SummaryPlants have been proposed as an attractive alternative for pharmaceutical protein production to current mammalian or microbial cell-based systems. Eukaryotic protein processing coupled with reduced production costs and low risk for mammalian pathogen contamination and other impurities have led many to predict that agricultural systems may offer the next wave for pharmaceutical product production. However, for this to become a reality, the quality of products produced at a relevant scale must equal or exceed the predetermined release criteria of identity, purity, potency and safety as required by pharmaceutical regulatory agencies. In this article, the ability of transient plant virus expression systems to produce a wide range of products at high purity and activity is reviewed. The production of different recombinant proteins is described along with comparisons with established standards, including high purity, specific activity and promising preclinical outcomes. Adaptation of transient plant virus systems to large-scale manufacturing formats required development of virus particle and Agrobacterium inoculation methods. One transient plant system case study illustrates the properties of greenhouse and field-produced recombinant aprotinin compared with an US Food and Drug Administration-approved pharmaceutical product and found them to be highly comparable in all properties evaluated. A second transient plant system case study demonstrates a fully functional monoclonal antibody conforming to release specifications. In conclusion, the production capacity of large quantities of recombinant protein offered by transient plant expression systems, coupled with robust downstream purification approaches, offers a promising solution to recombinant protein production that compares favourably to cell-based systems in scale, cost and quality.

SummaryRapid, large-scale manufacture of medical countermeasures can be uniquely met by the plantmade-pharmaceutical platform technology. As a participant in the Defense Advanced Research Projects Agency (DARPA) Blue Angel project, the Caliber Biotherapeutics facility was designed, constructed, commissioned and released a therapeutic target (H1N1 influenza subunit vaccine) in <18 months from groundbreaking. As of 2015, this facility was one of the world's largest plantbased manufacturing facilities, with the capacity to process over 3500 kg of plant biomass per week in an automated multilevel growing environment using proprietary LED lighting. The facility can commission additional plant grow rooms that are already built to double this capacity. In addition to the commercial-scale manufacturing facility, a pilot production facility was designed based on the large-scale manufacturing specifications as a way to integrate product development and technology transfer. The primary research, development and manufacturing system employs vacuum-infiltrated Nicotiana benthamiana plants grown in a fully contained, hydroponic system for transient expression of recombinant proteins. This expression platform has been linked to a downstream process system, analytical characterization, and assessment of biological activity. This integrated approach has demonstrated rapid, high-quality production of therapeutic monoclonal antibody targets, including a panel of rituximab biosimilar/biobetter molecules and antiviral antibodies against influenza and dengue fever.

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial. The system allowed rapid production and recovery of idiotypic single-chain antibodies (scFv) derived from each patient's tumor and immunization of patients with their own individual therapeutic antigen. Both low and high doses of vaccines, administered alone or co-administered with the adjuvant GM-CSF, were well tolerated with no serious adverse events. A majority (>70%) of the patients developed cellular or humoral immune responses, and 47% of the patients developed antigenspecific responses. Because 15 of 16 vaccines were glycosylated in plants, this study also shows that variation in patterns of antigen glycosylation do not impair the immunogenicity or affect the safety of the vaccines. Collectively, these findings support the conclusion that plant-produced idiotype vaccines are feasible to produce, safe to administer, and a viable option for idiotypespecific immune therapy in follicular lymphoma patients.phase I clinical trial ͉ plant-made pharmaceutical ͉ single-chain antibodies I n recent years, non-Hodgkin's lymphoma (NHL) has become the most common hematologic malignancy in the United States with an estimated 54,000 new cases each year. Approximately 30% of these cases are follicular B cell lymphoma (1), with a median survival of 8-10 years from diagnosis (2). Most patients subjected to standard treatments, such as chemotherapy, radiation, or antibodies (2-9), still relapse (10-12). Newer approaches have focused on active immunotherapy through vaccination. For B cell lymphoma, the cell-surface Ig (Ig), or idiotype, is the unique tumorspecific antigen. In contrast to passive therapy, an idiotype-induced immune response is (i) highly tumor specific, thus sparing normal cells; (ii) potentially more durable; and (iii) protective against tumor cell variants that might ''escape'' under selective pressure, because the response is polyclonal (13-15).There exists a 20-year history of idiotype vaccination for follicular lymphoma in animal models and clinical trials. The original vaccine manufacturing process, still used today in the majority of clinical research, used a patient's lymphoma B cells to derive a mouse/ human heteromyeloma cell line for production of the tumor's monoclonal idiotype (16). Once purified, the idiotype antibody was chemically coupled to a highly immunogenic carrier protein, keyhole limpet haemocyanin (KLH), and administered w...

Lysosomal acid lipase (LAL) is an essential enzyme that hydrolyzes triglycerides (TGs) and cholesteryl esters (CEs) in lysosomes.

A quantitative method is described for the determination of formaldehyde in biological tissues by stable isotope dilution using gas chromatography/mass spectrometry. (13C2H2)Formaldehyde is used as the isotopic diluent. After tissue homogenization, derivatization is carried out in situ with pentafluorophenylhydrazine, followed by extraction and analysis using selected ion monitoring. The sensitivity of the technique is higher than that of conventional methods of formaldehyde analysis, enabling endogenous formaldehyde to be quantitatively analyzed in tissues, even in samples as small as 20 mg wet weight. The effects of exposure to airborne formaldehyde or to airborne methyl chloride on the formaldehyde concentrations of several tissues of Fischer-344 rats are reported.