Investigation of purification process stresses on erythropoietin peptide mapping profile


1 Department of Recombinanit Biopharmaceutical Production, Pasteur Institute of Iran, Karaj, Iran

2 Department of Quality Control, Pasteur Institute of Iran, Karaj, Iran

3 Department of Pilot Biotechnology, Pasteur Institute of Iran, Tehran, Iran


Background: Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results.
Materials and Methods: Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h.
Results: No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation.
Conclusions: Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance.


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