Manufacturing Vaccines in Adherent Cell Lines Using Disposable Multi-tray Bioreactors

ABSTRACT

The recent growth in the vaccine market has led to renewed interest in using adherent human cell lines for vaccine production. Traditionally, small-scale adherent cell line production has been carried out in roller bottles or T-flasks. Over the past few years, however, a number of companies have found multi-tray disposable bioreactors an effective method for producing high-quality drug products using adherent cells. These disposable, expandable systems have also facilitated scale up from laboratory to clinical-scale.

For some time, the preferred format for producing biopharmaceuticals has been the suspension cell model using Chinese hamster ovary (CHO) cells). Recently, however, there has been a renewed interest in adherent cell production techniques. Researchers have observed that 95% of all human cell types are adherent, and that it may be more practical in the long run to culture human cell lines rather than to humanize an animal cell line for propagation in a suspension format.

THERMO FISHER SCIENTIFIC

In the field of vaccines, adherent cell culture is best known for its use in development, rather than as a production technique. Nevertheless, despite the popularity of large-scale cell suspension systems, many important vaccines, such as polio vaccines, have been produced using adherent cell lines.

Given the use of adherent cell lines in vaccine development in the 1970s, methods to facilitate the scale-up of adherent cell production were needed. First, roller bottles were developed as a logical progression from glass bottles. Roller bottles were then further refined by industry to include various sizes and shapes, and equipment for automated handling and processing was introduced. A significant drawback of a roller bottle, however, is that it is an open system that requires repeated opening and closing of individual bottles. For production, this is not an efficient process and requires increased deployment of labor and equipment. A better approach was needed.

In 1978, scientists at Rentschler Biotechnologie GmbH in Germany described a multi-tray cell-culture system designed to improve efficiencies for producing interferon beta with adherently growing human fibroblasts. In order to scale-up production without changing methods and format, they glued T-flasks together. This invention, referred to as a multi-tray bioreactor, later became a standardized industry device, and has been implemented by many major pharmaceutical manufacturers for the production of vaccines for polio and other diseases.

A Flexible Option

Today, multi-tray bioreactor technology may be a suitable solution for developing vaccines using human cell lines. This flexible, closed-system technology was developed as a way to easily scale up production directly from a single laboratory cell culture flask. An individual tray is similar to a commonly used tissue culture flask, but with an expanded surface area. A single tray can be used exactly like a common 175 cm² cell culture flask in preclinical work, and then, as more cells are needed, researchers can use the multiple tray versions to scale up adherent cell propagation. The system also facilitates quality control, because cells are easily examined by inverted microscope.

Because no cell line or equipment changes are needed when scaling up production with multi-tray bioreactors, using this technology can speed up new drug development. Cell propagation may be performed on a small- or large-scale, depending on the amount of cell mass required. Another benefit is that multi-tray systems are designed for single-use, and thus eliminate the need for efforts and infrastructure required for cleaning and validation.

Using adherent systems also can facilitate product quality when using human cell lines. Manipulating human cell lines for propagation in a suspension format heightens the risk of inadvertently producing antibodies and other proteins with distorted three-dimensional configurations that render the product useless. This risk is avoided with adherent systems.

Vaccines have been undergoing a resurgence over the past few years. Many institutions and companies are involved, and they must address the ever-present issues of productivity and cost-efficiency. Because preclinical studies can take 5 to 10 years, many companies try to keep multiple drug or vaccine candidates moving through the drug development pipeline at the same time. Multi-tray bioreactor systems provide a flexible and cost-efficient approach to implementing multiple projects simultaneously.

Case Studies: Multi-tray Bioreactors in Production

As the case studies below explain, several companies that are using multi-tray bioreactors have found the method allows for efficient production of high-quality products for preclinical and clinical studies.

Areta International

Researchers at Areta International, an Italian contract manufacturing organization, have used multi-tray bioreactors under good manufacturing practice (GMP) conditions to manufacture human T-lymphocytes (TALL-104 cell line) on a large scale for cell therapy. TALL-104 cells are human, IL-2 dependent cells derived from T cytotoxic lymphocytes. They are tumoricidal and not restricted by the major histocompatibility complex (MHC). The cells were initially isolated from a patient with acute lymphoblastoid leukemia. With the help of IL-2, they can be expanded and maintained in vitro.

A research group led by D. Santoli at the Wistar Institute in Philadelphia demonstrated that TALL-104 cells were able to control both spontaneous and induced malignant tumoral growth and increased survival rates in different animal models.¹ To carry out these studies, the Wistar group would require a commercial level of TALL-104 cell production to reach their target volume of cells.

According to the research group at Areta, commercial production levels of TALL-104 cells means that a propagation system must be able to guarantee cell expansions to a factor of at least 10⁹ in a homogeneous system.² Using a multi-tray system (Nunc Cell Factory, Thermo Fisher Scientific) consisting of 10 trays and a cell culture surface area of 6320 cm² , they achieved recovery of between 2.5 to 5 x 10⁹ TALL cells, meeting the criteria for commercial-scale cell propagation.

Maria Luisa Nolli, PhD, Areta's CEO, said the multi-tray bioreactors were an efficient method for manufacturing for early-stage clinical trials. "The speed of process development and low investment required to set up solid, standard operating procedures that we could scale up directly from a cell culture flask enabled us to get materials manufactured in a timely and affordable manner," she said. Members of the research team agreed. "The single-use, modular and sterile multi-tray bioreactor was a useful new method for industrial production of human cells for Phase 1 and Phase 2 clinical trials of cell therapy," said one member. "With this system, we were able to manufacture a high quality and reliable product in compliance with current good manufacturing practice guidelines."

Rentschler Biotechnologie

Another contract manufacturing operation that has used multi-tray disposable bioreactors is Rentschler Biotechnologie, the company that originally developed the multi-tray bioreactor format. Scientists there used human embryonic kidney cells (HEK293) and four multi-tray units comprising 40 tray units to develop a serum-free transient expression method for antibodies, as well as a standardized antibody purification protocol.³ The adherent nature of the HEK cells enabled repeated harvests, which increased antibody yields. Two types of IgGs were expressed in the HEK cells. For both types of antibodies, the researchers harvested the cells four different times after 3 to 4 days of growth. The volume used during production was 4 L per 40-tray bioreactor, which yielded a total harvest volume of 64 L for each IgG. IgG yields reached 12 mg/L in these serum-free conditions. The final yields achieved in this serum-free environment were 530 mg for one IgG type and 430 mg for the other IgG type (Table 1).

Table 1. Summary of the results obtained after large-scale transient expression of two different human IgGs. In each case, four harvests were performed repeatedly after 3 to 4 days of cultivation. The expression level reached 2 mg/L in serum-free culture conditions. The 1st IgG yielded 530 mg and the 2nd IgG, 430 mg.

After a highly purified product was obtained, the glycosylation patterns were analyzed by looking at N-glycan structures released from the antibodies (Table 2). This analysis confirmed the ability of the transiently transfected HEK293 cells to synthesize N-glycan structures similar to those obtained after stable expression (e.g., in CHO cells). This study showed that the antibodies produced in serum-free conditions using a multi-tray bioreactor were of equal quality to antibodies produced in suspension CHO cells.

Table 2. Analysis of N-glycan structures. The majority of N-glycan structures detected after desialylation were complex-type biantennary structures without terminal Gal (36.6%), with one terminal Gal (44.9%) and with two terminal Gal (15.1%). Approximately 2% of the N-glycan structures were still sialylated. This analysis shows that transiently transfected HEK293 cells have the ability to synthesize N-glycan structures similar to those obtained from CHO cells.

IQ Therapeutics BV

In a third study, IQ Therapeutics BV, a Dutch biotechnology company engaged in discovering and developing antibodies for therapeutic use in infectious diseases and for biodefense, uses multi-tray bioreactors (Nunc Cell Factory, Thermo Fisher Scientific) to quickly generate sufficient volumes of antibody material for animal studies. IQ has two monoclonal antibodies for anthrax in preclinical studies. The two antibodies are directed against two components of anthrax's lethal toxin: The antibody IQNPA is directed against protective antigen, and IQNLF is directed against lethal factor. Currently, IQ uses multi-tray bioreactors to produce these antibodies for preclinical studies in animal models. Production for clinical trials, however, is outsourced to a contract manufacturing operation to ensure compliance with current good manufacturing practices.

For in-house production using human hybridomas, IQ began to use multi-tray units instead of standard flasks. After investing some time to optimize the use of the multi-tray bioreactors, IQ researchers found conditions in which IQNPA antibody production improved by more than 30% and IQNLF antibody production more than doubled (+120%), compared to 175-cm² flasks.

The team at IQ found the system effective and easy to use. "The ten tray cell culture system is very effective in manufacturing 75 mg batches of antibody," said Herman Groen, PhD, the CSO of IQ Therapeutics. "That is sufficient material to do a lot of in vitro functional studies, as well as work in animal models." Hans Westra, responsible for manufacturing at IQ, notes that his team also benefited from simplified protocols when dealing with this type of technology. "The very short time we needed to become accustomed to handling the multi-tray bioreactors made the adaptation of the production process into our facility very easy," he said.

On the Horizon

The evolution of the multi-tray cell culture system for adherent cell line production was enabled by improving upon an existing device, the 175-cm² flask. The need to scale up adherent cell propagation pushed researchers and engineers to improve upon this technology to create the multi-tray format. The future success of this type of format, however, will be contingent upon continuous innovation and improvement. For example, the application of various surface treatments will help cells adhere to the inner walls of a tray in a stronger and more specific manner. Also, developing multi-tray bioreactors as completely closed systems with customized tubing configurations to facilitate and control the flow of liquids and gases within the interior environment is under way. A closed system like this would provide sterility, flexibility, and speed for vaccine development and production.

Edwin Schwander is the market development manager at Thermo Fisher Scientific, Roskilde, Denmark, +31.6.51541921, e.h.schwander@home.nl.

References:

1 Cesano A, Visonneau S, Jeglum KA, Owen J, Wilkinson K, Carner K, et al. Phase i clinical trial with a human major histocompatibility complex nonrestricted cytotoxic t-cell line (tall-104) in dogs with advanced tumors. Cancer Res. 1996; 56:3021–3029.

2. Schwander E, Ramusen, H. Manufacturing of lots of human T lymphocytes for cell therapy: A new process based on disposable bioreactors. Gen Eng News. 2005 Apr 15; 25(8).

3. Schlenke P, et al. Serum-free transient expression of antibodies using 40 tray cell factories. Poster. Laupheim, Germany: Renschler Biotechnologie Gmbh.