In recent years, biologics have become a continuously growing category in the pharmaceutical industry. In order to obtain regulatory approval and successfully enter the market, biologics must be produced under conditions that ensure their safety, quality and efficacy. Biologics are most commonly produced using living cells, which requires complex target product isolation and purification strategies. These isolation and purification strategies must remove several different types of impurities, such as different variants of the target product, host cell components, substances added or produced during the process, and various microbiological contaminants. Currently, most isolation and purification processes of modified therapeutic proteins are based on chromatographic techniques. These have many advantages over other techniques, such as high resolution, robustness and yield. Selection of chromatographic technique or chromatographic resin and the optimization of the working conditions are crucial for achieving the highest yield and purity.
As part of the master's thesis, we were determining the removal efficiency of product- (product aggregates) and process-related (HCPs, Protein A and DNA) impurities or the maximum reduction level of these impurities by affinity chromatography with protein A, multimodal and cation exchange chromatography. The removal efficiency of selected impurities at standard process parameter settings and standard performance of each chromatographic technique is known from the results of previous studies. Under such conditions, usually after the first or second chromatographic step, the content of selected impurities is below the quantification limit of the analytical methods, making it impossible to determine their actual removal capacity. To determine the latter, the chromatographic steps were performed under worst-case conditions and with significantly increased impurities content. The latter was achieved by adding impurities to the starting intermediates. Therefore, we wanted to determine whether such conditions, when the chromatographic steps are performed linked or separately, impact the efficiency and reliability of chromatographic steps in biologics production with a required high level of purity.
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