Cell-Culture–Based Manufacturing: Meeting the Challenge of 21st Century Influenza

ABSTRACT

Conventional influenza vaccines use an egg-based culture and harvest process. This method of production is slow and inflexible, compared with emerging cell-culture based approaches that can respond rapidly to the influenza virus' inherent ability to "drift," or more dangerously, to "shift"—a critical factor that would arise in the event of a pandemic. ImmBio's ImmunoBodies is a breakthrough vaccine under process development at Eden Biodesign that uses a baculovirus expression system for production. The vaccine is constructed from part of the flu hemagglutinin protein fused to the human dendritic cell-binding Fc region of human immunoglobulin, enabling it to efficiently trigger a broad, protecting response. In the event of a new strain tracking out of its protective range, the construct can be rapidly re-engineered. The baculovirus manufacturing capacity also can be augmented to produce large volumes.

The utility of influenza vaccination is well established, especially in "at risk" groups; nevertheless, infections result in some 300,000–500,000 deaths worldwide every year. Inoculation with traditional flu vaccine (delivered as an annual trivalent vaccine) leads to raised antibody response to virus hemagglutinin protein, which protects against anticipated major strains. However, the genome of the influenza virus is encoded as RNA, making it prone to mutation. Influenza thus frequently evolves into serotypes that evade the immune memory that has been raised in response to previous vaccinations or to formerly prevalent serotypes. In the event of a major "shift" in the strain, such as from a reassortment of avian influenza capable of human-to-human transmission, the lack of prior immunity in the population could result in a pandemic and a high mortality rate.

Of the three major pandemics in the last century, the "Spanish flu" was the most deadly, killing more people than in World War I, which had just preceded it. With the current prevalence of avian flu as a source of genetic reassortment, a significant elapsed time since the last major shift, and the recognition that egg-based flu vaccines cannot meet anticipated demand, concern has grown. Vaccines against strains originating from avian flu may achieve poor yields in egg-based systems. Hence, such vaccines may need modifying, potentially eroding their efficacy and extending their lead time to development. Both public and private interest in alternative systems is consequently high, with development focused on mammalian cell-based manufacturing approaches, but these may likewise have limitations.

ImmunoBiology ("ImmBio"), a biotech vaccine development company in Cambridge, UK, and Eden Biodesign, operating the UK's National Biomanufacturing Centre in Liverpool, are therefore developing an antibody-antigen fusion complex product manufactured in a baculovirus expression system.

Influenza

The influenza virus belongs to the Orthomyxoviridae family. It is an enveloped RNA virus with a segmented genome consisting of eight single-stranded negative RNA segments. The virus is split into three subtypes: A, B, and C, based on antigenic differences in two of its structural proteins: the matrix protein M2, and the nucleoprotein. As infection with the C subtype is relatively mild, vaccination is typically against the A and B subtypes.

Projecting from the viral envelope is a conglomeration of three proteins: hemagglutinin (HA), neuraminidase (NA), and the matrix (M2) protein. Influenza A viruses are subtyped according to the HA and NA proteins they display on their viral envelope. Although 14 HA proteins and 9 NA proteins have been identified, only a limited number are infectious to humans (H1, H2, H3, N1, and N2). The most common combination currently in circulation is the subtype H3N2. Distinct strains can be further identified by the virus type, geographic origin, and year of isolation.

To cope with this variety, current vaccines are trivalent, with two components against A and one against B. Through the World Health Organization (WHO) surveillance network, which tracks strain changes, manufacturers that supply vaccines to the US and Europe are advised around February what subtypes are expected to be prevalent through the following winter, when influenza incidence is highest. Typically, at least one component must be altered each year. The antigen composition recommended by the WHO may need modification if the strain grows poorly in eggs, a situation that may compromise efficacy. To begin the Northern hemisphere seasonal vaccination program, large volumes of vaccine product need to be available by September.

Egg Technology

Influenza vaccines are made by inoculating live flu virus into fertilized chicken eggs, then purifying and inactivating the resulting egg-adapted virus to produce trivalent inactivated virus, or TIV. TIVs represent the majority of the currently licensed and marketed flu vaccines worldwide. In addition, there is the recently licensed egg-derived live attenuated influenza vaccine (LAIV) produced by MedImmune.

Given the February-to-September time constraint, vaccine manufacturers have approximately six months to develop, manufacture, and release millions of doses of trivalent vaccine. In practice, this short timeline necessitates that manufacturers speculate on likely strains each year, so they can commence production of at least one, and probably two, strain-specific bulk antigens in advance of knowing actual strain requirements. This tight schedule leaves little or no time to optimize and validate the production process for the new virus strain. Consequently, the purification stream used for egg-derived flu vaccine involves a trade-off between the need to specifically purify the antigens from the crude egg harvest and the need to have a process that is not so specific that it no longer works if the properties of the HA and NA antigens change due to drifting or shifting. This trade-off does not lend itself to a stringent purification stream with predictable yields.

Although manufacturing capacity has been growing and the uptake of seasonal vaccination has increased, demand has outstripped capacity. Early assumptions were that the global capacity of around 300 million doses per annum could be tripled in a pandemic situation by producing only a monovalent rather than a trivalent vaccine. These assumptions have been modified by the recognition that low natural immunity in the population to a new virus may require a high dose, possibly in a boost regimen. Switching from seasonal flu capacity, built to meet elective seasonal vaccination in developed countries, is also grossly insufficient for potential global needs. Indeed, two of the last three pandemics originated in the Far East, where avian flu is most prevalent and where the cost of vaccines can be prohibitive. Antigen-sparing approaches, notably through the use of adjuvants, may enable reduced unit dosage, which could, in turn, increase vaccine coverage from a given manufacturing facility.

New Developments

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