Production Division
Centro de Ingenieria Genetica y
Biotecnologia
Ave 31 e/ 158 y 190
Cubanacan
Playa
Habana 0600
Cuba
Tel: 5372716022
Fax: 5372718675
E-mail Address:
luciano.hernandez{at}cigb.edu.cu
Quality Control Division, CIGB
Havana, Cuba
Quality Control Division, CIGB
Havana, Cuba
Analytical Division, CIGB
Havana, Cuba
Development Division, CIGB
Havana, Cuba
I would like to comment about Heberkinasa (recombinant streptokinase): one of the preparations included in the article written by Dr Hermentin et al.1
The quality control system of the Center of Genetic Engineering and Biotechnology has been successfully evaluated by the World Health Organization and another International Regulatory Organizations.
The biological activity of the Heberkinasa batch 80010 was approved by our quality control laboratory with a value of 688 773 IU, using the clot lysis assay described in the British Pharmacopoeia of 1998 for the determination of potency of streptokinase. A secondary standard of recombinant streptokinase previously calibrated against the Second International Standard for the streptokinase (code 88/826) was used.
We participated in the International Collaborative Study organized by the NIBSC for the establishment of the Third International Standard for Streptokinase (laboratory number 4). Our results2 were similar to the ones obtained by the rest of the participant laboratories. We have compared a batch of streptase and kabikinase and a batch of Heberkinasa using the method described in the Pharmacopoeia and all the cases fulfil the established specification (potency is not less than 90% and not greater than 111%).
Using other methods described in the literature to determine potency of several plasminogen activators, clear clot3 and the chromogenic substrate (it does not use fibrin in the assay and for this reason it is considered as an indirect method), we have determined the biological activity of two different preparations of recombinant streptokinase (one of them Heberkinasa) and streptase. The Third International Standard for streptokinase was used for both the methods.
Heberkinasa and streptase fulfil the specification of the British Farmacopea (90111%) using the method of clear clot, unlike the other recombinant streptokinase.
The same batches were evaluated with the chromogenic substrate and contradictory results were obtained with the recombinant streptokinase. With this method, Heberkinase does not pass; however, the other product containing recombinant streptokinase and streptase passes satisfactorily.
Our conclusions are as follows: a recombinant protein although differing by only one amino acid from the sequence of a natural protein is considered to be another product; therefore, it should be evaluated against standards of the same origin calibrated previously against the international standard using direct methods.
The discrepancies between the potency claimed and that measured for several streptokinase preparations in the results published in this article are due to the employment of a method that uses as standard a material of different nature to the one used in the preparation that was measured (at least for the recombinant preparations).
Heberkinasa is obtained by the isolation and cloning4 of the streptokinase gene of a strain of Streptococcus equisimilis group C (ATCC 9542). Regarding the protein primary structure, we have characterized the molecule by N-terminal sequencing, amino acid analysis, and mass spectrometry. We have demonstrated the identity, integrity, and consistency of Heberkinasa during the years of production.
The N-terminal sequencing of the protein matches with the sequence presented by Hermentin et al.1 We have also identified two populations, one of them beginning with methionine. This phenomenon has been reported for other recombinant proteins produced in Escherichia coli.
Heberkinasa contains five mutated amino acids in relation to the streptokinase of S. equisimilis of the C group. The theoretical isoelectric point of our product is 0.06 units lower than the streptase, meaning that the protein is more acidic. The molecule of Heberkinasa has two arginines substituted by histidine and glutamic acid, which changes the charge density, which surely modifies its mobility in native PAGE electrophoresis (Heberkinasa migrated slightly more rapidly than natural streptokinase).
Peptide mapping of the protein is frequently done, which allows the verification of 100% of its primary structure. Additionally, molecular mass determination of the intact protein with an accuracy lower than 1 Da assures the integrity of the protein. These amino acid changes in the molecule do not affect the safety, purity, and potency of the new drug produced by recombinant DNA technology.
No differences regarding tissue distribution and clearance mechanism were observed when comparing Heberkinasa with the natural product in pharmacokinetic studies.
References
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