Cell-Engineered Recombinant α-Synuclein: A Gage R&R Validated Protocol.

α-Synuclein (α-Syn) misfolding and its presence in Lewy bodies are observed in almost all Parkinson's disease (PD) patients. Basic biomedical research would benefit from a quick, low-cost approach to purifying α-Syn and developing in vitro and in vivo models for PD. Several research groups utilize PFF-based models, yet the production of α-Syn PFFs is inconsistent, resulting in nonconclusive findings. Some research laboratories prepare recombinant α-Syn (r α-Syn) by molecular cloning to overexpress α-Syn with various purifying techniques. Laboratory-to-laboratory protocols cause considerable variability and sometimes contradictory findings. PD researchers spend more on protein than solving α-Syn's riddles. This article uncovered a novel method for expressing and purifying r α-Syn validated through gage reproducibility and repeatability (Gage R&R). For the production of r α-Syn, we have employed the ability of a high-cell-density-based expression system to overexpress protein in BL21(DE3). A simple, high-throughput, nonchromatographical purification protocol has been devised to facilitate research with higher reproducibility, which was validated through Gage R&R. A crossover experimental design was utilized, and the purified protein was characterized using orthogonal high-end analytical methods, which displayed higher similarity between the isolated r α-Syn. Batch-to-batch variability was the least for produced protein and hence can be utilized for exploring the iceberg of PD.

Identification of Surfactant Impact on a Monoclonal Antibody Characterization via HPLC-Separation Based and Biophysical Methods.

PURPOSE OR OBJECTIVE: Surfactants, including polysorbates and poloxamers, play a crucial role in the formulation of therapeutic proteins by acting as solubilizing and stabilizing agents. They help prevent protein aggregation and adsorption, thereby enhancing the stability of drug substance and products., However, it is important to note that utilizing high concentrations of surfactants in protein formulations can present significant analytical challenges, which can ultimately affect the product characterization. METHODS: In our study, we specifically investigated the impact of elevated surfactant concentrations on the characterization of monoclonal antibodies. We employed various analytical techniques including size-exclusion chromatography (SEC), capillary electrophoresis (CE-SDS), a cell based functional assay, and biophysical characterization. RESULTS: The findings of our study indicate that higher levels of Polysorbate 80 (PS-80) have adverse effects on the measured purity, biological activity, and biophysical characterization of biologic samples. Specifically, the elevated levels of PS-80 cause analytical interferences, which can significantly impact the accuracy and reliability of analytical studies. CONCLUSIONS: Our study results highlight a significant risk in analytical investigations, especially in studies involving the isolation and characterization of impurities. It is important to be cautious of surfactant concentrations, as they can become more concentrated during common sample manipulations like buffer exchange. Indeed, the research presented in this work emphasizes the necessity to evaluate the impact on analytical assays when there are substantial alternations in the matrix composition. By doing so, valuable insights can be gained regarding potential challenges associated with assay development and characterization of biologics with complex formulations.

Analytical and Antimicrobial Characterization of Zn-Modified Clays Embedding Thymol or Carvacrol.

Carvacrol and thymol are broad-spectrum natural antimicrobial agents. To reduce their volatility and improve their antimicrobial performance, synergistic systems were prepared loading the active molecules in zinc-modified clays. Montmorillonite (MMT) and zeolite (ZEO) were modified with zinc ions (ZnMMT and ZnZEO), with well-known antimicrobial properties, and then with carvacrol or thymol, reaching the 26 ± 3% and 33 ± 2% w/w of loading, respectively. The resulting hybrid materials were characterized by FT-IR, XPS, XRD, TGA, and GC-MS to evaluate carvacrol/thymol release in simulating food matrices. Antimicrobial assays carried out using spoiler and pathogenic bacterial strains showed that the antimicrobial activity of both thymol and carvacrol was largely preserved once they were loaded into Zn-modified clays. However, MMT hybrids showed an antibacterial activity significantly higher than ZEO hybrids at 50 mg/mL of thymol and carvacrol. For this reason, deeper antimicrobial evaluations were carried out only for ZnMMT composites. ZnMMT loaded with thymol or carvacrol produced inhibition zones against most of the target strains, also at 3.12 mg/mL, while the positive controls represented by the single molecule thymol or carvacrol were not active. The hybrid materials can be useful for applications in which the antimicrobial activity of natural molecules need to be displayed over time as requested for the control of microbial pathogens and spoilage bacteria in different applications, such as active packaging, biomaterials, and medical devices.

Characterization of beta subunit variants of recombinant human chorionic gonadotrophin.

Human chorionic gonadotropin (hCG), an endogenous glycoprotein hormone, has been widely used for the treatment of infertility and corpus luteum defect in women. The biological specificity of hCG is essentially determined by its beta (ß-) subunit, whereas the alpha (α-) subunit is a common subunit shared among the gonadotropin family. In development of a therapeutic recombinant hCG, the purity analysis showed that the beta (ß-) subunit has two variants, ß1 and ß2. Structural characterization using a combination of analytical techniques has demonstrated that ß1-subunit is derived from non-glycosylation at Asn 13, whereas ß2-subunit is a normal species with complete N-glycosylation at both Asn 13 and Asn 30. In vivo Bioactivity evaluation of the r-hCG fractions with various ratios of ß1-and ß2-subunits showed that incomplete glycosylation at Asn 13 potentially reduced the biological activity of r-hCG to promote uterus growth. Although hCG has a long history of medicinal use, this is the first report to identify the structural difference of hCG ß-subunit variants, as well as to preliminary establish the structure-activity relationship of this variation. The obtained results also suggest the importance of variant characterization and necessary quality control of product variants during the development of recombinant protein therapeutics.

Reference Standards to Support Quality of Synthetic Peptide Therapeutics.

PURPOSE: Peptides are an important class of therapeutics. Their quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength. OBJECTIVE: Discuss approaches to producing peptide reference standards, including vialing, lyophilization, analytical testing and stability studies. METHODS: Case studies are used to illustrate analytical approaches to characterize reference standards, including methods for value assignment, content uniformity, and identity testing. Methods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities. RESULTS: This report describes the analytical strategy used to establish peptide reference standard and illustrates how results from multiple labs are integrated to assign a value to the final lyophilized vial. A two-step process for value assignment is described, which uses a mass balance approach to assign a quantitative value to a bulk peptide material. The bulk material is then used as a standard to assign a final value to the vialed material. Testing to confirm peptide identity and to ensure consistency of the vialed material is also described. Considerations for addressing variability, identifying outliers, and implementing stability studies are also presented. CONCLUSION: The methods and case studies described provide a benchmark for best practices in establishing the preparation, analytical testing, handling, and storage of peptide reference standards for the pharmaceutical industry. Some peptide features, such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization of the peptide reference standard.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Similarity demonstrated between isolated charge variants of MB02, a biosimilar of bevacizumab, and Avastin® following extended physicochemical and functional characterization.

The majority of recombinant mAb products contain heterogeneous charge variants, commonly the result of post-translational modifications occurring during cell culture and accumulated during production, formulation and storage. MB02 is a biosimilar mAb to bevacizumab. Similarity data of charge variants for biosimilars against its reference products must be generated to demonstrate consistency in product quality and to ensure efficacy and safety. The goal of this work was to isolate seven charge variants of MB02 and Avastin® by semi-preparative cation exchange chromatography followed by purity test and extended analytical characterization to prove similarity. Although poor purity obtained for minor variants complicated data interpretation, an in-depth insight into the charge variants pattern of MB02 compared to Avastin® was obtained, contributing to a better understanding of modifications associated to microheterogeneity. To our knowledge, this is the first comparative analytical study of individual charge variants of a bevacizumab biosimilar following a head-to head approach and the most comprehensive N-glycosylation assessment of IgG1 charge variants. Although modifications related to N- and C-terminal, N-glycans, size heterogeneity or deamidation were specifically enriched among low abundant charge variants, they did not affect binding affinity to VEGF or FcRn and in vitro potency compared with the main species or unfractionated material.

Secure and Sustainable Sourcing of Plant Tissues for the Exhaustive Exploration of Their Chemodiversity.

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.

Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics.

PURPOSE: To develop an analytical platform for the estimation as well as characterization of aggregates over the complete size spectrum (from invisible monomer to visible precipitates). METHODS: Two mAb samples were incubated at 30°C in different buffer systems of protein A chromatography for observing degradation due to aggregation. The aggregation in these samples was quantified by size exclusion chromatography (SEC), dynamic light scattering (DLS), and micro flow imaging (MFI). RESULTS: The results obtained from various characterization tools were analysed in various size ranges - size exclusion chromatography (SEC) (1 nm - 25 nm), dynamic light scattering (DLS) (10 nm - 5 µm), and micro flow imaging (MFI) (2 µm - 300 µm). Since each characterization tool covers a particular size range, data from multiple tools was collected in the "handover" regions to demonstrate accuracy of the platform. CONCLUSIONS: Based on the observations from the experiments, an analytical platform has been proposed covering the whole size spectrum that would be of utility to those engaged in formulation development as well as other aspects related to stability of biotherapeutic products.

Biosimilars: Key regulatory considerations and similarity assessment tools.

A biosimilar drug is defined in the US Food and Drug Administration (FDA) guidance document as a biopharmaceutical that is highly similar to an already licensed biologic product (referred to as the reference product) notwithstanding minor differences in clinically inactive components and for which there are no clinically meaningful differences in purity, potency, and safety between the two products. The development of biosimilars is a challenging, multistep process. Typically, the assessment of similarity involves comprehensive structural and functional characterization throughout the development of the biosimilar in an iterative manner and, if required by the local regulatory authority, an in vivo nonclinical evaluation, all conducted with direct comparison to the reference product. In addition, comparative clinical pharmacology studies are conducted with the reference product. The approval of biosimilars is highly regulated although varied across the globe in terms of nomenclature and the precise criteria for demonstrating similarity. Despite varied regulatory requirements, differences between the proposed biosimilar and the reference product must be supported by strong scientific evidence that these differences are not clinically meaningful. This review discusses the challenges faced by pharmaceutical companies in the development of biosimilars.

Natural or Artificial? Multi-Analytical Study of a Scagliola from Estoi Palace Simulating Imperial Red Porphyry.

In this paper the characterization of a gypsum plaster sample from the end of the 19th century simulating imperial red porphyry using a multi-analytical approach is presented and discussed. The results of X-ray diffraction (XRD), thermogravimetric and differential thermal analysis (TGA-DTA), physical and mechanical properties are summarized. In order to have further insight into the microstructure, polarized light microscopy (PLM), scanning electron microscopy coupled with energy dispersive X-ray spectrometer (SEM-EDS), and micro Raman spectroscopy analyzes were also made. They helped to clarify the main issues raised by the other complementary analytical techniques and allowed the establishment of interrelations between the different properties, providing important information about the materials, the skills, and the technological development involved in the art of imitating noble stones with gypsum pastes. This study also contributes to our knowledge concerning the preservation of these types of elements that are important in the context of European decorative arts and rarely reported in the literature.

The critical role of peptide chemistry in the life sciences.

Peptide chemistry plays a key role in the synthesis and study of protein molecules and their functions. Modern ligation methods enable the total synthesis of enzymes and the systematic dissection of the chemical basis of enzyme catalysis. Predicted developments in peptide science are described.

Understanding solid-state properties of triglycerides used in pharmaceutical and food microencapsulation.

Hydrophobic materials, in particular hydrogenated vegetable oils, HVO, are extensively used as coating materials in food and pharmaceutical systems. Correct application of these coatings requires an evaluation of their behaviour as a function of various parameters such as melting temperature, solubility, concentration and/or pH. The purpose of this study was to assess the physico-chemical properties of an HVO in terms of composition, crystallisation, phase transition and polymorphism using a variety of analytical techniques, such as electrospray mass spectrometry (ESI-MS), differential scanning calorimetry (DSC) and X-ray diffraction (XRD). High-resolution ESI-MS allowed establishment of the HVO main composition of long-chain triglycerides (average molecular weight 1183 Da). DSC results showed that thermal history determines the formation of at least two polymorphs of HVO, namely two different crystal forms, assigned as form α, melting point (m.p.) 48 °C, and form ß', m.p. 60 °C. A third polymorph, the more thermodynamically stable ß-form, having a melting point at 62 °C, is obtained by solution-mediated re-crystallisation. Phase transformation paths were investigated by isothermal DSC experiments, which evidenced that the α-form is kinetically stable at temperatures lower than 25 °C. These data are of particular interest in practical applications such as spray freezing or pan coating where significant heat transfer phenomena are involved.

Biochemical characterization of immobilized recombinant subtilisin and synthesis and functional characterization of recombinant subtilisin capped silver and zinc oxide nanoparticles.

This pioneering research explores the transformative potential of recombinant subtilisin, emphasizing its strategic immobilization and nanoparticle synthesis to elevate both stability and therapeutic efficacy. Achieving an impressive 95.25 % immobilization yield with 3 % alginate composed of sodium along with 0.2 M CaCl2 indicates heightened pH levels and thermal resistance, with optimal action around pH 10 as well as 80 °C temperature. Notably, the Ca-alginate-immobilized subtilisin exhibits exceptional storage longevity and recyclability, affirming its practical viability. Comprehensive analyses of the recombinant subtilisin under diverse conditions underscore its adaptability, reflected in kinetic enhancements with increased Vmax (10.7 ± 15 × 103 U/mg) and decreased Km (0.19 ± 0.3 mM) values post-immobilization using N-Suc-F-A-A-F-pNA. UV-visible spectroscopy confirms the successful capping of nanoparticles made of Ag and ZnO by recombinant subtilisin, imparting profound antibacterial efficacy against diverse organisms and compelling antioxidant properties. Cytotoxicity was detected against the MCF-7 breast cancer line of cells, exhibiting IC50 concentrations at 8.87 as well as 14.52 µg/mL of AgNP as well as ZnONP, correspondingly, indicating promising anticancer potential. Rigorous characterization, including FTIR, SEM-EDS, TGA and AFM robustly validate the properties of the capped nanoparticles. Beyond therapeutic implications, the investigation explores industrial applications, revealing the versatility of recombinant subtilisin in dehairing, blood clot dissolution, biosurfactant activity, and blood stain removal. In summary, this research unfolds the exceptional promise of recombinant subtilisin and its nanoparticles, presenting compelling opportunities for diverse therapeutic applications in medicine. These findings contribute substantively to biotechnology and healthcare and stimulate avenues for further innovation and exploration.

Molecular expression, purification and structural characterization of recombinant L-Glutaminase from Streptomyces roseolus.

The present research reports the anti-cancer potential of recombinant L-Glutaminase from Streptomyces roseolus. L-Glutaminase gene was synthesized by codon-optimization, cloned and successfully expressed in E. coli BL21 (DE3). Affinity purified recombinant L-Glutaminase revealed a molecular mass of 32 kDa. Purified recombinant L-Glutaminase revealed stability at pH 7.0-8.0 with optimum activity at 70 °C further indicating its thermostable nature based on thermodynamic characterization. Recombinant L-Glutaminase exhibited profound stability in the presence of several biochemical parameters and demonstrated its metalloenzyme nature and was also found to be highly specific towards favorable substrate (l-Glutamine) based on kinetics. It demonstrated antioxidant property and pronounced cytotoxic effect against breast cancer (MCF-7 cell lines) in a dose dependent behavior with IC50 of 40.68 µg/mL. Matrix-assisted laser desorption ionization-time of flight-mass spectroscopy (MALDI-TOF-MS) analysis of desired mass peaks ascertained the recombinant L-Glutaminase identity. N-terminal amino acid sequence characterization through Edman degradation revealed highest resemblance for L-glutaminase within the Streptomyces sp. family. The purified protein was characterized structurally and functionally by employing spectroscopic methods like Raman, circular dichroism and nuclear magnetic resonance. The thermostability was assessed by thermogravimetric analysis. The outcomes of the study, suggests the promising application of recombinant L-Glutaminase as targeted therapeutic candidate for breast cancer.

Elemental characterization of electronic waste: a review of research methodologies and applicability to the practice of e-waste recycling.

Economic and environmental considerations have elucidated research interests on the best approach to managing electronic waste (e-waste), which has increasing social, environmental, and economic impacts. Proper e-waste managementis essential for resource recovery, environmental sustainability, and public health protection, and effective management of e-waste necessitates analytical techniques to assess and characterize their elemental composition. Despite expansive literature published on the topic of e-waste, there is scarce coverage of the various analytical techniques employed to characterize the inorganic contents of e-waste. This review discusses the various e-waste characterization techniques used in studies published between 2013 and 2023. Specifically, this review covers the analytical approaches employed to characterize the inorganic content of e-waste, the electronic devices or their components analyzed, the elements identified, the sample preparation methods adopted, and the merits and demerits of the analytical procedures. This review highlights the disparate approaches to e-waste characterization and the need for reliable and repeatable e-waste analysis and sample preparation methods.

An Industry Perspective on the use of Forced Degradation Studies to Assess Comparability of Biopharmaceuticals.

Forced degradation, also known as stress testing, is used throughout pharmaceutical development for many purposes including assessing the comparability of biopharmaceutical products according to ICH Guideline Q5E. These formal comparability studies, the results of which are submitted to health authorities, investigate potential impacts of manufacturing process changes on the quality, safety, and efficacy of the drug. Despite the wide use of forced degradation in comparability assessments, detailed guidance on the design and interpretation of such studies is scarce. The BioPhorum Development Group is an industry-wide consortium enabling networking and sharing of common practices for the development of biopharmaceuticals. The BioPhorum Development Group Forced Degradation Workstream recently conducted several group discussions and a benchmarking survey to understand current industry approaches for the use of forced degradation studies to assess comparability of protein-based biopharmaceuticals. The results provide insight into the design of forced degradation studies, analytical characterization and testing strategies, data evaluation criteria, as well as some considerations and differences for non-platform modalities (e.g., non-traditional mAbs). This article presents survey responses from several global companies of various sizes and provides an industry perspective and experience regarding the practicalities of using forced degradation to assess comparability.

Comprehensive physicochemical and functional analysis of pembrolizumab based on controlled degradation studies: Impact on antigen-antibody binding.

Monoclonal antibodies-based medicines are widely used in the treatment of different diseases. These medicines are very sensitive to exposure to different environmental conditions and their handling in hospitals may affect their safety and efficacy. This is the case for pembrolizumab (Keytruda®, 25 mg/mL), for which there is not yet much information on its risk behaviour associated with routine handling or unintentional mishandling. Here we performed a wider physicochemical and functional analysis of pembrolizumab medicine including controlled degradation studies: heat, freeze/thaw, agitation, accelerated light exposure and high hypertonic solution. After that, the samples were analysed by a set of analytical techniques to evaluated critical quality attributes: Far-UV CD, IT-FS, DLS, RP/UHPLC(UV)-MS, SE/UHPLC(UV), RP/UHPLC(UV)-MS/MS and ELISA. The results provide an in-depth understanding of the biochemical and biophysical properties of pembrolizumab, showing that the medicine is affected by accelerated light exposure and temperature of 60 °C, demonstrated by the detection of non-natural dimers and HMWS. Light exposure also revealed different isoform profile and increase in oxidations. Regarding functionality by means of the interaction antigen-antibody binding, all the stressors promoted a decrease in pembrolizumab capacity to bind to PD-1 receptor, although the biological activity remained still high for all of them, being 60 °C and accelerated light exposure the most affected.

Analytical techniques for the characterization of nanoparticles for mRNA delivery.

Nanotechnology-assisted RNA delivery has gotten a tremendous boost over the last decade and made a significant impact in the development of life-changing vaccines and therapeutics. With increasing numbers of emerging lipid- and polymer-based RNA nanoparticles progressing towards the clinic, it has become apparent that the safety and efficacy of these medications depend on the comprehensive understanding of their critical quality attributes (CQAs). However, despite the rapid advancements in the field, the identification and reliable quantification of CQAs remain a significant challenge. To support these efforts, this review aims to summarize the present knowledge on CQAs based on the regulatory guidelines and to provide insights into the available analytical characterization techniques for RNA-loaded nanoparticles. In this context, routine and emerging analytical techniques are categorized and discussed, focusing on the operation principle, strengths, and potential limitations. Furthermore, the importance of complementary and orthogonal techniques for the measurement of CQAs is discussed in order to ensure the quality and consistency of analytical methods used, and address potential technique-based differences.

Comprehensive Stress Stability Studies Reveal the Prominent Stability of the Liquid-Formulated Biotherapeutic Asymmetric Monovalent Bispecific IgG1 Monoclonal Antibody Format.

The developed asymmetric monovalent bispecific IgG1 or Duet monoclonal antibody (Duet mAb) has two distinct fragment antigen-binding region (Fab) subunits that target two different epitope specificities sequentially or simultaneously. The design features include unique engineered disulfide bridges, knob-into-hole mutations, and kappa and lambda chains to produce Duet mAbs. These make it structurally and functionally complex, so one expects challenging developability linked to instability, degradation of products and pathways, and limited reports available. Here, we have treated the product with different sources of extreme stress over a lengthy period, including varying heat, pH, photo stress, chemical oxidative stress, accelerated stress in physiological conditions, and forced glycation conditions. The effects of different stress conditions on the product were assessed using various analytical characterization tools to measure product-related substances, post-translational modifications (PTMs), structural integrity, higher-order disulfide linkages, and biological activity. The results revealed degradation products and pathways of Duet mAb. A moderate increase in size, charge, and hydrophobic variants, PTMs, including deamidation, oxidation, isomerization, and glycation were observed, with most conditions exhibiting biological activity. In addition, the characterization of fractionated charge variants, including deamidated species, showed satisfactory biological activity. This study demonstrated the prominent stability of the Duet mAb format comparable to most marketed mAbs.