Removing viral contaminants from animal cell-culture derived biologicals is a major challenge of downstream purification because it involves laborious and time-consuming techniques that result in increased manufacturing costs. Updated regulatory guidelines demanding higher safety margins and enforcing good manufacturing practices are leading to tighter specifications. This stresses the need to implement robust and efficient orthogonal strategies for virus clearance to meet the requirements of a virus-clearance approach based on risk assessment. Such technologies can involve virus removal by nanofiltration, inactivation by ultraviolet C (UVC), and adsorption by membrane chromatography. Additionally, this three-tier platform should be characterized by using disposables to meet the flexibility and low capital requirements needed in early-stage process development. All of these new paradigms in virus clearance are scalable, economical,orthogonal, and disposable.

Today's downstream processing operations generally focus on two main areas: the initial recovery phase, when bulk purity is achieved, and the subsequent polishing phase, which adds safety through orthogonal strategies for impurity and pathogen clearance.

  Commercial manufacturing of therapeutic antibodies requires robust and reliable processes that are economical and deliver high yields of a product that is pure and safe for human use. One factor that poses a constant threat to product safety is the presence of viruses in the finished product. Virus contamination of products derived from human or animal cells can have disastrous clinical consequences causing diseases ranging from common colds and influenza, to acquired immune deficiency syndrome (AIDS), hepatitis, herpes, measles, and poliomyelitis. Some viruses like Epstein-Barr, human papillomavirus, and retroviruses are even oncogenic, causing the insertion of cancer-causing genes into cellular genomes.

  It is essential to review both the short- and long-term consequences of viral contaminants existing in biopharmaceutical products. In this context, it is worthwhile to understand some important aspects of current and state-of-the-art methods for inactivating and eliminating viruses from process streams that generate products intended for use by humans. 

Viruses are composed of small amounts of DNA or RNA, encapsulated by a protein coat, and may be enclosed in an envelope made of proteins, carbohydrates, and lipids. Viruses exploit the enzymes and other host-cell machinery to replicate themselves. The viral nucleic acid can be single- or double-strand DNA or RNA. A single virion is a completely developed virus particle made of 1–50% nucleic acid and 50–99% proteins or glycoproteins and lipids. Virions range from about 15 to 450 nm in size.  

Regulatory Requirements Regarding Viral Inactivation and Clearance 

Viral contamination is a risk to all biotechnology products derived from cell lines of human or animal origin. Contamination of a product with endogenous viruses from cell banks, or adventitious viruses from personnel can have serious clinical implications.

  To ensure maximum viral safety, the ICH Q5A regulatory guideline mandates that manufacturers of therapeutic biological products for human use implement adequate technologies in their manufacturing process and demonstrate the capability of their processes to remove or inactivate known or adventitious contaminants based on a process-specific virus clearance strategy.

  According to the latest draft on regulatory guidance from the European Agency for Evaluation of Medicinal Products (EMEA), potential contaminants may be enveloped or nonenveloped, small or large, DNA or RNA, labile or resistant viruses.

Viral safety of licensed biological products must be assured by three complementary approaches: (i) thorough testing of the cell line and all raw materials for viral contaminants, (ii) assessing the capacity of downstream processing to clear infectious viruses, and (iii) testing the product at appropriate steps for contaminating viruses.

  The first study is required before Phase 1 clinical trials, in which the process should be evaluated for inactivation or removal of an enveloped and a small nonenveloped virus and at least two orthogonal steps should be used for achieving the same.

  A second and more complex study is then conducted before manufacturing Phase 2/3 materials to provide evidence of the effective and adequate clearance of relevant and known viruses, as well as the removal of a range of novel and unpredictable viruses.

  A viral clearance study with at least four viruses for late stage is state-of-the-art and log-reduction values (LRV) of four or higher are perceived as robust and effective safety measures.

  At least one of these clearance steps evaluated in a validation study must be effective against nonenveloped viruses, such as porcine parvovirus (PPV), canine parvovirus (CPV), or minute virus of mice (MVM). As for enveloped retroviruses, although no cases of infection or transmission of Chinese hamster ovary (CHO) cell-related type A and C virus particles have been reported so far, retrovirus-like particles theoretically pose safety concerns to humans because of their morphological and biochemical resemblance to tumorigenic retroviruses, and therefore, have to be completely removed or inactivated.

Methods for Viral Inactivation and Clearance

The robust and reliable capability to eliminate viruses must be demonstrated by a risk-based approach.

  Today's requirements demand a statistically independent combination of methods (orthogonal technologies) for removing enveloped and nonenveloped viruses based on the different physical principles of removal and inactivation, and yet are complementary to each other.

Several methods can be used for virus clearance in bioprocessing. These include inactivation methods such as solvent and detergent (SD) or chemical treatments, low pH, microwave heating, adsorption by chromatography, and removal by mechanical or molecular sieving using normal and tangential-flow filtration methods. The first three of these, treatments with solvents and detergents, low pH, or microwave heating, all have significant limitations in their ability to inactivate small nonenveloped viruses. SD treatments were commonly used for plasma proteins and were considered the gold standard for inactivating enveloped viruses.

 It has been shown that SD treatments of a recombinant protein can completely and rapidly inactivate enveloped viruses like PI-3, XMuLV, IBR, and MCF.

  However, small nonenveloped viruses are not being eliminated substantially by this virus-clearance technology. Low pH inactivation of murine retroviruses is reported to be highly dependent on time, temperature, pH, and relatively independent of the recombinant protein type or conductivity conditions outlined.

  Heating is not considered one of the most reliable methods for virus inactivation because of the variation in stability of each viral genome to heat or temperature.  

Viral Clearance Capabilities of Membrane Chromatography 

Chromatography and filtration, on the other hand, are widely accepted methods for virus adsorption and removal respectively and act as orthogonal techniques in the viral-clearance platform. Membrane chromatography, a relatively newer technique gaining prominence in biomanufacturing, has proven to be efficient in removing small nonenveloped viruses.

  Ion-exchange membrane adsorbers, with ligand–virus-binding properties similar to those of anion-exchange (AEX) chromatography, have the disposable option as an added advantage. This not only reduces capital costs but also eliminates post-use cleaning, sterilization, validation, and risk of carry-over contamination, thereby simplifying adsorptive virus clearance.

Efficient clearance data between 4.41 log10 and 6.67 log10 for MVM has been determined for membrane chromatography.

  Additional studies have demonstrated that membrane chromatography meets and exceeds viral-clearance performance of Q resin chromatography.

  Clearance capabilities of Sartobind Q for nonenveloped viruses have been shown to be between 3.56 log10 for MVM and more than 6.92 log10 for PPV.

  It has been demonstrated that the platform tested membrane chromatography, has a process capacity greater than 3,000 g MAb/m

  or 10.7 kg MAb/L with a LRV >5 for four model viruses.

  Mass balance in viral-clearance study is another important parameter to demonstrate efficient virus removal by membrane adsorbers. A 100% recovery was demonstrated for PRV, Reo-3, and MVM, when the membrane was stripped with 1-M NaCl, illustrating efficient charge capture for the three model viruses while high salt treatment of the membrane showed 70% recovery for MuLV.

  The virus-clearance capability of such technology has been presented in Tables 1a and 1b. 

A second orthogonal technique in the virus-clearance platform is nanofiltration that has traditionally been accepted as a robust method for virus clearance.

  This is the most expensive downstream step, accounting for up to 40% of costs, and is the natural target for optimization.

  Initially, virus removal by filtration was found to be highly dependent on size of the virus, and less dependent on parameters like buffer composition, process time, protein type, and pressure.

  Earlier studies have shown the principal feasibility of PP7, a small nonenveloped 25 nm bacteriophage, to act as a model virus for small, nonenveloped viruses.

  The ability of commonly available nanofilters to retain bacteriophage has been clearly demonstrated in recent studies.

  Virus spiking trials using 20-nm retentive virus removal filters have also shown to clear both large and small viruses (Tables 2a and 2b).

The evaluation of a virus filter should not be limited only to its capacity. An ideal virus filter should retain all viruses and allow high protein transmission while maintaining a high flow rate without significant virus breakthrough. Unexpected virus passage during a virus-clearance step is undesirable from a good manufacturing practice (GMP) and validation standpoint.

  Virus passage through the filter could occur for many reasons, including accumulation of aggregates, high spike concentrations, and other impurities that may block the filter or result in a breakthrough.

  This is a serious safety concern that must be minimized. Although contaminants and other various parameters may be the main cause of filter breakdown, some nanofilters still efficiently remove viruses at high LRVs even when experiencing high flow decay. Earlier, detailed analysis of the retention characteristics of PP7 by a PESU-based 20-nm nanofilter underlined the principal capability of nanofiltration to act as a robust and effective virus removal step independent of flow decay or the nature of product being filtered (Figure 1).

  Additionally, a recent report outlined the various titer reduction capabilities of virus retentive nanofilters.

  The study showed that not all filters tested for their LRVs versus flow decay profile experienced a significant loss of titer reduction with increasing flow decay. To ensure the highest level of viral safety of biopharmaceuticals, it is important to understand and predict the efficiency of virus removal steps while also realizing that small virus-retentive filters should not be viewed as absolute in their capacity to clear viruses. 

Figure 1.  Log-reduction value (LRV) versus flow decay using monoclonal antibodies (MAbs) at 8 mg/mL in 20 mM Tris, 25 mM NaCl, at 7.2 pH, 2 bar, and upstream titer of 7.3 x 107/mL. All samples prefiltered through 0.1 mm membrane.31 

In this article, we propose implementing ultraviolet C (UVC) as a third orthogonal technology for virus clearance in the downstream purification process. UVC, newly developed as a virus-inactivation technology, targets small, nonenveloped viruses and offers another robust method for removing adventitious viruses. In UVC radiation, low-dose radiation at 254 nm destroys the viral nucleic acid while maintaining the structural and functional integrity of the protein of interest. IgG losses are <5%.

 The efficiency of viral inactivation and product recovery is sensitive to the viscosity and absorption coefficient of the protein solution and its residence time in the radiation chamber.

  However, further studies are on to demonstrate the effect of UVC, if any, on the protein-folding characteristics, disulphide bonds, glycosylation, and phosphorylation patterns of the protein of interest.  

Disposability, a New Paradigm in Viral Clearance

When evaluating the reliability and robustness of nanofiltration, membrane chromatography, and UVC technologies as orthogonal steps in virus clearance, the inclusion of single-use disposables to the above platform is also gaining prominence. In addition to reduced capital costs, disposables provide the flexibility and ease of operation in process optimization and early stages of manufacturing. Further, disposables eliminate cleaning, sterilization, process validation, and the risk of carryover contamination in viral-clearance studies. Various scenarios presented so far have provided evidence that the disposable option for both nanofiltration and membrane chromatography has proven to be technically possible, and furthermore, cost effective.

  To underline such statement, cost-model scenarios have been developed and are used to help evaluate the economic justification of sourcing disposable technologies.

Striking an optimum balance between ensuring high pathogen safety, achieving maximum product recovery, and meeting adequate regulatory expectations is a big challenge for the biopharmaceutical industry. Nanofiltration, membrane chromatography, and UVC, together used as a technology platform for virus clearance by removal, adsorption, and inactivation, provide robust and efficient clearance capability for all viruses with major focus on small, nonenveloped viruses such as PPV or MVM. Driven by regulatory guidance, technologies with the capability to remove or inactivate small, nonenveloped viruses should be implemented from an early stage into the downstream process of a biopharmaceutical to fulfill virus-clearance expectations. Furthermore, the disposable option, when integrated into this technology platform, offers higher flexibility in manufacturing, increased ease and speed of operation, and eliminates the risk of carry-over contamination. The present age recognizes several paradigm shifts in virus and contaminant clearance, which are scalable, economical, and orthogonal. Together with these, the trend toward disposables is a clear implication to combine steps and orthogonal strategies for every objective in bioseparation to realize the industry goals for yield and quality.  

 is a process development scientist, viral clearance and cell line development, Global Purification Technologies Group, and 

 is a senior product manager of purification technologies, both at Sartorius Stedim Biotech, Goettigen, Germany, +49.551.308.3959, 

 1. Gottschalk U. The renaissance of protein purification. BioPharm Int. 2007 Oct;Suppl:41–42. 

 3. International Conference on Harmonization. Q5A. Viral safety evaluation of biotechnology products derived from cell lines of human or animal origin. Geneva, Switzerland; 1998. 

 4. European Commission (Enterprise Directorate General). EMEA Guideline on virus safety evaluation of biotechnological investigational medicinal products. London: 2006 Jun 28. 

 5. Committee for Proprietary Medicinal Products (CPMP). Note for guidance on virus validation studies: the design, contribution and interpretation of studies validating the inactivation and removal of viruses. CPMP/BWP/268/95;1996 Feb. 

 6. Anderson KP, Lie YS, Low MA, Williams SR, Fennie EH, Nguyen TP, Wurm FM. Presence and transcription of intracisternal A-particle related sequences in CHO cells. 

 7. Anderson KP, Low MA, Lie YS, Keller GA, Dinowitz M. Endogenous origin of defective retrovirus-like particles from a recombinant Chinese hamster ovary cell line. 

 8. Dinowitz M, Lie YS, Low MA, Lazar R, Fautz C, Potts B, Sernatinger J, Anderson K. Recent studies on retrovirus-like particles in Chinese hamster ovary cells. 

 9. Shi L, Chen Q, Norling LA, Lau AS, Krejci S, Xu Y. Real-time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification. 

Articles

Peer-Reviewed Research

How to implement a risk-based approach to eliminate viruses using orthogonal technologies.

Virus Clearance Strategy Using a Three-Tier Orthogonal Technology Platform

Although the worldwide response to the swine flu outbreak of early 2009 has shown that we have come closer to being prepared for future flu pandemics, emerging diseases and new influenza strains still present a tremendous threat, with the potential for disastrous effects to global health and both local and global economies. A major limiting factor in preparedness is still the ability to manufacture an adequate number of vaccine doses, in a short period of time and in the right location, when an outbreak occurs. The following article discusses current developments and what could be done in the future from a technology and policy perspective to improve our capability to cope with pandemics. 

Each year, approximately 15 million deaths are directly caused by infectious diseases. The secondary effects of infections cause millions more. Two of the major killers are malaria and HIV, but influenza also represents a significant risk. In September 2008, the World Bank issued a report estimating that a flu pandemic of similar scope to that seen in 1918 could kill 71 million people worldwide and cost the global economy more than $3 trillion.

 Although this is a worst-case scenario based on past experiences, the differences in transmission and severity shown by the outbreaks of avian flu in South-East Asia in 2003 and this year's swine flu have highlighted how unpredictable such a virus can be. 

Although the effects of each pandemic will vary greatly, depending on the nature and severity of the disease, the threat applies to all corners of the globe, from major capital cities to rural areas of developing countries. In addition, pathogenic viruses such as HIV and influenza mutate rapidly, which is why the HIV virus we encounter today is very different from the strain that first emerged in the 1980s. Consequently, resistance to the antiviral drugs in circulation develops very quickly and as the speed of development of new treatments lags behind, we are left increasingly exposed to the effects of infection. If we also take into account the fact that infectious diseases are the second leading cause of death globally, then it is clear that we must have a robust strategy in place to address the threat of a pandemic. 

One of the best ways to combat the spread of infectious disease is through the implementation of an immunization program. Immunization has eradicated smallpox, reduced the global incidence of polio by 99%, and dramatically decreased many other causes of illness and death.

 Countries around the world are now looking to vaccines and immunization as the way forward, and the World Health Organization (WHO) Department of Immunization, Vaccines and Biologicals has set for itself the goal of attaining "a world in which all people at risk are protected against vaccine-preventable diseases."

Over the past eight to nine years, and especially since the SARS and avian flu outbreaks, many governments have concentrated their efforts on developing preparedness plans. Increased stockpiles of vaccines and antiviral drugs, finance set aside for vaccine development and research, and a focus on education of the public—all played their part in ensuring that when swine flu emerged earlier this year, we were more prepared. There is still a huge gap, however, between the level of vaccine availability and demand, and we still have a long way to go before we have the technology, manufacturing capacity, and responsiveness required to combat a pandemic on a scale based on the 1918 incident. Because vaccination is a key element in pandemic preparedness, what more could we do to ensure that the next global pandemic has as little impact as possible, both socially and economically? 

Although we are prepared for the next flu pandemic with stockpiles of antivirals and vaccines, these projects tend to be localized, with some countries holding enough to cover their own population and others falling well short. However, even those countries with large, carefully stored reserves of antivirals may not be protected in the event of an outbreak because of increasing viral resistance to currently available medicines. 

In addition, stockpiling of vaccines relies on being able to predict which virus, and which particular strain of that virus and with which traits, will strike next. Therefore, our biggest future pandemic threat comes from new emerging diseases and virus strains, which we know nothing about. In the case of the 2009 swine flu pandemic, many doses of vaccines were rendered useless because they were targeted at the avian flu strain H5N1, demonstrating that even here we need to adapt our approach. Also, vaccines are often relatively unstable because of their complexity, more so than any other class of biopharmaceuticals, and therefore, are difficult to transport and store becasuse they require controlled temperature conditions.  

Developing a rapid response capability is one way, and some argue the best way, to overcome the problems associated with stockpiling and being unable to predict what will cause the next pandemic. A rapid response would enable the production of a suitable, effective, and safe vaccine as close to the outbreak as possible, both in terms of time and geography. 

Currently, the investment required for a dedicated flu vaccine factory—from commissioning to validation—could run as high as $600 million for 50 million doses, depending on the vaccine technology used, and the time taken to build and start production could be as long as six years.

 In addition, although our existing vaccine manufacturing capacity is greater than it was a few years ago, we are still far from being able to produce sufficient vaccines to supply the whole world. We may be able to ramp up production to meet demand if given time, but in reality, in the event of a serious pandemic we would have neither the time nor capacity to cover even a small percentage of the world. 

To address this problem, suppliers have been working hard to develop platform and manufacturing process tools that could support novel ways of vaccine production, which would enable facilities to be rapidly deployed and set up as and where needed. A flexible platform process is currently under development on which any vaccine could theoretically be produced rapidly and in any given location. 

Another option is a mobile manufacturing facility, which involves the use of small-scale, modular manufacturing units that are able to work according to cGMP standards. Each unit could contain a flexible manufacturing structure, which is sufficiently adaptable to produce the particular vaccine required. Although such modular units are still in development, much of the technology contained in them is already established and widely used in the industry. Ready-to-use product platforms are ideally suited for these mobile units because they cut time spent on cleaning between runs and are prevalidated, radically shortening the manufacturing time. 

Borders are often closed in a pandemic, which would mean that a community could not rely on cross-border provisions and must have independent vaccine supplies. Also, large countries such as India, China, and the US have widely dispersed populations, often with very remote locations, and therefore would suffer greatly from the problems associated with transportation and storage of vaccines. Mobile manufacturing units would overcome these problems by allowing localized production to tackle an outbreak of disease in an affected country or region. The local manufacturing approach also contributes to long-term investment, creating local capacity, and helping to develop local skills and education, offering poorer countries the facility to manufacture their own vaccine rather than buying in from an outside supplier. However, vaccine-manufacturing solutions must also meet the specific local needs and consider restraints typical in a certain region, i.e., the availability of services such as water, which is vital in vaccine manufacture.  

With all of these solutions, it is essential to have a standardized approach and develop solutions that are validated and comply with all safety requirements. To achieve this, countries could benefit from working with a global company that also has the experience and infrastructure to work with partners locally.  

Although vaccine production methods are constantly improving, there are still many hurdles to overcome. The biggest limiting factor we currently face is the lack of sophisticated technology for rapid vaccine manufacture. One good example is the production method of influenza vaccines that still uses chicken eggs. This is widely considered to be an archaic system with a range of drawbacks including the logistics of egg procurement and a slow production process. As a result, production is shifting toward mammalian cell-based production and new technologies such as virus-like particles. Suppliers are working hard to develop new tools that will not only support existing production methods, but also push them forward to ensure that manufacturing capability is available when the methods are ready.  

In addition to significant levels of investment in vaccine-production processes, another area of focus for improving future ability to survive pandemic threats and mutating pathogens is in the research and development of universal vaccines. The universal vaccine approach may improve the chances of marginalizing pandemic diseases. There are many companies attempting to rise to this challenge.  

In the near future, it is also hoped that a "template" vaccine will be available that is easily adaptable when a new strain or new mutation arises. Although such template vaccines hold great promise, we are still far from having access to any technology that will protect us from our biggest potential threat—emerging diseases. These are pathogens of which we presently have no knowledge about and which would not behave in the same way as those we have been studying. 

Because we cannot know for sure what we might face in the future, investment in education is incredibly important. From informing the general public about how to prevent the spread of viruses and giving them the necessary knowledge to exert pressure on the authorities to facilitate preparedness, to building scientists' knowledge and ensuring the relevant expertise is available to run manufacturing facilities, it is crucial that both industry and government bodies work together for the future. 

For a country or region to be fully prepared, all stakeholders including government bodies, industry, and healthcare providers, must work together and coordinate their efforts. When swine flu emerged earlier this year in Mexico, it made evident the instrumental role the WHO has played over the past years by ensuring all countries had a preparedness plan in place. The WHO also has the important task of supporting the worldwide pandemic response infrastructure, ensuring access to data is available, coordinating stockpiling of drugs, and overseeing communications between stakeholders.  

Ensuring that all populations have access to safe vaccines and the corresponding level of supporting education are two important steps that must be achieved in the move toward true pandemic preparedness. Global companies have essential technological expertise and the required infrastructure to support governments and vaccines manufacturers so that everything possible is done to protect the global population from pandemic threats.  

Over the last few years, there has been improvement in the global level of preparedness and the ability to respond to global pandemic threats. This has been manifested in a united response that has been evident during the current swine flu pandemic. However, no stakeholders can afford to be complacent and there is a need to continue and enhance investment in developing technologies such as template vaccines and quicker, more flexible manufacturing approaches. Moreover, the next pandemic could be the next-generation HIV, malaria, or an unidentified emerging disease, which would be even more difficult to tackle, so investment in the ability to react quickly is vital.  

Developing true partnerships between governments, manufacturers, suppliers, and scientists is critical to create and support systems that will enable appropriate response to any emerging global threat to human health. 

 is a vaccine initiative marketing manager, both at GE Healthcare,  Uppsala, Sweden, +46.18.612.1636, 

 1. Burns A, van der Mensbrugghe D, Timmer H. Evaluating the economic consequences of avian influenza. World Bank Report; 2008 Sept. 

 2.  GAVI Fund. Washington, DC. Available from: 

http://www.gavialliance.org/about/in_technologies

 3.   World Health Organization. Geneva. Available from: 

4.  Robinson JM. An Alternative to the scale-up and distribution of pandemic influenza vaccine. BioPharm Int. Advances in vaccine development and manufacturing. 2009 suppl;22(1):12–20. 

The industry and government must collaborate to develop robust technologies and quicker, more flexible manufacturing approaches for vaccine development.

Pandemic Preparedness: What More Can We Do?

Current egg-based manufacturing methodologies for the production of influenza vaccines are slow and have an inherent lag period from variant identification to vial. In addition, seasonal influenza variants are predicted up to 12 months in advance by the World Health Organization but may only be confirmed five to six months before a vaccine reaches the market. Vaccine manufacturers have little time to produce and stockpile the selected candidates, and often have to manufacture one or more vaccine strains in advance at risk of the preferred strains altering. There is a need for a more flexible and rapid production methodology to produce cheap and effective influenza vaccines with minimal notice for pandemic variants. A new approach to combat this threat is required. There are several technologies in early development that may offer a more viable solution to the pandemic threat, such as the use of microbial-derived production processes and platform virus-like particle manufacturing strategies to alleviate some of these constraints and lead to a more rapid response time. 

As of June 2009, the World Health Organization updated the status of the current outbreak of influenza A H1N1 swine flu (Figure 1) to a global pandemic, highlighting the requirement for a cheap, effective prophylactic vaccine and manufacturing strategy to raise the current state of preparedness for such situations. In 1918, one such influenza pandemic swept the globe and an estimated 50–100 million people died as a direct consequence. This historical data suggests that a similar flu pandemic in the modern era would affect 30% of the global population (approximately 2 billion) and an estimated 60 million individuals (1%) would die.

 Combating the virus poses some significant and difficult challenges. Because the virus is an RNA virus, the lack of proof reading capability when the virus replicates can cause the virus to mutate and combine genetic material with another virus serotype resulting in variant forms, which may cross the species barrier wwAny vaccine must be specific to the current common strains of influenza virus to be effective; most vaccines are trivalent meaning they contain three inactivated virus strains to further increase the vaccine's efficiency and to overcome minor drifts in the virus morphology. Seasonal variants are predicted up to six months in advance with scientists selecting the virus strain and subtype that is most likely to protect against the virus in the first quarter of the year. Production of the vaccine by infecting live hen eggs, or more recently cells in culture, then begins and may take up to six months. The current production rate globally is approximately 300 million doses for each 6-month cycle in ideal conditions, providing vaccinations for only 5% of the global population.

There are several anti-viral drugs on the market, such as Roche's Tamiflu (oseltamivir phosphate), which can be used as an effective treatment for H1N1 influenza patients but have a different mode of action than a viral antigen vaccine. Tamiflu's mode of action is to prevent the release of the viral particles from infected cells, reducing the severity and duration of the infection; this is invaluable in reducing infection and controlling the spread of the disease but it has no prophylactic properties whatsoever. It has been estimated that Tamiflu production will be at 110 courses for 2009 alone.

Figure 1.  Images of H1N1 influenza virus. Image taken in the Centers for Disease Control and Prevention Influenza Laboratory2 

The original licensed influenza vaccines were inactivated versions of a mixture of three influenza strains produced by an egg-based manufacturing process. To give an idea of scale, on average, between one and two eggs are needed to produce one dose of vaccine. During the production process itself, fertile hen eggs are infected with the candidate influenza strain and incubated for several days. The vaccine is purified from the allantoic fluid of virus-infected chick embryos by a combination of tangential flow filtration (TFF), density centrifugation, and, more recently, by anion exchange column chromatography. The virus is also chemically inactivated through formaldehyde or  β-propiolactone treatment at some point during processing.  

More recently, licensed inactivated influenza vaccines are made in mammalian cell culture and purified by more modern techniques. The major hurdle facing the manufacturers for the production of a pandemic vaccine is the time of production with the entire production process for a season's influenza vaccine taking an average of six months. The challenge to the vaccine producers is to develop a production process with a rapid turnaround time with minimal notice to combat a pandemic threat. 

There are many approaches that may be taken for the development and production of a vaccine. An attractive prospect is the use of platform production systems which, in turn, would offer significant advantages for the production of pandemic vaccines, significantly reducing the lead in and production times and ultimately time to patient once the virus serotype has been identified. This approach will also allow for strategic manufacturing sites to be at a state of operational readiness for the production of the vaccine candidate with minimal notice. One such approach is the use of a microbial system for the production of virus-like particle (VLP) based vaccines.  

An Emerging Solution—Virus-Like Particles (VLPs) 

There are a number of expression technologies currently in use for the production of VLPs, including mammalian, insect and microbial-based systems. A microbial-based platform approach is ideally suited for this application. They are simple, cheap, and have a rapid turnaround time required for pandemic vaccine production. A microbial production system may also be favourable because, unlike insect and mammalian systems, they do not require incorporation of specific viral inactivation or reduction steps into the production process, which may be problematic when processing such large molecules as VLPs.  

VLPs for pharmaceutical applications typically exploit the inherent ability of many viral structural proteins to self assemble. For instance, VLPs are formed when the 16 kDa hepatitis B viral core proteins spontaneously self-assemble to form icosahedral VLP structures of approximately 30–50 nm in a host cell as shown in Figure 2. They can be engineered to display a range of surface antigens, such as the hemagglutinin (HA) surface antigen providing a useful carrier for vaccine platforms. By presenting antigens in a VLP form, where typically 240 copies of the same protein can be presented in a single particle, it may be possible to stimulate the immune system in a more appropriate way than can be achieved through presentation of dissociated antigens adjuvanted with other macromolecules. One such example is iQur Ltd's (London, UK) tandem core VLP expression technology.

Figure 2.  Scanning electron microscope image of a preparation of virus-like tandem core particles displaying (a) Hepatitis A virus complete P1 polypeptide or (b) Hepatitis B virus surface antigen from E. coli (picture courtesy of iQur Ltd.)4 

Vaccines are complex and diverse biomolecules ranging from recombinant subunit antigens to live attenuated organisms. Therefore, a range of production technologies and process formats are required to manufacture sufficient quantities of these varied products. However, this wide range of production methods can be problematic because each one requires specific capital expenditure and specialized development approaches coupled with operator expertise and concomitant high cost, which limits the availability of vaccines to developing nations. With current comparisons suggesting productivity in the range of 10-g/L for prokaryotic systems versus a realistic short- or medium-term 3-g/L goal from eukaryotic processes, there is a growing trend and focus toward a lower number of production platforms by manufacturing in similar expression systems including microbial backgrounds. 

These microbial platforms in particular offer an attractive solution to the often numerous challenges associated with the development of vaccine products. The rapid establishment of strains expressing the target recombinants is especially attractive to product developers, often facilitating early efficacy studies from multiple product variants for pandemic threats while paralleling process development and manufacturing activities.  

In addition to a reduction in process costs of goods and the process durations associated with microbial systems, the production scales required for low-dose, high-potency products have an associated reduction in capital expenditure for manufacturing facilities; thus an all round attractive proposition for reactive development of vaccine products.  

The final decision on the expression system of choice will be determined by the effect of post-translational modification (PTM) on product efficacy. However, where there are no specific PTM requirements, microbial-based production platforms gain an advantage. Previous resistance to using such systems, for instance in the production of Fab-based therapies have centred around the limitation for manipulation of vH to vL expression levels, which can affect both productivity and product efficacy. However, advances in transcriptional regulatory elements mean that coordinated control of these and other multicomponent products can be optimized to significantly increase process titers while assisting in the decrease of normally associated product impurities. 

In the VLP vaccine arena, various companies have focussed their attention on decreasing development timelines while increasing product titers through the use of alternative cell-culture–based systems. Novavax, Inc. (Rockville, MD), has focussed on a rapid development strategy for a VLP product targeted toward emerging influenza viruses, which has resulted in a concomitant increase in both product potency and significant relative dose yields compared to the traditional egg-based manufacturing systems. An effective vaccine can be manufactured in approximately 10–12 weeks from the point of strain identification while clearly the requirement for scale in response to required doses has been significantly reduced. However, typical process (raw material) cost of goods may still remain high. 

More recently, the development of effective vaccine treatments through yeast and 

-based VLP production platforms have further decreased development and manufacturing timelines and scale requirements while further increasing patient safety. With the development of platform downstream processing strategies and estimated recoveries in the region of 50–60%, even small-scale bioreactors remain a firm target for manufacturing campaigns using these systems. Indeed, estimated dose yields post purification are in the region of 500–1,000/L while development and manufacturing program timelines are expected to halve those associated with historical tissue-culture–based systems. 

A platform approach to the purification process can also be adopted. The size of the particles can be advantageous during the purification process allowing generic steps to be developed. One such purification strategy is outlined in Figure 3. 

Figure 3.  Platform purification strategy for the production of virus like particles 

The process broadly consists of cell harvest to separate the cells from the fermentation media, resuspension into a controlled, buffered environment, and cell lysis through high-pressure cell disruption. The bulk of the cellular debris is then cleared from the solution by microfiltration and the process solution concentrated and buffer exchanged using a high molecular weight cut-off TFF system. Further purification and polishing can then be achieved using anion exchange (AEX) and size exclusion chromatography.  

VLPs are produced as an intracellular product from a microbial production system. They must be released into the process stream to allow for subsequent purification. This can be achieved by several means, the most common being the use of high-pressure lysis. This is a process whereby the process solution is forced through a small fixed orifice at high pressure. The rapid transfer of the sample from a region of high pressure to one of low pressure causes cell disruption. 

This type of cell disruption is by far the most efficient and reproducible but does have a number of drawbacks in processing of VLPs. The extremely high shear not only lyses the cells but also micronizes the cellular debris; this coupled with the large size of the VLPs themselves can make the subsequent clarification step problematic. Careful attention must be paid during the development of such a step to strike a balance between cellular disruption and the release of the VLPs into the process medium and the micronization of cellular debris to avoid fouling issues further downstream in the production process.  

A number of options are available for clarification including centrifugation, depth filtration, or micro-filtration to name a few. Generally, microfiltration is the preferred option as it is well established in the biopharmaceutical industry as a scalable and robust clarification technique. Whichever technique is used, this clarification step may be problematic post high-pressure lysis.  

Because of the relatively large size of the VLP molecules, a high molecular weight cut off membrane such as 500 or 1,000 kDa may be used for this application. The large molecular weight cut off has the advantage of reducing the host cell protein (HCP) content while recovering the VLPs in the retentate fraction.   

An effective approach for the reduction of DNA and endotoxin levels is the use of AEX. Owing to the shear size of the particles, there are inherent mass transfer limitations that play a significant role during any chromatographic step. This reduces mass diffusion during column loading, reducing the effectiveness of the step and limiting of the column's overall dynamic binding capacity. Scale may then become an issue. There are several technologies available to negate this problem such as membrane adsorbtion technology, available from a number of suppliers such as Millipore, Pall, and Natrix Separations. Eden Biodesign has found the Poros HQ diffusion chromatography media (Applied Biosystems) to be effective. A typical chromatogram obtained during an anion exchange purification of a VLP is shown in Figure 4. 

Figure 4.  Anion exchange chromatogram for the purification of virus like particles (VLPs) 

Taking the platform approach, the size of the particles should be in the range of 30–50 nm. This is above the molecular exclusion limit for a number of size exclusion resins, and therefore, they may be used in a group separations mode whereby the large VLPs are above the upper exclusion limit for the resin to pass through in the void volume. The smaller HCPs and misformed VLPs pass through into the pores of the resin and are retained on the column for longer, eluting with a greater residence time.  

A typical group separations chromatogram is shown in Figure 5, peak one in the void volume being the VLPs and peak two being gross impurities such as residual HCPs and misformed particles. 

Figure 5.  Size exclusion chromatogram for the purification of virus-like particles (VLPs) 

There are a number of problems associated with the production of therapeutic products and vaccines from microbial systems, specifically from 

. These are centred mainly on the primary separations and reduction of lipopolysaccharides (LPS) from the process stream.  

A major challenge in the production of any therapeutic products from 

 is the requirement for the removal of LPS from the process stream, a specific processing step for this application may be required. LPS or endotoxins are a major component of gram negative bacteria such as 

. Endotoxins, even at relatively low doses can initiate an innate immune response resulting in a fever and in severe cases, septic shock and even death. Regulatory specifications exist for many parenteral products and are typically 2 IU per dose or less. There are many technologies specifically for this application such as LPS affinity chromatography and membrane adsorbtion technology. Membranes are a more attractive proposition because they are cheap, simple to operate, and require little or no cleaning validation. There are several options available for this application such as Pall's Mustang E and Sartorius's Q membrane. Membranes such as the Sartobind Q have a slightly different chemistry to the Mustang E but work on a similar charge effect, with the added advantage of a 3 μm pore size, so that few or no losses are seen because of the size exclusion effect that may be observed when processing such large molecules as VLPs. 

However, this is not the complete picture; mechanically removing free endotoxins from the process stream is only half the challenge. Given the typically hydrophobic nature of VLPs, there is the additional problem of endotoxins adhering to the particles themselves, offering additional and significant further challenges to reduction. Bound endotoxins may be released by treatment with solvents, detergents, or a combination of both, further complicating the manufacturing process and adding expense. A combination of endotoxin reduction techniques is the more favourable approach. A production strategy in yeast such as 

 may also be the favoured approach to avoid this problem.  

New technologies such as virus-like particles are promising weapons in the battle against pandemic influenza.

Advancing Vaccine Technology to Combat Global Pandemic Threats

The commercial preparation of acellular pertussis vaccine depends on the effective recovery and purification of the antigens pertussis toxin (PT), filamentous haemaglutinin (FHA), and pertactin (PRN) from 

fermentation. This study describes the recovery of the antigens using an open channel 0.45-μm tangential flow filtration module with optimization of the process parameters of transmembrane pressure, cross flow, and flux. Under the optimized conditions, greater than 98% recovery of FHA and PT was obtained. 

Pertussis or whooping cough is an acute infectious disease of the respiratory tract caused primarily by 

 Pertussis still continues to cause significant morbidity and mortality globally. "Per" meaning intensive and "tussis" cough: describes the clinical manifestation of the disease characterized by coughing and minor systemic complaints predominant in children.

 less than 1 μm in width and length. Diptheria toxoid and tetanus toxoid combined with whole-cell pertussis (DTP vaccine) has long been in use for childhood immunization programs. Whole-cell DTP vaccines are associated with local adverse reactions like erythrema, swelling, fever, and mild systemic events, e.g., convulsions and hypotonic hyporesponsive events.

 The reactogenecity of the whole cell pertussis vaccine has evoked public controversy in several countries and prompted the development of the acellular pertussis vaccine, which has a lower rate of adverse events and is effective in preventing pertussis. 

Pathogensis of pertussis is a complex process and not completely understood. It includes pertussis toxin, filamentous hemagglutinin, pertactin, tracheal colonization factor (TCF), serum bactericidal resistance factor (BrkA), tracheal cytotoxin (TCT), adenyl cyclase toxin-hemolysin (ACT), lipoplooligosaccharide (LOS), heat labile toxin (HLT), and fimbriae 2 and 3 (Fims 2 & 3).

 Acellular pertussis vaccines contain inactivated pertussis toxin (PT), 105 kD, and may contain one or more of the bacterial components: filamentous hemagglutinin (FHA), 220 kD, a 69 kD outer membrane protein pertactin (PRN), and Fims 2 & 3. 

To keep the disease under control large amounts of PT, FHA, and PRN must be prepared for mass immunization. The first step toward purification is the clarification of the fermentation broth for the recovery and further downstream processing of the antigens. The traditional method available is centrifugation. The scale-up of the process using centrifugation may lead to lower recovery of the antigens and a huge capital investment with low scalability. Therefore, an efficient bioseparation technique that can be easily scaled up with lesser capital investment is necessary. Membrane-based technology for biopharmaceuticals has been widely suggested and used.

 Tangential flow filtration (TFF) technology using an open channel, steam-sterilizable device allows microorganisms to be washed and concentrated in an efficient, rapid manner by using a closed recirculating filtration system, where both the concentrated cell mass and the filtrate can be collected under aseptic conditions. The factors that must be considered for successful scale-up of TFF technology are purity, yield, and reproducibility, for which the process parameters of the TFF must be optimized accurately. This article describes the optimization of the TFF process for high recovery of the acellular pertussis vaccine antigenic components: PT and FHA. 

 strain Tohama-I was used in this study. The strain was procured from the American Type Culture Collection (Manassas, VA) as a freeze-dried ampoule. 

Purified pertussis toxin, purified FHA, anti-PT serum (mouse), anti-FHA serum (mouse), anti-PT serum (sheep), and anti-FHA serum (sheep) were obtained from National Institute for Biologicals and Control (Hertfordshire, UK). Anti-mouse polyvalent immunoglobins peroxidase conjugate (GAMHRP) was obtained from Sigma (St. Louis, MO). Sodium-L-glutamate, glutamic acid, and CaCl

O were from Merck. All other chemicals were obtained from Sigma.  

 Tohama strain was grown and maintained on Bordet-Gengou (B.G.) agar medium containing defibrinated horse blood. The strain was stored in a lyophilized state at 2–8 °C until grown on B.G. agar medium. The solid bacterial growth on B.G. agar slant, incubated for 48 h at 35 °C, was transferred into 500-mL Erlynmeyer flasks containing 200 mL of liquid medium similar to the defined medium described by Stainer and Scholte.

 One liter of medium contained 10.7 g of monosodium glutamate, 0.24 g of L-proline, 2.5 g of NaCl, 0.5 g of KH

O, 1.5 g of Tris base, 10 g of casamino acids, and 5 g of yeast extract. The medium was supplemented with the following amount of supplements per liter: 0.04 g of L-cysteine monohydrochloride, 0.01 g of FeSO

O, 0.15 g glutathione (reduced), 0.4 g of L-ascorbic acid, 0.004 g of niacin, and 0.02 g of CaCl

O. The supplement was prepared in a concentrated form (100x) and filter sterilized. Cultures were incubated on an orbital shaker (170 rev/min) for 24 h at 35 °C.  

This 200-mL culture was transferred to 10-L flask containing 3 L of liquid medium having the same composition as the 200-mL medium. The 3-L culture was incubated on a rotary shaker (190 rev/min) for 24 h at 35 °C. When optical density (OD) at 580 nm of the 3-L culture was more than 1.0, it was transferred into a fermenter containing 30 L of defined medium. Heptakis solution was sterilized by filtration and added into the fermenter at a concentration of 1 g/L of liquid medium. The bacteria were grown in batch mode in a fermenter of 50 L operating volume at 35 °C. The fermentation was maintained at a temperature of 35 °C, a dissolved oxygen level of 25% by cascading with an aeration of 3 L/min to 21 L/min and the agitation rate from 130 rpm to 290 rpm. The pH was controlled by a solution of glutamic acid and FeSO

O in hydrochloric acid. Foaming was controlled by adding antifoam 204 as needed. The culture was harvested after reaching an OD 7.5±0.1. 

The TFF was carried out using a microporous membrane of pore size 0.45 μm. The construction material of the membrane was Durapore, i.e., hydrophilized polvinyledene difluoride (PVDF). The Prostak TFF module (Millipore, MA) consists of pre-assembled, pretested, and prebonded PVDF membranes on polysulfone plates and available in filtration area. This open channel module is designed to operate up to 5.6 bar, is steam sterilizable, and can be regenerated by cleaning. 

An automated microfiltration (MF) system (Millipore, Bangalore, India) consisting of a 75-L jacketed tank was used in this study. The fluid to be clarified entered from the tank to a rotary lobe pump of capacity 1.8 m

/h at 4 bar pressure and 750 rpm, after the inlet pneumatic diaphragm valve was opened. The feed temperature and pressure were monitored by transmitters. Differential pressure across the installed device was controlled by throttling the motorized diaphragm valve on retentate (fluid that is retained by the membrane) line and retentate flow was measured by an electromagnetic flow meter. The permeate (fluid that passed through the membrane) line was equipped with a flow meter, a pressure and conductivity transmitter, and a peristaltic pump to control the permeate flow. The total system was steamed-in-place (SIP) and a steam trap was used at drain points to remove the condensate after SIP. The flow rate, volume of fluid at permeate and retentate line were monitored continuously during TFF pressures at the feed, permeate, and retentate line. 

The TFF system was installed with a 0.45-μm open channel Prostak device (PVDF membrane, Millipore). The MF filtration module consisted of four stacks with total filtration area 0.34 m

. The system, fitted with the membrane, was flushed with 20-L water for injection (WFI). Integrity of the MF membrane and installation was checked by a diffusion test using an automated integrity testing machine (IT4, Millipore). The system was then drained. SIP of the system was conducted, steam pressure inside the system was maintained at 1.2 bar and 123 °C for 30 min, and then the system was left at a positive pressure of 1 bar. The next day, it was observed that the system was at a positive pressure of 0.4 bar at 28 °C.  

 culture was transferred aseptically into the system tank and clarification was carried out at constant transmembrane pressure (TMP). A secondary peristaltic pump in the permeate side was used for permeate flow control. The feed was concentrated 10 times (30 to 3 L). Temperature, pressure, and volume were monitored at the feed, permeate, and retentate line during the operation. 

The PT and FHA were quantized by ELISA with purified antigens as standards. Microtiter plates (Nunc Maxisorp, Roskilde, Denmark) were coated with either sheep anti-PT antibody (PT ELISA) at a dilution of 1:4,000 or sheep anti-FHA antibody (FHA ELISA) at dilution of 1:12,000 in 50-mM sodium carbonate buffer (pH 9.6). Plates were incubated overnight at 2–8 °C. Volumes of 0.1 mL were used in all steps and the plates were washed four times between incubations by using 100-mM phosphate-buffered saline containing 0.05% (w/v) Tween 20. The plates were blocked for 1 h at 37 °C with 2% (w/v) bovine serum albumin (BSA) in phosphate-buffered saline (PBS; pH 7.4). The plates were washed and culture supernatants were serially diluted and added to the wells, and then the plates were incubated at 37 °C for 1 h. Mouse anti–PT at dilution of 1:1,000 and mouse anti-FHA at dilution of 1:500 in 1% (w/v) BSA in phosphate-buffered saline (PBS; pH 7.4) were added to the plates. The plates were incubated for 1 h at 37 °C. Bound antigen was detected by using goat antimouse horse reddish peroxidase conjugate with a dilution of 1:7,000 (PT ELISA) and 1:10,000 (FHA-ELISA) in 1% BSA in phosphate-buffered saline. The plates were incubated for 1 h at 37 °C and were washed and developed with tetramethylbenzidine and 0.006% hydrogen peroxide in 100 mM citrate-phosphate buffer, pH 5.0. The reaction was stopped by adding 2NH

. The enzyme substrate reaction that developed at 37 °C was measured at 450 nm on a Universal Microplate reader (EL-800 with KC4 data analysis software, BioTek Instruments, Inc., Vermont, MA). 

There are different options for clarification and acellular pertussis antigen recovery. Centrifugation is the most commonly used tool in primary clarification. It can handle considerably high concentrations of insoluble material in the feed. A critical drawback of centrifugation is that cell disruption can occur because of shear, resulting in the generation of smaller particles, which cannot be separated by a centrifuge efficiently. The precipitation efficiency of a centrifuge decreases with increasing deposition of solid sludge in the bowl resulting in frequent discharge, which in turn, decreases product yield and increases process time. Buffer or water used for bowl flush can create osmotic differences between culture broth and flush fluid, resulting in cell lysis and further downstream complication. Dead end or normal flow filtration is unable to take high particle load and the residue on the filter is not recoverable, and hence, is not a feasible option for the clarification of 

fermentation broth, where the whole cells (containing PRN) and the filtrate containing PT and FHA are the products of interest. 

In this microfiltration study, recirculation is accomplished in a closed loop (Figure 1) using a TFF module. The feed pump allows the product present in the feed tank into the TFF module tangentially across the membrane. Materials smaller than the pore size of the membrane are able to pass through the membrane (

 fermentation media containing the antigens PT and FHA), and are collected aseptically. The membrane retains the larger particles (retentate) containing 

Figure 1.  A schematic diagram of microfiltration flow pattern 

The retained components do not build up at the surface of the membrane. Instead, they are swept along by the tangential flow. This feature of TFF makes it an ideal process for finer sized-based separations. 

The pump at the permeate line is used to control polarization across the membrane by offering permeate flow control and low pressure from the permeate side. This type of control ensures that the flux and TMP are low and stable across theflow channel and enables microfiltration to be carried out causing a very low shear to the cells.  

Throughout the TFF experiment, pressure at the feed (P1), permeate (P2), and retentate (P3) were monitored and the volume of permeate collected per unit time (V

) was noted. The permeate flux was calculated by measuring the permeate flow rate per unit membrane area. Details of the process parameters are presented in Table 1. 

Table 1. Microfiltration process parameters 

The inverse relationship of flux and TMP is commonly observed in a microporous TFF operation. Higher TMP causes greater compaction of the cake layer deposition against the membrane, resulting in a higher resistance to filtrate flow rate and a lower flux. Therefore, the flux and TMP control offered by TFF system in this investigation helps to optimize the flux while also preventing cell damage because of  low shear, and thereby, potentially maximizing the passage of soluble FHA and PT components through the membrane. 

Flux and TMP were constant throughout the MF operation, with a high yield  (Table 2)  of cell mass and FHA, PT antigens in the permeate (results were mean value of five studies). The initial high flux was because of ramping up of the pump to appropriate feed flow rate, which was stabilized after 20 min. The flux remained steady and the average flux obtained in the experiments was 37.75 L/m

/h at an average TMP of 0.135 bar. The relationship of Flux and TMP is shown in Figure 2. 

Figure 2.  Flux and TMP curve during the unit operation 

The optimal maintenance of cross flow (retentate flow rate) and well regulation of TMP enhanced and maintained consistent recovery in all the five experiments. The next goal was the establishment of an effective cleaning regime ensuring repeated usage of the membrane to reduce the downstream cost. Twenty liters of WFI was recirculated for 5 min and then drained off. WFI recirculation was done twice. The system was sanitized with sodium hypochlorite solution (200 ppm of chlorine), recirculated for 30 min and drained off. Normalized water permeability (NWP) was calculated before and after cleaning. After each experiment, the NWP was restored to its original value, thereby establishing the cleaning effectiveness. 

 fermentation broth must be clarified quickly to increase the recovery of the acelullar pertussis vaccine antigenic components. Conventional methods like centrifugation are cumbersome, time consuming, and have limited scalability. Membrane-based technology in TFF mode can ensure easy and conventional scale-up with a high recovery percentage. An effective protocol suitable for scale-up is presented in this article. Because of the same channel geometry in different modules of Prostak, i.e., 2, 4, 10, and 20 stacks of effective filtration area 0.17, 0.33, 0.84, and 1.7 m

 respectively, it provides a suitable technology for manufacturing at each scale. After a clarification step, the Prostak modules can be regenerated and sanitized, providing reusability with validated steam sterilization capacity for 20 cycles. The higher recovery is mainly because of the low hold up volume of the system, the low protein binding nature of the membrane, and also because the operation takes palce under low shear condition. The Prostak module's design allows aseptic processing without cross contamination or aerosol generation. 

The authors are thankful to Lalit Saxena and Gajanan Joshi of Millipore India Pvt. Ltd. for technical assistance during the study. 

 is an executive director of the Serum Institute of India, Ltd., Pune, India, 

 is a technical manager of the biomanufacturing sciences network at Millipore Singapore Pte. Ltd., Singapore, +65.6403.5308, 

 is a manager of process applications and 

 is a vice president of the bioprocess division at Millipore India Pvt. Ltd., Bangalore, India. 

 1. Cherry JD, Heininger U. Pertussis and other Bordetella infections, In: Feigin RD, Cherry JD, eds. Textbook of Pediatric Infectious Diseases, 4th edn. WB Saunders; Philadelphia, PA. 1998:1423–40. 

 2. Conly JM, Johnston BL. The role of the acellular pertussis vaccine and the demise of the 'Pertussis Pete'. Can J Infect Dis. 2001:98:15–17. 

 3. Pertussis Vaccination : Use of Acellular Pertussis vaccines among infants and young children, Recommendations of the advisory committee on immunization practices (ACIP); Morbidity and mortality weekly report, US Department of Health and Human Services. 1997;22: Mar 28. 

 4. Corbel MJ, Corbel MJ, Toxicity and potency evaluation of pertussis vaccine. Vaccines. 2004:89–100. 

 5. Sundaran B, Palianiappan C, Rao YUB, Boopathy R, Bhau RLN. Tangential flow filtration technology applicable to large scale recovery of diptheria toxin. J Biosci Bioeng. 2002;94:93–98. 

Membrane-based TFF technology can ease scale-up and provide a higher recovery percentage compared to conventional purification methods.

Tangential Flow Filtration for the Recovery of Acellular Pertussis Vaccine Components

The development of new vaccines has progressed from an empirical approach to more sophisticated vaccine design. This has been enabled by advances in our understanding of the immune system and by improvements in immunological tools, particularly the use of new adjuvant combinations. It is now possible to combine an antigen with a specific adjuvant system to design effective vaccines that provide an immune response tailored for each vaccine. The clinical application of the antigen–adjuvant system concept has shown benefits for diseases like pandemic influenza and human papillomavirus (HPV) cervical cancer. Observed benefits include higher and sustained immune response (humoral and cell mediated) and immune memory, cross-type immunity, and antigen-sparing, along with a favorable safety profile. The advantages of using an adjuvant system in the preventive field also have generated interest in the context of therapeutic vaccines, particularly in cancer immunotherapy. Strong signals of clinical activity of MAGE-A3 antigen-specific cancer immunotherapeutics (ASCI), demonstrated in proof-of-concept studies in melanoma and non-small cell lung cancer (NSCLC), have prompted the start of two large pivotal Phase 3 trials. The tailored antigen–adjuvant system combination approach is thus opening new possibilities for designing effective vaccines against unmet medical needs.

The future of vaccine development is based on the ability to address remaining unmet medical needs linked to challenging pathogens or challenging populations. There are no vaccines available yet to fight leading infectious killers including challenging pathogens such as parasites requiring complex, multistage immune responses (malaria), highly variable viruses that evade or subvert the immune response (hepatitis C, AIDS), or mycobacteria (tuberculosis). In addition, novel vaccine strategies are needed to target new pathogens with pandemic potential such as new influenza strains, because during a pandemic, large populations will have to be immunized in short time. Other challenges include the improvement of currently established vaccine strategies to address the aging of the immune system (immunosenescence) in the elderly population or a better protection of individuals with impaired immune response caused by chronic conditions or immunodeficiency. Finally, new approaches are needed when using antigens that have weak immunogenicity (highly purified proteins and peptides, polysaccharides etc.) or are prone to genetic drifting (seasonal influenza). 

A unique feature of the human immune system is its ability to recall an encounter with a disease-causing pathogen for decades, even over the course of a lifetime. This fundamental property of the immune system is the basis for vaccination. The goal of any successful vaccine is to induce a strong priming of the immune system. This should translate to a high and sustained immune response, as well as strong stimulation of the immune memory that should provide long-term protection against a specific disease. Vaccine design begins with the identification of the immune pathway responsible for protecting against a particular disease. The antigen, i.e., the targeting component of the vaccine, is then selected for its ability to elicit a desired immune response. The second step is the selection of adjuvant(s), because vaccines with inactivated pathogens or purified subunit antigens are usually less immunogenic compared to vaccines with live-attenuated pathogens. Therefore, the addition of compound(s) able to enhance the immune response towards the administered antigen is required. The added value of adjuvants in vaccine development was discovered more than 80 years ago when aluminium salts were first used in vaccine formulation.

The better understanding of the immune system, especially the interaction between the innate and adaptive immune response, and the use of adjuvants, have enabled the formulation of vaccines better tailored to the needed immune response.

Adjuvant systems are designed to enhance protection in cases where the classical single adjuvant approach has proven to be insufficient or incapable of providing optimal protection, i.e., for specific populations or challenging diseases. The aim in designing a vaccine adjuvanted with an adjuvant system is to optimize the vaccine's interaction with the immune system's response to the vaccine through synergy between the antigen and the selected adjuvant system.  

The target population, the antigen, the route of administration, the type of desired immune priming, and the required duration of immunity all influence the choice of the most appropriate adjuvant system for a given vaccine. 

Adjuvant Systems: Maximizing Vaccine Potential

Adjuvant systems are designed to enhance vaccine-induced protection by providing a strong and sustained immune response. They can facilitate the development of novel vaccines for challenging diseases which formerly were out of reach. Moreover, they can offer individuals broader protection against multitype pathogens. In some instances, adjuvant systems may even make it possible to reduce the amount of antigen needed and hence to increase vaccine manufacturing capacity, which may be necessary in a public health emergency such as an influenza pandemic. Several clinical studies have confirmed that the use of adjuvant system technology offers the capacity to develop potent vaccines that can address currently unmet medical needs.

It is now possible to combine antigens with specific adjuvant systems to create more effective vaccines.

BioPharm International-10-02-2009

Virus Clearance Strategy Using a Three-Tier Orthogonal Technology Platform

Pandemic Preparedness: What More Can We Do?

Advancing Vaccine Technology to Combat Global Pandemic Threats

Tangential Flow Filtration for the Recovery of Acellular Pertussis Vaccine Components

Addressing Unmet Medical Needs Through Tailored Vaccine Design: The Importance of Adjuvant Systems

Innovations in Intranasal Vaccine Delivery Technology