Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: effect of pH, buffers, and excipients.

A successful parenteral formulation can be developed by studying stability and compatibility of biopharmaceuticals as a function of solution composition. Here, we evaluate the influence of pH, buffers, ionic strength, protein concentration and presence of excipients on recombinant human epidermal growth factor (rhEGF) stability. The stability was accessed by reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), enzyme-linked immunosorbent assay (ELISA), Far-UV circular dichroism (CD) and light scattering. The overall maximal stability was obtained in pH near to 7.0 in phosphate, Tris and histidine buffers as the results of the different methods revealed. The CD results revealed that this protein is stable in an extensive pH range. Aggregation of rhEGF was minimized at pH values ranged from 6.0 to 8.0 as indicated the SEC-HPLC and light scattering results. Nor the ionic strength neither the rhEGF concentration had significant effect on the reaction rate constants. Most rhEGF-excipient instability occurs among this protein and reducing sugars. Polymers like poly(ethylene glycol) (PEG) and polysorbates increased methionine oxidation. The rhEGF oxidation and deamidation were the most common degradation pathways. This research identified critical solution factors to be considered for the development of a successful rhEGF parenteral formulation.

Optimisation of the coupled monoclonal antibody density for recombinant hepatitis B virus surface antigen immunopurification.

Using immunosorbents based upon cyanogen bromide-Sepharose CL-4B, we have examined different ligand densities in coupling of monoclonal antibody (MAb) to find the best performance, for recombinant hepatitis B surface antigen (rHBsAg) purification. Three replicates of 5 and 15 cycles of densities ranges: 2.17-2.19, 3.18-3.62, 4.06-4.17, and 5.13-5.40 mg/ml (control); or 1.81-2.47, 3.17-3.41, 4.16-4.28, and 5.16-5.19 mg/ml (control), respectively were evaluated in terms of binding capacity, antigen recovery, ligand leakage and purity of antigen, and compared to the control. Adsorption and antigen recovery of immunosorbents manufactured were not different statistically, eventhough increased 8.08 and 9.90% at a range of 3.17-3.41 mg/ml. At this range, efficiency expressed as productivity and MAb saving was optimal. Ligand leakage and purity of antigen showed similar behaviour among all densities. Aspects related to ligand density in antigen immunoaffinity purification are discussed.

Enzymic, spectroscopic and calorimetric studies of a recombinant dextranase expressed in Pichia pastoris.

Conformational stability and structural characterization of an rDex (recombinant dextranase) expressed in Pichia pastoris were studied by enzymic assays, fluorescence, CD and DSC (differential scanning calorimetry). We also identified two disulphide bridges (Cys9-Cys14, Cys484-Cys488) and two free Cys residues (Cys336, Cys415) that are not conserved between bacterial and fungal dextranases of GH-49 (glycoside hydrolase family 49) by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS. Enzymic and fluorescence studies revealed that rDex is biological and conformationally stable at acidic pH, with maximum activity at pH 4.5-5.0, while CD spectra indicated a secondary structure basically composed of beta-sheets. rDex loses biological activity at neutral pH without total disruption of its conformation. In addition, rDex preserves its conformation close to 60 degrees C, but it is thermally denatured with appreciable aggregation at temperatures above 75 degrees C. DSC studies always displayed irreversible transitions and a strong dependence on the scan rate. Our combined analysis suggested that the denaturation process of rDex is under kinetic control, which is described reasonably well by the two-state kinetic scheme.