The biopharmaceuticals market has experienced a rapid growth over the last few decades and this trend is expected to continue for the foreseeable future. One of the main challenges in developing a new biopharmaceutical is to assure long-term stability of a therapeutic protein in the final dosage form. Due to their complex structure and unpredictable nature, proteins in formulations can be affected by every constituent. Since these effects vary with concentrations, we must be able to quantitatively determine each excipient. Ionic properties of some excipients, e.g. salts and buffers, can be exploited with ion chromatography.
Our purpose in this master's thesis was to develop analytical methods for determination of the most common ionic excipients in biopharmaceuticals with ion chromatography, using suppressed conductometric detection. The development of two methods, for anions and cations respectively, was performed separately, though the process was identical in both cases. First, we optimized the chromatographic conditions in a way that would allow a successful separation of all ions within the shortest possible time. We achieved this mostly by varying the ionic strength of the mobile phase. After the final chromatographic conditions were established, we began validating the method in accordance with the intern validation procedure. We assessed linearity, accuracy and intra-assay precision in the selected concentration range for each individual ion. Later, we investigated the method's intermediate precision as well. One-week stability of all analytical solutions at 2–8 °C was also confirmed.
The analytical method for anions was fully validated for determination of acetate, chloride, succinate and phosphate. For their successful separation, a gradient of potassium hydroxide had to be utilised. The method for cations was fully validated for determination of sodium. Isocratic conditions with methanesulfonic acid were sufficient. We failed to separate tris(hydroxymethyl)aminomethane, also known as tromethamine, from sodium. The final method was thus deemed unsuitable for samples containing both sodium and tromethamine.
After validation we also performed a few regular analyses within the scope of several project studies at our analytical department. The determined ion contents in all of the samples were within expectations. All acceptance criteria, which were used to examine the instrument's performance and standard solution preparation, were met.
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