Scale-Up to 1000-Liter Perfusion Cell Culture in a Single-Use, Stirred-Tank Bioreactor

by Matt Niloff

Overview

Bioprocess scientists have long sought to expand the application of perfusion cell culture in order to achieve the theoretical productivity gains compared to batch and fed-batch approaches. The perfusion process is based on continuously adding nutrient solutions to the bioreactor while removing wastes from it. This keeps the organism in optimal growing conditions. However, this simple process presents practical challenges that often thwart those efforts.

Perfusion operations enable biologics manufacturers to achieve production volumes with smaller bioreactors which results in reduced capital costs and reduced footprint for the equipment. In some cases, smaller seed bioreactors can also be eliminated. Perfusion enables productive processing of unstable products by limiting the exposure of products to damaging proteases. Slow-growing or difficult-to-grow cell lines can often be productively grown only in perfusion operations.

The advent of bioprocessing based on single-use components is creating opportunities to broaden and simplify the use of perfusion, primarily due to the elimination of complex and time-consuming cleaning and sterilization steps, as well as the reduction in the risk of contamination. Recent advances in the supply of single-use bioprocess equipment such as bag assemblies, bioreactors, mixing systems, centrifuges, and filter cartridges make implementation of single-use processes a feasible alternative to traditional biomanufacturing processes.

This article presents the results from the evaluation of a perfusion-based process utilizing single-use bioreactors, a single-use mixing system, and a single-use centrifuge. The combined operation of these systems permitted successful 1000L/day harvesting. The project resulted in successful development, scale-up and demonstration of a perfusion mode operation at the 200L (160L working volume) and 1000L scale using exclusively single-use process equipment. The 1000L process was operated for one month, with 12 harvests over 19 days, and was only terminated at that time as proof of concept had been achieved. The program concluded with a successful demonstration of process viability and reliability that exceeded the initial performance expectations.

Equipment Description

Equipment is shown in the Process Layout photo.

Bioreactor
Single-use bioreactor systems with nominal working volumes of 200 liters and 1,000 liters. These systems included the following components with associated features:

Single-use Assembly

• All product contact surfaces single-use except for sensors.
• No assembly required within sterile barrier.
• USP Class VI films & components, low endotoxin, low particulate, non-cytotoxic materials.

Bioreactor Hardware and Controls

• Turnkey, fully integrated system with jacketed vessel, integrated temperature control and process controller.
• Stirred tank design - bottom magnetic drive agitation with no rotating seals, integrated sparger with open pipe.
• 5:1 turndown ratio (systems able to operate at 20 percent of maximum volume).

The remainder of the perfusion train consisted of:

Media Prep System: Prototype 1000L media mixing system that produced 1000 liters/day.

Single-use External Cell Retention Device: Pneumatic Scale single-use centrifuge system.

Chilled Harvest Collection System: Chilled mixing/storage system.

Process Description

Cells were grown in a 1000L single-use bioreactor operating at 1000L working volume. The bioreactor was installed on a load cell to monitor system weight and was operated at a constant 1000KG. Supernatant was removed at a fixed rate and sent to the single-use based centrifuge. A concentrated stream of cells was returned directly from the centrifuge to the bioreactor, and the load cell/controller loop governed a pump that would add new media based on the system weight readings in order to maintain constant volume in the bioreactor.

The primary objective was to assess the advantages offered by a single-use based process, including:

• Very fast process setup
• No need for CIP/SIP
• Less space required
• Short turnaround time between batches
• Easy product changeover
• Facility flexibility

The criteria to determine success included:

• Bioreactor working volume: up to 1000 L
• High cell density: be able to reach 10 million cells/ml
• Product comparability
• Reliability: be able to support one-month process

The application evaluated was:

• Cell line: Human cell line
• Culture mode: suspension perfusion cell culture
• Media: animal-product-free media
• Perfusion rate: 1.0 v.v.d.

Results

Process runs conducted:

10 L glass bioreactor run	27 days with 22 harvests
160 L single-use run	28 days with 22 harvests
1000 L single-use run	19 days with 12 harvests

The charts below illustrate the outcomes of the 1000L perfusion run compared with a 200L (at 160L working volume) run and a 10L bench-top run. The results demonstrate successful perfusion process scale-up and continual cell culture at 1000L as measured by cell density, cell viability, protein production/yield and other criteria. The process demonstrated equivalent control of:

• Temperature
• pH
• DO

These results show the single-use based process delivered performance comparability of cell viability, viable cell density, and product yield (data not shown) at 200L and 1000L. The overall system reliability also exceeded expectations.

As the current trend in disposables expands throughout the industry, many bioprocess operations will continue to be simplified. Implementation of disposable technology in the process can result in reductions in footprint, set-up time, risk of cross contamination, capital costs, and operating costs. The example here for a perfusion-based process demonstrates the equivalent performance of single use disposable equipment at different bioreactor volumes. This flexibility along with the increase in protein titers and drug potency, are contributing factors to the potential that commercial-scale manufacturing at 2000L will be a reality in the near future for new therapeutics coming to market. Such a scale for disposable bioreactors is currently available.

About the Author
Matt Niloff is Senior Director of Products at Xcellerex, Inc., a leader in the development and deployment of innovative single-use bioprocessing technologies. Matt is responsible for Xcellerex’s XDR line of single-use bioreactors and XDR and XTM single-use mixing systems. Prior to joining Xcellerex, Matt held various positions at Stedim Biosystems and Millipore Life Sciences. He also worked previously at the MIT Center for Genome Research on the Human Genome Project. Matt holds a Master of Science in Biology from McGill University. Matt can be reached at 508-683-2296 or MNiloff@xcellerex.com.