytes (polycation polyelectrolytes): include chitosan, polyvinylpyriddines, primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers (Moya, US 8362217), polyarginine (PLA, including poly-L-arginine hydrochloride and poly-L-arginine sulfate, polylysine (e.g., poly-L-lysine hydrochloride, polyomithine, polyvinylguanidine (poly-(vinyguanidine), polymethylacrylamidopropyltrimethylammonium chloride, polyvinylbenzyltrimethylammonium chloride and polyhistidine (Fahrner, 2008/0193981).

Specific Types of Cationic Polyelectrolytes

Chitsan is inexpensive and available in a highly purified form that is low in heavy metals, volatile organics and microbial materials. It is a cationic linear polymer of beta-(1-4) linked D-glucosamine monomers generated by the chemical deacetylation of chitin and has been used fro the removal of nucleic acid and endotoxin and the flocculation of cell debris of yeast and bacteria. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010).

Fahrner (US2008/0193981; see also WO/2008/091740) teaches a method of purifying antibodies by adjusting the acidity or salt concentration of a mixture containing the antibody, adding a positively charged polycation polyelectrolyte such as polyargiine (PLA) to the mixture whereby a precipitate is formed comprising the positively charged polycation polyelectrolyte and impurities, and separating the the precipitate from the mixture comprising the antibody. 

Polyamines: Polyamines are cationic polyelectroytes with multiple repeating amine functional groups. Due to this nature of these amine functional groups, they are protonated under a wide pH range and thus pisitively charged. The polyamines can be used to precipitate negatively charged proteins in solution. Ma (J. Chromatography B, 878 (2010) 798-806) discloses polyamines, which are cationic polyelectrolytes, which can be used to precipitate hamster ovary (CHO) host cell proteins (CHOP). If precipitation is carried out at the appropriate pH, at which the most basic monoclonal antibody of interest has net positive charges, the polyamines selectively precipitate and remove the negatively charged CHOP impurities leaving the monoclonal antibody intact and soluble in HCFF.

Jaber, Moya, etc. (US8,691,918) disclose separating a target molecule by providing a sample containing the target molecule, contacting the sample with a soluble stimulate responisve polymer comprising a cationic polyelectrolyte backbone which can be a polyallylamine (BzMPAA) backbone modified with a hydrophobic groups attached to the backbone to form a complex of the polymer and one or more impurities, adding a stimulus to the sample to precipitate the complex out of solution to thereby separate the target molecule from one or more impurities.

Ethacridine lactate: Lester discloses a method for purifying a desired polypeptide such as an antibody by adding to the broth 6.9-diamino-2-ethoxyacrdine lactate (ethacridine lactate) to precipitate host cell impurities under conditions wherein the majority of polypeptide remains soluble and then separating the desired polypeptide.

PVP and Co-Polymer of PVP (Polyvinyl pyridine-containing polymers):

 Moya (US 8,362,217 and and WO2008/079280A1) (See also Moya, US2008/0255027 and US 13/747495) discloses a Ph dependent polymer such as poly(4-vinylpyridine-co-styrene) which has an affinity for a desired biomolecule such as an antibody in the insoluble state. In a first step, a mixture is harvested from cell broth, then the mixture is conditioned to the correct pH (e.g., below 5) to maintain the polymer in solution, then the mixture conditions are changed to cause the polymer to precipitate out of solution by altering the pH (e.g., 7.0) and the antibody recovered such as by elution. 

Moya (US2013/0123476) also disclosess a method purifying a biomolecule of itnerest from negative charged impurities by adjusting the pH of the mixture, adding a cationic polyelectrolyte selected from PVP and a co-polymer of PVP, creasing the pH of the mixture to precipitate the polymer and form a supernatant wehre the precipitated polymer binds the impurities and recovering the supernatant containing the biomoecule of interest. Examples of copolymers of PVP are poly(2 vinylpyridine)(P2VP) or poly(4 vinylpyridine) (P4VP), polyvinylpyridine-co-styrene (PVP-A), polyvinylpyridine-co-butyl methacrylate (PVP-BMA) as well as other primary, secondary and tertiary amine containing polymers. These polymers are soluble at a pH lower than about 6-7 and are insoluble at a pH greater than about 5-7. When in solution, these polymers will precipitate if the pH is raised above this critical range (pH=5-7). Such a cationic polyelectroyte can be added to a fluid containing the biomolecule either as a solution in a carrier liquid at a pH of about 4.5 or as a solid particulate in which the fluid is either modified to a pH of about 4.5 either before, during or after the introduction of the polymer to it so that the polymer binds all negatively charged impurites such as cells, cell fragmetns, nucleic acids, ciruses, endotoxins, etc. The biomoecule of interest does nto interact with teh polymer given its postive net charge due to its basic pI. The pH is then raised to 5-7 or more if desired and the polymer precipitates out of solution, carrying with it all the impurities as well as any excess polymer. The precipitate can then be easily removed by centrifugation or filtration resulting in a purified biomolecule containing solution.

Anionic polyelectrolytes (polyanion polyelectrolytes)  include copolymers of acrylic acid, methacrylic acid and methyl methacrylate (US 8362217), polyacrylic acid (PAA), polystyrenesulfonic acid (PSS, poly(4-vinylbenzenesulfonate metal soat)), polymethacrylate (PMA), polyacrylamidomethylpropanesulfonate (PAMPS), carboxymethylcellulose (CMC), maleic anhydride-styrene copolymer (MAS, maleic anhydride-vinylmethyl ether copolymer (MAVE), polyaspartate, polyglutamate, dextrane sulfate, pectin, alginate and glycosaminoglycans such as chondroitin sulfate, heparin/heparan sulfate and hyaluronic acid (Fahrner 2008/0193981). 

Precipitation of antibodies using anionic polyelectrolytes

McDonald (Biotechnology and bioengineering, 102(4), 2009) discloses antibody precipitation using polyelectrolytes such as poly-acrylic acid (PAA) and polystyrenesulfonic aicd (PSS). McDonald also evaluted the impact of solution pH, ionic strengh and polyelectrolyte molecular weight on the degree of presicpitation using the polyelectrolytes. At a given pH, increasing solution ionic strenght prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH, there was an increase in impurity clearance. Increasing polyelectrolyte MW allowed the precipitation to be performed under conditions of higher ionic strenght. 

Moya (US 2008/0255027) also teaches a method for purifying a biomolecule such as an antibody by providing the mixture at a set of conditions, adding one or more polymers which can consist of anionic polyelectrolytes and precipitating the polymer and bound biomolecule out of solution by changing the set of conditions in the mixture followed by spearating the precipitated polymer and bound biomolcule from the mixture.

Fahrner (US2008/0193981; see also WO/2008/091740) teaches a method of purifying antibodies by adjusting the acidity or salt concentration of amixture containg the antibody, adding a negatively charged polyelectrolyte such as polyacrylic acid (PAA) and polyvinylsulfonic acid (PVS) and their anions, polyacrylate and polyvinylsulfonate, separting the antibody-polyelectrolyte precipitate from impurities and resuspending the precipitte in an aqueous solution. 

Van Alstin (WO2010/082894) discloses a method of isolating a biomolecule such as an antibody by mixing an aqueous sample containing the antibody with a negatively charged polymer in the presence of a salt under conditions such that the polymer selectively complex and flocculate the antibody to form a mixture of precipitate including the antibody. The antibody precipitate is then separated from the aqeuous liquid and resuspended in buffer. Negatively charged, carboxyl group containing polymers. Specific polymers include polycarboxylic acid (PCA), polyacrylic acid or carboxymethyldextran polymers of various molecular weights. Other polymers include CM cellulose or CM starch. In preferred embodiments, the flocculate (precipitate) containing the biomolecule is formed in the presence of the polymer at around neutral pH and relatively high conductivity (e.g., >50mM NaPhosphate). 

Precipitation of impurities using anionic polymers

Itol (US 15/748736, published as US 2019/0010188) discloses a method for purifying an antibody which includes preapring a composition which includes the antibody so that the omcomposition includes an anionic polymer at pH lower than the pI of teh antibody and removing an inpurity insolubilized by teh antionic polymer such as by filtration.