Cannabis is an herbaceous flowering plant of the Cannabis genus (Rosale) that has been used for its fibre and medicinal properties for thousands of years^1,2. In recent decades medicinal cannabis has become legal in several jurisdictions and the possibility of legalisation is being explored in many more^3–8. The chemistry of cannabis is rich and varied; it includes phytocannabinoids, terpenes and phenolics, and each of these classes contain compounds with biological activity⁹. The cannabinoids, in particular, Δ⁹-tetrahydrocannabinol (THC) and cannabidiol (CBD) have been the main focus of bioactive research. These cannabinoids are naturally present in planta as their acid analogues Δ⁹-tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). THCA and CBDA are the major cannabinoids found in cannabis although more than 90 cannabinoids have been reported⁹. The acids degrade naturally to the corresponding neutral species at a slow rate via non-enzymatic processes¹⁰. Decarboxylation is facilitated by heating (e.g. when smoked) (Citti et al. 2018). Many medicinal cannabis preparations are consumed in an oil or are made from an extracted resin and not smoked. In this case, the dried cannabis material must be ‘cured’ prior to, or during, extraction (i.e. heated for a specific time and temperature to ensure maximum decarboxylation). THCA is the main psychoactive cannabinoid followed by cannabigerolic acid (CBGA).

Medicinal cannabis is consumed by patients using a variety of methods including smoking, vaping and consuming infused oils or other edible products. One of the issues associated with smoking or vaping is that the dose the patient receives is not reliable due to both the variability in plant product and the way patients inhale and hold vapour¹¹. Vaping and smoking also have well known negative health effects and is particularly undesirable for juvenile patients. For this reason, the production of an extract that can be formulated in various ways is becoming more popular. The cannabinoids are non-polar compounds with low water solubility and can be extracted using a range of different organic solvents including hydrocarbons (e.g. hexane) and alcohols (e.g. ethanol). Extractions using these solvents can be efficient but depending on the final product, can impact regulation and require additional testing. For instance, residual solvent must be defined for medicines under good manufacturing practice (GMP). These solvents can also be costly and due to their toxicity, environmental risk and flammability are less desirable for large scale extractions.

Supercritical fluid extraction (SFE) can eliminate the need for these organic solvents during the manufacture of medicinal cannabis extracts. Carbon dioxide (CO₂) is the main solvent in SFE. It has GRAS (generally recognised as safe) status and evaporates from the extract when exposed to normal atmospheric conditions. The majority of the published studies on SFE of cannabis are either at small/analytical scale¹² or on hemp material that contains low levels of cannabinoids and very little THC^13–18. There have been studies investigating the solubility of THC and CBN (cannabinol, a degradation product of THC) in supercritical CO₂ suggesting that temperature and pressure are critical parameters^19,20. A more recent study evaluated the use of the Waters Bio-botanical extraction system with the use of ethanol as a co-solvent with CO₂ extraction using four cannabis samples (500 g each) with THCA:CBDA content of 15:1 or more. The authors noted extraction efficiency was highly linked to pressure as well as cannabinoid content of the plant material²¹.

This study was undertaken as part of our efforts to obtain a GMP license to produce pharmaceutical grade medicinal cannabis. In order to simplify the regulatory process, and produce a cleaner product, we chose to avoid the use of co-solvents such as ethanol. Process development (PD) is part of GMP. The aim of PD to ensure that the factors underlying any manufacturing protocols are well understood so that any potential source of product variation can be better managed to ensure a safe and consistent pharmaceutical product. As part of GMP PD we used design of experiments (DOE) methodology to develop an extraction method for medicinal cannabis. The aim of DOE was to explore the factors influencing the extraction of cannabinoids and optimise the process for extraction of cannabis material at a pharmaceutical scale.

DOE is commonly used to optimise engineering processes and to determine which process inputs have a significant effect on process outputs. In a factorial design each variable, or factor, is investigated at predefined levels. With a two-level factorial design each factor can be one of two values. For continuous variables, this is a high and a low level of the variable under investigation²². Importantly, the methodology can also determine if there are interactions between the factors in the process. This allows for more rapid optimisation of processes than if single variables were considered independently. This methodology has been used to investigate factors important in extraction of biomaterials. For example, Prasad et al. explored the effect of five factors (pH, ethanol concentration, temperature, time and liquid to solid ratio) on four responses (extract yield, antioxidant capacity, phenolics and flavonoids) for the extraction of the Malaysian fruit, Mangifera pajang²³. Lee et al. analysed similar factors to optimise the recovery of phenolics, flavonoids and anti-oxidants from palm-kernel by-products²⁴. A review of the literature by Reverchon and De Marco demonstrated that CO₂ flow rate, extraction pressure and extraction time are the critical parameters in SFE for the extraction of natural materials²⁵. Therefore, these were the factors considered in this study.