HIV inactivation in plasma products.

This is a review of therapeutic plasma products viz., coagulation factor concentrates, immunoglobulins, volume expanders, and protease inhibitors, and the progress made in rendering them free from transmitting infectious virus. Problems associated with the development of plasma products and the risks associated with their use are addressed. The recent technical advances designed to inactivate the hepatitis virus which led to the inactivation of HIV while preserving most of the biologic activity are reviewed. Today manufactured plasma products are essentially free from transmitting viral diseases of any significant clinical consequences.

Virus inactivation/elimination in therapeutic protein concentrates.

Concern for virus transmission from intravenous biological products, derived from natural and biotechnological sources, has prompted us to investigate various methods for virus inactivation while preserving the biological activity of the therapeutic entity. This concern is currently acute regarding the presence of HIV in purified human plasma protein concentrates. Four different methods of virus removal (1) heating in solution; (2) use of lipid solvents; (3) low pH treatment, and (4) partitioning during purification, are reported for various plasma proteins that have been spiked with model viruses. General virus inactivation strategies for therapeutic protein concentrates are discussed.

Inactivation of viruses in therapeutic products derived from human plasma.

Alpha-1-proteinase inhibitor concentrates have been prepared from pooled human plasma for therapeutic applications. Pasteurization (60 degrees C, 10 hours) conditions have been defined to reduce risks of transmission of viral agents without significant loss of biologic activity of the purified product. Human clinical data collected to date support the model virus inactivation studies regarding viral safety.

Elimination of infectious retroviruses during preparation of immunoglobulins.

Safety concerns for immunoglobulin preparations have led us to study partition/inactivation of two prototype retroviruses, mouse xenotropic type C and lymphadenopathy-associated virus (LAV) of the acquired immunodeficiency syndrome (AIDS), during manufacture and storage of immunoglobulins. Reduction of infectious retrovirus titers were 10(5) to 10(8)-fold through Cohn-Oncley cold ethanol fractionation from plasma to fraction II, 10(3) to 10(5)-fold through incubation at pH 4.0 and another 10(4)-fold through incubation of the purified liquid immunoglobulin preparations at 27 degrees C or 45 degrees C. The results support the clinical and epidemiological evidence that therapeutic immunoglobulin preparations do not transmit AIDS virus.

Recovery and inactivation of infectious retroviruses from factor VIII concentration.

The ability of infectious retroviruses to withstand the procedures used for factor VIII concentration was investigated. Mouse retroviruses added to human plasma survived these procedures and remained infectious in lyophilised samples of factor VIII. Lyophilised material had to be heated at 68 degrees C for several hours before substantial quantities of infectious virus became inactivated. These findings support the possible role of retroviruses in AIDS, and indicate that factor VIII concentrates must be heated to inactivate these infectious viruses.

[A new immune serum globulin preparation for intravenous administration (author's transl)]. / Ein neues Immunserumglobulinpräparat zur intravenösen Verabreichung.

A new immune serum globulin preparation has been developed which is suitable for i.v. administration. This product was prepared from Cohn fraction II by selective reduction with dithiothreitol of one or more of the interchain disulfide bonds between the heavy chains and subsequent alkylation of the formed sulfhydryls (about 8--10 per 160 000 daltons) with iodoacetamide. The of these chemical reactions has been termed MISG (modified immune serum globulin). It is free of anticomplement activity, contains essentially all of the antibody potency of immune serum globulin, sediments as essentially a single entity with a sedimentation coefficient of about 7 S, has been freed of all reactants, is non-toxic, and in rabbits is antigenically with normal human immune serum globulin. MISG with its Fc fragment portion intact maintains its complement mediated function as evidenced by its opsonic activity against bacteria. MISG had been tested extensively in vitro, in animals, and in humans. Half-life measurements in humans averaged 22.4 days (range 15.5--28.7 days) and efficacy was demonstrated in a 2-year crossover clinical study in which MISG was compared with normal immune serum globulin administered i.m.

Hypotension associated with prekallikrein activator (Hageman-factor fragments) in plasma protein fraction.

Thirteen lots of plasma protein fraction made by one manufacturer were implicated in 23 recent reports of hypotension in surgical patients. Four of these patients required resuscitation after rapid administration of the product in the postoperative period. All implicated lots had prekallikrein-activator activity but low levels of bradykinin and kallikrein. The prekallikrein activator was identified as Hageman-factor fragments by molecular weight (35,000 as estimated by gel chromatography), isoelectric point (4.2 to 4.4), and inhibition by antibody to Hageman factor. These data suggest that Hageman-factor fragments are potent hypotensive agents, presumably because they trigger the generation of bradykinin in recipients. Prekallikrein-activator activity, usually at levels lower than those in the initial 13 implicated lots, was frequently detected in plasma protein fraction made by other manufactures. Several of these lots were associated with additional reports of hypotension. Prekallikrein-activator activity rarely occurred in albumin.

Preparation and standardization of an Australia antigen antibody of equine origin.

A horse has been immunized with Australia antigen (Au/SH) purified 20-fold by a procedure employing gel filtration of Cohn fraction IV derived from an Au/SH-positive human plasma pool. Hyperimmunization was initiated by the intramuscular injection of 20 ml of a mixture of equal parts of purified Au/SH and complete Freund's adjuvant. The 20-ml volume was divided into four 5-ml doses, two of which were administered on each side of the horse's neck. Booster doses of antigen alone were given as follows: 10 ml intravenously 30 days later and 5 ml intramuscularly on each of days 77 and 205. Au/SH antibody formed readily, beginning on day 17, and was demonstrated by the agar gel double-diffusion technique and the complement fixation test during the subsequent 6 months. Antihuman plasma protein antibodies were effectively removed from the horse serum by one absorption with 1 to 3 volumes of normal human plasma. Abrupt rises in anticomplementary activity observed shortly after the third and fourth antigen injections, when the horse had developed elevated and steady levels of Au/SH antibody, could possibly be due to formation of antigen-antibody complexes. After optimal conditions were determined, an Au/SH antibody reagent pool which met official requirements was prepared. It was found equally suitable for the agar gel double-diffusion, complement fixation, and counterimmunoelectrophoresis test procedures.

Australia antigen: distribution during Cohn ethanol fractionation of human plasma.

When plasma containing a hepatitis-associated antigen (Au/SH) is fractionated, the antigen is localized in fractions III and IV with none in fraction II and only small amounts in fractions I and V. The amount of antigen found in each of these fractions is probably not predictive of clinical infectivity of Cohn ethanol fractions from normnal pooled plasna.