Cell Culture Technology and Main Development
The cell culture process can be served as the process of recombinant protein production. In cell culture, the change of technological parameters will directly influence the quality attribute and consistency of the product. Moreover, in some culture modes, the culture environment changes over time, so cell density, specific productivity, and protein mass may be affected accordingly. Some culture environment or parameter changes may even affect key quality attributes of proteins, such as folding errors, amino acid sequence changes, and glycosylation modification types. Therefore, it's necessary to select an appropriate culture mode and optimize the process parameters to ensure its stability, consistency, and scalability.
At present, the main culture modes used in industrial production include batch culture, fed-batch culture, and perfusion culture.
Fed-batch Cell Culture
The batch culture mode has no nutrient supplement, so its production cycle is short and the yield is low. It's mostly used for seed chain amplification. In fed-batch mode, nutrients are added in batches to maintain cell growth and viability, which can greatly prolong the culture cycle and increase production. In terms of perfusion culture, cells are blocked in the bioreactor by a cell interception device, and a relatively stable culture environment is maintained by a continuous replacement of fresh medium at a constant rate, to further increase cell density, improve cell viability, maximize specific production rate, and prolong the culture cycle.
Regardless of the culture mode, a stable feedback loop must be used to control key process parameters during cell culture in a bioreactor, including temperature, dissolved oxygen content (DO), pH value, and stirring. In addition, cell interception and recycling must be considered in perfusion culture. Contamination and excessive shear stress need to be avoided during cell interception, otherwise, the efficiency of the perfusion system may decrease over time. And cell viability and density should be constantly monitored as well to enable productivity. In addition, nutrient supply determines the growth rate, physiological state, and productivity of cells. Therefore, the perfusion rate matching the metabolic waste removal rate should be set according to the cell consumption rate, which not only needs to keep the culture volume constant but also needs to ensure enough nutrition to meet the needs of cells without waste.
Perfusion Culture
In perfusion culture systems, the most commonly used cell interception device is a porous membrane. In general, with the extension of culture time, the expected flow rate of the filter may be different from the actual flow rate due to blockage. To prevent clogging, the aperture of the filter is appropriately increased, but this could lead to product loss. Currently, popular self-cleaning membranes such as tangential flow filtration (TFF) and alternating tangential flow filtration (ATF) membrane systems can reduce the possibility of filter contamination or clogging to some extent. The two systems share a similar principle in that the medium is injected tangentially into a membrane with a specific pore size that allows only the medium to flow through (filtrate), while the cells (residue) continue to flow along the feed fluid. As a result, the cell-containing holdup is returned to the bioreactor while the filtrate is continuously harvested. In both membrane systems, the culture flows tangentially across the membrane, reducing the risk of contamination and clogging of the filter. The difference is that in TFF, the feed fluid travels through the film in a single direction while the ATF system includes a pump that generates a two-way flow across the membrane, further reducing the potential for filter contamination and clogging. Therefore, the latter has a lower rate of product interception and higher efficiency of product separation. In short, both methods have less risk of contamination and clogging and are simple to operate and scale up.
By integrating the upstream perfusion culture system with the downstream multilayer column unit, an integrated production platform can be established. Its advantages lie in the following aspects: it can shorten the residence time of the proteins in the culture medium so that the quality of the product is more uniform. The protease and secretory enzyme in the culture medium can be quickly separated from the proteins to avoid affecting the quality properties of the protein. The scale of the integrated molding continuous process is generally small, so disposable technology is often used to replace stainless steel equipment for production. To complement the upstream continuous process, a continuous purification process must be designed to treat the upstream harvest solution. Multiple chromatographic columns are often utilized, switching during the process to prevent resin overload and product loss. Compared with the traditional batch capture cycle, this operation mode can greatly improve resin utilization. However, virus inactivation in continuous systems remains a major challenge, especially because of the high risk of contamination and clogging of virus filters, which still need to be optimized and improved.
Major considerations in scaling up/down of Cell Culture
In large-scale cell culture, the process of seed-train expansion usually requires continuous passage many times. If the design of the seed chain is not reasonable, it may lead to slow cell growth. Moreover, seed chains need to be adjusted and optimized for specific cells and processes since they are not immutable. In addition, the change in equipment structure and volume will also affect the culture environment, so it is necessary to adjust the relevant control parameters accordingly. Therefore, high-throughput experimental design methods should be developed to screen, analyze, and optimize process variables to ensure consistent cell passage and growth conditions at all scales.
In traditional production, the manufacturing capacity needs are generally met by scaling up. Stainless steel bioreactors of 20000 liters and above and related equipment are often used for scale-up. However, too large a scale often leads to lower productivity and a heavy burden of process development. Moreover, due to the flow rate, processing capacity, hydrostatic pressure, and other factors, the upstream and downstream equipment requirements are also high. In addition, additional preparation time and cleaning steps are required. As a result, the use of relatively flexible disposable technologies has become increasingly common in recent years. Because the scale of disposable bioreactors is smaller, generally about 1/10 of the stainless steel reactors, it is sometimes necessary to reduce the scale to meet the needs of disposable production.
In addition, to better proceed with scale-up, a suitable small-scale culture model should be selected to simulate large-scale production conditions, and optimize the process to reduce the risk of scale-up. When designing the scaling-down model, the scale differences caused by velocity, hydrostatic pressure, mixing, mass transfer, and other factors should be fully considered as well.
Process Monitoring in Cell Culture
Proper control strategies have to be adopted in the cell culture process. High-precision detection equipment or sensors are used to monitor data in real-time to identify changes in process conditions and conduct corresponding feedback control accordingly. Currently, some highly sensitive probes have been able to continuously monitor DO, pH, and temperature, which can meet the needs of real-time control. In the future, next-generation manufacturing processes are designed to automate analysis with robust process analysis techniques (PAT), eliminating manual sampling operations and off-line testing, and facilitating real-time adjustment of process control parameters, nutrient supply strategies, and other process conditions.
Cell density and cell viability are key indicators of cell culture and are detected by the trypan blue exclusion method in most cases. Although this detection method can meet the needs of fed-batch culture, it may require real-time detection of cell density and viability in some processes. For example, in continuous perfusion culture, it may be necessary to measure cell density in real time to determine cell release and culture medium perfusion rates to prevent excessive cell loss and meet cell nutrient requirements. Sensors that can detect cell biomass or density online are often used to enable real-time feedback control. The biomass is generally characterized by cell capacitance, and the results are consistent with Trypan blue exclusion method in the exponential growth phase. In addition, the optical system can also capture cell images in real time and conduct an in-depth analysis of morphological changes to determine the physiological state of cells.
