Optimized Vaccine Development and Manufacturing: A Technology Overview

January 2, 2008
Silke Fetzer

BioPharm International, BioPharm International-01-02-2008, Volume 2008 Supplement, Issue 1

The replacement of a IgG ELISA assay with an SPR-based test can shorten the production workflow by one day.


In the current biopharmaceutical market, companies must move quickly from discovery to patent, and then on to trials and efficient production to increase competitiveness and maximize revenue during the patent life of a drug. Time-to-market and flexibility are some of the key issues that the biomanufacturing industry needs to address to improve its cost-effectiveness. In the light of these pressures, it is increasingly important to make use of the latest technologies to simplify vaccine development and manufacturing. Many opportunities to optimize vaccine development and manufacture can be found in the analytical and processing technologies. This articles gives an insight into technology solutions that will address the needs of vaccine manufacturers by ensuring quality and reducing time.

Vaccine producers share many development and manufacturing problems with colleagues who commercialize other types of biopharmaceuticals. Smooth progress through R&D and clinical trials, plus cost-effective production and quick time-to-market are much sought-after. The nature and use of vaccines nevertheless presents some special issues with which producers must contend, particularly regarding scale, affordability, change-of-pace, and safety.

The urgent need to increase stocks of the influenza vaccine means that producers face scale challenges that are somewhat different from those seen with other therapeutics. In the advent of a pandemic, currently existing global manufacturing capacity would only suffice to vaccinate the US population.1 This scenario has stimulated the mobilization of billions of dollars of public money and grants, and has motivated influenza vaccine-producing companies to invest in a shift in technology from eggs to cells, and to build additional manufacturing capacity.

A second issue confronting manufacturers is cost. In developing countries, the mortality rate for children as a result of infectious diseases such as pneumonia, malaria, and HIV is extremely high, but even the most basic immunizations cannot be afforded by those who need it most.

Thirdly, new vaccines for cancer or other therapeutic vaccines will open a new, highly competitive, and probably high-price market, where the leader will shape the market so that time-to-clinic and time-to-market will become a very important issue.

Finally, from a technical perspective, vaccine production is much like any other biopharmaceutical production workflow in that it generally requires about six steps: fermentation, clarification, purification, sterilization, formulation, and final filling. All of the steps need to be robust and reproducible, which is a particular challenge in biopharmaceutical manufacture because of the complexities and natural variability of biological systems. One important difference between the production of vaccines and other biopharmaceuticals, however, is the risk and safety considerations related to working with pathogens and pathogenic antigens.

The development of vaccines and manufacturing capability must therefore follow four ground rules:

  • Vaccines must be developed, produced, and delivered in very large volumes.

  • Process costs must be kept down.

  • Development and delivery times must be short.

  • Patient and employee safety may not be compromised.

In the light of these pressures, it is increasingly important to make use of the latest technologies to simplify vaccine development and manufacturing. The following sections offer insight into technology solutions that will address the needs of vaccine manufacturers.

Analytical Method Development That Drives Time-Savings

Process development should always start by defining the required target purity goals and selecting analytical methods with appropriate detection limit that are sufficiently accurate and time-efficient. As with all biomolecules purified from crude biological material, the removal of contaminants, e.g., derivatives from the host cell such as DNA, host cell protein, or leachables, must be documented. The removal or inactivation of adventitious viruses is a special challenge.

Of the in vitro and in vivo assays currently applied to vaccine production processes, some take weeks to run, although the accuracy of others, such as the single radial immunodiffusion (SRID) test used for batch-release, is limited.2

Toward Shorter, More Accurate Testing Times

Improving analytical methods during development and production will aid in optimizing process parameters, improve safety and efficacy, and reduce batch release times. Label-free interaction analysis based on surface plasmon resonance (SPR) provides rapid product characterization and unique data to support critical decisions at every step of the vaccine development and manufacturing process. For example, much emphasis is now being placed on developing effective vaccines against many influenza types and this requires analysis of the product and control of the breadth of immune responses elicited by such vaccines.

SPR-based interaction systems deliver kinetic data (on and off rates) by monitoring binding events in real-time, providing greater insights into protein function than from end point assays such as enzyme linked immunosorbent assay (ELISA). Label-free interaction analysis also can be used to provide accurate concentration measurements. The interaction occurs close to a sensor surface on which changes in mass concentration are detected, eliminating the need for labels, which may interfere with the interaction properties.

These systems have also been applied to immunogenicity studies using untreated serum where the build-up of a strong antibody response mechanism is desired. In one study, an immunization regime designed to elicit an anti-IgE response was optimized during the development of an immunotherapeutic against allergy and asthma.3 Data from SPR-based analysis have been shown to be more reproducible than techniques such as ELISA and more information (kinetic quality assessment) can be derived from a single interaction. The ability to detect low–medium affinity antibodies means that immune responses can be detected earlier.

SPR-based systems are widely applicable throughout the vaccine development and production process (see, for example, the work from NVI),6 potentially replacing more tedious methods such as bioassays and in vivo tests.

Rapidly Acquired High Data Quality

The benefits of SPR-based systems have led many pharmaceutical and vaccine producers to consider these systems for product characterization tests to reduce testing times. The replacement of a traditional mouse IgG ELISA assay with an SPR-based test system can reduce the time per assay run from 7 to 2 hours and ultimately shorten the production workflow by one day.4 More importantly, studies comparing the accuracy of IgG ELISA assays with an SPR-based system in the analysis of monoclonal antibody (MAb) response in serum revealed a 10-fold improvement in terms of the coefficients of variance.5 These numbers emphasize the potential and relevance of SPR-based systems in batch-release testing of vaccines where a higher accuracy in testing the final vaccine efficacy would significantly contribute to improved process economics and dose-saving strategies—both of which are a particular concern in influenza batch-release.

Upstream Process Development That Reduces Capital Expense

The motivation to shift from egg-based to cell-based production capacity results primarily from the fact that egg-based capacity cannot be scaled up further in times of emergency, largely because of its reliance on specially prepared and treated eggs. In contrast, cells can be frozen in advance and large numbers may be grown quickly. Capacity can also be increased by adding fermenting equipment. The footprint for cell culture-based vaccine production is considerably smaller, and processing takes place in closed systems. Cell-based influenza vaccines also provide an option for people who are allergic to eggs and are therefore unable to receive currently-licensed vaccines.7,8,9

The productivity of large-scale cell culture can be increased either by scaling up to larger volumes with cell densities of 2–3 x 106 /mL, or by intensifying the process in smaller volumes but with higher cell densities (up to 2 x 108 cells/mL). When intensifying cell densities, more frequent media changes are needed and perfusion is eventually applied.10

Many alternative technologies are available. Cross-linked dextran beads (microcarriers) provide an extended surface and a stable environment for optimal cell growth. Microcarrier culture of anchorage-dependent or entrapped cells reduces volume and thus belongs to the latter of the options cited above.1 The technique in general has many advantages for the commercial manufacturer. It can be operated in batch or perfusion modes during cell culture and is well-suited to efficient process development and smooth scale-up. Washing and changing culture media just before viral infection is easier. The reactors can also be modified to grow other organisms.

For the majority of cell-based seasonal and pandemic influenza vaccines, virus titers obtained from adherent cell lines are superior to those obtained from cells in suspension. GlaxoSmithKline Biologicals (Wavre, Belgium) recently presented data on the impact of this technology on capital expenditure and time-of-construction.11 Because adherent cell lines produce more virus per volume, microcarriers help lower the volume of fermentation and the size of the tanks from 5,000 to 1,000 L. In this example, the use of smaller fermentation vessels eliminated the need to construct special room heights and thus avoided additional engineering expenses. According to the presented data, the overall savings can add up to hundreds of thousands of dollars and significantly shorten lead-times for engineering plant construction.

Other examples have documented the process economics and operational cost savings regarding microcarrier-based rabies vaccine production.12 As the size of such operations increases, so does investment in resources and personnel—and any failure becomes more costly. Scalability of cell culture unit operations is therefore mandatory, and Baxter (Wien, Austria) has demonstrated the successful operation of a 6,000-L scale mammalian cell culture based on microcarriers for the economic production of influenza vaccine.13

Process Development Strategies That Drive Time and Cost-Efficiency

Portfolio management dictates increased throughput of development candidates through the laboratory, preferably at constant or even lower operating costs. A two to three-fold increase in projects per year compared to five years ago is not uncommon. This challenge is reflected by the formats, tools, technologies, and workflows used. Statistical tools such as design of experiments, and high-throughput tools like microtiter plates and robots, are being used to run large series of experiments in a short time.

In process development for chromatography, this approach is used not only to select media for specific steps,14 but also to develop and optimize the parameters of a purification step. For MAbs, about 400 conditions for two chromatographic steps could be tested in a one-day screen. Furthermore, less than one gram of MAb was consumed. The conditions arrived at with these robotic systems were later confirmed with laboratory-scale columns.15

Another way to eliminate bottlenecks in process development is to use technology platforms. These may be defined as a standard set of conditions and methods applied to all molecules of a given class.16

The biopharmaceutical manufacturing industry has communicated time savings of 3–8 months using such a fast-track development approach when technology platforms are applied in all key aspects of development; cell line development, cell culture, downstream processing, analytical concepts, and even filling. A head start of only three months into clinical trials can increase a product's net present value (NPV) by tens of millions of dollars.17

If the platforms used for clinical manufacturing also can be used on a commercial scale with little or no modification, then similar or even higher value gains can be achieved for projects and a lower risk from comparability issues can be assumed.

Technology and Product Choices That Drive Safety

The purification of modern biological therapeutics generally involves both membrane and chromatographic separations. Membrane separations complement chromatography and offer a number of key benefits. They are fast, robust, and can bring greater effectiveness to the key stages of bioprocessing by concentrating and washing feed streams before chromatography, for example. Cross-flow filters (tangential-flow filters) are best suited to higher solid contents, more viscous feed solutions, or where concentration, recovery, or purification of cells or target species is desired.

For vaccine manufacturing, particularly virus purification, fully scalable macrovoid-free hollow-fiber technology applied to ultrafiltration and microfiltration offers great advantages. The defect-free surface allows the use of more open porosity ratings (such as 500 and 750 kd), thereby enabling the use of ultrafiltration membranes as part of the virus purification process. Moreover, the open-flow path design of hollow-fibers gently processes cell suspensions and other particulate feed streams and reduces shear forces, thereby helping maintain the integrity of the virus. As a result, recovery rates of the virus target and overall process economics are improved.18

Unlike single-pass or dead-end filtration, cross-flow methodology continuously sweeps the membrane surface by circulating the feedstream across it. This minimizes blinding the membrane and promotes consistent, long-term productivity. It also allows units to be cleaned, stored, and re-used as needed. While the feedstream is pumped through the cassette or cartridge, the retentate (materials excluded by the membrane pores) continues through the circulation loop, whereas the permeate, including the solvent and solutes, is transported through the membrane.

Chromatography media are also widely used for manufacturing viruses. These unit operations need to be robust, produce high yields, achieve high purity and withstand cleaning-in-place with low ligand leakage from the media. A particular challenge for chromatography is productivity, because viruses, viral vectors, and plasmids are considerably larger than traditional biopharmaceuticals and cannot therefore easily enter the pores of microporous media, which is a prerequisite for achieving high capacities for the target of interest. Alternative strategies must be applied to achieve larger surfaces, for example, by reducing the bead size of chromatography media.

Adsorptive technologies, such as, affinity chromatography, can be used to purify adeno-associated viruses for clinical trials, thereby replacing low-yield and poorly scalable density gradient centrifugation.19 The same principle can be applied to isolate and concentrate influenza virus. Anion exchange chromatography can be used to polish viruses such as influenza or adenovirus because the charges of these targets and those of contaminants differ, depending on the pH and conductivity during binding and elution. Group separation on agarose-based gels is also a commonly-used technology to separate viruses from host cell protein and small DNA molecules based on the different sizes of target and contaminant. The target virus will elute in the void volume, but the contaminants will be delayed when applying isocratic elution conditions.20

Disposable Manufacturing Solutions That Cut Time and Cost

In the current biopharmaceutical market, companies must move quickly from discovery to patent, and then on to trials and efficient production to maintain or increase competitiveness and maximize revenue during the patent life of a drug.21

The cost of delay in R&D can be more than one hundred thousand dollars per hour.22 Time-to-market and flexibility are thus key issues that the biomanufacturing industry needs to address to improve its cost-effectiveness. One strategy that offers a good compromise in this respect is the use of disposable hardware and flexible facility designs.23 Tanks can be replaced with bags and piping with tubing; disposable pump heads; and detection cells can be used, and both cleaning and certain validation efforts can be simpler to operate in the pilot facility compared to full-scale manufacture.

Installation lead times can be minimized and hardware can be moved based on day-to-day needs. Disposable solutions not only offer indirect cost benefits to flexibility and validation, but also a direct running cost advantage. The latter, however, is strictly dependent on scale; at the 500-L cell culture scale, for example, cost advantage has been calculated to be in the range of 8–14%.24

According to the 3rd Annual Report and Survey of Biopharmaceutical Manufacturing, Capacity and Production,23 the most often cited advantages associated with use of disposable components include reduced risk of cross-contamination (57%), elimination of cleaning requirements (55%), minimization of cost of down-time for cleaning, sterilization, and corresponding validation procedures (44%), and the labor and running costs associated with these operations. Reductions in operating cost are related mainly to smaller installations for process quality water systems and to the complete elimination of the handling and discarding of hazardous chemicals. Equipment maintenance is also minimized.

If we consider the preparation required for a pandemic influenza outbreak, disposable solutions would drive the speed of response and manufacturing economics, i.e., quick change of target molecule, flexible batch volumes, and the relocation of manufacturing facilities. These benefits would facilitate the mandatory quick response needed in countries not producing a seasonal influenza vaccine.

Secure Supply Ensures Deployment of Capacity

One aspect to consider outside the technology itself is that of the supply chain. Secure manufacturing processes are dependent on technology providers having quality systems and contingency plans in place. Some producers may need to evaluate options for alternative supplies should their primary supplier be unable to deliver under exceptional or unforeseen circumstances (such as during a pandemic or an accident). Qualified suppliers with a broad range of products and services can usually help minimize dependency on alternatives.


Many opportunities to optimize vaccine development and manufacture can be found in the analytical and processing technologies used by commercial producers of other types of biopharmaceuticals. Today's technologies for various steps of vaccine manufacture can help ensure quality and reduce time. Some key technologies include analytical methods such as surface plasmon resonance, upstream technologies such as microcarrier beads for adherent cell lines, high-throughput screening methods for process development steps such as chromatography media selection, and downstream technologies such as cross-flow filtration. In all areas, disposable technologies are increasingly being adopted because they make it possible to develop and scale up processes quickly and to facilitate manufacturing by reducing the cleaning validation burden.

SILKE FETZER, PHD, is the head of product management cell culture, and vaccine operational marketing at GE Healthcare, Freiburg, Germany, silke.fetzer@ge.com


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