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Development and Validation of a Method for Quantifying HER1 Extracellular Domain in Culture Supernatant by RP-HPLC

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Abstract

Techniques based on high-resolution liquid chromatography are currently widely used to quantify recombinant proteins from culture supernatants. Such assays can be easily and rapidly developed. In this paper, we describe the development and validation of an analytical technique for quantifying HER1 extracellular domain (HER1 ECD) in bioreactor supernatant of HEK 293 transfectomes using reversed-phase chromatography with a C8 column in an HPLC system. The HER1 ECD protein was quantified by monitoring the absorbance of the sample at 214 nm. The resulting analytical methodology was found to provide precise and accurate results for a wide range of concentrations (10–120 µg/mL) of HER1 ECD. The accuracy of the method varied from 86 to 109 % depending on the protein concentration, while the repeatability and the day-to-day intermediate precision were less than 7.25 and 7.85 %, respectively. This methodology constitutes a useful tool that can be applied during the production of the HER1 ECD vaccine.

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Acknowledgments

We thank Loany Calvo M.Sc. from the Center of Molecular Immunology for his invaluable help with the validation assays and for his careful reading of the manuscript.

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Authors and Affiliations

  1. Center for Molecular Immunology, 216th Street to 15th, P.O. Box 16040, Havana, 11600, Cuba

    Yadira Prieto, Katia García & Dianelys Ochoa

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  1. Yadira Prieto
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  2. Katia García
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  3. Dianelys Ochoa
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Correspondence to Yadira Prieto.

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The authors declare no conflict of interest and/or competing financial interest.

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Prieto, Y., García, K. & Ochoa, D. Development and Validation of a Method for Quantifying HER1 Extracellular Domain in Culture Supernatant by RP-HPLC. Chromatographia 79, 311–318 (2016). https://doi.org/10.1007/s10337-016-3032-1

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  • DOI: https://doi.org/10.1007/s10337-016-3032-1

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