IgM are immunogloublins secreted upon the body’s first contact with an antigen. They are the first type of immunoglobuilins released by plasmocytes. The presence of IgM in the blood indicates a current infection (Schmitthaeusler (US7771725).

IgM by nature is a vigorous activator of complement after the binding of antigens. Thus unspecific anti-complementary activity of denatured IgM molecules is far more dangerous to patients than denatured IgG molecules.

Initial Purification of IgM by Cohn Fractionation

The initial purification of human IgM solutions has been carried out by classical Cohn plasma fractionation methods or its well known modification. Using cold ethanol precipitation processes the IgM fraction is recovered in fraction III or fraction I/III (also called B or B+1). Starting from fraction III or I/III the following methods have been descirbed for purification of protein solution enriched in IgM. (Moeller US2013/0052208).

Isolation from Cohn Fraction III precipitate: Plasma IgM can be recovered from the byproducts of the production of IVIG. An example of such a byproduct is Cohn fraction III precipitate. The IgM is most easily solubilized from the Cohn fraction III precipitate by 20 mM sodium acetate. Other pleasma proteins are similarly solubilized along with the IgM. (Brown US 14.476,559)

Treatments with Various Agents

B-propiolactone

Pentaglobin® described in EP 13901 is a commerical example of an intravenously tolerable IgM preparation. This immunoglobulin preparation which is treated with 0.01 to 0.15% of beta-propiolactone to make it intravenously tolerable, contains, in addition to 10% of IgM, 80% of IgG and 10% of IgA. (Moller, US 5,190,752). 

Moller (US5,190,752) discloses an immunoglobulin preparation that contains at least 50% by weight of IgM, has low anticomplementary activity. The prepration is manufactured form an IgM containing fraction such as Cohn Fraction III obtained from plasma by treatment with an ion exchanger, eluting the exchanger with a saline gradient or pH gradient and gelfiltraiton where treatment with beta-propiolactone, precipitation with PEG 4000 and optionally heating are conducted either before or after chromatography. 

Beta-propiolactone is a well known chemical used in sterilization steps in order to inactivate viruses which are potentially present. However, it is very reactive which causes the chemical modification of proteins and there is also substantial loss of the anti-viral and anti-bacterial activities of the immunoglobulins. (Moeller US2013/0052208).

–Colloidal silica – AEX – Beta-Propiolactone: 

Stephan,( US4,318,902; EP0013901 equivalent in German) discloses a process for the preparation of IgM by treating an IgM containing fraction obtained by conventional fractionation (ie.., starting material can be Cohn fraction III  from blood plasma or serum with beta-propiolactone. Prior to the treatment, the IgM containing protein can be freed of lipids by treatment with colloidal silica gel and with crosslinked dextrans or diethylaminoethyl cellulose.  

–AEX-Beta-Propiolactone – UVC light irradiation: EP035255 described the preparation of an IgM concentrate for intravenous application with a reduced anti-complementary activity by using AEX, bet-propiolactone, UVC light irradiation and an incubation step at increased temperature. 

Octanoic acid (also known as Caprylate Acid)

–Octanoic acid (mixing with a vibrating agitator or stirring or vigorous mixing): 

Moeller (13/655,649 and US2013/0052208) describe a process for the preparation of an IgM immunoglobulin from plasma fraction by providing a plasma fraction adn mixing it with a C7 to C9 carboxylic acid such as octanoic acid with a vibrating agitator to precipitate containating proteins and then separating the precipitated proteins from the solution to yield the IgM containing immunoglobulin composition. 

Ng (Vox Sang 1993, 65: 81-86) discloses solubilization of a fraction III past with acetate buffer followed by stirring with 1.5 (v/w) octanoic acid, pH 4.66, for 4 h. Precipitate was then removed by centrifugation and supernatant was clarified by passed through filter.

Perosa (J. Immunological Methods, 128 (1990) 9-16) discloses a two step procedure for purification of IgG, IgA and IgM by deluting serum with acetate buffer and then (1) adding caprylic acid dropwise with vigorous mixing, centrigugation and then filtering the supernatant folowed by(2) addition of ammonium sulfate. The preciptiated Ig is collected by centrigutation.

–Octanoic acid – protease treatment: 

Rentsch (EP0835880, see also US 6,136,312) describes the preparation of an IgM containing protein solution for intravenous application by using octanoic acid precipitation followed by protease (pepsin) treatment ti redyce the anticomplementary activity. 

–Octanoic acid and AEX: 

The preparation of proteins solutions enriched in IgM without chemical modification by beta-propiolactone has been described.

Chtourou (US7,186,410) describes prepurification by precipitation of lipidic and proteic contamics using known agents such as octanoic acid, tricalcium phosphate or bentonite and a single AEX at alkaline pH thereby retaining immunoglobulins on the support. 

Miles (EP0345543) discloses starting with fraction III, suspending it in sodium acetate and adding caprylic acid. After aging at 5C for aobut 16 hours, the insoluble impurities are removed by centrifugation and filtration. The filtrate was was adjusted to 0.16 M sodium potassium phosphate at pH 6.5 and the IgM absorbed on an AEX such as Q Sepharose. 

Moller (US5,410,025) discloses  starting with a fraction such as Cohn II/III or III that contains immunoglobulin which can be dissolved in buffer and then eliminating most impurities by preciptiation with 0.5 to 5% octanoic acid at a pH of 4-6 and preferably 5 and then treating the solution at low conductivity with an AEX that binds most of the IgA and IgM. If the desired product is to contain IgA nand IgM, elution is carried out with a salt gradient that will elave about 10-20% of the IgM on the matrix. If the product is intended to contain IgA and IgM, the elution is carried out at a lower osmolarity and the IgM will remain absorbed onto th matrix. A pure IgM, extensively free of IgA, can be obtained by washing the AEX ahead of time with a buffer to elute the IgA before the IgM.

EP0413187 (the octanoic acid treatment is carried out by stirring for 15 in in order to remov lipids in the Cohn fraction III) and EP0413188 described subjecting a suitable protein solution to octanoic acid and AEX starting fro Cohn fraction III or II/III. 

–Octanoic Acid (by Stiring) – active charcoal – AEX – Beta-propiolactone:

Kotitschke (US 5,075,425) discloses a process for the preparation of immunogoglobulin suitable for intraenous administration from a human blood protein fraction containing IgG, IgA and IgM by adding octanoic acid, centrifuging, subjecting the supernatant to AEX (DEAE-Sephadex A), and filtering the solution. 

Moller (US 5,190,752) disclsoes preparation of IgM by starting with fraction that contains IgM such as Cohn Fraction III obtained by Cohn’s alcohol fractionation or the IgM fraction that occurs during the chromatographic isolation of IgG from blood is precipitated with 1-5% and preferably 2.5% caprylic acid. The supernatant that contains the IgM is applied to an AEX with DEAE, QAE or AMA groups for example, at a pH of 5.5-7.5. The IgM fraciton bound is eluted with a saline or pH gradient. Subjsequent to concnetraiton by UF, the IgM eluate is treated with 0.05 to 0.5 ml of beta-propiolactone per 100 ml of IgM solution. 

–Caprylic acid – PEG/AEX – virus inactivation: discloses preparation of globulin composition (56.8% IgM, 21.6% IgG and 21.7% IgA) from human plasma sarting with Fraction III from the Cohn/Oncley cold ethanol method or alternatively precipitate B from the method of Kistler and Nitschmann. The Fraction III paste is suspeneded in sodium acetate and caprylic acid is added. The filtrate which is adjusted with PEG, the IgM paste solubilized and virus inactivation. In a separate embodiment, AEX follows the caprylic acid treatmen

Miles (EP0345543) discloses starting with fraction III, suspending it in sodium acetate and adding caprylic acid. After aging at 5C for aobut 16 hours, the insoluble impurities are removed by centrifugation and filtration. The filtrate was adjusted to 7.5 PEG and the resulting IgM precipitate collected by centrifugation.

–Aerosil – Octoanoic acid beta-propiolactone -active charcoal:

Stephan,( US4,318,902; EP0013901 equivalent in German) teaches dissovling Cohn fraction III of human plasma, freeying it of lipids with 3#% aerosil, treating with DEAE-Sephadex A-50 and then treating with octanoic acid, then beta-orpiolactone. 

–Octanoic acid and Affinity chromatography:

EP0345543 describes a hihgly concentrated IgM preparation with at least 33% IgM for therapeutic use wehre octanoic acid precipitation is carried out by adding the actonoic acid and the isoagglutinins are removed by Synsorb affinity chromatography. 

Polyethylene glycol (PEG): It is known that IgM, IgA and alpha2 macroglobulin are present in Cohn fraction III. Precipitation of IgM is complete at 10% PEG and alpha2 macroglobulin at 20% while both IgA and IgG are partially soluble even at 20% PEG concentration. Based on this information, Wickerhauser (Vox Sang, 23: 119-125 (1972) prepared 2 sub-fractions: a 4-10% and 10-20% PEG fractions. IgM was present mainly in the first fraction and alpha2macroglobulin in the second fraction, while IgA was present in both. To prepare IgM from this, certain impurities were precipitated by zinc sulfate adn further purificaiton of the zinc sulfate supernatant was done by gel filtration on a Sephadex G-200 column. The IgM fraction was eluted about in the first quarter of the total protein eluate. Thsi fraction, recovered form the eluate by ammonium sulfate precipitation, contained 45% IgM at a yeild of 25%. It also contained about 22% IgA and a small amount of IgG, races of alpha2 macroglobulin of C-=3 complement and several other proteins.

Chromatography for the Purification of IgM

Affinity chromatography:

Brown (US 14.476,559) discloses where plasma IgM from a soublized Cohn fraction III precipitate (see above) is covalently bound to recombinant histidine tagged secretory component in vitro forming a secretory IgM within the protien mixture. The secretory IgM that is now tagged can be purified by affinity binding of the tag to an immobilized nickel or other divalent metal ion or other suitable binding moeity.

Corthesy (US 2015/0056180) disclosses preparation of human plasma IgM by affinity chromatography using CaptureSelect Human IgA resin using 3 different sources of plasma IgA; cryo-depleted plasma, resolubilised cold ethanol fractionation paste or a strip fraction from ACX. Secretory Ig was latter obtained by combining in vitro IgA with recombinant human secretory component.

AEX-heat treatment:

EP0413188B1 describes the preparation of an IgM enriched preparation for intravenous administration by using an anion exchange chromatography in order to reduce anti-complementary activity. Additionaly, a heat treatment at pH 4-4.5 at 40-60C was used to reduce the anticomplementary activity. 

Reduction of Non-specific Complementary Activity

Heat-treatment: Tsay (US 5,612,033 and EP0450412A1) disclsoes mild heat-treatment (40-62D)  in a solution having acid pH (preferablye 4-5) for at least about 10 minutes of IgM antibody concentrates diminishes non-specific complement activaiton without significant loss of normal immunologic effector functions. 

Commercial preparations

Pentaglobin: is a commercial immunoglobulin preparation which is enriced specifically for IgG (klingemann, Bone Marrow Transplant 1990 6(3) 199-202.

Purification of dimeric/polymeric IgA containing Secretory Component (SC)

Simon (US 15/205, 359, published as US 2016/0319039) discloses a method of purifying IgM with recombinant secretory component. According to the procedure a fraction III precipitate that is produced as a byproduct from production of IgM by ethanol fractionation of pooled human plasma is further purified by ion exchange adsorption purificaiton follwed by incubation with immobilized hydrolases to inactivate viruses and vasoactive substances. From 5-10% of plasma IgM is dimeric and polymeric IgA. The resulting dimeric IgAMis further coupled to recombinant secretory comonent that is produced by recombinant techniques. the coupling is accomplished by forming disulfide bonds under mildly oxidizing conditions. Dimeric IgM cotnaining both J chain and SC is again purified by ion-exchange and size exclusion chromatography and/or UF. The purified dimeric and polymeric IM  containing SC is iptionally stablized for example by the addition of human serum albumin to a final concentraiton of 5%.