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2023

Ana Ferjančič Budihna, Anže Martinčič Celjar, Sergeja Lebar, Andreja Gramc Livk and Aleš Štrancar

Cell & Gene Therapy Insights 2023; 9(9), 1231–1247 | DOI: 10.18609/cgti.2023.161

Rapid advancement of mRNA technology, as a response to the COVID-19 crisis, prompted an increased need for precise analytical methods to support the fast-paced mRNA process development. Accurate and robust analytics are required to support modifications in the mRNA production process, protocols, raw materials, in vitro transcription reaction, purification methods, scale-up, or final formulation processes, to ensure high quality and safety of the final product. This Innovator Insight demonstrates the application of an ion pair reverse phase chromatographic analytical method as a robust analytical tool to determine mRNA fragmentation while also separating in vitro transcription components from the main product. The method’s efficacy is assessed through a comprehensive stability study of a mRNA standard at different temperatures. The chromatographic analytical results are compared to the ones obtained by the capillary gel electrophoresis, a well-established method for the analysis of fragmented mRNA.

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Irena Trbojević-Akmačić, Frano Vučković,Tea Pribić, Marija Vilaj, Urh Černigoj, Jana Vidič, Jelena Šimunović, Agnieszka Kępka, Ivana Kolčić, Lucija Klarić, Mislav Novokmet, Maja Pučić-Baković, Erdmann Rapp, Aleš Štrancar, Ozren Polašek, James F. Wilson and Gordan Lauc 

Communications Biology volume 6, Article number: 312 (2023)

Human plasma transferrin (Tf) N-glycosylation has been mostly studied as a marker for congenital disorders of glycosylation, alcohol abuse, and hepatocellular carcinoma. However, inter-individual variability of Tf N-glycosylation is not known, mainly due to technical limitations of Tf isolation in large-scale studies. Here, we present a highly specific robust high-throughput approach for Tf purification from human blood plasma and detailed characterization of Tf N-glycosylation on the level of released glycans by ultra-high-performance liquid chromatography based on hydrophilic interactions and fluorescence detection (HILIC-UHPLC-FLD), exoglycosidase sequencing, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We perform a large-scale comparative study of Tf and immunoglobulin G (IgG) N-glycosylation analysis in two human populations and demonstrate that Tf N-glycosylation is associated with age and sex, along with multiple biochemical and physiological traits. Observed association patterns differ compared to the IgG N-glycome corroborating tissue-specific N-glycosylation and specific N-glycans’ role in their distinct physiological functions.

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Lucija Rebula, Andrej Raspor, Mojca Bavčar, Aleš Štrancar and Maja Leskovec

Journal of Chromatography B, Volume 1217, 15 February 2023

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.
In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.

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2022
  • Can IVT yields be increased beyond 5-8 g/L?
  • Does feeding nucleotides into the IVT reaction increase its yield?
  • Is there a fast analytical method to quantify NTPs in IVT in real-time?
  • Can production of mRNA be automated?
Transitioning from batch to fed-batch IVT can increase IVT yield to 12 g/L resulting in 50 % reduction in cost per gram of mRNA. Integrating HPLC monitoring of IVT reaction can allow real-time decisions on feed additions.

Janja Skok, Polona Megušar, Tina Vodopivec, Domen Pregeljc, Nina Mencin, Matevž Korenč, Andreja Krušič, Anže Martinčič Celjar, Nejc Pavlin, Jana Krušič, Matthias Mueller, Kevin McHugh, Aleš Štrancar, and Rok Sekirnik

Chemie Ingenieur Techik, October 2022

The COVID-19 pandemic triggered an unprecedented surge in development of mRNA-based vaccines. Despite the need to increase process productivity and thus decrease the cost of mRNA vaccines, limited scientific literature is available on strategies to increase the yield of in vitro transcription (IVT) reaction, the unit operation with highest cost of goods, which has traditionally been performed as a batch reaction. Single-use bioreactors are traditionally used for cell-based production of biopharmaceuticals, but some core functionalities, such as controlled and automated feed addition, are potentially useful for cell-free mRNA processes. We report the production of 2 g mRNA in an Ambr® 250 Modular bioreactor system with a starting volume of 100 mL, reaching a maximum mRNA concentration of 12 g L−1 by a fed-batch IVT approach, and demonstrate the feasibility of continuous fed-batch production, paving the way towards continuous manufacturing of mRNA.

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Ana Ferjančič Budihna, Nejc Pavlin, Anže Martinčič Celjar, Andreja Gramc Livk and Aleš Štrancar

BioProcess International eBook, September 14, 2022

Robust and precise chromatographic analytical methods are key for the efficient development of the mRNA production process.

Three different analytical methods, which utilize three different column chemistries, are embedded in a ready-to-use PATfix™ analytical platform to support mRNA process development and product quantification and characterization.

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Katarina Markovič, Maja Cemazar, Gregor Sersa, Radmila Milačič and Janez Sčančar

Journal of Analytical Atomic Spectrometry

Ceruloplasmin (Cp) is the major copper-carrying (Cu) protein in human plasma. Due to copper's important physiological functions and its role in various diseases, there is a need to quantify the concentration bound to Cp and the exchangeable form of Cu. In the present work, conjoint liquid chromatography (CLC) on short-bed convective interaction media (CIM) monolithic disks was used to separate the Cu bound to low molecular mass (LMM) species, and the Cu bound to Cp and albumin (HSA) in human serum. Two immunoaffinity CIMmic albumin depletion (α-HSA) disks and one CIMmic weak anion-exchange diethylaminoethyl (DEAE) disk were assembled in a single housing, forming a CLC monolithic column. By applying isocratic elution with a 50 mmol L−1 MOPS buffer (pH 7.4) in the first 3 min, followed by gradient elution with 1 mol L−1 NH4Cl (pH 7.4) in the next 9 min, HSA was retained by the α-HSA disk, allowing subsequent separation of the LMM-Cu from the Cu bound to the Cp on the DEAE disk. Further elutio with 0.5 mol L−1 acetic acid in the next 4 min rinsed the HSA from the α-HSA disk. The separated Cu species were quantified by post column isotope dilution inductively coupled plasma mass spectrometry (ID-ICP-MS), while the elution profile of the proteins was followed by UV detection at 278 nm. Quantitative column recoveries were obtained. Good repeatability of the measurement was achieved for Cu-Cp (±1%), while for Cu-HSA and Cu-LMM species the repeatability of the measurements was slightly worse, due to the much lower Cu concentrations (±6% and ±9%, respectively). The developed method required only 20 μL of a 15-times diluted sample. Low limits of detection for the Cu-Cp, Cu-HSA and Cu-LMM species (6.1, 5.3 and 3.3 ng mL−1 Cu, respectively) were obtained. The technique was successfully applied in the determination of Cu-Cp, Cu-HSA and a fraction that most probably corresponds to the Cu-LMM species in the human serum of healthy individuals, kidney transplant patients and cancer patients.

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2021

by Nejc Pavlin, Blaž Bakalar, Janja Skok, Špela Kralj, Andreja Gramc Livk, and Aleš Štrancar

BioProcess International, October 2021

Abstract:

Plasmid DNA (pDNA) has become a crucial component in the production of next generation therapeutics such as messenger RNA (mRNA) and viral vectors.

As companies ramp up their production capabilities and move towards clinical applications, obtaining cGMP grade pDNA has become a production bottleneck, leading to lengthy production delays.

There is a growing market demand for solutions that can streamline the production of cGMP pDNA and help optimize down-stream processes (DSP) for better yields & purity.

The key step in this process is having quantifiably reliable analytics that give rapid results
for process optimization and scale-up, as well as production runs.

Establishing and expanding inhouse pDNA production platform in a quick and efficient manner will be a key differentiator between more and less successful next generation therapeutics projects.

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Michael Winkler, Mikhail Goldfarb, Shaojie Weng, Jeff Smith, Susan Wexelblat, John Li, Alejandro Becerra, Sandra Bezemer, Kevin Sleijpen, Aleš Štrancar, Sara Primec, Romina Zabar, April Schubert, Akunna Iheanacho, and David Cetlin

BioProcess International, April 2021

Abstract

Over the past decade, adenoassociated virus (AAV) vectors have become established as leading gene-delivery vehicles. In 2017, the pipeline for gene therapies included 351 drugs in clinical trials and 316 in preclinical development. As those candidates advance, significant efforts are being made in process development and manufacturing for viral vectors, with the overall goal of reducing process impurities while maintaining the highest possible process yield.

Sartorius BIA Separations has developed and commercialized CIMmultus QA monoliths, which have been cited in several AAV downstream processes for their ability to separate empty and full virus particles effectively. Monolithic supports represent a unique type of stationary phase for liquid chromatography, bioconversion, and solid-phase synthesis. Aside from increased processing speed, monolithic flow-through pores (channels) also provide easy access for large molecules, which supports both purification and depletion of nanoparticles such as plasmid DNA (pDNA) molecules and AAV particles.

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Petrović T, Alves I, Bugada D, Pascual J, Vučković F, Skelin A, Gaifem J, Villar-Garcia J, Vicente MM, Fernandes Â, Dias AM, Kurolt IC, Markotić A, Primorac D, Soares A, Malheiro L, Trbojević-Akmačić I, Abreu M, Sarmento E Castro R, Bettinelli S, Callegaro A, Arosio M, Sangiorgio L, Lorini LF, Castells X, Horcajada JP, Pinho SS, Allegri M, Barrios C, Lauc G.

Glycobiology. 2020 Nov.

Abstract:

A large variation in the severity of disease symptoms is one of the key open questions in COVID-19 pandemics. The fact that only a small subset of people infected with SARS-CoV-2 develop severe disease suggests that there have to be some predisposing factors, but biomarkers that reliably predict disease severity have not been found so far. Since overactivation of the immune system is implicated in a severe form of COVID-19 and the IgG glycosylation is known to be involved in the regulation of different immune processes, we evaluated the association of inter-individual variation in IgG N-glycome composition with the severity of COVID-19. The analysis of 166 severe and 167 mild cases from hospitals in Spain, Italy and Portugal revealed statistically significant differences in the composition of the IgG N-glycome. The most notable difference was the decrease in bisecting Nacetylglucosamine (GlcNAc) in severe patients from all three cohorts. IgG galactosylation was also lower in severe cases in all cohorts, but the difference in galactosylation was not statistically significant after correction for multiple testing. To our knowledge, this is the first study exploring IgG N-glycome variability in COVID-19 severity.

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2019

Calef Sánchez-Trasviña, Marco Rito-Palomares, and José González-Valdez

Advances in Polymer Technology, Volume 2019, December 12 2019, 10 pages

Abstract

PEGylated or polyethylene glycol-modified proteins have been used as therapeutic agents in different diseases. However, the major drawback in their procurement is the purification process to separate unreacted proteins and the PEGylated species. Several efforts have been done to separate PEGylation reactions by chromatography using different stationary phases and modified supports. In this context, this study presents the use of chromatographic monoliths modified with polyethylene glycol (PEG) to separate PEGylated Ribonuclease A (RNase A). To do this, Convective Interaction Media (CIM) Ethylenediamine (EDA) monolithic disks were PEGylated using three PEG molecular weights (1, 10, and 20 kDa). The PEGylated monoliths were used to separate PEGylated RNase A modified, as well, with three PEG molecular weights (5, 20, and 40 kDa) by hydrophobic interaction chromatography. Performance results showed that Bovine Serum Albumin (BSA) can bind to PEGylated monoliths and the amount of bound BSA increases when ammonium sulfate concentration and flow rate increase. Furthermore, when PEGylated RNase A was loaded into the PEGylated monoliths, PEG-PEG interactions predominated in the separation of the different PEGylated species (i.e., mono and di-PEGylated). It was also observed that the molecular weight of grafted PEG chains to the monolith impacts strongly in the operation resolution. Interestingly, it was possible to separate, for the first time, isomers of 40 kDa PEGylated RNase A by hydrophobic interaction chromatography. This technology, based on PEGylated monoliths, represents a new methodology to efficiently separate proteins and PEGylated proteins. Besides, it could be used to separate other PEGylated molecules of biopharmaceutical or biotechnological interest.

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Katarina Marković, Radmila Milačič, Janja Vidmar, Stefan Marković, Katja Uršič, Martina Nikšić Žakeljc, Maja Cemazar, Gregor Sersa, Mojca Unk, Janez Ščančar

Journal of Trace Elements in Medicine and Biology, Volume 57, January 2020, Pages 28-39.

Abstract

Monolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run. Here, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. The data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. Low-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.

Keywords: low-pressure and high-pressure conjoint liquid chromatography, anion-exchange and affinity monolithic disks, inductively coupled plasma mass spectrometry, Pt-based chemotherapeutics, serum of cancer patients

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J. R. Lorsch, A. M. Munoz, J. S. Nanda, V. Rajagopal, P. Yourik, S. E. Walker

RNA Biology (2017), volume 14 (2), pp. 188–196.
Published online 2016 Dec 16.

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

KEYWORDS: Eukaryotic translation, in vitro translation, ribosome, ribosome purification, yeast

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Thanaporn Liangsupree, Evgen Multia, Jari Metso, Matti Jauhiainen, Patrik Forssén, Torgny Fornstedt, Katariina Öörni, Aleš Podgornik & Marja-Liisa Riekkola 

Scientific Reports, volume 9, August 2019

Low-density lipoprotein (LDL) is considered the major risk factor for the development of atherosclerotic cardiovascular diseases (ASCVDs). A novel and rapid method for the isolation of LDL from human plasma was developed utilising affinity chromatography with monolithic stationary supports. The isolation method consisted of two polymeric monolithic disk columns, one immobilized with chondroitin-6-sulfate (C6S) and the other with apolipoprotein B-100 monoclonal antibody (anti-apoB-100 mAb). The first disk with C6S was targeted to remove chylomicrons, very-low-density lipoprotein (VLDL) particles, and their remnants including intermediate-density lipoprotein (IDL) particles, thus allowing the remaining major lipoprotein species, i.e. LDL, lipoprotein(a) (Lp(a)), and high-density lipoprotein (HDL) to flow to the anti-apoB-100 disk. The second disk captured LDL particles via the anti-apoB-100 mAb attached on the disk surface in a highly specific manner, permitting the selective LDL isolation. The success of LDL isolation was confirmed by different techniques including quartz crystal microbalance. In addition, the method developed gave comparable results with ultracentrifugation, conventionally used as a standard method. The reliable results achieved together with a short isolation time (less than 30 min) suggest the method to be suitable for clinically relevant LDL functional assays.

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2018

The purpose of this book is to provide you with a guide to developing monoclonal antibody purification procedures taht meet the requirements of both research and commercial applications. It is based on successful purifications developed for over 250 monoclonal-based products, addressing a wide range of diagnostic and therapeutic applications. it is supported by nearly 1000 citations from the scientific literature and enriched by the insights of skilled practitioners from throught the industry. It incorporates over 100 figures and tables to illustrate key concepts.

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Tsutomu Arakawa, Pete Gagnon

Journal of Pharmaceutical Sciences 107 (2018) 2297-2305

The concept of cosolvent exclusion was developed by a group of Timasheff's laboratory in 1970-1990 and is currently used widely to explain the effects of a variety of cosolvents on the stability and solubility of macromolecules. Not surprisingly, these concepts have had substantial influence in the fields of formulation, protein folding and unfolding, but they have perhaps more surprisingly found their way into the field of chromatography. A variety of excluded cosolvents have been used to enhance binding and resolution of proteins and other macromolecules in ion exchange, hydroxyapatite, affinity, and hydrophobic interaction chromatography. These cosolvents include salting-out salts, amino acids and polymers, and frequently polyethylene glycol (PEG). A new mode of chromatography, termed “steric exclusion chromatography,” was recently introduced. It employs hydroxylated solid phase surfaces. Steric exclusion of the PEG stabilizes the association of macromolecules with the solid phase. Elution is achieved by reducing the PEG concentration. Magnetic particles are also used in this chromatography. This review summarizes the concepts of preferential cosolvent exclusion and its applications in column chromatography.

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2017

V.Rajamanickam, D.Wurm, C.Slouka, C.Herwig, O.Spadiut

Anal Bioanal Chem (2016)

The bacterium Escherichia coli is a well-studied recombinant host organism with a plethora of applications in biotechnology. Highly valuable biopharmaceuticals, such as antibody fragments and growth factors, are currently being produced in E. coli. However, the high metabolic burden during recombinant protein production can lead to cell death, consequent lysis, and undesired product loss. Thus, fast and precise analyzers to monitor E. coli bioprocesses and to retrieve key process information, such as the optimal time point of harvest, are needed. However, such reliable monitoring tools are still scarce to date. In this study, we cultivated an E. coli strain producing a recombinant single-chain antibody fragment in the cytoplasm. In bioreactor cultivations, we purposely triggered cell lysis by pH ramps. We developed a novel toolbox using UV chromatograms as fingerprints and chemometric techniques to monitor these lysis events and used flow cytometry (FCM) as reference method to quantify viability offline. Summarizing, we were able to show that a novel toolbox comprising HPLC chromatogram fingerprinting and data science tools allowed the identification of E. coli lysis in a fast and reliable manner. We are convinced that this toolbox will not only facilitate E. coli bioprocess monitoring but will also allow enhanced process control in the future

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Marina Naldi, Urh Černigoj, Ales Štrancar, Manuela Bartolini

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5 mm×5.2 mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90 ± 9 s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200 ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.

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Sebastijan Peljhan, Tina Jakop, Dunja Šček, Vid Skvarča, Blaž Goričar, Romina Žabar, Nina Mencin. Electrophoresis 2017 July 20

The plasma-derived IgG used either for diagnostic purpose or intravenous application (in form of IVIG) in various medical therapies is certainly gaining more and more attention on annual basis. Different manufacturing processes are used to isolate immunoglobulins from human plasma. However, a quest for alternative paths in IgG isolation not only requires development of the most efficient isolation process, but also a rapid and reliable analytics to track the purification. Fast and reliable fingerprint based method for characterization of IgG prepared from Cohn I+II+III paste is presented in this paper. The fingerprint method bases on partial separation of proteins in linear gradient on CIMacTM quaternary amine, strong anion exchange group (QA) 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG isolation from Cohn I+II+III fraction paste, but very accurate qualitative information about the composition of the sample can be obtained in less than 5 min. From the differences in the chromatograms of various samples, the ratio between IgG and impurities in each sample can be easily assessed. The method is suitable for input material control, in-line monitoring of the downstream processing, final control of the products, as well as in stability studies and enables taking fast and accurate decisions during fractionation process.

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Antonio M. Munoz, Paul Yourik, Vaishnavi Rajagopal, Jagpreet S. Nanda, Jon R. Lorsch, Sarah E. Walker

RNA Biology, 2017, VOL. 14, NO. 2, 188–196

In vitro studies of translation provide critical mechanistic details, yet purification of large amounts of highly active eukaryotic ribosomes remains a challenge for biochemists and structural biologists. Here, we present an optimized method for preparation of highly active yeast ribosomes that could easily be adapted for purification of ribosomes from other species. The use of a nitrogen mill for cell lysis coupled with chromatographic purification of the ribosomes results in 10-fold-increased yield and less variability compared with the traditional approach, which relies on sedimentation through sucrose cushions. We demonstrate that these ribosomes are equivalent to those made using the traditional method in a host of in vitro assays, and that utilization of this new method will consistently produce high yields of active yeast ribosomes.

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2016

M. Naldi, M. Baldassarre, M. Domenicali, F. A. Giannone, M. Bossic, J. Montomoli,T. D. Sandahl, E. Glavind, H. Vilstrup, P. Caraceni, C. Bertucci
Journal of Pharmaceutical and Biomedical Analysis, Volume 122 (2016) 141-147

Human serum albumin (HSA) is the most abundant plasma protein, endowed with several biological properties unrelated to its oncotic power, such as antioxidant and free-radicals scavenging activities, binding and transport of many endogenous and exogenous substances, and regulation of endothelial function and inflammatory response. These non-oncotic activities are closely connected to the peculiarly dynamic structure of the albumin molecule. HSA undergoes spontaneous structural modifications, mainly by reaction with oxidants and saccharides; however, patients with cirrhosis show extensive post-transcriptional changes at several molecular sites of HSA, the degree of which parallels the severity of the disease. The present work reports the development and application of an innovative LC–MS analytical method for a rapid and reproducible determination of the relative abundance of HSA isoforms in plasma samples from alcoholic hepatitis (AH) patients. A condition of severe oxidative stress, similar to that observed in AH patients, is associated with profound changes in circulating HSA microheterogeneity. More interestingly, the high resolution provided by the analytical platform allowed the monitoring of novel oxidative products of HSA never reported before.

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