A chimeric Fab was expressed in Chinese hamster ovary cells under the control of the CMV promoter in a two-stage production process. Cells were first grown to 90% confluence at 37 degrees C in a proliferation phase, followed by a production phase at either 37 degrees C or 28 degrees C. Medium supplemented with serum and medium free from serum was tested in the production phase at both temperatures. Comparison of Fab expression revealed that reducing the temperature to 28 degrees C resulted in a 14-fold increase in product yield when cells were cultivated in serum-containing medium, and in a 38-fold increase in product yield when serum-free medium was applied.

During hemostasis the zymogen factor X (FX) is converted into its enzymatically active form factor Xa by the intrinsic FX-activating complex. This complex consists of the protease factor IXa (FIXa) that assembles, together with its cofactor, factor VIIIa, on a phospholipid surface. We have studied the functional properties of a FIXa-specific monoclonal antibody, 224AE3, which has the potential to enhance intrinsic FX activation. Binding of the antibody to FIXa improved the catalytic properties of the intrinsic FX-activating complex in two ways: (i) factor VIIIa bound to the FIXa-antibody complex with a more than 18-fold higher affinity than to FIXa, and (ii) the turnover number (k cat ) of the enzyme complex increased 2-to 3-fold whereas the K m for FX remained unaffected. The ability of 224AE3 to increase the FXa-generation potential (called the "booster effect") was confirmed in factor VIII (FVIII)-depleted plasma, which was supplemented with different amounts of recombinant FVIII. In the presence of antibody 224AE3 the coagulant activity was increased 2-fold at physiological FVIII concentration and up to 15-fold at low FVIII concentrations. The booster effect that we describe demonstrates the ability of antibodies to function as an additional cofactor in an enzymatic reaction and might open up a new principle for improving the treatment of hemophilia.One of the key events during normal hemostasis is the conversion of the zymogen factor X (FX) 1 into its enzymatically active form factor Xa (FXa) via the intrinsic coagulation pathway, a process that subsequently leads to activation of prothrombin and the formation of a fibrin clot (1). The FX-activating protease factor IXa (FIXa) assembles (together with FVIIIa as well as Ca 2ϩ ions) on a phospholipid surface to form the intrinsic FX-activating complex (2). In this complex FVIIIa serves as a cofactor for the enzyme FIXa and increases the turnover number (k cat ) of FIXa by 4 to 5 orders of magnitude (3, 4). Binding of the stoichiometric FVIIIa-FIXa complex and the substrate FX to a phospholipid surface strongly reduces the K m for the substrate (3, 4), probably because the protein-protein interactions are limited to two dimensions (5).The physiological importance of the intrinsic FX-activating complex is well defined because the severe bleeding disorders hemophilia A and hemophilia B are characterized by very low plasma levels (Ͻ 5%) or by the absence of the pro-cofactor FVIII and the pro-enzyme FIX, respectively. Treatment of hemophilia is successfully achieved by supplementing the blood of patients with either human FVIII or human FIX. Both proteins can be derived from human plasma or produced as recombinant protein in mammalian cell culture (6, 7). Although progress in the production of coagulation factors to ensure their purity, efficacy, and viral safety has been made over the last 20 years, treatment of hemophilia is still beset with several difficulties. First, production of recombinant and plasma-derived, therapeutic coagulation factors is expensive, an...