Automated Counterflow Centrifugal System for Small-Scale Cell Processing

Summary

Automation is key to upscaling and cost management in cell manufacturing. This manuscript describes the use of a counterflow centrifugal cell processing device for automating the buffer exchange and cell concentration steps for small-scale bioprocessing.

Abstract

Successful commercialization of gene and cell-based therapies requires manufacturing processes that are cost-effective and scalable. Buffer exchange and product concentration are essential components for most manufacturing processes. However, at the early stages of product development, these steps are often performed manually. Manual dead-end centrifugation for buffer exchange is labor-intensive, costly, and not scalable. A closed automated system can effectively eliminate this laborious step, but implementation can be challenging. Here, we describe a newly developed cell processing device that is suitable for small- to medium-scale cell processing and aims to bridge the gap between manual processing and large-scale automation. This protocol can be easily applied to various cell types and processes by modifying the flow rate and centrifugation speed. Our protocol demonstrated high cell recovery with shorter processing times in comparison to the manual process. Cells recovered from the automated process also maintained their proliferation rates. The device can be applied as a modular component in a closed manufacturing process to accommodate steps such as buffer exchange, cell formulation, and cryopreservation.

Introduction

The landscape of modern medicine has transformed rapidly through recent developments in gene and cell-based therapies (GCT). As one of the fastest growing fields in translational research, the GCT sector also faces unique and unprecedented challenges. In addition to robust clinical outcomes, efficient and cost-effective manufacturing processes are essential for the commercial success of GCT, which is particularly difficult to achieve in small-scale manufacturing¹. The cost of time, labor, and quality assurances are magnified when each batch of cells only produces a few doses for one patient instead of hundreds or thousands. Unlike allogeneic cell therapies in which the manufacturing processes are more akin to the production of antibodies and recombinant proteins, autologous cell therapies are typically produced as small-scale operations¹. As a relatively new phenomenon in biopharmaceutical manufacturing², options for small-scale cell processing are currently quite limited.

Buffer exchange is essential to cell manufacturing. It is one of the downstream processes where cells are removed from culture media and concentrated for cryopreservation or infusion. Currently, small-scale cell manufacturing often applies processes similar to those in the academic research setting and relies on specialized clean rooms to maintain sterility³. Manual downstream processes often use benchtop centrifuges to pellet and resuspend cells for volume reduction and buffer exchange. These open processes are costly (i.e., labor and clean room maintenance) and have limited manufacturing capacity, which are not ideal for commercial production^2,3.

Implementing automation has been proposed as a solution to improve manufacturing efficiency and achieving commercial scale productions². Sterility cannot be achieved in cell-based products through traditional methods used for biologics, such as gamma irradiation or terminal end filtration. Instead, an automated closed system is deployed to reduce risks of contamination and operators relying on clean rooms to maintain sterility⁴. Process automation also addresses the issue of scalability by either having multiple systems running in parallel (scale-out) or increasing the processing capacity of an individual device (scale-up), which in turn minimizes the variability between operators. Furthermore, cost modelling analysis of autologous therapies suggests that automation may reduce the cost of manufacturing^5,6. However, no cost benefit was found in an autologous stem cell clinical trial where an automated manufacturing platform was used⁷, suggesting that the cost benefit of automation may depend on the individual manufacturing process.

There are different strategies in which automation can be introduced into an existing manufacturing process. This can be achieved either by implementing a fully integrated platform or a modular-based processing chain. There are several fully integrated platforms commercially available for autologous cell manufacturing, such as CliniMACS Prodigy (Miltenyi Biotec), Cocoon (Octane Biotech), and Quantum (Terumo BCT). These integrated platforms, which are often described as "GMP-in-a-box", have low demands on infrastructure and are easy to operate. However, the manufacturing capacity of a fully integrated setup may be restricted by the incubator attached to the system. For example, the culturing capacity of Prodigy is limited to its 400 mL chamber⁸ and the Quantum cartridge has a limiting surface area set to 2.1 m² (equivalent to 120 T175 flasks)⁷, which may not be sufficient for patients requiring higher cell doses^9,10. Additionally, Prodigy and Quantum have a common attribute that limits their use: the operational unit is occupied by a single batch of cells throughout the cell expansion period, thus limiting the number of batches that can be manufactured by each unit¹¹. The modular approach to automation is to create a manufacturing chain with multiple modular units that simulates the commercial manufacturing process^12,13. This approach, which separates the culture device from the cell washing device, can thereby maximize manufacturing efficiency. An ideal processing device would be one that is adaptable and scalable to manufacturing needs¹².

Counterflow centrifugation (CFC) technology, which dates back to the 1970s, has had a long history in cell processing¹⁴. It achieves cell concentration and separation by balancing centrifugal force with a counterflow force. Typically, a cell suspension enters from the narrow end of a cell chamber under a constant flow rate while subjected to a centrifugal force (Figure 1A). The flow of the fluid is exerted in the opposite direction to the centrifugal force. This is referred to as the counterflow force, which forms a gradient within the cell chamber. The counterflow force then decreases as the cell chamber widens away from the tip of the cone-shaped cell chamber. Cells with higher density and larger diameter have a higher sedimentation rate, and thus they reach force equilibrium towards the tip of the cone-shaped cell chamber. Smaller particles may reach equilibrium towards the base of the chamber or be too small to be retained in the chamber and will be washed away. The CFC technology is mostly known for its application in processing blood apheresis products, such as isolating monocytes for dendritic cell therapies^15,16. In terms of buffer exchange, the CFC technology has only been applied in large-scale manufacturing¹⁷ and has yet to be used for the smaller scale manufacturing of autologous cell therapies.

To address the need of a suitable device for small-scale cell manufacturing, an automated CFC device (See Table of Materials), was recently developed¹⁸. The automated cell processing device uses counterflow centrifugation technology to remove cell debris and facilitate buffer exchange. The device performs buffer exchange with a single-use kit that can be sterile-connected to a cell transfer bag, which allows the cells to be processed within a sterile, enclosed system. Here, we investigate the use of a counterflow centrifugal device to perform buffer exchange in mammalian cell cultures in automated protocols. In this study, we tested the buffer exchange protocol using Jurkat cells and mesenchymal stromal cells (MSCs) to model nonadherent and adherent cell types, respectively. Jurkat cells are immortalized T cells often used for the study of acute T cell leukemia^19,20. MSCs are adult stem cells that have been studied in human clinical trials for a wide range of diseases⁹.

Protocol

1. Preparation of reagents and cells for buffer exchange

1. Prepare buffers (see Table of Materials) in a class 2 laminar flow hood. Using a syringe and needle assembly, remove 50 mL of saline solution from a 500 mL saline bag. Replace this with 50 mL of 20% human serum albumin (HSA) to make 2% HSA in saline, which will serve as the wash buffer.
2. Remove the cells from the culture vessels and perform a cell count to determine the starting cell quantity and viability by trypan blue exclusion. Load the cells into a transfer bag.
   NOTE: In this protocol Jurkat cells were cultured in RPMI and MSCs were cultured in DMEM:F12. Both media were supplemented with 10% fetal bovine serum (FBS) and 1% Antibiotic-Antimycotic solution. Cells were cultured at 37 °C with 5% CO₂. For MSCs or adherent cells, add a digestion enzyme (e.g., trypsin) into the culture vessels in order to detach the cells, and quench with culture media once the cells are detached. A transfer bag was used in this protocol because the cells were expanded in culture vessels. However, if the cells are cultured in a cell culture bag, it can be directly connected to the single-use kit via sterile tube welding. In this protocol, either 3 x 10⁷ Jurkat cells or 1 x 10⁷ MSCs were used for each run, where cells were suspended in 40 mL of culture media.
   1. Load cells into a transfer bag (see Table of Materials) using a syringe and needle assembly if a sterile tube welder is available to connect the transfer bag to the single use processing kit.
   2. Alternatively, use a pair of autoclaved scissors to cut the connecting tube with around 10 cm remainining attached to the transfer bag and add a female Luer connector to the end of the tubing. Use a syringe to aspirate cells and media, then connect the syringe to the Luer connector and load cells to the transfer bag.
3. Connect the bag containing the cells, wash buffer bag containing 500 mL of wash buffer prepared in step 1.1, waste bag, and collecting syringe to the single-use kit (Figure 1B).
   NOTE: The connecting position of each bag depends on the settings in the program. In this protocol, we connected the waste bag at position A, wash buffer at position B, cell bag at positions D and F, and collection syringe at position H (Figure 1C).

2. Program for automated buffer exchange protocol

1. Open the Graphic User Interface (GUI) of the device and press the "START" button on a laptop PC or tablet. Then "Open" an existing protocol or press "New" to create a new one.
2. Use the "Forward" and "Backward" button to navigate through each step, select the valves to be opened/closed, centrifugal speed, pump speed, pump direction, and action triggers. The settings for each step are summarized in Table 1. Then save the protocol.

3. Setting up the machine

1. Place the single-use processing kit on the machine and hang the connected bags accordingly. Press the red "Stop/Reset" button to reset all valves into the default closed position.
   NOTE: The machine will perform a test when the door is closed to ensure the kit has been placed correctly and that all the valves are functioning appropriately.
2. Connect the GUI to the processing device and press the "Connect" button. Then download the saved program. When the green "Play" button lights up, it indicates that the protocol is ready to start.
3. Open the manual clamps for the transfer bag tubing.
   NOTE: If the operator forgets to open the clamps, the device will stop and the pressure sensor warning will appear. Press the "Stop" button to reset the device and open the clamps to start again.

4. Automated buffer exchange

1. Press "Start" to initiate the buffer exchange program.
   NOTE: To manually alter the automated process, press the "Next" button to move to the next step before reaching the "Trigger" or press "Pause" to put the process on hold. This will recirculate the cells through the kit while keeping only valves J and K open (Figure 1C).
2. At the end of automation step 6 (Table 1), the process will pause when air in the now emptied bag triggers the bubble sensor. Press "Next" to move the process to the next step if the bag is indeed empty, or press "Pause" to resume the processing if the sensor was triggered by a bubble in the line.

5. Collecting and sampling the cells

1. When the automated process is finished, close all tubing clamps. Open the door and take the single-use kit out of the device. Disconnect the syringe to collect the cells for subsequent processes. Take a sample of the collected cells for a cell count and viability check (see step 6.2).

6. Process validation

1. Perform control experiments using a manual buffer exchange protocol.
   1. Centrifuge cell suspension at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 30 mL of cell wash buffer. Centrifuge the cell suspension again at 200 x g for 5 min. Discard the supernatant and resuspend the cells with 10 mL of cell wash buffer.
2. Perform cell counting via the trypan blue exclusion assay to assess cell viability using an automated cell counter.
3. Perform an MTS assay to quantify cell proliferation at 2, 24, and 48 h.
   NOTE: The MTS assay was performed on a 96 well tissue culture plate according to the manufacturer's instructions. A 100 µL aliquot of cell culture media (containing 10,000 Jurkat cells or 1,500) per well were plated after the buffer exchange process. At each time point, 20 µL of MTS reagents were added into each well and incubated for 2 h at 37 °C. The absorbance was assessed at 490 nm using a plate reader.
4. Perform functional assays to compare the impact of the automated process versus the manual process on the Jurkat cells and MSC function.
   1. Culture Jurkat cells in a 96-well plate at a density of 1 x 10⁶ cells/mL stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1 µg/mL ionomycin (cell stimulation cocktail) in DMEM:F12 media containing 10% fetal bovine serum (FBS) for 24 h. Incubate the cells at 37 °C with 5% CO₂.
   2. Measure interleukin-2 production from culture supernatant using bead-based ELISA assay (see Table of Materials) according to the manufacturer's instructions.
   3. Culture MSCs in a 12 well plate at the density of 10,000 cells/cm² in 1 mL of DMEM:F12 media containing 10% FBS supplemented with interferon-γ 50 units/mL for 48 h. Collect supernatant for the kynurenine concentration assay to measure indoleamine 2,3-dioxygenase (IDO) enzyme activity of MSCs as previously described²¹.

Results

In this protocol, we used Jurkat cells and MSCs as representative examples to demonstrate the automated buffer exchange process. During the process, Jurkat cells and MSCs shared the same processing steps with differences in centrifugal force and pump speed that control the flow rate (Table 1). Figure 2 shows representative images captured by the camera of how the fluidized cell bed may appear during the buffer exchange process. Typically, the fluidized cell bed will resemble...

Discussion

The automated buffer exchange protocol described is simple and user friendly. Nevertheless, there are a few key steps in this protocol that are critical and require particular attention. In our experience, when processing larger cells such as MSCs (average diameter 10–15 µm) each run should include at least 1 x 10⁷ cells to achieve optimal cell recovery (Figure 4B). Processing smaller cells, such as Jurkat cells (average ~10 µm diameter), requires ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
20 ml Luer lock syringes	BD	302830
20% Human serum albumin (HSA)	CSL Behring	AUST R 46283
4-(Dimethylamino)benzaldehyde	Sigma-Aldrich	156477-25g
500ml IV saline bag	Fresenius Kabi	K690521
Antibiotic-Antimycotic	Thermo Fisher Scientific	15240112
Automated cell counter (Countess)	Thermo Fisher Scientific	N/A
Cell counting chamber slides	Thermo Fisher Scientific	C10228
Cell stimulation cocktail (500x)	Thermo Fisher Scientific	00-4970-93
Cell transfer bags	Terumo	T1BBT060CBB
CellTiter AQueous One Solution Cell Proliferation Assay (MTS)	Promega	G3582
Centrifuge	Eppendorf	5810R
DMEM: F12 media	Thermo Fisher Scientific	11320082
EnVision plate Reader	Perkin Elmer	N/A
Fetal bovine serum (FBS)	Thermo Fisher Scientific	10099141
Human Interleukin 2 (IL2) Kit	Perkin Elmer	Al221C
Luer (female) fittings	CPC	LF41
PC laptop or PC tablet device	ASUS	N/A
Plate reader (SpectraMax i3)	Molecular Device	N/A
Recombinant Human IFN-γ	PeproTech	300-02
Rotea counterflow centrifuge cell processing device	Scinogy	N/A
Rotea single-use processing kit	Scinogy	N/A
RPMI media	Thermo Fisher Scientific	11875119
Surgical scissors	ProSciTech	420SS
Trichloroacetic acide	Sigma-Aldrich	T6399-250g
Trypan Blue stain	Thermo Fisher Scientific	T10282
Trypsin digestion enzyme (TrypLE Express Enzyme)	Thermo Fisher Scientific	12604013