A Charged Aerosol Detector That Reduces Vaccine Development Time

The Infectious Disease Research Institute (IDRI, Seattle, WA) is a not-for-profit corporation that has developed the first defined vaccine for leishmaniasis, which is now in clinical trials, in partnership with Corixa Corp. (Seattle, WA). IDRI is currently involved in the development of adjuvants that increase the effectiveness of this vaccine with a focus on monophosphoral lipid A (MLA), which has proved safe and effective when used with other vaccines.

The development process requires frequent analysis of various forms of MLA. The traditional analysis method, which uses HPLC with a fluorescence detector, has long been a bottleneck because of lengthy required sample preparation procedures. IDRI researchers Darrick Carter, Director of Process Sciences, and Mary Wallace, Development Associate, are in the process of validating an HPLC detector— the Corona® charged aerosol detector (CAD) (ESA Biosciences, a division of Magellan Biosciences, Chelmsford, MA)—that has demonstrated the potential to substantially reduce analysis times. IDRI researchers expect the detector to reduce the time required to prepare 200 MLA samples from between one and two weeks to just one day.

Fighting a deadly tropic disease

Figure 1 - Phlebotamine sand fly, which transmits leishmaniasis.

Members of the genus Leishmania infect many vertebrates, including humans, dogs, and rodents. The life cycles of members of the genus involve a vertebrate host and a vector, a sand fly, which transmits the parasite between vertebrate hosts (see Figure 1). The most common forms are cutaneous leishmaniasis, which causes skin sores, and visceral leishmaniasis, which affects some of the internal organs of the body, such as the spleen, liver, and bone marrow. The number of new cases of leishmaniasis each year in the world is thought to be about 1.5 million of the cutaneous and 500,000 of the visceral form. Hundreds of thousands of people, mostly children, die from the disease every year. Leishmaniasis is found in parts of about 88 countries. Most of the affected countries are in the tropics and subtropics. The settings in which leishmaniasis is found range from rain forests in Central and South America to deserts in West Asia. More than 90% of the world’s cases of visceral leishmaniasis are in India, Bangladesh, Nepal, Sudan, and Brazil.

Drugs are available to treat leishmaniasis, but the cost, at about $100 per dose, is high enough to put it out of reach of most residents of the affected areas. IDRI’s goal is to develop a vaccine that can be delivered at a much lower cost to developing areas. Protective immunity against leishmaniasis requires the specific induction of T-helper type 1 (Th1) cells that recognize the parasite. IDRI researchers have identified proteins of Leishmania that stimulate protective Th1 responses and are effective as vaccines in animal models. In fact, these proteins have even been used successfully to treat mucosal leishmaniasis—one of the most disfiguring and difficult forms to cure—in humans. Three vaccine proteins have been genetically engineered for production as a trifusion protein.

Adjuvants aid antigen delivery

Appropriate antigen delivery to induce the right type of immune response against Leishmania is another critical component of an effective vaccine. The two adjuvants approved for human use, alum and squalene, induce potent antibody responses but are poor inducers of antigen-specific Th1 responses. MLA has been used as an adjuvant in several trials for vaccines for malaria, genital herpes, and allergy desensitization as well as being approved for use in hepatitis B. Recently, scientists at Corixa developed synthetic lipid A mimetics that are chemically unique, acylated monosaccharides called aminoalkyl glucosaminide 4-phosphates (AGPs). In contrast to the complex family of lipid A congeners found in MLA, synthetic AGPs represent mimetics of MLA that can be synthesized as highly pure chemical entities. The AGPs can be designed to accommodate molecular changes for improved biological and pharmacological activities. These unique molecules are well suited for use as adjuvants for the Leishmania and other vaccines. IDRI researchers have determined that combining their trifusion proteins with an MLA adjuvant produced an even more potent vaccine. This is the first defined candidate vaccine developed for leishmaniasis. Clinical development of the vaccine is being supported by a generous grant from the Bill & Melinda Gates Foundation.

Importance of qualitative and quantitative analysis

Understanding the importance of the adjuvant to the vaccine effectiveness and safety, IDRI researchers are continuing to develop new MLA molecules in an effort to deliver further improvements. The development of novel adjuvants naturally requires frequent qualitative analysis during the development process. When the time comes to develop manufacturing processes, quantitative analysis will be required as well. The current state-of-the-art for analysis of MLA and similar lipids involves the use of HPLC with fluorescence detectors. The first step is a conjugation process that involves attachment of hydrazone to the lipid sample. This manual process takes about a half-day for a single sample or about a week for 200 samples. Then the sample is run on an HPLC system with fluorescence detection. Interpretation of the results is complicated by the fact that some forms of MLA fluoresce more than others, primarily depending on the number of acyl chains. This means that correction factors need to be applied to each peak, which adds about half a day to the time required to analyze a typical batch of samples.

Recognizing that conventional analytical methods represent perhaps the most time-consuming aspect of the adjuvant development process, IDRI researchers considered several alternate detector technologies. MLAs have boiling points that are too high to separate and analyze accurately with GC. The commonly used UV detector is not quantitative for lipids because analytes must possess at least one carbon–carbon double bond to be detected, while many lipids are saturated and invisible to UV or have varying levels of saturation. Refractive index detectors work well in a number of HPLC quantitative analysis applications; however, they are limited to isocratic analysis with a single solvent, while gradient analysis with multiple solvents works better with most lipids because it provides superior separation. Evaporative light scattering detectors (ELSDs) have become the standard method of quantitative analysis of lipids on HPLCs, and IDRI researchers seriously considered their use.

Charged aerosol detector technology

Figure 2 - Corona CAD.

The Corona CAD promises to deliver in a single package the performance benefits of RI, UV, and ELSD (Figure 2). In the detector, the HPLC column eluent is first nebulized with nitrogen, and the droplets are dried to remove the mobile phase, generating an aerosol stream composed of particles of all nonvolatile constituents. A secondary stream of nitrogen becomes positively charged as it passes a high-voltage, platinum corona wire. The secondary stream merges with the primary particle stream to turbulently mix the gas ions with the aerosol particles. The secondary stream’s charge transfers to the opposing stream of analyte particles. The charge is transferred to a collector, where it is measured by a highly sensitive electrometer, generating a signal in direct proportion to the quantity of analyte present.

Figure 3 - Twenty microliters of monophosphoryl lipid A in water/triethanolamine, pH = 7.2, from vendor 1 were injected and run over a reversed-phase C-18 column with a gradient from 90% Buffer A (95% acetonitrile/0.1% triethanolamine/0.25% acetic acid/water) to 85% Buffer B (95% isopropanol/0.1% triethanolamine/0.25% acetic acid/water) over 90 min followed by a ramp down to 10% Buffer B over 30 min (total time = 120 min). The stream was fed into a Corona detector under nitrogen at 35 psi, and data were collected using Agilent Chemstation software (Agilent Technologies, Santa Clara, CA). A heterogeneous mix of hexa-, penta-, and tetraacyl congeners was detected using the Corona detector.

Figure 4 - Conditions the same as in Figure 3, except that 20 μL of monophosphoryl lipid A in water/triethanolamine, pH = 7.2, from vendor 2 were injected and run over a reversed-phase C-18 column with a gradient from 90% Buffer A (95% acetonitrile/0.1% triethanolamine/0.25% acetic acid/water) to 85% Buffer B (95% isopropanol/0.1% triethanolamine/0.25% acetic acid/water) over 90 min followed by a ramp down to 10% Buffer B over 30 min (total time = 120 min). The stream was fed into a Corona detector under nitrogen at 35 psi, and data were collected using Agilent Chemstation software. A single peak was detected corresponding to the hexaacyl form of MPL.

IDRI researchers are currently in the process of developing methods for MLA analysis using the Corona CAD and HPLC. The detector is simple to set up and operate. One of the few parameters that need to be adjusted on the detector is gas flow. The researchers are currently working with two different forms of MLA. They are seeing a dramatic reduction in sample preparation time, primarily due to the elimination of conjugation. For an aqueous formulation, there is no sample preparation at all. The lipid sample is merely mixed with solvent and injected into the HPLC. Solid-phase extraction may still be required for oil-based samples, and that will take another half-day or so depending on the number of samples. The calculation of correction factors has also been eliminated (see Figures 3 and 4).

The result is that IDRI researchers have demonstrated the ability to analyze a single sample in about 2 hr and can prepare 200 samples in one day. This reduction in preparation time has the potential to substantially decrease both the development and manufacturing costs of the leishmaniasis vaccine. IDRI is currently investigating different excipients that could be added to the MLA adjuvant, and is also considering reengineering the organism or switching to a synthetic source to reduce costs. The researchers are still in the process of validating the CAD and have taken note of results from Dr. Robert A. Moreau of the Eastern Regional Research Center of the Agricultural Research Service, U.S. Department of Agriculture (Washington, DC). Dr. Moreau evaluated the CAD with several normal-phase and reversed-phase HPLC systems commonly used for the quantitative analysis of lipid classes and lipid molecular species. The CAD detected common lipids such as triacylglycerols, cholesterol esters, and free sterols to a minimum limit of detection of about 1 ng per injection.

Mr. Fireman is President, Structured Information, 363 Massachusetts Ave., Ste. 206, Lexington, MA 02420, U.S.A.; tel.: 781-674-2300; fax: 408-228-8750; e-mail: [email protected]. Dr. Carter is Vice President for R&D, Dharma Therapeutics Inc., Seattle, WA, U.S.A. Ms. Wallace is Research Assistant, IDRI, Seattle, WA.