Biopharmaceuticals are mostly protein molecules that have a much higher molecular weight and more complex structure compared to small synthetic active pharmaceutical ingredients. Production process and characterization of the structure is crucial in development of these molecules. Therapeutic monoclonal antibodies are becoming one of the fastest growing groups of biopharmaceuticals. Monoclonal antibodies are heterogeneous molecules in both size and charge. Charge variants of monoclonal antibodies are one of the critical quality attributes. They have to be characterized, quantified, and their impact on drug safety and efficacy has to be determined. Capillary isoelectric focusing (cIEF) is one of the methods used to analyze and characterize charge variants that are separated into main, acidic and basic groups based on their isoelectric point and are presented as seperated peaks on the electropherogram. In the master's thesis we tested the suitability of the generic isoelectric focusing method in the development of three biological molecules. For one of the molecules we did not achieve a good resolution, therefore we optimized the method by changing composition of reaction mixture and focusing conditions. In the generic method we use pharmalyte, which covers a wide pH range (3-10), urea concentration 2.0 M and focusing time 6 minutes. In the optimized method, resolution was improved by adding a different pharmalyte, which covers a narrower pH range (8-10.5) and by extending the focusing time to 10 minutes. For all three molecules we determined the range in which the method is linear and tested desalting of samples. We also confirmed that the method is suitable for detecting changes in charged variants of a protein when exposed to stress contiditons.
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