Milan Wickerhauser

Development of Large‐Scale Fractionation Methods

Abstract. A large scale method for preparation of antithrombin III (AT III) concentrate from plasma or from Cohn fraction IV‐1 (Fr. IV‐1) has been described. It consists of the following steps: (a) partial purification by precipitation of impurities with 20% polyethylene glycol (PEG) 4000; (b) isolation of AT III from the PEG supernatant by batch adsorption and elution on heparin‐Sepharose at a ratio corresponding to 45 vol of plasma or 80 vol of 10% Fr. IV‐1 solution to 1 vol of gel; (c) concentration and desalting of the eluted AT III on a Pellicon ultrafiltration system; (d) pasteurization of AT III concentrate by heating for 10 h at 60°C in the presence of 0.5 M sodium citrate at pH 7.5; (e) removal of excess citrate by gel filtration on Sephadex G‐50; and (f) sterile filtration, filling and lyophilization. The recovery by activity was 32% from a 113‐liter plasma batch and 16% from a 42‐kg Fr. IV‐1 batch.

A single-step method for the isolation of antithrombin III.

Abstract. A single‐step method is described for the isolation of a highly purified antithrombin III (AT III) concentrate at a recovery of over 30% using affinity chromatography on heparin‐Sepharose (HS). The polyethylene glycol precipitation step frequently employed in the preparation of AT III concentrates for clinical use has been eliminated and purification is accomplished entirely by optimizing the salt concentration in the HS washing buffer to enhance the desorption of impurities prior to elution of AT III. Pasteurization of the AT III concentrate in the presence of 0.5 M sodium citrate to minimize the risk of hepatitis decreases the recovery by about 20% and induces changes in the patterns obtained by polyacrylamide gel electrophoresis and by crossed immunoelectrophoresis in heparinized agarose gel.

Large scale preparation of nonthrombogenic prothrombin complex.

Abstract A method has been developed to prepare a nonthrombogenic prothrombin complex concentrate. Human plasma after the separation of cryoprecipitate was adsorbed on kaolin and then fractionated by polyethylene glycol. The supernatant obtained after precipitation with 10% polyethylene glycol was batchwise adsorbed on DEAE-cellulose. Prothrombin complex was eluted with buffer of higher ionic strength (0.5M) containing a small amount of antithrombin III. The eluate was concentrated, gel filtered on a Sephadex G-50 column, sterilized and lyophilized under aseptic conditions. The final product was found nonthrombogenic by the Wesslers stasis model and by the in vitro nonactivated partial thromboplastin time test.

Contact factors are responsible for the thrombogenicity of prothrombin complex.

Abstract In order to determine the factors responsible for the thrombogenicity of prothrombin complex (PC), preparations of PC were prepared from normal plasma and from plasmas deficient in Factor XII, Factor XI, prekallikrein and high molecular weight kininogen. PC isolated from normal or Factor XI deficient plasma was thrombogenic unless the plasma was treated with kaolin prior to the isolation procedure. PC isolated from plasmas deficient in Factor XII, prekallikrein, or high molecular weight kininogen were not thrombogenic even when the kaolin adsorption step was omitted. These results indicate that Factor XIIa activation of prekallikrein is the initial reaction responsible for the thrombogenicity of PC.

Large-scale preparation of a highly purified solvent-detergent treated factor VIII concentrate.

Abstract. Large‐scale adaptation of a recently reported glycine precipitation method for the production of factor VIII (FVIII) concentrate is described. Scaling up of the method required some modification including the addition of aluminum hydroxide to the glycine buffer to reduce the level of contaminating proteins in the final preparation and the use of centrifugation to replace filtration by glass beads. Furthermore, the resultant product was virus inactivated by incorporation of the organic solvent and detergent technique. At industrial level, the modified method gave a good recovery of FVIII activity (230 IU/l plasma) with high purity (4 IU/mg protein). The final product, after virus inactivation and lyophilization, yielded 185 IU of FVIII activity per liter of starting plasma and was considered to be suitable for clinical evaluation.

Pasteurization of C1 inactivator in the presence of citrate salts

Conditions have been determined under which the C1 inactivator (C1‐INA) can be pasteurized to reduce the risk of transfusion hepatitis associated with its use for replacement therapy in patients with genetic or acquired deficiencies. Recovery of 90% of the biological and immunological activity of a C1‐INA concentrate was achieved following heat treatment for 10 h at 60°C in the presence of 3 M potassium citrate. Crossed immunoelectrophoresis in heparinized agarose was used to demonstrate the ability of the pasteurized C1‐INA to bind heparin and to form a precipitation pattern with antibody which was almost indistinguishable from that of an unheated control. High pressure liquid chromatography and enhancement of the fluorescence of 1,8‐anilinonaphthalene sulfonate were used to show that other proteins present in the concentrate were also stabilized.

Heat stability of lyophilized C1 inactivator concentrates

Abstract. Heat stability of lyophilized C1 inactivator (C1‐INA) concentrates of intermediate and high purity has been investigated under several heat treatment protocols that include heating for 96 and 192 h at 68°C and for 10 h at 80, 90 and 100°C. Both types of concentrate showed high stability in functional activity, with not more than 5% loss in any of the time‐temperature combinations evaluated. However, the C1‐INA antigen from both concentrates showed small but progressive changes in crossed immunoelectrophoretic pattern, in proportion to the intensity of heat treatment. High‐pressure size‐exclusion chromatography revealed only minimal signs of aggregation in the high‐purity concentrate, but a significant and progressive aggregation of nonspecific protein contaminants present in the intermediate‐purity concentrate, making the high‐purity concentrate preferable for heat treatment.

A simplified method for the preparation of immune-serum globulin.

Abstract. This report describes the recovery of highly purified immune‐serum globulin (ISG) from the 42% ethanol precipitate fraction (42% P) generated during the first step of albumin purification. The method consists of three purification steps: (1) reprecipitation of the 42% P at 20% ethanol, pH 7.2; (2) solubilization of ISG at 17% ethanol, pH 5.2, and (3) isoelectric precipitation of ISG at 25% ethanol, pH 7.2. ISG prepared by this method was homogeneous when subjected to immunoelectrophoresis and polyacrylamide gel electrophoresis, and gave a yield equivalent to 4.7 g/l plasma, corresponding to a final recovery of over 48%.