Eminence’s High-Performance Perfusion Medium, Empowering Perfusion Process Development and Optimization

Over the past 30 years, the biopharmaceutical industry has experienced rapid growth, with the market expanding from approximately 4.4 billion USD in 1990 to over 180 billion USD in 2017. It is projected that the global biopharmaceutical market will reach nearly 500 billion USD by 2025. Currently, the production of biologics such as recombinant proteins, vaccines, and monoclonal antibodies relies primarily on mammalian cell expression systems. As the demand for biologics continues to rise, the development and optimization of mammalian cell culture processes have become a major focus of research. Continuous production, due to its advantages such as higher efficiency and flexibility, has gradually become a technology trend for biopharmaceuticals. As early as 2011, the U.S. FDA encouraged traditional batch production companies to transition to continuous production. In February 2019, the FDA issued a statement promoting continuous production, emphasizing that "continuous production is a key step in improving drug quality and drug production efficiency". Additionally, the FDA issued a draft guidance on quality considerations for continuous production. Although end-to-end continuous drug production still needs refinement, continuous production undoubtedly will be a significant direction for the future development of the pharmaceutical industry.

I. Perfusion Culture

Perfusion culture is an essential component of continuous bioproduction. In perfusion culture mode, nutrient-depleted medium within the reactor is continuously replaced with fresh medium through a cell retention device, while cells are retained inside the reactor. Compared to traditional batch or fed-batch culture, perfusion culture uses smaller-sized equipment to achieve comparable or higher yields to large-sized equipment, and to some extent, reduce costs while increasing operational flexibility. The continuous process based on perfusion technology has garnered increasing attention in the industry due to its advantages in the development of molecules such as recombinant proteins and multispecific antibodies that are prone to degradation and difficult to express. Currently, over 20 commercial products are produced using perfusion culture.

II. Optimization Directions for Perfusion Culture

1. Cell retention devices for perfusion culture

Cell retention is one of the major process elements for suspended cell perfusion culture, and its core challenge lies in effectively protecting cells from damage during retention. Cell retention devices are mainly designed based on principles such as filtration, sedimentation, and centrifugation. In large-scale perfusion culture, cell retention devices developed based on hollow fiber tangential flow filtration have become mainstream, with the advantage of achieving 100% cell separation and linear scale-up. There are two main types of cell retention devices in this category: tangential flow filtration (TFF) and alternative tangential filtration (ATF).

In TFF, cell suspension passes through a peristaltic pump to form a continuous circular flow direction. After entering the fiber membrane, waste fluid is expelled outside the system through the membrane, while cells return to the culture system along the loop. TFF technology changes the filtration direction using a peristaltic pump or other pressurization methods to make it perpendicular to the liquid flow direction, thereby reducing the risk of filter clogging. However, TFF may generate significant shear forces on cells, increasing the risk of cell lysis.

Alternative tangential flow (ATF) connects hollow fiber column, high-pressure air pump, and diaphragm vacuum pump. By adjusting the pressure difference between air pump and vacuum pump, cells and medium pass through the hollow fiber column alternately, achieving cell retention inside the hollow fiber column. Compared to traditional TFF, ATF uses a diaphragm pump instead of a peristaltic pump, reducing shear forces on cells. The periodic concave-convex movement of the diaphragm pump causes alternating reciprocating flow of the medium in the hollow fiber column, which slows down the rate of column clogging to some extent. ATF not only maintains high cell density but also improves product yield and quality, making it more favorable in perfusion culture processes.

2. Control of process parameters during perfusion

• Cell specific perfusion rate (CSPR)

CSPR is a critical process parameter, typically defined as the volume of fresh medium perfused per unit cell per unit time. The corresponding formula is:

The CSPR reflects the nutritional level provided to the cells by perfusion and does not represent the metabolic state of the cells themselves. In the development and optimization of perfusion culture processes, CSPR acts as a bridge between PR and VCD, providing important guidance. Reducing the CSPR value is an important factor in process design, as it significantly reduces production costs and improves product quality and concentration.

• Bleeding rate

In perfusion culture, when a specific cell density is reached, a portion of the cell-containing medium can be withdrawn by bleeding to maintain a constant working volume and cell density inside the reactor, while avoiding clogging of membrane of the cell retention device due to cell debris and released nucleic acids and proteins from dead cells.

During the stable phase of perfusion culture, while maintaining a sufficiently high cell density to ensure a high space-time yield (STY, g/(L·d)) of the target product, it is necessary to optimize towards reducing the bleeding rate to further improve product yield.

The basic principle of reducing the bleeding rate is to provide suboptimal growth conditions for cells through nutrient or environmental restrictions, thereby reducing the growth rate of cells. Typically, by arresting cells in the G0/G1 phase (preparation phase of the cell cycle), cell division can be delayed. During this phase, cellular metabolic activity, including synthesis of the target product, remains high.

• Cell-specific productivity (Qp)

Qp is typically defined as the yield of antibody protein per unit cell per unit time, representing the ability of cells to express recombinant proteins heterologously. It serves as a key technical indicator during the process of cell line construction, including primary and secondary screening. The corresponding calculation formula is:

Viable cell density, Qp, and culture period (depending on the maintenance of cell viability under high cell density) are often key factors influencing the space-time yield. From the perspective of cell lines, those with excellent growth, metabolism, and substantial yield can be obtained through cell line construction and clone screening for use in production. From the perspective of cell culture processes, various strategies can be adopted to optimize media and control parameters to improve space-time yield. Firstly, optimizing media formulations (including concentrations and ratios of nutrients) according to specific cell line characteristics and types of target products is essential to meet the needs of cell growth and product synthesis. Secondly, precise control and adjustment of various parameters during the culture process are crucial. For example, environmental factors such as temperature, dissolved oxygen, and pH can significantly affect cell growth and metabolic activity. By optimizing control strategies for these parameters, cells can be provided with the most suitable growth environment to promote their growth and product synthesis.

3. Perfusion medium formulation development

In the entire development and optimization process of perfusion culture technique, the design and optimization of perfusion media play a decisive role. Since continuous supply of media is required during perfusion, the formulation and performance of media directly affect cell growth and product synthesis. Today, with the dominance of fed-batch processes, there is still a lack of specialized platform media for perfusion available in the market. When exploring perfusion processes, biopharmaceutical companies often have to use a mixture of basal media and a small amount of feed media as perfusion media. These media have not undergone specialized perfusion formulation optimization and often fail to fully utilize the potential of perfusion media. For example, components in media that stimulate cell growth are essential in fed-batch processes, but they may not be necessary or even harmful during the production phase of perfusion processes. Therefore, the development of perfusion media must also be based on the analysis of media components. A common approach is to use MVA to identify key components and then utilize Design of Experiments (DOE) methods to rapidly optimize the concentrations of key components, thereby enhancing the performance of the media. Currently, the requirements for perfusion media in the market not only include supporting culture at lower cell specific perfusion rates (CSPR) to reduce media consumption but also maximizing cell culture time and protein yield. To achieve these goals, optimization of perfusion media formulation is necessary.

III. EmCD CHO^® 906 Perfusion Medium

Leveraging rich experience in CHO cell medium formulation development, Eminence proudly presents the independently developed EmCD CHO^® 906 perfusion medium. EmCD CHO^® 906 perfusion medium can support high-density growth and expression of CHO cells under perfusion culture. Stable inter-batch quality, rapid and flexible delivery, and professional after-sales service ensure smooth production for customers. EmCD CHO^® 906 perfusion medium is fully chemically defined and does not contain any animal-derived components, ensuring safety and stability throughout the cell culture process.

IV. EmCD CHO^® 906 Perfusion Medium Data Sharing

EmCD CHO^® 906 perfusion medium, independently developed by Eminence, is a high-performance Chemical Defined (CD) medium suitable for high-density perfusion culture of CHO-K1 cells. CHO-K1 cells can maintain a density of 50-60*10⁶ cells/mL for a long period with stable growth and expression, while critical quality attributes such as SEC and CEX remain stable throughout the cell growth process.

(Figure 1) Growth of CHO-K1 Cells in EmCD CHO^® 906 Perfusion Medium

(Figure 2) Lactic Acid Metabolism of CHO-K1 Cells in EmCD CHO^® 906 Perfusion Medium

(Figure 3) Antibody Expression Level of CHO-K1 Cells in EmCD CHO® 906 Perfusion Medium

(Figure 4) Antibody Quality of CHO-K1 Cells in EmCD CHO^® 906 Perfusion Medium