Abstract
The aim of this study was to demonstrate the reliability of the protein determination methods used in the process of recombinant human insulin development before its scale up. The total protein content was measured by Bradford, molar extinction coefficient, and dry weight methods. The standards were analyzed using Mono-Q, Aquapore RP300, and Kromasil columns to calculate the concentrations of the proteins using the theoretical extinction coefficient and peak area. The following highly purified standards were used: batches B4-258 and QS009-010 of sulfonated fusion protein; batches B4-267, B4-268, RALF-018, HGUT-042, HGUT-043, and HGUT-045 of renatured fusion protein; the United States Pharmacopeia reference; and batch B4-253 of bulk insulin crystals. The results were analyzed using ANOVA or Student’s t-test at 95 % significance. The Bradford method showed up to 60 % variation for all evaluated standards, while the remaining two methods were consistent with each other. The chromatographic parameters were used to validate the analytical methods, and all results met the current guidelines of Brazilian regulatory agencies. The use of quality parameters and the statistical evaluation of the data demonstrated that analytical methods used in the in-process control are suitable for the intended purpose, which certifies the reliability of the generated data.
Highlights
The hormone insulin has been prescribed in the treatment of diabetes mellitus since it was discovered in 1922 (Karamitsos, 2011), and it became one of the most important products in the biopharmaceutical industry
The use of quality parameters and the statistical evaluation of the data demonstrated that analytical methods used in the in-process control are suitable for the intended purpose, which certifies the reliability of the generated data
The technology for producing recombinant human insulin crystals can be used on an industrial scale, with quality standards that meet the requirements of the World Health Organization (WHO) and Food and Drug Administration (FDA)
Summary
The hormone insulin has been prescribed in the treatment of diabetes mellitus since it was discovered in 1922 (Karamitsos, 2011), and it became one of the most important products in the biopharmaceutical industry. With the discovery of and the advances in the technology of recombinant DNA in the 1970s, insulin was the first product that was developed and produced by recombinant DNA technology and approved by the Food and Drug Administration (FDA), allowing great advances in medicine as well as in the biopharmaceutical industry (Astolfi-Filho et al, 2004; Baeshen et al, 2014; Hwang et al, 2016; Polez et al, 2016; Mikiewicz et al, 2017). The technology for producing recombinant human insulin crystals can be used on an industrial scale, with quality standards that meet the requirements of the World Health Organization (WHO) and FDA. Multiple methods, qualitative and quantitative, are available for determining the protein amount and the purity and impurity profile
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