title: Variability and viral safety of cell culture media to ensure process performance in the biopharmaceutical industry
Creators: name: Djemal, Leila
Publisher: name: UNSPECIFIED
Year: 2022

description: This PhD project focuses on two topics related to the optimization of the cell culture media for monoclonal antibody (mAb) production in the biopharmaceutical industry. The first topic aims at developing a better understanding of the batch-to-batch variability of soy protein hydrolysates used in cell culture media to ensure robustness of the mAb production process. The second topic is about mitigation of viral contamination risk of cell culture media using high-temperature short to ensure safety of mAb production processes. A challenging aspect with the use of protein hydrolysates in the manufacturing processes of recombinant therapeutic proteins is their impacts on the protein production due to a lack of understanding of batch-to-batch variability. Soy hydrolysates variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 37 batches from the same supplier. The batch-to-batch variability of soy hydrolysates impacted cell growth, mAb titer and mAb quality. Physico-chemical characterization of batches confirmed that soy hydrolysates are mainly a source of amino acids and peptides containing lower amounts of other components such as carbohydrates and chemical elements in cell culture media. Soy hydrolysates composition of different batches was consistent except for trace elements. Statistical analyses identified iron as a potential marker of a poor process performance. To verify this correlation, two forms of iron, ferric ammonium citrate and ferrous sulfate, were added to a batch of soy hydrolysates associated to a low level of iron during cell culture. Both forms of iron significantly reduced cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Excess of iron in the process might lead to significant negative impacts on process performance and product quality. This lethal process, named ferroptosis in the literature, is described by the iron Fe(II)-dependent accumulation of lipid reactive oxygen species and the peroxidation of polyunsaturated fatty acids leading to their depletion and the accumulation of toxic lipid reactive oxygen species (ROS). To counterbalance the negative effect of excess of iron, an iron chelating agent and a specific inhibitor of ferroptosis were used in our assays. Increasing levels of the iron chelating agent in the cell culture media led to a significant increase of the mAb titers for batches of soy hydrolysates containing high level of iron. Ferrostatin-1 supplementation, a known inhibitor of ferroptosis, did not offer cell protection from the excess of iron during cell culture in our assay. Two main potential causes of increased iron were investigated in close collaboration with the vendor of soy protein hydrolysates: the soy starting material and the manufacturing process steps. Trace elements in soy starting material and all along the manufacturing process were measured to identify the source of the higher level of iron in the soy hydrolysates. The iron content in soy starting material remained steady, while during the manufacturing process iron content tended to increase. The manufacturing step causing iron increase in soy hydrolysates was identified. Consequently, we highlighted how the manufacturing process may impact the final trace elements composition of soy hydrolysates and finally mAb production. Those observations allowed to define an additional critical quality attributes of soy hydrolysates to reach a better management of soy hydrolysates batch-to-batch variability for the monoclonal antibody production. It is important to notice that variable levels of trace elements can occur for other raw materials used in cell culture media, including chemically defined cell culture raw materials. Therefore, it becomes important to control trace elements levels in raw materials used in cell culture media to ensure process performance. In addition, fingerprinting analysis of soy hydrolysates combined with chemometric did not allow to build a model that predicts the performance of a given batch of soy hydrolysates using our dataset. Only LC-MS analysis brought to light some potential markers of a good performance. In addition to batch-to-batch variability concern associated to cell culture medium raw materials, cell culture medium raw materials are also known as the major source of viral contamination in the biotechnology industry. The prevention strategy of viral contamination events can be reinforced by eliminating or inactivating adventitious viruses in cell culture media during the manufacturing process. Filters of 0.1 μm porosity are commonly used to protect bioreactors from bacteria and mycoplasma contaminations but do not offer protection from viral contaminations. High-temperature short-time (HTST) treatment of cell culture media is known to be an efficient barrier to inactivate viruses. One major challenge of HTST application is the compatibility with medium composition. Media may contain heat-labile components that can precipitate or get degraded during heating. Here, the compatibility of HTST treatment with media and feed solutions of two fed-batch processes for monoclonal antibody (mAb) production were assessed. Media were heated at least at 95°C for 5 or 10 seconds using the FT74-20-MkIII-UHT/HTST system. HTST treatment did not affect the measured physico-chemical properties of the media and the feed solutions except for one complex medium for which heating induces an increase of the medium turbidity. A precipitate was also collected on the surface of the HTST heat exchanger plates identified as a mix of hydroxyapatite and iron oxides. A fluidized sand batch was used to collect, analyse, and troubleshoot the precipitate formation associated with high temperature treatment of this cell culture medium. Calcium phosphate precipitation was identified as the cause of the increased turbidity of the complex medium. The presence of iron chelators in the medium in contact with stainless steel was pointed out to be the cause of iron oxides formation. Nevertheless, for both fed-batch processes, the use of HTST system neither impacted the cell culture performance negatively, nor the product quality. Therefore, HTST treatment is compatible with media except for those containing high ratio of calcium and phosphate or, in this example, iron chelating agents.