Recombinant human interferon alpha2b (rhIFN-2b) protein is FDA approved for treatment of many tumors and viral diseases. A rhIFN-2b isoform has been produced and purified from the refolding reaction using high-resolution anion ion exchange chromatography. This isoform has a proper MW (19kDa) and high purity and homogeneity. The conservation of native linear and conformational epitopes in this isoform was immunologically confirmed by Western blot and ELISA. Mass spectrometry assessment of its intact mass showed average mass (19,337Da) equivalent to that of the expressed rhIFN-2b protein without any chemical modification and without the first methionine. Peptide mapping of rhIFN-2b through tryptic digestion of reductive/alkylated protein using urea as a denaturing agent gave the best pattern. The rhIFN-2b had a high specific antiviral activity (2.5x10(8)+/- 1.1x10(8)IU/mg protein). In vivo clearance study of rhIFN-2b in female SD rats (500g/kg, intramuscularly) revealed rapid clearance (elimination half-life 0.54h with a maximum plasma concentration of 33,792pg/ml) compared with the commercial rhIFN-2 (elimination half-life 0.75-0.96h). In conclusion, the prepared rhIFN-2b isoform has high purity, homogeneity, native like chemical and structural composition, high antiviral activity, and proper biological stability, which reduce its immunogenicity and raise its therapeutic efficiency.