Seed Train Intensification Using High Cell Density Cryopreservation

In upstream bioprocessing, a seed train scales a small volume of cell culture to a larger volume to inoculate intermediate bioreactors, and eventually the production bioreactor. This step, if using the traditional expansion process, is often time consuming. However, the development of seed train intensification techniques such as high cell density cryopreservation (HCDC) offer several benefits.

The Traditional Fed-Batch Seed Train

The traditional seed train process begins with a 1 mL vial of cells thawed from a master cell bank and expanded to a level sufficient to inoculate the bioreactor. This population of cells is then expanded to up to 15,000 L stainless steel bioreactors or 2,000 L single-use bioreactors (Figure 1, top). This process takes between 20-30 days before inoculating the production bioreactor and requires open cell culture operations that increases the risk of contamination and reproducibility issues.

High Cell Density Cryopreservation: An Alternative to Traditional Seed Train Expansion

In high cell density cryopreservation, or HCDC, cells banked at high density (ex: 50-100 x 10⁶ VC/mL) and high volume (150 mL) can intensify the seed train expansion process. HCDC removes the need for traditional seed train expansion and begins with frozen seed train intermediates in a single-use bag. These cells then undergo one seed train expansion and can then be used to inoculate the bioreactor (N-x stage) (Figure 1, bottom).

Figure 1. The traditional seed train process (top) vs. HCDC (bottom). HCDC enables one seed train expansion step at the central site to support several production campaigns, including decentralized manufacturing facilities.

Benefits of HCDC

Quicker Seed Train Expansion

In the example shown in Table 1, a 150 mL bag of 50 x 10⁶ cells/mL (total cell count: 7.5 x 10⁹) reduced the standard process by ten days as compared to starting with a 1 mL vial of 10 x 10⁶ cells/mL. This time saving is achieved by starting the expansion in N-3.

Table 1. Comparison of process using inoculation with a vial versus a bag. HCDC cells significantly reduced the process time.

Bioreactors	Production Bioreactor	Volume (L)	Cell count for Inoculation (10 VS)	Run time (without tag-phase) (d)
	N	15,000	7,500,000	3.5
	N-1	1,250	625,000	6.9
	N-2	105	52,083	10.4
	N-3	8.70	4,340	13.8
	N-4	0.72	361	17.1
	N-5	0.06	30	20.7
	N-6	0.005	2.5	24.2

Closed Processing

In HCDC, there are no open cell culture operation steps in manufacturing, which significantly reduces contamination risks and can lead to a lower room classification for GMP manufacturing.

Better Reproducibility Between Batches in Seed Train Expansion

HCDC also offers better reproducibility in seed train expansion, which is not only important for manufacturing, but also in R&D and process development. Creating a reproducible manufacturing process can be difficult when working with living organisms. Overall expansion time can vary between batches due to differences in recovery time of cell vials after thawing. Freezing cells in larger volumes using cryobags provides the same starting point for all experiments and because these cryobags are created further along in the expansion process, there is less time for larger deviations in recovery time.

Decoupling Cell Expansion and Batch Production

HCDC also allows decoupling of cell expansion and batch production activities so that distribution of cells from a central expansion facility to global decentralized production facilities is possible. In the conventional production process, every production campaign starts with a vial of cells. If HCDC is incorporated into the process, one vial is thawed and expanded, but then several bags are generated that can be used in multiple production processes (Figure 1).

Regulatory Advantages of HCDC

Furthermore, HCDC can offer advantages from a regulatory perspective. The traditional seed train expansion process involves creating a master cell bank (MCB) and a working cell bank (WCB), from which manufacturing campaigns can be started. A WCB is a highly regulated aspect of each registered biopharmaceutical manufacturing process, requiring specific documentation for the cell line to be registered and characterized. Each vial used in traditional cell expansion processes comes from a working cell bank, which is expensive to create, test, and maintain.

With HCDC, one expansion from a single WCB vial is performed, and the cells are then filled into cryobags in a closed manner. These cells are then classified as a process intermediate instead of a cell bank, which simplifies the registration of such intermediates in existing manufacturing processes.

Implementing an HCDC Process

Both the cell culture medium and the bag assembly are essential for successful HCDC. Below is an outline of the steps used to implement the HCDC process, from choosing the cell culture medium and assembly and to filling and thawing the cryobag.

Choosing the Cell Culture Medium

While different cryomedium formulations can be evaluated for use in HCDC, it's best to ensure the chosen formulation for expansion, freezing, and subsequent expansion works well with the production medium. The goal is to maximize growth, minimize any lag phase when moving from one medium to the other, and minimize cell damage during freeze and thaw. Learn more about some guidelines and best practices for DMSO concentration and freezing technique in our accompanying technical article.

The Single-use Assembly for HCDC

A single-use assembly designed for the HCDC process (Figure 2) contains a cell suspension line and a one liter waste bag (for flushing the tubes when switching from filling the bags with medium to filling the bags with the cell suspension). The bag can be sealed and disconnected with a crimping tool to ensure a closed and sterile environment. A line on each cryobag can be connected to a bioreactor after thawing for inoculation.

Figure 2. Parts of the HCDC single-use assembly.

Parts of a single-use assembly for HCDC:

1. Cryo medium filling line
2. Cell suspension line / connection to bioreactor
3. 1 L waste bag for flushing lines
4. Cryobags
5. Metallic pinch pipe for sterile cutting with a crimping tool
6. Line for connection to bioreactor after thaw

Filling the HCDC Bag Assembly

Figure 3. Filling HCDC bag assembly.

To fill the bag assembly, a container with the cryomedium is connected. This medium should contain a concentrated cryoprotectant, which is diluted with the addition of the cell suspension to bag (Figure 3). Once the bag is filled, it can be disconnected and placed into a freezer in a supportive case.

Inoculating the Bioreactor

Figure 4. Process for inoculating the bioreactor.

To use a bag for inoculation, remove it from the freezer, thaw it at 37 °C, and connect it to the bioreactor (Figure 4). The cells can then be added either with a pump or gravity.

Similar Viable Cell Density and Production in Bioreactors Seeded from HCDC Bags vs. Control Vials

We evaluated the impact of the bag container itself on viable cell density, viability, and IgG concentration. CHO-K1 cells were grown in a bioreactor seeded from traditional vials. The bioreactor was run in batch mode until day three at which time perfusion mode was initiated. When the cells reached densities of 15x10⁶, 37.5x10⁶, 75x106, 112.5x10⁶, and 150x10⁶ cells/mL, cell suspensions were used to generate cryobags and vials, which would serve as a control. The viable cell density, viability, and IgG concentration from cultures initiated either from vials or cryobags. Cryobags were thawed and used to inoculate shaketubes which were compared in a batch. No major differences could be observed between conditions (Figure 5).

Figure 5. Comparison of viable cell density, viability, and IgG concentration from cells seeded from vials or cryobags.

In a proof-of-concept experiment, we found that cells arising from HCDC bags vs. control vials generated comparable viable cell density and titer in production bioreactors. After thaw, the bags were used to simulate a production campaign in lab-scale, using a frozen intermediate for inoculation of the perfused N-1 bioreactor, followed by a steady-state perfusion bioreactor step. A standard expansion starting with thawing of a vial was used as control. Results showed that growth and productivity were comparable (Figure 6), confirming that the HCDC application can be implemented without any negative effects on cell performance.

Figure 6. Growth and titer in simulated production bioreactor for CHO-K1. Growth and titer were comparable for both bioreactors during steady state.

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