Conditions/Parameters for CEX

Bind and Elute Mode:

If a CEX is used at pH 6.5-6.6, IgG binds to the resin whereas negatively charged protein fraction is unbound. An almost pure IgG can be isolated in one step this way. Falksveden “Ion exchange and Polyethylene Glycol Precipitaiton of Immunoglobulin G” ).

Hodler (WO/1995/018155) disclsoes a method of producing anti-D immunogolobulin of the IgG subclasses  1 and 3 from human plasma where plasma plasma from Rh-negative blood of donors sensitized to rhesus factor D or a plasma fraction containing anti-D IgG is subjection to CEX with carboxymethyl groups, the anti-D IgG being bound to the adsorbent, then washing with solution at a pH in the range of 5-8 and conductivity 2-4 mS/cm and subsequently eluting with pH 6-8 and conductivity 2-4 and then finally ocncentrating the anti-D IgG.

Teschner (US 2013/0058961) discloses methods for reducing the amidolytic activity (e.g.,FX1 and FX1a) and anti-complement activity (ACA) content of immunoglobulin composition through the use of CEX which includes eluting the IgG from the CEX with an elution buffer that includes a pH of at least 7.5 and a conductivity of at least 1.5 mS/cm to form and eluate that includes a leading porition and lagging protion and collecting the leading portion of a CEX eluate. The majority of the amidolytic activity present in the composition elutes off the resin in the lagging porition of the eluate. 

Flow through Mode:

Maneg (US 16/320,900, published as US 2019/0161533) discloses a prcoess for reducingthe properdin content of a properdin-containing IgG composition which includes CEX under conditions of pH and conductivity where properdin is bound to the column to obtain an IgG coomposition having a reduced preoprdin content. In one embodiment, the CEX is contacted withthe IgG composiiton at a pH in the range of 5-6 and a conductivity in the range of from 16-30 mS/cm and IgG is recoved in the flow-through.. 

Mintz (US2012/0294847) disclosea a method for remvoing a thrombogenic agent from an immunoglbulin solution by pvodiign the solution in the range of higehr than 3.81 to lower than 5.3, contacting with a support comprising immobilized negatively charged groups (CEX) and collecting the unbound fraction I (flow through).

CEX in Combination with other Purification Techniques

CEX-Virus Inactivation: Andersson (US6,835,379) teaches producing IgG from plasma by removal of albumin resulting in an IgG fraction and adsorbing IgG to a CEX and collecting the adsorbed IgG fraction which is subjected to virus inactivation. 

-Plasma-citrate-viral inactivation – CEX:

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. In one embodiment a weak CEX in bind and elute mode Toyopearl CM-60C resin is sued. 

CEX-AEX: 

Laursen (WO/1999/064462) discloses a process for preparating IgG from a crude immunoglobulin containing plasma protein fraction which includes the steps of preparing an aqueous suspension of crude immunoglublin containing plasma protein fraction, adding a water soluble protein precipitant to said suspension to cause precipitation of the non-immunoglobulin G proteins, applying a clarified IgG containing supernatant to CEX, washing out protein contaminants, eluting the IgG from the CEX, UF/DF, virus inactivation and then AEX and subsequently CEX, washing and leuting from the CEX and then UF/DF.

(Olas, Clinical Exp. Immun. 140, 2005, 478-490, human serum IgA prepared from plasma pool (Cohn fractions II and III) by ethanol precipitation and cation-exchange, followed by anion-exchange.)

Sarno (US 5,177,194) discloses a process for purifying an immune serum globulin fraction from a crude plasma protein fraction which includes precipitating non-serum globulin proteins from an aqueous suspension of the crude plasma protein faction, adding a virus inactivating agent, absorbing the immune serum globulins onto a CEX and subjecting the eluate to UF and then conctacting the concentrate with an AEX to absorbe non-serum globulin contaminants. 

PEG-CEX-PEG-AEX

Falksveden (US3,869,436) discloses a method of fractionating plasma proteins by removing blood corpuscles and cell fragments from the plasma by centrifugation, precipitating the globulins with PEG having a MW of about 6000 in a concentration of 10-15% by weight and at a pH of 6.5-8.0 at room temperature, centrifuging out the prcipitate which contains practically all the globulins (the solution contains albumin) and then dissolving the precipitate at pH 5.8 and centrifuging out undissolved fibrinogen, plasminogen and IgM, adsorbing the globulins in the clear solution on a carboxy methyl dextran CEX at pH 5.8, eluting the CEX to obtain the absorbed globulins including IgG, precipitate the eluate with additional PEG at pH 6.5-8.0, centrifuging and dissolving the precipitate and adsorbing all the globulins in the solution excluding IgG on an AEX at pH 6.6, cooling the IgG and precipitating with ethanol in a concentration of 25% by volume at -5 to -10C.

Lars-Gunnar (GB1344340) disclose a method of fractionating plasma proteins by precipitating the gloublins with PEG, dissolving the precipitate and centrifuging out undissolved fibrinogen, plasminogen and IgM, then adsorbing the globulins onto a cation exchanger and eluting with sodium chloride followed by precipitation with PEG and dissolving the prcipitate in buffer followed by adsorbing the globulins excluding IgG on an anion exchanger, cooling the IgG and precipitating with ethanol, separating the precipitate, followed by another ethanol step and finally dissolving in glycine.

Precipitation (ETOH)-Silicon Dioxide-CEX-AEX-NF-UF-DF:

Bruckschwaiger (US2013/0224183) disclsoes a method for preparing an enriched IgG composition by precipitating from 95-100% of IgG and A1pI from a Cohn pool in a first precipitation step by adding ethanol to a final concetnration of between 20 and 30% at a pH from 5-6 to form a first precipitate and first supernatant, suspending the first preciptiate to form a first suspension, treating the first suspension with finely divided silicon dioxide, separating the soluble porition of the suspension from the insoluble porition, where the soluble porition contains immunoglobulins and the insoluble contains A1pI, fibrinogen, Factor H and IaIp, binding the immunoglobuilins to CEX, eluting the Igs and contacting the eluate with an AEX and recvoering the IgGs that do not bind to the resin.

Bruckschwaiger (US2013/0224184) also disclose a method for prearing an enriched IgG from plasma by adjusting the pH of a cryopoor plasma fraction to about 7, the ETOH to about 25% at -7C–9C, spearating liquid from precipitate, resupsending precipitate, mixing finely divided SiO2 with the suspension, filtering the suspension, washing the filter press with at least 3 filter press dead volumes buffer, combining the filtrate with the wash and treating the solution with a detergent, adjusting the pH to about 7 and adding ETOH to about 25% , spearating the precipitate and disolving it in a solution containing a detergent for at least 60 minutes, passing the solution through a CEX and eluting, passeing the eluate through an AEX and passing this effluent through a nanofilter and then UF/DF.

Teschner (US12/789,345 and US20100330071; see also 13/949565) discloses a method of the following steps: 1) Separation of cryoprecipitates: separate liquid and precipitate form plasma by centrifugation, 2) Obtain Supernatant of Fractionation I: mix precooled ethanol with the liquid form (1) to form a mixture, (3) separate liquid and precipitate as by centrifugation (4) Precipitate of Fractionation II+III: adjust pH and ethanol concentraiton of liquid from (3) (5) separate liquid and precipitate from the mixture by centrifugation (6) Extraction from Fractionations II and III Precipitate: resuspend precipitate of (5) & Fumed Silica Treatment and Filtration: (7), mix silicon dioxide with suspension form (6) and obtain a filtrate by filtration, Fractionation of Precipitate G: (8) mix detergent and cold alcohol with the filtrate of (7) and obtain a precipitate by centrifugation, Suspension of Precipitate G: (9) dissolve precipitate Cation Exchange chromatography: (10) pass solution after (9) through a cation exchange chromatography column and elute proteins absorbed on the column Anion exchange chromatography: (11) and eluate from (1) through anion exchange to generate an effluent Nanofiltration: (12) pass effluent through a nanofilter Ultrafiltration and Diafiltration: (13) pass nanofiltrate through an ultrafiltration membrane Formulation: (14) diafiltrate the ultrafiltrate against a diafiltration buffer to generate a solution having a protein concentration of about 20% (w/v) (15) sterilize solution from 14 by filtering through a filter of 0.2 um or less.

Teschner (WO2013/033042A1) also discloses methods for reducing the amidolytic and anti-complement activity (ACA) contact of an immunoglublin composition by CEX where the Fraction II precipitate may be res-suspended in water or a low ionic strengh buffer and subjected to CEX. Typically, the pH of the re-suspended Fraction II precipitate is 5.2 and conductivity is low, ytpically no more than 1 mS/cm. The Fraction II suspension is then filtered to remove non-solubilized materail prior to loading onto a CEX. To elute the immunoglubilins, the CEX is then contacts with an elution buffer having a conductivity of at least 15 mS/cm (e.g., with a salt concentration of at least 150 mM NaCL) and a pH of greater than 7. In one embodiment, after loading a clarified Fraction II suspension onto the CEX, the resin is washed with a buffer have a sufficiently low conductivity such that the immunoglbuilinbs are not eluted form the resin and a Ph between 5.1-5.9. The pH of the elution buffer is greater than 75. and the ocnductivity of the eltuion buffer is at least 10 mS/cm.

Precipitation (PEG)-Virus inactivation -CEX-UF-AEX: Sarno (US5,177,194) disclsoes a multi step process for purifying an immune serum globulin fraction from a crude plasma prtoein fraction by adding a virus inactivating agetn to the clarified immuen serum globulin contining liquied, absorbing the immune serum globulins onto a CEX and subjecting the eluate to UF to concentrate the immune globulins and separate low MW species and contacting the concentrate with an AEX to absorb non-serum globulin contaminants. 

Disolving fraction I+II+III or II+III plasma fraction-Precipitation (caprylic acid)-AEX-Inactivate viruses (solvent/detergent) – CEX – DF-AEX-DF/UF

Park (US 15/123925, published as US 2017/0015732) discloses purification of an immunoglobulin by dissovling fraction I+II+III of II+III protein fraction followed by precipitation such as with carpylic acid, removing the precipitate and concentrating the supernatant followed by AEX in flow through mode, inactivating viruses as with solvent/detergent followed by CEX to remove the solvent/detergent, DF/UF and EX in flow through mode and then DF/UF.

Reduction of Particular Impurities

Amidolytic content (e.g., FXI/FXIa/proteases):

Teschner (US2013/0058961) discloses methods for reducing the amidolytic content of IgG immunoglobulin compositions (Factor XI and/or Factor XIa) by contacting a plasma derived immunoglobulin composition with a CEX under a first solution having a pH of no more than 6 and conductivity of no more than 11 mS/cm to bind the IgG and at least a fraction of the FXI and/or FXIa and eluting the IgGs from the resin by using an elution buffer having a pH of at least 7.5 and doncutivity of at least 15 mS/cm to form an eluate having a leading and lagging portion and collecting the elading porition of the eluate separately form the lagging porition of the eluate, whereas the leading porition of the eluate comprises no more than 80% of the eluate.