Inactivation of lipid-enveloped viruses in proteins by caprylate.

The use of caprylate for the inactivation of lipid-enveloped viruses in biologically active proteins both plasma derived and produced by cell culture was evaluated. Viruses consisted of herpes simplex virus type I, vesicular stomatitis virus, vaccinia virus, and Sindbis virus. Utilizing the dissociation reaction and varying the concentration of the ionized form of caprylate, a specific amount of the nonionized form of caprylate was maintained over a wide pH range. Virus-spiked protein solutions contacted with caprylate provide rapid virus inactivation under a variety of conditions while maintaining the integrity of the respective protein or activity. With the exception of coagulation factor AHF, protein and biological activity yield were essentially quantitative. Caprylate is removed after treatment by size exclusion chromatography or anion/cation exchange adsorption of the protein, followed by buffer wash.

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.

Characterisation of various intravenous immunoglobulin preparations.

Five commercially available immunoglobulin preparations are characterised with regard to molecular weight distribution, immunoglobulin subclass distribution and antibody content. Preparations processed utilising enzymic or chemical modification demonstrate considerable variance of molecular weight species and subclass distribution when compared to native preparations. Similarly, cytomegalovirus and anti-streptolysin O antibody content is dramatically reduced in the modified preparations. Possible reasons for the differences are discussed. In-process inactivation of Human Immunodeficiency Virus is reviewed with regard to one of the native preparations.

Comparative studies of impurities in intravenous immunoglobulin preparations.

In a comparative study, seven commercially available immunoglobulin preparations and one experimental preparation, processed by various manufacturers in the United States and Europe, were investigated for non-IgG proteins and impurities. The basic fractionation methodology affects the purity of subsequently processed intravenous preparations. Five of the seven products manufactured in Europe contained varying amounts of plasma proteins other than IgG. A residual heterologous protein was detected and quantitated in one product whose processing includes porcine pepsin treatment. Only one of the eight products was low in aggregates (as judged by both anticomplement activity and nephelometric turbidity), while three products contained anticomplement activity at levels high enough to be a potential cause of reactions in patients. In general, the level of prekallikrein activator activity was proportional to that of amidolytic activity; four products had only trace levels of amidolytic activity as well as prekallikrein activator. Only one product contained significant isoagglutinin levels, while six products had detectable IgA levels.

Fibronectin enhances the opsonic and protective activity of monoclonal and polyclonal antibody against group B streptococci.

We have investigated the opsonic and protective effects of fibronectin (FN) against type III group B streptococci. When used by itself, the FN failed to promote actual internalization of group B organisms. The addition of FN to group B streptococci that had been preopsonized in an immunoglobulin preparation modified for intravenous use ( IgIV ) or a type-specific, murine monoclonal antibody of IgG isotype markedly enhanced interaction with human polymorphonuclear leukocytes (PMN). A similar enhanced effect was observed when the FN was combined with type-specific monoclonal antibody preparations of IgM and, surprisingly, IgA isotype. Preincubation experiments indicated that the major effect was upon the PMN rather than directly on the bacteria, but we could not demonstrate an effect of FN on cell surface receptors for the Fc fragment of Ig or C3b using rosetting techniques. In addition to enhancing the in vitro opsonic activity of Ig, the FN significantly increased the protective effect of the polyclonal and monoclonal Ig preparations in an animal model of neonatal group B streptococcal disease. Thus, FN appears to have a critical role in the host defense mechanisms against group B streptococci.

Kinetics of thermal denaturation of antithrombin III.

Purified antithrombin III (AT III), a single-chain human plasma glycoprotein, molecular weight 58,000 daltons, and one of the major serine protease inhibitors, was heated in the 60-70 degrees C range for inactivating possible contaminations by hepatitis B virus (HBV). Loss of inhibitory activity, unfolding of tertiary structure, and the rate of aggregate formation of AT III were monitored experimentally during heatig. Sucrose and sodium citrate were demonstrated to stabilize the protein. From the rate data the calculated activation energies (E) showed E(tert. struct.) < E(biol. act.) < E(aggreg.) indicating the order (lower activation energy process first) in which heat causes these changes in the protein molecule. The activation energy corresponding to denaturation of HBV was estimated to be at least fourfold lower than that associated with the unfolding of the tertiary structure of the protein. Purified AT III, thus stabilized and pasteurized, should be therapeutically effective, and the risk for transmission of hepatitis B should be decreased significantly.

Plasma protein fraction. Product improvement studies.

The vasodilative effects associated with the use of sodium acetate have been reported in the literature. Preparations of plasma protein fraction (PPF) substantially free of this vasodilator were developed. This paper presents and discusses experimental work on the improved product.

Studies on ultrafiltration of antihemophilic factor.

Ultrafiltration as an alternative approach to reprecipitation was studied in the production of an intermediate-purity antihemophilic factor concentrate suitable for clinical use. Almost complete recoveries of factor-VIII-related activities have been demonstrated across the processing step resulting in increased net yield of the product. Several important processing parameters are discussed. The product thus prepared is stable and has improved VIII:C/mg fibrinogen ratio.

A new preparation of modified immune serum globulin (human) suitable for intravenous administration. I. Standardization of the reduction and alkylation reaction.

Immune serum globulin (ISG) prepared by Cohn cold alcohol fractionation of pooled human plasma was reduced with dithiothreitol (DTT) and alkylated with iodoacetamide and other alkylating agents. Our results show that there are a few labile interheavy chain disulfide bonds in ISG which react rapidly under mild, nondissociating conditions. The extent of disulfide cleavage is controlled primarily by the ratio of DTT to ISG until about 4-5 disulfide bonds have been reduced. We report detailed studies on the variables of ISG concentration, DTT to ISG ratio, pH, and time, leading to a chemically modified ISG that has a controlled and limited number of reduced and alkylated disulfide bonds.

A new preparation of modified immune serum globulin (human) suitable for intravenous administration. II. Functional characterization.

The commercial immune serum globulin (ISG) product is suitable for use in passive immunization only by intramuscular administration; intravenous use results in vasomotor side reactions. We report the functional characterization of a chemically modified ISG (MISG) that is essentially free of nonspecific complement-binding activity and contains no new antigenic determinants. The half-life values for MISG given intravenously to rabbits, guinea pigs, and humans are shown not to be different from that of ISG. The MISG we describe is shown to fulfill the in vitro and in vivo criteria of stability, safety, function, and half-life, making it suitable for human intravenous administration.

Preparation of a stable intravenous gamma-globulin: process design and scale-up.

Intravenous gamma-globulin has been prepared on a pilot scale by the process of reduction with dithiothreitol and alkylation with iodoacetamide. The spent reagents are removed by diafiltration followed by ultrafiltration. The product is stabilized with maltose to minimize precipitation. Process design concepts as well as product characteristics are discussed.

Quantitation of sodium in human albumin solutions.

An osmolatity method is described for quantitating sodium in isotonic solutions of diafiltered albumin and diafiltered plasma protein fractions. The proposed method shows comparable results with a reference procedure bases on atomic absorption spectrometry.

In vitro assay for prekallikrein activator (PKA).

The assay measures the action of prekallikrein in generating kallikrein. The prekallikrein activator is the limiting reagent. The product kallikrein is an esterase which can be quantitated using benzoyl arginine ethyl ester. The assay is reproducible and results are comparable with other methods. The large scale preparation of prekallikrein is described.

Simultaneous salt and ethanol removal from human serum albumin.

Diafiltration of salt and ethanol from human serum albumin was shown to be a competitive alternative to freeze drying. At least 99% of the original alcohol content could be removed in approximately five volume changes. Data on changes in ionic strength, bacteriological buildup, permeate flux, and dimer contents are presented.