Pasteurization of C1 Inactivator in the Presence of Citrate Salts

Abstract

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.

Biological and Physical Properties of Fibronectin Pasteurized in the Presence of Stabilizers

“…Although precipitation problems interfered with the evaluation of citrate by the ANS technique, this compound has previously been shown to be effective in stabilizing antithrombin III [28,40,41], Cl inactivator [42] and a,-proteinase inhibitor [43], High concentrations of citrate also precipitate Cl inactivator, but prevent aggregation. In such cases, nonspe cific stabilizers which exert their influence via effects on solvent properties are likely to be more useful.…”

“…A similar increase in opsonic activity has been reported to occur when purified Fn is incubated at 37 °C in the ab sence of stabilizers [49], A major reason for pasteurization of plasma derivatives is to decrease the inci dence of transmission of viral hepatitis. However, in vivo studies in chimpanzees are required to assess the effectiveness of hepa precipitation apparently does not interfere with, and is not required for, stabilization [42]. Citrate was therefore included in a group of seven protective compounds and mixtures which were then further tested for their ability to stabilize Fn during pasteuri zation for 10 h at 60 °C.…”

“…Citrate and tricarballylate (dehydroxycitrate) alone could not be evaluated by the ANS technique because of their tendency to precipitate Fn. However, they were included in this study because of their ability to sta bilize other plasma proteins [40][41][42][43][44], Su crose, although less effective than other sug ars in shifting the Td (table I), was recently reported by Wallace et al [45] to protect Fn during pasteurization, and was therefore also included in the study. Elution volume, ml fragments were visible, reflecting either en zymatic proteolysis or non-enzymatic frag mentation [46], By contrast, the stabilized samples resembled the unheated control, showing no evidence of fragmentation or aggregation except in the case of citrate/cluconate where slight aggregation was evi dent.…”

Heat Stability of Lyophilized C1 Inactivator Concentrates

“…High-purity Cl-INA concentrate was prepared from the interme diate-purity concentrate by fractional precipitation with ammonium sulfate [31,32] Crossed immunoelectrophoresis (CIEP) was carried out as pre viously described [22], with antiserum to Cl-INA (Behring Diagnos tics) incorporated in the gel for the second dimension.…”

“…However, in later studies [20,21], the presence of sucrose was found to significantly reduce the virus inactivation rate during heat treatment. We reported that Cl-INA could with stand pasteurization treatment in the presence of 3 M potassium citrate [22], but a subsequent test using chim panzees revealed that under these conditions, the HBV was stabilized as well [23], Several investigators reported the use of dry heat for viral inactivation in human factor VIII [24][25][26] and factor IX [27], The dry-heat approach minimizes the risk of reinfection because it is performed in the final containers, …”