- Email: [email protected]
Anti-counterfeiting DNA molecular tagging of pharmaceutical excipients: An evaluation of lactose containing tablets
International Journal of Pharmaceutics 571 (2019) 118656
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Anti-counterfeiting DNA molecular tagging of pharmaceutical excipients: An evaluation of lactose containing tablets
T
Mohamad Jamal Altamimia,b, Joanna C. Greenwoodc, Kim Wolffa,d, Michael E. Hoganc, ⁎ Ahuti Lakhania, Gary P. Martina, Paul G. Royalla, a
Institute of Pharmaceutical Science, King’s College London, London SE1 9NH, United Kingdom Department of Forensic Evidence and Criminology, U.A.E., Dubai, Dubai Police HQ, Al Tawar 1, United Arab Emirates c Applied DNA Sciences, Stony Brook, NY 11790, USA d Department of Pharmacy & Forensic Science, King’s College London, London SE1 9NH, United Kingdom b
ARTICLE INFO
ABSTRACT
Keywords: Anti-counterfeiting Medicine authentication DNA molecular tag DNA amplification Solid dosage form Tableting Lactose
The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting, but it remains a very difficult task to differentiate between counterfeit and real products. Moreover, there is a lack of techniques available for providing a batch specific molecular bar code for tablets that has the required traceability, specificity and sensitivity to be fit for purpose. The aim of this study was to evaluate DNA molecular tags as a potential anti-counterfeiting technology in tablets. Lactose tablets (400 mg) were used as a model to investigate incorporation DNA molecular tag into a solid dosage form: DNA authentication was carried out on an Applied DNA SigNify® qPCR instrument. Tablet batches were subjected to accelerated stability conditions (40 °C and 75% RH) for up to 6 months. All batches passed the monograph specifications of the British Pharmacopoeia (hardness, friability and mass uniformity) throughout the storage period. In all of recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected. It would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new solid dosage form is brought to the market so as to mitigate the risk and dangers of counterfeiting.
1. Introduction The problems associated with counterfeit medicines are well known. For example, in 2014 there was evidence of least 69 of medicines manufactured by one of the larger pharmaceutical companies being falsified in 107 countries (Perks, 2018) and such fake medicines have been estimated to cost the global economy approximately US$75bn annually (Blackstone et al., 2014). Counterfeit medicines may have little or no efficiency against the diseases that they have been taken to treat. When treating malaria and TB for instance, the use of falsified medicines has been implicated in over 700,000 deaths every year (Mackey and Liang, 2011). The pharmaceutical industry has always been at the forefront of safety practices given the consequences of a breach of purity in its products (Ferenczi-Fodor et al., 2006). The equipment to manufacture unlicensed pharmaceutical products is readily available, enabling the easy replication of an authentic medicine such as a tablet or capsule (Glass, 2014). Current digital printing technology may replicate the graphics and serialization on a
⁎
package containing counterfeit drug products (Dégardin et al., 2018). The availability of online “pharmacy” stores is a growing issue, as these vendors are not subject to the checks carried out in licensed pharmacies (Lavorgna, 2015). Counterfeiting not only harms the manufacturer but it can especially harm the patient that is unknowingly not taking their medications (Gaudiano et al., 2016). Potential risks may include the wrong dose, the inclusion of toxic impurities, or in some cases the replacement of the labeled active ingredient with another inappropriate compound, all of which may result in poor patient outcomes, hospitalization and, in critical cases, death (Johnston and Holt, 2014). The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting; however, it is a very difficult task since pharmacists and consumers cannot differentiate between the counterfeit and real products. All national pharmacopeias outline the approved approaches for determining the uniformity of content and dose (e.g. British Pharmacopoeia Commission, 2019). However, there is a lack of techniques available for providing a batch specific molecular bar code on each tablet which is regarded as safe and has the required
Corresponding author. E-mail address: [email protected] (P.G. Royall).
https://doi.org/10.1016/j.ijpharm.2019.118656 Received 4 April 2019; Received in revised form 30 August 2019; Accepted 31 August 2019 Available online 06 September 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 1. Experimental plan to validate DNA tagging of lactose powder and tablets.
2. Materials and methods
traceability, specificity and sensitivity to be fit for purpose. Food and Drug Administration (FDA) Guidance defines a Pharmaceutical Chemical Identifier (PCID) as “a substance or combination of substances possessing a unique physical or chemical property that unequivocally identifies and authenticates a drug product or dosage form”. Examples of substances that may be incorporated into solid oral dosage forms (SODFs) as PCIDs include inks, pigments, flavors and molecular taggants. Such PCIDs are an important feature of current anti-counterfeiting technology and they allow product authentication by their presence alone or they may be used to code the product identity into or onto SODF. There are various available means for presentation and detection of traditional PCIDs (e.g., photolithography, holography, optical microscopy, laser scanning devices, excitation/ fluorescence detection). Some identifying characteristics, such as pigments or flavors, can be easily observed by patients, healthcare practitioners and pharmacies. Others could require the use of a detection instrument e.g., a scanner, photometric detector, mass spectrometry (Andres et al., 2014; Hayward et al., 2018). However, for the labelling of excipients such approaches are not applicable because the detection methods are destructive in nature. They also lack the versatility should a combination of tagged excipients be included in the SODF. Molecular deoxyribonucleic acid (DNA) tags have showed promise in the food sectors and are emerging as a solution for supply chain authentication for fast moving and high value manufactured goods (Barcaccia et al., 2015). However, while there is a groundswell of industry interest in ensuring non-counterfeit packaging (Cozzella et al., 2012), there has not been a breakthrough on the use of technologies on either a tablet or capsule dosage form. The use of DNA as a molecular taggant or, in simpler terms, as a “molecular bar code” is one of the most recent introductions for function as a PCID (Hayward et al., 2018). There is great potential in developing this technology because unique DNA sequences that can unambiguously identify samples will provide evidence at the highest level of possible certainty in court, as has been demonstrated when analysing DNA samples of individuals (OsbornGustavson et al., 2018). It is surprising that DNA molecular tags have not been already adopted for labelling of pharmaceutical excipients to date. It is possible that this is a consequence of the lack of a thorough study into the stability of DNA tags stressed in pharmacopeia recommended conditions. The aim of the work reported here was to evaluate DNA molecular tags for their use as a potential anti-counterfeiting technology in tablets. Our study used lactose tablets containing non active pharmaceutical ingredient (API) as a model to investigate whether a DNA molecular tag could be incorporated into a solid dosage form. Once formed our model formulation was used as a platform for stability testing of the DNA tag under International Council for Harmonisation (ICH) guided accelerated storage conditions. Lactose was chosen as an excipient on the basis of an audit of the British National Formulary where it was a named component of 55% of all the tablet formulations listed (Joint Formulation Committee, 2009; Altamimi et al., 2019).
2.1. Materials Disintequick MCC 25, a co-processed mixture of 75:25 lactose/MCC and magnesium stearate was purchased from Kerry Group (Naas, Ireland) and Sigma-Aldrich Co. (Dorset, UK) respectively. A patented and coded product containing the DNA tag, reference MD-125-15-50, a synthetic DNA clone comprising a fragment of less than 200 base pairs manufacture using purified enzymes (Jung et al., 2019) and proprietary reagents (polymerase chain reaction (PCR) mix containing primers and probe) were supplied by Applied DNA Sciences Inc. (Stony Brook, NY). An ELGA water purifying instrument was used to produce ultra-pure water with a resistance of 18.2 MΩ. HPLC grade Ethanol was purchased from Fisher. 2.2. Methods Several methodologies were used sequentially in this study to prepare the pre-tablet mix, manufacture lactose tablets, carry out stability studies and authenticate the lactose samples. Fig. 1 provides an overview of the experimental methods that were used to complete this study. 2.2.1. Preparation of tableting mix A tableting mix of 99% w/w Disintequick MCC 25 lactose and 1% w/w magnesium stearate was prepared by first weighing the two powders, and then transferring the powder mix (202 g) into a Wahl ZX669 mini chopper with modified blades (to reduce shear stress). The powder was mixed for 2 min. 2.2.2. DNA tag integration First, the entire mass of pre-tablet mix comprising Disintequick MCC 25 lactose and magnesium stearate (202 g) was placed in an electric Kenwood major mixer (5 L bowl). The MD-125-15-50 DNA clone was pipetted into 20 mL 75% v/v ethanol solution contained in a plastic spray bottle. The bottle was shaken, and the total 20 mL of the solution sprayed onto the powder bed while the paddle was turning. This was designed to deliver a concentration of the DNA clone of ≤1 ng per tablet based on the weight of tableting mix used. The top of the mixer was covered to minimize any potential powder loss of the sample and the mixing step carried out for 2 min. The tagged powder was then transferred to a beaker. All the excess powder was recovered from the powder mixing bowl. The sample was reweighed and only approximately 1 g of the initial powder could not be recovered. The target tablet size was 400 mg and the amount of tagged powder (just above 200 g) was sufficient to produce approximately 500 tablets. 2.2.3. Tablet manufacture A target mass of 400 mg of the powder mix discussed above was weighed to prepare each tablet. 2
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
2.2.6. Hardness and friability Following the British Pharmacopoeia (BP) (British Pharmacopoeia Commission) recommendations ten tablets each were used to test the friability and the hardness of a batch. Friability testing was carried out using an automated friability testing F1/F2 instrument (SOTAX friability tester). The tablets were dusted using a brush and the total weight of the tablets was recorded before initiating 100 rotations of the chamber (25 rpm). After the test, the tablets were examined for breaking or crumbling and their weight was recorded and compared to the original weight. The hardness testing was carried out using a C50 hardness tester (Engineering Systems, Nottingham): the samples were placed in a compartment in an orientation that allows the instrument to break the tablets between a stationary metal surface and a metal press with flat surfaces.
Table 1 Summary of BP uniformity of mass (mg) measurement (n = 20 for each batch).
Mean SD Lower limit Upper limit
Batch 1
Batch 2
Batch 3
390.2 2.8 370.7 409.7
393.2 1.9 373.5 412.8
393.6 1.1 373.9 413.2
The 400 mg of powder was transferred into a tableting 1 cm (diameter) stainless punch and die set. Each tablet was prepared individually using a manual hydraulic disc press (Specac, Kent) by applying fourteen-tons (137.3 kN) of force. The pressure was held for 1 min before it was released using the pressure valve. The formed tablet was ejected from the die and placed into a sealed glass vial.
2.2.7. Uniformity of mass of tablets Following BP recommendations (the mass should vary by ≤ ± 5% when the tablets are 250 mg or more), 20 tablets were weighed and their average mass calculated (British Pharmacopoeia Commission).
2.2.4. DNA tag analysis/sample authentication For tablets, a small area of the surface was swabbed with the tip of a sterile cotton swab that had been dipped in ultra-pure water. Swabbing was carried out using ten ‘up and down’ swabbing actions. This was repeated on two other areas of the same tablet with new swabs to ensure that each sample was assayed in triplicate. The swab tips were then cut and placed directly into tubes containing 18 μL PCR mix. For the powder samples, 100 mg powder was partially dissolved in 5 mL ELGA purified water (conductance − 18.2 MΩ) by vortex mixing the suspended particles. It was assumed that the un-dissolved material comprised a high proportion of magnesium stearate and a fraction of MCC, with the lactose fully dissolved, due to the high aqueous solubility of the sugar. The sample was then centrifuged for 5 min at 10,000 RPM (≅7000g) and 2 µL of the supernatant was transferred to a new tube containing 18 µL of the PCR mix. The samples were analysed and thus authenticated by the Applied DNA SigNify® qPCR instrument. For all DNA authentications, the samples were run directly on the portable Applied DNA SigNify® qPCR instrument. The instrument was set to heat up at 3 °C/min to 95 °C, hold for 10 s, cool at 1.5 °C/s to 60 °C and hold for 30 s. Heating cycles are used to allow temperature-dependent reactions to occur, amplifying the amount of DNA present. Thirty heating cycles were used to reduce run time since all the samples that have been tested could be quantified within less than 30 cycles. The Cq value represents the cycle at which the fluorescence passes the limit of detection, that is, the threshold where the signal to noise ratio is exceeded. Thus, if the concentration of the DNA tag recovered is high, the number of required cycles for detection (Cq value) is low. The number of cycles could be increased if required to detect lower concentrations of the DNA tag. Standards used were 0.1 pg/mL, 1 pg/mL, 10 pg/mL and 100 pg/mL created by serial dilution of the supplied MD125-15-50 clone. This clone was also used as a positive control. The Cq value (number of cycles to reach the detection threshold) was calculated by the qPCR instrument’s analysis programme. Negative controls comprised a representative aliquot of PCR mix. The latter were included to ensure that the reagents were not contaminated with the target DNA clone. If contamination had occurred, the negative controls would be expected to show a Cq value (i.e. amplification of the DNA present would occur). Where DNA was not present above the threshold levels at the end of the PCR cycles performed, no Cq value was recorded.
3. Results 3.1. Pharmacopoeia testing of lactose tablets Three batches of lactose tablets were manufactured each comprising of sixty tablets. The uniformity of weight, hardness and friability of each batch was determined. The uniformity of content was determined by weighing all sixty tablets of each batch (Table 1) and each batch passed the BP specifications. The tablets also passed the friability test (Table 2) where none of the batches of ten tablets lost more than 1% of their weight. The hardness of the tablets was also measured and summarised below (Table 3) The manufactured lactose tablets, having an average hardness of 22–28 kg, could be designated as ‘hard’ (British Pharmacopoeia Commission). In summary, the tablets were deemed to be of a high standard and passed the monograph specifications of the British Pharmacopoeia. The same tests were repeated for samples that were used in the stability study and all batches remained within the specified limits. 3.2. DNA tag recovery from swabbed lactose tablets The DNA tag recovery data comprised a series of plots showing the resultant fluorescent intensity of the replicated DNA tag as a function of the number of enzyme reactions, i.e. DNA replication cycles. In all of the recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected, (Fig. 2). Negative controls, whereby no DNA tag was included in the sample under investigation clearly showed no reaction as evidenced by the lack of fluorescent detection. Therefore, no cross contamination was observed during this work. The Cq values obtained from the swabbing experiments displayed a high repeatability between batches as a function of time over the six month study period
2.2.5. Stability testing Three batches of 60 tablets were manufactured using the manual press. All the samples were stored in glass vials for 3 and 6 months without lids following the ICH accelerated stability conditions set at 40 °C and 75% RH using a Memmert HPP110 controlled temperature and humidity chamber. The temperature and humidity of the samples during storage were recorded. All samples were authenticated using the qPCR instrument (Section 2.2.4). Results were analysed for statistical difference using a Student’s t-test.
Table 2 BP friability test results for manufactured lactose tablets (n = 10 for each batch).
3
Batch
Weight of tablets before test (g)
Weight of tablets after test (g)
Weight loss (g)
Weight loss (%)
1 2 3
3.87 3.91 3.93
3.86 3.90 3.92
0.01 0.01 0.01
0.17 0.20 0.20
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
indicating a decrease in DNA recovery. In addition, there was some loss in the precision of the assay. The differences between the assay values taken over the period of the assay proved to be statistically significant (p < 0.05).
Table 3 BP hardness (kg) test results for lactose tablets (n = 10) for each batch. Sample
Batch 1
Batch 2
Batch 3
mean SD
28.1 4.9
24.2 2.5
22.0 2.1
4. Discussion This study has presented the first report of the use of a DNA as a PCID to tag an excipient which was subsequently employed to manufacture a tablet. Lactose frequently occurs in tablets and many other formulations and was used in this study as a ‘model’ excipient because it was found in the top 200 prescriptions of tablets and capsules, with the disaccharide present as a filler in 77 out of the 200 (Dave, 2008; Gabbott et al., 2003). Applying a DNA tag to lactose as it is manufactured would allow the lactose to be easily traced confirming the authenticity of the product by the commercial manufacturer. In this study, lactose tablets were manufactured to be used as models for API-containing solid-dosage forms. Using the DNA tag to monitor the authenticity of a medicine by mixing with tagged powdered excipients, would be less chemically demanding than if the marker were simply mixed with the individual API. Finding optimum mixing parameters and techniques to fully incorporate the DNA tag into a test excipient powder was an integral part of the preliminary work for this study. The high precision and inter-batch repeatability showed that the developed methodology allowed the DNA to be apparent and equally distributed within the whole tablet. Some of the high stress environments routinely experienced during processing, such as mixing, drying, and compression during tableting were replicated in this study. Pharmacopeia tests were conducted at intervals to determine whether tablets remained within specified official limits. It is apparent that the DNA tag can be amplified and detected after storage of the tablets at elevated temperatures and humidities for 6 months, demonstrating the viability of the methods as a possible anti-counterfeiting strategy.
with low coefficient of variation (COV = 2.4–3.7%). There was an increase in the Cq value by about 5 cycles in the month 3 samples compared to those obtained initially (month 0); however, the difference was found to be less between the 3 and 6 month samples (around 1 Cq value). 3.3. DNA tag recovery from crushed lactose tablets Similar to the swabbed tablet results, there was found to be an increase in the Cq value that was higher between the 0 and 3 month samples than the increase between the 3 and 6 month samples (Fig. 3). The Cq repeatability was greatest when the tablets were crushed immediately after manufacture (COV = 1.3%) but this increased after storage for 6 months at 40 °C and 75% RH (COV = 3.6%). 3.4. Evaluation of the swabbed and crushed tablet methods A difference was apparent in the reproducibility between the swabbing method and the crushed tablet method of analysis immediately after the tablet manufacture (Fig. 4). The narrow distribution of data obtained using the methodology for analyzing crushed tablets clearly exhibited a greater precision than those from the method which involved swabbing the tablets. From the histogram, the range of the results from swabbing the tablets was much larger (19.5–22.3 Cq) than when the tablets were crushed prior to assay (20.2–21.2 Cq). Both sets of data show good precision for the methodology used. Fig. 5 depicts the combined batch data as a histogram to show the differences in Cq as a function of storage time for the crushed tablets. As storage time was increased, clearly the Cq value increased but the longer the stability study, the slower the rate at of increase,
4.1. Analysis of DNA tags Despite the use of DNA as a tag being in its relative infancy, several applications of the technology have been outlined (Bar-Or et al., 2006). Fig. 2. Amplification curves for all swabbed tablets analysed at 0 (red), 3 (black) and 6 (green) months were overlayed, including negative controls (grey) and the summary of DNA analysis of all swabbed tablets carried out in triplicate from 3 batches (n = 3) at month 0, 3 and 6 months in embedded table (mean ± SD). Storage conditions were 40 °C & 75% RH. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 3. Amplification curves for all crushed tablets analysed at 0 (red), 3 (black) and 6 (green) months, including negative controls (grey) and the summary of DNA analysis of all swabbed tablets carried out in triplicate from 3 batches (n = 3) at month 0, 3 and 6 months in embedded table. Storage conditions were 40 °C & 75% RH. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
target DNA sequence concentration in the starting solution. As the starting DNA concentration increased, less cycles were required until the DNA concentration could be ‘visualised’, resulting in a lower Cq value being obtained in the analysed sample.
For example, it has been advocated as a method of labeling milk along its production pathway in the manufacture of secondary dairy products (e.g. cheese and yoghurt) and has a practical use in security tagging (using spray solutions) with a view to combat theft, robbery and vandalism (e.g. SelectDNA®, 2019; Applied DNA Sciences, 2019). The central concept underpinning these applications is that by synthesising or isolating specific sequences of DNA and making them sufficiently stable to withstand the environment for a prolonged time, they can be used as a unique code that can be amplified with the use of PCR so that they can subsequently be detected and authenticated. However, no studies using DNA tagging technology have been published with regards to labelling formulated medicines. One reason for the lack of published material is likely to be the commercially sensitive nature of anti-counterfeiting work. Any approach requires both a specific and unique DNA sequence and an analytical technique that can subsequently identify that sequence. PCR is routinely used to amplify, or increase, the amount of a specific DNA sequence for analysis. Here, the PCR mix comprising Taq polymerase, sequence specific primers and a fluorescent probe, is heated and cycled between ~65 and 95 °C. As the sample approaches boiling temperatures, the hydrogen bonds between the nucleotides in the DNA break down causing the DNA template to unwind from its double helix. Then the temperature is cooled and held at 55–65 °C, the exact temperature depending upon the primer length, where the DNA is annealed. During the annealing process, the primers, which are short DNA strands designed to attach to specific sequences in the DNA template, attach to the DNA strands. The nucleotide bases are each tagged with fluorescent markers that can be monitored using a fluorescent photometer or via a quantitative PCR (qPCR) instrument. Theoretically, each cycle will double the amount of DNA in the sample and this is usually repeated multiple times until there is sufficient fluorescence for a signal to be detected. Techniques such as qPCR, or real-time PCR, incorporate lasers and light sensing equipment to monitor the production of DNA fragments labelled with fluorescent tags as the number of such fragments produced increase with each cycle. The measurement method employed was based on the number of cycles needed for the DNA to be amplified to a concentration in the solution that allowed it to be detected by the qPCR instrument (Fraga et al., 2008). The Cq value represented the number of cycles that needed to be effected until the DNA amplification could be detected via the incorporated fluorescent markers, also termed the inflection point (Section 2.2.4). The Cq value can vary or change depending on the
4.2. Stability of DNA tags In this study SigNature® DNA was used as the molecular taggant. SigNature® DNA is manufactured by PCR, purified by anion exchange chromatography and provided in 10 mM Tris-hydroxymethyl amino methane plus 1 mM Ethylene diamine tetraacetic acid, pH 7.8 (Tris-EDTA or “TE”) buffer at a DNA concentration of ≥4.0 µg/mL. Although this may appear to be a dilute solution, it could be even further diluted at least one million-fold to provide a solution ≤100 ng/dose for inclusion in the batch, as a PCID. TE buffer is a commonly used in molecular biology to solubilize DNA while protecting it from degradation from heavy metals. The ≥4.0 µg/mL solution was aliquoted into the ethanolic solution and sprayed into the tableting mix (Section 2.2.2), the resulting concentration of the DNA clone was below 1 ng per tablet, at approximately 0.4 ng/tablet. The current study did not test specifically for the minimal amount of DNA required, but employed a standard concentration typically used for research purposes (Jung et al., 2019). Based on the results obtained, the concentration of DNA per tablet could be reduced by at least 10 times, and still produce satisfactory results. This value is likely to fall further in future studies as the tag and its concentration can easily be tailored for each new pharmaceutical application. It should be noted that either by swabbing or crushing, because of the required dilution steps within the methodology, the amount of DNA analysed by PCR is much less that the total present in the individual tablet. The DNA standards used as successful controls had a lower limit of 0.1 pg/mL (Section 2.2.4), thus in this study the minimal amount of DNA needed to allow identification falls within the 1 pg range. DNA can be degraded by the hydrolysis of the phosphodiester bonds. These bonds are the backbone of the DNA strands as they are the linkage bond between the 3′ carbon on one sugar and the 5′ carbon. Alternatively, DNA degradation pathway can occur by the oxidation of nucleotide bases. Despite the primary structure of DNA being susceptible to damage by elevated heat, temperatures for this to occur markedly are those above 100 °C (Karni et al., 2013), although biochemical catalyzed degradation can occur at lower temperatures (Lindahl, 1993). Such limitations have been overcome by developing coatings or treatments that allow DNA to be 5
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 4. A histogram that groups the samples according to the Cq values for all swabbed and crushed tablets at month 0 and the original amplification curves for swabbed (A) and crushed (B) tablets at month 0.
much less susceptible to such damage. For example, the addition of silica has been shown to act as an ideal material for encapsulation and provide exceptional barrier properties (Paunescu et al., 2013, 2016). Adding such silica-encapsulated DNA to milk before manufacturing cheese resulted in the barcode maintaining its integrity during the manufacturing process (Bloch et al., 2014). DNA robustness may also be increased by appropriate sequence selection and design. However, in this study, the addition of silica could have affected the tableting process and in addition its use might pose problems in justifying its inclusion in licensed commercial formulations. In any case, the results presented here demonstrated that the DNA tag employed showed high durability, which would be a prerequisite for utilization in anti-counterfeiting technology.
manufacture counterfeit dosage forms. It is possible that DNA could interact with components of the tablet i.e. the lactose, MCC or magnesium stearate and future studies should investigate whether this does occur. However, the DNA analysis method used in this study is not oriented around DNA quantification per se. The success of a run was determined by the Cq values of the samples which were compared with positive and negative results. It is possible to add specific concentrations of DNA to each assay set so as to generate a relevant calibration curve. However, the repeated calibration before each set of samples can be costly and counterproductive as it is not required to confirm whether DNA is successfully detected. In a pharmaceutical setting, crushing tablets before carrying out the authentication assay may be more suitable as this method of preparation would be expected to create a more homogeneous sample with less variation in the results as compared with the swabbing method. There was an increase in the Cq values after the tablets were stored at 40 °C and 75% RH. Some of the surface powder may interact with the surrounding moisture and cause the degradation of some DNA. In addition some possible loss of DNA tag could be due to the interaction with lactose (Figs. 2 and 3). It is possible that such an interaction might account for the results depicted in Fig. 6, where depurination may have
4.3. Tagging lactose tablets The uniformity of content and weight tests proved that tablets manufactured using this technique were of sufficient quality to pass the specifications indicated in the BP. The aspect ratio of the powder bed to the rotational end of the mixing device as well as a high energy output ensured the powdered components were well mixed. Such widely available domestic mixers can easily be employed by illicit suppliers to 6
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 5. A histogram that groups the samples according to the Cq values for all crushed lactose tablets. 54 data points were available in each batch where six tablets were sampled 3 times and each assay carried out in triplicate. Storage conditions were 40 °C & 75% RH.
occurred, resulting in higher Cq values after storage. DNA-lactose tagging tests proved that a very small amount of DNA tag is very sensitive and detected easily as shown by the technique of swabbing tablets. Although a small amount (pg) of DNA was at the surface of the tablet, the Cq value remained below 30, which provided a clear positive result using the portable qPCR instrument. Such swabbing methodology may be useful for in-field testing and for forensic authentications. Crushing the tablet would be expected to provide a more efficient method of ensuring tablet authentication and so it proved (lower Cq values being obtained). Such a method would be advantageous for inhouse assessment of tablets. Molecular tagging technologies can impart identity and traceability to solid oral dosage forms and could also be employed to tag associated
packaging (Bar-Or et al., 2006) to be used as layered security throughout a supply chain. Such approaches are important strategies to counter the increasing problem of counterfeit medicines. Lactose does appear to be a good model excipient with which to associate the DNA tag and further development of this method is likely to be achievable at relatively small cost due to the very high sensitivity of the method (pg/ mL levels of DNA being required). The pharma industry would require different “bar codes” for different types of excipient, manufacturer, time and date of manufacture and also for different batches of the excipient in question. In the current example, the number of possible combinations of DNA analysis by PCR is very high. The combinations available are equal to four (=bases of DNA) raised to the power of x, whereby x is the total number of bases of
Fig. 6. Using adenine as an example, the interaction between lactose and DNA resulting in the loss of the adenine structure. 7
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
the primer and probe sequences. This high versatility of the coding allows the implementation of cryptography for the protection of the barcode and hence provides exceptional promise. Separate DNA molecular taggants might be added to different APIs, fillers, coatings, disintegrants, lubricants, glidants, inks, capsule shells etc. of an SODF to uniquely identify a medicine’s complete authenticity and traceability. The DNA molecular tags used to infuse goods with unique identification for traceability in pharmaceuticals are orders of magnitude smaller than the human genome, typically less than 200 bp in length (Murrah, 2010). This PCID identifier could be used like a license plate, referring to much more contextual information in a database that might, for example, include producer, manufacturing facility, year of manufacture or other relevant information. DNA molecular taggants offer several benefits over other forms of PCIDs. DNA is a common molecule that is highly resolvable due to the fact that while it can be used in femtogram-level (10−15 g) trace amounts in pharmaceutical applications, either in-lab or in-field devices supporting PCR processes can use one molecule extracted from the SODF or packaging to amplify to millions of molecules and analyse within 30 to 45 min. In addition, cost-effective large-scale production is achievable using the same PCR technology to support the potentially large amounts of DNA required by the industry (Murray et al., 2012). DNA molecular tags can be formulated to be compatible with their host carriers and to be protected against environmental or process challenges, yet also to be extractable for authentication purposes.
References Altamimi, M.J., Wolff, K., Nokhodchi, A., Martin, G.P., Royall, P.G., 2019. Variability in the a and beta anomer content of commercially available lactose. Int. J. Pharm. 555, 237–249. Andres, J., Hersch, R.D., Moser, J.E., Chauvin, A.S., 2014. A new anti-counterfeiting feature relying on invisible luminescent full color images printed with lanthanide-based inks. Adv. Funct. Mater. 24, 5029–5036. Applied DNA Sciences, 2019. https://adnas.com/security-and-asset-marking-crime-prevention/ (accessed 11.03.19). Bar-Or, Y.A., Scheibe, P.O., Plaxco, K.W., Thomas, A.J., 2006. Method, apparatus, and system for authentication using labels containing nuleotide sequences US patent application: US 2006/0286569 A1. Barcaccia, G., Lucchin, M., Cassandro, M., 2015. DNA barcoding as a molecular tool to track down mislabeling and food piracy. Diversity 8, 2. Blackstone, E.A., Fuhr Jr, J.P., Pociask, S., 2014. The health and economic effects of counterfeit drugs. Am. Health Drug Benefits 7, 216. Bloch, M.S., Paunescu, D., Stoessel, P.R., Mora, C.A., Stark, W.J., Grass, R.N., 2014. Labeling milk along its production chain with DNA encapsulated in silica. J. Agric. Food Chem. 62, 10615–10620. British Pharmacopoeia Commission, 2019. British Pharmacopoeia 2019. TSO, London. Code of Federal Regulations, 2018. 21 CFR 170.30, April 1, 2018. Chapter 1 – Food and Drug administration, Department of Health and Human Services, Subchapeter B – Food for human comsumption. Food Additives. Title 21, Volume 3. https://www.accessdata.fda. gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=170.30 (accessed 04.02.19). Cozzella, L., Simonetti, C., Spagnolo, G.S., 2012. Drug packaging security by means of whitelight speckle. Opt. Lasers Eng. 50, 1359–1371. Dave, R.H., 2008. Overview of pharmaceutical excipients used in tablets and capsules. Drug Topics 24, 1–13. Dégardin, K., Guillemain, A., Klespe, P., Hindelang, F., Zurbach, R., Roggo, Y., 2018. Packaging analysis of counterfeit medicines. Forens. Sci. Int. 291, 144–157. https://doi.org/10.1016/ j.forsciint.2018.08.023. FDA Federal Register, 1992. Statement of Policy – Foods Derived from New Plant Varieties. Volume 57-1992 https://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/biotechnology/ucm096095.htm (accessed 04.02.19). Ferenczi-Fodor, K., Végh, Z., Renger, B., 2006. Thin-layer chromatography in testing the purity of pharmaceuticals. TrAC Trends Anal. Chem. 25, 778–789. Fraga, D., Meulia, T., Fenster, S., 2008. Real-time PCR. In: Current Protocols Essential Laboratory Techniques. John Wiley & Sons Inc, pp. 10.3.1–10.3.34. https://doi.org/10. 1002/9780470089941.et1003s00. Gaudiano, M.C., Manna, L., Bartolomei, M., Rodomonte, A.L., Bertocchi, P., Antoniella, E., Romanini, L., Alimonti, S., Rufini, L., Valvo, L., 2016. Health risks related to illegal and online sale of drugs and food supplements: results of a survey on marketed products in Italy from 2011 to 2013. Annali dell'Istituto superiore di sanita 52, 128–132. Glass, B.D., 2014. Counterfeit drugs and medical devices in developing countries. Res. Rep. Trop. Med. 5, 11–22. Gabbott, P., Clarke, P., Mann, T., Royall, P., Shergill, S., 2003. A high-sensitivity, high-speed DSC technique: measurement of amorphous lactose. Am. Lab. 35, 16–17. Hayward, J. A., Hogan, M. E., Liang, M. B., Jung, L., 2018. Method for authenticating active pharmaceutical ingredients: Patent Application US20180142294A1. Joint Formulation Committee, 2009. British National Formulary (BNF) September 2009. Pharmaceutical Press, London. Johnston, A., Holt, D.W., 2014. Substandard drugs: a potential crisis for public health. Brit. J. Clin. Pharmacol. 78, 218–243. Jung, L., Hogan, M.E., Sun, Y., Liang, B.M., Hayward, J.A., 2019. Rapid authentication of pharmaceuticals via DNA tagging and field detection. PLoS One 14, 1–11. https://doi.org/ 10.1371/journal.pone.0218314. Karni, M., Zidon, D., Polak, P., Zalevsky, Z., Shefi, O., 2013. Thermal degradation of DNA. DNA Cell Biol. 32, 298–301. Lavorgna, A., 2015. The online trade in counterfeit pharmaceuticals: new criminal opportunities, trends and challenges. Eur. J. Criminol. 12, 226–241. https://doi.org/10.1177/ 1477370814554722. Lindahl, T., 1993. Instability and decay of the primary structure of DNA. Nature 362, 709–715. Mackey, T.K., Liang, B.A., 2011. The global counterfeit drug trade: patient safety and public health risks. J. Pharm. Sci. 100, 4571–4579. Murrah, J., 2010. DNA molecular taggants: a hidden key to pharmaceutical patient safety. Tablets Capsules 16, 33–36. Murray E, Gregg DA, Norton ML, Swick JT, Towler WI., 2012. Method for a continuous rapid thermal cycle system. US Patent 8,163,489. Awarded April 24, 2012. Osborn-Gustavson, A.E., McMahon, T., Josserand, M., Spamer, B.J., 2018. Chapter 12 – the utilization of databases for the identification of human remains A2 – Latham, Krista E. In: Bartelink, E.J., Finnegan, M. (Eds.), New Perspectives in Forensic Human Skeletal Identification. Academic Press, pp. 129–139. Paunescu, D., Fuhrer, R., Grass, R.N., 2013. Protection and deprotection of DNA—high-temperature stability of nucleic acid barcodes for polymer labeling. Angew. Chem. Int. Ed. 52, 4269–4272. Paunescu, D., Stark, W.J., Grass, R.N., 2016. Particles with an identity: tracking and tracing in commodity products. Powder Technol. 291, 344–350. Perks, B., 2018. Pharmaceutical fakes: a dangerous pandemic. Lung Cancer 15, 05. SelectDNA®, 2019. https://www.selectadna.co.uk (accessed 04.02.19). Taylor, C., 2015. Marking material (pp. 1-4): US Patent: US9171443B2.
5. Conclusion DNA tagging is a novel prospect that could revolutionise law enforcement and indeed has already been used in a multitude of other fields (Applied DNA Sciences, 2019, Taylor, 2015; SelectDNA®, 2019). Many studies have used DNA tagging as an authentication method, but none have used the direct inclusion of a DNA tag into a powder excipient mix. In the current study, the DNA tag could be extracted and characterized after application of fourteen-tons (137.3 kN) of force to form a tablet, and withstood storage at storage at high temperature and humidity. DNA tags are Generally Recognized as Safe (GRAS) through “experience based on common use in food” since before January 1, 1958 (Code of Federal Regulations, 2018). The FDA’s Statement of Policy “Food Derived from New Plant Varieties (FDA Federal Register, 1992)” addresses its view that transferred genetic material is considered GRAS based upon DNA's omnipresence in food. The DNA molecular tags, and the processing technology investigated here, produced traceable tablets that could withstand accelerated ICH recommended storage conditions. On the basis of these results it would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new medicine is brought to the market so as to mitigate the risk and dangers of counterfeiting. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.118656.
8
Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Anti-counterfeiting DNA molecular tagging of pharmaceutical excipients: An evaluation of lactose containing tablets
T
Mohamad Jamal Altamimia,b, Joanna C. Greenwoodc, Kim Wolffa,d, Michael E. Hoganc, ⁎ Ahuti Lakhania, Gary P. Martina, Paul G. Royalla, a
Institute of Pharmaceutical Science, King’s College London, London SE1 9NH, United Kingdom Department of Forensic Evidence and Criminology, U.A.E., Dubai, Dubai Police HQ, Al Tawar 1, United Arab Emirates c Applied DNA Sciences, Stony Brook, NY 11790, USA d Department of Pharmacy & Forensic Science, King’s College London, London SE1 9NH, United Kingdom b
ARTICLE INFO
ABSTRACT
Keywords: Anti-counterfeiting Medicine authentication DNA molecular tag DNA amplification Solid dosage form Tableting Lactose
The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting, but it remains a very difficult task to differentiate between counterfeit and real products. Moreover, there is a lack of techniques available for providing a batch specific molecular bar code for tablets that has the required traceability, specificity and sensitivity to be fit for purpose. The aim of this study was to evaluate DNA molecular tags as a potential anti-counterfeiting technology in tablets. Lactose tablets (400 mg) were used as a model to investigate incorporation DNA molecular tag into a solid dosage form: DNA authentication was carried out on an Applied DNA SigNify® qPCR instrument. Tablet batches were subjected to accelerated stability conditions (40 °C and 75% RH) for up to 6 months. All batches passed the monograph specifications of the British Pharmacopoeia (hardness, friability and mass uniformity) throughout the storage period. In all of recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected. It would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new solid dosage form is brought to the market so as to mitigate the risk and dangers of counterfeiting.
1. Introduction The problems associated with counterfeit medicines are well known. For example, in 2014 there was evidence of least 69 of medicines manufactured by one of the larger pharmaceutical companies being falsified in 107 countries (Perks, 2018) and such fake medicines have been estimated to cost the global economy approximately US$75bn annually (Blackstone et al., 2014). Counterfeit medicines may have little or no efficiency against the diseases that they have been taken to treat. When treating malaria and TB for instance, the use of falsified medicines has been implicated in over 700,000 deaths every year (Mackey and Liang, 2011). The pharmaceutical industry has always been at the forefront of safety practices given the consequences of a breach of purity in its products (Ferenczi-Fodor et al., 2006). The equipment to manufacture unlicensed pharmaceutical products is readily available, enabling the easy replication of an authentic medicine such as a tablet or capsule (Glass, 2014). Current digital printing technology may replicate the graphics and serialization on a
⁎
package containing counterfeit drug products (Dégardin et al., 2018). The availability of online “pharmacy” stores is a growing issue, as these vendors are not subject to the checks carried out in licensed pharmacies (Lavorgna, 2015). Counterfeiting not only harms the manufacturer but it can especially harm the patient that is unknowingly not taking their medications (Gaudiano et al., 2016). Potential risks may include the wrong dose, the inclusion of toxic impurities, or in some cases the replacement of the labeled active ingredient with another inappropriate compound, all of which may result in poor patient outcomes, hospitalization and, in critical cases, death (Johnston and Holt, 2014). The licensed pharmaceutical industry and regulators use many approaches to control counterfeiting; however, it is a very difficult task since pharmacists and consumers cannot differentiate between the counterfeit and real products. All national pharmacopeias outline the approved approaches for determining the uniformity of content and dose (e.g. British Pharmacopoeia Commission, 2019). However, there is a lack of techniques available for providing a batch specific molecular bar code on each tablet which is regarded as safe and has the required
Corresponding author. E-mail address: [email protected] (P.G. Royall).
https://doi.org/10.1016/j.ijpharm.2019.118656 Received 4 April 2019; Received in revised form 30 August 2019; Accepted 31 August 2019 Available online 06 September 2019 0378-5173/ © 2019 Elsevier B.V. All rights reserved.
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 1. Experimental plan to validate DNA tagging of lactose powder and tablets.
2. Materials and methods
traceability, specificity and sensitivity to be fit for purpose. Food and Drug Administration (FDA) Guidance defines a Pharmaceutical Chemical Identifier (PCID) as “a substance or combination of substances possessing a unique physical or chemical property that unequivocally identifies and authenticates a drug product or dosage form”. Examples of substances that may be incorporated into solid oral dosage forms (SODFs) as PCIDs include inks, pigments, flavors and molecular taggants. Such PCIDs are an important feature of current anti-counterfeiting technology and they allow product authentication by their presence alone or they may be used to code the product identity into or onto SODF. There are various available means for presentation and detection of traditional PCIDs (e.g., photolithography, holography, optical microscopy, laser scanning devices, excitation/ fluorescence detection). Some identifying characteristics, such as pigments or flavors, can be easily observed by patients, healthcare practitioners and pharmacies. Others could require the use of a detection instrument e.g., a scanner, photometric detector, mass spectrometry (Andres et al., 2014; Hayward et al., 2018). However, for the labelling of excipients such approaches are not applicable because the detection methods are destructive in nature. They also lack the versatility should a combination of tagged excipients be included in the SODF. Molecular deoxyribonucleic acid (DNA) tags have showed promise in the food sectors and are emerging as a solution for supply chain authentication for fast moving and high value manufactured goods (Barcaccia et al., 2015). However, while there is a groundswell of industry interest in ensuring non-counterfeit packaging (Cozzella et al., 2012), there has not been a breakthrough on the use of technologies on either a tablet or capsule dosage form. The use of DNA as a molecular taggant or, in simpler terms, as a “molecular bar code” is one of the most recent introductions for function as a PCID (Hayward et al., 2018). There is great potential in developing this technology because unique DNA sequences that can unambiguously identify samples will provide evidence at the highest level of possible certainty in court, as has been demonstrated when analysing DNA samples of individuals (OsbornGustavson et al., 2018). It is surprising that DNA molecular tags have not been already adopted for labelling of pharmaceutical excipients to date. It is possible that this is a consequence of the lack of a thorough study into the stability of DNA tags stressed in pharmacopeia recommended conditions. The aim of the work reported here was to evaluate DNA molecular tags for their use as a potential anti-counterfeiting technology in tablets. Our study used lactose tablets containing non active pharmaceutical ingredient (API) as a model to investigate whether a DNA molecular tag could be incorporated into a solid dosage form. Once formed our model formulation was used as a platform for stability testing of the DNA tag under International Council for Harmonisation (ICH) guided accelerated storage conditions. Lactose was chosen as an excipient on the basis of an audit of the British National Formulary where it was a named component of 55% of all the tablet formulations listed (Joint Formulation Committee, 2009; Altamimi et al., 2019).
2.1. Materials Disintequick MCC 25, a co-processed mixture of 75:25 lactose/MCC and magnesium stearate was purchased from Kerry Group (Naas, Ireland) and Sigma-Aldrich Co. (Dorset, UK) respectively. A patented and coded product containing the DNA tag, reference MD-125-15-50, a synthetic DNA clone comprising a fragment of less than 200 base pairs manufacture using purified enzymes (Jung et al., 2019) and proprietary reagents (polymerase chain reaction (PCR) mix containing primers and probe) were supplied by Applied DNA Sciences Inc. (Stony Brook, NY). An ELGA water purifying instrument was used to produce ultra-pure water with a resistance of 18.2 MΩ. HPLC grade Ethanol was purchased from Fisher. 2.2. Methods Several methodologies were used sequentially in this study to prepare the pre-tablet mix, manufacture lactose tablets, carry out stability studies and authenticate the lactose samples. Fig. 1 provides an overview of the experimental methods that were used to complete this study. 2.2.1. Preparation of tableting mix A tableting mix of 99% w/w Disintequick MCC 25 lactose and 1% w/w magnesium stearate was prepared by first weighing the two powders, and then transferring the powder mix (202 g) into a Wahl ZX669 mini chopper with modified blades (to reduce shear stress). The powder was mixed for 2 min. 2.2.2. DNA tag integration First, the entire mass of pre-tablet mix comprising Disintequick MCC 25 lactose and magnesium stearate (202 g) was placed in an electric Kenwood major mixer (5 L bowl). The MD-125-15-50 DNA clone was pipetted into 20 mL 75% v/v ethanol solution contained in a plastic spray bottle. The bottle was shaken, and the total 20 mL of the solution sprayed onto the powder bed while the paddle was turning. This was designed to deliver a concentration of the DNA clone of ≤1 ng per tablet based on the weight of tableting mix used. The top of the mixer was covered to minimize any potential powder loss of the sample and the mixing step carried out for 2 min. The tagged powder was then transferred to a beaker. All the excess powder was recovered from the powder mixing bowl. The sample was reweighed and only approximately 1 g of the initial powder could not be recovered. The target tablet size was 400 mg and the amount of tagged powder (just above 200 g) was sufficient to produce approximately 500 tablets. 2.2.3. Tablet manufacture A target mass of 400 mg of the powder mix discussed above was weighed to prepare each tablet. 2
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
2.2.6. Hardness and friability Following the British Pharmacopoeia (BP) (British Pharmacopoeia Commission) recommendations ten tablets each were used to test the friability and the hardness of a batch. Friability testing was carried out using an automated friability testing F1/F2 instrument (SOTAX friability tester). The tablets were dusted using a brush and the total weight of the tablets was recorded before initiating 100 rotations of the chamber (25 rpm). After the test, the tablets were examined for breaking or crumbling and their weight was recorded and compared to the original weight. The hardness testing was carried out using a C50 hardness tester (Engineering Systems, Nottingham): the samples were placed in a compartment in an orientation that allows the instrument to break the tablets between a stationary metal surface and a metal press with flat surfaces.
Table 1 Summary of BP uniformity of mass (mg) measurement (n = 20 for each batch).
Mean SD Lower limit Upper limit
Batch 1
Batch 2
Batch 3
390.2 2.8 370.7 409.7
393.2 1.9 373.5 412.8
393.6 1.1 373.9 413.2
The 400 mg of powder was transferred into a tableting 1 cm (diameter) stainless punch and die set. Each tablet was prepared individually using a manual hydraulic disc press (Specac, Kent) by applying fourteen-tons (137.3 kN) of force. The pressure was held for 1 min before it was released using the pressure valve. The formed tablet was ejected from the die and placed into a sealed glass vial.
2.2.7. Uniformity of mass of tablets Following BP recommendations (the mass should vary by ≤ ± 5% when the tablets are 250 mg or more), 20 tablets were weighed and their average mass calculated (British Pharmacopoeia Commission).
2.2.4. DNA tag analysis/sample authentication For tablets, a small area of the surface was swabbed with the tip of a sterile cotton swab that had been dipped in ultra-pure water. Swabbing was carried out using ten ‘up and down’ swabbing actions. This was repeated on two other areas of the same tablet with new swabs to ensure that each sample was assayed in triplicate. The swab tips were then cut and placed directly into tubes containing 18 μL PCR mix. For the powder samples, 100 mg powder was partially dissolved in 5 mL ELGA purified water (conductance − 18.2 MΩ) by vortex mixing the suspended particles. It was assumed that the un-dissolved material comprised a high proportion of magnesium stearate and a fraction of MCC, with the lactose fully dissolved, due to the high aqueous solubility of the sugar. The sample was then centrifuged for 5 min at 10,000 RPM (≅7000g) and 2 µL of the supernatant was transferred to a new tube containing 18 µL of the PCR mix. The samples were analysed and thus authenticated by the Applied DNA SigNify® qPCR instrument. For all DNA authentications, the samples were run directly on the portable Applied DNA SigNify® qPCR instrument. The instrument was set to heat up at 3 °C/min to 95 °C, hold for 10 s, cool at 1.5 °C/s to 60 °C and hold for 30 s. Heating cycles are used to allow temperature-dependent reactions to occur, amplifying the amount of DNA present. Thirty heating cycles were used to reduce run time since all the samples that have been tested could be quantified within less than 30 cycles. The Cq value represents the cycle at which the fluorescence passes the limit of detection, that is, the threshold where the signal to noise ratio is exceeded. Thus, if the concentration of the DNA tag recovered is high, the number of required cycles for detection (Cq value) is low. The number of cycles could be increased if required to detect lower concentrations of the DNA tag. Standards used were 0.1 pg/mL, 1 pg/mL, 10 pg/mL and 100 pg/mL created by serial dilution of the supplied MD125-15-50 clone. This clone was also used as a positive control. The Cq value (number of cycles to reach the detection threshold) was calculated by the qPCR instrument’s analysis programme. Negative controls comprised a representative aliquot of PCR mix. The latter were included to ensure that the reagents were not contaminated with the target DNA clone. If contamination had occurred, the negative controls would be expected to show a Cq value (i.e. amplification of the DNA present would occur). Where DNA was not present above the threshold levels at the end of the PCR cycles performed, no Cq value was recorded.
3. Results 3.1. Pharmacopoeia testing of lactose tablets Three batches of lactose tablets were manufactured each comprising of sixty tablets. The uniformity of weight, hardness and friability of each batch was determined. The uniformity of content was determined by weighing all sixty tablets of each batch (Table 1) and each batch passed the BP specifications. The tablets also passed the friability test (Table 2) where none of the batches of ten tablets lost more than 1% of their weight. The hardness of the tablets was also measured and summarised below (Table 3) The manufactured lactose tablets, having an average hardness of 22–28 kg, could be designated as ‘hard’ (British Pharmacopoeia Commission). In summary, the tablets were deemed to be of a high standard and passed the monograph specifications of the British Pharmacopoeia. The same tests were repeated for samples that were used in the stability study and all batches remained within the specified limits. 3.2. DNA tag recovery from swabbed lactose tablets The DNA tag recovery data comprised a series of plots showing the resultant fluorescent intensity of the replicated DNA tag as a function of the number of enzyme reactions, i.e. DNA replication cycles. In all of the recovery plots, the number of cycles required for DNA detection (Cq values) increased as a function of storage time, which indicated a reduction in tag levels, but it should be noted for all storage experiments the tag was clearly detected, (Fig. 2). Negative controls, whereby no DNA tag was included in the sample under investigation clearly showed no reaction as evidenced by the lack of fluorescent detection. Therefore, no cross contamination was observed during this work. The Cq values obtained from the swabbing experiments displayed a high repeatability between batches as a function of time over the six month study period
2.2.5. Stability testing Three batches of 60 tablets were manufactured using the manual press. All the samples were stored in glass vials for 3 and 6 months without lids following the ICH accelerated stability conditions set at 40 °C and 75% RH using a Memmert HPP110 controlled temperature and humidity chamber. The temperature and humidity of the samples during storage were recorded. All samples were authenticated using the qPCR instrument (Section 2.2.4). Results were analysed for statistical difference using a Student’s t-test.
Table 2 BP friability test results for manufactured lactose tablets (n = 10 for each batch).
3
Batch
Weight of tablets before test (g)
Weight of tablets after test (g)
Weight loss (g)
Weight loss (%)
1 2 3
3.87 3.91 3.93
3.86 3.90 3.92
0.01 0.01 0.01
0.17 0.20 0.20
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
indicating a decrease in DNA recovery. In addition, there was some loss in the precision of the assay. The differences between the assay values taken over the period of the assay proved to be statistically significant (p < 0.05).
Table 3 BP hardness (kg) test results for lactose tablets (n = 10) for each batch. Sample
Batch 1
Batch 2
Batch 3
mean SD
28.1 4.9
24.2 2.5
22.0 2.1
4. Discussion This study has presented the first report of the use of a DNA as a PCID to tag an excipient which was subsequently employed to manufacture a tablet. Lactose frequently occurs in tablets and many other formulations and was used in this study as a ‘model’ excipient because it was found in the top 200 prescriptions of tablets and capsules, with the disaccharide present as a filler in 77 out of the 200 (Dave, 2008; Gabbott et al., 2003). Applying a DNA tag to lactose as it is manufactured would allow the lactose to be easily traced confirming the authenticity of the product by the commercial manufacturer. In this study, lactose tablets were manufactured to be used as models for API-containing solid-dosage forms. Using the DNA tag to monitor the authenticity of a medicine by mixing with tagged powdered excipients, would be less chemically demanding than if the marker were simply mixed with the individual API. Finding optimum mixing parameters and techniques to fully incorporate the DNA tag into a test excipient powder was an integral part of the preliminary work for this study. The high precision and inter-batch repeatability showed that the developed methodology allowed the DNA to be apparent and equally distributed within the whole tablet. Some of the high stress environments routinely experienced during processing, such as mixing, drying, and compression during tableting were replicated in this study. Pharmacopeia tests were conducted at intervals to determine whether tablets remained within specified official limits. It is apparent that the DNA tag can be amplified and detected after storage of the tablets at elevated temperatures and humidities for 6 months, demonstrating the viability of the methods as a possible anti-counterfeiting strategy.
with low coefficient of variation (COV = 2.4–3.7%). There was an increase in the Cq value by about 5 cycles in the month 3 samples compared to those obtained initially (month 0); however, the difference was found to be less between the 3 and 6 month samples (around 1 Cq value). 3.3. DNA tag recovery from crushed lactose tablets Similar to the swabbed tablet results, there was found to be an increase in the Cq value that was higher between the 0 and 3 month samples than the increase between the 3 and 6 month samples (Fig. 3). The Cq repeatability was greatest when the tablets were crushed immediately after manufacture (COV = 1.3%) but this increased after storage for 6 months at 40 °C and 75% RH (COV = 3.6%). 3.4. Evaluation of the swabbed and crushed tablet methods A difference was apparent in the reproducibility between the swabbing method and the crushed tablet method of analysis immediately after the tablet manufacture (Fig. 4). The narrow distribution of data obtained using the methodology for analyzing crushed tablets clearly exhibited a greater precision than those from the method which involved swabbing the tablets. From the histogram, the range of the results from swabbing the tablets was much larger (19.5–22.3 Cq) than when the tablets were crushed prior to assay (20.2–21.2 Cq). Both sets of data show good precision for the methodology used. Fig. 5 depicts the combined batch data as a histogram to show the differences in Cq as a function of storage time for the crushed tablets. As storage time was increased, clearly the Cq value increased but the longer the stability study, the slower the rate at of increase,
4.1. Analysis of DNA tags Despite the use of DNA as a tag being in its relative infancy, several applications of the technology have been outlined (Bar-Or et al., 2006). Fig. 2. Amplification curves for all swabbed tablets analysed at 0 (red), 3 (black) and 6 (green) months were overlayed, including negative controls (grey) and the summary of DNA analysis of all swabbed tablets carried out in triplicate from 3 batches (n = 3) at month 0, 3 and 6 months in embedded table (mean ± SD). Storage conditions were 40 °C & 75% RH. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 3. Amplification curves for all crushed tablets analysed at 0 (red), 3 (black) and 6 (green) months, including negative controls (grey) and the summary of DNA analysis of all swabbed tablets carried out in triplicate from 3 batches (n = 3) at month 0, 3 and 6 months in embedded table. Storage conditions were 40 °C & 75% RH. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
target DNA sequence concentration in the starting solution. As the starting DNA concentration increased, less cycles were required until the DNA concentration could be ‘visualised’, resulting in a lower Cq value being obtained in the analysed sample.
For example, it has been advocated as a method of labeling milk along its production pathway in the manufacture of secondary dairy products (e.g. cheese and yoghurt) and has a practical use in security tagging (using spray solutions) with a view to combat theft, robbery and vandalism (e.g. SelectDNA®, 2019; Applied DNA Sciences, 2019). The central concept underpinning these applications is that by synthesising or isolating specific sequences of DNA and making them sufficiently stable to withstand the environment for a prolonged time, they can be used as a unique code that can be amplified with the use of PCR so that they can subsequently be detected and authenticated. However, no studies using DNA tagging technology have been published with regards to labelling formulated medicines. One reason for the lack of published material is likely to be the commercially sensitive nature of anti-counterfeiting work. Any approach requires both a specific and unique DNA sequence and an analytical technique that can subsequently identify that sequence. PCR is routinely used to amplify, or increase, the amount of a specific DNA sequence for analysis. Here, the PCR mix comprising Taq polymerase, sequence specific primers and a fluorescent probe, is heated and cycled between ~65 and 95 °C. As the sample approaches boiling temperatures, the hydrogen bonds between the nucleotides in the DNA break down causing the DNA template to unwind from its double helix. Then the temperature is cooled and held at 55–65 °C, the exact temperature depending upon the primer length, where the DNA is annealed. During the annealing process, the primers, which are short DNA strands designed to attach to specific sequences in the DNA template, attach to the DNA strands. The nucleotide bases are each tagged with fluorescent markers that can be monitored using a fluorescent photometer or via a quantitative PCR (qPCR) instrument. Theoretically, each cycle will double the amount of DNA in the sample and this is usually repeated multiple times until there is sufficient fluorescence for a signal to be detected. Techniques such as qPCR, or real-time PCR, incorporate lasers and light sensing equipment to monitor the production of DNA fragments labelled with fluorescent tags as the number of such fragments produced increase with each cycle. The measurement method employed was based on the number of cycles needed for the DNA to be amplified to a concentration in the solution that allowed it to be detected by the qPCR instrument (Fraga et al., 2008). The Cq value represented the number of cycles that needed to be effected until the DNA amplification could be detected via the incorporated fluorescent markers, also termed the inflection point (Section 2.2.4). The Cq value can vary or change depending on the
4.2. Stability of DNA tags In this study SigNature® DNA was used as the molecular taggant. SigNature® DNA is manufactured by PCR, purified by anion exchange chromatography and provided in 10 mM Tris-hydroxymethyl amino methane plus 1 mM Ethylene diamine tetraacetic acid, pH 7.8 (Tris-EDTA or “TE”) buffer at a DNA concentration of ≥4.0 µg/mL. Although this may appear to be a dilute solution, it could be even further diluted at least one million-fold to provide a solution ≤100 ng/dose for inclusion in the batch, as a PCID. TE buffer is a commonly used in molecular biology to solubilize DNA while protecting it from degradation from heavy metals. The ≥4.0 µg/mL solution was aliquoted into the ethanolic solution and sprayed into the tableting mix (Section 2.2.2), the resulting concentration of the DNA clone was below 1 ng per tablet, at approximately 0.4 ng/tablet. The current study did not test specifically for the minimal amount of DNA required, but employed a standard concentration typically used for research purposes (Jung et al., 2019). Based on the results obtained, the concentration of DNA per tablet could be reduced by at least 10 times, and still produce satisfactory results. This value is likely to fall further in future studies as the tag and its concentration can easily be tailored for each new pharmaceutical application. It should be noted that either by swabbing or crushing, because of the required dilution steps within the methodology, the amount of DNA analysed by PCR is much less that the total present in the individual tablet. The DNA standards used as successful controls had a lower limit of 0.1 pg/mL (Section 2.2.4), thus in this study the minimal amount of DNA needed to allow identification falls within the 1 pg range. DNA can be degraded by the hydrolysis of the phosphodiester bonds. These bonds are the backbone of the DNA strands as they are the linkage bond between the 3′ carbon on one sugar and the 5′ carbon. Alternatively, DNA degradation pathway can occur by the oxidation of nucleotide bases. Despite the primary structure of DNA being susceptible to damage by elevated heat, temperatures for this to occur markedly are those above 100 °C (Karni et al., 2013), although biochemical catalyzed degradation can occur at lower temperatures (Lindahl, 1993). Such limitations have been overcome by developing coatings or treatments that allow DNA to be 5
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 4. A histogram that groups the samples according to the Cq values for all swabbed and crushed tablets at month 0 and the original amplification curves for swabbed (A) and crushed (B) tablets at month 0.
much less susceptible to such damage. For example, the addition of silica has been shown to act as an ideal material for encapsulation and provide exceptional barrier properties (Paunescu et al., 2013, 2016). Adding such silica-encapsulated DNA to milk before manufacturing cheese resulted in the barcode maintaining its integrity during the manufacturing process (Bloch et al., 2014). DNA robustness may also be increased by appropriate sequence selection and design. However, in this study, the addition of silica could have affected the tableting process and in addition its use might pose problems in justifying its inclusion in licensed commercial formulations. In any case, the results presented here demonstrated that the DNA tag employed showed high durability, which would be a prerequisite for utilization in anti-counterfeiting technology.
manufacture counterfeit dosage forms. It is possible that DNA could interact with components of the tablet i.e. the lactose, MCC or magnesium stearate and future studies should investigate whether this does occur. However, the DNA analysis method used in this study is not oriented around DNA quantification per se. The success of a run was determined by the Cq values of the samples which were compared with positive and negative results. It is possible to add specific concentrations of DNA to each assay set so as to generate a relevant calibration curve. However, the repeated calibration before each set of samples can be costly and counterproductive as it is not required to confirm whether DNA is successfully detected. In a pharmaceutical setting, crushing tablets before carrying out the authentication assay may be more suitable as this method of preparation would be expected to create a more homogeneous sample with less variation in the results as compared with the swabbing method. There was an increase in the Cq values after the tablets were stored at 40 °C and 75% RH. Some of the surface powder may interact with the surrounding moisture and cause the degradation of some DNA. In addition some possible loss of DNA tag could be due to the interaction with lactose (Figs. 2 and 3). It is possible that such an interaction might account for the results depicted in Fig. 6, where depurination may have
4.3. Tagging lactose tablets The uniformity of content and weight tests proved that tablets manufactured using this technique were of sufficient quality to pass the specifications indicated in the BP. The aspect ratio of the powder bed to the rotational end of the mixing device as well as a high energy output ensured the powdered components were well mixed. Such widely available domestic mixers can easily be employed by illicit suppliers to 6
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
Fig. 5. A histogram that groups the samples according to the Cq values for all crushed lactose tablets. 54 data points were available in each batch where six tablets were sampled 3 times and each assay carried out in triplicate. Storage conditions were 40 °C & 75% RH.
occurred, resulting in higher Cq values after storage. DNA-lactose tagging tests proved that a very small amount of DNA tag is very sensitive and detected easily as shown by the technique of swabbing tablets. Although a small amount (pg) of DNA was at the surface of the tablet, the Cq value remained below 30, which provided a clear positive result using the portable qPCR instrument. Such swabbing methodology may be useful for in-field testing and for forensic authentications. Crushing the tablet would be expected to provide a more efficient method of ensuring tablet authentication and so it proved (lower Cq values being obtained). Such a method would be advantageous for inhouse assessment of tablets. Molecular tagging technologies can impart identity and traceability to solid oral dosage forms and could also be employed to tag associated
packaging (Bar-Or et al., 2006) to be used as layered security throughout a supply chain. Such approaches are important strategies to counter the increasing problem of counterfeit medicines. Lactose does appear to be a good model excipient with which to associate the DNA tag and further development of this method is likely to be achievable at relatively small cost due to the very high sensitivity of the method (pg/ mL levels of DNA being required). The pharma industry would require different “bar codes” for different types of excipient, manufacturer, time and date of manufacture and also for different batches of the excipient in question. In the current example, the number of possible combinations of DNA analysis by PCR is very high. The combinations available are equal to four (=bases of DNA) raised to the power of x, whereby x is the total number of bases of
Fig. 6. Using adenine as an example, the interaction between lactose and DNA resulting in the loss of the adenine structure. 7
International Journal of Pharmaceutics 571 (2019) 118656
M.J. Altamimi, et al.
the primer and probe sequences. This high versatility of the coding allows the implementation of cryptography for the protection of the barcode and hence provides exceptional promise. Separate DNA molecular taggants might be added to different APIs, fillers, coatings, disintegrants, lubricants, glidants, inks, capsule shells etc. of an SODF to uniquely identify a medicine’s complete authenticity and traceability. The DNA molecular tags used to infuse goods with unique identification for traceability in pharmaceuticals are orders of magnitude smaller than the human genome, typically less than 200 bp in length (Murrah, 2010). This PCID identifier could be used like a license plate, referring to much more contextual information in a database that might, for example, include producer, manufacturing facility, year of manufacture or other relevant information. DNA molecular taggants offer several benefits over other forms of PCIDs. DNA is a common molecule that is highly resolvable due to the fact that while it can be used in femtogram-level (10−15 g) trace amounts in pharmaceutical applications, either in-lab or in-field devices supporting PCR processes can use one molecule extracted from the SODF or packaging to amplify to millions of molecules and analyse within 30 to 45 min. In addition, cost-effective large-scale production is achievable using the same PCR technology to support the potentially large amounts of DNA required by the industry (Murray et al., 2012). DNA molecular tags can be formulated to be compatible with their host carriers and to be protected against environmental or process challenges, yet also to be extractable for authentication purposes.
References Altamimi, M.J., Wolff, K., Nokhodchi, A., Martin, G.P., Royall, P.G., 2019. Variability in the a and beta anomer content of commercially available lactose. Int. J. Pharm. 555, 237–249. Andres, J., Hersch, R.D., Moser, J.E., Chauvin, A.S., 2014. A new anti-counterfeiting feature relying on invisible luminescent full color images printed with lanthanide-based inks. Adv. Funct. Mater. 24, 5029–5036. Applied DNA Sciences, 2019. https://adnas.com/security-and-asset-marking-crime-prevention/ (accessed 11.03.19). Bar-Or, Y.A., Scheibe, P.O., Plaxco, K.W., Thomas, A.J., 2006. Method, apparatus, and system for authentication using labels containing nuleotide sequences US patent application: US 2006/0286569 A1. Barcaccia, G., Lucchin, M., Cassandro, M., 2015. DNA barcoding as a molecular tool to track down mislabeling and food piracy. Diversity 8, 2. Blackstone, E.A., Fuhr Jr, J.P., Pociask, S., 2014. The health and economic effects of counterfeit drugs. Am. Health Drug Benefits 7, 216. Bloch, M.S., Paunescu, D., Stoessel, P.R., Mora, C.A., Stark, W.J., Grass, R.N., 2014. Labeling milk along its production chain with DNA encapsulated in silica. J. Agric. Food Chem. 62, 10615–10620. British Pharmacopoeia Commission, 2019. British Pharmacopoeia 2019. TSO, London. Code of Federal Regulations, 2018. 21 CFR 170.30, April 1, 2018. Chapter 1 – Food and Drug administration, Department of Health and Human Services, Subchapeter B – Food for human comsumption. Food Additives. Title 21, Volume 3. https://www.accessdata.fda. gov/scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?fr=170.30 (accessed 04.02.19). Cozzella, L., Simonetti, C., Spagnolo, G.S., 2012. Drug packaging security by means of whitelight speckle. Opt. Lasers Eng. 50, 1359–1371. Dave, R.H., 2008. Overview of pharmaceutical excipients used in tablets and capsules. Drug Topics 24, 1–13. Dégardin, K., Guillemain, A., Klespe, P., Hindelang, F., Zurbach, R., Roggo, Y., 2018. Packaging analysis of counterfeit medicines. Forens. Sci. Int. 291, 144–157. https://doi.org/10.1016/ j.forsciint.2018.08.023. FDA Federal Register, 1992. Statement of Policy – Foods Derived from New Plant Varieties. Volume 57-1992 https://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/biotechnology/ucm096095.htm (accessed 04.02.19). Ferenczi-Fodor, K., Végh, Z., Renger, B., 2006. Thin-layer chromatography in testing the purity of pharmaceuticals. TrAC Trends Anal. Chem. 25, 778–789. Fraga, D., Meulia, T., Fenster, S., 2008. Real-time PCR. In: Current Protocols Essential Laboratory Techniques. John Wiley & Sons Inc, pp. 10.3.1–10.3.34. https://doi.org/10. 1002/9780470089941.et1003s00. Gaudiano, M.C., Manna, L., Bartolomei, M., Rodomonte, A.L., Bertocchi, P., Antoniella, E., Romanini, L., Alimonti, S., Rufini, L., Valvo, L., 2016. Health risks related to illegal and online sale of drugs and food supplements: results of a survey on marketed products in Italy from 2011 to 2013. Annali dell'Istituto superiore di sanita 52, 128–132. Glass, B.D., 2014. Counterfeit drugs and medical devices in developing countries. Res. Rep. Trop. Med. 5, 11–22. Gabbott, P., Clarke, P., Mann, T., Royall, P., Shergill, S., 2003. A high-sensitivity, high-speed DSC technique: measurement of amorphous lactose. Am. Lab. 35, 16–17. Hayward, J. A., Hogan, M. E., Liang, M. B., Jung, L., 2018. Method for authenticating active pharmaceutical ingredients: Patent Application US20180142294A1. Joint Formulation Committee, 2009. British National Formulary (BNF) September 2009. Pharmaceutical Press, London. Johnston, A., Holt, D.W., 2014. Substandard drugs: a potential crisis for public health. Brit. J. Clin. Pharmacol. 78, 218–243. Jung, L., Hogan, M.E., Sun, Y., Liang, B.M., Hayward, J.A., 2019. Rapid authentication of pharmaceuticals via DNA tagging and field detection. PLoS One 14, 1–11. https://doi.org/ 10.1371/journal.pone.0218314. Karni, M., Zidon, D., Polak, P., Zalevsky, Z., Shefi, O., 2013. Thermal degradation of DNA. DNA Cell Biol. 32, 298–301. Lavorgna, A., 2015. The online trade in counterfeit pharmaceuticals: new criminal opportunities, trends and challenges. Eur. J. Criminol. 12, 226–241. https://doi.org/10.1177/ 1477370814554722. Lindahl, T., 1993. Instability and decay of the primary structure of DNA. Nature 362, 709–715. Mackey, T.K., Liang, B.A., 2011. The global counterfeit drug trade: patient safety and public health risks. J. Pharm. Sci. 100, 4571–4579. Murrah, J., 2010. DNA molecular taggants: a hidden key to pharmaceutical patient safety. Tablets Capsules 16, 33–36. Murray E, Gregg DA, Norton ML, Swick JT, Towler WI., 2012. Method for a continuous rapid thermal cycle system. US Patent 8,163,489. Awarded April 24, 2012. Osborn-Gustavson, A.E., McMahon, T., Josserand, M., Spamer, B.J., 2018. Chapter 12 – the utilization of databases for the identification of human remains A2 – Latham, Krista E. In: Bartelink, E.J., Finnegan, M. (Eds.), New Perspectives in Forensic Human Skeletal Identification. Academic Press, pp. 129–139. Paunescu, D., Fuhrer, R., Grass, R.N., 2013. Protection and deprotection of DNA—high-temperature stability of nucleic acid barcodes for polymer labeling. Angew. Chem. Int. Ed. 52, 4269–4272. Paunescu, D., Stark, W.J., Grass, R.N., 2016. Particles with an identity: tracking and tracing in commodity products. Powder Technol. 291, 344–350. Perks, B., 2018. Pharmaceutical fakes: a dangerous pandemic. Lung Cancer 15, 05. SelectDNA®, 2019. https://www.selectadna.co.uk (accessed 04.02.19). Taylor, C., 2015. Marking material (pp. 1-4): US Patent: US9171443B2.
5. Conclusion DNA tagging is a novel prospect that could revolutionise law enforcement and indeed has already been used in a multitude of other fields (Applied DNA Sciences, 2019, Taylor, 2015; SelectDNA®, 2019). Many studies have used DNA tagging as an authentication method, but none have used the direct inclusion of a DNA tag into a powder excipient mix. In the current study, the DNA tag could be extracted and characterized after application of fourteen-tons (137.3 kN) of force to form a tablet, and withstood storage at storage at high temperature and humidity. DNA tags are Generally Recognized as Safe (GRAS) through “experience based on common use in food” since before January 1, 1958 (Code of Federal Regulations, 2018). The FDA’s Statement of Policy “Food Derived from New Plant Varieties (FDA Federal Register, 1992)” addresses its view that transferred genetic material is considered GRAS based upon DNA's omnipresence in food. The DNA molecular tags, and the processing technology investigated here, produced traceable tablets that could withstand accelerated ICH recommended storage conditions. On the basis of these results it would appear that DNA molecular tags could feasibly be applied within the pharmaceutical development cycle when a new medicine is brought to the market so as to mitigate the risk and dangers of counterfeiting. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2019.118656.
8