Not such a good egg?

Vaccine production has never been more in the limelight and traditional egg-based production has proved it’s self to be robust – but is it time to take on the adaptability of a cell-culture based approach?    

Organisations across the globe are continuously looking for ways to increase capacity for the manufacturing of vaccines while streamlining the production process. The mass-production of vaccines has been proven to be extremely safe. However, the use of the traditional egg-based systems has presented a number of challenges, including flexibility, supply and cost. The desire to transition from egg-based production to the more robust cell-based platforms has resulted from periodic shortages of vaccine supply as well as the growing concerns of a pandemic outbreak. Recent developments have brought significant advances in in vitro cell culture technology, which include the development of new applications and products to meet the increasing demand for animal-free solutions. The move to implement cell-based systems has become more feasible and more easily justified.

In this article, we look at the implementation of cell-based systems and its associated benefits, and discuss the solutions available from Thermo Fisher Scientific; specifically the offering of commercially available disposable bioreactor systems for attachment–dependent mammalian cells. This includes the Thermo Scientific Nunc Cell Factory system and the HyClone Single-Use Bioreactor (S.U.B.), HyQShperes, and HyClone SFM4MegaVir medium.

The traditional method of egg-based production uses fertilized chicken embryos for virus infection and replication, from which, after several days of incubation, the virus is harvested, purified and chemically inactivated. The use of one or two eggs per vaccine dose dictates that not only is great planning required, but there must also be a reliable, long-term supply of eggs from biosecure flocks.  The entire production process takes approximately six months and the production of vaccines in eggs commonly requires a commitment of one year. Embryonated egg supply can thus pose a bottleneck, limiting the ability to stockpile vaccines, and adjust production with sudden increases in demand (bioterrorism attack/pandemic outbreak). More importantly, potential contaminants in the eggs (antibiotics, other viruses) may cause purity problems, and the risk of allergy to egg albumin can present possible health risks. Egg passage antigen drift can also present issues of antigen variability, production efficiency and lot-to-lot variability.

Cell-based systems commonly use mammalian cells lines - e.g., MDCK or Vero cells - which are initially infected with virus. Following virus inoculation, cells are disrupted and the viral particles are harvested. Such systems require optimisation and validation to maintain consistency of production between batches. However, the benefits of cell-based systems include rapid production scale-up which easily provides flexibility that can accommodate mid-season shifts in strain (Influenza), or global stockpiling in times of emergency. Furthermore, increased purity from the raw harvest simplifies downstream purification steps. The upstream and downstream processes of cell-based systems can easily be automated. Automation provides many benefits including the simplification of the production process, batch to batch consistency and a reduction of labor, time and cost. There is an elimination of viable/non-viable contamination and, with the use of animal-free constituents; there is no risk of allergies related to, e.g., egg albumin. Concerning matters of efficacy, the strains grown in cell cultures are closer to the original clinical isolates, and thus, more representative of the circulating wild type virus than that grown in eggs.

Solution providers have invested heavily in R&D to ensure its products and technologies meet the demands and stringent regulatory requirements necessary for vaccine production. Maximising safety and cost efficiency, and providing efficient scalable processes are common aims. However, achieving competitive virus yields, and reduced clonal variation, using systems that reduce or negate the requirements for animal derivatives are paramount. Thermo Fisher Scientific has optimised conditions for cell attachment using serial passage scale-up within scalable culture vessels and single-use bioreactors.

The company has developed production platforms that incorporate animal derived component free (ADCF) media together with scalable, disposable culture vessels, microcarriers, and fully controlled, single-use bioreactor systems. These systems serve to reduce start-up times and increase production efficiency, providing an attractive alternative to the legacy egg-based systems. They also decrease the capital investment commonly associated with the larger less flexible, stainless steel bioreactors and control systems. These platforms facilitate the sub-passaging of cells in controlled growth conditions along with simplifying the harvesting and downstream processing. Most importantly, the process results in increased titers and the flexibility to rapidly scale-up production on demand.

The Thermo Scientific NUNC Cell Factory system is comprised of a collection of

single-use multi-layer culture trays (Figure 1), providing a wide range of surface areas in one compact unit. Based on the format of the traditional tissue culture flask, the Cell Factory is a sterile, ready-to-use platform that promotes ease of use by offersing linear scalability, while eliminating the need for expensive and time-consuming cleaning validations. The utilisation of the Cell FactoryIt  supports process development from R&D through to commercial scale production. A 40-layer Cell Factory unit provides 25,280cm² of cell culture surface. Well established and referenced for the industrial scale production of traditional human vaccines, the Cell Factory it continues to offer an economical and a reliable commercial solution.

Thermo Fisher Scientific is uniquely positioned to offersr a full range of equipment to support the use of Cell Factories, which include an Automated Cell Factory Manipulator (ACFM), Cell Factory shaker for cell release applications, a portable CO2 incubator and shelving and lift systems. The versatile cart and rack system integrates each piece of equipment for ease of use as well as manufacturing expansion. Each cart can accommodate 4 – 40 layer Cell Factories or 12 – 10 layer Cell Factories. Tubing manifolds can be employed to connect the individual Cell Factory units within a rack to achieve a closed system. One cart of 40 layer Cell Factories can replace approximately 120 standard polystyrene roller bottles. In addition to its space saving format, Cell Factories reduce the risk of contamination by significantly minimising the number of operator interventions when compared to the typical roller bottle.

For further scalable production, the Thermo Scientific HyClone S.U.B. is a fully

controlled disposable bioreactor system that provides working volumes from 25-1000L. Since its introduction, the HyClone S.U.B. has become the leading disposable bioreactor system by incorporating stirred-tank technology with a proprietary, ADCF film. Offering equivalent functionality to conventional stirred-tank reactors, the S.U.B uses single-use contact surfaces for containment, mixing, venting, sparging and temperature sensing. The S.U.B. offers the ability to dramatically improve production economics. It can be retrofitted to existing control systems, reducing capital investment and operating costs, or can be fully integrated with the control system of choice. Utilisation of this disposable technology also minimiszes the sterilisation, cleaning and validation requirements associated with reusable surfaces. Additionally, the use of disposables in manufacturing facilities eliminates the need for equipment dedication to particular production processes.

The Thermo Scientific HyQSphere microcarriers are particles with a surface chemistry that facilitate the attachment and growth of anchorage-dependent cells (Figure 2). Their primary function is to provide an increased surface area to volume ratio to support efficient, scalable processes for cell culture within a relatively small footprint, i.e. from a simple spinner flask to a stirred-tank bioreactor. The application of HyQSpheres enables the use of traditional suspension cell culture systems for adherent cell culture applications. They  come in a variety of options including completely ADCF microcarriers to those with traditional animal origin surface coatings.

In the past, serum-free cultures have commonly precluded the use of microcarriers due to inadequate attachment. However, media such as the Thermo Scientific HyClone SFM4MegaVir medium, has been designed for the productive culture of attachment-dependent cells (including MDCK, Vero cells) within multiple cell culture systems. Furthermore, cell harvesting using proteases such as trypsin can prove problematic both practically and in terms of cell viability. Using the Thermo Scientific HyQTase non-trypsin reagent for dissociation, cell handing is reduced as it does not need to be removed before reseeding the culture vessels and there are no detrimental effects to the cells.  In a variety of cell culture systems, SFM4MegaVir has shown an ability to maintain cell growth kinetics similar to serum-containing media, while dramatically improving viral expression levels (Figure 3).

Recent investments by large Biopharm companies in new cell-based vaccine production facilities are a clear indicator of the transition away from egg-based production. As a result, the demand for reliable, industrialised animal-free solutions for the production of cell-based vaccines is increasing rapidly. With single-use, animal-free cell-culture production solutions readily available, the impact of a mid-season shift in viral strain or a supply shortage of vaccines can be minimised while reducing cost and increasing efficiency in parallel.