Characterization of murine monoclonal antibody to tumor necrosis factor (TNF-MAb) formulation for freeze-drying cycle development.

PURPOSE: This study was designed to characterize the formulation of protein pharmaceuticals for freeze-drying cycle development. Thermal properties of a protein formulation in a freezing temperature range are important in the development of freezing and primary drying phases. Moisture sorption properties and the relationship between moisture and stability are the bases for the design of the secondary drying phase. METHODS: We have characterized the formulation of TNF-MAb for the purpose of freeze-drying cycle development. The methods include: DTA with ER probes, freeze-drying microscopy, isothermal water adsorption, and moisture optimization. RESULTS: The DTA/ER work demonstrated the tendency to "noneutectic" freezing for the TNF-MAb formulation at cooling rates of -1 to -3 degrees C/min. The probability of glycine crystallization during freezing was quite low. A special treatment, either a high subzero temperature holding or annealing could promote the maximum crystallization of glycine, which could dramatically increase the Tg' of the remaining solution. The freeze-drying microscopy further indicated that, after the product was annealed, the cake structure was fully maintained at a Tp below -25 degrees C during primary drying. The moisture optimization study demonstrated that a drier TNF-MAb product had better stability. CONCLUSIONS: An annealing treatment should be implemented in the freezing phase in order for TNF-MAb to be dried at a higher product temperature during primary drying. A secondary drying phase at an elevated temperature was necessary in order to achieve optimum moisture content in the final product.

Characterization of an endotoxemic baboon model of metabolic and organ dysfunction.

An anesthetized endotoxemic baboon model has been developed by infusing 2.0 mg E. coli endotoxin/kg i.v. over 1 hr (n = 7). Animals were monitored for 5-7 days with analyses of: cardiovascular, metabolic, and organ dysfunction; acid base, hemostatic, and hematological alterations; as well as tumor necrosis factor (TNF) and interleukin-6 (IL-6) levels. Pathophysiologies detected at 2 hr included transient decreases in vascular resistance and blood pressure, a 157% increase in blood lactate, and a 90% decrease in circulating neutrophils. Organ dysfunction was not observed until 24 hr and, although thrombocytopenia was prevalent (-72% at 48 hr), disseminated intravascular coagulation (DIC) was not a major pathology. Hematocrit fell 21% by 24 hr and was -41% at 5-7 days. Serum TNF peaked at 90 min (7.8 +/- 0.2 ng/mL) and was undetectable after 3 hr. IL-6 also increased early, peaked at 3 hr (3872 +/- 846 U/mL) and was still detectable at 24 hr. A low mortality primate model of gram-negative sepsis has been developed that is characterized by early cardiovascular and metabolic dysfunction (2-6 hr), late organ dysfunction (24-48 hr), sub-clinical DIC, a prolonged anemia, and a 29% mortality between 48 and 72 hr.