Note: the following studies show that ISG is safe IF all other processes for preparation are sterile. That was not the case until the 90's. The products were not pasteurized until the eighties. Prior to this, the Fractionation process, at all four plants, including Connaught in Canada, were constantly sited by the FDA for violations of Good Manufacturing procedures for sanitary processes and equipment.

Three generations of immunoglobulin G preparations for clinical use. 

McCue JP, Hein RH, Tenold 

R Rev Infect Dis 1986 Jul-Aug;8 Suppl 4:S374-81

The first purified human immunoglobulin G (IgG) preparation used clinically was immune serum globulin (ISG), which was prepared in the 1940s by E. J. Cohn's group. It was originally formulated in water with 0.3 M glycine at pH 6.8 and was 70%-80% monomeric. ISG was safe when given intramuscularly and efficacious for measles and hepatitis prophylaxis. The next generation of purified IgG began in the 1960s with chemically modified preparations suitable for intravenous administration. The first such IgG intravenous preparation (IGIV) in the United States was IGIV pH 6.8 (Gamimune, Cutter Biological), in which the anticomplement activity found in ISG was removed by reduction and alkylation of disulfide bridges. This product was originally formulated as a 5% IgG solution in water (pH 6.8) with 0.2 M glycine in 10% maltose for stabilization. It remained stable for at least 2.5 years at 5 degrees C, was 80%-90% monomeric, had virtually no anticomplement activity, was safe given intravenously, and was efficacious for prophylaxis in agammaglobulinemic patients. A third generation of purified IgG has since been developed; IGIV pH 4.25, (Gamimune N, Cutter Biological), which was isolated by the Cohn method from human plasma and is safe for intravenous use, is a 5% solution of IgG in water (pH 4.25) with 10% maltose. The product is greater than 99% IgG, greater than 95% monomeric, and has greater than 90% less anticomplement activity than ISG. 

PMID: 3092303, UI: 86315374

Plasma Fractionation

Blood plasma contains a mixture of hundreds of different kinds of proteins, only a few of which are of therapeutic interest. To make plasma derivative products, plasma can be treated with a variety of substances to separate the desired proteins from others, in a process called fractionation. Fractionation of plasma, from pools often derived from thousands of donors, was developed during World War II by Cohn and co-workers at Harvard Medical School. Today, most plasma derivative manufacturers use a modified Cohn method developed by Oncley (Cohn-Oncley fractionation process) or further variants of this method, that permit manufacture of additional products.

Fractionation by the Cohn-Oncley method relies on precipitation of plasma proteins by a combination of cold alcohol (usually ethanol)-water mixtures and adjustments of pH, ionic strength, temperature, and protein concentration. Alternatively, some manufacturers separate plasma derivatives by column chromatography using ion exchange, gel filtration, or affinity methods, without alcohol. In all cases, fractions of plasma are separated sequentially, with the product from one step, such as the precipitate and/or supernatant, becoming the starting material for the next step in the fractionation process. If each step is not done properly, subsequent fractions can be adversely affected. Thus, the integrity of each final product is dependent on all of the preceding steps in the process.

After fractionation, derivatives undergo further processing to purify and concentrate proteins and to inactivate or remove (clearance) any bacterial or viral contaminants. While early steps in the manufacturing process are not performed aseptically, all final products are sterile. Types of viral clearance include those steps that are part of the fractionation process itself, e.g., pH4/pepsin or polyethylene glycol (PEG) fractionation, or those steps that are deliberately added, e.g., solvent/detergent treatment or viral filtration. In some instances more than one viral clearance step is used for a given product. Plasma derivatives are similar to other biological products in that they are protein-based and subject to denaturization at high temperatures. These products are usually filled by using aseptic processing techniques, and cannot be terminally sterilized, although in some instances they can be heat-treated in the final container to effect viral or bacterial inactivation.

A few plasma proteins may also be manufactured by recombinant DNA methods.

Fractionation Products

Each plasma fraction is enriched in specific protein components and is used for a different purpose. In the Cohn-Oncley method, Fraction I contains mostly fibrinogen (not a licensed product), the main protein component of blood clots. Fraction II+III has a high concentration of immunoglobulins (antibodies). Some manufacturers use Fraction IV to prepare licensed products; others consider it a by-product. Fraction IV-1 is the source material for Alpha-1-proteinase Inhibitor (Human); Fraction IV-4+V is the source of Plasma Protein Fraction (Human). Fraction V is the source of Albumin (Human). Most of these products, but not all are intravenously administered. A description of some of the major plasma derivatives follows:

Antihemophilic Factor (Human) (AHF, Factor VIII). AHF protein, one component of the cryoprecipitate fraction of plasma, is used to treat classical hemophilia (hemophilia A). Cryoprecipitate is the solid material that remains after frozen plasma is thawed at a near freezing temperature; it serves as the source of AHF. After the cryoprecipitate dissolves upon warming, the AHF in it can be purified to a high degree, subjected to various viral clearance procedures, and prepared as a lyophilized concentrate. AHF is administered intravenously. NOTE: Even though the clinically active ingredient is the same, AHF is not the same product as Cryoprecipitated AHF, a single donor product prepared in blood banks.

Factor IX Complex (Human) is adsorbed from the plasma fraction remaining after cryoprecipitate removal. It is a heat- or solvent/detergent-treated, lyophilized preparation containing factors II, VII, IX, and X. It is administered intravenously for the prevention and control of bleeding caused by Factor IX deficiency (hemophilia B), and other coagulation disorders.

Coagulation Factor IX (Human) is a highly purified factor IX product that contains negligible amounts of other coagulation factors, and is used to treat hemophilia B.

Immune Globulin (Human) (IG) is a solution of immunoglobulin G (IgG) indicated for prophylaxis of hepatitis A, prevention or modification of measles (Rubeola), and for immunoglobulin deficiency. It is administered intramuscularly.

Additional specific immune globulins for intramuscular administration are obtained from donors whose plasma contains selected high titer antibodies. Products are available for use in the passive prophylaxis of varicella-zoster, tetanus, hepatitis B, rabies, and other infections. Another product, Rho(D) Immune Globulin (Human), is for the prevention of sensitization to the Rho(D) antigen and hemolytic disease of the newborn. Some of the intramuscular immunoglobulin products have been subjected to heat- or solvent/detergent-treatment.

Immune Globulin Intravenous (Human) (IGIV) is a lyophilized preparation that contains intact, unmodified, immunoglobulin. It is often stabilized with monosaccharide (sucrose, glucose, or mannose) and/or Albumin (Human) or glycine. It is indicated for patients with primary immunodeficiency, immune thrombocytopenia and Kawasaki's disease. Additional specific IGIV products are also available and used for such indications as prevention of hemolytic disease of the newborn, or passive prophylaxis of cytomegalovirus or respiratory syncytial virus. All IGIV products have been subjected to viral inactivation/removal procedures by either fortuitous or deliberate methods.

http://www.fda.gov/ora/cpgm/42_006.html

Immunoglobulin Transmits Hepatitis C. True or False?

Hepatology, January 1999, p. 299-300, Vol. 29, No. 1 Correspondence

To the Editor:The safety of HCV-RNA-positive intramuscular immunoglobulin preparations can be attributed to several factors: (1) partitioning of viruses away from immunoglobulin, (2) inactivation of viruses by the fractionation process, and (3) a high concentration of neutralizing antibodies.^7,9,12 
Read

Partitioning of hepatitis C virus during Cohn-Oncley fractionation of plasma.

Yei S, Yu MW, Tankersley DL.

Laboratory of Plasma Derivatives, Food and Drug Administration, Bethesda, Maryland.

Because of concern about the safety of immune globulins with respect to transmission of hepatitis C, the partitioning of hepatitis C virus (HCV) during alcohol fractionation of a plasma pool prepared exclusively from anti-HCV-reactive donations was examined. Quantitation of HCV RNA was accomplished by nested polymerase chain reaction (PCR) at limiting dilutions. One PCR unit was arbitrarily defined as the minimum amount of HCV RNA from which an amplified product could be detected. The starting plasma pool contained 1.4 x 10(5) PCR units per mL. Most of the HCV RNA was found in cryoprecipitate and in Cohn fractions I and III, but it was also detected in fraction II, which is used for immunoglobulin G preparations. A 3.4-percent solution of IgG prepared from this fraction II contained 30 PCR units per mL. The fractionation process leading to immune globulin resulted in overall reduction in HCV RNA by a factor of 4.7 x 10(4). Although the presence of HCV RNA in the final product does not necessarily imply the presence of infectious virus, this work suggests that the safety of immune globulins with respect to HCV transmission is not due solely to the partitioning of HCV away from the immunoglobulin fraction.

PMID: 1335184 [PubMed - indexed for MEDLINE]

Biotechnol Appl Biochem 1998 Oct;28 ( Pt 2):169-78

Non-A, non-B hepatitis occurring in agammaglobulinaemic patients after intravenous immunoglobulin.

Lever AM, Webster AD, Brown D, Thomas HC.

Acute non-A, non-B hepatitis developed in twelve patients with primary hypogammaglobulinaemia during treatment with intravenous gammaglobulin prepared by Cohn fractionation of pooled plasma. The illness was clinically and histologically identical to the short-incubation non-A, non-B, hepatitis observed in haemophilic patients receiving factor VIII concentrates. Most of the patients were symptomless, but 10 months after onset ten of the twelve still had abnormal liver function. The occurrence of non-A, non-B hepatitis in agammaglobulinaemics indicates that humoral mechanisms are not essential for production of hepatocyte necrosis in this infection. This outbreak emphasises the need for a screening test to identify the agent in blood products, and shows that Cohn fractionation of plasma does not always inactivate the agent. Furthermore, the finding that the virus can be transmitted in IgG concentrates suggests either that the general population has a very low level of antibodies to the putative virus or that such antibodies are not virus-neutralising.

Publication Types:

• Clinical Trial
• Controlled Clinical Trial

PMID: 6150140 [PubMed - indexed for MEDLINE]
 

Veterans Administration cooperative study

A randomized, double blind controlled trial of the efficacy of immune serum globulin for the prevention of post-transfusion hepatitis. A

Seeff LB, Zimmerman HJ, Wright EC, Finkelstein JD, Garcia-Pont P, Greenlee
HB, Dietz AA, Leevy CM, Tamburro CH, Schiff ER, Schimmel EM, Zemel R, Zimmon DS, McCollum RW

Gastroenterology 1977 Jan;72(1):111-21 

A double blind, randomized, controlled trial has been conducted in 11 Veterans Administration hospitals during a 49-month period to compare the relative efficacies of immune serum globulin (ISG) and an albumin placebo for the prevention of post-transfusion hepatitis (PTH). A total of 2204 patients, of whom 1094 received ISG, participated in the study. The results indicate that ISG significantly reduced the incidence of icteric type non-B hepatitis only (inferred to be also type non-A hepatitis). Adverse reactions were rare, and the ISG did not significantly alter the incubation period or duration of the disease. 

The data suggest, however, that a similar reduction in type non-A, non-B hepatitis would have occurred had commercial blood been excluded from use. Analysis of the 241 patients who developed hepatitis indicates that type B hepatitis constituted less than 20% of the cases each year of the study. Furthermore, the efficacy of the ISG, manufactured in 1944, against apparent type non-A, non-B hepatitis suggests that this overlooked disease has existed from at least that time. Host- and transfusion-related factors that might have modified the development of PTH were examined. The use of commercial blood was observed to be the most important risk factor.

It is concluded that the PTH incidence can be most effectively reduced by eliminating commercial donor blood, and continuing to screen volunteer donors for hepatitis B surface antigen (HBsAg) by sensitive procedures. Of prime importance is the need to define the agent(s) responsible for type non-A, non-B hepatitis.

Publication Types:
Clinical trial
Randomized controlled trial 
 

Detection and characterization of hepatitis C virus RNA in immune globulins.

Yu MY, Mason BL, Tankersley DL
Transfusion 1994 Jul 34:7 596-602

BACKGROUND: Hepatitis C virus (HCV) RNA was measured in immune globulins and its chemical and physical properties were characterized. STUDY DESIGN AND METHODS: The study examined 69 immune globulin lots from 7 manufacturers, including 44 intravenous and 25 intramuscular immune globulin preparations. In addition, 8 experimental intravenous immune globulin preparations were investigated. Detection and quantitation of HCV RNA were achieved by reverse transcription and nested polymerase chain reaction at limiting dilution. A multi-antigen anti-HCV enzyme immunoassay was also used to test these immune globulins. 

RESULTS: The highest level of HCV RNA was found in an experimental immune globulin lot derived from a plasma pool made up of 186 anti-c100-3-reactive units. HCV RNA was detected only in 1 of 7 manufacturers' experimental intravenous immune globulin preparations derived from a pool made up of 2887 anti-c100-3-negative units. It was also detected in commercial intravenous immune globulin lots prepared by the same manufacturer from source plasma, but not from recovered plasma. More than half of the commercial intramuscular immune globulin lots, including specific immune globulin products, were HCV RNA positive. All immune globulin products examined were reactive for anti-HCV. Certain similarities were found for HCV RNA present in an immune globulin product and plasma. Ethanol at 20 or 25 percent had no effect upon the buoyant density of HCV RNA. CONCLUSION: Many immune globulin preparations contained HCV RNA, with levels depending upon both the type of starting plasma and the manufacturing process. Exposure to ethanol did not appear to affect the physical characteristics of HCV RNA.

Laboratory of Plasma Derivatives, Food and Drug Administration, Bethesda, Maryland.

Subcutaneous injections of a drug containing human immunoglobulins 

Infect Dis 1977 Feb;135(2):252-8 Petrilli FL, Crovari P, De Flora 

SInce late 1974 and early 1975, several cases of viral hepatitis were reported in Italy among subjects who had received subcutaneous injections of a drug containing human immunoglobulins that was prescribed for the treatment of allergies. Epidemiologic and laboratory investigations provided evidence that the original immunoglobulins, the series of the drug containing these immunoglobulins, and sera from a number of patients were all positive for hepatitis B surface antigen (HBs Ag) of the adw subtype, which is relatively rare in Italy. Some sera from patients and healthy subjects treated with the HBs Ag-positive drug were also found to be positive for antibody to HBs Ag of the adw subtype. The clinical course of the disease was consistent with typical forms of icteric hepatitis in all patients examined. The average length of the possible incubation period was 111-143 days, and an inverse relation was observed between the number of doses administered and the length of the incubation period. The possibility that immunoglobulins can be responsible for the transmission of viral hepatitis raises a number of theoretical and practical problems concerning control and use of these blood products.

Vox Sang 1974;27(4):302-9
Berg JV, Berntsen KO, Bjorling H, Holmstrom B, Vyas GN
PMID: 4213354, UI: 75014376

Low Frequency of Cirrhosis in a Hepatitis C (Genotype 1b) Single-Source Outbreak in Germany: A 20-Year Multicenter Study.

Hepatology 2000 Jul;32(1):91-96

Wiese M, Berr F, Lafrenz M, Porst H, Oesen U
University Affiliated Hospital St. Georg of Leipzig, Departments of Medicine
of the Universities, Germany.


>From August 1978 until March 1979, 14 batches of anti-D immune globulin contaminated with hepatitis C virus (HCV) genotype 1b (20,000-480,000 copies/dose) from a single erythrocyte donor had been administered for prophylaxis of rhesus isoimmunization throughout East Germany.

All 2,867 women involved had been recalled after January 12, 1979 for repeated screening of alanine transaminase (ALT). They were prospectively followed in regional centers. We have reexamined a cohort of 1,018 women (median age 24, range 16-38 years at infection) on follow-up for 20 years in 9 representative centers. Within 6 months after anti-D administration, 10% of these women had no evidence of disease and 90% had acute hepatitis C (n = 917) including 49% with symptomatic and 22% with icteric course. After 20 years, 85% of the 917 affected women still tested positive for HCV antibodies (among them 3% responded to interferon treatment) and 55% were positive for HCV RNA (among them 7% were nonresponders to interferon and 3% were apparent HCV carriers). Only 4 (0.4%) had overt cirrhosis. Two (0.2%)
died of superinfected fulminant hepatitis B or alcoholism and cirrhosis, respectively. Histology obtained in 44% of the viremic women showed hepatitis of minimal to moderate grade in 96%, portal fibrosis in 47%, and septal fibrosis in 3% of the cases.

In conclusion, formerly healthy young women, without hepatic comorbidity, may clear HCV (1b) infection in half of the cases or develop mild chronic hepatitis C with low risk of progression to cirrhosis within 20 years.

PMID: 10869294

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