John L. Lundblad

A New Preparation of Modified Immune Serum Globulin (Human) Suitable for Intravenous Administration

Abstract. Immune serum globulin (ISG) prepared by Cohn cold alcohol fractionation of pooled human plasma was reduced with dithiothreitol (DTT) and alkylated with iodoace‐tamide and other alkylating agents. Our results show that there are a few labile inter heavy chain disulfide bonds in ISG which react rapidly under mild, non dissociating 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.

Optimization of Solute Separation by Diafiltration

Abstract Preliminary consideration suggests that process time in diafiltration can be optimized. A mathematical derivation of the optimum time gives a surprisingly simple general relationship between the bulk concentration and the membrane surface concentration. Experimental values confirm that an optimum value can indeed be obtained.

Inactivation of Lipid‐Enveloped Viruses in Proteins by Caprylate

Abstract. 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.

Preparation of a Stable Intravenous Gamma-Globulin: Process Design and Scale-Up

Abstract. Intravenous γ‐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.

Biological activity and conformational stability of the domains of plasma fibronectin

Abstract As measured by two assays of biological activity, fibronectin was readily denatured by heat. Both by the rat liver slice assay and by gelatin-latex agglutination, 90% of the activity disappeared in about 10 min at 60 °C. In contrast, immunological activity, as measured by microcomplement fixation, showed little change at 10 min and was at least 60% as great as unheated fibronectin after 20–50 min at 60 °C. Binding of heparin was unaffected by heating up to 52 min, but at very long times (48 hr at 60 °C), it also was lost. Differential scanning calorimetry of native fibronectin showed three endothermal denaturing transitions, at 68, 82, and 119 °C. Enthalpies of denaturation for the three transitions are approximately 2.6, 0.4, and 0.7 cal/g of flbronectin. These results are consistent with a three-domain structure for fibronectin. The domain which unfolds at 68 °C is associated with gelatin binding and cell. binding. The 82 °C domain appears to be associated with much of the immunological activity, and the 119 °C domain with heparin binding, as well as with some immunological activity. Residual immunological activity after loss of heparin binding may reside in nonordered portions of the molecule.

The Antigenic Nature of Heat-Treated Human Plasma Protein Fractions

The Ouchterlony and Grabar technics have been applied to a study of three fractionated, heated plasma protein solutions. Two of these products, namely, Chalb albumin and SPPS, have been shown previously to produce reactions or antibodies in humans. It was confirmed that Chalb albumin possesses at least one antigen not present in normal unheated human serum. SPPS was shown to be atypical when compared to human serum protein.

Plasma Protein Fraction (Human)

Abstract. Plasma Protein Fraction, USP (PPF) has largely replaced preparations of pooled plasma as a plasma expander. Three commercial lots of PPF (Plasmanate ®‐Cutter) prepared in 1959 were subjected to a planned stability study extending over a nine year period with the following findings: 1. Motion lability was not adversely affected by storage for periods up to 7 years at temperatures of 25° or below. 2. With extended time, there was a small gradual decrease in the albumin component and a corresponding increase in a component with an electrophoretic mobility of alpha‐globulin but with a sedimentation rate equivalent to that of albumin; this rate of change was greatly accelerated at 40°. 3. Viscosity increased at a rate of approximately 0.3% per year at 5° and 25°; at 40° viscosity increased sharply with extended time. 4. Turbidity was little affected by time and temperature except for periods in excess of 2 years at 40°. 5. No antigenic changes were detectable after 2 years at 40° or 8 years at 25°. 6. Clinical studies in normal volunteers with PPF stored 9 years at 25°, and in ill patients with PPF stored 2 years at 40° and 9 years at 25° showed no adverse reactions. It is concluded that Plasma Protein Fraction can be safely used after storage for 5 years at temperatures not exceeding 30°.

Survival of native structure and biological activity in fibronectin pasteurized in the presence of sucrose.

Abstract When fibronectin was heated at 60°C in isotonic buffer, all biological activity was lost in less than 1 hr. Structural changes also occurred, as evidenced by the appearance of aggregates on polyacrylamide gels and by a reduction in immunological activity. In contrast, when subjected to the same heat conditions in the presence of 54 to 57% sucrose ( w v ), fibronectin remained virtually in a native state for periods up to 17 hr. After dialysis to remove sucrose, pasteurized fibronectin (10 hr, 60°C) was tested for biological activity by the rat-liver-slice assay, using labeled gelatin-latex particles and by a gelatin-latex agglutination assay. By both methods, sucrose-pasteurized fibronectin exhibited activity nearly as great as unheated controls. When sucrose-pasteurized fibronectin was examined by electrophoresis a single band with no evidence of aggregation was observed. By the method of microcomplement fixation, sucrose-pasteurized fibronectin did not show significant immunological changes compared with unheated fibronectin. When differential scanning calorimetry (DSC) was applied to sucrose-pasteurized fibronectin, there was little or no difference between it and unheated fibronectin in number, temperature, or enthalpy of endothermic transitions. Other substances, which bind to fibronectin (such as heparin and arginine) were shown not to stabilize fibronectin to heat.

Studies on Ultrafiltration of Antihemophilic Factor

Abstract. 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.