Scalable multimodal weak anion exchange chromatographic purification for stable mRNA drug substance.

Messenger RNA (mRNA) has emerged as a modality with immense therapeutic potential. Recent innovations in production process of mRNA call for procedures to isolate pure mRNA drug substance (DS) with high yield, high capacity, scalability, and compatibility with GMP production systems. Novel RNA modalities, such as circular RNA (circRNA), have further driven the need for non-affinity capture possibilities which are already widely used in the biopharmaceutical industry, for example, in monoclonal antibody processing. The principle that multimodal ion exchange/hydrogen bonding chromatography can be used to separate mRNA from in vitro transcription components has recently been demonstrated. Here, we apply and refine this approach to be suitable for scalable purification of multiple mRNA constructs with sufficient yields, purity, and stability, for use in mRNA production process. Binding capacity of the PrimaS-modified monolithic chromatographic column for mRNA enabled up to 7 mg/mL product isolation in a single chromatographic run, with 98% recovery and room temperature stability of the eGFP mRNA demonstrated for up to 28 days. This approach is independent of construct size or the presence of polyadenylic acid tail and is applicable for capture of a wide variety of RNAs, including mRNA, self-amplifying RNA, circRNA, and with optimization also smaller RNAs such as transfer RNA and others.

High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.

Thermal and Rheological Properties of Gluten-Free, Starch-Based Model Systems Modified by Hydrocolloids.

Obtaining good-quality gluten-free products represents a technological challenge; thus, it is important to understand how and why the addition of hydrocolloids influences the properties of starch-based products. To obtain insight into the physicochemical changes imparted by hydrocolloids on gluten-free dough, we prepared several suspensions with different corn starch/potato starch/hydroxpropyl methyl cellulose/xanthan gum/water ratios. Properties of the prepared samples were determined by differential scanning calorimetry and rheometry. Samples with different corn/potato starch ratios exhibited different thermal properties. Xanthan gum and HPMC (hydroxypropyl methyl cellulose) exhibited a strong influence on the rheological properties of the mixtures since they increased the viscosity and elasticity. HPMC and xanthan gum increased the temperature of starch gelatinization, as well as they increased the viscoelasticity of the starch model system. Although the two hydrocolloids affected the properties of starch mixtures in the same direction, the magnitude of their effects was different. Our results indicate that water availability, which plays a crucial role in the starch gelatinization process, could be modified by adding hydrocolloids such as, hydroxypropyl methyl cellulose and xanthan gum. By adding comparatively small amounts of the studied hydrocolloids to starch, one can achieve similar thermo-mechanical effects by the addition of gluten. Understanding these effects of hydrocolloids could contribute to the development of better quality gluten-free bread with optimized ingredient content.

Immunonutritional Bioactives from Chenopodium quinoa and Salvia hispanica L. Flour Positively Modulate Insulin Resistance and Preserve Alterations in Peripheral Myeloid Population.

Innate immunity plays a determinant role in high fat diet (HFD)-induced insulin resistance. This study compares the effects of immunonutritional bioactives from Chenopodium quinoa (WQ) or Salvia hispanica L. (Ch) when used to partially replace wheat flour (WB) into bread formulations. These flours were chosen to condition starch and lipid content in the products as well as because their immunonutritional activity. To be administered with different bread formulations, HFD-fed C57BL/6J mice were distributed in different groups: (i) wild type, (ii) displaying inherited disturbances in glucose homeostasis, and (iii) displaying dietary iron-mediated impairment of the innate immune TLR4/TRAM/TRIF pathway. We analyze the effects of the products on glycaemia and insulin resistance (HOMA-IR), plasmatic triglycerides, intestinal and hepatic gene expression and variations of myeloid (MY), and lymphoid (LY) cells population in peripheral blood. Our results show that feeding animals with WQ and Ch formulations influenced the expression of lipogenic and coronary risk markers, thus attaining a better control of hepatic lipid accumulation. WQ and Ch products also improved glucose homeostasis compared to WB, normalizing the HOMA-IR in animals with an altered glucose and lipid metabolism. These positive effects were associated with positive variations in the peripheral myeloid cells population.

Aqueous Extracts of Wild Mushrooms Show Antimicrobial and Antiadhesion Activities against Bacteria and Fungi.

Mushrooms represent promising sources of novel bioactive compounds and can be applied as innovative strategies to control microbial contamination and infection via the food chain. We characterized aqueous extracts from 21 wild basidiomycete mushrooms and the cultivated oyster mushroom, Pleurotus ostreatus, as putative sources of antimicrobial and antiadhesive compounds. Broth microdilutions and adhesion to a polystyrene surface were evaluated on Gram-positive and Gram-negative bacteria and on fungi. The aqueous extracts tested showed antimicrobial and antiadhesive activities against these microorganisms. Biochemical analyses of the P. ostreatus extract indicated the involvement of several compounds with different molecular masses. Copyright © 2017 John Wiley & Sons, Ltd.