Production and separation of antibody-drug conjugates with a dye as drug substitute by continuous reactive chromatography

Antibodies, such as the most common immunoglobulin G (IgG) target antigens foreign to the body as a immune response to prevent diseases from spreading. The conjugation of monoclonal antibodies with active drug adducts is a promising way in modern healthcare to target and eliminate affected cells very precisely. Especially in the area of cancer treatment, antibody drug conjugates (ADCs) can enable through their improved selectivity the use of more effective drugs that are too harmful for healthy tissue otherwise, e.g. in conventional chemotherapy. The synthesis of ADCs, however, is not trivial: the efficiency is related to the nature of charge isoforms and the degree of conjugation on the multiple potential sites on the protein. In order to achieve better control of the product, reactive chromatography offers an improved production pathway. Adsorption of protein on a solid support prior to conjugation with a drug in liquid phase hinders the accessibility of most sites on a protein and allows only fewer sites to be active, thus leading to a more homogeneous degree of conjugation. Furthermore, the ADC can be recovered in a more pure form due to controlled release and capture from the column. In this work, IgG was conjugated in solution and in an on-column process using a dye as drug substitute, to study the influence of reaction parameters such as pH, conjugate ratio and column type on the conversion, selectivity and yield of the reaction. In solution conjugation, conversion of native IgG increases up to 70\% with a dye-to-protein (d/p) ratio from 1 to 3. Selectivity towards mono-conjugation, as the target defined in this work, decreases with d/p and over time due to the favoring of the formation of multi-conjugates. The best trade-off in terms of mono-conjugate yield here was defined as d/p = 1.5. Furthermore, in order to understand the reaction kinetics, a simple population balance model was developed as well, taking into account conjugation and dye hydrolysis. A fitted model can describe well the conversion and predict the individual conjugate evolutions, however, the limited capability to analytically distinguish mono-conjugates in the multi-conjugate peak lead to discrepancies in fitting the feed presenting charge heterogeneity. Finally, conjugation on column was investigated. From several options for adsorbates, a YMC-BioPro SmartSep column was chosen due to its performance in separation of the conjugates and native proteins. The optimized conditions in terms of pH = 7 and d/p ratio = 1.5 were applied comparing the reaction in condition of dye flow and not. The calculated performance parameters were compared along all the carried experiments. Comparable and promising results were exhibited in the reaction on-column in terms of conversion, selectivity and yield.

In questo lavoro viene simulata la coniugazione di un anticorpo con un farmaco citotossico mediante l'utilizzo di un fluorocromo. L'obiettivo del processo è massimizzare il prodotto monoconiugato. La reazione viene effettuata in soluzione e analizzata in diverse condizioni per trovare la performance ottimale. La cinetica della reazione è stata analizzata mediante un modello. Quindi è stata realizzata la coniugazione in colonna, testando diverse condizioni di lavoro, in modo da studiarne il possibile vantaggio e applicazione. Quindi vengono confrontati i risultati ottenuti per quanto riguarda la conversione, la selettività e il rendimento nei due diversi casi in esame.