In a previous post, I outlined the basic departments and specialties, along with some of the easily accessible transition points between them, and will now begin a deeper dive into each of those departments in this post series. First in line is the Upstream department, which comprises several vital functions in process development, and without which the biotech industry wouldn’t have any products at all!
Cell Line Development – Where it All Begins
Once a candidate molecule has been selected, it’s time for the Upstream team to begin the process of producing it in a living cell line. This begins in the Cell Line Development (CLD) group, and starts off with the selection of the type of cell line for production. Biotech products most often use Chinese Hamster Ovary (CHO) mammalian cell lines, but E. Coli microbial cell lines are fairly common, and occasionally other options including yeast or insect cell lines are selected. After the cell line type is selected, the blank cell line is modified to produce the molecule of interest via transfection, which typically uses a viral vector to insert the genetic code for the new molecule into that of the cell.
Transfection produces varying rates of success in the cells that it is successful in, and this moves to the second step of CLD, which is clone selection. Often a round or two of “pool” cells are grown up to produce some initial product for early screening purposes, and these pools are also then sorted and the best cells selected for further evaluation during clone selection. Once some promising clones are isolated, they are grown in small quantities (50 – 200 mL) for further evaluation by both upstream and downstream criteria. The upstream team is looking for a cell line that has good viability, good cell growth, reasonable doubling times, and high product production (titer). The downstream team is looking for a cell line that produces high titer but with low aggregation, low host-cell protein (HCP), and a minimum of any other product-related impurities. Data from both teams is evaluated, and the top performing clone is selected for further development. If resources allow, a back up clone is often banked for insurance.
After the final clone is selected and stabilized, the CLD group’s final task is to lay down the Master Cell Bank (MCB), which is what will supply all needs for cell culture for the life of the product. This is done by growing up the cells in a GLP environment, performing various quality tests (such as ensuring they are mycoplasma-free), and then carefully vialing them aseptically and freezing them down in liquid nitrogen, where they will be stored until needed. MCB’s typically produce hundreds of vials, and the effort to lay down a quality MCB is not insignificant but is critical to the long-term success of the production cell line.
Bioreactor Development – Deep Thoughts for Deep Tanks
Initial bioreactor development may begin at the pool stage of clone selection, depending on the timeline and the resources available, but most of the work will be carried out after clone selection is completed. Once the final clone has been selected, the bioreactor team will get to work to determine the tweaks to the upstream process will produce the best results. This process is ongoing and involves scaleup throughout the work, starting with shake flasks, moving to small bioreactors (250 mL – 15 L), then to larger bioreactors or wave bags (10 L – 40 L), and then to the largest bioreactors typically used in process development (100 L, occasionally 500 L). The cell culture production will almost always end up in a Single Use Bioreactor (SUB) these days, although there are still older facilities and processes that run in reusable stainless steel bioreactors.
The bioreactor team will monitor cell growth and product production, taking care to ensure that the cells are being fed adequately, that the pH and osmolality of the culture is maintained as the cells require, and that mixing within the tank is sufficient for gas/nutrient/waste transfer but not so vigorous as to damage the cells in suspension. Most cell lines operate with chemically defined media, although there are still a few holdouts using soy isolates, and that includes defined glucose and other nutrient feeds throughout the culture. Duration of the cell culture between tank inoculation and final harvest is set during this work, as is any temperature shifts required to induce product production. A key challenge for this team is often the different geometry and mixing behavior of the different size tanks or SUBs used, which must be accounted for not only during development but for scaleup to manufacturing. The final cell culture conditions, from thaw through expansion and the N-stage (final) reactor, will be set by the end of this part of the development process.
Harvest Development – Product Payoff
All the cell line and bioreactor development in the world won’t help if the harvest isn’t successful, so while this is often a last-step (or even an afterthought), having a well-designed harvest process is key to a successful production run. Most smaller reactors used in early development are harvested via centrifugation followed by sterile filtration, but centrifugation is not typically a solution used at manufacturing scale. The most common harvest method is depth filtration, which uses progressively finer media in filters to remove the cells and cell debris while allowing the cell culture liquid containing the product of interest to flow through. There are several different manufacturers of depth filtration trains available, and within each vendor there are several options for which filters to include in the harvest train depending on the product and its needs. A high cell density process will require different filters than a low cell density process, for example, and some recent innovations including charged depth filters can provide additional clarification and even some removal of process-related impurities such as host cell proteins (HCP). Ensuring that the capacity of the filter train is sufficient to handle the volume of cell culture is critical, as is determining the filter flushing procedure (both pre and post harvest) required for the best product quality.
Once the harvest is in the bag, it is handed off to the downstream team for the purification into the final drug substance – the topic of the next post!
Upstream Development – Other Considerations
Several of the product quality attribute ranges are often set by the upstream process, as that’s where the molecule of interest is being produced. The cell line and cell culture are responsible for the proportion of acidic/basic variants, glycosylation of the final product, folding errors that lead to aggregation, proteases from the cells that lead to clips, and more. The downstream process can clear product and process related impurities, but often is unable to make significant impacts to acidic/basic variants, folding errors, or glycosylation without significant and unusual steps such as a refold. In the case of bispecific molecules, the cell culture can also impact how much of the desired heterodimer is produced as opposed to either of the homodimers.
When a project makes it into late-stage process characterization, a Working Cell Bank (WCB) may be set up and the process to go from MCB to WCB defined for the future. This allows the vials from the MCB to last even longer, as each MCB vial can be used to generated a WCB of another few hundred vials, providing support for commercial runs beyond the number of original MCB vials. Process characterization for upstream will also involve the Limit of In-Vitro Cell Age (LIVCA) study, which defines how long the cells can be passaged and used to inoculate tanks, which is particularly important for commercial processes that will be produced in multi-run campaigns where it is desired to inoculate those multiple runs from a single MCB or WCB vial thaw.
So Many Skills, Just One Department
The Upstream development team requires a wide range of skills for the different aspects of development, and knowing exactly what expertise is needed to ensure a robust and successful process is key. Bringing together cell line development, media development, reactor sampling and analysis, bioreactor development, and harvest development is necessary for a solid commercial upstream process that will pass regulatory standards and produce safe product for the lifetime of the project.