In the 1970s, chromatography was also found to be useful in the separation and purification of plasma proteins, particularly for the final purification of gamma globulin after separation from the plasma by Cohn or modified Cohn methodologies. Immunoglobulin purified from normal human plasma has proved effective in the treatment of various diseases when adminsitered intravenously.

The ion-exchangers consist of ligands of strong anionic type (quaternary ammonia-ethyl: AQE) and weak anionic type (diethmlamino etyl: DEAE), or strong cationic (sulphopropyl: SP) and weak cationic (carboxymethyl: CM). The ligands are covalently immobilised on insoluble supports or matirices such as silica (ceramics), acrylic (polyacrylamides, polystyrene), carbohydrate (cellulose, dextran, agarose). Those formed by destran or agarose are most efficient and most used. (Ristol Debart (EP1225180B1))

Affinity Chromatography

Preparative steps can be used to enrich a particular isotype. For example, protein A chromatoaphy can be used to enrich a mixture of immunoglobulins for IgG (US5180810).

Protein A – AEX: Merlino (Giorn Batt. Virol. Immun. LXXVIII, 77-85, 1985) (abstract only) discloses loading serum on a Protein A sepharose column, IgG3 is recovered as they do not bind to the Protein A. IgG of the other subclasses are eluted, equilibrated at pH 6 and loaded on a AEX wehre IgG4 is retained and then recovered by lowering the pH wehreas IgG1 and IgG2 pass through the column and can be recoved.

Multi-column Immuno Affinity columns

Typically, protein A columan can only be loaded to about 30-50% of their maximum binding capacity, because at higher loads, product is lsot in the breakthrough. This means that much of the resin resmins unused, leading to a production cost increase. A straight forward solution is to capture teh mAb that is breaking through in a second column. This very simple principle is the basis of a novel multi-column chromatography capture process, teh Capture SMB or 2-column periodic countercurrent chromatography (2C-PCC) process. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Bataille (US 15/125483, published as US 2017/0073396) discloses using a an IgSelect gel from GE Healthcare (ligand si from CAC, BioAffinityCompany) which specifically binds the Fc fragments of human IgGs and  Capture Select FcXL affinity gel from Life Technologies which specifically binds the CH3 domain of the 4 human IgG subclasses on 4 columns, controlled sequentially an an automated system followed by diafiltration concentraiton of the eluate. Elution with the IgSelect was carreid out with 0.1 M glycine solution, pH 3. 

Burton (US 8,198,407) discloses sequential protein isolation and purificaiton from a biological sample such as plasma by affinity chromatography which is conducted using ligands or ligand support complexes that selectively and specifically bind to proteins in the sample. The ligands/support complexes are contacted sequentally in a predetermeind order with the sample to sequentially bind a protein from the sample. For example, the target protein may be fibrinogen, immunoglobulins and albumin such that albumin is siolated from the plasma prior to or after the IgG. 

Naylor (US 2005/0042772) discloses methods for depletion of proteins suchas albumin, IgG and a third abundant protein such as fibrinogen from a plasma sample using affinity columns such as Protein A columns and anti-fibrinogen columns. The removal of abundant proteins facilitates the detection of lower conentraiton proteins. 

Ion Exchange Chromatography Generally

Generally: Cohn Fraction (I+)(II)+III ethanol precipitation (Tanaka, Braz J Med Biol Res 2000 (33)37-30) coupled to column chromatography.

Teschner (Vox Sang, 2007 (92):42-55) have described a method for production of a 10% IVIG product in which cryo-precipitate is first removed from pooled plasma and then a modified Cohn-Oncley cold ethanaol fractionation is performed, followed by S/D treatment of the intermediate, ion exchange chromatography, nanofiltatration, and optionally ultrafiltration/diafiltration.

Ion Exchange – Filtration (activated carbon) – Affinity: Hou (US4639513) discloses a method for producing IVIG comprising passing prefiltered human plasma through an ion exchange column (IgG passes through the column and other components remaining behind), the IgG stream then passes to an affintiy column. Hou (US 4,639,513 see also EP0180766) also discloses a process for isolation of IgG from plasma where after a first dilution step using deionized water to insolubilize lipis, plasma is subjected to filtration with preferred materials such as activated carbon to remove micron particles such as lipids and then the filtrate is passed to IEX where IgG passes through the column while other large molecule proteins remain absorbed to the column. Proteoytic enzymes are then removed from the IgG by affinity chromatography. 

Cohn Fractionation – CEX – AEX: Sarno (US 5, 177,194)  teaches starting with a plasma protein fraction such as the Fraction I+II+III precipitate from a Cohn fractionation proecdures (this is usually obtained by subjecting a conventional cyroprecipitate supernatant to cold ETAOH at pH 6.9. In addiition to the immune serum globulins, the Cohn Fraction I+II+III contains fibrinogen, various lipoproteins, several proteins involved in the hemostatic and fibrinolytic systems and numerous mino components) or in the alternative a Cohn Fraction II+III or Cohn II fraction. The first step involves suspending the plasma fraction in water at acidic pH, pH 4.5-5.5, precipitating out the non-serum globulin proteins from the suspension using a precipitant such as pEG and recovering the clarified immune gloublin containing liquid (as by centrifugation and filtration), inactivating viruses, contacting the solution with a CEX to remove the virus inactivating agent and other non-serum globulin contaminants, eluting the immune serum globulins, UF, and then contacting with AEX. 

Anion Exchange  See outline (also see virus inactivation)

Cation Exchange See outline (see also virus inactivation) 

Multi-Ion Exchange (in series)

Laursen (WO 99/64462) dcloses a process for purifying IgG from a crude IgG containing plasma protein fraction using AEX (DEAE Sepharose resin in flow through mode) and CEX (CM Sepharose in bind and elute mode) connected in series. The eluted IgG fraction from the CEX is desalted by UF/DV against a buffer that includes sorbitol. 

Hydrophobic Interaction Chromatography (HIC)

Kothe (US7,041,798) discloses fractionation of plasma or serum into at least one albumin fraction and one immunoglobuilin fraction by HIC using an incremental salt gradient such as ammonium sulfate buffer. The separation is based on the interaction of hydrophobic domains of the proteins with hydrophobic groups on the media. Under physiological conditions, the hydrophobic groups of the proteins are not freely accessible so that binding to the HIC does not take place. By adding the salt, the hydrate sheath of the proteins is decreased so that the hydrophobic domains are available for HIC. The first fraction from the HIC is an albumin fraction and is similar in composition to a supernatant II/III of the Cohn alcohol fractionation. It contains albumin, transferrin, antithrombin II and alpha-antitrypsin. All immunoglobuilins are contained in fraction 2 which is similar to the composition of a past II/II obtained by cold thenaol precipitation. A lipoprotein fraction remains bound to the media and can be eluted by reducing the ammonium sulfate concentration to 0 moles/L.  Fractions 1 and 2 obtained from the HIC can be processed further into therapeutically usable plasma protein solutions by known methods. For example, IVIG can be produced form the fraction 2 using anion exchange chromatography, optional virus filtration, treatment with actanoic acid, CEX and the usual concentrating, filtering and sterilizing steps. 

In combination with Expanded/fluidized Bed chromatography  See definitions for definition of EBA

Lihme (US2007/0299251) discloses a process for the isolation of protein(s) from a solution by applying the solution to eitehr a packed bed or expanded bed comprising an adsorbent which comprises a functionalised matrix polymer carrying a plurality of covalently attached funcitonal groups comprising an aromatic or heteroaromatic ring system and one or more acidic groups. The absorbent also comprises a particle density of at least 1.5 gml and a mean volume particle diameter of at most 150 um. EBA offers a robust process comprising fewer steps and can be scaled up to industrial scale without any significant considerations regarding increased back pressures or breakdown of the process due to clogging of the system which often is a problem when using packed bed columns. The source of the protein for the procedure maybe by croy-poor plasma, sueprnatant I, supernatant II+III, supernatant IV-1, supernatant Iv-4, resolubilised cryo-preipitate, resolubilised fraction 1, resolubilised fraction II_III, resolubilised frraciton IV-1, resolubilised fraction IV-4, resolubised fraction V. 

–Caprylic acid – HIC (on fluidized bed): 

(Chtourou, US14/131944) discloses a method for preapring human polyvalent immunoglobulins from blood plasma by removing protein contaminants with caprylic acid to obtain a solution free of proteases and a fluidized bed chromatography step which may be of of the ion exchange, affinity or mixed-mode type. 

Ultrafiltration/diafiltration: 

The last manufacturing steps of IVIG before the formulation is an ultra/diafiltration step or washing out unwanted salts present in the IgG preparation and for adjusting the desired protein concentration (Ahrer (J.Membrane Science, 274 (2006) 108-115).