Development of a thermostable microneedle patch for polio vaccination
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The aim of this study was to develop a dissolving microneedle (MN) patch for administration of inactivated polio vaccine (IPV) with improved thermal stability when compared with conventional liquid IPV. Excipient screening showed that a combination of maltodextrin and D-sorbitol in histidine buffer best preserved IPV activity during MN patch fabrication and storage. As determined by D-antigen ELISA, all three IPV serotypes maintained > 70% activity after 2 months and > 50% activity after 1-year storage at 5 °C or 25 °C with desiccant. Storage at 40 °C yielded > 40% activity after 2 months and > 20% activity after 1 year. In contrast, commercial liquid IPV types 1 and 2 lost essentially all activity within 1 month at 40 °C and IPV type 3 had < 40% activity. Residual moisture content in MN patches measured by thermogravimetric analysis was 1.2–6.5%, depending on storage conditions. Glass transition temperature measured by differential scanning calorimetry, structural changes measured by X-ray diffraction, and molecular interactions measured by Fourier transform infrared spectroscopy showed changes in MN matrix properties, but they did not correlate with IPV activity changes during storage. We conclude that appropriately formulated MN patches can exhibit thermostability that could enable distribution of IPV with less reliance on cold chain storage.
KeywordsDissolving microneedle patch Inactivated polio vaccine Thermostability Thermogravimetric analysis Differential scanning calorimetry X-ray diffraction
Poliomyelitis is a highly infectious disease which mainly affects children. The disease can be caused by any of the three serotypes of inactivated polio vaccine (IPV) (type 1, type 2, or type 3) and can be prevented by immunization . Due in large part to the Global Polio Eradication Initiative (GPEI), the number of polio cases has dropped from 350,000 in 1988 to only 22 reported cases in 2017. IPV type 1 is the only serotype that is currently endemic to three countries (Afghanistan, Nigeria, and Pakistan), and wild-type IPV type 2 has been eradicated . However, WHO predicts that failure to eradicate this disease can reverse these advances and cause as many as 200,000 new cases every year in 10 years .
The progress made so far is predominantly due to mass vaccination using the oral polio vaccine (OPV) which is a live-attenuated virus given by mouth . OPV offers advantages such as low cost, ease of oral administration, no sharps, and generation of mucosal immunity. However, a major concern with OPV is the possibility of genetic reversion to a virulent form that can cause vaccine-associated paralytic polio (VAPP)  and can be transmitted to others. A safer alternative is IPV, which does not carry the risk of paralysis or transmission, but is administered as an intradermal (ID)  or intramuscular (IM) injection .
To eliminate the risks of VAPP, OPV was replaced by IPV in USA and UK in 2000 and 2004, respectively , and plans to eventually discontinue use of OPV worldwide are underway . However, the biggest drawbacks of switching to IPV are the need for trained medical professionals to administer IM or ID injections, generation of sharps waste, need for cold chain storage, high cost of the vaccine, poor mucosal immune responses, and need for multiple doses for a protective immune response [10, 11, 12].
In this study, we propose the use of a dissolving microneedle (MN) patch for IPV vaccination. When a MN patch is applied to the skin, micron-scale, solid needles made of safe, water-soluble excipients that encapsulate vaccine penetrate the outer barrier layer of the skin, where they dissolve and release their vaccine payload [13, 14, 15]. MN patches can be self-administered painlessly [16, 17, 18], generating no sharps waste, and have been used previously for various vaccinations in research [14, 19], including IPV , and a recent clinical trials on influenza . Encapsulation of vaccines in appropriately formulated MN patches has been shown to increase vaccine thermostability [22, 23, 24]. It was therefore our objective to formulate a MN patch for IPV vaccination that is thermostable, has reduced reliance on cold chain, is easy to administer and eliminates sharps waste. This approach could facilitate mass vaccination campaigns to achieve polio eradication, especially in developing countries .
Materials and methods
Concentration of inactivated polio vaccine
Monovalent, bulk IPV (Mahoney strain of type 1, Middle East Forces (MEF) of type 2 and Saukett of type 3) was kindly provided by Bilthoven Biologicals (Bilthoven, Netherlands). The initial antigen concentrations were 1675, 963, and 950 D-antigen units (DU)/ml for IPV types 1, 2, and 3, respectively. The bulk IPV was concentrated using Amicon Ultra centrifuge spin filters with 100 kDa MW cutoff (EMD Millipore, Billerica, MA) either ~10-fold by volume to a final antigen concentration of 1.7 × 104, 9.6 × 103, and 9.5 × 103 DU/ml for IPV types 1, 2, and 3, respectively, or ~47-fold by volume to a final antigen concentration of 7.9 × 104, 4.8 × 104, and 4.5 × 104 DU/ml for IPV types 1, 2, and 3, respectively. All D-antigen values were determined by ELISA, as described below.
Vaccine stability screening
To increase throughput of formulation screening, candidate vaccine-excipient formulations were dried on polydimethylsiloxane (PDMS) chips, which are representative of the surface of a MN patch mold. PDMS chips were prepared by curing a thin film of Sylgard 184 (Dow Corning, Midland, MI) and preparing 6-mm diameter circular discs using a hammer punch. IPV was concentrated 10-fold by volume and mixed with selected excipients. A 10 μl drop of formulated vaccine solution was placed on each chip and allowed to dry at 5 °C in a desiccator filled with desiccant (Drierite, W A Hammond Drierite, Xenia, OH) overnight. After reconstituting the dried IPV in 999 μl media M199, the D-antigen content of the IPV was determined by ELISA, as described below. All excipients were purchased from Sigma-Aldrich (St. Louis, MO).
Microneedle patch fabrication
PDMS molds consisting of a 10 × 10 array of cavities that form the MNs were fabricated as described previously . The antigen casting solution was prepared by mixing 47-fold concentrated vaccine with a solution consisting of a 10% w/v maltodextrin and D-sorbitol in 0.35 mM histidine buffer at pH 6.3. Fifty microliters of the antigen solution was cast onto a MN mold, which was then centrifuged for 1 h at 5 °C to fill the mold cavities with solution and partially dried in the mold, following a procedure previously described . This resulted in MN patch loading of 71 ± 1.8 DU, 14 ± 0.6 DU, and 47 ± 3.2 DU of IPV types 1, 2, and 3, respectively, per patch.
The polymer matrix solution used to form the MN patch backing consisted of 35% w/v fish gelatin and 10% w/v D-sorbitol in 0.15 mM histidine buffer. The solution was mixed for 1 h at 35 °C until completely dissolved. Two hundred microliters of polymer matrix solution was cast onto a mold pre-filled with partially dried antigen solution. The mold was then placed under vacuum for 2 h at room temperature (20–25 °C) followed by storing in a desiccator at room temperature for 2 days before demolding. Demolded patches were packaged with desiccant in aluminum pouches to maintain 0% RH (relative humidity) and then stored at a given temperature (i.e., 5, 25, or 40 °C) in stability chambers. For experiments involving storage at ~ 50% RH, patches were stored in a desiccator containing a saturated solution of magnesium nitrate in distilled water .
The composition of MNs (i.e., just the MNs without the patch backing) was determined by preparing placebo patches (i.e., with no IPV, but otherwise fabricated in the same way) and carefully cutting off the MNs. The MNs were reconstituted in water, and the protein content was determined using micro BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), which was interpreted to be roughly equal to the gelatin content, since the only protein in a placebo patch should be gelatin from 2nd cast solution which could migrate into the MN during the fabrication process. In this way, the composition of the MNs was estimated to be fish gelatin, D-sorbitol, and maltodextrin in a mass ratio of 55:38:7. This formulation was used when studying IPV stability on chips that simulate MN composition. The chips were stored at a given temperature (i.e., 5, 25, or 40 °C) for specified times in stability chambers.
To study the effect of lyophilization on IPV stability, demolded MN patches and dried IPV-excipient chips were subjected to lyophilization (VirTis AdVantage 2.0 BenchTop lyophilizer, SP Industries, Warminster, PA). The lyophilization process involved a primary drying at − 45 °C (10 mTorr) for 3 h followed by a secondary drying at 25 °C (10 mTorr) for 24 h. The samples were removed from the lyophilizer and stored in a desiccator until tested.
Microneedle patch characterization
MN patches were imaged using brightfield microscopy (Hirox KH-8700, Tokyo, Japan). To assess MN insertion into skin, patches were pressed by thumb with a force of ~ 20 N onto stretched porcine cadaver skin. The MNs were left in the skin at room temperature for 15 min (without further manipulation of the MN patch), and then removed. The skin was stained for 5 min, with gentian violet (2% solution, Humco, Texarkana, TX), which was then wiped off to show preferentially stained sites of MN penetration into skin . The removed MN patches were imaged to determine extent of MN dissolution.
ELISA assay measurements
The D-antigen content of IPV from chips and MN patches was determined by antigen-capture sandwich ELISA, as previously described . Polio-specific monoclonal antibodies were used for both capture and detection. The capture antibody solutions were prepared by adding type 1 (HYB295-15-02), type 2 (HYB294-06-02), or type 3 (HYB300-05-02) (Thermo Fisher Scientific) specific antibodies to 0.05 M carbonate-bicarbonate buffer, pH 9.6. Type 1 and type 3 antibodies were diluted at 1:1000, and type 2 was diluted at 1:500.
Thermogravimetric analysis (TGA, Q50, TA Instruments, New Castle, DE) was used to determine the residual moisture content of the MN patches. Samples obtained from MN patch (each weighing 15–20 mg) were heated at 10 °C/min from room temperature to 110 °C and then maintained isothermally for 10 min. The residual moisture content (%) was determined by the stable weight loss (%) at 110 °C using TA Instruments Universal Analysis 2000 software.
Differential scanning calorimetry
Differential scanning calorimetry (DSC Q200, TA Instruments) was used to measure the glass transition temperature (Tg) of the samples. Samples obtained from MN chips (each weighing 10–15 mg) were placed in hermetically sealed aluminum pans (TA Instruments, cooled from room temperature to 0 °C at a rate of 10 °C/min and subsequently heated to 200 °C at a heating rate of 10 °C/min). The sample chamber was purged with dry nitrogen at 50 ml/min. Empty pans were used as a reference control. The onset temperature of the discontinuities in the heat flow versus temperature curve was taken as the glass transition point, which was determined using TA Instruments Universal Analysis 2000 software.
X-ray diffraction (XRD, X’pert Pro Multi-Purpose Diffractometer, PANalytical, Westborough, MA) analysis was used to study structural changes of MN chips stored at specified temperatures over time. The measurements were made in the θ-2θ mode on the MN chip samples using a bracket sample holder with a Cu Kα radiation source (Cu Kα = 1.54059 Å) at room temperature. Data were collected between 2θ values of 5° and 50° using a step size of 0.013° and an acquisition time of 30 s per step. Samples were measured at 45 kV and 40 mA. X-ray diffraction patterns were analyzed using Jade 8 software (MDI Materials Data, Livermore, CA) and shown after background correction. Diffraction peaks were fitted to a Gaussian profile.
Fourier transform infrared spectroscopy
Fourier transform infrared spectrometry (FTIR, Nicolet iS 50 FT-IR, Thermo Fisher Scientific, Waltham, MA) was used to study structural changes of MN chips stored at specified temperatures over time. The FTIR spectra of the samples were recorded at room temperature from 4000 to 600 cm−1 and shown after background correction.
Statistics and Design of Experiments
The design of experiments model for excipient screening was developed using MiniTab software version 18 (MiniTab, State College, PA). Statistics were calculated using either MiniTab or Excel (Microsoft, Redmond, WA). All listed averages represent the arithmetic mean of the samples. Comparison between three or more samples was performed by one-way ANOVA or the data were fit to a general linear model. Comparisons between individual samples were done using an unpaired t test. Probability (p) values of < 0.05 were considered to be significant.
Results and discussion
Excipient screening for IPV stabilization
To identify formulations that stabilize IPV during MN patch fabrication, we studied excipients including maltodextrin, which was previously shown to stabilize IPV , and D-sorbitol, sucrose, and trehalose, which are widely used to stabilize proteins during drying or lyophilization [31, 32]. Using a design of experiments model [see Table S1 in Supplemental Information (SI)], we studied the effect of individual excipients as well as combinations of up to four excipients, because multiple stabilizers have often been shown to provide better stability than individual excipients .
Trivalent concentrated IPV was formulated with combinations of different excipients in histidine buffer and dried on PDMS chips at 5 °C overnight. The samples were then lyophilized and stored at 25 °C with desiccant for 1 week. Each sample was reconstituted and assayed for D-antigen binding activity by ELISA.
It is notable that all three serotypes of unformulated IPV lost ~ 75–90% activity after air drying and lost ~ 90–100% activity after lyophilization (and storage). In contrast, none of the formulated vaccine samples lost more than 60% activity after drying, lyophilization and storage of IPV type 1, 2, or 3. The optimal formulation was the combination of maltodextrin and D-sorbitol (50:50 mass ratio), which maintained at least 80% activity after drying, lyophilization, and storage.
Buffer and pH screening
Optimization of polio MN patches
Initial formulation studies above were performed by casting onto flat PDMS chips to facilitate higher throughput. We next assessed formulation optimization in the context of MN patches fabrication by a two-cast process.
Optimization of 1st cast formulation
Optimization of 2nd cast formulation
No significance differences in IPV type 1 and type 2 activities were observed between GS78 and GS55 formulations (ANOVA, p > 0.05), but GS78 was better in stabilizing IPV type 3 (ANOVA, p < 0.05). However, MN patches prepared with GS55 formulation were not mechanically strong enough to penetrate the skin. GS88 had adverse effects on all three IPV serotypes (ANOVA, p < 0.05), especially after extended storage at 40 °C.
Hence, a fish gelatin:D-sorbitol ratio of 77:22 (on a dry basis, when cast as 45% w/v solids in solution) was selected as the final 2nd cast formulation because it resulted in the best stabilization of all three IPVs types and produced MN patches with strong microneedle tips.
Effect of drying conditions
Solid MN patches are formed by drying the cast formulations in the MN mold. As the vaccine is removed from an aqueous environment during drying and subsequent storage, it undergoes changes that can decrease its specific antigenicity . Formulation with polyols such as D-sorbitol and polysaccharides such as maltodextrin, as well as drying at controlled temperature and/or by lyophilization can help maintain antigen activity during drying [36, 37].To study the effect of drying temperature and lyophilization, the residual moisture content of MN patches were measured by TGA and the IPV activity was measured by ELISA after air drying, after subsequent lyophilization, after storage at 40 °C (without lyophilization), and after storage at 40 °C (with lyophilization).
Concerning kinetics, residual moisture content of MN patches depended on temperature during initial air drying with desiccant for 2 days, where drying at 40 °C, 25 °C, and 5 °C resulted in significantly different residual moisture contents of 3.6%, 6.0%, and 10.9%, respectively (one-way ANOVA, p < 0.0004) (Fig. 5a). Lyophilization after air drying reduced residual moisture content after air drying at 5 °C (Student’s t test, p < 0.015), but had no significant effect on residual moisture content after air drying at 25 °C or 40 °C (Student’s t test, p > 0.7).
Air drying at 5 °C or 25 °C resulted in similar IPV activities (one-way ANOVA, p > 0.05), whereas drying at 40 °C resulted in lower IPV activity, especially for IPV types 1 and 3 (one-way ANOVA, p < 0.05) (Fig. 5b and Fig. S5 in SI). This is consistent with literature, which reports that heating IPV results in loss of D-antigenicity . After storage at 40 °C for 1 week with desiccant (with or without prior lyophilization), IPV stability was similar when previously air dried at 5 °C or 25 °C, but was lower when previously dried at 40 °C. We therefore selected air drying at 25 °C (since it dries faster than at 5 °C) without lyophilization (since it adds time, cost, and complexity with no apparent advantage in IPV stability) as the preferred MN patch drying method.
Polio MN patch characterization
Long- term stability of polio MN patches
The next step was to determine stability of MN patches during long-term storage up to 1 year at up to 40 °C. Extended storage at elevated temperatures was studied because reduced need for a cold chain of storage is desirable, especially in polio-endemic countries which often have poor infrastructure. Consistent with literature ,storage of commercial IPV at an elevated temperature of 40 °C led to complete loss of activity within days to weeks (Fig. S6 in SI).
Correlation of residual moisture content in patch to IPV stability
Residual moisture content of MN patches immediately after fabrication was 6.6 ± 0.2%. Storage at different temperatures for different times produced patches with residual moisture contents as low as 1.3 ± 0.1%. Storage at 5, 25, or 40 °C for 1 month with desiccant resulted in moisture loss of 2%, 4%, or 5%, respectively. Storage at 40 °C and 50% RH resulted in minimal moisture loss of 0.5%.
At these different times and temperatures, IPV activity loss ranged between 91 and 7%. Correlation between IPV activity and residual moisture content (independent of temperature) was poor (Fig. 8b and Fig. S7 in SI), with R2 values of < 0.05. In contrast, IPV activity and temperature (independent of residual water content) were correlated (R2 values of 0.38–0.67) (Fig. S8 in SI). This shows that IPV destabilization processes are driven by elevated temperature with little influence from residual moisture content over the range of conditions studied.
Characterization of changes in MN matrix material properties during patch storage
We determined the composition of MNs (i.e., without the patch backing) to determine the effect of IPV-excipient interaction in IPV activity loss. We then studied the changes in material properties in MNs during storage at 5 °C, 25 °C, or 40 °C with desiccant and then characterized them as described below.
Determination of glass transition temperature of MNs by DSC
Determination of chemical composition changes of MNs by FTIR
Determination of matrix structural changes in MN by XRD
The WHO has recommended replacing OPV with IPV worldwide to eliminate the risk of vaccine-derived polioviruses . However, some limitations with the current IPV vaccination are the need for trained healthcare personnel, risk of needle stick injuries , and the need for cold chain to maintain IPV potency. MN patches can improve IPV delivery by enabling simple administration by minimally trained personnel and avoiding generation of biohazardous sharps waste . In this study, we optimized MN patch formulation to improve IPV stability during extended storage at elevated temperatures.
Initial formulation optimization studies showed that a combination of maltodextrin and sorbitol was optimal for minimizing IPV loss during drying. Buffer screening showed that histidine buffer at pH 6 was optimal for IPV stability. Guided by these findings, MN patch optimization studies identified a 1st cast composition of maltodextrin:D-sorbitol ratio of 20:80 (on a dry basis) and a 2nd cast composition of fish gelatin:D-sorbitol ratio of 77:22 (on a dry basis, when cast as 45% w/v solids in solution) that provided the best stabilization of all three types of IPVs and produced MN patches with strong microneedle tips. Drying temperature optimization studies showed that air drying of the MN patches at 25 °C was effective, and subsequent lyophilization did not show any significant improvement in IPV stability.
The main components of the MN patch fabrication are: preparation of two casting solutions, sequential casting of the solutions, drying and packaging. With further development, this process could be scaled up to meet the potential demands for many millions of patches per year required by the polio eradication program. The IPV patches were tested in pig cadaver skin and found to insert into skin by thumb without the need for an applicator. This simple application process could enable administration by minimally trained personnel [50, 51] thereby reducing costs by reducing the need for healthcare professionals. This simplified administration could facilitate more efficient mass IPV vaccination campaigns and routine IPV vaccination too. The inserted patches also showed near-complete MN dissolution in the skin within 15 min. This MN dissolution has the potential to eliminate the risks of biohazardous sharps. Finally, the MN patch demonstrated significant thermostability even at 25 °C for up to 1 year (> 50% activity) or at 40 °C for up to 2 months (> 40% activity). This thermostability could aid reduce reliance on cold chain storage, especially for short periods of time (days to weeks) during vaccination campaigns and transport to remote locations. This collection of capabilities represents a significant advancement that can overcome many of the hurdles posed by the transition from OPV to IPV. Future studies will need to assess mechanical properties of MN patches, IPV immunogenicity in animals and people, and other quality attributes after extended storage. Although not addressed in this study, vaccination by MN patch has enables dose sparing for some vaccines [52, 53], including IPV , which could enable vaccine costs savings. Remaining questions include determining the cost of IPV vaccination by MN patch , the possibility of skin vaccination by MN patch inducing mucosal immunity  and the number of vaccine doses needed for protective immunity.
Mechanistic studies were performed to understand the effect of MN matrix properties on IPV activity during storage. Residual moisture content of the patches decreased with increase in storage temperature but no correlation was seen between moisture content and IPV stability. MN patches showed an increase in Tg with increase in storage temperature, which was consistent with the moisture loss determined by TGA (i.e., where moisture loss correlates with increased Tg). This suggests that the MN matrix become more amorphous at elevated storage temperature and over time. XRD results also showed an increase in amorphousness of the matrix upon extended storage. Even though a more amorphous (i.e., glassy) matrix should retard diffusion-controlled reactions such as protein unfolding, and aggregation, IPV was less stable at elevated temperature and longer storage times (both of which promote diffusion-controlled, and most non-diffusion-controlled reactions). Because none of these trends in macroscopic MN matrix changes correlated with changes in IPV activity, we hypothesize that intramolecular interactions in IPV virus particles and inter molecular IPV-excipient interactions could be the source of IPV instability.
This study presents the development of a dissolving MN patch for IPV vaccination. Formulation and process optimization studies were performed to identify an excipient combination and drying condition during MN patch fabrication that minimized IPV activity loss. The MN patch was simple to administer, generated no sharps waste, and maintained significantly improved thermostability during extended storage at elevated temperature compared to conventional liquid IPV. When stored with desiccant, IPV in MN patches maintained > 70% activity after 2 months and > 50% activity after 1 year at 5 °C or 25 °C, and maintained > 40% activity after 2 months and > 20% activity after 1 year at 40 °C. Changes in MN matrix properties such as residual moisture content, Tg and crystallinity were measured, but did not correlate with changes in IPV activity. These findings suggest that a MN patch could enable increased thermostability and other advantages important to IPV vaccination and eradication programs.
We thank Dr. Jeong Woo Lee for helpful discussions, Dr. Mohammad Mofidfar for performing the XRD testing, Dr. Meisha Shofner for providing us access to the TGA and DSC instruments in her lab, and Donna Bondy for administrative support. This work was supported by the Bill and Melinda Gates Foundation (Grant OPP1112700). XRD and FTIR studies were performed at the Georgia Tech Institute for Electronics and Nanotechnology, a member of the National Nanotechnology Coordinated Infrastructure, which is supported by the National Science Foundation (Grant ECCS-1542174).
Compliance with ethical standards
Conflict of interest
Mark Prausnitz is an inventor of patents that have been or may be licensed to companies developing microneedle-based products, is a paid advisor to companies developing microneedle-based products and is a founder/shareholder of companies developing microneedle-based products, including Micron Biomedical. These potential conflicts of interest have been disclosed and are being managed by Georgia Tech and/or Emory University.
- 3.Eichner M, Dietz K. Eradication of poliomyelitis: when can one be sure that polio virus transmission has been terminated? Am J Epidemiol. 1996;143(8):816–22.Google Scholar
- 5.Kohler KA, Banerjee K, Gary Hlady W, Andrus JK, Sutter RW. Vaccine-associated paralytic poliomyelitis in India during 1999: decreased risk despite massive use of oral polio vaccine. Bull W H O. 2002;80:210–6.Google Scholar
- 8.Hamborsky J, Kroger A, Wolfe S, Epidemiology and prevention of vaccine-preventable diseases. US Department of Health & Human Services, Centers for Disease Control and Prevention; 2015.Google Scholar
- 9.Davis R, Biellik R. Inactivated polio vaccine: its proposed role in the final stages of polio eradication. Pan Afr Med J. 2013;14(1).Google Scholar
- 21.Rouphael NG, Paine M, Mosley R, Henry S, McAllister DV, Kalluri H, et al. The safety, immunogenicity, and acceptability of inactivated influenza vaccine delivered by microneedle patch (TIV-MNP 2015): a randomised, partly blinded, placebo-controlled, phase 1 trial. Lancet. 2017;390(10095):649–58.CrossRefGoogle Scholar
- 33.Schlehuber LD, McFadyen IJ, Shu Y, Carignan J, Duprex WP, Forsyth WR, et al. Towards ambient temperature-stable vaccines: the identification of thermally stabilizing liquid formulations for measles virus using an innovative high-throughput infectivity assay. Vaccine. 2011;29(31):5031–9.CrossRefGoogle Scholar
- 38.Beale A, Mason P. The measurement of the D-antigen in poliovirus preparations. Epidemiol Infect. 1962;60(1):113–21.Google Scholar
- 39.Milstien JB, Galazka AM, Kartoglu Um, Zaffran M, Organization WH. Temperature sensitivity of vaccines. 2006.Google Scholar
- 44.da Almeida PF, da Silva Lannes SC, Calarge FA, da Brito Farias TM, Santana JCC. FTIR characterization of gelatin from chicken feet. J Chem Chem Eng. 2012;6(11):1029.Google Scholar
- 45.Sibik J, Korter TM, Zeitler JA, editors. Combined infrared and terahertz analysis of amorphous sorbitol. Infrared, Millimeter, and Terahertz waves (IRMMW-THz), 40th International Conference; 2015: IEEE.Google Scholar
- 47.Cullity B. Elements of X-ray diffraction. 2nd ed. San Francisco: Addision-Wesley Pub. Co Inc.; 1978.Google Scholar
- 48.Simonsen L, Kane A, Lloyd J, Zaffran M, Kane M. Unsafe injections in the developing world and transmission of bloodborne pathogens: a review. Bull W H O. 1999;77(10):789–800.Google Scholar
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