Anion Exchange

Almost pure IgG can be isolated in one step by AEX on DEAE ion exchangers at pH 6.5-6.6. Under these conditions, about 90% of the plasma proteins are negatively charged and consequently bound to the AEX. Positively charged, residual protein, mainly conisting of the IgG fraction is left unbound. (Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” , from “methods of plasma Protein Fractionation” by J.M. Curling, Academic Press 1980).  

Baumstart (Archives of Biochemistry and Biophysics 108, 414-522 (1964) dicloses separation of gamma globulin in high yield from undialyzed normal human serum by a rapid two stage procedure employing DEAE-Sephadex at pH 6.5. The chromatography on DEAE cellulose appears to be the method of choice for the samll scle or noncommercial production of gamma globuilin from human serum with Cohn alcohol precipitation being preferred for large scale. Web (Vox Sang 23: 279-290 (1972) also disclose a 30 minute preparative method for isolation of IgG from human serum with DEAE Sephadex. 

(US6093,324, subjecting plasma to macroporous anion exchange). (US7186410, purification and a single anion-exchange carried out at alkaline pH, thereby enabling the immnoglobulins to be retains on the support). (US7553938, caprylate and/or heptanoate ions and one or more anion exchange resins).

Bertolini (WO/1998/005686 discloses a method for the purification of immunoglobulins from plasma which comprises subjecting the plasma to macro-porous anion exchange. Preferred starting materials are plasma or plasma fractions obtained by the Cohn fractionation process such as Cohn Supernatant I (fibrinogen depleted plasma) or solubilised and clarified Fraction II + III. Where the starting material is plasma which contains lipoproteins, preferably a pretreatment step where lipoproteins are removed prio to the chromatographic factionation step.

Chtourou (US7,186,410) discloses praring immunoglobulin (IgG, IgA and IgM) from plasma by prepurifcation and a single AEX carried out at alkaline pH. 

Laursen, (US7138120, “process for producing immunoglobulins for intravenous administration and other immunoglobulin products”). Almost pure IgG can be isolated in one step by ion exchange chromatography on DEAE-ion exchangers at pH 6.5-6.6 and suitable ionic strength. Under these conditions, about 90% of the plasma proteins are negatively charged and consequently bound to the anion exchanger. Positively charged, residual protein, mainly consisting of the IgG fraction is left unbound. (If a cation exchange material is used instead, an opposite effect obtained, leaving the negatively charged protein faction unbound (Falksveden, “Ion exchange and polyethylene glycol precipitation of Immunoglobulin G, p. 95).

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

Plasma-citrate-viral inactivaiton -AEX:

Zurlo (US 2007/0049733) discloses large scale alcohol free plasma fractionation which produces human IgG product. The process employs sodium citrate in two initial fractionation steps, followed by diafiltration to remove the sodium citrate. For the first fractionation, sodium citrate is added to plasma by gentle stiring such that the supernatant contains virtually of the IgG. For the second fractionation, additional soidum ictriate is added to the spernatant. The resulting supernatant contains virtually no IgG and virtually all the IG and other serum proteins are now found in the paste. Next remal of sodium citrate is done by diafiltration, viral inactivation is accomplished such as by solvent/detergent (S/D). Finally purificaiton is done by chromatographic purificaiton techniques. A strong AEX Toyopearl AQE-550C resin is performed in flwo through mode

Anion Exchange in combination with other techniques

Cohn Fraction I+II+III – Glycine extraction + AEX: Levy (WO03/034982) teaches a method for purification of immune globulins from Cohn’s fraction I+II+III or II+III prepared from plsma by precipitation of the paste at 20% ethanol and pH 6.7-6.8, followed by glycine extraction and the AEX. 

–Cohn Fractionation – caprylate acid -AEX:   (see also fatty acids used as precipitation agent)

Alred (US20030152966) discloses a process for the purification of antibodies from human plasma by addition of caprylic acid and a pH shift to pH 5.0 to 5.2. A precipitate of contaminating proteins, lipids and cprylate forms is removed while antibodies remain in solution. Sodium caprylate is against added. Anion exchange is used to obtain an exceptionally pure IgG.

Buchacher (US 7,553,938) discloses preparing a preufied virus inactived antibody prepraration from a starting solution by adjusting the pH to about 4.6-4.95, adding caprylate, diafiltrating the filtered solution, applying the filtered solution with at least one AEX. 

Lebing (6,307,028) discloses the purification of antibodies from plasma by addition of caprylic acid where the precipitate of contamination proteins, lipids and caprylate forms are removed followed by anion exchange chromatography using two different resins to obtain a high yield of IgG.

Lebing (US5,886,154; see also Lebing, “Properties of a new intravenous immuhnoglobuiolin (IGIV-C, 10%) produced by virus inactivation with caprylate and column chromatography, Vox Sanguinis, 2003, 84, 193-201) discloses supsending a composition containing precipitated immunoglobulins such as a fraction II+III past, dissolving the immunogloublins into solution by lowering the pH 3.8-4.5, preferably 4.2 by addition of an acid like acetic acid, adding a source of caprylate ions and adjusting Ph 5.0-5.2, remove precipitated proteins, lipids and caprylate by filtration, addition of further caprylate and passing the solution through two AEX.

Parkkinen (WO2005073252) teaches a process for preparing a purified Ig preparation by subjecting a crude Ig solution to cprylic acid treatment, removing protein aggregates and viruses from the Ig solution and subjecting the Ig solution to AEX.

–PEG treatment – AEX: 

Hirao (US 6,159,471) discloses starting with a gamma globulin fraction and subjecting it to low concentration of PEG, recovering the supernatant, treating the suspension with PEG have a MW of from about 1 to 10k, centrifugation and then treating the supernatant thus separated with a high concentration of PEG, centrifugation and then disoolving the gamma-globulin containing fraction and contacting it with an AEX to recover the non-adsorbed fraction.

Anion-Cation exchange: 

(Bertolini (US2011009244) discloses Large scale separataion of alpha-1 proteinase; US7879800).

Burnouf-Radosevich (US6069236) using a series of chromatographic separation steps without ethanol precipitation to produce plasma derived IgG. In one embodiment, the process includes a deslating step, AEX, viral inactivation, CEX.

Mamidi (WO 00/76534) discloses producing IVIG substantially free of viruses by starting with a Cohn Fraction II + III or Cohn Fraction II paste or in the alternative the Fraction II + III paste can be subjected to a preliminary washing procedure for form Fraction II + IIIs (done by suspending Fraction II + III precipitate in cold water and adding sodium phosphate solution) heat treating a gamma globulin solution for viral inactivation, PEG fractionation, and then a second viral inactivation using solvent-detergent which can be carried out prior to or following AEX, inacitvation of viruses using a a solvent detergent solution containing a mixture of tri-n-buyl phosphate (TNB) and polysorbate 80, then CM Sepahdex C0-50 (weak CEX) was added to the solution which was mixed and filtered. The resin contining the adsorbed IgG was washed and eluted with 1.4 M sodium chloride and the eluate was clarified, concnetrated and diafiltered with cold water. D-sorbitol was added. 

Tanaka “High quality human immunoglobulin G purified form Cohn fractions by liquid chromatography” Brazilian J of Medical and Biological Research 2000, 33: 27-30) discloses using Q-Sepharose FF AEX followed by CM-Sepharose FF CEX and then gel filtration for purificaiton of intravenous IgG from F-I+III+III or F-II+III pastes preared by the Cohn method. 

–Precipitation (salt)-AEX-CEX (low capacity):

Zurlo (US Patent Application 17/560,163, published US 2022/0204556) discloses a method of isolating an antibody from a solution such as plasma from contaminants such Factor IX or Factor XI which includes the steps of adding a salt to the solution to generate a supernatant and a precipitate, dissolving the precipitate and appplying the dissolved precipitate to a frist ion exchange media such as AEX in flow-through mode and applying the first flow-through to a second IEX such as CEX also in flow through mode where second exchange media has a limited capacity such that greater than 70% of the content of the target protein is recvoed in the second flow-through. The size of thsi small or low capacity CEX is selected so that it is near or slightly greater than the amout or capacity for breakthrough of a contaminating protein found in the flow-through of the AEX. The inventors believed that the contaminating proteins displace any IgG that may temperarily bind the the CEX media. Careful selection of the amount/capacity of the CEX provides efficient removal of contaminating protein while also providing high yields of IgG in the flow-through fraction. 

–Fraction I+II+III or fraction II+III – precipitation (PEG/ caprylic acid or ammonium sulfate) -AEX- solvent/detergent – CEX-AEX

Maneg (US 16/320,900, published as US 2019/0161533) disclsoes preparing an immunogloublin composition from a plasma dervied fraction that includes Cohn fraction I/II/III or Kistler-Nitchmann fraction A+1 which includes the steps of  resuspending the Cohn fraction I/II/III fraction under conditions to adjust the conductivity to at least 1 mS/cm to obtain a suspension contining resolubilized IgG, IgM and IgA, subjecting the resolubilized immunogloublin to precipitation with as with octanoic acid and removing contaminating protection form the suspension as by filtration (the impurity depleted immunoglobulin composition typically has an IgG content 8.5-94% by weight, IgA content 3-9% by weight and IgM 3-9% by weight at this point), subjecting the impurity depleted immunoglobulin composition to AEX under conditions of pH and conductivity to substantially bind IgM and IgA and optionally IgG to the AEX (so IgG enriched immunoglobulin may be obtaiend int he flow-through fraction and/or by eluting the IgG), and then subjecting the IgG enriched immunoglobulin to CEX under conditions where properdin is bound and recovering the IgG in the flow-through fraction and/or by eluting the IgG. . Following the procedure the IgG content was at least 45 g/l, 95% by weight, a properdin content of not mroe than 001 ug/mg and a IgG polymer content of not more than 0.05%.  In a specific example, Maneg outlines the procedure as follows: To obtain the Cohn fraction I/II/III a cryopprecipitation step was first perfromed to separate factors such as Factor Viii, von Willebrand Factor and Fibrinogen by adjusting temperatrue under gentle stirring at 2 C such that the cryoprecipitate remains undissolved in the thawed plasma which could be separated by centrifugation, from the sueprnatn of the cryoprecipitation step the Cohn fraction I/II/III was precipitated by ethanol precipitated (temp -5C, 20% ETOH), the Cohn fraction I/II/III precipitate which includes all the immunoglobulins (IgG, IgA, IgM) was resuspended with sodium acetate buffer, pH 4.8 and treated with octanoic acid and calcium phosphate treatment, the filtrate was diafiltered and subjected to virus inactivation at pH 4.0 and then subjected to AEX using a macroporous POROS 50 HQ AEX with the IgG enirched composition obtained in the flow-through fraction (the IgM/IgA) enriched fraction could also be obtained by eluting by increasing conductivity). The IgG enirched flow through fraction was then subjected to CEX with the flow through and wash fraction collected and further processed by nanofiltration. 

Park, (US 15/123925, published as US 2017/0015732) discloses preparing an intravenous immunogolbulin by dissolving with distilled water an immunoglobulin containing plasma protein fraction I+II+III or fraction II+III followed by precipitation using polyethylene glycol (PEG), carpylic acid or ammonium sulfate, , removing the precipitate, filtering the supernatant and subjecting the concentrated filtrate to AEX such asdiethylaminoethyl (DEAE) in flow through mode to remove caprylate and other plasma proteins, treatinghte recovered fraction with asolvent/detergent to inactivate viruses, following by CEX and then dialyzing and/or concentrating the eluate from the CEX and subjeting the eluate to AEX in flothrough mode followed by filtering the recvoered fraction through a virus filter and dialyzing and/or concentrating the filtrate. 

–Fraction II Paste –dissolvaing –dialyzing/concentration –AEX –solvent/detergent –CEX –dialyzing/concentration

Son (US 15/123,869, published as US 2017/0022248) discloses a method for purifying an immunogloublin by dissolving a fraction II paste, followed by filtration to obtain a fraction II solution, dialyzing/concentration the fraction II solution and subjecting this to AEX in flow through mode, treating the recovered fraction with a solvent/detergent to inactivate viruses followed by subjecting the fraction to CEX to remove the solvent and/or the detergent and thrombotic substances, dialyzing/concentraiton the eluate from the CEX and then filtering.

–AEX-CEX-AEX-CEX (connected in series):

Laursen (US 2001/0051708; see also WO 99/64462) also discloses a process for purifying IgG from crude plasma by adding a water soluble precipitant to cause precipitation of non-immunoglobulin G proteins, applying the IgG to an anion exchange resin and subsequently a cation exchange resin, performing a dia-UF and performing a virus inactivation step. Laursen (US6281336) also disclose purifying IgG from a plasma fraction which includes adding a water soluble non-denaturating protein precipitant (e.g., PEG, caprylic acid and ammonium sulphate) to a crude immunoglobulin containing plasma protein fraction to precipitate non-immunoglobulin G proteins, recvovering the IgG containing supernatant and applying the supernatant to an anion exchange chromatography followed by cation exchange chromatography. Laursen (US7138120 and US2001/0051708) also teaches a process for purifying immunoglobulin G from a crude plasma protein fraction in which AEX operated in flow through mode and CEX operated in bind and elute mode are preferably connected in series. The use of two serially connected chromatography columns makes the operation more practical in that there is no need for intermediary steps of collecting the IgG containing fraction between the two IEX. Laursen (EP2272870) also discloses preaparing an aqueous suspension of crude immunoglobulin containing plasma protein fraction, adding a water soluble non-denaturating protein precipitate to cause precipitation of non-immunoglobulin G proteins, recovering the clarified IgG supernatant and applying it to AEX and subseqeuntly a CEX, washing the protein contaminants from the CEX with a buffer having pH and ionic strenght sufficient to remove the contaminats from the resin without causeing elution of IgG, eluting IgG from the CEX with buffer ahving a pH and ionic strenght sufficient to cause elution of the IgG, performing DF/UF and adding a stabilzing agent, virus inactviation, applying again to an AEX and then CEX.