Assessment of biological reagents for presence of human immunodeficiency virus-1 (HIV-1) antigens.

We tested 122 biological reagents including laboratory quality assurance sera and therapeutic human immune serum globulin products using an immunoenzymometric assay (IEMA) for HIV-1 antigens. Biological reagents tested were 64 HIV-1 antibody non-reactive and 44 HIV-1 antibody reactive quality assurance samples, and 14 HIV-1 antibody reactive human immune serum globulins from 21 manufacturers. Twenty-one of these biological reagents were previously reported by us as Western blot reactive. All 122 samples tested were non-reactive for HIV-1 antigens. Low incidence of HIV-1 antigen in these biological reagents should not alter laboratory safety practices in which all samples are considered infectious. Use of HIV-1 antigen measurements, either alone or with HIV-1 antibody determinations does not increase the likelihood of detecting HIV-1 reactive samples.

HIV antibody screening of commercially available blood products in India.

In a screening of 182 vials of various commercially available blood products in the Indian market, 32 (17%) were found to contain HIV-1 antibodies. The contamination with HIV-1 antibodies was detected in all types of blood products including immunoglobulin preparations, cryoprecipitates as well as albumin. Interestingly, the contamination was seen almost exclusively in the products manufactured in the Bombay-Pune area (32 of 92, 34%). It was very low in products manufactured near Delhi (1 of 90). A single vial of imported Factor VIII concentrate tested negative.

Immunoglobulin preparation: safe from virus transmission?

The incidence of non-A, non-B hepatitis associated with the administration of immunoglobulin preparations, especially intravenous preparations, which had been considered to be free from virus transmission, is reported. Research efforts to improve the safety of intravenous immunoglobulin preparations which could be administered in a large volume must be continued. Adding sorbitol under weakly acidic conditions, heat treatment of IgG at 60 degrees C for 10 h is possible. Intravenous immunoglobulin preparation manufactured by polyethylene glycol fractionation followed by the heat treatment is not only intact, but also much closer to the ideal intravenous immunoglobulin preparation in the safety and stability.

Immunoglobulin G subclass distribution in three human intravenous immunoglobulin preparations.

In immunodeficiency patients the lack of immunoglobulins (Ig) can be total or partial with a specific IgG subclass imbalance masked by normal values for total IgG. In the latter case therapy with intravenous IgG preparations (IVIG) is generally beneficial, provided the IVIG preparations used originate from large pools of normal blood donors and exhibit a normal IgG subclass distribution. We have analyzed the subclass distribution of three IVIG products: Sandoglobulin (SAGL), GamimuneN (GI), Gammagard (GG), 6-10 lots each, in four different laboratories. The competitive enzyme immunoassays and radial immunodiffusion methods used different monoclonal and polyclonal antibodies specific for IgG1, IgG2, IgG3, and IgG4, respectively. Despite minor interlaboratory differences, the results show that the slightly lower IgG1 content of SAGL versus GI and GG was quantitatively compensated by a higher proportion of IgG2, that no differences existed in IgG3 levels, but that one preparation (SAGL) contained 2-3% of IgG4 compared to 0.5-1.5% in GI and below 0.5% in GG. This difference was significant, the two latter preparations being at or below the lower limit of what are considered to be normal values found in human adults. Such differences may have important clinical consequences.

Clinical safety of intravenous immune globulin and freedom from transmission of viral disease.

Although rare, side-effects have been associated with the administration of iv immune globulins (IVIG). While the clinical presentation may be similar, several different mechanisms account for these adverse reactions. There are those effects in the hypogammaglobulinaemic patient which are probably due to antigen-antibody interaction (so-called inflammatory reactions), those due to spontaneous activation of the complement system (possibly caused by IgG aggregates), those due to true hypersensitivity (for example, hypersensitivity to IgA), and those due to possible contaminants or even stabilizers or preservatives which might have been used. The incidence and nature of clinical side-effects seen in 37 patients who were treated with a native IVIG is shown. In addition, data are provided which support the absence of transmission of human immune deficiency virus in idiopathic thrombocytopenia (ITP) patients, and the agents of non-A, non-B hepatitis in 41 immune-deficient patients having periods of follow-up ranging up to 18 months. Finally, evidence of the inactivation of human immunodeficiency virus during the manufacturing procedure is provided.

The effect of processing methods on intravenous immune globulin preparations.

In this comparative study we investigated five commercially available immunoglobulin preparations manufactured by various processing methods. Two enzyme-modified preparations and one chemically modified preparation demonstrated appreciable variation in molecular weight species when compared with native preparations utilizing gentle processing conditions. One native preparation, processed with DEAE Sephadex, was low in IgG4 subclass, while the enzymic and chemical processing methods provided products demonstrating substantial variation in subclass distribution. The other native preparation, formulated at low pH, demonstrated particularly low turbidity values, indicating greater IgG stability at pH 4.25 than at pH 7.0. Certain antibody levels were seen to be greatly reduced in the modified preparations. These differences are discussed with reference to effects of the processing conditions employed.

Safety aspects of intravenous immunoglobulins.

Seven commercially available intravenous immunoglobulins (IVIG) preparations, a gamma globulin prepared by ethanol fractionation, and an experimental IgG isolated by a chromatographic procedure were compared in several tests. Split products were present in preparations manufactured by procedures involving protease treatment and in a sulphitolysed IgG. The same preparations and another chemically modified product displayed a loss in their capacity to bind staphylococcal protein A. None of the preparations exerted a high anticomplementary activity using concentrated human serum as a complement source. No strict correlation between aggregate content and anticomplementary activity could be established. None of the commercial IVIG preparations tested displayed a sizeable hypotensive action as assessed by a rat model involving potentiation of bradykinin action by an angiotensin convertase inhibitor. The chromatographically purified IgG and an intramuscular IgG prepared by Rivanol fractionation contained high endogenous protease and prekallikrein activator (PKA) activity, respectively and both were found markedly hypotensive. Neither endogenous protease nor PKA activity was detected in the Cohngammaglobulin fraction. However, it was very strongly hypotensive even without any previous blocking of angiotensin convertase. Our data support the view that immunoglobulin preparations may affect blood pressure without inducing bradykinin generation.

Potential contribution of mild pepsin treatment at pH4 to the viral safety of human immunoglobulin products.

Different manufacturers use several different processes for the production of intravenous immunoglobulin. Several manufacturers include a production step where the immunoglobulin is treated with low levels of pepsin at pH 4. A series of experiments were undertaken to assess whether or not pH 4/pepsin treatment could inactivate a range of test viruses. Acid-labile viruses such as vaccinia, herpes simplex, mumps and Semliki Forest virus were found to be susceptible to pH 4/pepsin treatment whereas poliovirus type 2, an acid-stable virus, was completely resistant to this treatment. In immunoglobulin preparations, viral contaminants are likely to be present as antibody/virus complexes and such complexing was found to help protect the test viruses from inactivation by pH 4/pepsin treatment. Despite this protection, at least 99% of the test inoculum of two susceptible viruses (vaccinia and herpes simplex) was found to be inactivated after treatment and the subsequent dissociation of virus/antibody complexes. It is concluded that pH 4/pepsin treatment may contribute to the safety of intravenous IgG by inactivating potential viral contaminants.

A strategy for testing established human plasma protein manufacturing procedures for their ability to inactivate or eliminate human immunodeficiency virus.

The manufacturing procedures used for the preparation of human plasma proteins that were established before AIDS was first described may reasonably be expected to provide AIDS safe products. Such manufacturing procedures are heat treatment at 60 degrees C in solution for ten hours, described as pasteurization, preparation of human immunoglobulins by ethanol precipitation, pepsin treatment, and sulfonation. To test whether these methods effectively inactivated and/or eliminated the AIDS causing human immunodeficiency virus (HIV), nine volumes or more of plasma or a plasma fraction taken from a production lot were spiked with HIV using one volume of a HIV concentrate and were then subjected to exactly the same procedure as that specified for the manufacturing process. HIV infectivity titres of the initial HIV/plasma protein mixtures and of the resulting products after treatment were determined by the H9 cell assay. In all cases studied complete inactivation/elimination of the added HIV was achieved. We therefore conclude that pasteurization of human plasma proteins or the manufacturing procedure used for the isolation of immunoglobulins from plasma pools result in final products which do not contain any infectious HIV and which are thus safe in that they cannot be vehicles for the transmission of AIDS.

Quality criteria for i.v. immunoglobulins: importance of tests and product properties.

Quality criteria for i.v. immunoglobulins (i.v. Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Quality criteria for i.v.-immunoglobulins: importance of tests and product properties.

Quality criteria for i.v.-immunoglobulins (i.v.- Igs) are critically discussed laying emphasis on spontaneous anticomplementary activity (aca) and size-dependent composition. Considering the percentage of fractions obtained by gel filtration (Ultrogel AcA 34), however, the monomeric IgG containing fraction contributed the major part of aca under the experimental conditions chosen. Subclass IgG3 seems to contribute considerably to aca. No correlation was found between aca and the percentage of fractions containing components larger in size than IgG dimers in various commercially available Igs. Moreover, the subclass distribution in different batches of individual products showed considerable variations. The amount of IgG4 is correlated with that of IgA in chemically unmodified products relatively poor in IgA (approx. less than or or equal to 10 mg/5 g Ig), indicating that attempts to reduce IgA consequently result in removal of IgG4.

Standardization of U.S. Reference Rh0 (D) Immune Globulin by quantitative automated hemagglutination.

An automated hemagglutination procedure was used to assess the relative potency of US Reference Rh0 (D) Immune Globulin, Lot 3, with respect to the International Reference Preparation, WHO Anti-D immunoglobulin, Lot 68/419. A value of 300 international units (IU) of anti-D per ampoule has been assigned to Lot 68/419. In 25 assays, the mean value for Lot 3 was 820 IU anti-D per milliliter when tested in parallel with Lot 68/419.