Downstream Purification Steps Research Articles

Exploring the Impact of In Vitro-Transcribed mRNA Impurities on Cellular Responses.

Advances in mRNA technology have enabled mRNA-based therapies to enter a new era of medicine. Such therapies benefit from a single, standardized in vitro transcription (IVT) manufacturing process applicable to a wide range of targets. This process includes several downstream purification steps, which aim to eliminate impurities that potentially affect safety and efficacy. However, it is not fully understood which impurities are the most critical; hence, some efforts are still needed to establish the correlation between RNA impurities and their role in limiting therapeutic efficacy. To study this relationship, we produced in vitro-transcribed mRNAs using several bacteriophage T7 RNA polymerases, including one wild-type and four engineered variants. Important attributes of the mRNA such as integrity, purity, and functional activity were then measured using advanced physicochemical and cellular assays. For impurities including abortive transcripts, mRNAs containing partial poly(A) tails, and double-stranded (ds)RNA byproducts, structure-function relationships have been established by tracking cellular responses (i.e., protein expression, reactogenicity) in multiple cell models. By varying the T7 RNA polymerase, different levels of sense-antisense dsRNA byproducts were measured by mass photometry, contributing directly to immunological reactogenicity in bone marrow-derived dendritic cells. T7 RNA polymerase differences with regard to short (<20 nucleotides) 3'-loopback dsRNA byproducts were also further investigated using native mass spectrometry by precisely resolving these impurities at the nucleotide level. Overall, this study highlights the importance of developing sensitive and advanced analytical methods to characterize IVT mRNA impurities and understand their interaction with cellular machinery in order to ensure quality control of RNA-based therapies.

High-yield, plant-based production of an antimicrobial peptide with potent activity in a mouse model.

Plants offer a promising chassis for the large-scale, cost-effective production of diverse therapeutics, including antimicrobial peptides (AMPs). However, key advances will reduce production costs, including simplifying the downstream processing and purification steps. Here, using Nicotiana benthamiana plants, we present an improved modular design that enables AMPs to be secreted via the endomembrane system and sequestered in an extracellular compartment, the apoplast. Additionally, we translationally fused an AMP to a mutated small ubiquitin-like modifier sequence, thereby enhancing peptide yield and solubilizing the peptide with minimal aggregation and reduced occurrence of necrotic lesions in the plant. This strategy resulted in substantial peptide accumulation, reaching around 2.9 mg AMP per 20 g fresh weight of leaf tissue. Furthermore, the purified AMP demonstrated low collateral toxicity in primary human skin cells, killed pathogenic bacteria by permeabilizing the membrane and exhibited anti-infective efficacy in a preclinical mouse (Mus musculus) model system, reducing bacterial loads by up to three orders of magnitude. A base-case techno-economic analysis demonstrated the economic advantages and scalability of our plant-based platform. We envision that our work can establish plants as efficient bioreactors for producing preclinical-grade AMPs at a commercial scale, with the potential for clinical applications.

Selective Metal Recovery: Innovating Leaching from Lfp-NMC Cathode Mixtures in Lithium-Ion Batteries

Lithium-ion batteries lie at the heart of the energy transition and electric mobility. In the coming years, numerous gigafactories and recycling plants will be constructed worldwide to produce and recycle batteries for electric vehicles. While recycling plants currently focus on batteries for electric vehicle, there will also be a need to develop the recycling industry for smaller batteries, such as those used in electric bicycles, a market that is rapidly expanding. The chemical processes required to recycle these batteries may not necessarily be the same as those for batteries in electric vehicle due to differences in the composition of the materials to be processed. For electric vehicles, the composition will be well established as the materials will mainly come from either NMC (LiNixMnyCozO2) or LFP (LiFePO4) technologies. However, for batteries in electric bicycle and electric scooter, the materials to be processed will be a mix of NMC and LFP technologies. Therefore, it will be necessary to develop a flexible process capable of extracting metals from the feed which the composition will vary. Furthermore, the hydrometallurgical processes used to treat these materials must be capable of recovering cobalt, nickel, manganese, and lithium despite the presence of varying and significant concentrations of iron, which is generally challenging in hydrometallurgy.This talk will demonstrate how we can take advantage of the physicochemistry of transition metals in the presence of phosphate to develop an effective leaching process that selectively dissolves cobalt, nickel, manganese, and lithium from mixtures of NMC and LFP, resulting in a sufficiently pure leachate to facilitate downstream purification steps through liquid-liquid extraction after leaching.

A case study application of AQbD to the re-development and validation of an affinity chromatography analytical procedure for mAb titer quantitation

Protein A (ProA) high-performance liquid chromatography (HPLC) is a common analytical procedure for measuring monoclonal antibody (mAb) titers due to its high specificity and efficiency. Accurate and reliable results of this procedure are imperative, as the quantitation of the total mAb present for in-process samples directly impacts downstream purification steps related to the removal of process-related impurities. This study aimed to improve a platform ProA HPLC analytical procedure which was previously developed using traditional approaches and was not always reliable. By retrospectively applying Analytical Quality by Design (AQbD) principles and statistical assessments of performance, a bias in the calibration standard due to protein-adsorption to common sample vial materials was identified. The inclusion of Tween® 20 into the mobile phase used as sample diluent was optimized to ensure procedure performance and improve analytical range. The resulting procedure robustness was evaluated using Design of Experiment (DoE) approaches and performance was verified against Analytical Target Profile (ATP) criteria as recommended by regulatory agencies. The resulting linearity displayed R2 values of 1.00 with intercept biases of 1.2 % (analyst 1) and 0.8 % (analyst 2), accuracy across all levels was reported at 99.2 % recovery, and intermediate precision was reported as 3.0 % RSD. Application of this new platform procedure has since reduced development timelines for new mAb products by 50 % and allowed for accurate titer determination to support >5 early phase product-specific process decisions without requiring extensive analytical procedure development. This work demonstrates the utility and relative ease of adopting AQbD concepts, even for established procedures, and supporting them with a lifecycle approach to managing procedure performance.

Safety evaluation of the food enzyme cellobiose phosphorylase from the genetically modified Escherichia coli strain LE1B109-pPB130.

The food enzyme cellobiose phosphorylase (cellobiose: phosphate α-d-glucosyltransferase; EC 2.4.1.20) is produced with the genetically modified Escherichia coli strain LE1B109-pPB130 by c-LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is considered free from viable cells of the production organism and its DNA. It is intended to be used in combination with a sucrose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of total organic solids are removed by downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Safety evaluation of the food enzyme sucrose phosphorylase from the genetically modified Escherichia coli strain LE1B109-pPB129.

The food enzyme sucrose phosphorylase (sucrose: phosphate α- d-glucosyltransferase; EC 2.4.1.7) is produced with the genetically modified Escherichia coli strain LE1B109-pPB129 by c-LEcta GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme was free from viable cells of the production organism. It is intended to be used in combination with a cellobiose phosphorylase in the production of the specialty carbohydrate cellobiose. Since residual amounts of food enzyme-total organic solids are removed by the downstream purification steps, the Panel considered that toxicological studies other than assessment of allergenicity were unnecessary and a dietary exposure was not estimated. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Absolute Quantification of Hepatitis B Core Antigen (HBcAg) Virus-like Particles and Bound Nucleic Acids.

Effective process development towards intensified processing for gene delivery applications using Hepatitis B core Antigen (HBcAg) virus-like particles (VLPs) relies on analytical methods for the absolute quantification of HBcAg VLP proteins and bound nucleic acids. We investigated a silica spin column (SC)-based extraction procedure, including proteinase K lysis and silica chromatography, for the absolute quantification of different species of nucleic acids bound to HBcAg VLPs analyzed by dye-based fluorescence assays. This revealed load-dependent nucleic acid recoveries of the silica-SC-based extraction. We also developed a reversed-phase high-performance liquid chromatography (RP-HPLC) method to separate and quantify the HBcAg proteins and the bound nucleic acids simultaneously without prior sample treatment by dissociation reagents. The method demonstrated sufficient linearity, accuracy, and precision coefficients and is suited for determining absolute protein and nucleic acid concentrations and HBcAg protein purities at various purification stages. Both the silica-SC-based extraction and the RP-based extraction presented overcome the limitations of analytical techniques, which are restricted to relative or qualitative analyses for HBcAg VLPs with bound nucleic acids. In combination with existing analytics, the methods for an absolute quantification of HBcAg VLPs and bound nucleic acids presented here are required to evaluate downstream purification steps, such as the removal of host cell-derived nucleic acids, concurrent protein loss, and efficient loading with therapeutic nucleic acids. Hence, the methods are key for effective process development when using HBcAg VLP as potential gene delivery vehicles.

A quick, easy and efficient protocol for extracting high-quality RNA from Mycobacterium tuberculosis using a spin column commercial kit

RNA extraction from Mycobacterium tuberculosis has been a historically challenging task for researchers due to the thick lipids associated with the cell wall of this “notorious” pathogen that is responsible for Tuberculosis (TB) outbreaks. Several studies have successfully extracted RNA from M. tuberculosis using a Trizol reagent combined with organic solvents. Recently, our laboratory has successfully extracted high quality total RNA using a commercial kit from clinical strains belonging to F15/LAM4/KZN, Beijing and F11 strain families and H37Rv laboratory strain by exploiting high speed homogenizer for cell lysis and spin columns for RNA purification. The quality and integrity of the extracted RNA was analyzed and confirmed through the Nanodrop, Bioanalyzer and RNA 3-(N-morpholino) propanesulfonic acid (MOPS) gel electrophoresis. Furthermore, to confirm the integrity of small RNA (sRNA) molecules due to their vulnerability to degradation, the RNA samples were converted to cDNA and sRNAs were amplified and confirmed through PCR. This detailed RNA extraction protocol proposes to carve a new path into TB transcriptome research without the use of organic solvent for downstream purification steps while yielding high quality RNA that can be used to understand M. tuberculosis transcriptome regulation.

Host cell protein identification in monoclonal antibody high molecular weight species

High molecular weight (HMW) species are product-related variants that may impact therapeutic product safety and efficacy. Therefore, HMW species and aggregates are considered critical quality attributes and their levels should be closely monitored and controlled during drug development, commercial manufacturing, and shelf-life storage period for therapeutic monoclonal antibody drug products. Various biophysical and analytical methods have been developed to characterize the HMW species to understand their mechanisms of formation and assess potential product risk. However, host cell protein (HCP) analysis has seldom been conducted to characterize the impurities in aggregates. In this work, HCP analysis of enriched HMW species and drug substance (DS) from five different monoclonal antibodies (mAbs) was performed. More HCPs are identified in the enriched HMW than in the DS, thus demonstrating a potential interaction between HCPs and HMW. Certain HCPs, including commonly detected HCPs and problematic HCPs, were enriched in HMW fractions. Especially, the most abundant HCP from mAb1, CC motif chemokine, was 46 times more abundant in enriched HMW than DS. The enriched HMW was further fractionated into enriched dimers and enriched very HMW (vHMW) fractions. The CC motif chemokine was found to interact mainly with mAb1 dimer species rather than vHMW fraction. Removing the HMW species from mAb1 significantly decreased the CC motif chemokine level in the final mAb1 DS. Our findings demonstrate that some HCPs are more preferentially bound to HMW species and this finding may provide a new opportunity for removing HCPs in downstream purification steps.

Population balance modelling captures host cell protein dynamics in CHO cell cultures.

Monoclonal antibodies (mAbs) have been extensively studied for their wide therapeutic and research applications. Increases in mAb titre has been achieved mainly by cell culture media/feed improvement and cell line engineering to increase cell density and specific mAb productivity. However, this improvement has shifted the bottleneck to downstream purification steps. The higher accumulation of the main cell-derived impurities, host cell proteins (HCPs), in the supernatant can negatively affect product integrity and immunogenicity in addition to increasing the cost of capture and polishing steps. Mathematical modelling of bioprocess dynamics is a valuable tool to improve industrial production at fast rate and low cost. Herein, a single stage volume-based population balance model (PBM) has been built to capture Chinese hamster ovary (CHO) cell behaviour in fed-batch bioreactors. Using cell volume as the internal variable, the model captures the dynamics of mAb and HCP accumulation extracellularly under physiological and mild hypothermic culture conditions. Model-based analysis and orthogonal measurements of lactate dehydrogenase activity and double-stranded DNA concentration in the supernatant show that a significant proportion of HCPs found in the extracellular matrix is secreted by viable cells. The PBM then served as a platform for generating operating strategies that optimise antibody titre and increase cost-efficiency while minimising impurity levels.

Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

BackgroundThe use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar.ResultsThe pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production.ConclusionsThe current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

The reactivity of iron oxides and hydroxide during low-temperature sulfation

Sulfation is a technique commonly applied to the extraction of mineral commodities, such as rare earths, lithium, nickel, and titanium, as a preparation stage for leaching. The behavior of iron compounds during sulfation/dissolution will directly impact acid consumption and downstream purification steps. This work shows the marked differences in the sulfation/dissolution of hematite (granular, specular, and one produced by pyrite roasting), goethite, and magnetite samples over increased temperature and time. At 80 °C, the solubilized iron fraction varies within 2% to 70% accordingly specular hematite < calcine hematite < granular hematite < magnetite << goethite. Sulfation increases over time, from 5 to 15 min, when it reaches a plateau, ascribed to the formation of a ferric sulfate product layer. Increase in temperature from 80 °C to 160 °C increases the sulfation of magnetite (42.0 to 95.0%), calcine hematite (16.0 to 55.5%), and specular hematite (2.0 to 13.0%), with small or negative effect on goethite (70.0 to 76.0%) and granular hematite (33.0 to 25.0%). The influence of the crystalline structure, porosity, and phase transformation on the sulfation behavior of the different iron oxides and hydroxide, and the possible implications on the selective leaching of metals, are discussed in this article.

Recovery of succinic acid from whey fermentation broth by reactive extraction coupled with multistage processes

Abstract Fermentative production of succinic acid (SA) from renewable resources such as whey is environmentally sustainable compared to petroleum-based synthesis. However, a major drawback of fermentation is the concurrent production of SA with byproducts such as lactic acid (LA), formic acid (FA) and acetic acid (AA). Therefore, appropriate downstream SA recovery and purification steps are significant in ensuring sustainable SA production. In this study, SA was fermented by Actinobacillus succinogenes and recovered in an integrated process consisting of ultrafiltration, vacuum distillation and reactive extraction. The extractant used was tri-n-octylamine (TOA) with 1-octanol as a diluent for both liquid-liquid (LLE) extraction and supported liquid membrane (SLM). The produced SA titer and yield was 11.16 g/L and 0.44 g/g, respectively. The steady state ultrafiltration permeate flux ranged from 31.18 to 33.42 L/m2h, and complete decolorization of the fermentation broth was achieved with 10 % (w/v) of powdered activated carbon. The extraction efficiency for LLE was 51.5 %, whereas SLM achieved 57.3 % recovery. SA exhibited transport and permeability coefficient of 0.00697 h−1 (R2 > 0.92) and 0.08605 cm h−1, respectively. Extraction of SA tremendously decreased as the aqueous pH was increased from 2 to 5. In SLM, initial SA flux was calculated as 9.65 g/m2h and doubled that of lactic acid. Selective extraction of only SA was not achieved; however, residue biological material and macromolecular substances were effectively removed. Herein, we clearly demonstrated that process integration applied in reactive extraction is a promising approach for recovery of SA from fermentation broth.

Bio-production of high-purity propionate by engineering L-threonine degradation pathway in Pseudomonas putida.

Propionic acid (PA) is widely used in the food, agricultural, and pharmaceutical industries. Since the petrochemical PA is unsustainable, biological production of PA from renewable substrates is gaining attention. In this study, we engineered the strain Pseudomonas putida KT2440 to transform L-threonine to PA with only CO2 released as by-product. The cell factory was created by chromosomal incorporation of heterologous L-threonine deaminase, permease, and acyl-CoA thioesterase, deletion of branch pathways as well as overproduction of the endogenous branched-chain alpha-keto acid dehydrogenase complex. The final engineered strain could produce 399mM PA from 400mM L-threonine in a batch biotransformation process, with a molar yield of 99.8% under the optimized conditions in 48h. The PA titer further reached to 50.3g/L (679mM) with a productivity of 0.6g/L/h in a fed-batch conversion process. No obvious by-products, such as acetate and succinate, were detected in the broth, which would significantly facilitate downstream purification steps. Thus, this study offers an alternative route for biological production of PA.

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth.

Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process. In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation. Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD). The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators. The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Recovery of lithium from mineral resources: State-of-the-art and perspectives – A review

Lithium (Li), as a new energy metal, is becoming a “hot” topic in both academia and industry due to the rapid vehicle electrification and grid storage. Although brines have been the major Li sources, Li-bearing minerals, owing to the wider distribution and more rapid pathway to market, have also attracted much attention in recent years, with a number of new industrial projects launched and various novel methods proposed. The present study provides a start-of-the-art review of Li recovery from different mineral resources (i.e. excluding brines), and gives perspectives and outlook towards various recovery methods. In this study, the major mineral deposits of Li are summarised and illustrated, which shows its high abundance and wide distribution around the global. Various methods of Li recovery reported so far are then summarised with flowsheets and discussed by different type of minerals, covering spodumene, lepidolite, zinnwaldite, amblygonite and clays. It is predicted that spodumene will continue being dominantly used as Li source over other minerals with sulfuric acid (H2SO4) roasting as a major method of processing. However, other novel methods including direct processing of natural spodumene and the process that favours the direct production of LiOH will be the trends of future research. Fluoride-based methods can achieve low energy consumption and high extraction efficiency but still need to be further investigated for a sustainable, economical and safe application. To compete with spodumene, the comprehensive utilization of all the valuable elements contained in lepidolite and zinnwaldite is crucial. In most of the recovery processes, more attention should be paid to the treatment of voluminous residue and waste for a safe disposal or further reuse. In addition, this study not only presents the methods for Li recovery, but also includes various downstream separation and purification steps to make the process integrated. It is expected that, as a review specialising in mineral resources of Li, the present study can provide insights for the development of this particular area.

Cation exchange chromatography performed in overloaded mode is effective in removing viruses during the manufacturing of monoclonal antibodies.

Viral safety is a critical concern with regard to monoclonal antibody (mAb) products produced in mammalian cells such as Chinese hamster ovary cells. Manufacturers are required to ensure the safety of such products by validating the clearance of viruses in downstream purification steps. Cation exchange (CEX) chromatography is widely used in bind/elute mode as a polishing step in mAb purification. However, bind/elute modes require a large volume of expensive resin. To reduce the production cost, the use of CEX chromatography in overloaded mode has recently been investigated. The viral clearance ability in overloaded mode was evaluated using murine leukemia virus (MLV). Even under high-load conditions such as 2,000 g mAb/L resin, MLV was removed from mAb solutions. This viral clearance ability was not significantly affected by resin type or mAb type. The overloaded mode can also remove other types of viruses such as pseudorabies virus and reovirus Type 3 from mAb solutions. Based on these results, this cost-effective overloaded mode is comparable to the bind-elute mode in terms of viral removal.

Ready-to-use green polyphenolic extracts from food by-products

To establish environmentally friendly polyphenolic extracts from grape and olive pomace, natural deep eutectic solvents (NADES) were used coupled with alternative energy sources – ultrasound and microwave irradiation. Obtained extracts were characterized by HPLC analysis, while antioxidant capacity was determined by ORAC method. Furthermore, in vitro cytotoxicity of prepared extracts was assessed by antiproliferation assay on two tumour cell lines, whereas for investigation of type of cell death or cell cycle arrest a flow cytometric analysis was applied. In addition, a detection of compounds with DNA/RNA-bindingaffinity in extracts was investigated by UV/Vis and circular dichroism spectroscopy. Grape pomace extract in NADES showed to be the best of all extracts tested, with regard to extraction of total polyphenolic compounds (p < 0.05) and related biological activities such as antioxidant and antiproliferative activity. Prepared polyphenolic extracts in NADES could be considered as ready-to-use in food and pharmaceutical industry without demanding and expensive downstream purification steps.

Bispecific antibody process development: Assembly and purification of knob and hole bispecific antibodies.

Production of knob and hole dual light chain bispecific antibodies poses several unique challenges for development of a feasible industrial scale manufacturing process. We developed an efficient process for the assembly and purification of knob and hole dual light chain bispecific antibodies. Two distinct half-antibodies targeting two different antigens were expressed separately in Escherichia coli cells and captured independently using Protein A chromatography. When combined, the knob and hole mutations in the CH3 domains promoted heterodimer formation. The hinge region disulfides were reduced and reoxidized to form the disulfide bridge between the two complementary half antibodies. Unreacted half antibodies, noncovalently linked homodimers, covalently linked homodimers, and noncovalently linked heterodimers are impurities closely related to the product of interest and are challenging to remove by standard processes. Characterization of the molecular properties of the half antibodies and high-throughput screening predicted column chromatography performance and allowed for rapid development of downstream purification steps for removal of unique product-related and process-related impurities. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:397-404, 2018.

Synthesis of value added fatty alcohols and FAMEs by non-catalytic supercritical methanol transesterification of jojoba (Simmondsia chinensis) wax

Nowadays jojoba (Simmondsia chinensis) wax production with an industrial or energetic objective can be focused on a bio-refinery processing perspective where the fatty alcohols are the main products and fatty acid methyl esters (FAMEs) the by-products. To date, jojoba wax methanolysis has been performed using catalytic processes where long reaction times and downstream separation and purification steps are required. However, jojoba wax transesterification using a non-catalytic process such as the supercritical methanolysis has not been reported yet. The yield of jojoba fatty alcohols and FAMEs was determined in the temperature and reaction time ranges of 250–350 °C (12–42 MPa) and 15–90 min, respectively, at a methanol-to-wax molar ratio of 15:1. The maximum experimental yield of fatty alcohols and FAMEs (95.3 and 95.4 mol%, respectively) was reached at 350 °C after 90 min of reaction. Therefore, no thermal decomposition of jojoba alcohols and FAMEs was observed under the most severe experimental reaction conditions studied, which can be explained by the highly monounsaturated nature of the products obtained and the protective antioxidant effect of β-sitosterol present in jojoba wax. The rate constants obtained for this process were: 1 × 10−5, 3 × 10−5, 1.1 × 10−4, 4.1 × 10−4 and 6.5 × 10−4 s−1, at 250, 275, 300, 325 and 350 °C, respectively, with an activation energy of 115.5 kJ/mol. In addition, response surface methodology was used to determine the optimal reaction conditions that maximize the yield of fatty alcohols and FAMEs: 338.9 °C and 90 min to obtain yields of 96.8 mol% and 97.0 mol%, respectively. Thus, it can be concluded that jojoba wax methanolysis under non-catalytic supercritical conditions is an alternative way to obtain high-added value compounds and biodiesel.