Development of methods for quality control of recombinant human Tenecteplase

Objective To develop methods for quality control of recombinant human Tenecteplase(TNK-tPA).Methods The bulk of TNK-tPA was determined for biological activity by bubble assay,for purity by non-reduced SDS-PAGE and RP-HPLC,for relative molecular mass by reduced SDS-PAGE,for single chain content by HPLC-SEC,for peptide map by trypsin digestion,for amino acid sequence at N-terminus by Edman degradation,for isoelectric point by capillary isoelectric focusing electrophoresis,for protein content by Lowrry method,and for residual exogenous DNA content by Digoxin method.Identity,appearance,pH value,moisture content,sterility,filling quantity and abnormal toxicity of final product of TNK-tPA were tested according to the requirements in Chinese Pharmacopoiea(Volume III,2005 edition).Results The biological activities of three batches of bulks of TNK-tPA were(2.09 ~ 2.16) × 106 U / ml,while the specific activities were more than 3.5 × 105 U / mg,thus the specific activity of bulk of TNK-tPA was defined as not less than 3.0 × 105 U / mg.All the non-reduced SDS-PAGE purities of three batches of bulk of TNK-tPA were more than 98.0%,while the RP-HPLC purities were more than 97.0%.Two bands were observed on reduced SDS-PAGE profile,with relative molecular masses of 68 000 and 35 000 respectively.The single chain content in bulk was 75.7%.All the peptide maps of three batches of bulks were consistent with those of physiochemical references.The amino acid sequence at N-terminus,isoelectric point,protein content,residual exogenous DNA content of bulk,as well as the test results of Identity,appearance,pH value,moisture content,sterility,filling quantity and abnormal toxicity of final product,met the requirements in Chinese Pharmacopoiea(Volume III,2005 edition).Conclusion The developed methods ensured the safety,effectiveness and controllability of TNK-tPA,which might be used for routine control tests.