See also Agents of Fractionation on

Commercial Sources of IVIG and their preparation

Intravenous infusion of immunoglobulins of the IgG class prepared from the pooled serum of humans (IVIG) is currently employed for treatment of several diseases. Commercial sources include Pentaglobin (Biotest Pharma GmbH which is a mixture of IgG, IgM and IgA, Intratect (Biotest Pharma GmbH), Gammagard S/D (Baxter Healthcare Corporation), Inveegam EN (Baxter Healthcare Corporation) and Carimune NF (ZLB Bioplasma Ag), all of which are IgG preparations containing only trace amounts of other immunoglobulin classes. While the majority of marketed IVIG preprations are composed of purified IgG antibodies, a more complete IVIG preparation composed of IgG, IgM and IgA formulated in about the same proportion as found in human serum is also avaiable. (Paul, US 11988761).

Hooper (“intraenous immunoglobuilins: evolution of commercial IVIG preparations” Immunol. Allergy Clin N Am 28 (2008) 764-778) sets out a nice chart of commercially available IVIG and their preparation. A brief summary is presented below:

Trade Name                Manufacturer               Manufacturing Procedure                                                                                                                Composition/Formulation

Gammagard S/D       Baxter HealthCare Cor    Cold ethanol, DEAE chromatography, S/D, pH 6.8, freeze dried         50 mg/ml; 8.5 mg/mL NaCl, 0.3 M glycine, 20 mg/mL PEG, 3 mg/mL albumin, 20 mg/mL glucose

Gammagard Liquid    Baxter HealthCare Corp   Cold ethanol, DEAE chromatography, S/D nanofiltration, pH 4.8        100 mg/mL; 0.25 M glycine

Intratect                   Biotest                           Cold ethanol, octanoic acid/calcium acetate treat, S/D. liquid             50 mg/mL; 0.3 M glycine

Vigam                      Bio Products Laboratory                                                                                                        50 mg/mL; IgG 20 mg/mL human albumin, sucrose, glycine, pH 4.8-5.1

Carimune NF            CSL Behring AG               cold ethanol, pepsin treat, nanofiltration, pH 6.5, freeze dried             30, 60, 90 or 120 mg/mL; 100 mg/mL sucrose, 1.2 mg/mL NaCl

Sandoglobuioin NF liquid  CSL Behring AG        cold ethanol, pepsin treat, DEAE Sephadex batch adsorption, NF, pH 5.3    120 mg/ml; 100 mM L-isoleucine, 120 mM L-proline, 80 mM Nicotimamide

Privigen                  CSL Behring AG                cold ethanol, octanoic acid fractionation, AEX, NF, pH 4.8                     100 mg/mL; 0.25 M proline

Vivaglobin              CSL Behring AG                 cold ethanol, fatty alcohol, DEAE Chrom, activated C, heated 10 h at 60, pH 6.4         100 mg/mL; 3 g/L NaCl, 0.25 N glycine  formulated for subcutaneous inject

Febogamma 5%     Instituto Grifols, SA            cold ethanol, PEG precip. IEX, 10 h at 60C, pH 5.5                               50 mg/mL; 50 mg/mL, D-sorbitol, <6 mg/mL PEG

Flebogamma 5% DIF   Instituto Grifois, SA       cold ethanol, PEG prec, IEX, pH 4 at 37C, 10 h at 60C, S/D, NF, pH5.5     50 mg/mL; 50 mg/mL, D-sorbitol, <3 mg/mL, PEG    4 virus liminations steps

Octagam          Octapharma                            cold ethanol, S/D, 24 h at pH 4, pH 5.5, liquid                                      50 mg/mL; 100 mg/mL maltose

Omr-IgG-am     Omrix Biopharmaceuticals Ltd  cold ethanol, S/D, 24 h at pH 4, pH 5.5                                                 50 mg/mL; 100 mg/mL maltose

Gamunex        Talecris Biotherapeutics            cold ethanol, carylate precipt, Q Sepharose-ANX, Sepharose chromatography, pH 4.2     100 mg/mL, 0.2 M glycine

Purification schemes for IVIG, Generally

Plasma-dervied intravenous immunoglobulin (IVIG) has become the major plasma product on the global blood products market. IgG is one of the most abundant plasma proteins and has a plasma concentration of 6.6 to 14.5 mg/mL. 

Immune globulin products from human plasma were first used in 1952 to treat immune deficiency. Initially, intramuscular (IMIG) or subcutaneous administration of IgG were the methods of choice. For injecting larger amounts of IgG necessary for effective treatment of various diseases, however, intravenous administration products with lower concentrated IgG (50mg/mL) were developed. Usually intravenous immunoglobulin (IVIG) contains the pooled IgG from the plasma of more than a thousand blood donors. 

Separation of immune globulins (IgG) from blood plasma depends upon early work by Edwin J. cohn. As found in US Pat 5,177,194, issued in 1993, one scheme in widespread use is the well known Cohn fractionation method, which is based on differential preciptaition using cold ethanol (Cohn, J. Am. Chem. Soc. 68, 459 (1946). 

US Pat No. 2,390,074 issued in 1946 to Cohn discloses use of alcohol, acetone and dioxane as precipitants in such factionation processes. Continued dependence upon alcohol as a precipitant is further demonstrated in US 6,893,639B2 issued in 2005 to joshua Levy.

In comparison with other methods of separation such as chromatography, fractional precipitation has the advantages of simplicity and applicability to mass production. It requires the proper control of (1) temperature, pH, concentration of protein, ionic strength and the dielectric constant. The purificaiton priciples of IVIG have not changed frammatically in the last two decades, with ethanol factionation still considered the main method (Buchacher, Bkotechnol. J. 2006, 1, 148-163).

Most IVIG were, until recently, extracted based on a complete ethanol fractionation process, comprising 3-4 precipitation steps and leading to the isoaltion of fraction II. Under those conditions, the precipitate is subjected to a lwo pH/low pepsin conentration treatment or to a combination fo CEX and AEX steps to reduce ACA andor remove protein contaminants. See Ethanol Fractional Precipitation  right hand panel.  Methods to improve IgG recovery include modified processes to initiate the IgG finishing pruificaiton steps from upstream fractions, avoiding the IgG losses associated with the preciptiations of Cohn fractions III and II. For example, in one procedure, the upstream fraciton II+III is subjected to modified ethanol/pH precipitation, S/D treatment and HIC. However, as these reusulted in 10% loss in IgG and required long processing time, ethe process was subsituted for two stages of caprylic acid treatment (to precipitate non-IgGcontaminants) and two AEX steps. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Other Fractionation Methods

Chromatography: See right hand Panel 

Other Procedures Involved in the Isolation of IVIG

Proteolytic enzymes, etc., intended to eliminate the aggregates of immunoglobulin polymers (liable to activate the complement system and to lead to anaphylactic reactions) (DE1148037), (US3966906, pepsin).

Viral Inactivation:  See right hand panel

Formulations of IVIG: 

IVIG products of different companies are either liquid formulations with an IgG content ranging from 5-12% or lyphilized products. The shelf life is between 1-3 years depending on the manufacture process and the formulation.

IVIG has enhanced stability in the liquid state at pH 4.25. There is a trend for modern IVIG preparation to be formulated as liquid at high protein concentrations (typically 100 mg/ml) within a low pH range (pH 4.5-5.5) in the presence of stabilizers like polyols (sorbitol), sugars (maltose, glocose) or amino acid (glycine, proline, isoleucine) and without sodium chloride addition. (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Applications

Doses: 

A standard dose of IVIG is 400 mg/kg/day x 5 days ((Knezveic-Maramica, Transfuaion, 43, 2003). 

IVIG are among the most complex plasma products in their mechanisms of action. Their clinical use encompasses replacement therpay in primary immune deficiencies (e.g., X-linked agammaglobulinaemia, severe combined immunodeficiency and hypogammaglobulinaemia) and in secondary immune deficiencies resulting from diesease or disease therapy and has gradually expanded in the last 15 years to the treatment of several inflammatory and autoimmune diseases (Burnouf, “Intravenous immunoglobulin G: trends in production methods, quality control and quality assurance. Vox Sanguinis (2010) 98, 12-28. 

Diseases:

Bacterial & Viral Infections:  

Intravenous immune globulin (i.e., IGIV or IVIG) has been used to treat bacterial infections. 

Buckheit (US2006/0292162) discloses a plasma or serum fraction derived form a mammal exposted to an inoculant which has been depleted of one or more HMW proteins in the plasma or serum for treatment of bacerial infections. 

Davis (WO 01/60156) teaches methods and compositions for treating viral and bacterail infections using nuetralizing antibodies produced in gotas. The goats are immunized with a virus (e.g., HIV) or bacterail (e.g., Staphylococcus, E. coli Streptococus). The blood of the animal is then collected and enriched for neutralizing antiobdies. 

Paul (US11/988761) discloses isolation and purifcation of a pooled immunoglobuilin prepration of a define class having catalytic activity. The immunoglobuilins can be isolated from mucosal secretions, saliva, milk, blood, plasma or serum by affinity chromatography using antibodies to human IgA, IgM or IgG, instead of harsh chemical treatments that result in loss of catalytic activity. The defined class may be IgA, IgM, GgG or mixtures thereof. The immunoglobuilins catalyze amide bond cleavage, peptide bond clevage and are useful in prevention or therapy of viral diseases like HIV or for treatment of bacterial infection. 

–Necrotizing enterocolitis: is a major casue of morbidity and mortality in preterm infants and those of low brith weight. An oral immunoglobulin preparation (73% IgA and 26% IgG) has been shown effective in reducing the incidence of necrotizing enterocolities in infants of low birth weight for whom breast milk from their mothers was not available. (Eibl, NEJM, 319(1), 1-7, 1988).

HJypogammaglobulinemia/Agammaglobulinemia/Thrombocytopenia: IVIG has been used to treat hypogammaglobulnemia and concomitant thrombocytopenia. IVIG treatment of hypogammaglobulnemia results in an increase in platelets. (Knezveic-Maramica, Transfuaion, 43, 2003). 

Autoimmune conditions: IVIG have been described for the treatment of certain autoimmune conditions (US2002/0114802). Paradoxically, IgG can exert both pro and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low dose IVIg requires complement activation. In contrast, when adminsitered in high concentrations, IVIg has anti-inflammatory properties. Durandy, Clinical & Experimental Immunology, 2009, 158. 

IVIg can be used in all age ranges for primary immunodeficiency syndromes with imparied antibody production, hypogammaglobulinaemia and recurrent bacterial infection sin patients with chronic lymphocytic leukaemia (CLL), in whom prophylactic antibiotics have fialed and in children and adolescents with congenital AIDS, hyopgrammablobulinaemia in patients after allogeneic haematopoietic stem cell transplantation (HSCT). EMA Guideline on the clinical investigation of human normal immunoglobulin for intravenous adminsitration (IVIg, Jully 22 2010). 

Teschner (US2010/0330071) teaches that the FDA has approved the use of IVIG to treat various indications including idiopathic thrombocytopenic purpura (ITP).