Characterization of lipoprotein supplement and influence of its oxidized lipid content on cell culture performance and monoclonal antibody production by a SP2/0 hybridoma cell line.

A challenging aspect with the use of the Sp2/0 hybridoma cell line in commercial manufacturing processes of recombinant therapeutic proteins is their exogenous lipids requirement for cell proliferation and optimal protein secretion. Lipids are commonly provided to the culture using serum or serum-derivatives, such as lipoprotein supplement. The batch-to-batch variability of these non-chemically defined raw-materials is known to impact cell culture process performance. Lipoprotein supplement variability and its impact on fed-batch production of a recombinant monoclonal antibody (mAb) expressed in Sp2/0 cells were studied using 36 batches from the same vendor. Several batches were associated with early viability drops leading to low process performance during fed-batch production. Increased caspase-3 activity (an indicator of apoptosis) was correlated to viability drops when low-performing batches were used. Addition of an antioxidant to the culture limited the increase in caspase-3 activity. Physicochemical characterization of batches confirmed that lipoproteins are mainly composed of lipids and proteins; no clear correlation between low-performing batches and lipoprotein supplement composition was observed. Controlled lipoprotein oxidation leads to lipoprotein solution browning, increasing absorbance at 276 nm and results in poor process performance. Because low-performing batches absorb more at 276 nm than other batches, oxidized lipids were suspected to be the root cause of low-performing batches. This study increased the understanding of lipoprotein supplement composition, its sensitivity to oxidation and its impact on process performance.

Characterization of soy protein hydrolysates and influence of its iron content on monoclonal antibody production by a murine hybridoma cell line.

A challenging aspect with the use of protein hydrolysates in commercial 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 vendor. The batch-to-batch variability of soy hydrolysates impacted cell growth, titer and product quality. Physicochemical 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 reduced significantly cell growth, mAb titer and increased level of the acidic charge variants of the mAb. Consequently, trace element composition of soy hydrolysates or of all incoming raw materials might lead to significant impacts on process performance and product quality and therefore need to be tightly controlled.

Review: High temperature short time treatment of cell culture media and feed solutions to mitigate adventitious viral contamination in the biopharmaceutical industry.

Events of viral contaminations occurring during the production of biopharmaceuticals have been publicly reported by the biopharmaceutical industry. Upstream raw materials were often identified as the potential source of contamination. Viral contamination risk can be mitigated by inactivating or eliminating potential viruses of cell culture media and feed solutions. Different methods can be used alone or in combination on raw materials, cell culture media, or feed solutions such as viral inactivation technologies consisting mainly of high temperature short time, ultraviolet irradiation, and gamma radiation technologies or such as viral removal technology for instance nanofiltration. The aim of this review is to present the principle, the advantages, and the challenges of high temperature short time (HTST) technology. Here, we reviewed effectiveness of HTST treatment and its impact on media (filterability of media, degradation of components), on process performance (cell growth, cell metabolism, productivity), and product quality based on knowledge shared in the literature.

Application of Quality by Design to the characterization of the cell culture process of an Fc-Fusion protein.

The production bioreactor step of an Fc-Fusion protein manufacturing cell culture process was characterized following Quality by Design principles. Using scientific knowledge derived from the literature and process knowledge gathered during development studies and manufacturing to support clinical trials, potential critical and key process parameters with a possible impact on product quality and process performance, respectively, were determined during a risk assessment exercise. The identified process parameters were evaluated using a design of experiment approach. The regression models generated from the data allowed characterizing the impact of the identified process parameters on quality attributes. The main parameters having an impact on product titer were pH and dissolved oxygen, while those having the highest impact on process- and product-related impurities and variants were pH and culture duration. The models derived from characterization studies were used to define the cell culture process design space. The design space limits were set in such a way as to ensure that the drug substance material would consistently have the desired quality.