In a press release published on Monday, the American Association for Laboratory Accreditation (A2LA), announced the renewal of accreditation for Global Laboratory Services, Inc. for ISO 17025:2017 in cannabis testing. The laboratory, based in Wilson, North Carolina, becomes first cannabis testing laboratory accredited in that state by adding the industrial hemp testing to their chemical scope of accreditation.
According to Kim Hesse, business development manager at Global Laboratory Services, they plan to expand their services in the hemp market with additional types of hemp testing. “At Global Laboratory Services, we always strive to keep pace with industry needs,” says Hesse. “We saw the need for an accredited laboratory in the hemp industry and therefore added CBD and THC testing to our scope. Our next step is to expand our service offerings to include agrochemical analysis of industrial hemp.”
Adam Gouker, general manager at A2LA, says accreditation plays a vital role in the cannabis industry and its regulatory requirements. “We congratulate Global Laboratory Services on becoming the first cannabis testing laboratory accredited in the state of North Carolina, specifically for industrial hemp,” says Gouker. “A2LA realizes the vital role that accreditation plays in the cannabis industry to support compliance with regulatory requirements, and we are thrilled to see that our service has been adopted in a new state. We look forward to our continued relationship with Global Laboratory Services in the provision of their accreditation needs.”
It is easy to forget as one steps inside this world-class medical conference (held this year in Berlin), that cannabis is disputed as medicine anywhere in the world.
Inside a packed conference hall in an upscale hotel in East Berlin, international researchers presented evidence that when taken as a drug, this simple plant can make a world of difference to patients suffering from a range of illnesses.
There were also doctors who talked about prescribing this as medicine (even to children), with dramatic and affirming results (if not heart-warming pictures).
In sum, as always, the IACM is the best place to find facts if not evidence galore to convince even the most hard-boiled egghead that this drug works – and across a broad range of so far “other” drug-resistant medical conditions.
As a participant in the IACM said after the opening remarks on the very first morning, it is so easy to ask the question – “Why are doctors still so afraid of if not resistant to this drug?”
Medical efficacy is no longer an unanswered question…
For those seeking affirmation and evidence, this year’s IACM did not disappoint. There were presentations on the drug’s impact on neurological, oncological and inflammatory conditions that while not all new, are increasingly impactful in an aging planet.
A full house of attendees for the patients panel
But that is not all that was discussed. The broader implications of adding cannabis into skincare, diets and medicine chests were also presented – from cannabis’ impact on lowering obesity and positively affecting acne to impacting the opioid epidemic.
Also intriguing this year was a far-reaching study on how polluted the CBD supply chain is in Europe, even for non-medical and nutraceutical products. Not to mention a socio-political plea for legalization of personal use in South Africa.
And that was just the presentations from the stage and in the poster hall.
The conversations swirling around were just as interesting. Because of course, nobody at this three-day gathering, for all the normalization on display, did forget that this gathering of doctors, scientists, cannabis companies and patients is still an anomaly.
The fact is that there are still too few doctors prescribing. And too few trials. And too many fights over efficacy still in the room.
As Alice O’Leary Randall (wife and former partner in activism with her late husband, Professor Randall who initiated the medical efficacy fight in the U.S. in 1975 over glaucoma) said to Cannabis Industry Journal, “It is hard to believe that we are still fighting the same fights all over again.”
Another “AIDS” Crisis?
There is a more dramatic sense of urgency at the IACM than other conferences that focus just on the “business.” In part, this is because the conference is made up of not only doctors and researchers who fight to prescribe the drug or get trials funded, but also patients on the front lines in a country where the drug is supposed to be covered by health insurance.
Dr. Franjo Grotenhermen (seen on the table) and others during a panel discussion
The patient panel, as a result, was an international face of accusation: To national authorities who still refuse to mandate cannabinoid care – across Europe and beyond. To medical establishments who are not demanding cannabinoid treatment be made mandatory in hospitals and emergency rooms in every country in the EU and beyond. To individual doctors who refuse to come to such conferences, where, if they wanted to, could learn how to begin prescribing the “next penicillin.” To payers and insurers who are still too slow to pick up the message if not the tab.
Indeed, one of the best panels of the conference was a gaggle of doctors, led by Grotenhermen, who discussed the particulars of approaching a new drug – for the very first patient and first time.
Act Up, Speak Out, Silence Equals Death
As the conference wrapped up with its awards dinner, there was of course, a sense of needing to go home with not wanting this to end. For those in the thick of this multi-generational fight, there of course were words of encouragement to colleagues from the industry, internationally. But there was also a new sense of needing to up the pace, if not create faster change.
The battles are far from over – in fact, they are just beginning in many places. As one questioner said of a panel about halfway through the conference – “We need to pick up the fight the same way the AIDS community did on this drug.”
That remark perhaps means less today than it did 20 to 30 years ago when an embattled LGBTQ subculture was the organized point of the spear that fought the early state legalization battles as pioneers for a cause that sought equality as much as it sought a cure.
The plea did not fall on deaf ears.
In the midst of studies, statistics and scientific evidence, in other words, there was a new sense of a need for a renewed fight – and from the medical and scientific community as well as patients.
Across the country and across the world, governments that legalize cannabis implement increasingly rigorous requirements for laboratory testing. Helping to protect patients and consumers from contaminants, these requirements involve a slew of lab tests, including quantifying the levels of microbial contaminants, pathogens, mold and heavy metals.
Cannabis and hemp have a unique ability to accumulate elements found in soil, which is why these plants can be used as effective tools for bioremediation. Because cannabis plants have the ability to absorb potentially toxic and dangerous elements found in the soil they grow in, lab testing regulations often include the requirement for heavy metals testing, such as Cadmium, Lead, Mercury, Arsenic and others.
In addition to legal cannabis markets across the country, the USDA announced the establishment of the U.S. Domestic Hemp Production Program, following the enactment of the 2018 Farm Bill, essentially legalizing hemp. This announcement comes with information for hemp testing labs, including testing and sampling guidelines. While the information available on the USDA’s website only touches on testing for THC, required to be no greater than 0.3% dry weight concentration, more testing guidelines in the future are sure to include a discussion of heavy metals testing.
Table 1. ICP-MS operating conditions (shaded parameters were automatically optimized during start up for the HMI conditions).
In an application note produced by Agilent Technologies, Inc., the Agilent 7800 ICP-MS was used to analyze 25 elements in a variety of cannabis and hemp-derived products. The study was conducted using that Agilent 7800 ICP-MS, which includes Agilent’s proprietary High Matrix Introduction (HMI) system. The analysis was automated by using the Agilent SPS 4 autosampler.
Instrumentation
The instrument operating conditions can be found in Table 1. In this study, the HMI dilution factor was 4x and the analytes were all acquired in the Helium collision mode. Using this methodology, the Helium collision mode consistently reduces or completely eliminates all common polyatomic interferences using kinetic energy discrimination (KED).
Table 2. Parameters for microwave digestion.
As a comparison, Arsenic and Selenium were also acquired via the MassHunter Software using half-mass correction, which corrects for overlaps due to doubly charged rare earth elements. This software also collects semiquantitative or screening data across the entire mass region, called Quick Scan, showing data for elements that may not be present in the original calibration standards.
SRMs and Samples
Standard reference materials (SRMs) analyzed from the National Institute of Standards and Technology (NIST) were used to verify the sample prep digestion process. Those included NIST 1547 Peach Leaves, NIST 1573a Tomato Leaves and NIST 1575 Pine Needles. NIST 1640a Natural Water was also used to verify the calibration.
Figure 1. Calibration curves for As, Cd, Pb, and Hg.
Samples used in the study include cannabis flower, cannabis tablets, a cannabidiol (CBD) tincture, chewable candies and hemp-derived cream.
Sample Preparation
Calibration standards were prepared using a mix of 1% HNO3 and 0.5% HCl. Sodium, Magnesium, Potassium, Calcium and Iron were calibrated from 0.5 to 10 ppm. Mercury was calibrated from 0.05 to 2 ppb. All the other elements were calibrated from 0.5 to 100 ppb.
Table 3. Calibration summary data acquired in He mode. Data for As and Se in shaded cells was obtained using half mass correction tuning.
After weighing the samples (roughly 0.15 g of cannabis plant and between 0.3 to 0.5 g of cannabis product) into quartz vessels, 4 mL HNO3 and 1 mL HCl were added and the samples were microwave digested using the program found in Table 2.
HCI was included to ensure the stability of Mercury and Silver in solution. They diluted the digested samples in the same acid mix as the standards. SRMs were prepared using the same method to verify sample digestion and to confirm the recovery of analytes.
Four samples were prepared in triplicate and fortified with the Agilent Environmental Mix Spike solution prior to the analysis. All samples, spikes and SRMs were diluted 5x before testing to reduce the acid concentration.
Calibration
Table 4. ICV and CCV recovery tests. Data for As and Se in shaded cells was obtained using half mass correction tuning.
The calibration curves for Arsenic, Cadmium, Lead and Mercury can be found in Figure 1 and a summary of the calibration data is in Table 3. For quality control, the SRM NIST 1645a Natural Water was used for the initial calibration verification standard. Recoveries found in Table 4 are for all the certified elements present in SRM NIST 1640a. The mean recoveries and concentration range can also be found in Table 4. All the continuing calibration solution recoveries were within 10% of the expected value.
Internal Standard Stability
Figure 2 highlights the ISTD signal stability for the sequence of 58 samples analyzed over roughly four hours. The recoveries for all samples were well within 20 % of the value in the initial calibration standard.
Figure 2. Internal standard signal stability for the sequence of 58 samples analyzed over ~four hours.
Results
In Table 5, you’ll find that three SRMs were tested to verify the digestion process. The mean results for most elements agreed with the certified concentrations, however the results for Arsenic in NIST 1547 and Selenium in both NIST 1547 and 1573a did not show good agreement due to interreferences formed from the presence of doubly-charged ions
Table 5. Mean concentrations (ppm) of three repeat measurements of three SRMs, including certified element concentrations, where appropriate, and % recovery.
Some plant materials can contain high levels of rare earth elements, which have low second ionization potentials, so they tend to form doubly-charged ions. As the quadrupole Mass Spec separates ions based on their mass-to-charge ratio, the doubly-charged ions appear at half of their true mass. Because of that, a handful of those doubly-charged ions caused overlaps leading to bias in the results for Arsenic and Selenium in samples that have high levels of rare earth elements. Using half mass correction, the ICP-MS corrects for these interferences, which can be automatically set up in the MassHunter software. The shaded cells in Table 5 highlight the half mass corrected results for Arsenic and Selenium, demonstrating recoveries in agreement with the certified concentrations.
In Table 6, you’ll find the quantitative results for cannabis tablets and the CBD tincture. Although the concentrations of Arsenic, Cadmium, Lead and Cobalt are well below current regulations’ maximum levels, they do show up relatively high in the cannabis tablets sample. Both Lead and Cadmium also had notably higher levels in the CBD tincture as well.
Table 6. Quantitative data for two cannabis-related products and two cannabis samples plus mean spike recovery results. All units ppb apart from major elements, which are reported as ppm.
A spike recovery test was utilized to check the accuracy of the method for sample analysis. The spike results are in Table 6.
Using the 7800 ICP-MS instrument and the High Matrix Introduction system, labs can routinely analyze samples that contain high and very variable matrix levels. Using the automated HMI system, labs can reduce the need to manually handle samples, which can reduce the potential for contamination during sample prep. The MassHunter Quick Scan function shows a complete analysis of the heavy metals in the sample, including data reported for elements not included in the calibration standards.
The half mass correction for Arsenic and Selenium allows a lab to accurately determine the correct concentrations. The study showed the validity of the microwave sample prep method with good recovery results for the SRMs. Using the Agilent 7800 ICP-MS in a cannabis or hemp testing lab can be an effective and efficient way to test cannabis products for heavy metals. This test can be used in various stages of the supply chain as a tool for quality controls in the cannabis and hemp markets.
Disclaimer: Agilent products and solutions are intended to be used for cannabis quality control and safety testing in laboratories where such use is permitted under state/country law.
Secretary Perdue made the announcement in a YouTube video titled “USDA’s Hemp Policy.” Later in the week, an interim final rule formalizing the program will be published in the Federal Register, according to the USDA’s website. “The rule includes provisions for the U.S. Department of Agriculture (USDA) to approve hemp production plans developed by states and Indian tribes including: requirements for maintaining information on the land where hemp is produced; testing the levels of delta-9 tetrahydrocannabinol; disposing of plants not meeting necessary requirements; and licensing requirements,” reads the press release. “It also establishes a federal plan for hemp producers in states or territories of Indian tribes that do not have their own approved hemp production plan.” The interim final rule will go into effect as soon as it is published in the Federal Register, which should be by the end of this week.
You can find a preview of the rule here. The agency has also developed guidelines for sampling and testing procedures, which you can find here. Those documents are meant to provide more information for hemp testing laboratories.
You can watch the YouTube video and read the announcement he made below:
Hello everyone, as I travel across this great country of ours, I hear a lot about a strong interest in a new economic opportunity for America’s farmers: the production of hemp. Which is why today I am pleased to announce the USDA has published the rule establishing the US domestic hemp production program. We said we’d get it done in time for producers to make planning decisions for 2020 and we followed through. We have had teams operating with all hands-on-deck to develop a regulatory framework that meets Congressional intent while seeking to provide a fair, consistent and science-based process for states, tribes, and individual producers who want to participate in this program. As mandated by Congress, our program requires all hemp growers to be licensed and includes testing protocols to ensure that hemp grown under this program is hemp and nothing else. The USDA has also worked to provide licensed growers access to loans and risk management products available for other crops. As the interim final rule, the rule becomes effective immediately upon publication in the federal register. But we still want to hear from you. Help us make sure the regulations meet your needs. That’s why the publication of the interim final rule also includes a public comment period continuing a full and transparent rulemaking process that started with a hemp listening session all the way back in March 2019. At USDA, we are always excited when there are new economic opportunities for our farmers and we hope the ability to grow hemp will pave the way for new products and markets. And I encourage all producers to take the time to fully educate themselves on the processes, requirements and risk that come with any market or product before entering this new frontier. The Agricultural Marketing Service will be providing additional information, resources and educational opportunities on the new program. And I encourage you to visit the USDA hemp website for more information. As always, we thank you for your patience and input during this process.
Both labs are located in Streetsboro, Ohio, becoming A2LA’s first accredited labs in the state. North Coast Testing does cannabis testing for Ohio’s medical cannabis industry, whereas North Coast Analytical does testing for the hemp industry.
Carolyn Friedrich, Ph.D., scientific director at North Coast Testing, says they are excited to help ensure the safety of patients for Ohio’s medical cannabis program. “We are extremely proud of the work of our entire team in rapidly developing and implementing a comprehensive quality management program that can give all participants in the Ohio Medical Marijuana Control Program confidence in the quality and safety of products tested in our laboratory,” says Friedrich.
Nick Szabo, laboratory director at North Coast Analytical, says A2LA went “above and beyond at every step, we greatly appreciate their efforts. Our accreditation by A2LA is a testament to our ability to meet the most rigorous quality management standards in analytical testing of hemp products, and a vote of confidence in our team’s ability to perform at the highest levels.”
According to a press release published back in September, Steep Hill announced their expansion to the state of Oklahoma. Steep Hill, a cannabis science company that started with cannabis testing labs in California, has been on an impressive expansion trajectory over the past few years.
Kandice Faulkenberry, co-owner and CEO of Steep Hill Oklahoma, says they hope to raise the bar for cannabis lab testing in Oklahoma. “With Oklahoma being one of the fastest-growing medical markets in the nation, we are excited and honored to be a part of our state’s growth,” says Faulkenberry. “We hope to be a valuable resource in our community and Oklahoma’s cannabis industry. Through our partnership with Steep Hill, the world’s leading cannabis science company, we aim to raise the bar in laboratory services, education, and product safety for the medical cannabis industry in the Sooner State.”
Dr. Chris Orendorff, the other co-owner of Steep Hill Oklahoma, is a family physician based in Sallisaw, Oklahoma. “As a physician, I understand that safety and regulations are critical to patient outcomes and I look forward to providing the same assurance for my patients and fellow Oklahoma residents in the cannabis industry,” says Dr. Orendorff. “I am excited to partner with Steep Hill to provide the highest quality testing in the State of Oklahoma.”
Project Yosemite, a cannabis product innovation and brand development company, announced earlier this month the appointment of Jennifer Raeder-Devens as their new Chief Scientific Officer. Raeder-Devens is a veteran of the MedTech industry, working for companies like Becton Dickinson, Cardinal Health, Medtronic and 3M.
Prior to joining Yosemite, she was the Vice President of Research & Development at Becton, Dickinson, where she oversaw product development and technology strategies to launch infection prevention products including the ChloraPrep first-in-the-US sterile solution patient preoperative topical antiseptic. She was previously the Vice President of R&D, Strategy and Innovation at Cardinal Health. She’s also held roles at Medtronic, 3M Drug Delivery Systems and 3M Skin Health Division and she has a number of patents in drug delivery and medical devices.
Jennifer Raeder-Devens, Chief Scientific Officer at Project Yosemite
In November of 2018, Project Yosemite launched their first product, OLO, which is an infused, controlled-release sublingual strip. Part of Raeder-Devens’ new role at the company is the continued development and expansion of the OLO sublingual strip technology platform. Andrew Mack, CEO and founder of Project Yosemite, says he’s thrilled to have Raeder-Devens on the team. “Jennifer is an extremely accomplished scientist and engineer with extensive experience driving innovation and R&D in the pharmaceutical and medical device industries,” says Mack.
We caught up with Jennifer over the phone to talk about her background in the MedTech space, why she decided to jump ship to join the cannabis industry and what she’s excited to work on now.
Cannabis Industry Journal: Can you tell us about your background, including your work with 3M and Medtronic?
Jennifer Raeder-Devens: I’m coming directly from Becton Dickinson, a global med tech company, where I supervised the development of drug-device combination products for topical antiseptics. I spent about 10 years there, mostly in topical drug and combination product development. Prior to that, I was at 3M and Medtronic working in drug-device combination products. At 3M, I was supervising a team of technology developers for the 3M Drug Delivery Systems business. I had experience working with designing and manufacturing transdermal, nasal, buccal and inhalation drug delivery mechanisms for pharmaceutical partners.
I worked on implantable drug delivery systems at Medtronic, which included working on the biocompatibility of things like pacemakers and drug infusion pumps and optimizing them to reduce infection and enhance healing after the implantation procedure.
CIJ: What made you consider joining the cannabis industry?
Jennifer: With my work in topicals, transdermal and inhalation drug delivery, I had an easy understanding of the different routes of administration we see today in the cannabis industry. And so, from the technology standpoint, I thought this was a place I could contribute to immediately. And then what got me really excited about it was thinking about cannabis, and just like any other drug, with oral drug delivery, you’ve got first class metabolism and side effects from the 11-Hydroxy-THC that are undesirable and you’d rather not have delivered through the gut.
OLO sublingual strips have a 10-minute onset time
I got excited when I saw the development of things like sublingual strips that were focusing on alternatives to smoking that would preserve that relatively fast onset and mitigate some of the side effects of edibles.
The other thing I really like about the cannabis industry: Previously I have been very focused on known drugs that are already approved and repurposing them into a new delivery system. What really interests me about the cannabis industry is the active cannabinoids and terpenes are somewhat known and somewhat unknown, so there is this really interesting challenge there of trying to separate the wheat from the chaff in terms of producing therapeutic effects.
It is a really interesting space where the indications of certain molecules are evolving along with the delivery technology. So, it is a really exciting and eye-opening way to take the next step in my career and have this wide-open space in front of me, both in terms of the different cannabinoids, their effects and the delivery systems we can use.
CIJ: How might you be prepared, given your background, for some of the challenges in the cannabis space?
Jennifer: I think the challenges in cannabis delivery are not different from the challenges in pharmaceutical drug delivery. It’s just that we have this additional complexity of the entourage effect. We can be engineering not just the main ingredient of THC, but also all the other cannabinoids and terpenes. So, for example, with my background in infection prevention, we build a product that we know reduces the risk of infection, but we are really challenged to actually prove it reduces the risk of infection. We have a similar situation in the cannabis industry, where we can get the THC, or CBG or CBN where we want it to go, but then we are really challenged to figure out how we can find, what we call in the pharmaceutical industry, a surrogate end point for efficacy, so that we can test that product and really believe that when we put the product on the market, even though we haven’t tested thousands of users or conducted large randomized clinical trials, that the effect will be shown. We are networking and partnering with a good scientific community to build the right product and do some testing at a small scale that really demonstrates the product achieves the effect that we are really looking for.
CIJ: Can you tell us a little about your new role with Project Yosemite?
Jennifer: My job description falls into three buckets: The first part is that we are forming a scientific advisory board and we are working with some of the leading cannabinoid researchers around the country and around the world. These are the people identifying whether or not certain cannabinoids could reduce cancer cell metabolism or whether cannabinoids contribute to weight loss or diabetes control and other things of that nature. We are trying to reach as far upstream as we can to grasp the emerging understanding of the performance of cannabinoids and terpenes in the endocannabinoid system. So, part of my job is to chair that scientific advisory board, get the thought leaders together in the room and have them bring their knowledge and explore with our own knowledge what cannabis can really do.
The OLO sublingual strips
I have worked in topical, transdermal, buccal, nasal, inhalation drug delivery. In the second bucket of my job, we are trying to understand a given indication or experience that our users want to have, what would be the right route for them. We are challenging our sublingual delivery mechanism to see how fast of an onset we can really get. Right now, we are at 10 minutes for drug delivery in sublingual and we are still trying to get an even faster onset time for the sublingual strip.
For other indications, like chronic pain, we may want to think about a sustained release, so sort of aligning the different indications with which different cannabinoids and terpenes will work for it and see which delivery platform will work for what we are trying to accomplish in each indication. So, we do not plan to remain solely a sublingual strip company, but will build out additional delivery platforms as we develop new indications.
Right now, we are working upstream with the growers and the processors to get cannabis oil and extracts. Some of the growers are working on different genetics in their cultivars to grow plants that have different ratios of different cannabinoids that we know from the emerging research will have an impact on people’s experience. Now we are working with growers to really get ahead of the curve on how to formulate products with various cannabinoids.
We have an R&D team in house that I supervise. We are always working with our production team to make small improvements such as the faster onset and the dissolution rate and things like flavors, which covers a downstream focus as well.
In 1887, Julius Petri invented a couple of glass dishes, designed to grow bacteria in a reproducible, consistent environment. The Petri dish, as it came to be known, birthed the scientific practice of agar cultures, allowing scientists to study bacteria and viruses. The field of microbiology was able to flourish with this handy new tool. The Petri dish, along with advancements in our understanding of microbiology, later developed into the modern field of microbial testing, allowing scientists to understand and measure microbial colonies to detect harmful pathogens in our food and water, like E. coli and Salmonella, for example.
The global food supply chain moves much faster today than it did in the late 19th century. According to Milan Patel, CEO of PathogenDx, this calls for something a little quicker. “Traditional microbial testing is tedious and lengthy,” says Patel. “We need 21st century pathogen detection solutions.”
Milan Patel first joined the parent company of PathogenDx back in 2012, when they were more focused on clinical diagnostics. “The company was predominantly built on grant funding [a $12 million grant from the National Institute of Health] and focused on a niche market that was very specialized and small in terms of market size and opportunity,” says Patel. “I realized that the technology had a much greater opportunity in a larger market.”
Milan Patel, CEO of PathogenDx Photo: Michael Chansley
He thought that other markets could benefit from that technology greatly, so the parent company licensed the technology and that is how PathogenDx was formed. Him and his team wanted to bring the product to market without having to obtain FDA regulatory approval, so they looked to the cannabis market. “What we realized was we were solving a ‘massive’ bottleneck issue where the microbial test was the ‘longest test’ out of all the tests required in that industry, taking 3-6 days,” says Patel. “We ultimately realized that this challenge was endemic in every market – food, agriculture, water, etc. – and that the world was using a 140-year-old solution in the form of petri dish testing for microbial organisms to address challenges of industries and markets demanding faster turnaround of results, better accuracy, and lower cost- and that is the technology PathogenDx has invented and developed.”
While originally a spinoff technology designed for clinical diagnostics, they deployed the technology in cannabis testing labs early on. The purpose was to simplify the process of testing in an easy approach, with an ultra-low cost and higher throughput. Their technology delivers microbial results in less than 6 hours compared to 24-36 hours for next best option.
The PathogenDx Microarray
Out of all the tests performed in a licensed cannabis testing laboratory, microbial tests are the longest, sometimes taking up to a few days. “Other tests in the laboratory can usually be done in 2-4 hours, so growers would never get their microbial testing results on time,” says Patel. “We developed this technology that gets results in 6 hours. The FDA has never seen something like this. It is a very disruptive technology.”
When it comes to microbial contamination, timing is everything. “By the time Petri dish results are in, the supply chain is already in motion and products are moving downstream to distributors and retailers,” Patel says. “With a 6-hour turnaround time, we can identify where exactly in the supply chain contaminant is occurring and spreading.”
The technology is easy to use for a lab technician, which allows for a standard process on one platform that is accurate, consistent and reproduceable. The technology can deliver results with essentially just 12 steps:
Take 1 gram of cannabis flower or non-flower sample. Or take environmental swab
Drop sample in solution. Swab should already be in solution
Vortex
Transfer 1ml of solution into 1.5ml tube
A look at how the sample is added to the microarray
Conduct two 3-minute centrifugation steps to separate leaf material, free-floating DNA and create a small pellet with live cells
Conduct cell lysis by adding digestion buffer to sample on heat blocks for 1 hour
Conduct Loci enhancement PCR of sample for 1 hour
Conduct Labelling PCR which essentially attaches a fluorescent tag on the analyte DNA for 1 hour
Pipette into the Multiplex microarray well where hybridization of sample to probes for 30 minutes
Conduct wash cycle for 15 minutes
Dry and image the slide in imager
The imager will create a TIFF file where software will analyze and deliver results and a report
Their DetectX product can test for a number of pathogens in parallel in the same sample at the same time down to 1 colony forming unit (CFU) per gram. For bacteria, the bacterial kit can detect E. coli, E. coli/Shigella spp., Salmonella enterica, Listeria and Staph aureus, Stec 1 and Stec 2 E.coli. For yeast and mold, the fungal kit can test for Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus.
Their QuantX is the world’s first and only multiplex quantification microarray product that can quantify the microbial contamination load for key organisms such as total aerobic bacteria, total yeast & mold, bile tolerant gram negative, total coliform and total Enterobacteriaceae over a dynamic range from 100 CFU/mL up to 1,000,000 CFU/mL.
Not all of the PathogenDx technology is designed for just microbial testing of cannabis or food products. Their EnviroX technology is designed to help growers, processors or producers across any industry identify areas of microbial contamination, being used as a tool for quality assurance and hazard analysis. They conducted industry-wide surveys of the pathogens that are creating problems for cultivators and came up with a list of more than 50 bacterial and fungal pathogens that the EnviroX assay can test for to help growers identify contamination hotspots in their facilities.
Using the EnviroX assay, growers can swab surfaces like vents, fans, racks, workbenches and other potential areas of contamination where plants come in contact. This helps growers identify potential areas of contamination and remediate those locations. Patel says the tool could help growers employ more efficient standard operating procedures with sanitation and sterilization, reducing the facility’s incidence of pathogens winding up on crops, as well as reduction in use of pesticides and fungicides on the product.
Deploying this technology in the cannabis industry allowed Milan Patel and the PathogenDx team to bring something new to the world of microbial testing. Their products are now in more than 90 laboratories throughout the country. The success of this technology provides another shining example of how the cannabis market produces innovative and disruptive ideas that have a major impact on the world, far beyond cannabis itself.
There is certainly, in retrospect, much to be proud about in Canada – home of one of the most disruptive international cannabis industries in the world. And certainly an early mover.
That starts with having the national mojo to begin this journey in the first place, not to mention pivot and even admit faults along the way. For all the complaints and whinges, however on the ground, most Canadians are proud that they tackled the canna question at a federal level.
As the industry now does a bit of an annual review and revisit, what are some of the largest accomplishments, takeaways (and let’s be honest, major f*ckups) so far? And where is this all headed as the industry at least tries to gear up for another year, if not quite Cannabis 2.0?
The Big Bravos
Launching in the first place. Yes Full Monty Recreational was scary, and delayed a few months last year. And even though there have been many problems (retail outlets, online sales, privacy, supply chain issues in every direction, ex im, foreign markets and etc.), it is up and running.
In comparison, the Brits have been haranguing over Brexit for the last three years and are still not really there.
Further, it is also apparent that the agencies in charge of the new industry are themselves giving a bit of a shake after CannaTitanic (CannTrust). That was embarrassing for them too, although of course, while a bit of a negative compliment, the recall system seems to work.
Even if it needs a few jump starts via whistleblowing.
That in and of itself is a fact that is still in the room, although perhaps the pancaking of the stock price of most of the public industry of late was also another much needed wakeup call.
The Devil In The Details
Domestic Requirements. Health Canada is getting hip to the fact that the industry needs a bit more of a heavy hand. See the book thrown at CannTrust. No matter what, Canadians are demanding to know where their cannabis comes from, and further are also demanding that it be at least free of pesticides that can harm them.
Licensing. Many cannapreneuers are complaining, still, about the delays in licensing, particularly for retail outlets in the provinces who are taking the cannabull by the horns. That said, there are still lots of enterprises who are perfectly happy to dodge the requirements all together and sell to the black or gray market. No licensing fees, and no taxes is a wonderful dream, but that is not exactly how regulated democratic capitalism works – at least at this level.
Supply Chain Logistics and Related Technologies. Canadians are struggling to implement a regulated industry in a country where patient home grow is constitutionally protected, and in an environment where who can sell what, and to whom including online, is still evolving. Predictably, no matter how groovy the solution works at home, (or the U.S.), no it will not fly in Europe. See GDPR regs, for starters.
Seed Culture (Aka Strain Protection). No matter how much the lawyers in the colonies are gearing up to sue each other over Huey’s Half Baked, in Europe, there are tomato and pepper farmers who are laughing, literally, all the way to the bank on this one. While hip to be a “strain defender,” the reality in a medical market looking for cheap cannabinoids is rather different. Effective, clean product, which can be reproduced reliably and cheaply, is the name of the game. Girl Scout Cookies, and such ilks will be a long time in coming as anything but highly expensive, niche products you can find in a Dutch Coffee Shop.
Domestic Requirements Vs International Export. Canadian standards, so far, have been widely divergent in an environment where exports to Europe in particular are part of the story for the biggest companies. That said, GMP, and in particular EU GMP, has become at least a buzzword if not a standard to live up to.
Privacy. California might be considering its own form of GDPR (European privacy legislation) but so far, the industry has largely failed to protect consumers (from themselves). Ideas about owning huge data troves on cannabis users for someone else’s profit are still very much in the room. After all, data is the new oil, whether people know their data is being harvested or not. And just like big oil has done for most of its existence, those in the driver’s seat so far show little compunction about harvesting personal information, to become in the words of the now departed CEO of Canopy Growth Bruce Linton, “the Google of Cannabis.” Won’t happen. Starting with the fact that in not just Europe but now even California, people, far beyond pot users are tired of a world where privacy is a second class right.
While the issue first hit in Canada on the recreational side, the reality is that companies know who their clients and patients are in a way that is not only disturbing but increasingly being challenged.
Microbial contamination on cannabis products represents one of the most significant threats to cannabis consumers, particularly immunocompromised patients who are at risk of developing harmful and potentially fatal infections.
As a result, regulatory bodies in the United States and Canada mandate testing cannabis products for certain microbes. The two most popular methods for microbial safety testing in the cannabis industry are culture-based testing and quantitative polymerase chain reaction (qPCR).
When considering patient safety, labs should choose a method that provides an accurate account of what is living on the sample and can specifically target the most harmful microbes, regardless of the matrix.
1. The Method’s Results Must Accurately Reflect the Microbial Population on the Sample
The main objective of any microbial safety test is to give the operator an indication of the microbial population present on the sample.
Figure 1: MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.
Culture-based methods measure contamination by observing how many organisms grow in a given medium. However, not all microbial organisms grow at the same rate. In some cases, certain organisms will out-compete others and as a result, the population in a post-culture environment is radically different than what was on the original sample.
One study analyzed fifteen medicinal cannabis samples using two commercially available culture-based methods. To enumerate and differentiate bacteria and fungi present before and after growth on culture-based media, all samples were further subjected to next-generation sequencing (NGS) and metagenomic analyses (MA). Figure 1 illustrates MA data collected directly from plant material before and after culture on 3M petrifilm and culture-based platforms.
The results demonstrate substantial shifts in bacterial and fungal growth after culturing on the 3M petrifilm and culture-based platforms. Thus, the final composition of microbes after culturing is markedly different from the starting sample. Most concerning is the frequent identification of bacterial species in systems designed for the exclusive quantification of yeast and mold, as quantified by elevated total aerobic count (TAC) Cq values after culture in the total yeast and mold (TYM) medium. The presence of bacterial colonies on TYM growth plates or cartridges may falsely increase the rejection rate of cannabis samples for fungal contamination. These observations call into question the specificity claims of these platforms.
The Live Dead Problem
Figure 2: The enzyme is instantaneously inactivated when lysis buffer is added
One of the common objections to using qPCR for microbial safety testing is the fact that the method does not distinguish between live and dead DNA. PCR primers and probes will amplify any DNA in the sample that matches the target sequence, regardless of viability. Critics claim that this can lead to false positives because DNA from non-viable organisms can inflate results. This is often called the Live-Dead problem. However, scientists have developed multiple solutions to this problem. Most recently, Medicinal Genomics developed the Grim Reefer Free DNA Removal Kit, which eliminates free DNA contained in a sample by simply adding an enzyme and buffer and incubating for 10 minutes. The enzyme is instantaneously inactivated when lysis buffer is added, which prevents the Grim Reefer Enzyme from eliminating DNA when the viable cells are lysed (see Figure 2).
2. Method Must Be Able to Detect Specific Harmful Species
Figure 3: The team spiked a known amount of live E. coli into three different environments
Conversely, qPCR assays, such as the PathoSEEK, are designed to target DNA sequences that are unique to pathogenic Aspergillus species, and they can be run using standard qPCR instruments such as the Agilent AriaMx. The primers are so specific that a single DNA base difference in the sequence can determine whether binding occurs. This specificity reduces the frequency of false positives in pathogen detection, a frequent problem with culture-based cannabis testing methods.
Additionally, Medicinal Genomics has developed a multiplex assay that can detect the four pathogenic species of Aspergillus (A. flavus, A. fumigatus, A. niger, and A. terreus) in a single reaction.
3. The Method Must Work on Multiple Matrices
Figure 4: The team also placed TSB without any E. coli onto a petrifilm to serve as a control.
Marijuana infused products (MIPs) are a very diverse class of matrices that behave very differently than cannabis flowers. Gummy bears, chocolates, oils and tinctures all present different challenges to culture-based techniques as the sugars and carbohydrates can radically alter the carbon sources available for growth. To assess the impact of MIPs on colony-forming units per gram of sample (CFU/g) enumeration, The Medicinal Genomics team spiked a known amount of live E. coli into three different environments: tryptic soy broth (TSB), hemp oil and hard candy. The team then homogenized the samples, pipetted amounts from each onto 3M™ Petrifilm E. coli / Coliform Count (EC) Plates, and incubated for 96 hours. The team also placed TSB without any E. coli onto a petrifilm to serve as a control. Figures 3 and 4 show the results in 24-hour intervals.
Table 1: DNA was spiked into various MIPs
This implies the MIPs are interfering with the reporter assay on the films or that the MIPs are antiseptic in nature.
Many MIPs use citric acid as a flavoring ingredient which may interfere with 3M reporter chemistry. In contrast, the qPCR signal from the Agilent AriaMx was constant, implying there is microbial contamination present on the films, but the colony formation or reporting is inhibited.
Table 3: SenSATIVAx DNA extraction can successfully lyse the cells of the microbesTable 2: Different numbers of DNA copies spiked into chocolate
This is not an issue with DNA-based methods, so long as the DNA extraction method has been validated on these matrices. For example, the SenSATIVAx DNA extraction method is efficient in different matrices, DNA was spiked into various MIPs as shown in Table 1, and at different numbers of DNA copies into chocolate (Table 2). The SenSATIVAx DNA extraction kit successfully captures the varying levels of DNA, and the PathoSEEK detection assay can successfully detect that range of DNA. Table 3 demonstrates that SenSATIVAx DNA extraction can successfully lyse the cells of the microbes that may be present on cannabis for a variety of organisms spiked onto cannabis flower samples.
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According to Kim Hesse, business development manager at Global Laboratory Services, they plan to expand their services in the hemp market with additional types of hemp testing. “At Global Laboratory Services, we always strive to keep pace with industry needs,” says Hesse. “We saw the need for an accredited laboratory in the hemp industry and therefore added CBD and THC testing to our scope. Our next step is to expand our service offerings to include agrochemical analysis of industrial hemp.”
