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.

  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.

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.  

 assays currently applied to vaccine production processes, some take weeks to run, although the accuracy of others, such as the single radial immunodiffusion (SRID)

    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.

  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),

  potentially replacing more tedious methods such as bioassays and 

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.

  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.

  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.

The productivity of large-scale cell culture can be increased either by scaling up to larger volumes with cell densities of 2–3 x 10

 /mL, or by intensifying the process in smaller volumes but with higher cell densities (up to 2 x 10

  cells/mL). When intensifying cell densities, more frequent media changes are needed and perfusion is eventually applied.

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.

  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.

  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.

  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.

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,

  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.

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.

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.

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.

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.

  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.

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.

The cost of delay in R&D can be more than one hundred thousand dollars per hour.

  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.

  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%.

According to the 3rd Annual Report and Survey of Biopharmaceutical Manufacturing, Capacity and Production,

  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. 

Articles

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

Optimized Vaccine Development and Manufacturing: A Technology Overview

A new recombinant protein vaccine against leishmaniasis has been produced by fusing portions of three 

-derived proteins. To simplify manufacturing, process improvements included removal of a 6-histidine sequence near the amino terminus, and the mutation of a proteolytic hot spot. The improved manufacturing method consists of fermenting an engineered version of the HMS-174 strain of 

 following current good manufacturing practice regulations at the 30-L scale. A purification scheme yields purified protein product at greater than 100 mg/L. The purified protein then yields a stable cake that can safely be sent to clinical trials. The product was tested with the adjuvant monophosphoryl lipid A in stable emulsion (MPL-SE) in a murine potency assay. The Leish-110f recombinant protein product reported here is an improved version of the original leishmaniasis vaccine. 

Leishmaniasis is a devastating disease that causes untold human suffering and numerous deaths worldwide.

  Leishmaniasis is a spectrum of diseases ranging from a fatal visceral form to a localized self-healing cutaneous lesion. The global prevalence of leishmaniasis, caused by the intracellular protozoan 

 species, is estimated to be approximately 12,000,000 cases total, with 500,000 new reports of visceral leishmaniasis and 1,500,000 million new reports of cutaneous leishmaniasis each year.

  Although much progress has been made in developing drug therapies for leishmaniasis, a number of clinical problems, such as length of treatment and prohibitive expense, are associated with these therapies. Therefore, new vaccines and improvements to existing clinical products are valuable additions to the fight against this terrible disease.  

The first-line clinical therapy, antimonial drug, was developed almost a century ago. This clinical regimen requires multiple injections (four weeks of daily injections for the visceral form of leishmaniasis), is costly, often associated with unpleasant side effects, and is becoming ineffective in many endemic locales. With resistance to therapeutic antimonials increasing to 60% in certain parts of India, the mortality rate from visceral leishmaniasis has been very high.

  Recent progress has been made by an oral anti-leishmaniasis drug, miltefosine, with the current clinical recommendation to use this drug as frontline treatment for some forms of leishmaniasis, including cutaneous leishmaniasis. Miltefosine, however, is teratogenic and requires an extended therapeutic regimen. Another therapeutic, liposomal amphotericin B, also has considerable efficacy against clinical leishmaniasis, but the expense of the drug renders it impractical in all but severe cases of the disease. Both miltefosine and amphotericin B function independently of the immune system, therefore, when the drug is withdrawn before completion of the therapeutic regimen, parasite loads re-emerge.

  Control of the phlebotomine sand fly vectors is also not feasible given the prohibitive expense associated with insecticide use.  

The development of a safe, effective, and economical vaccine product is possible, given that controlled vaccination with live parasites has been practiced for centuries in the Middle East, and individuals who recover from clinical leishmaniasis develop immunity against re-infection. Additionally, recent trials with killed parasites demonstrate good efficacy of such a vaccine in post-infection settings of cutaneous leishmaniasis,

 and numerous studies have demonstrated the effectiveness of such vaccines in experimental models of leishmaniasis.

The Infectious Disease Research Institute (IDRI) has developed a prophylactic vaccine for cutaneous leishmaniasis by constructing a recombinant fusion protein consisting of portions of three 

 proteins: thiol-specific antioxidant (TSA),

  The schematic of this construct is shown in Figure 1. The recombinant approach was preferable to live vaccine or a killed parasite vaccine. Live vaccination is being discontinued because of undesirable side effects,

  and the manufacturing process of a killed parasite vaccine presents many difficulties relating to reproducibility, the need for animal-derived products in the culture medium, and variability in potency. The recombinant approach lends itself to the production of an inexpensive and effective vaccine at large scale, and if efficacious, would drastically reduce the morbidity and mortality caused by 

The original form of this vaccine was manufactured under current good manufacturing practice (cGMP) conditions, is currently undergoing human clinical testing, and will ideally provide a lasting protective immunological response. To simplify manufacturing, several subtle process improvements included removal of a 6-histidine sequence near the amino terminus, and the mutation of a proteolytic hot spot. The improved manufacturing method consists of fermenting an engineered version of the HMS-174 strain of 

) following cGMP regulations at the 30-L scale. A purification scheme combining anion exchange chromatography with hydroxyapatite chromatography was developed that yielded purified protein product at greater than 100 mg/L. The purified protein was formulated and lyophilized to yield a stable cake that can safely be sent to clinical trials worldwide. The cGMP-manufactured product was tested with the adjuvant monophosphoryl lipid A in stable emulsion (MPL-SE) in a murine potency assay. The Leish-110f recombinant protein product reported here is an improved version of the original leishmaniasis vaccine.  

Master and working cell banks were manufactured under cGMP conditions at Charles River Laboratories (Malvern, PA). The cell banks were tested for purity, viability, and identity and are stored as frozen stocks in glycerol at –80° C. The flow chart of fermentation and purification is shown in Figure 2. 

Both the inoculums and the fermentation used 2XYS as the growth media, with dextrorotatory glucose (D-glucose) as the primary carbon source, to grow the recombinant 

in a fed-batch fermentation. The fermentor was programmed to control the pH at 7.0 and dissolved oxygen at 30%. When the optical density at 600 nanometers had increased to 8 + 1, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the fermentor to a final concentration of 1 mM to induce expression of the recombinant gene encoding the Leish-110f protein. At three hours post-induction, the culture was harvested by centrifugation. 

Following fermentation harvest, the wet cell paste was resuspended in lysis buffer (150 mM NaCl, 20 mM Tris pH 8.0, 5 mM EDTA) at a defined volume-to-mass ratio of 5 mL of lysis buffer to 1 g of cell paste. The resuspended 

were disrupted with an M-110S microfluidizer (Microflluidics, Newton, MA). The resulting lysate was centrifuged at 18,000 grams for 30 min at 4° C, which pelleted the insoluble inclusion bodies (IBs). The IBs were washed with a 2% CHAPS (3[(3 cholamidopropyl) di-methylammonio]-propanesulfonic acid) wash, followed by an isopropanol wash. The washed IB pellets were stored at –80° C until needed.  

The inclusion body pellets were solubilized with 8 M urea, 50 mM Tris pH8, and 25 mM dithiothreitol at 150 mL/g of inclusion body pellet. The solubilized inclusion bodies were clarified using centrifugation. The first column, Amersham Biosciences' Q Sepharose Fast Flow resin (QFF), was equilibrated using buffer A (8 M urea, 50 mM Tris pH8). The clarified inclusion body preparation was loaded onto the anion exchange column at a linear velocity of approximately 120 cm/h. The full-length Leish-110f protein was eluted in a single peak from the resin, using buffer B (8 M urea, 50 mM Tris pH8, 150 mM NaCl). The second column was a BioRad Macro-Prep ceramic hydroxyapatite (CHT), type I, 40 mM. The CHT column was equilibrated using buffer B at a linear velocity of 120 cm/h. The reduced QFF elution peak was loaded onto the CHT column and washed with buffer B for 10 column volumes. The Leish-110f protein was eluted using buffer C (8 M urea, 100 mM potassium phosphate, pH 7.5, 150 mM NaCl).  

Diafiltration was performed on the CHT elution peak to remove the urea and replace the buffer with 20 mM Tris pH 8.0. The diafiltration was performed using a tangential flow filtration device with a 30 kDalton molecular weight cut off (MWCO) polyethersulfone ultra filtration membrane, and with a final protein concentration of 0.5 mg/mL as determined by A280. The purified Leish-110f bulk protein was then filter-sterilized using a 0.2 µm filter. The final purified bulk protein was stored at –80° C until formulation.  

The final configuration was a lyophilized cake, containing sucrose mannitol and polysorbate 80. The formulated product was then loaded into a Virtis lyophilizer and lyophilized.  

Testing for Process-Related Contaminants 

General safety and residual amounts of process-related contaminants, such as kanamycin, urea, and endotoxins, were measured using standard procedures. In the case of general safety and residual kanamycin, United States Pharmacopeia (USP) methods were applied. For urea, a modification of the method of Knorst, Neubert, and Wohlrab was used, adapting the test to a 96-well format.

  For endotoxins, the limulus amebocyte assay was used.  

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) was performed according to the Laemmli method.

  Samples were diluted 1:1 in reducing 2x Laemmli sample buffer (Sigma) and boiled for 5 min. Proteins were then loaded on 4–20% acrylamide gradient gels (Bio-Rad Laboratories, Hercules, CA) and were run at 30 milliamperes (mA) constant for 60 min. The gels were stained using Coommassie blue, with standard techniques.  

Female BALB/c (Bagg albino color locus genotype c/c) mice were purchased from Charles River Laboratories (Wilmington, MA) and were maintained in specific-pathogen-free conditions. Eight- to 12-week-old mice at the beginning of experiments were used. Three mice per group were immunized with 10 µg of Leish-110f or Leish-111f formulated with 20 µg of an adjuvant MPL-SE (GlaxoSmithKline Biologicals, Rixensant, Belgium) in a volume of 0.1 mL. The mice were immunized subcutaneously once at the base of the tail. Blood was taken from the mice one week after the immunization to determine immunoglobulin G1 (IgG1) and immunoglobulin G2a (IgG2a) antibody titers. Plates were coated with 200 ng per well of Leish-110f or Leish-111f diluted in coating buffer, followed by blocking with phosphate-buffered saline containing 0.05% Tween 20 and 1% bovine serum albumin. Mouse serum samples were diluted to 1:100 and applied to the plates in two-fold serial dilutions. The plates were incubated with horseradish peroxidase (HRP) conjugated goat anti-mouse IgG1 or IgG2a (SouthernBiotech, Birmingham, AL), were developed with 3,3',5,5'-Tetramethylbenzidine (TMB) substrate, and were read by a microplate reader at a 450 nanometer wavelength. Endpoint titers were calculated with GraphPad Prism software using an optical density (OD) value of 0.1 as a cutoff. 

The fed-batch fermentation of the HMS-174/L110f recombinant system was consistent in its growth profile and production of the vaccine product Leish-110f. The Leish-110f protein was produced in the inclusion bodies. A two-step orthogonal purification procedure post-inclusion body washing achieved significant purification of the vaccine candidate. A chromatogram of the QFF anion exchange column separation (Figure 3) shows a single major peak that contained the Leish-110f polyprotein. The second step in the purification used ceramic hydroxyapatite, with the Leish-110f protein eluted as a single major peak (Figure 4) at low concentrations of phosphate. The majority of the contaminants remained bound to the resins used. The in-process SDS-PAGE gel shown in Figure 5 illustrates purification as additional purification steps were added. The utility of coupling these methods is that the eluted protein from the first step need not be conditioned between chromatographies, as the protein binds to the hydroxyapatite even in the presence of NaCl. SDS-PAGE of the eluted material illustrates the purity of the final Leish-110f protein product (Figure 6). Yield of purified protein is more than 150 mg/L and is a tremendous improvement over the original L-111f protein yield of 30 mg/L. 

Following purification, urea was removed by diafiltration, and the protein was filled and lyophilized as described in the materials and methods section, above. The protein formed a stable white cake in the vials after lyophilization, and that quickly redissolved upon addition of water. Real-time stability studies at several temperatures are ongoing, but preliminary data suggests that the purified bulk protein and the lyophilized cake are stable at all temperatures tested (data not shown). 

The final product was tested for endotoxins, residual moisture, pH, concentration, physical appearance, identity, and quality as measured by SDS-PAGE. These data are shown in Table 1. 

Immunogenicity of the cGMP Material in a Murine Model

We compared the Leish-111f protein with the improved version Leish-110f in a murine potency assay. Mice were immunized with a single dose of 10 µg of Leish-111f or Leish-110f adjuvanted with MPL-SE. Seven days post-injection, mice were bled and antigen-specific IgG1 and IgG2a antibody titers were measured by enzyme-linked immunosorbent assay (ELISA) (Figure 7). The two forms of the protein induced comparable titers of both IgG1 and IgG2a antigen cross-reactive antibody. These results suggest that Leish-110f made using our new process and construct induced antibody responses comparable to that obtained with Leish-111f in mice. 

Table 1.  Summary of in-process contaminant clearance using CEX for capture and HCI for polish 

Researchers at IDRI have chosen to develop a 

 vaccine that is a fusion of three distinct, conserved 

 antigens. These three antigens—TSA, LmSTI1, and LeIF—were selected for the development of a subunit vaccine based on their demonstrated abilities to induce at least partial protection in the BALB/c mouse model of L. major in either prophylactic (TSA and LmSTI1) or therapeutic (LeIF) applications.

  All three antigens are present in the amastigote and the promastigote forms of the parasite and are highly conserved among 

 species, a requisite for ensuring cross-species protection. Immunization with a single recombinant antigen, in addition to having a simplified manufacturing process, may insure equivalent uptake of the components by individual antigen-presenting cells; in turn, it may generate an immune response that is broadly specific for all the immunogenic epitopes spanning the polyprotein. 

Creating a vaccine composed of three or more independently produced recombinant proteins would be prohibitively expensive to manufacture and formulate, compared to the single polypeptide vaccine described here. The manufacturing cost of a vaccine is particularly important in developing countries, and it was one of the considerations in producing Leish-110f. The original Leish-111f fermentation was different from that used in the new process. Conditions were developed for Leish-110f to attain a higher cell density before induction and thus, a higher product yield. This outcome was accomplished by using a richer, semi-defined media, and a controlled feeding strategy to manage the growth of the culture. These two changes allowed for a substantial increase in recombinant protein production per liter of culture.  

The purification procedures for the two recombinant products were similar, in that both processes used an anion exchange resin followed by the hydroxyapatite column purification steps. However, the previous process used Amersham Pharmacia Biotech's Source 30Q resin, and linear gradients were used to elute the product. In the new process the Q-Sepharose Fast Flow resin was used, and step-wise elutions were used for ease in scale-up and manufacturing. The process described above makes a consistent product at acceptable yields for the current stage of development. IDRI is producing nearly 200,000 doses per lot of vaccine based on the highest dose level currently envisioned. This level is satisfactory for the amounts needed to conduct current clinical trials, but scale-up of the process to the 300-liter scale is already ongoing. Assuming no loss in unit production, this scale would deliver 2,000,000 doses per lot and could enable large-scale clinical trials of the vaccine even without further process improvements.  

An additional encouraging fact is the stability of the lyophilized protein. When performing trials or vaccinations in field settings where a cold chain cannot be guaranteed, stability of the product is crucial to successful deployment of the trial and the final product. This lyophilized vaccine product has been shown to be stable for more than three years. 

IDRI has developed a process to manufacture a new recombinant protein product for the treatment and prophylaxis of leishmaniasis. This candidate vaccine consisting of Leish-110f formulated in MPL-SE will be tested in a spectrum of diseases caused by the various 

 species. A robust, scalable procedure will allow rapid progress in bringing this vaccine to patients and will be a step in the fight against leishmaniasis.  

 is director of process sciences, 206.330.2530, 

 is founder and head of research and development, and 

 is director of formulations, all at at the Infectious Disease Research Institute, Seattle, WA. Dr. Carter is also the vice president of research and development at Dharma Therapeutics, Seattle, WA. 

The recombinant approach lends itself to the production of an inexpensive and effective vaccine at large scale.

An Improved Manufacturing Process for a Recombinant Polyprotein Vaccine

Aside from clean water, vaccination has proven to be the most effective means of controlling infectious diseases. Current vaccines technologies include live attenuated vaccines, inactivated vaccines, vectored vaccines, and DNA vaccines. Many challenges still remain including immunization of the neonate, delivery of the vaccine via the mucosal surfaces, and vaccination against chronic infections. Some of the novel concepts to overcome these challenges are based on the use of adjuvants that can induce both innate and acquired immunity or that can target the vaccine to specific immune cells, such as dendritic cells. This article highlights some of these strategies to develop novel vaccine formulations for the neonate. 

Background image: Vaccine & Infectious Disease Organization  

 The use of vaccines dates back to Jenner and Pasteur, who first described the concept of vaccination more than 200 years ago. Since then, vaccines have been used successfully in humans and animals, and aside from clean water represent the most effective means of controlling systemic, respiratory, and gastrointestinal infections in both humans and animals. 

 Adjuvants: Important Components of Vaccines

Adjuvants are important components of vaccines. They provide an additional stimulus for the immune system, which typically results in stronger immune responses, and can assist in delivering or even targeting of the vaccine. Adjuvants represent a wide variety of molecules and formulations including cell-wall components, alum, QuilA, immunostimulatory complexes (ISCOMs), carbomers, liposomes, and oil-in water emulsions, to name a few. Many of these adjuvants have been developed empirically without an exact understanding of their mechanisms of action. Recent research, however, has provided insight into the activation of the immune system by pathogen recognition receptors, including toll like receptors, C-type lectins, mannose-receptors, and nucleotide-binding oligomerization domain (NOD)-like receptors. These receptors typically recognize highly conserved molecular pattern in pathogens such as bacterial DNA, lipopolysaccharide, or flagellin

  and activation often leads to increased NF-κB and/or type I interferon (IFN) production, which can result in up-regulation of chemokines and cytokines needed for a variety of immune functions including recruitment of immune cells to the site of immunization, activation and maturation of dendritic cells, and improved presentation of the antigen.

  These events constitute the innate immune response phase, which serves to 1.) contain and eradicate pathogens, and 2.) induce an appropriate adaptive immune response to eliminate infection and prevent reccurrence. Thus, by signaling through pathogen recognition receptors, adjuvants can stimulate both innate and acquired immunity resulting in stronger and longer lasting immune responses to vaccination. 

Immune responses elicited by vaccines can be divided into innate and acquired immune responses. Innate immune responses occur within hours of infection or vaccination and are characterized by upregulation of chemokines and cytokines, typically providing a pro-inflammatory environment at the site of immunization. Other innate immune molecules include host defense peptides, also called cationic antimicrobial peptides, complement, and acute phase proteins, which together can recruit and activate dendritic cells to provide a link with the acquired immune response.

  The latter is characterized by the presence of antibodies and cytotoxic T cells, both of which are highly antigen-specific and which recognize very specific structures of the pathogen.  

While vaccination in the past traditionally has  focused on the induction of the acquired immune response, more recent approaches are focused on stimulating both innate and acquired immunity. In fact, it has become clear now that the innate immune response sets the stage for the acquired immune response, and thus, novel immunomodulators are being used as vaccine adjuvants that can link both innate and specific immunity to enhance the immune responses following vaccination.  

Enhancing and modulating immune responses is particularly important in neonates, as infants and young children are highly susceptible to many diseases. When compared to the adult immune system, the neonatal immune system displays a number of functional differences, including its bias towards a Th2-type immune response, delayed germinal center development, shorter lifespan of plasma cells, fewer T-helper cells, and functionally impaired antigen-presenting cells. Together, these functional differences represent a great challenge for the development of effective vaccines for the newborn. Furthermore, potential interference with maternally-derived antibodies makes most vaccines less effective in the neonate.  

We recently developed a novel disease model for pertussis in neonatal pigs.

  Using this model we are currently developing novel vaccine formulations against pertussis, also called whooping cough. Pertussis is an important disease of infants and young children, causing more than 50 million cases and about 300,000 deaths every year.

  Current vaccines are based on recombinant proteins (acellular vaccine) or inactivated bacteria (whole cell vaccines), and require multiple immunizations between 2–60 months of age. While this is possible in developed countries, access to multiple immunizations is limited in developing countries resulting in lower vaccine coverage and less effective protection in many regions of the world. Funded by the Bill & Melinda Gates Foundation, the Krembil Foundation, and the Canadian Institutes of Health Research, our project aims at developing a variety of immunomodulators that link innate and acquired immunity and therefore can provide more effective immune protection in the neonate after fewer immunizations. 

Innate immune stimulators function as adjuvants that can enhance both innate and acquired immunity. The most often used stimulator of the innate immune system is bacterial DNA or synthetic DNA containing CpG motifs (CpG DNA). CpG DNA activates the immune system by signaling via toll like receptor 9, which typically results in activation of both innate and acquired immunity. Treatment with CpG oligonucleotides (ODNs) has been successfully used in a variety of experimental infectious and non-infectious diseases and clinical studies are in various stages to evaluate CpG ODN therapy against infectious disease, cancer, asthma, and allergy.

  As an adjuvant, CpG ODNs promote predominantly Th1-type immune responses, a quality needed for optimal protection against intracellular pathogens. Because of the Th2 bias in neonates, however, CpG ODN also represent potential adjuvants for balancing the immune response in the neonate or skewing it toward a Th1 response, which is needed for a number of diseases including pertussis.

  In this regard, neonatal mice immunized with HBsAg in the presence of CpG developed a predominantly Th1-type immune response, while immunization with only HBsAg alone induced a Th2-type response.

 Contradicting results have been obtained from 

 stimulation studies with CpG using neonatal peripheral blood mononuclear cells or dendritic cells. Some studies demonstrated that stimulation with CpG DNA could elicit Th1 responses in the neonate, high levels of IgM 16 upregulation of co-stimulatory markers on dendritic cells, and proliferation of cord blood B cells. Other studies, however, showed that innate responses in the neonate including the production of IFN-α by plasmacytoid dendritic cells were impaired and Th-2 responses to allergens were increased following addition of CpG DNA to house dust mite allergens.

   Despite the controversy, encouraging results have been obtained in our own laboratory, which have confirmed that CpG DNA is highly active in cord blood cells and can induce similar levels of interferons when compared to adults (Gong, et al.,unpublished results). However, more research is necessary to fully evaluate the potential of CpG DNA for the use in neonates.  

Host defense peptides (HDPs), also called cationic antimicrobial peptides, are another example of innate immune stimulators. These molecules are found in virtually every life form and can be grouped as defensins and cathelicidins. HDPs are fundamental components of the innate immune response.

  Their wide spectrum of functions includes direct antimicrobial activities, immunostimulatory functions of both innate and acquired immunity, and involvement in wound healing, cell trafficking, and vascular growth.

  The antimicrobial activities of HDPs have been known for a long time, but recent evidence suggests that under physiological concentrations the immunomodulatory functions of mammalian HDPs outbalance the direct antimicrobial activities. These functions can include the recruitment of immature dendritic cells and T-cells, glucocorticoid production, macrophage phagocytosis, mast cell degranulation, complement activation, and interleukin-8 production by epithelial cells.

 HDPs also have been shown to up-regulate gene expression in epithelial cells and monocytes, and neutralization of pro-inflammatory cytokine induction and lethality in response to lipopolysaccharide/endotoxin.

  Moreover, recent studies have shown that host defense peptides can also adjuvant  vaccines.

  For example, ovalbumin-specific immune responses were enhanced after intranasal co-administration of ovalbumin and the human neutrophil peptides 1–3. The  fusion of the gene encoding the murine beta-defensin 2 to the gene encoding the human immunodeficiency virus glycoprotein 120 resulted in not only stronger but also mucosal immune responses in immunized mice.

  Other examples include B-cell lymphoma vaccines, which were enhanced by fusion of the tumor epitope with genes encoding murine beta defensins.

  These examples illustrate that HDPs have been successfully used as adjuvants to enhance vaccine-specific immunity.  

Polyphosphazenes are synthetic polymers, which are water soluble and biodegradable. They can function both as innate immune stimulators as well as delivery vehicles for vaccines. Polyphosphazenes are made from polymers with alternating nitrogen and phosphorus atoms, with side groups attached to each phosphorus.

  Polyphosphazenes are inexpensive to produce, can be lyophilized and stored for a long time at room temperature, and are safe to use. Polyphosphazenes have been used to enhance the magnitude of the immune response against viral and bacterial antigens in mice and to modulate the quality of immune responses, resulting in a more balanced or even Th1-type of immunity.

  For example, poly[di(sodium carboxylato-ethylphenoxy)phosphazene] (PCEP) altered the magnitude and quality of influenza antigen X:31-specific immune responses in mice resulting overall in higher and more balanced immune responses.

  Similarly, PCEP induced a balanced Th1/Th2-type immune response with hepatitis B surface antigen (HBsAg), and compared to the conventional adjuvant alum induced much higher immune responses (Mutwiri, et al., unpublished results). Moreover, polyphosphazenes can be formulated in microparticles, which facilitate immunization by the mucosal routes, an important feature of modern vaccines. 

Particle-based delivery of vaccine antigens has proven to be a very effective way of delivery, especially when compared to the delivery of soluble protein antigens. Microparticles can protect the vaccine antigen from degradation while increasing uptake by specialized immune cells such as dendritic cells.

  They therefore represent effective ways of delivering vaccines, and large efforts are currently underway to develop effective particulate delivery systems. Microparticles including polymers such as PLGA particles, polyphosphazenes, liposomes, and ISCOMs facilitate phagocytosis by antigen-presenting cells.

  Once inside the cell, they release the antigen into the phago-endosome, where it is processed and subsequently loaded onto MHC molecules for antigen-presentation. Pathogen recognition receptors such as TLR9 are present in the same compartment, suggesting the incorporation of CpG DNA as an effective method of stimulating antigen presenting cells. Furthermore, microparticles also facilitate uptake across the mucosal surfaces, which predominantly occurs by M cells which are overlying the Peyer's patches.

 Dendritic cells have been shown to extend their dendrites between epithelial cells to sample antigen in the lumen, but the significance of this pathway for uptake of particles is unclear.

  The use of microparticles in the delivery of mucosal vaccines has been modestly successful. However, novel approaches such as targeting to specific cells as well as addition of adjuvants such as CpG ODN or HDPS have the potential to improve the potency of microparticles as delivery vehicles for the induction of mucosal immune responses.

Because most invading pathogens enter the host via the mucosal surfaces, induction of strong mucosal immunity is often crucial for disease protection.

  Prime examples of such infections include gastrointestinal infections with enterotoxigenic 

(ETEC), rotavirus, or calicivirus, and respiratory infections with Mycoplasma, influenza virus, or respiratory syncytial virus. Effective vaccines therefore need to target the mucosa-associated lymphoid tissues (MALT), ideally at the potential site of infection. The MALT represent the largest immune compartment in the body and is required to (1) protect the mucosal membranes against infection; (2) to tolerate antigens derived from ingested food, airborne matter, and commensal microorganisms; and (3) to prevent the development of any potentially harmful immune responses against these antigens in case they breach the mucosal lining.

 To induce strong mucosal immune responses vaccines must be delivered to the mucosal site, where the vaccine antigen is taken up by either M cells, dendritic cells, or epithelial cells and subsequently passed on to antigen presenting cells. Subsequently, these cells migrate into specialized tissues such as Peyer's patches, lymph nodes, and tonsils, in which the antigen is presented to effector cells of the immune system. During this process the effector cells are imprinted to home back to the mucosal surfaces, a process mediated by interaction between locally expressed chemokines and integrins and chemokine receptors found on the surface of the effector cells. It is this interaction that allows effector cells to specifically home to the site of induction. For example, expression of the chemokine CCL25 and recognition by the chemokine receptor CCR9 allow specific homing to the small intestinal mucosa. This demonstrates the importance of delivering future vaccines by the mucosal surfaces. Delivery systems and effective mucosal adjuvants are needed that not only facilitate delivery of the vaccine to mucosal sites, but also enhance antigen uptake and provide a pro-inflammatory environment resulting in recruitment and activation of immune cells.  

Vaccines are the most effective means of controlling infectious diseases. Novel approaches have been developed to improve existing vaccines and to develop novel vaccines. In this regard, adding of potent adjuvants that link innate and acquired immunity as well as delivery systems for mucosal delivery that eliminate the risk of needles, are promising approaches to make vaccines more effective in humans and animals. The development of vaccines for the newborn will greatly benefit from these novel technologies, as immunization of the newborn has proven to be a major challenge for today's vaccines.   

Funding in the investigators' laboratories was provided by a grant through the Grand Challenges in Global Health Initiative by the Bill and Melinda Gates Foundation, the Krembil Foundation, the Canadian Institutes for Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Saskatchewan Health Research Foundation (SHRF), and the Agriculture Development Fund Saskatchewan (ADF Saskatchewan). Published with permission of the Director of the Vaccine & Infectious Disease Organization as article number 480.    

 is a program manager at Vaccine & Infectious Disease Organization Saskatoon, Canada, +1.306.966.1513, 

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Potential interference with maternally-derived antibodies makes most vaccines less effective in the neonate.

Strategies to Improve Vaccines for the Neonate

Yeasts are distinguished by a growing track record as expression platforms for the production of pharmaceuticals. Commercially available, yeast-derived, recombinant pharmaceuticals include, among others, insulin, the anti-coagulant hirudin, interferon-alpha-2a, and various vaccines against the hepatitis B virus and papillomavirus infections. The vaccines are produced in either baker's yeast (

. In this article, we focus on a production process for hepatitis B vaccines in methylotrophs. Methylotrophs provide  highly balanced production of both the membrane and the protein component of a recombinant viral particle. A brief outlook is given for the development of yeast strains designed for the production of other vaccine candidates. 

 The advent of gene technology has provided new and powerful methods for the safe, efficient production of pharmaceuticals, with the bacterium 

, mammalian cells, and various yeasts as preferred platforms for the production of such recombinant compounds.

  Early examples include human growth hormone

. Among the most important available recombinant pharmaceuticals are yeast-derived vaccines against papillomavirus

  and hepatitis B infections. Hepatitis B vaccines are based on particles containing the hepatitis B surface antigen (HBsAg) inserted into host-derived membranes.

  The success of current vaccination programs against hepatitis B is a result of the development of effective, yeast-derived recombinant hepatitis B surface proteins. Initially, the production of such vaccines was restricted to baker's yeast, 

, but with improvements in biotechnological methods, alternative yeast expression systems have been identified and developed. In particular, the methylotrophic yeast 

 has been found to exhibit many superior expression characteristics, and is currently being used in the production of several vaccines against different subtypes of the hepatitis B virus.

In this article, we briefly describe the hepatitis B virus, its subtypes, and the disease it causes. Subsequently, recombinant protein production is discussed, focusing in particular on the application of the 

 expression system. We describe how a heterologous 

 strain expressing HBsAg is constructed and how efficient vaccine production is developed based on such recombinant strains. 

Finally, we will provide an outlook for alternative vaccine strategies and  the development of alternative vaccine candidates. 

Hepatitis B virus (HBV) was identified as the causative agent of serum hepatitis in the 1970s

  after B. Blumberg discovered the Australia antigen.

  Blumberg first recognized this antigen as a serum protein specific for aborigines in Australia. It was only later that the infectious nature of the Australia antigen was identified; it turned out to be the surface protein of HBV that is secreted into the bloodstream of infected patients in large excess over viral particles.

   HBV was found to be endemic in many parts of the world, with more than 2 billion people having had contact with the virus and more than 350 million chronic carriers of the virus.

Human HBV can be grouped into eight genotypes (A–H) which differ by at least 8%.

  Genotype F, which is found in Brazil, Colombia, and Polynesia is the most divergent genotype

  and is grouped into genotypes F1 and F2.

  The genome of hepadnaviruses codes for four groups of proteins on the minus strand, namely (1) the core protein (HBcAg) which forms the nucleocapsid, and a shorter form (HBeAg), which is secreted by the host cell; (2) the three hepatitis B surface antigen (HBsAg) proteins of different size having different initiation sites of transcription but share the sequence of the small surface protein (SHBs) of 24 kDa at their carboxy-terminus which is the most abundant representative of the HBsAg proteins; minority proteins are the middle HBs of 32 kDa and the large surface protein (LHBs) of 39 kDa (3) the DNA polymerase, which is also a reverse transcriptase with a primer function and an RNAse H domain; and (4) a protein X (HBx) with unknown function for the virus and a plethora of reported properties 

Human HBV can be grouped into eight genotypes, which differ by at least 8%.

BioPharm International-01-02-2008

Optimized Vaccine Development and Manufacturing: A Technology Overview

An Improved Manufacturing Process for a Recombinant Polyprotein Vaccine

Strategies to Improve Vaccines for the Neonate

Recombinant Vaccine Production in Yeast

Needle-Free Methods of Vaccination