A stable process requires proper control of the medium feeding and harvesting rate, i.e., the perfusion rate. By real-time monitoring of viable cell concentration, maintaining a stable and optimal cell specific perfusion rate (CSPR) and adjusting cell harvest volume through parameters such as perfusion rate, a constant working volume and cell density can be maintained in the bioreactor.
In the absence of an online cell density control system, the semi-continuous cell bleeding method is most commonly used to control cell quantity. Continuous cell bleeding can be achieved through online and offline control systems to maintain stable cell density. However, offline control levels have a certain impact on production process performance and product quality, so this semi-continuous method must be used with caution in commercial-scale production. Volumetric productivity and product quality are two key performance indicators in biopharmaceutical cell culture processes.
Optimizing capacity utilization and production efficiency by increasing volumetric productivity while maintaining or improving product quality is crucial. In the production of recombinant human interferon-β (β-IFN) using CHO cells, compared with batch culture, low-temperature perfusion culture can increase productivity by 3.5 times, improve volumetric productivity by 7 times, reduce aggregates by 39%, and enhance biological activity to a certain extent.
Short-term perfusion in a bioreactor followed by conventional fed-batch culture with highly concentrated feed results in nearly double the overall productivity compared to traditional fed-batch processes. An integrated system of a perfusion bioreactor and four-column periodic counter-current chromatography (PCC) for continuous target protein capture achieves much higher volumetric productivity than current perfusion or fed-batch cultures.
Medium Screening
Medium development is one of the most important aspects in cell culture development and optimization. This is partly because the medium is primary for process performance and partly for safety considerations. The first cell culture media used animal-derived products, exposing patients using the corresponding products to many risks such as viruses. The concept of decoupled perfusion medium (i.e., feeding through different ports to better control specific nutrients and perfusion rates) has provided new insights for medium development.
A concentrated medium is obtained by mixing the basal medium and concentrated feed based on the fed-batch culture process in a certain ratio, on the basis of which an effective it medium is developed. After obtaining the optimal ratio and adjusting the concentration of some nutrients and osmotic pressure, the resulting medium only requires half the perfusion rate to maintain a density of 30×10⁶ cells/ml. This provides a reference for the development of it media.
Aeration Efficiency
During high-density cell perfusion culture, special attention should be paid to the effectiveness of the bioreactor’s aeration system to ensure a certain oxygen transfer rate (OTR) for sufficient oxygen supply to the culture medium. A bioreactor with excellent OTR can reduce aeration requirements, which in turn lowers the risk of exhaust filter clogging and high pressure inside the tank. At the same time, the bioreactor must be able to timely remove the large amount of carbon dioxide produced by high cell density to avoid its inhibitory effect on production and potential negative impact on the product.
Another issue that cannot be ignored is that due to the high gas flow rate and protein content in concentrated perfusion, excessive aerosols may form in the exhaust gas. Attention should be paid to reducing the risk of exhaust filter clogging and improving process stability.
Batch-to-Batch Stability Control & Critical Quality Attributes (CQAs)
There are two driving forces for the transition from batch production to continuous production: one is to save operational costs, and the more important one is to improve target protein quality. In discontinuous production systems, such as batch culture and fed-batch culture, accumulated toxic substances and reaction by-products have an adverse effect on the quality of the target product. In perfusion culture processes, a stable culture environment can be maintained throughout the culture period.
All kinetic parameters in the bioreactor, including those related to impurities or post-translational modifications, do not change over time. Therefore, almost identical products are produced in the bioreactor without the cumulative effects of batch and fed-batch cultures. Among quality attributes, galactosylation, non-fucosylation, and aggregates are comparable between shake tubes and small-scale bioreactor models. Other quality attributes are more dependent on product residence time. For example, deamidation, C-terminal lysylation, and cleavage differ in semi-continuous mode. Thus, perfusion culture can improve quality attributes by reducing the residence time of the product in the medium.
Metabolite Control & Cell Retention System
The robustness of the separation device is crucial for effective separation and achieving high cell density in perfusion culture processes. To ensure timely harvest of the target product without loss of the cultured suspension cells during perfusion, the cell retention device has become an essential element of this process.
Currently, there are two main types of cell retention systems used in perfusion culture: tangential flow filtration (TFF) and alternating tangential flow filtration (ATF).
Tangential Flow Filtration (TFF): In TFF, the cell suspension forms a continuous circular flow through the action of a peristaltic pump. After entering the fiber membrane, the waste liquid is discharged out of the system through the membrane, while the cells return to the culture system along the loop.
Alternating Tangential Flow Filtration (ATF): ATF achieves the reciprocating flow of the medium in the tank within the retention device through the reciprocating blowing and suction of a diaphragm pump. Meanwhile, metabolic wastes are discharged through the membrane along with the medium, and the cells are retained in the bioreactor. When using ATF, the alternating movement generates a scouring effect in the filter membrane, helping to prevent fiber membrane clogging. Additionally, ATF has advantages in improving volumetric productivity. When ATF is used instead of the internal spin filter (ISF), volumetric productivity can be increased by 50% to 70%.
About Ferbio
Ferbio has extensive experience in the manufacturing of various bioreactors and pressure vessels. We have a team of experts in the fields of bioreaction, fermentation process, mechanical manufacturing, and automatic control. Our scientific research and process levels are always at the domestic leading and international first-class standards, providing you with a comfortable, reliable, and reassuring product experience.