Electronic Journal of Biotechnology ISSN: 0717-3458 |
Vol.
10 No. 1, Issue of January 15, 2007 |
© 2007 by Pontificia Universidad Católica
de Valparaíso -- Chile |
Received November
14, 2004 / Accepted November 10, 2006 |
DOI: 10.2225/vol10-issue1-fulltext-15 |
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DNA removal from a purification process of recombinant hepatitis B surface antigen
Alejandro Beldarraín Iznaga*
Planta de Producción
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022. Ext. 2119
Fax: 537 2713208
E-mail: alejandro.beldarrain@cigb.edu.cu
Mayda Candelario Frontera
Laboratorio de Biología Molecular
Control de Calidad
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022, Ext 3253
Fax: 537 2713208
E-mail: maida.candelario@cigb.edu.cu
Javier Rodríguez Uramis
Planta de Producción
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022, Ext 2119
Fax: 537 2713208
E-mail: javier.uramis@cigb.edu.cu
José Blas Tejera González
Planta de Producción
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022, Ext 7279
Fax: 537 2713208
E-mail: jose.blas@cigb.edu.cu
Yodelis Calvo Parra
Laboratorio de Biología Molecular
Control de Calidad
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022, Ext 3253
Fax: 537 2713208
E-mail: yodelis.calvo@cigb.edu.cu
Yoel Madruga González
Departamento de Producción de Hepatitis B
Planta de Producción
Centro de Ingeniería Genética y Biotecnología
P.O. Box 6162
Habana 10600, Cuba
Tel: 537 2716022, Ext 2122
Fax: 537 2713208
E-mail: yoel.madruga@cigb.edu.cu
*Corresponding author
Financial support: This research was supported by CIGB grants.
Keywords: DNA-clearance factor, process characterization, rHBsAg purification process, spiking experiments.
Abbreviations: |
AP:
acid precipitation
API: active pharmaceutical ingredient
API-rHBsAg: active pharmaceutical ingredient of recombinant
hepatitis B surface antigen
C-D: concentration-diafiltration by tangential flow filtration
DS: desalting
GF: gel filtration
IAF: Immunoaffinity
NAE: negative anion-exchange
PAE: positive anion-exchange
PCR: polymerase chain reaction
PP: primary purification
rHBsAg: recombinant hepatitis B surface antigen
RF: reduction factor
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We
studied the capacity of an API-rHBsAg purification process to eliminate
DNA contamination from yeast-host cell. Firstly, was demonstrated
consistency of manufacturing purification process to remove DNA, from
(3.9 ± 1.9)108 pg/dose in starting material to (3.4 ± 1.6)
pg/dose, equivalent to 8.2 log in Active Pharmaceutical Ingredient
(API), measuring DNA quantity in several unit operations along manufacturing
process for twenty batches, five consecutive in 2000, 2001, 2003 y
2005. These values for API, lower than 10 pg/dose, accomplish current
WHO requirements for Hepatitis B vaccines obtaining by recombinant
DNA technology (WHO, 1989; European
Pharmacopoeia, 2001a). The main removal factor for manufacturing
process, equivalent to 6.4-log, was reached in negative anion-exchange
chromatography. Then, the
capacity of immunoaffinity chromatography and positive anion-exchange
chromatography to remove chromosomal DNA purified from yeast-host
cell was assessed using a scaled-down chromatographic process which
was shown to yield product meeting purity criteria set for the manufacturing
process. Log10 reductions for DNA through the immunoaffinity
chromatography and positive anion-exchange chromatography were 7.3
± 0.1, and 5.8 ± 0.1 respectively. Overall, these studies indicate
that total DNA clearance factor for API-rHBsAg manufacturing process
was 19.4 log, 2.4 times higher than the real DNA contamination, indicating
that API-rHBsAg manufacturing as described here have sufficient DNA
reducing capacity to achieved a high margin of DNA safety.
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