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A powder formulation of measles vaccine for aerosol delivery
Vaccine 19 (2001) 2629– 2636 www.elsevier.com/locate/vaccine
A powder formulation of measles vaccine for aerosol delivery Cynthia LiCalsi *, Michael J. Maniaci, Troy Christensen, Elaine Phillips, Gary H. Ward, Clyde Witham Dura Pharmaceuticals, 7475 Lusk Bl6d., San Diego, CA 92121, USA
Abstract Both the mortality rate for measles and the risks associated with injection continue to be high in the developing world. In response to the need for safe, cost-effective vaccine delivery technologies, a powder formulation of measles vaccine has been developed to test the feasibility of administering measles vaccine as an aerosol. The first challenge in aerosol formulation development is to produce fine particles without damaging the activity of the virus or inducing physical changes. In this study, live attenuated measles vaccine is micronized by jet milling to generate particle sizes appropriate for pulmonary delivery (1–5 mm). Milling does not induce detectable physical changes and significant viral potency is maintained. Potency retention of milled vaccine ranges from 31 to 89%, demonstrating that the standard dose of vaccine can easily be achieved. Following size reduction, particles are blended with an inert carrier to improve handling and aerosol dispersion. The measles vaccine formulation is dispersible, as shown by laser light particle size analysis of vaccine aerosols. Thus, evaluation of both the potency retention and the aerosol characteristics of the current formulation clearly demonstrates the feasibility of delivering measles vaccine as a powder aerosol for immunization. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Measles vaccine; Aerosols; Dry powder inhalation
1. Introduction Pulmonary delivery using powder vaccine aerosols is an approach to immunization that offers advantages over injection in terms of both delivery technology and vaccine formulation [1]. The technology advantages include increased safety and ease of administration. The formulation advantages when using dry powders are the potential reduction of refrigeration requirements and increased stability during transport and administration. Additionally, there is potential for enhanced biological efficacy since pulmonary delivery may produce mucosal immunity superior to that which is produced by parenteral administration of vaccines. Each of these factors is pertinent to the battle against measles in developing countries, where the disease continues to be a leading cause of mortality among children. First, in those countries where mass
* Corresponding author. Tel.: +1-858-3207625; fax: + 1-8585584120. E-mail address: [email protected] (C. LiCalsi).
immunization campaigns are most needed, the potential contamination risks associated with needles are also the highest. Second, while cold-chain maintenance and stability are concerns associated with vaccine delivery in general, these issues are particularly significant in the case of measles vaccine. Although the lyophilized vaccine is relatively thermostable, measles vaccine loses potency rapidly at both 25 and 37°C when reconstituted. Thus, vaccine reconstituted from multi-dose vials must be kept cold during administration. Finally, measles is highly contagious and, therefore, the potential to induce both mucosal and systemic immunity via pulmonary delivery may contribute significantly to measles elimination/eradication. To date, many studies have shown both the safety and efficacy of measles vaccine delivered as a liquid aerosol from a nebulizer [2,3]. Elimination of the reconstitution requirement would significantly strengthen the aerosol approach. This paper describes the development of a dry powder aerosol vaccine formulation and establishes the feasibility of delivering a standard dose of measles vaccine as a powder aerosol from a dry powder inhaler.
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2. Materials and methods
analysis was conducted using 5–8 mg of material in a crimped pan.
2.1. Milling and blending 2.5. Thermogra6imetric analysis Lyophilized measles vaccine (Edmonston-Zagreb BIRMEX, Mexico; Moraten Berna, Swiss Serum and Vaccine Inst., Bern, Switzerland; Attenuvax, Merck & Co., West Point, PA) was micronized using a one inch jet mill (Micron-master model c01-506, Jet Pulverizer Co., Palmyra, NJ) with compressed nitrogen as the gas stream. Injection nozzle pressure was 100 psig for milling pressures of 18 or 35 psig and 180 psig for a milling pressure of 65 psig. Vaccine was fed manually into the mill. Milling was carried out under low relative humidity conditions (B4% RH) in a Nitrogen-purged glove box. Blends of micronized measles vaccine and Pharmatose lactose (100 M, DMV International, Veghel, The Netherlands) were prepared in glass vials using a 3-D tumble-in-container blender (Turbula® T-2C, Glen Mills Inc., Clifton, NJ).
2.2. Scanning electron microscopy (SEM) SEM samples were prepared using a standard gold sputter technique and then imaged on an ESEM (XL30 ESEM TMP, Philips Electron Optics, Hillsboro, OR) at 10 – 15 kV with average working distances of 8 – 15 mm. Neat and blended samples were imaged at several magnification levels ranging from 50× to 8000× .
2.3. Particle size measurement Particle size was measured using a laser diffraction particle size analyzer (Malvern Mastersizer-S, San Bernardino, CA) with dynamic, small volume sample cell. A reverse-Fourier lens (detection range: 0.1 –3000 mm) was used to size material in liquid suspension of polydimethylsiloxane. A Sympatec HELOS KF laser diffraction particle size analyzer (Sympatec Inc., Princeton NJ) utilizing the RODOS dry powder disperser and Vibri vibratory feeder was used to measure the size distribution of the free powder. The RODOS module was operated with a primary air dispersion pressure of 4 bar and a depression value of 103 mbar. The Vibri feeder was operated at 75%. An R2 focusing lens (0.25 –87.5 mm) was used for the micronized material and an R5 lens (4.5 – 875 mm) for the 50% vaccine blend.
2.4. Differential scanning calorimetry (DSC) Samples were first dried at 30°C in a thermogravimetric analysis (TGA) (Hi-Resolution TGA 2950, TA Instruments, New Castle, DE) for 120 min, then scanned from −10 to 275°C at a scan rate of 10°C/min with a TA Instruments dual cell model 2920 DSC. Each
Approximately 5–8 mg samples of material in an open pan were scanned from 25 to 250 or 300°C at a scan rate of 10°C/min on a Thermal Analysis model AutoTGA 2950.
2.6. Moisture sorption analysis Step isotherms of measles vaccine at 25°C were obtained using a nitrogen flow-based, automated, microbalance system (Model MB-300W, VTI Corporation). Samples were dried under a stream of dry nitrogen prior to exposure to various RH conditions. Approximately 10–15 mg of vaccine were scanned between 5 and 95% RH in increments of 5% RH. Each increment was programmed to satisfy the following equilibrium criteria: sample exposure to the next humidity level occurred when the change in sample weight was 5 0.005% of the beginning sample weight in 5 min, for three consecutive measurements, or after 120 min, whichever occurred first.
3. Results
3.1. Particle size measurement and solid state characterization of measles 6accine Live, attenuated measles vaccine was micronized by jet milling at a milling pressure of 35 psig. This method uses high pressure, high velocity jets of gas to cause particle to particle impaction and thus particle attrition [4]. A qualitative evaluation of the results was made using scanning electron microscopy. Fig. 1 shows micrographs of measles vaccine taken before and after size reduction. Unmilled vaccine appears to consist of many large (up to :400 mm) and small (down to : 10 mm) irregularly shaped plates. In contrast, milled material shows particles of more uniform shape with sizes primarily smaller than 10 mm. To quantify the effectiveness of the milling process, particle size distribution of vaccine powder was measured in liquid suspensions using a laser diffraction instrument (Table 1). Both unmilled and milled vaccine have broad unimodal particle size distributions as measured by this technique. Particle size is typically reported as volume diameter, thus, the parameters D(v, 0.1), D(v, 0.5), and D(v, 0.9) refer to particle diameters below which 10, 50, or 90% of the particle volume is contained. Consistent with the microscopy results, the D(v,0.5) determined for milled vaccine ranges from 3.2 to 4.2 mm, values significantly smaller than the D(v,0.5) of 21.6 mm measured for the
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Fig. 1. Scanning electron micrographs of measles vaccine before (left) and after (right) micronization by jet milling. 369 × magnification; 50 mm size marker.
unmilled material. Thus, 56 – 71% of the milled vaccine has particle sizes less than 5 mm, an appropriate size for pulmonary delivery. For delivery of a live viral vaccine, it is perhaps more pertinent to consider the number of appropriately sized virus-containing particles. When the volume measurement is translated to reflect particle numbers, approximately 99% of the particles in the milled vaccine have sizes less than 5 mm. Because the intended use of the formulation is as a dry powder aerosol, it is essential that the powder be dispersible. Therefore, particle size distribution of aerosolized powder was measured using a Sympatec laser diffraction instrument. The Sympatec uses a gas shear stream to overcome adhesive forces between particles. Sample introduction takes place with the aid of a vibratory conveyor feeder. The size distribution of an aerosol of milled measles vaccine is shown in Fig. 2. The cumulative distribution curve shows a D(v,0.5) of 4.390.3, which agrees well with the particle size measurements made using liquid suspensions. The density distribution curve is not unimodal, possibly indicating that the powder is not completely deagglomerated by the dispersion pressure used. However, under the conditions used, the results show that 57% of the aerosolized sample volume consists of particle sizes considered appropriate for pulmonary delivery (5 5 mm). While particle sizes of 5 5 mm target the deep lung, larger particles (up to 10 mm) deposit preferentially in the tracheobronchial region. No assessment has yet been made regarding optimal size distribution for measles immunization. While the success of micronization is determined in part by the resultant particle size distribution, it is also important in each case to confirm that the physical characteristics of the material are maintained. Measles vaccine is a mixture of live attenuated virus with various excipients that presumably function to stabilize the virus and/or aid in cake formation during lyophiliza-
tion. The physical characteristics of the unmilled and milled vaccines were compared using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and moisture sorption analysis. Differential scanning calorimetry thermograms of measles vaccine reveal several energetic transitions (Fig. 3). The various endotherms and exotherms represent glass transitions, melting, and recrystallization events of crystalline and amorphous materials formed upon heating. Due to the complexity and variety of excipients in the vaccine formulation, individual peaks cannot be assigned definitively to a compound or event. However, most notable is the observation that all of the transitions present in the unmilled material are seen in the thermogram of the milled vaccine, thus, no change is observed in the processed vaccine. Thermogravimetric analysis quantifies weight change in a sample as a function of temperature. Weight losses detected may represent loss of moisture or solvent, volatilization, or evolution of gaseous by-products. The TGA of unmilled and milled measles vaccine shows a weight loss of 4.3 and 3.1%, respectively (Fig. 4). Weight loss takes place in more than one step and the shape of the two curves is virtually identical. As with the DSC traces, the complexity of excipients used in the vaccine formulation likely account for multiple transi-
Table 1 Summary of particle size (mm) of jet milled measles vaccine (35 psig) Vaccine
D(v, 0.1)
D(v, 0.5)
D(v, 0.9)
Attenuvax (Merck)
1.4
3.4
6.9
EZ Vaccine (Birmex)
1.2 1.1 1.3 1.3 1.3
3.3 3.2 4.3 4.3 4.2
9.2 8.2 10.5 13.5 12.8
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Fig. 2. Particle size distribution of milled measles vaccine aerosol.
Fig. 3. Comparison of physical properties of unmilled and milled measles vaccine by differential scanning calorimetry (curves are offset).
tions (peak maxima from 50 to 175°C) observed in the corresponding derivative TGA trace. No changes occur after :220°C for either DSC or TGA, indicating that denaturation and degradation is complete at this temperature. Moisture adsorption isotherms for unmilled and milled vaccine at 25°C are compared in Fig. 5. The isotherms are superimposable for relative humidity (RH) values of 5–60%. At high relative humidities, adsorption is so great that the samples fail to reach
equilibrium at each step, thus causing the data to be less comparable in the upper range. Overall, this particular vaccine formulation adsorbs large amounts of moisture. By 90% RH, unmilled vaccine picks up a total moisture content of : 70% and milled vaccine takes up :57%. These results call attention to the extremely hygroscopic nature of the excipients present in the formulation, a characteristic that is expected since the vaccine, as currently provided by manufacturers, is intended for reconstitution. Desorption isotherms
C. LiCalsi et al. / Vaccine 19 (2001) 2629–2636
show no significant hysteresis for either the unmilled or milled material (data not shown) and are also superimposable below 60% RH. The samples do not lose all adsorbed moisture upon re-equilibration at 5% RH, indicating the possible formation of a hydrate in at least one of the filler components.
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3.2. Process control and potency retention Fig. 6 shows the effect of milling pressure on particle size reduction of measles vaccine. The approximately linear relationship between the variables, when plotted on a log normal scale, indicates a degree of predictabil-
Fig. 4. Thermogravimetric analysis of milled and unmilled measles vaccine.
Fig. 5. Water vapor sorption measurement of milled and unmilled measles vaccine.
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Fig. 6. Volume median diameter for measles vaccine milled under different milling pressures.
ity that is a useful and powerful tool for process optimization. Potency of vaccine milled at different pressures was analyzed by the viral plaque assay. Table 2 shows that micronization of live, attenuated measles vaccine by jet milling generates particle sizes appropriate for pulmonary delivery while maintaining viral potency at levels up to as high as 89%, depending on the milling conditions. While significant viral potency is retained using milling pressures of 18 and 35 psig, a loss in activity is observed when vaccine is milled at 65 psig. Since the vaccine is heat sensitive, this result may be due to increased heat generated at the higher pressure. This effect could potentially be minimized by cooling the mill collection vessel. In the case of measles vaccine, however, milling pressures of 18 – 35 psig are sufficient to produce particles of respirable size. In six different experiments, EZ measles vaccine from Birmex micronized at a milling pressure of 35 psig yielded potency retention values ranging from 49 to 89% with an average value of 67%. Similar experiments using Moraten Berna (Swiss Serum and Vaccine Institute, Bern) (n = 4) and Attenuvax (Merck & Co.) (n = 2) yielded values in a range of 31–45%.
dose of a blend is measured using the Andersen Cascade Impactor (ACI) technique. The ACI consists of a stack of stainless steel plates set on stages with systematically varied configurations of holes. Aerosolized particles introduced into the ACI predictably settle on specific plates according to aerodynamic particle size. Thus, larger particles impact on upper plates while smaller particles travel further and deposit on plates further down in the stack. In the case of measles vaccine, the plates are recovered with tissue culture medium and tested in the plaque assay. Despite the fact that significant deposition was repeatedly observed on Plates 2–7, (particle sizes 5 5.8 mm,) only minimal viral activity was reported by the plaque assay. In addition, blend samples assayed directly (i.e. without introduction to the ACI) also showed values lower than expected based on the potency of the milled vaccine and the blend concentration. Two possible explanations for these results include interference of the lactose with some aspect of the plaque assay, perhaps Vero cell adhesion or growth, or chemical interaction between the lactose and viral particles. Both are currently being investigated. In the absence of ACI data, a test to
3.3. Blending and aerosol performance testing
Table 2 Retention of viral potency for milled measles vaccine
In the next step of powder formulation development, micronized particles are blended with an inert carrier, typically lactose. The lactose particles are relatively large (: 100 mm) and serve to improve handling and aerosol dispersion of smaller vaccine particles. Measles vaccine milled at 35 psig was subsequently blended with lactose to a final concentration of 50%. Fig. 7 shows an image of a lactose particle covered with micronized vaccine particles in the 50% blend. Typically, respirable
Milling conditions (psig)
% Potency retained vs. unmilled control
Volume median diameter (VMD) (mm)
18 35 35 (cool mill collection vessel) 65
75 81 89
6.4 3.2 4.3
8
2.2
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Fig. 7. Scanning electron micrograph of a 50% blend of milled measles vaccine and lactose. Shown is a single lactose particle covered with micronized vaccine particles. 500 × magnification; 50 mm size marker.
Fig. 8. Particle size distribution of a 50% measles vaccine:lactose blend.
determine whether the respirable vaccine particles in the blend are effectively aerosolized was performed using laser diffraction. Two peaks are shown in the density distribution curve (Fig. 8). These primarily represent
milled vaccine (left) and lactose (right), however, the presence of lactose fines and some mid-sized lactose particles does contribute to a small degree to the volume measurements across the entire range. The peak
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value of the left curve is approximately 5.1 mm. This value is consistent with the 4.3 mm D(v, 0.5) measured for milled vaccine alone (Fig. 2) and suggests that a high percentage of the micronized particles are effectively dispersed from the lactose carrier.
4. Discussion Measles is a particularly useful model for studying the potential of powder aerosol immunization, specifically because it is a live viral vaccine and therefore poses significant challenges at the stage of size reduction. While other methods of size reduction exist, jet milling was chosen in this study because it generates the smallest particles while maintaining the lowest heat rise. The discovery that a large (:150 – 200 nm), lipid-enveloped virus with functionally important transmembrane surface proteins can withstand milling implies that other vaccines, more robust than the paramyxoviruses, should be capable of being processed similarly. This observation suggests that particle size reduction by jet milling is likely to be a suitable approach for other vaccines. The data in this study demonstrate that a standard dose of measles vaccine is easily achievable by powder aerosol delivery, even without thorough formulation optimization. Measles vaccine is available from manufacturers in titers of 104.5 – 105.6 at 30 – 50 mg dose weights. Up to 89% viral potency retention following micronization is achievable. However, with even a conservative assumption of 50% percent potency retention and 25% aerosol efficiency, delivery of 103 TCID50 in fill weights of 5–25 mg requires blend concentrations between 3 and 96%. These numbers offer a wide range of working values for pulmonary delivery of an aerosol. The 50% measles vaccine:lactose blend was shown to be readily dispersible. A 25 mg fill weight is also conservative since precedent exists for delivering up to 50 mg of powder aerosol to humans [5] and this mass has been shown to be well-tolerated. One important consideration in this feasibility study is the fact that the vaccines used were formulated by the manufacturers specifically with a view towards optimizing properties that aid in reconstitution. The vaccine formulations tested are highly hygroscopic. Thus, while feasibility has been demonstrated with currently available measles vaccines, reformulation of the vaccine with less hygroscopic excipients should further improve results, perhaps dramatically. A less hygroscopic formulation may be more stable and more dispersible, thus requiring a smaller mass to achieve the same dose or eliminating the need to blend with lactose carrier particles. The feasibility of a dry powder aerosol approach to immunization extends beyond formulation challenges alone and depends also on the development of appropri-
ate delivery technology. The technology must be durable, portable, easy-to-use, and highly cost-effective. A single-use, disposable device avoids any potential cross contamination from patient to patient. In addition, a foil powder storage system may be used to protect moisture-sensitive vaccines. Systems with these attributes are currently under development. Finally, the fact that the aerosol route holds promise for efforts towards measles eradication was further demonstrated in a recent study [3] which showed that a nebulized liquid aerosol of measles vaccine evokes better humoral immunity than the same vaccine administered by injection. A shift from liquid to powder formulation coupled with appropriate DPI technology will potentially strengthen the advantages already realized by the aerosol approach by providing more stable formulations, reproducible unit dosing, and significantly less release of vaccine into the environment. As a next step, in vivo demonstration of the safety and efficacy of the measles vaccine dry powder aerosol is required. Towards that end, the powder formulation will be tested for safety and efficacy in a monkey model this year.
Acknowledgements This project is supported in part by grant No. V21/ 181/113 from the WHO, Geneva, Switzerland. We thank Paul A. Rota and Bruce Newton at the Centers for Disease Control and Prevention, Atlanta, GA, USA for providing analytical support and technical input, John Bennett of Emory University for his expert advice throughout this study, Jorge Fernandez de Castro for generously donating Edmonston-Zagreb measles vaccine produced by the Instituto Nacional de Virologia, BIRMEX (Mexico), and R. Gluck of Swiss Serum and Vaccine Institute, Bern for generous donation of Moraten Berna vaccine. The authors also thank William Colby for his excellent technical assistance in aerosol testing.
References [1] LiCalsi C, Christensen T, Bennett JV, Phillips E, Witham C. Dry powder inhalation as a potential delivery method for vaccines. Vaccine 1999;17:1796– 803. [2] Cutts FT, Clements CJ, Bennett JV. Alternative routes of measles immunization: a review. Biologicals 1997;25:323– 38. [3] Dilraj A, Cutts FT, Fernandez de Castro J, Wheeler JG, Brown D, Roth C, Coovadia HM, Bennett JV. The Lancet 2000;355:798– 803. [4] Phillips E, Allsopp E, Christensen T, Fitzgerald M, Zhao L. Size reduction of peptides and proteins by jet-milling. In: Dalby RN, Byron PR, Farr SJ, editors. Respiratory Drug Delivery, vol. VI. Buffalo Grove: Interpharm, 1996:197– 208. [5] Patient package insert, Relenza product information 4116429, GlaxoWellcome, Research Triangle Park, NC 1999.
A powder formulation of measles vaccine for aerosol delivery Cynthia LiCalsi *, Michael J. Maniaci, Troy Christensen, Elaine Phillips, Gary H. Ward, Clyde Witham Dura Pharmaceuticals, 7475 Lusk Bl6d., San Diego, CA 92121, USA
Abstract Both the mortality rate for measles and the risks associated with injection continue to be high in the developing world. In response to the need for safe, cost-effective vaccine delivery technologies, a powder formulation of measles vaccine has been developed to test the feasibility of administering measles vaccine as an aerosol. The first challenge in aerosol formulation development is to produce fine particles without damaging the activity of the virus or inducing physical changes. In this study, live attenuated measles vaccine is micronized by jet milling to generate particle sizes appropriate for pulmonary delivery (1–5 mm). Milling does not induce detectable physical changes and significant viral potency is maintained. Potency retention of milled vaccine ranges from 31 to 89%, demonstrating that the standard dose of vaccine can easily be achieved. Following size reduction, particles are blended with an inert carrier to improve handling and aerosol dispersion. The measles vaccine formulation is dispersible, as shown by laser light particle size analysis of vaccine aerosols. Thus, evaluation of both the potency retention and the aerosol characteristics of the current formulation clearly demonstrates the feasibility of delivering measles vaccine as a powder aerosol for immunization. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Measles vaccine; Aerosols; Dry powder inhalation
1. Introduction Pulmonary delivery using powder vaccine aerosols is an approach to immunization that offers advantages over injection in terms of both delivery technology and vaccine formulation [1]. The technology advantages include increased safety and ease of administration. The formulation advantages when using dry powders are the potential reduction of refrigeration requirements and increased stability during transport and administration. Additionally, there is potential for enhanced biological efficacy since pulmonary delivery may produce mucosal immunity superior to that which is produced by parenteral administration of vaccines. Each of these factors is pertinent to the battle against measles in developing countries, where the disease continues to be a leading cause of mortality among children. First, in those countries where mass
* Corresponding author. Tel.: +1-858-3207625; fax: + 1-8585584120. E-mail address: [email protected] (C. LiCalsi).
immunization campaigns are most needed, the potential contamination risks associated with needles are also the highest. Second, while cold-chain maintenance and stability are concerns associated with vaccine delivery in general, these issues are particularly significant in the case of measles vaccine. Although the lyophilized vaccine is relatively thermostable, measles vaccine loses potency rapidly at both 25 and 37°C when reconstituted. Thus, vaccine reconstituted from multi-dose vials must be kept cold during administration. Finally, measles is highly contagious and, therefore, the potential to induce both mucosal and systemic immunity via pulmonary delivery may contribute significantly to measles elimination/eradication. To date, many studies have shown both the safety and efficacy of measles vaccine delivered as a liquid aerosol from a nebulizer [2,3]. Elimination of the reconstitution requirement would significantly strengthen the aerosol approach. This paper describes the development of a dry powder aerosol vaccine formulation and establishes the feasibility of delivering a standard dose of measles vaccine as a powder aerosol from a dry powder inhaler.
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 5 0 3 - X
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2. Materials and methods
analysis was conducted using 5–8 mg of material in a crimped pan.
2.1. Milling and blending 2.5. Thermogra6imetric analysis Lyophilized measles vaccine (Edmonston-Zagreb BIRMEX, Mexico; Moraten Berna, Swiss Serum and Vaccine Inst., Bern, Switzerland; Attenuvax, Merck & Co., West Point, PA) was micronized using a one inch jet mill (Micron-master model c01-506, Jet Pulverizer Co., Palmyra, NJ) with compressed nitrogen as the gas stream. Injection nozzle pressure was 100 psig for milling pressures of 18 or 35 psig and 180 psig for a milling pressure of 65 psig. Vaccine was fed manually into the mill. Milling was carried out under low relative humidity conditions (B4% RH) in a Nitrogen-purged glove box. Blends of micronized measles vaccine and Pharmatose lactose (100 M, DMV International, Veghel, The Netherlands) were prepared in glass vials using a 3-D tumble-in-container blender (Turbula® T-2C, Glen Mills Inc., Clifton, NJ).
2.2. Scanning electron microscopy (SEM) SEM samples were prepared using a standard gold sputter technique and then imaged on an ESEM (XL30 ESEM TMP, Philips Electron Optics, Hillsboro, OR) at 10 – 15 kV with average working distances of 8 – 15 mm. Neat and blended samples were imaged at several magnification levels ranging from 50× to 8000× .
2.3. Particle size measurement Particle size was measured using a laser diffraction particle size analyzer (Malvern Mastersizer-S, San Bernardino, CA) with dynamic, small volume sample cell. A reverse-Fourier lens (detection range: 0.1 –3000 mm) was used to size material in liquid suspension of polydimethylsiloxane. A Sympatec HELOS KF laser diffraction particle size analyzer (Sympatec Inc., Princeton NJ) utilizing the RODOS dry powder disperser and Vibri vibratory feeder was used to measure the size distribution of the free powder. The RODOS module was operated with a primary air dispersion pressure of 4 bar and a depression value of 103 mbar. The Vibri feeder was operated at 75%. An R2 focusing lens (0.25 –87.5 mm) was used for the micronized material and an R5 lens (4.5 – 875 mm) for the 50% vaccine blend.
2.4. Differential scanning calorimetry (DSC) Samples were first dried at 30°C in a thermogravimetric analysis (TGA) (Hi-Resolution TGA 2950, TA Instruments, New Castle, DE) for 120 min, then scanned from −10 to 275°C at a scan rate of 10°C/min with a TA Instruments dual cell model 2920 DSC. Each
Approximately 5–8 mg samples of material in an open pan were scanned from 25 to 250 or 300°C at a scan rate of 10°C/min on a Thermal Analysis model AutoTGA 2950.
2.6. Moisture sorption analysis Step isotherms of measles vaccine at 25°C were obtained using a nitrogen flow-based, automated, microbalance system (Model MB-300W, VTI Corporation). Samples were dried under a stream of dry nitrogen prior to exposure to various RH conditions. Approximately 10–15 mg of vaccine were scanned between 5 and 95% RH in increments of 5% RH. Each increment was programmed to satisfy the following equilibrium criteria: sample exposure to the next humidity level occurred when the change in sample weight was 5 0.005% of the beginning sample weight in 5 min, for three consecutive measurements, or after 120 min, whichever occurred first.
3. Results
3.1. Particle size measurement and solid state characterization of measles 6accine Live, attenuated measles vaccine was micronized by jet milling at a milling pressure of 35 psig. This method uses high pressure, high velocity jets of gas to cause particle to particle impaction and thus particle attrition [4]. A qualitative evaluation of the results was made using scanning electron microscopy. Fig. 1 shows micrographs of measles vaccine taken before and after size reduction. Unmilled vaccine appears to consist of many large (up to :400 mm) and small (down to : 10 mm) irregularly shaped plates. In contrast, milled material shows particles of more uniform shape with sizes primarily smaller than 10 mm. To quantify the effectiveness of the milling process, particle size distribution of vaccine powder was measured in liquid suspensions using a laser diffraction instrument (Table 1). Both unmilled and milled vaccine have broad unimodal particle size distributions as measured by this technique. Particle size is typically reported as volume diameter, thus, the parameters D(v, 0.1), D(v, 0.5), and D(v, 0.9) refer to particle diameters below which 10, 50, or 90% of the particle volume is contained. Consistent with the microscopy results, the D(v,0.5) determined for milled vaccine ranges from 3.2 to 4.2 mm, values significantly smaller than the D(v,0.5) of 21.6 mm measured for the
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Fig. 1. Scanning electron micrographs of measles vaccine before (left) and after (right) micronization by jet milling. 369 × magnification; 50 mm size marker.
unmilled material. Thus, 56 – 71% of the milled vaccine has particle sizes less than 5 mm, an appropriate size for pulmonary delivery. For delivery of a live viral vaccine, it is perhaps more pertinent to consider the number of appropriately sized virus-containing particles. When the volume measurement is translated to reflect particle numbers, approximately 99% of the particles in the milled vaccine have sizes less than 5 mm. Because the intended use of the formulation is as a dry powder aerosol, it is essential that the powder be dispersible. Therefore, particle size distribution of aerosolized powder was measured using a Sympatec laser diffraction instrument. The Sympatec uses a gas shear stream to overcome adhesive forces between particles. Sample introduction takes place with the aid of a vibratory conveyor feeder. The size distribution of an aerosol of milled measles vaccine is shown in Fig. 2. The cumulative distribution curve shows a D(v,0.5) of 4.390.3, which agrees well with the particle size measurements made using liquid suspensions. The density distribution curve is not unimodal, possibly indicating that the powder is not completely deagglomerated by the dispersion pressure used. However, under the conditions used, the results show that 57% of the aerosolized sample volume consists of particle sizes considered appropriate for pulmonary delivery (5 5 mm). While particle sizes of 5 5 mm target the deep lung, larger particles (up to 10 mm) deposit preferentially in the tracheobronchial region. No assessment has yet been made regarding optimal size distribution for measles immunization. While the success of micronization is determined in part by the resultant particle size distribution, it is also important in each case to confirm that the physical characteristics of the material are maintained. Measles vaccine is a mixture of live attenuated virus with various excipients that presumably function to stabilize the virus and/or aid in cake formation during lyophiliza-
tion. The physical characteristics of the unmilled and milled vaccines were compared using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and moisture sorption analysis. Differential scanning calorimetry thermograms of measles vaccine reveal several energetic transitions (Fig. 3). The various endotherms and exotherms represent glass transitions, melting, and recrystallization events of crystalline and amorphous materials formed upon heating. Due to the complexity and variety of excipients in the vaccine formulation, individual peaks cannot be assigned definitively to a compound or event. However, most notable is the observation that all of the transitions present in the unmilled material are seen in the thermogram of the milled vaccine, thus, no change is observed in the processed vaccine. Thermogravimetric analysis quantifies weight change in a sample as a function of temperature. Weight losses detected may represent loss of moisture or solvent, volatilization, or evolution of gaseous by-products. The TGA of unmilled and milled measles vaccine shows a weight loss of 4.3 and 3.1%, respectively (Fig. 4). Weight loss takes place in more than one step and the shape of the two curves is virtually identical. As with the DSC traces, the complexity of excipients used in the vaccine formulation likely account for multiple transi-
Table 1 Summary of particle size (mm) of jet milled measles vaccine (35 psig) Vaccine
D(v, 0.1)
D(v, 0.5)
D(v, 0.9)
Attenuvax (Merck)
1.4
3.4
6.9
EZ Vaccine (Birmex)
1.2 1.1 1.3 1.3 1.3
3.3 3.2 4.3 4.3 4.2
9.2 8.2 10.5 13.5 12.8
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Fig. 2. Particle size distribution of milled measles vaccine aerosol.
Fig. 3. Comparison of physical properties of unmilled and milled measles vaccine by differential scanning calorimetry (curves are offset).
tions (peak maxima from 50 to 175°C) observed in the corresponding derivative TGA trace. No changes occur after :220°C for either DSC or TGA, indicating that denaturation and degradation is complete at this temperature. Moisture adsorption isotherms for unmilled and milled vaccine at 25°C are compared in Fig. 5. The isotherms are superimposable for relative humidity (RH) values of 5–60%. At high relative humidities, adsorption is so great that the samples fail to reach
equilibrium at each step, thus causing the data to be less comparable in the upper range. Overall, this particular vaccine formulation adsorbs large amounts of moisture. By 90% RH, unmilled vaccine picks up a total moisture content of : 70% and milled vaccine takes up :57%. These results call attention to the extremely hygroscopic nature of the excipients present in the formulation, a characteristic that is expected since the vaccine, as currently provided by manufacturers, is intended for reconstitution. Desorption isotherms
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show no significant hysteresis for either the unmilled or milled material (data not shown) and are also superimposable below 60% RH. The samples do not lose all adsorbed moisture upon re-equilibration at 5% RH, indicating the possible formation of a hydrate in at least one of the filler components.
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3.2. Process control and potency retention Fig. 6 shows the effect of milling pressure on particle size reduction of measles vaccine. The approximately linear relationship between the variables, when plotted on a log normal scale, indicates a degree of predictabil-
Fig. 4. Thermogravimetric analysis of milled and unmilled measles vaccine.
Fig. 5. Water vapor sorption measurement of milled and unmilled measles vaccine.
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Fig. 6. Volume median diameter for measles vaccine milled under different milling pressures.
ity that is a useful and powerful tool for process optimization. Potency of vaccine milled at different pressures was analyzed by the viral plaque assay. Table 2 shows that micronization of live, attenuated measles vaccine by jet milling generates particle sizes appropriate for pulmonary delivery while maintaining viral potency at levels up to as high as 89%, depending on the milling conditions. While significant viral potency is retained using milling pressures of 18 and 35 psig, a loss in activity is observed when vaccine is milled at 65 psig. Since the vaccine is heat sensitive, this result may be due to increased heat generated at the higher pressure. This effect could potentially be minimized by cooling the mill collection vessel. In the case of measles vaccine, however, milling pressures of 18 – 35 psig are sufficient to produce particles of respirable size. In six different experiments, EZ measles vaccine from Birmex micronized at a milling pressure of 35 psig yielded potency retention values ranging from 49 to 89% with an average value of 67%. Similar experiments using Moraten Berna (Swiss Serum and Vaccine Institute, Bern) (n = 4) and Attenuvax (Merck & Co.) (n = 2) yielded values in a range of 31–45%.
dose of a blend is measured using the Andersen Cascade Impactor (ACI) technique. The ACI consists of a stack of stainless steel plates set on stages with systematically varied configurations of holes. Aerosolized particles introduced into the ACI predictably settle on specific plates according to aerodynamic particle size. Thus, larger particles impact on upper plates while smaller particles travel further and deposit on plates further down in the stack. In the case of measles vaccine, the plates are recovered with tissue culture medium and tested in the plaque assay. Despite the fact that significant deposition was repeatedly observed on Plates 2–7, (particle sizes 5 5.8 mm,) only minimal viral activity was reported by the plaque assay. In addition, blend samples assayed directly (i.e. without introduction to the ACI) also showed values lower than expected based on the potency of the milled vaccine and the blend concentration. Two possible explanations for these results include interference of the lactose with some aspect of the plaque assay, perhaps Vero cell adhesion or growth, or chemical interaction between the lactose and viral particles. Both are currently being investigated. In the absence of ACI data, a test to
3.3. Blending and aerosol performance testing
Table 2 Retention of viral potency for milled measles vaccine
In the next step of powder formulation development, micronized particles are blended with an inert carrier, typically lactose. The lactose particles are relatively large (: 100 mm) and serve to improve handling and aerosol dispersion of smaller vaccine particles. Measles vaccine milled at 35 psig was subsequently blended with lactose to a final concentration of 50%. Fig. 7 shows an image of a lactose particle covered with micronized vaccine particles in the 50% blend. Typically, respirable
Milling conditions (psig)
% Potency retained vs. unmilled control
Volume median diameter (VMD) (mm)
18 35 35 (cool mill collection vessel) 65
75 81 89
6.4 3.2 4.3
8
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Fig. 7. Scanning electron micrograph of a 50% blend of milled measles vaccine and lactose. Shown is a single lactose particle covered with micronized vaccine particles. 500 × magnification; 50 mm size marker.
Fig. 8. Particle size distribution of a 50% measles vaccine:lactose blend.
determine whether the respirable vaccine particles in the blend are effectively aerosolized was performed using laser diffraction. Two peaks are shown in the density distribution curve (Fig. 8). These primarily represent
milled vaccine (left) and lactose (right), however, the presence of lactose fines and some mid-sized lactose particles does contribute to a small degree to the volume measurements across the entire range. The peak
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value of the left curve is approximately 5.1 mm. This value is consistent with the 4.3 mm D(v, 0.5) measured for milled vaccine alone (Fig. 2) and suggests that a high percentage of the micronized particles are effectively dispersed from the lactose carrier.
4. Discussion Measles is a particularly useful model for studying the potential of powder aerosol immunization, specifically because it is a live viral vaccine and therefore poses significant challenges at the stage of size reduction. While other methods of size reduction exist, jet milling was chosen in this study because it generates the smallest particles while maintaining the lowest heat rise. The discovery that a large (:150 – 200 nm), lipid-enveloped virus with functionally important transmembrane surface proteins can withstand milling implies that other vaccines, more robust than the paramyxoviruses, should be capable of being processed similarly. This observation suggests that particle size reduction by jet milling is likely to be a suitable approach for other vaccines. The data in this study demonstrate that a standard dose of measles vaccine is easily achievable by powder aerosol delivery, even without thorough formulation optimization. Measles vaccine is available from manufacturers in titers of 104.5 – 105.6 at 30 – 50 mg dose weights. Up to 89% viral potency retention following micronization is achievable. However, with even a conservative assumption of 50% percent potency retention and 25% aerosol efficiency, delivery of 103 TCID50 in fill weights of 5–25 mg requires blend concentrations between 3 and 96%. These numbers offer a wide range of working values for pulmonary delivery of an aerosol. The 50% measles vaccine:lactose blend was shown to be readily dispersible. A 25 mg fill weight is also conservative since precedent exists for delivering up to 50 mg of powder aerosol to humans [5] and this mass has been shown to be well-tolerated. One important consideration in this feasibility study is the fact that the vaccines used were formulated by the manufacturers specifically with a view towards optimizing properties that aid in reconstitution. The vaccine formulations tested are highly hygroscopic. Thus, while feasibility has been demonstrated with currently available measles vaccines, reformulation of the vaccine with less hygroscopic excipients should further improve results, perhaps dramatically. A less hygroscopic formulation may be more stable and more dispersible, thus requiring a smaller mass to achieve the same dose or eliminating the need to blend with lactose carrier particles. The feasibility of a dry powder aerosol approach to immunization extends beyond formulation challenges alone and depends also on the development of appropri-
ate delivery technology. The technology must be durable, portable, easy-to-use, and highly cost-effective. A single-use, disposable device avoids any potential cross contamination from patient to patient. In addition, a foil powder storage system may be used to protect moisture-sensitive vaccines. Systems with these attributes are currently under development. Finally, the fact that the aerosol route holds promise for efforts towards measles eradication was further demonstrated in a recent study [3] which showed that a nebulized liquid aerosol of measles vaccine evokes better humoral immunity than the same vaccine administered by injection. A shift from liquid to powder formulation coupled with appropriate DPI technology will potentially strengthen the advantages already realized by the aerosol approach by providing more stable formulations, reproducible unit dosing, and significantly less release of vaccine into the environment. As a next step, in vivo demonstration of the safety and efficacy of the measles vaccine dry powder aerosol is required. Towards that end, the powder formulation will be tested for safety and efficacy in a monkey model this year.
Acknowledgements This project is supported in part by grant No. V21/ 181/113 from the WHO, Geneva, Switzerland. We thank Paul A. Rota and Bruce Newton at the Centers for Disease Control and Prevention, Atlanta, GA, USA for providing analytical support and technical input, John Bennett of Emory University for his expert advice throughout this study, Jorge Fernandez de Castro for generously donating Edmonston-Zagreb measles vaccine produced by the Instituto Nacional de Virologia, BIRMEX (Mexico), and R. Gluck of Swiss Serum and Vaccine Institute, Bern for generous donation of Moraten Berna vaccine. The authors also thank William Colby for his excellent technical assistance in aerosol testing.
References [1] LiCalsi C, Christensen T, Bennett JV, Phillips E, Witham C. Dry powder inhalation as a potential delivery method for vaccines. Vaccine 1999;17:1796– 803. [2] Cutts FT, Clements CJ, Bennett JV. Alternative routes of measles immunization: a review. Biologicals 1997;25:323– 38. [3] Dilraj A, Cutts FT, Fernandez de Castro J, Wheeler JG, Brown D, Roth C, Coovadia HM, Bennett JV. The Lancet 2000;355:798– 803. [4] Phillips E, Allsopp E, Christensen T, Fitzgerald M, Zhao L. Size reduction of peptides and proteins by jet-milling. In: Dalby RN, Byron PR, Farr SJ, editors. Respiratory Drug Delivery, vol. VI. Buffalo Grove: Interpharm, 1996:197– 208. [5] Patient package insert, Relenza product information 4116429, GlaxoWellcome, Research Triangle Park, NC 1999.