Modified penicillin acylase signal peptide allows the periplasmic production of soluble human interferon-γ but not of soluble human interleukin-2 by the Tat pathway in Escherichia coli

"Production of periplasmic human interferon-γ (hINF-γ) and human interleukin-2 (hIL-2) by the Tat translocation pathway in Escherichia coli BL21-SI was evaluated. The expression was obtained using the pEMR vector which contains the Tat-dependent modified penicillin acylase signal peptide (mSPpac) driven by the T7 promoter. The mSPpac-hINF-γ was processed and the protein was transported to periplasm. Up to 30.1% of hINF-γ was found in the periplasmic soluble fraction, whereas only 15% of the mSPpac-hIL-2 was processed, but hIL-2 was not found in the periplasmic soluble fraction.

Optimization of human interferon gamma production in Escherichia coli by response surface methodology

"The production of human interferon gamma (hIFN-γ) using a synthetic gene in Escherichia coli BL21-SI was optimized by response surface methodology (RSM) and a Box-Behnken design. The process variables studied were temperature, bio-mass concentration at induction time and the NaCl concentration as inducer. According to the Box-Behnken design, a second order response function was developed. The optimal expression conditions were a temperature of 32.6°C, induction biomass of 0.31 g/L and 0.3 M NaCl in minimal medium. The model prediction for the maximum hIFN-γ production was 77.3 mg/L, which corresponded satisfactorily with the experimental data. The hIFN-γ concentration attained under optimized conditions was 13-times higher than that obtained using the non-optimized conditions. We conclude that RSM is an effective method for the optimization of recombinant protein expression using synthetic genes in E. coli.

Direct Magnetic Evidence, Functionalization, and Low-Temperature Magneto-Electron Transport in Liquid-Phase Exfoliated FePS3

Magnetism and the existence of magnetic order in a material is determined by its dimensionality. In this regard, the recent emergence of magnetic layered van der Waals (vdW) materials provides a wide playground to explore the exotic magnetism arising in the two-dimensional (2D) limit. The magnetism of 2D flakes, especially antiferromagnetic ones, however, cannot be easily probed by conventional magnetometry techniques, being often replaced by indirect methods like Raman spectroscopy. Here, we make use of an alternative approach to provide direct magnetic evidence of few-layer vdW materials, including antiferromagnets. We take advantage of a surfactant-free, liquid-phase exfoliation (LPE) method to obtain thousands of few-layer FePS3 flakes that can be quenched in a solvent and measured in a conventional SQUID magnetometer. We show a direct magnetic evidence of the antiferromagnetic transition in FePS3 few-layer flakes, concomitant with a clear reduction of the Néel temperature with the flake thickness, in contrast with previous Raman reports. The quality of the LPE FePS3 flakes allows the study of electron transport down to cryogenic temperatures. The significant through-flake conductance is sensitive to the antiferromagnetic order transition. Besides, an additional rich spectra of electron transport excitations, including secondary magnetic transitions and potentially magnon-phonon hybrid states, appear at low temperatures. Finally, we show that the LPE is additionally a good starting point for the mass covalent functionalization of 2D magnetic materials with functional molecules. This technique is extensible to any vdW magnetic familyE.B. acknowledges funds from Ministerio de Ciencia e Innovación in Spain (RTI2018-096075-A-C22, RYC2019- 028429-I). E.M.P. thanks the Spanish Ministerio de Ciencia e Innovación (PID2020-116661RB-I00) and Comunidad de Madrid (P2018/NMT-4367). M.G.H. and A.C.-G. acknowledge funds from European Union Horizon 2020 research and innovation program (Graphene Core3-Grant agreement no. 881603 Graphene-based disruptive technologies), EU FLAGERA through the project To2Dox (JTC-2019-009), and Comunidad de Madrid through the project CAIRO-CM project (Y2020/NMT-6661). A.C.-G. also acknowledges funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 755655, ERC-StG 2017 project 2D-TOPSENSE) and the Ministry of Science and Innovation (Spain) through the project PID2020-115566RB-I00. M.L.R.G. acknowledges support by the Spanish Ministry of Science and Innovation through Research Project PID 2020- 113753RB-100. The National Centre for Electron Microscopy (ELECMI National Singular Scientific Facility) is also acknowledge for provision of access to corrected aberration microscopy facilities. CzechNanoLab Research Infrastructure supported by MEYS CR (LM2018110) is acknowledge

Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology: the case of human interferon beta

"A human interferon beta (hINF-β) synthetic gene was optimized and expressed in Escherichia coli BL21-SI using a vector with the T7 promoter. To determine the best culture conditions such as culture medium, temperature, cell density and inducer concentration, we used the response surface methodology and a Box-Behnken design to get the highest hINF-β production. The maximum hINF-β production of 61 mg l−1 was attained using minimum medium and the following predicted optimal conditions: temperature of 32.5 °C, cell density of 0.64, and inducer concentration of 0.30 M NaCl. This is the first report showing the successful performance of the BL21-SI system in a minimum medium. The response surface methodology is effective for the optimization of recombinant protein production using synthetic genes.

Regulatory Pathway for Licensing Biotherapeutics in Mexico

Biotherapeutic products which are derived from living organisms using recombinant DNA technology significantly contribute to the progress in the treatment of life-threatening and chronic diseases. The worldwide sale of biological drugs in 2016 was near US $263,700 million. In Latin America, where monoclonal antibodies market was worth US$7000 million, being Mexico the second largest market. Approval is one of the key aspects which influences the market of medicinal products, thus it is responsibility of the regulatory authority to establish a regulatory framework that ensure safety and efficacy of the products, and it is responsibility of the applicants to provide a high quality dossier in accordance with the registration requirements of the country. The applicants submitting registration requests in Mexico need to be aware of the requirements. Similar to many other countries, Mexico has adopted the Common Technical Document (CTD) structure for organizing dossier of the medicinal product for submission into main modules (i.e., quality, non-clinical, and clinical). This facilitates the submission process of medicinal products following a logical sequence aligned to the International Council on Harmonisation (ICH) guidelines. Moreover, this structure improves the transparency and clarity of the dossier in process of evaluation of medicinal products. In Mexico, the Ministry of Health has published a regulation, NOM-257-SSA1-2014, which established the general requirements to be followed by applicants to complete the registration of biotherapeutics. This regulation stipulates that the evaluation process is supported by a regulatory framework involving Good Manufacturing Practices, labeling, stability, clinical trials, biocomparability studies, pharmacovigilance, and a technical evaluation performed by a multidisciplinary team of experts in biotherapeutics development. Additionally, the Mexican regulatory agency, COFEPRIS, has published specific guidelines to facilitate the application process. Despite the availability of this information, the scope is limited to regulatory and administrative purposes, rather than technical-scientific supporting knowledge. The aim of this article is to provide concise information to improve and promote communication between industry and regulatory agencies. Herein, we describe the current process of COFEPRIS in regulating biotherapeutics in Mexico. This process explains the basis for the organization and structure of the technical-scientific information of biotherapeutics required for registration application

Mycorrhiza induced resistance against pests: from the lab to the field

1 página - Conferencia invitada presentada en Iberian Plant Biology 2023. XVIII Portuguese-Spanish Congress on Plant Biology and the XXV Meeting of the Spanish Society of Plant Biology. 9-12 Julio 2023, Braga, PortugalArbuscular mycorrhizal fungi (AMF) can prime plant defences increasing their resistance against pathogens and insect herbivores. Using tomato as a model, we have shown that inoculation with different AMF reduces the performance of the chewing herbivore Spodoptera exigua and the leaf miner Tuta absoluta. Transcriptomic and metabolomics analyses revealed that this Mycorrhiza Induced Resistance (MIR) is associated to boosted activation of plant direct and indirect defences in response to the attackers. We found primed accumulation in attacked leaves of antiherbivore metabolites, including alkaloids and polyamine conjugates, and functional analyses demonstrated that some of the identified compounds significantly inhibit herbivore development. In addition, the symbiosis altered the volatile blends released by the plant, and enhanced the attraction of natural enemies of the pests (Nesidiocoris tenuis, commonly used in biocontrol programs). Finally, networks analyses allowed the identification of key regulators of the primed response within the jasmonic acid and ethylene signalling pathways. Despite the many studies showing induced resistance by microorganisms in different plant‐pest systems, the variability in the protection achieved under agronomic settings is hindering the application of this strategy in agriculture. Plant‐microbe‐herbivore interactions are highly context dependent, with multiple biotic and abiotic factors influencing the final output. Identifying such factors is essential to optimize the application of microbial inoculants for crop protection in agriculture. We found that the plant genotype and nutrient availability are important drivers of the context dependency of MIR in tomato. Despite of the variability, comparisons across different experimental scales, from controlled lab set‐ups to commercial production conditions, confirmed that MIR can be achieved under crop production conditions and is compatible with other biocontrol methods. Accordingly, MIR can be a relevant addition to current Integrated Pest Management Programs

Physicochemical Characteristics of Transferonƒ Batches

Transferon, a biotherapeutic agent that has been used for the past 2 decades for diseases with an inflammatory component, has been approved by regulatory authorities in Mexico (COFEPRIS) for the treatment of patients with herpes infection. The active pharmaceutical ingredient (API) of Transferon is based on polydispersion of peptides that have been extracted from lysed human leukocytes by a dialysis process and a subsequent ultrafiltration step to select molecules below 10 kDa. To physicochemically characterize the drug product, we developed chromatographic methods and an SDS-PAGE approach to analyze the composition and the overall variability of Transferon. Reversed-phase chromatographic profiles of peptide populations demonstrated batch-tobatch consistency from 10 representative batches that harbored 4 primary peaks with a relative standard deviation (RSD) of less than 7%. Aminogram profiles exhibited 17 proteinogenic amino acids and showed that glycine was the most abundant amino acid, with a relative content of approximately 18%. Further, based on their electrophoretic migration, the peptide populations exhibited a molecular mass of about 10 kDa. Finally, we determined the Transferon fingerprint using a mass spectrometry tool. Because each batch was produced from independent pooled buffy coat samples from healthy donors, supplied by a local blood bank, our results support the consistency of the production of Transferon and reveal its peptide identity with regard to its physicochemical attributes

Pharmacokinetic Comparability of a Biosimilar Trastuzumab Anticipated from Its Physicochemical and Biological Characterization

Comparability between a biosimilar and its reference product requires the evaluation of critical quality attributes that may impact on its pharmacological response. Herein we present a physicochemical characterization of a biosimilar trastuzumab focused on the attributes related to the pharmacokinetic response. Capillary isoelectrofocusing (cIEF) and cation exchange chromatography (CEX) were used to evaluate charge heterogeneity; glycosylation profiles were assessed through hydrophilic interaction liquid chromatography (HILIC); aggregates content was evaluated through size exclusion chromatography (SEC) while binding affinity to FcRn was evaluated using isothermal titration calorimetry (ITC). The biosimilar trastuzumab and its reference product exhibited a high degree of similarity for the evaluated attributes. In regard to the pharmacokinetic parameters, randomized, double blind, and two-arm parallel and prospective study was employed after the administration of a single intravenous dose in healthy volunteers. No significant differences were found between the pharmacokinetic profiles of both products. Our results confirm that similarity of the critical quality attributes between a biosimilar product, obtained from a different manufacturing process, and the reference product resulted in comparable pharmacokinetic profiles, diminishing the uncertainty related to the biosimilar's safety and efficacy

Characterization and comparability of biosimilars: A filgrastim case of study and regulatory perspectives for Latin America

Background: Developing countries have an estimate of ten times more approved biosimilars than developed countries. This disparity demands the need of an objective regulation that incorporates health policies according to the technological and economical capabilities of each country. One of the challenges lies on the establishment of comparability principles based on a physicochemical and biological characterization that should determine the extent of additional non-clinical and clinical studies. This is particularly relevant for licensed biosimilars in developing countries, which have an extensive clinical experience since their approval as generics, in some cases more than a decade. To exemplify the current status of biosimilars in Mexico, a characterization exercise was conducted on licensed filgrastim biosimilars using pharmacopeial and extended characterization methodologies. Results: Most of the evaluated products complied with the pharmacopeial criteria and showed comparability in their Critical Quality Attributes (CQAs) towards the reference product. These results were expected in accordance with their equivalent performance during their licensing as generics. Accordingly, a rational approval and registration renewal scheme for biosimilars is proposed, that considers the proper identification of CQAs and its thoroughly evaluation using selected techniques. Conclusions: This approach provides support to diminish uncertainty of exhibiting different pharmacological profiles and narrows or even avoids the necessity of comparative clinical studies. Ultimately, this proposal is intended to improve the accessibility to high quality biosimilars in Latin America and other developing countries

Effectiveness, safety/tolerability of OBV/PTV/r ± DSV in patients with HCV genotype 1 or 4 with/without HIV-1 co-infection, chronic kidney disease (CKD) stage IIIb-V and dialysis in Spanish clinical practice - Vie-KinD study

Limited data are available on the effectiveness and tolerability of direct-acting antivirals (DAAs) therapies in the real world for HCV-infected patients with comorbidities. This study aimed to describe the effectiveness of OBV/PTV/r ± DSV (3D/2D regimen) with or without ribavirin (RBV) in HCV or HCV/HIV co-infected patients with GT1/GT4 and CKD (IIIb-V stages), including those under hemodialysis and peritoneal dialysis in routine clinical practice in Spain in 2015.Non-interventional, retrospective, multicenter data collection study in 31 Spanish sites. Socio-demographic, clinical variables, study treatment characteristics, effectiveness and tolerability data were collected from medical records.Data from 135 patients with a mean age (SD) of 58.3 (11.4) years were analyzed: 92.6% GT1 (81.6% GT1b and 17.6% GT1a) and 7.4% GT4, 14 (10.4%) HIV/HCV co-infected, 19.0% with fibrosis F3 and 28.1% F4 by FibroScan®, 52.6% were previously treated with pegIFN and RBV. 11.1%, 14.8% and 74.1% of patients had CKD stage IIIb, IV and V respectively. 68.9% of patients were on hemodialysis; 8.9% on peritoneal dialysis and 38.5% had history of renal transplant. A total of 125 (96.2%) of 135 patients were treated with 3D, 10 (7.4%) with 2D and 30.4% received RBV. The overall intention-to-treat (ITT) sustained virologic response at week 12 (SVR12) was 92.6% (125/135) and the overall modified-ITT (mITT) SVR12 was 99.2% (125/126). The SVR12 rates (ITT) per sub-groups were: HCV mono-infected (91.7%), HCV/HIV co-infected (100%), GT1 (92.0%), GT4 (100%), CKD stage IIIb (86.7%), stage IV (95%) and stage V (93%). Among the 10 non-SVR there was only 1 virologic failure (0.7%); 4 patients had missing data due lost to follow up (3.0%) and 5 patients discontinued 3D/2D regimen (3.7%): 4 due to severe adverse events (including 3 deaths) and 1 patient´s decision.These results have shown that 3D/2D regimens are effective and tolerable in patients with advanced CKD including those in dialysis with GT 1 or 4 chronic HCV mono-infection and HIV/HCV coinfection in a real-life cohort. The overall SVR12 rates were 92.6% (ITT) and 99.2% (mITT) without clinically relevant changes in eGFR until 12 weeks post-treatment. These results are consistent with those reported in clinical trials