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BioPharm International, October 2010 Supplement (PDF)

Virus-like particle (VLP) vaccines are currently used for the prevention of hepatitis B and human papillomavirus infections. Influenza VLPs have been produced using a baculovirus–insect cell production system and shown to be safe and immunogenic in multiple Phase 2 clinical trials. These vaccines present unique challenges in their development in terms of the design of product release assays, product characterization, and stability testing. Methods that have been used to characterize simpler biological drugs (monoclonal antibodies, protein therapeutics) to ensure their potency and consistency have been applied to release and stability testing of VLPs and will be discussed here.  

The H1N1 pandemic influenza outbreak has served as a reminder that current approaches to the development and production of vaccines are not sufficient to meet future unexpected needs. There have been three vaccine technologies used for the prevention of human viral disease. Classical live attenuated and inactivated or split vaccines have been used successfully to protect against multiple viruses including smallpox, influenza, measles, mumps, poliovirus, hepatitis A, herpes zoster, and rotavirus. These vaccines have been safe and effective but in some cases their use has been limited, particularly in developing countries, because of cost and requirements for a cold storage chain. Challenges are also seen in the industrialized world, because these types of vaccines require the growth of viral strains which in many cases is carried out in chicken eggs, and the strains do not always grow well in that platform. More recently, recombinant technology has allowed the development of new types of vaccines (subunit proteins, virus-like particles) that do not require the growth of viral agents and are non-infectious. 

Live attenuated and inactivated whole virus vaccines are the progenitors of VLPs that are in use today to prevent hepatitis B (Recombivax HB, Engenrix B) and human papillomavirus infections (Gardasil, Cervarix). VLPs contain viral proteins required to generate protective immune responses but are non-infectious and lack the potential to recombine with circulating virus because they lack viral nucleic acid. These vaccines have potential immunologic advantages over split or purified protein vaccines because: 1) VLPs contain multiple copies of antigens presented in an organized array, thus allowing activation of the innate immune system and 2) the size of VLPs allows efficient uptake and processing by dendritic cells, which are the antigen presenting cells of the immune system and stimulate lymphocytes in lymph nodes. 

Through the use of a recombinant baculovirus–insect host cell system, Novavax has developed a platform technology to produce VLP vaccines to multiple targets including influenza, SARS, and HIV. Influenza VLPs have been biochemically characterized and GMP-produced materials have been tested in five clinical trials, with a total of more than 5,000 subjects.

The establishment of a recombinant baculovirus containing the influenza genes for the hemagglutinin (HA) and neuraminidase proteins (NA) together with the gene for Matrix (M1) protein is the key step in this process.

  The HA and NA genes are obtained from viral RNA of relevant circulating seasonal or pandemic influenza strains by molecular means from isolated RNA. Alternately, they can be chemically synthesized and then, through a series of cloning steps, placed in a single plasmid (bacmid) under the control of baculovirus-specific transcription initiation and translation elements along with a constant M1 gene sequence shown to allow enhanced VLP formation. Transfection of the bacmid into 

 (Sf9) insect cells generates baculoviruses that encode and express the three influenza genes. Baculovirus stocks are further amplified on Sf9 cells to generate master and working virus stocks. Infection of Sf9 cells with the recombinant baculovirus allows expression of the HA, NA, and M1 proteins which form pleomorphic spherical membrane-containing particles with HA and NA protein spikes on their surface and an M1 core. An example of an influenza VLP is shown in Figure 1. These particles are released into the cell culture supernatant and then purified, resulting in the VLP vaccine. The membrane nature of these particles is different from currently licensed VLP vaccines and presents purification and characterization challenges. 

Figure 1. Recombinant influenza virus-like particles (VLPs) and pleomorphic spherical particles  

Production and purification of influenza VLPs has been performed for Phase 2 clinical trial materials. Sf9 insect cells from a working cell bank are amplified to seed a 100-L disposable wave bioreactor cell culture, which is then infected with a master baculovirus seed. During harvest, cells are removed by tangential flow filtration (TFF) and the filtrate is concentrated and diafiltered to remove media components and cell debris. 

VLPs are separated from baculovirus (BV) particles and contaminating RNA and DNA using a flow-through ion exchange (IEX) chromatography step in which BV, RNA, and DNA are bound to the column while VLP is allowed to flow through (Figure 2). This process step takes advantage of the charge difference between BV and VLP; BV is highly negatively charged by virtue of its DNA content, whereas VLP lacks this charge given the absence of DNA. Allowing VLP to be separated from BV without binding to the column matrix eliminates the need to modify this purification step from season to season in response to changes in the HA protein. 

Figure 2. Purification of virus-like particles (VLPs) using an ion exchange (IEX) column. Host cell contaminants-baculovirus (BV), DNA, and RNA-bind, whereas VLPs flow through. 

Residual BV is inactivated by treatment with beta-propiolactone (BPL) and residual host contaminants are removed by size exclusion chromatography (SEC), as shown in Figure 3. The VLP preparation is sterile filtered to produce the bulk vaccine. 

Figure 3. Residual host contaminants are removed by size exclusion chromatography. 

Release testing is designed to monitor the safety, identity, purity and potency of the VLP vaccine (Table 1). Many of the methods are typical of those used to release licensed split-virion and whole-virus influenza vaccines and were adapted to the VLP product. Additional methods were developed to specifically characterize the VLPs and to demonstrate removal of host and baculovirus DNA and protein as well as materials used in the manufacturing process. 

Table 1. Analytical testing of influenza VLP bulk vaccine 

Single Radial Immunodiffusion (SRID) Potency Assay

Single radial immunodiffusion (SRID) is the standard potency assay used by all manufacturers and appropriate regulatory agencies to determine the concentration of immunologically active HA in all licensed inactivated influenza vaccines.

  The assay requires two standards for each influenza vaccine strain: 1) a reference HA protein whose concentration is determined by an alternative method and 2) a polyclonal antibody source that recognizes the reference HA protein. The method is based on diffusion of the HA protein into an agarose gel containing a prequalified amount of specific antibody, raised against the HA protein, which forms precipitation rings proportional to a fixed concentration of the reference HA. The potency for a vaccine lot is computed by the parallel line bioassay method using the reference standard and test vaccine dose response curves. 

Table 2 compares the source of reagents used by regulatory agencies to release standard inactivated influenza vaccines with reagents developed by Novavax to determine the potency of our baculovirus-derived product. Whereas regulatory agencies use HA antigen and corresponding antisera derived from egg-grown influenza virus, Novavax uses HA protein derived from baculovirus and prepares a corresponding antisera to that antigen. 

Table 2. Comparison of the source of the single radial immunodiffusion (SRID) reagants used by regulatory agencies to release standard inactivated influenza vaccines and the reagants developed by Novavax to determine the potency of the baculovirus-derived VLP product.  

Multiple VLPs have been tested for potency in the SRID assay comparing the egg- and baculovirus-derived reagents, and results for H5N1 pandemic vaccine lots are presented as a representative example in Table 3. Baculovirus- and egg-derived SRID reagents give comparable values for HA concentration for multiple lots of pandemic VLP vaccines.  

Table 3.  Comparison of the HA concentration results for H5N1 VLPs from single radial immunodiffusion (SRID) potency assays using egg-derived and baculovirus-derived reagents. The two different reagents give comparable results for multiple lots of vaccines.  

The 2008–2009 inactivated influenza vaccine used a like H3N2 strain (A/Uruguay instead of the recommended A/Brisbane) because of problems with production of the recommended strain. Because VLP development does not require the growth of the actual influenza virus, the protein coding sequence for the A/Brisbane H3N2 HA was used to generate the baculovirus recombinant and subsequently the purified VLP vaccine containing the recommended HA. Potency of trivalent VLP and inactivated 2008–2009 influenza vaccines was tested with baculovirus- and egg-derived reagents in the SRID assay. Table 4 demonstrates that the homologous A/H1N1 and B/Florida components of both vaccines gave comparable potency values when tested by baculovirus- or egg-derived reagent where the results with homologous reagents (baculovirus-derived for VLP and egg-derived for inactivated egg produced vaccine) were considered the expected value. However, when the heterologous H3N2 HA potencies were determined, it was seen that accurate potency measurements required homologous reagents, i.e., H3N2 HA potency in VLP was accurately measured by baculovirus reagents but not by egg-derived reagents and the opposite was seen with egg-derived vaccine. The table also demonstrates that the source of the antisera was not an issue because baculovirus- and egg-derived antisera could be interchanged with the source of antigen and resulted in accurate HA concentration determinations. However, the source of HA reference protein was crucial; only baculovirus-derived A/Brisbane protein was accurate for measuring VLP H3N2 HA content and the egg-derived A/Uruguay HA protein was necessary to accurately test egg-derived vaccine for this strain. 

Table 4. Matched and heterologus strain testing with egg- and baculovirus-derived reagents. The homologous A/H1N1 and B/Florida components of both Novavax (NVAX) virus-like particle and inactivated egg-produced influenza vacccines gave comparable potency values when tested with either baculovirus- (NVAX) or egg-derived (CBER) reagants. However, when the heterologous H3N2 HA potencies were tested, accurate potency measurements required homologous reagants (baculovirus for VLPs, and egg-derived for inactivated egg-produced vaccine). 

In conventional inactivated influenza vaccines, no attempt is made to quantify or maintain NA activity. A number of preclinical and clinical studies have pointed to a significant role for NA-inhibiting (NAI) antibodies in protective immunity.

  NA-specific responses observed in human and mouse studies are responsible for a reduction of viral replication and disease prevention. In primates, NA is known to induce NAI, virus-neutralizing, and HAI antibodies. Immunity toward NA is also known to affect primary viral outcomes (disease progression and viral titers) and prevention of secondary bacterial infections. Moreover, NA-specific responses protect against lethal doses of highly pathogenic avian influenza by eliciting NAI and virus-neutralizing antibodies. NA-specific responses are also known to have reduced the impact of the 1968 H3N2 pandemic. 

VLPs contain quantifiable levels of active NA protein and we have shown that VLP-associated NA is very similar to the NA of the influenza virus. The enzymatic properties, Vmax (maximal velocity) and Km (substrate concentration required to achieve ½ maximal velocity) for VLP and viral NA in H3N2 and H5N1 strains are similar. In addition, VLP NA is found to be inhibited by oseltamivir carboxylate (Tamiflu) at a concentration similar to corresponding virus with a <3-fold difference in IC50. Moreover, in the clinical trials summarized above, we have demonstrated that immunization with VLPs elicits NAI antibodies in addition to HA-specific responses, demonstrating the broader immunological response of this vaccine. 

The immunologic advantage of VLPs results from their particle nature, which allows efficient uptake, processing, and presentation by dendritic cells. VLPs are membrane particles with a diameter of 141–192 nm containing the influenza HA and NA proteins as surface spikes. Measurement of VLP particle size by a Malvern Zetasizer is an important release criteria to demonstrate consistency of the manufacturing process and ensures the absence of aggregation. 

The goal of the purity assessment is to determine the levels of baculovirus (BV) and Sf9 cell DNA and proteins associated with each lot of VLP vaccine. These are determined by specific assays, as summarized in Table 1. 

There are two sources for BV and Sf9 preparations in VLP vaccine. Although the current purification process for VLPs completely inactivates BV, it does not completely remove BV particles. Because VLPs bud from the insect cells, there is a likelihood that they acquire cellular and BV proteins during this process. Thus, determining the true host and BV protein contaminants in VLP vaccine is a considerable challenge. 

Multiple batches of a single type of VLP must be analyzed to determine potential BV and Sf9 contamination. Two orthogonal approaches are used to determine VLP purity and the identity of BV and Sf9 proteins. SDS–PAGE followed by Coomassie staining allows us to visualize the proteins in each VLP batch. Scanning the stained sample lane and determining the percentage of each specific protein band allows the percentage of HA, NA, and M1 proteins to be determined and enables us to assign a relative purity value to the VLP (Table 3). LC–MS-MS of either the whole VLP sample or specific bands isolated from gels has been used to identify the BV and Sf9 proteins associated with different VLP lots. A typical VLP protein band profile is shown in Figure 4. The proteins detected were predominantly the expected influenza HA, NA, and M1 proteins engineered into the B/Florida VLP. Baculovirus gp64 envelope protein and p39 major capsid protein were identified through SDS–PAGE analysis of this vaccine along with viral ubiquitin detected by MS analysis. Sf9 proteins detected by MS analysis included tubulin, actin, Hsp70 chaperone, and small amounts of several housekeeping proteins. 

Figure 4. Identification of proteins in virus-like particle influenza vaccines. This SDS-PAGE band profile of the B/Florida/4/06 VLPs, obtained by LCâMS-MS, shows that the proteins detected were predominantly the expected HA, NA, and M1 proteins, along with two baculovirus proteins (the gp64 envelope protein and the p39 major capsid protein). 

ELISA assays have been developed as a second method to measure the concentration of BV and Sf9 proteins in VLP lots. As seen in Table 5, the purity of VLP lots based on the baculovirus ELISA is generally in good agreement with the values seen using the SDS-PAGE method. 

Table 5. Comparison of VLP purity determined by SDSâPAGE and BV ELISA 

An alternative method based on reverse-phase HPLC of VLP samples is being developed to identify the proteins in VLP lots. The protein peaks are identified either by Western blot using specific antisera or by fraction collection and analysis by mass spectrometry. A typical VLP profile with currently identified protein peaks is presented in Figure 5. Currently, this method can identify the influenza HA, NA, and M1 proteins as well as the baculovirus gp64 protein. 

Figure 5. Reverse-phase HPLC peak identification of proteins in the A/Brisbane H1N1 strain of a virus-like particle 

During clinical development, a stability testing program is established to 1) demonstrate vaccine stability during the time it is administered to volunteers and 2) begin to collect data to allow the assignment of vaccine shelf life upon approval for marketing. 

Table 6 presents 12-month stability data generated for the 60 µg HA/mL VLP lot of 2008–2009 trivalent seasonal VLP vaccine administered to healthy adults in a clinical trial (Table 2). The analytical testing parameters that were followed were 1) particle size, 2) SRID HA value for each strain in the vaccine, 3) vaccine purity/identity by SDS–PAGE and Western blot and 4) total NA activity. These measurements allow us to track potency and physical characteristics of the vaccine over the course of the study. The results demonstrated no change in the physical properties of the vaccine over 12 months and showed stability of the HA potency for all three components and consistent NA activity over the 12-month study. 

Table 6. Stability testing results of 60 Âµg HA/mL dose of trivalent 2008â2009 influenza VLP vaccine 

Peer-Reviewed Research

Release testing involves both standard potency assays and unique assays (particle size, NA activity) developed to ensure the physical, chemical, and biological stability of this type of vaccine.

Release and Stability Testing Programs for a Novel Virus-Like Particle Vaccine

Oligonucleotide sequences known as CpG ODNs are potent TLR9 agonists. The literature shows that formulation of CpG ODNs elicits more potent immune responses than the formulation or the adjuvant molecule alone. Given the importance of formulation on adjuvant biological activity and toxicity, formulation parameters should be well characterized and rationally designed. An understanding of the biophysical phenomena related to adjuvant interactions with formulation components can be obtained through complementary analytical techniques. Microeletrophoresis, UV spectrophotometry, and HPLC are used to characterize the association of CpG ODNs with aluminum hydroxide and oil-in-water emulsion formulations. 

Synthetic oligodeoxynucleotides containing cytosine-phosphorothioate-guanine sequences (CpG ODNs) are designed to mimic bacterial genomes that contain a higher proportion of unmethylated CpG motifs compared to vertebrate genomes. CpG ODNs have demonstrated potent vaccine adjuvant activity as TLR9 agonists, inducing higher antibody titers and an array of cellular immune responses.

(SCIENCE PHOTO LIBRARY/TEK IMAGE/SPL/GETTY IMAGES) 

Several studies have demonstrated the importance of appropriate formulation of CpG ODNs to maximize adjuvant response, including a recent special issue dedicated to this topic.

  For example, CpG ODN mixed with aluminum hydroxide or MF59 elicited higher hemagglutination inhibition antibody titers compared to CpG or either formulation alone, and a more Th1-biased cytokine response compared to either formulation alone in a mouse influenza model.

  Similarly, adding CpG ODN to MF59 induced higher total antibody levels compared to either component alone and increased IgG2a/IgG1 ratios in mice compared to MF59 alone on immunization with HIV p55 gag antigen.

It was also shown that CpG ODN formulated with aluminum hydroxide or an oil-in-water emulsion induced significantly higher antibody titers than either component alone in a rabbit model with various antigens or a piglet model with swine streptococcic septicemia killed vaccine.

  In addition, total antibody levels and CTL activity induced in mice by a hepatitis B surface antigen formulated with CpG ODN and aluminum hydroxide were significantly higher than CpG or alum alone.

  Finally, encapsulation of CpG ODNs in polymer microparticles has been shown to increase antibody titers to a meningitis antigen in mice compared to non-encapsulated CpG.

Given that the combination of adjuvant molecules such as CpG ODNs with particulate formulations translates into significantly more potent immune responses or reduced toxicity, it is important to optimize the formulation and presentation of the adjuvant to the immune system. Improving adjuvant formulation design requires an understanding of the basic biophysical principles underlying adjuvant association with the particulate formulation. This topic will be discussed using several specific examples of the formulation of CpG ODNs with particulate formulations commonly employed as vaccine adjuvants, namely aluminum hydroxide gel and oil-in-water emulsion. A particular emphasis has been placed on practical aspects of material properties and characterization methodology. 

CpG oligodeoxynucleotides (ODNs) were obtained from Avecia Biotechnology and Coley Pharmaceutical Group. Several different sequences are available and efficacy varies between animal models depending on the sequence. The ODNs described here are a modified CpG ODN similar to 10103 (the order of two sequential base pairs were switched from CpG ODN 10103): TCG TCG TTT TTC GGT GCT TTT, CpG 2395: TCG TCG TTT TCG GCG CGC GCC G, and CpG ODN 1826: TCC ATG ACG TTC CTG ACG TT.

  CpG ODN (similar to 10103) from Avecia is supplied as a dry powder and stored at –20 °C. CpG 1826 was purified with an ethanol precipitation step and reconstituted in 10 mM pH

  Tris with 1 mM EDTA. CpG ODN absorbance values were measured using a Beckman Coulter DU 640i or a Hitachi 3900H UV-Vis spectrophotometer. 

 Alhydrogel "85" was manufactured by Brenntag Biosector. Alhydrogel "85" consists of aluminum hydroxide gel containing small aggregates (~1 to 10 mm) of high surface area and positive charge.

  X-ray diffraction and infrared spectroscopy data indicate that the structure of aluminum hydroxide adjuvant resembles boehmite with the chemical formula AlO(OH), instead of the implied formula of Al(OH)

  The aluminum content in Alhydrogel "85" is 1% or 10 mg/mL.

  Alhydrogel is stored at room temperature and settles with time, so vigorous shaking is required before use. Aluminum hydroxide zeta potential measurements were measured using a Zetasizer Nano-ZS and an MPT-2 multi-purpose titrator from Malvern Instruments.  

 An oil-in-water stable emulsion (SE) was manufactured at the Infectious Disease Research Institute. Squalene (Sigma-Aldrich), purified from shark liver, was used for the oil source and was present at 10% (v/v) in the final product. High purity (99%) phosphatidylcholine (Avanti Polar Lipids) from chicken egg yolks, was used as a natural emulsifier and was present at 1.92% (w/v) in the final product. Poloxamer 188 (Pluronic F68 from BASF), a synthetic triblock copolymer nonionic surfactant, was used as a coemulsifier at a final concentration of 0.09% (w/v). To create an isotonic formulation, glycerol (Sigma-Aldrich) was included in the SE at 1.8% (v/v). Alpha-tocopherol (Spectrum Chemical) was used as an antioxidant and present at 0.05% (w/v). A 25 mM ammonium phosphate buffer was used to maintain the aqueous phase at pH 5.1 ± 0.05. 

The oil phase, consisting of the squalene, phosphatidylcholine (PC), and alpha-tocopherol, was prepared by sonication in a heated (~50 °C) water bath until the PC was fully dissolved (typically 1–2 h). The aqueous phase was prepared by combining deionized water with glycerol, Pluronic F68, and ammonium phosphate buffer. The buffered aqueous phase was added at 90% (v/v) to the oil phase and then mixed with a Silverson heavy-duty laboratory mixer emulsifier (3/4 inch tubular square hole high shear screen attachment) at ~8,000–10,000 rpm for several minutes. The mixture was subjected to high-pressure homogenization using the Microfluidics M-110EH-30 for 12 passes at ~207 MPa (~30,000 psi).  

Particle diameter was ~100–110 nm as measured by dynamic light scattering using the Malvern Instruments Zetasizer Nano-S. Visually, the SE had the appearance of homogenized milk. The SE formulation was stored in rubber-stoppered glass vials at 2–8 °C and demonstrated good stability (minimal change in particle size and no visual phase separation) for at least 1 year. The aqueous phase separation procedure described in the text was carried out using Ficoll PM400 obtained from GE Healthcare. In addition to the manufacture of SE, an MF59-like emulsion was manufactured using similar procedures to produce an oil-in-water formulation containing 5% v/v squalene and 0.5% v/v each of Tween 80 and Span 85 as surfactants; the water in this case was unbuffered.  

CpG-aluminum hydroxide adsorption isotherm.

 Because of the negatively charged phosphate groups that make up the CpG ODN backbone, these molecules are electrostatically attracted to the positively charged Alhydrogel. Besides electrostatic considerations, other mechanisms, including phosphate ligand exchange and hydrophobic interactions, also can attract adjuvants or antigens to the aluminum adjuvant.

The following illustrates two complementary procedures for determining the extent of CpG ODN adsorption to Alhydrogel "85". These techniques also are applicable to antigen adsorption studies.

  Aliquots of 100 mL Alhydrogel "85" (10 mg/mL aluminum) were pipetted into each of 10 microcentrifuge tubes. One hundred mg of CpG ODN (1 mg/mL) were added to the first microcentrifuge tube, 200 mg to the second tube, etc., with the last tube containing 1 mg of CpG ODN. Each tube contained the same total solution volume (1.1 mL). After briefly vortexing, the samples were incubated for 1 h at room temperature to allow for binding of CpG ODN to Alhydrogel "85". The samples were then centrifuged for 9 min at 16,000

 to separate Alhydrogel "85" from aqueous solution (Alhydrogel "85" settles). The absorbance of supernatant at 260 nm was measured in each sample. The non-adsorbed concentration of CpG ODN using a standard curve was determined and subtracted from the original CpG ODN concentration to calculate adsorbed CpG ODN. Adsorbed CpG ODN versus original CpG ODN concentration in solution is plotted in Figure 1. 

Figure 1. Adsorption isotherm of a modified CpG ODN (similar to CpG 10103) on aluminum hydroxide (Alhydrogel "85") as measured by centrifugal separation and absorbance at 260 nm.  

A CpG ODN-aluminum hydroxide adsorption isotherm also can be constructed by measuring the zeta potential of aluminum hydroxide particles during CpG ODN titration. Increased negative charge on the particles reflected in lower zeta potential represents more bound CpG ODN, whereas a plateau in zeta potential represents maximum CpG ODN adsorption. To measure the zeta potential of CpG ODN molecules adsorbed to Alhydrogel "85", a stock solution of aluminum hydroxide was prepared at 1 mg/mL aluminum and CpG ODN at 4 mg/mL. The titrator was configured to inject 0.05 mg/mL aliquots of CpG ODN and collect duplicate measurements of zeta potential after each injection. Of course, if a titrator is not available, the zeta potential measurements can be carried out by preparing separate solutions, as was done for the UV absorption experiment above. The titration was continued until the final CpG concentration was 1.25 mg/mL, corresponding to 1.8 mg CpG ODN/mg aluminum. Zeta potential versus CpG ODN/aluminum w/w is plotted in Figure 2. Notice that the agreement between both adsorption isotherms is quite good (Figures 1 and 2). 

Figure 2. Adsorption isotherm of a modified CpG ODN (similar to CpG 10103) on aluminum hydroxide as measured by zeta potential titration. Error bars represent duplicate measurements at each concentration in three separate experiments. 

Adsorption Effects of Excipients, Other Adjuvants, or Antigen

The excipients present in some antigen or adjuvant stock solutions can alter the binding behavior of the CpG-Alhydrogel complex. One must be especially cautious of ligand exchange with phosphate-containing solutions. Before the effects of the antigen on the adsorption of CpG and vice versa can be monitored, the effects of the excipients present in the antigen preparation must be evaluated. To determine excipient effects on CpG-Alhydrogel adsorption behavior, the binding capacity protocol from above was repeated using a CpG ODN solution containing the excipients from a lyophilized antigen preparation instead of water. Thus, for a 100 mL

 injection consisting of 10 mg CpG and 100 mg aluminum, a sample phosphate buffer containing EDTA and sucrose was mixed with the adjuvant and aluminum. The sample was centrifuged as described above and the absorbance of supernatant was measured at 260 nm. For this example, it was found that the adsorption of CpG ODN to Alhydrogel was not diminished in the presence of the excipient mixture (Table 1). It should be noted that order of addition of each component and time to bind also may affect adsorption behavior. The effects on CpG-Alhydrogel adsorption of several different buffers and excipients has been discussed elsewhere.

Table 1. Adsorbtion of CpG ODN to aluminum hydroxide gel 

A similar procedure can be followed to determine the effects of other adjuvants or antigens on CpG-Alhydrogel adsorption. However, detection using spectrophotometry becomes more complicated since other adjuvants and especially antigens can interfere with the 260 nm absorbance signal. In these cases, other detection methods may be desirable. Supernatant CpG ODN can be detected by SDS-PAGE/laser densitometry, in addition to spectrophotometry.

  Size exclusion HPLC can detect CpG ODN present in the supernatant after centrifugation.  

The mobile phase for size exclusion HPLC methods often is the same as the sample buffer, making these methods simple to perform. Other HPLC methods for ODNs or DNA, including reversed phase and ion exchange, also have been demonstrated.

  In addition, a dissociation step may be necessary before HPLC analysis to separate CpG ODN bound to protein or other materials.

  In this case, because the CpG ODN was stored in TE buffer, a mobile phase was prepared consisting of 10 mM Tris buffer at pH 8. The flow rate was set to 0.5 mL/min (TSKgel G3000PWXL column), with an injection volume of 20 μL, and the UV-Vis absorbance detector set to 260 nm. To develop the detection range for this method, samples of CpG 2395 were prepared at 1, 0.1, and 0.01 mg/mL, with a blank of TE buffer. Detection of CpG ODN was possible at a concentration of 0.01 mg/mL (Figure 3). 

Figure 3. HPLC detection of CpG 2395 in TE buffer 

Association of CpG ODN with Emulsion Formulations

The association of CpG with the oil (squalene) phase is thought to be weak because of the unfavorable energetics of hydrophobic interactions. The partitioning of oligonucleotides is dominated by strong solvation in the aqueous phase, though surface or interfacial effects may be occurring through mediation of surfactants. However, even weak association with particles may have an effect on biological activity. Because of the small difference in density of the two phases, it is necessary to increase the density gradient by adding a high MW sucrose polymer such as Ficoll PM400 (GE Healthcare). Ficoll can be dissolved in water to create a solution that has a significantly higher density than water because of its high molecular weight and excellent water solubility. This allows separation of the phases in an emulsion using a standard microcentrifuge. Alternatively, an ultracentrifuge may be used to separate the phases without adding the sucrose polymer.

To determine the extent of CpG ODN associated with the oil particulates in the SE formulation, a 30% (w/v) solution of Ficoll in water was mixed at a 1:1 ratio with the SE formulation (10% oil v/v and 1.25 mg/mL CpG ODN) in a microcentrifuge tube and vortexed briefly. The sample was centrifuged for 30 min at 16,000

. After centrifugation, the sample was carefully removed and the tube bottom carefully punctured with a 27g half-inch needle to extract an aliquot of the lower aqueous phase. The absorbance at 260 nm was then measured and compared to a CpG ODN standard. The difference in absorption represents the amount of CpG ODN associated with the oil phase (Table 2). In this example, all of the CpG ODN was in the aqueous phase, as was the case for a CpG-MF59 formulation that underwent ultracentrifugation.

  This probably is because these emulsions use nonionic surfactants or neutral phospholipids as emulsifiers. Emulsions made with charged emulsifiers show higher CpG ODN association with the oil phase and somewhat better immune activity; however, strong oil association is not necessary for synergistic adjuvant effects demonstrated with neutral emulsion-CpG ODN formulations.

Table 2. Association of CpG ODN with oil-in-water emulsion  

Because CpG ODNs are efficiently ionized at physiological pH, a change in electrophoretic mobility (zeta potential) of the emulsion can be observed if the CpG ODN molecules associate preferentially into the interface between the oil and water phases. Differences in the composition and local chemical potentials at the interface can adjust to balance the electrostatic–hydrophobic interactions that shift as the activity of CpG grows with increasing solution concentration. To measure the change in emulsion interfacial charge caused by CpG ODN interactions, a titration of zeta potential versus CpG ODN concentration was performed. The emulsion used here was the MF59-like emulsion described above. The emulsion was diluted 10-fold in water to permit light beam transmission of the scattering solution, as well as lowering the ionic strength to decrease the conductivity and increase the Debye length, permitting a more accurate measurement.  

The diluted emulsion was titrated against 4.0 mg/mL CpG ODN and changes in emulsion surface charge and curvature were measured by measuring both zeta potential and particle size as described above. In this example, the effects were subtle, with a gradual downward slope in zeta potential from 0 to ~0.1 mg/mL CpG ODN, a gradual upward slope from ~0.1 to 0.25 mg/mL (Figure 4), and an abrupt shift of ~2 mV at ~0.25 mg/mL CpG ODN, possibly caused by a reorganization of the molecules in the interfacial Stern layer to accommodate the changes in the aqueous phase as solvation of the added CpG becomes more extensive. At higher CpG ODN concentrations up to 0.9 mg/mL, the zeta potential is relatively constant. 

Figure 4. Zeta potential measurement of the titration of CpG 1826 in an MF59-like emulsion. Error bars represent triplicate measurements from a single experiment. 

Appropriate formulation of CpG ODN can significantly improve vaccine adjuvant activity. Rational design of CpG ODN formulations is enabled by thorough characterization of fundamental formulation parameters. In addition, changes in vaccine excipient composition should be monitored to determine effects on CpG ODN formulation. In this work, simple but informative physicochemical analytical techniques have been effectively used to determine the extent of CpG ODN association with particulate formulations, namely aluminum hydroxide and oil-in-water emulsion formulations.  

This work was supported in part by Grant 42387 from the Bill & Melinda Gates Foundation. The authors would like to thank Martin Freide, PhD, for helpful discussions regarding method development.  

 is the process development specialist II, 

 is the head of research and development, and 

 is the director of process sciences, all at the Infections Disease Research Institute, Seattle, WA, 206.330.2527, 

, formerly of the Infections Disease Research Institute, currently is the account consultant—northern California, at Chemical Abstracts Service, Columbus, OH. 

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Adjuvant activity can be greatly improved by appropriate formulation of cytosine-phosphorothioate-guanine oligodeoxynucleotides (CpG ODNs).

Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs

Adjuvants are substances added to vaccine formulations to enhance, accelerate, and prolong the immune response to a vaccine. Despite these notable benefits, vaccine adjuvants also can add to concerns related to clinical efficacy, safety, and dose stability. In the manufacturing environment, adjuvants can create challenges for sterilizing membrane filter capacity and bacterial retention validation. The particulate character of some adjuvants can cause premature plugging of filter membranes, and the low surface tension of many adjuvant solutions may contribute to reduced bacterial retention efficiency and potential non-sterility. In this article, we provide some recommendations for membrane filter selection and process conditions that may enhance filter capacity and increase the probability of successful bacterial retention validation for sterilizing filtration of adjuvanted vaccines.  

Immune potentiators or immunomodulators, commonly referred to as adjuvants, are gaining increased attention from vaccine manufacturers. Adjuvants are used to enhance, accelerate, and prolong the efficacy of a vaccine. They are increasingly necessary now that simple, highly purified antigens, such as recombinant proteins or DNA, which do not elicit strong immune responses on their own, are replacing more antigenic intact pathogens in vaccine preparations. Interest in adjuvants also has increased because of their dose-sparing effect, facilitating a quicker and broader response during a pandemic.

Adjuvants are a heterogeneous group of compounds (Table 1), including single compounds with intrinsic immunostimulating properties, for example, monophosphoryl lipid A (MPL) or the saponin QS21, and antigen carriers such as liposomes, microparticles, aluminum salts, but often have a combination of both immunostimulatory and carrier characteristics (e.g., emulsions and immune-stimulating complexes).

  Aluminum salts have been widely used and accepted in vaccines produced globally for several decades, whereas approval of new vaccine adjuvants has, until recently, been limited to Europe, for example, H1N1 pandemic influenza vaccines Focetria (Novartis) and Pandemrix (GSK). In the US, FDA approved the vaccine Cervarix (GSK), containing MPL, in late 2009. 

Table 1. Examples of vaccine adjuvants3,6 

The properties of these adjuvants or adjuvanted vaccines, in combination with specific process operating conditions, may create some challenges during sterilizing filter selection and sizing (filterability) studies and during sterilizing filter bacterial retention validation. 

The Challenges of Filter Qualification Filterability

The particulate size of some adjuvants (aluminum salts, liposomes, and microparticles) may be too large to pass through the pores of a sterilizing grade filter membrane (with a typical mean pore diameter ~0.2 μm, i.e., 200 nm) and aseptic preparation of an adjuvant vaccine formulation may have to be carried out in lieu of final sterilizing filtration. The membrane pore rating and material of construction also may influence the integrity of the liposomes or microparticles or reduce the mean particle size of the emulsion.

  Typical sizes of emulsions and liposome solutions can range from <100 nm up to 600 nm (0.6 μm), as measured using dynamic light scattering (DLS). For example, the squalene oil-in-water emulsion MF59 (Table 1) has a particle size of ~165 nm, while emulsion SB62 has a particle size of ~155 nm, both with a broad particle size distribution.

It has been observed that a complete transmission of liposomes can be achieved through 0.2 μm rated membranes, including liposomes that were estimated to be larger in size than the mean pore rating.

  A comparison of liposome size distributions by DLS and transmission electron microscopy (TEM) imaging, however, has revealed that DLS may not be very accurate in predicting liposome size relative to filterability. Specifically, liposomes examined by TEM were found to be ~3 times smaller than those obtained by DLS, suggesting that liposome size measurements using dynamic light scattering may, at times, overestimate the effective liposome size in regard to filtration.

  This could potentially explain the observations of large liposome transmission through 0.2 μm rated membranes.

Even when sterilizing filtration is an option based on antigen size, adjuvants may cause premature plugging of the filter membrane, reducing filter capacity. This can be linked to the solution viscosity or to the particulate character of the adjuvant in the formulation, as described above. For a model oil-in-water emulsion, it has been reported that membrane capacity is increased with decreasing adjuvant particle size.

  This suggests a known mechanism where membrane capacity may be overchallenged with particles similar in size to the filter's effective pore size rating. The flux decays seen with such adjuvants or adjuvanted vaccines may be a function of the membrane structure, its effective pore size distribution, and total porosity.  

Prefiltration can be a useful procedure for avoiding overchallenging a membrane with particles larger than, or in the same size range as the effective mean pore size of the membrane. Prefiltration typically helps in improving throughput by removing larger emulsion droplets or liposomes that otherwise plug the sterilizing membrane. Prefiltration or bioburden reduction membranes rated at 0.2 μm (mean effective pore size) can represent a fairly wide range of pore size distributions and retention efficiencies for nm or sub-micron–sized particles. Hence, the decision of whether to use coarser or finer 0.2 μm rated prefilters, is dependent on feed characteristics as well as overall process economics.  

Another challenge that adjuvanted solutions pose for sterilizing grade membranes is a potential reduction in bacterial retention efficiency. During the process-specific bacterial retention validation of a sterilizing grade filter membrane, worst-case conditions are simulated. In rare cases, under the high bacterial challenge levels imposed, poor filtration properties and reduced bacterial retention may be observed. Investigations are currently in progress to identify key process parameters that may affect bacterial retention in the presence of adjuvants. Some parameters that might reduce bacterial retention have been described for a model oil-in-water emulsion.

  Amongst them were the plugging behavior of the model emulsion, the membrane properties, temperature, and operating pressure. For this model oil-in-water emulsion, and for a selected sterilizing grade membrane filter, performing filtration at ambient temperature (versus at cold temperature), lowering operating pressure, and using a prefiltration step before sterilizing grade filtration may improve bacterial retention and the probability of demonstrating a sterile effluent. 

Given that more controlled studies are not yet available, we evaluated our extensive database of field studies and sterilizing filtration validation studies to identify process parameters that may affect bacterial retention. We offer our observations as preliminary recommendations to minimize the risk of bacterial penetration during validation studies and thus reduce efforts and costs to repeat studies, as well as to avoid delays in time-to-market. 

In general, adjuvants and adjuvanted vaccines are most often solutions containing oils or surfactants, formulated as such, in emulsions or as liposomes with a reduced surface tension (Table 1). For example, squalene-based emulsions like SB62 or MF59 typically have a surface tension of ~33 dynes/cm

In the process of validating numerous sterilizing filtration steps through various 0.2 μm rated filters, under a multitude of conditions, we have observed that test fluids with a "low" surface tension (<68 dynes/cm

) present a higher risk of reducing bacterial retention of sterilizing grade filters than fluids with a "high" surface tension (~70 dynes/cm

, comparable to that of water). This categorization was based on surface tension data when available or rational assumptions based on the chemical composition of the test fluid solution when surface tension data were not available (for example, the presence of a surfactant or lipid suggested the likelihood of reduced surface tension in the test fluid).  

We further analyzed formulation data of low surface tension test fluids and categorized them (where information was available) as 1) liposome solutions, 2) lipid and lipid-like solutions (i.e. emulsions, proteins with long chain fatty acids, sterols, etc.), and 3) surfactant solutions. In the interest of a broad analysis, we didn't limit ourselves to adjuvants or adjuvanted vaccines, but considered all low surface tension solutions to better understand what parameters might increase the risk of bacterial penetration during validation challenges of sterilizing grade membranes.  

Following the categorization of low surface tension test fluids, we analyzed events of bacterial penetration in each category and observed that the risk, qualitatively defined here as the likelihood of an occurrence of bacterial penetration during retention validation challenge, was the highest for liposome solutions, followed by lipid and lipid-like solutions and then by surfactant-containing solutions. 

Figure 1. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in low surface tension solutions as a function of total challenge bacteria load (CFU/cm2), relative to baseline, i.e., risk at lowest bacterial load. 

With these higher penetration risk test fluids, challenge parameters associated with bacterial penetration were identified as the total 

 EFA) and the rate of load (CFU/min) during the 

 challenge test (Figures 1 and 2). Although the minimum total load cannot be changed (because of the regulatory requirement for achieving ≥1 x 10

  EFA during bacterial retention validation), our data suggest that 

 may significantly increase the risk of validation issues (either premature plugging or bacterial penetration) when conducting bacterial challenges of sterilizing grade membranes with these potentially higher-risk solutions. If possible, the 

 bacteria challenge loading rate should be minimized. 

Figure 2. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in low surface tension solutions as a function of bacterial load rate (CFU/min), relative to baseline, i.e., risk at lowest bacterial load rate. 

Bacterial challenge studies are conducted either under constant flow or constant inlet pressure conditions, so the categories were analyzed with regard to these challenge conditions (Figure 3). The data suggest that with higher risk solutions containing liposomes, lipids, or surfactants, a constant flow challenge condition may have a higher risk of a penetration event than constant pressure conditions. In a situation of constant flow, the pressure inevitably varies in an attempt to maintain a constant flow rate. This changing (generally increasing) pressure may result in increased potential for penetration. Similar observations have been previously reported.

Figure 3. Risk of Brevundimonas diminuta penetration events with 0.2 Î¼m sterilizing grade membranes in categories of low surface tension solutions according to challenge method, relative to baseline, i.e., risk for surfactants solutions and constant pressure challenge method. 

These categories were further compared with regard to the flux across the filter. To determine flux (flow per unit area), the maximum recorded flow rate (if available) or the average flow rate (if available) was divided by the effective filter area (EFA) of the test filter. For each category, two histograms of flux were generated; one for validated sterilizing filtrations and one for filter challenges where 

 bacteria penetration was reported. The peak values of these histograms provide insight into the differences between the retentive and penetrative filtration populations, as seen in Table 2. The data suggest that increased flux (or flow rate) does not necessarily increase the risk of penetration. In fact, when all data are considered, higher flux appears to suggest a correlation with a reduced risk of penetration, and particularly in the case of lipid solutions. This however, may only be relative to the positive association between rapid plugging of the membrane and bacterial penetration. 

Table 2. The peak value of flux histograms (mL/min/cm2) within a category (the shape of the histograms are skewed such that most of the test fluids have flux rates less than or equal to the peak).  

Achieving Successful Sterilizing Grade Filter Qualification

A review of field and laboratory bacterial retention validation data for a variety of fluids and challenge conditions suggests that low surface tension fluids, such as many adjuvants and adjuvanted vaccines, present a higher risk of the occurrence of a bacterial penetration event during sterilizing filter validation (Table 3). Among the classified solutions examined, liposome solutions represent the highest risk, followed by lipid and finally surfactant solutions. 

  (>10 times the minimum required challenge density) increases the chance of a penetrative event, as does a loading rate of greater than 1 x 10

  CFU/min. For liposome, lipid, and surfactant solutions, bacterial challenge using constant flow may present a higher risk and we would hence recommend conducting validation studies and operating processes of low surface tension adjuvants and adjuvanted vaccines at constant pressure. 

Table 3. Summary of the results of metadata analysis. Risk is qualitatively defined here as the likelihood of an occurrence of bacterial penetration during retention validation challenge. 

The analysis discussed herein, along with published work by other filter manufacturers, strongly suggests that although bacterial retention by sterilizing grade filters is most commonly achieved by size exclusion, it occasionally can be strongly influenced by the nature of some feed solutions (surface tension, particle size), processing conditions (constant pressure versus constant flow, temperature, operating pressure), membrane structure, and bacterial challenge test conditions (bacterial challenge load, loading rate). Based on the reviewed data, clearly no single membrane is successful in all applications.  

For the successful sterilizing filtration of adjuvants and adjuvanted vaccines, we recommend filter users consider the risk of reduced bacterial retention very early in the process, i.e., when designing the formulation, the filtration operating conditions and sequence, and when drafting the sterilizing filtration validation bacterial challenge test protocol. Several membrane candidates for bacterial retention should be tested along with filterability studies, to arrive at the candidate offering both complete bacterial retention and providing the highest filtration capacity for superior process economy. Because of the impact of operating conditions on bacterial retention, it is important to maintain process consistency during scale-up and to design the process with the end in mind.   

We would like to thank our colleagues from the Pall Scientific Laboratory Services group and Vincent Guercio in particular, for their efforts in collecting and analyzing field data for this article. 

 is a senior vice president of global scientific affairs, all at Pall Life Sciences, Fribourg, Switzerland, +41 26 350 5360, 

 1.  The European Medicines Agency. Guideline on adjuvants in vaccines for human use. Brussels, Belgium. 2004 Mar. Available from: 

http://www.emea.europa.eu/docs/en_GB/document_library/Scientific_guideline/2009/11/WC500015469.pdf

 2.  Van Nest G. Trends in developing new vaccine adjuvants. Vaccine Europe; 2009 Nov 17–18; Brussels, Belgium. 

 3.  Guy B. The perfect mix: recent progress in adjuvant research. Nature Rev Microbiolog. 2007;5:505–17. 

 4. Goldbach P, Brochart H, Wehrle P, Stamm A. Sterile filtration of liposomes: retention of encapsulated carboxylfluorescin. Int J Pharma. 1995;117:225–30.  

 5. Orsello CD. Novel vaccine adjuvants: What's the future? What are the challenges? A3P conference; 2010 Apr 28; Brussels, Belgium. 

 6. Fox CB. Squalene emulsion for parenteral vaccine and drug delivery. Molecules. 2009;14:3286–312. 

 7. Richard A, Delvaux J, Bonnet LB. Effect of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles. Int J Pharma. 2006;312:144–50. 

 8. Tsukada Y, Hara K, Bando Y, Huang CC, Kousaka Y, Kawashima Y, Morishita R, Tsujimoto H. Particle size control of poly (DL-lectide-co-glycolide) nanospheres for sterile applications. Int J Pharma. 2009;370:196–201. 

 9. Carbrello C, Rogers M. Optimizing vaccine adjuvant filtration. BioPharm Int suppl. Vaccine development and manufacturing: pandemics and beyond. Jan 2010; pp. 21–6. 

10. FDA CDER Perspective on Isolator Technology. ISPE Barrier Technology Conference; Rockville, MD; 1995 Dec. 

Filterability and bacterial retention must be verified very early in process development to ensure successful sterilizing filtration validation.

Sterilizing Filtration of Adjuvanted Vaccines: Ensuring Successful Filter Qualification

, has recently emerged as a novel manufacturing platform for recombinant subunit vaccines. It combines ease of growth with facile genetics in a complex unicellular eukaryote that can be grown rapidly in inexpensive media on an industrial scale. 

 devotes a large part of its metabolism to membrane protein production, and can release proteins to the extracellular space by constitutive and stimulus-dependent pathways of secretion. In this article, we show high-level expression of correctly folded parasite and viral proteins in a 

system and provide direct evidence that regulated secretion can be harnessed as an effective pathway for producing influenza hemagglutinin (HA). HA can be targeted to dense core granules 

 and be recovered following stimulus-dependent secretion in association with a proteinaceous gel termed PRISM. PRISM offers a convenient matrix for protein purification, but at the same time, has intrinsic properties with the potential to induce potent immune responses to co-administered antigens. 

Although cell-mediated immunity can play a significant role in clearing microbial pathogens, antibodies targeting secreted proteins or exposed antigens on microbial surfaces are often sufficient to generate protection against infectious agents. In such cases, vaccines that elicit strong, long-lasting antibody responses are highly effective in preventing disease. When compared with live and killed whole pathogens, subunit vaccines have distinct advantages particularly when dealing with newly emerging infectious agents such as pandemic influenza virus that have the potential to cause widespread disease. Subunit antigens can be produced rapidly on a large-scale as recombinant proteins without relying on attenuation or killing as steps in the production process. Subunit proteins are typically less reactogenic than live or killed pathogens and can be delivered at high antigenic mass. Furthermore, their production obviates the need to work directly with pathogens, which can be difficult to grow and dangerous. Finally, because recombinant antigens can be manufactured in microbial systems, the potential for contamination by adventitious agents that infect mammalian cells is minimized. 

Despite the advantages of recombinant subunit proteins, their protective efficacy may be lower than that of native antigens because of a number of factors. Primary among these is absence of pathogen-associated molecules and other "danger signals" required to stimulate robust immune responses to purified antigens.

  Although these can be provided by adding immuonstimulatory substances, or adjuvants, to the vaccine formulation, there are few such substances currently approved by the FDA.

  Equally important is the problem of protein folding and post-translation processing. In the case of viruses and parasites, the preponderance of vaccine candidates are membrane or secreted proteins that must fold properly to elicit protective antibodies in the host. This requires accurate disulfide bond formation, which is often difficult to achieve in bacterial expression hosts.

  Some microbial systems (including yeast) also contain rigid cell walls that impede downstream protein purification, and contain endogenous pyrogens that must be removed in the production process.  

To address some of the challenges surrounding potency, investigators have turned to eukaryotic expression hosts for improved protein folding (especially insect cells and fungi),

  and using virus-like particles (VLPs) that can present antigens in a repetitive, high-density format that can generate strong B- and T-cell responses.

  Still, VLPs are not applicable to all vaccine antigens and can be difficult or slow to produce in high yield.  

As an alternative to these approaches, we have focused on 

, a eukaryotic microbe that is capable of rapid, scalable growth. 

 lacks a cell wall and adds mammalian-like post-translational modifications (PTMs) onto proteins.

devotes a large-part of its metabolism to membrane protein production owing to the hundreds of cilia that extend from its surface. Moreover, 

 not only constitutively secretes proteins, but also stores large amounts of protein in hundreds-to-thousands of dense core granules that can be induced to secrete at will.

  Indeed, the material released from these granules takes the form of a proteinaceous gel (termed PRISM), which can easily be harvested from cells by low-speed centrifugation, providing a natural matrix for streamlined protein purification. PRISM has an underlying crystalline structure, and like VLPs, offers the opportunity to present antigens in a repetitive format that is optimal for cross-linking of the immunoglobulin (Ig) receptor on B-cells. In this article, we demonstrate the expression of correctly folded viral and eukaryotic vaccine antigens in the 

 system and provide preliminary evidence that PRISM may be an ideal matrix for rapid production of highly potent, low-cost vaccines. 

The gene encoding the H5 hemagglutinin from H5N1 (Vietnam) subtype avian influenza virus (accession number: EF541402.1) was codon-optimized for expression in 

 and introduced into the macronuclear genome, either on high-copy number rDNA vectors, or in the somatic MTT1 metallothionein gene locus by homologous recombination.

  In both instances, the transgene was under the control of the endogenous MTT1 promoter.

  For the constitutively secreted gene product, the region encoding 48 amino acids at the C-terminus of the protein was deleted. For targeting to mucocysts, the truncated H5 gene was tagged with a sequence encoding one of several granule lattice proteins of 

. The gene encoding the IAG4B[G1] i-antigen gene of 

 (accession number: AAD31283) was introduced into cells, either at the b-tubulin 1 (BTU1) locus under MTT1 promoter control, or on high copy number rDNA vectors as above. For the truncated gene product, the region encoding the C-terminal 19 amino acids of the IAG48[G1] gene was removed.  Cells were transformed biolistically and positive transformants selected by growth in paromomycin.

  cells/mL in Neff medium and treated with 2 μg/mL CdCl

 for varying periods of time to induce expression of the transgenes. In the case of granule-targeted H5, cells were washed and mucocyst discharge induced by adding dibucaine to a final concentration of 2 mM.

  The PRISM matrix was then harvested for further analysis by centrifugation at 6,000

 for 5 min and transferred to fresh tubes. 

To prepare monoclonal antibodies (MAbs) recognizing H5, BALB/c mice were injected intraperitoneally with killed reassortant A/Vietnam/1203/2004 X PR8 (H5N1; 105 HAU) mixed with Freund's incomplete adjuvant (FIA). One to four months later, mice were injected intravenously with 6–9 x 10

  HAU virus in phosphate-buffered saline (PBS). After three days, mice were euthanized and spleens were collected. Lymphocytes were harvested and fused to SP2/0-AG14 mouse myeloma cells using conventional methods.

  Mice were purchased from Harlan (Indianapolis, IN) and housed in the James A. Baker Institute vivarium according to the guidelines of the American Association of Laboratory Animal Care. Hybrid cells secreting antibodies specific for virus were identified by testing supernatants in hemagglutination inhibition (HI) assays, according to Barret and Inglis.

  Supernatants from positive wells were tested in ELISA for binding to virus using a peroxidase-conjugated anti-mouse IgG as previously described.

BioPharm International-10-04-2010

BioPharm International, October 2010 Supplement (PDF)

Release and Stability Testing Programs for a Novel Virus-Like Particle Vaccine

Characterization of Aluminum Hydroxide Gel and Oil-in-Water Emulsion Formulations Containing CpG ODNs

Sterilizing Filtration of Adjuvanted Vaccines: Ensuring Successful Filter Qualification

An Alternative Platform for Rapid Production of Effective Subunit Vaccines