See also precipitation agents used,  See also Chromatography for removal of aggregates from plasma

Removal of Anti-complmentary Aggregates from IVIG Preparations

Isolated IgG preparation have marked anticomplementary activities (ACA). It has been shown that the components responsible for these activities are aggregates of IgG which form either spontaneously or as a result of the isolation procedure. These anticomplementary aggregates have been shown to be harmful such as anaphylactic shock. Accordingly varous solutions have been proposed to covercome the problem (Anderson, US 2003/0143222).

In Cohn’s plasma protein fractionation method, IgG is obtained from Cohn fraction II or II+III. However, the recovered IgG may contain a significant amount of aggregates because IgG tends to aggregate spontaneouly during storage of human blood plasma or tends to aggregate in contact with alcohol or other chemical during the fractionation procedure, or also tends to aggregate during lyophilization. If Igg containing aggregates is adminsitered intravenously, the aggregates may exert an anticomplement effect to cause serious anaphylactoid reacitons such as hypotension, chill and deverl. Methods used to remove aggregates from IgG include (1) the polyethyene glycol precipitation method (i.e., PEG is added to an aqueous IgG solution containing aggregates and the resulting precipitate of aggregates is separated by filtration), (2) the method of dissociating aggregates by reducing the pH of an aggregate contianing aqueous IgG solution to a low value such as 4 and allowing to stand so as to dissociate the aggregates, (3) or a combination of (2) above and addition of a slight amount of a proteolytic enzymes, (4) the removing aggregates by adsorption to an ion exchange resin, (5) adsorption to an adsorbent such as activated charcol 6) gel filgration method such as by using sephadex G-200 and (7) the membrane separation method such as by using a porous polymethyl methacrylate membrane which permits the passage of IgG monomer and dimer but blocks the passage of aggregates (Suzuki US 5,219,999). 

Four basic procedures exist for further processing of immunoglobulin obtained by Cohn fractioantion to prevent IgG aggregates. These are 1. enzymatic degradation by plasmin or pepsin, 2. chemical modification of the IgG molcule by beta-propiolactone or by cleavange of the interchain disulfide bridges by sulfonation or reduction and akylation, 3. slective elimination of aggregates by precipitation with PEG and hydroxyethyl starg (HES) and 4. adsorption of aggregates by DEAE gels such as Sephadex C50 Hou, EP0180766).

Enzymatic degradation

–Pepsin: US 3,966,906 describe treating a crude gamma globulin fraction with pepsin to aggregate IgG and reduce anticomplement activity.

See also (DE1148037), (US3966906, pepsin).

A drawback is that the immungolbulin Fc binding capacity is lower than for native immunoglobulins (Anderson, US 2003/0143222)

—-Stabilization of pepsin treated IgG preparation with PEG: has been disclosed (WO86/06993).

–plasmin: treatment of gamma globulin with human plasmin results in cleavage into 3 components of about 50k MW. When sufficiently low levels of plasmin are used, only about 15% of the molecules are cleaved, with 85% remianing as intact gamma globulin which show little anti-complement activity (J, Hinman, et al. Vox Sang 13:85 (1967).

Reduction and alkylation of SH groups: has also been disclosed to solve the problem of high ACA activity (US 3,902,262).

Precipitation with PEG: Prior patents directed to selective elimination of aggregates by precipitation with PEG in clude Coval (US 3,763,135),  (US 4,093,606) (US4,165,370) and Eibl (US4,276,283).

Acid Treatment:

–Caprylic (octanoic) acid: 

Kothe (US 5,164,487) discloses a method for manufacturing IVIG free of aggregates, vasoactive substances and proteolytic enzymes form a starting materials treated with 0.4-1.5% vy volume of octaonic acid and then chromatographed on an ion, CEX or hydrophobi matrix. 

–Low pH: 

When dissociation of IgG aggregates and denaturation of IgG mononmers contained in IgG preparation obtained using ethanol fractionation was examined at various pH values, treatment for 60 min at 28C at pH 3.8-4 was found to be optimum for obtaining such monomeric IgG. (Uemura, Tohoku J. Exp. Med. 1983, 141, 337-349. 

Other methods to remove aggregate removal

 DEAE Gels: Selective adsorption of aggregates by DEAE gels such as SephadexC50 after Cohn fractionation are reported by Curling, J.M et al. Vox Sang 33:97 (1997) and Suomela H., et al. Vox Sang 33:37 (1977), Ppier (US 3,664,994), Kandi (US4,136,094), Yokoo (US4,256,631) and Zuffi (US4,272,521).

Coppola (WO00/67789) discloses a method for producing an immunoglobulin preparation by subjecting an immunoglobulin solution to conditions which enhance the formation of immunoglobulin aggregates as by incubating the immunoglobulin solution at a pH of about 5.8 to about 8.0 at temperature of about 4-27 for at least 6 hours, then removing the aggregates as by filtration.

Reducing Amidolytic Content (e.g., FXI/FXIa)

Studies have shown that adminsitration of high levels of amidolytic activity may result in unwanted thromboembolic events. The EDQM (European Directorate on the Quality of Medicines & Health Care) provides for steps that have been shown to remove thrombosis generating agents. Emphasis is given to the identification of activated coagulation factors and their zymogens and process steps that may cause their activation. Serine proteases, generically known as coagulation factors, are integral components of both the contact activation and tissue factor pathways of the coagulation cascade. Upon a stimulus of the coagulation pathways, erine protease zymogens, which are inactive enzyme precursors, become activated proteases that catalyze the activaiton of the next protease zymogen, resulting in an activation cascade. Thsi coagulation cascade culminates in the activaiton of Thrombin (Factor IIa) and Factor XIIIa, which funciton to convert Fibrinnogen (Factor I) into Fibrin (Factor Ia) and cross-link fibrin to form a fibrin cot, respectively. The contact activation pathway, also known as the intrinsic coagulation pathway, begins with the activation of Kallikrein and Factor XIIa (FXIIa) from Prekallikrein and Factor XII, respectively. The activated serine proteiase FXIIa cleaves Factor XI (FXI), covnerting the zymogen into Factor XIa (FXI1), an active serine proteinase which participates in the subsequent activaiton of Factor Xa (FXa).  (Teschner (US2013/0058961)

Use of Cation Exchange to reduce levels of amidolytic activity: See Cation Exchange